JP2019510506A - Anti-podocalyxin-like protein precursor subtype 2 monoclonal antibody, method for producing the same and use thereof - Google Patents
Anti-podocalyxin-like protein precursor subtype 2 monoclonal antibody, method for producing the same and use thereof Download PDFInfo
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Abstract
胃癌細胞の表面に機能的に発現するポドカリキシン様タンパク質前駆体サブタイプ２（ＰＯＤＸＬ−ｖ２）に対するモノクローナル抗体とその生成方法及び使用を提供し、前記抗体のＬ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：２又はその保存的変異体配列であり、Ｈ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：４又はその保存的変異体配列である。
The present invention provides a monoclonal antibody against podocalyxin-like protein precursor subtype 2 (PODXL-v2) functionally expressed on the surface of gastric cancer cells, a method for producing the same and use thereof, wherein the amino acid sequence in the light chain variable region of said antibody is SEQ ID The amino acid sequence in the H chain variable region is SEQ ID NO: 4 or a conservative variant sequence thereof.
Description
本発明はバイオファーミング分野に関し、特に、胃癌細胞の表面に機能的に発現するポドカリキシン様タンパク質前駆体サブタイプ２（ＰＯＤＸＬ−ｖ２）に対するモノクローナル抗体（ＭＳ１７−３８と命名）とその生成方法及び使用に関する。 The present invention relates to the field of biopharming, and in particular to a monoclonal antibody (designated as MS17-38) against podocalyxin-like protein precursor subtype 2 (PODXL-v2) functionally expressed on the surface of gastric cancer cells, a method for producing the same and use thereof .
発明者である陸梅生らが過去に出願した「細胞表面の異所性発現に抵抗するモノクローナル抗体ＭＳ１７−５７とその生成方法及び使用（特許文献１。公開・許可済）の背景知識に記載されているように、胃癌（Ｇａｓｔｒｉｃ Ｃａｎｃｅｒ，ＧＣ）はヒトにとって最も一般的な消化器系の悪性腫瘍であって、腫瘍関連死の主な原因の一つとなっている。中国、日本などは世界で最も胃癌発症率の高い国である。これらの国では毎年新たに６０〜７０万近い発症例が確認されており、世界の症例の多数を占めている。また、胃癌患者の多くは初診の段階ですでに臨床末期となっている。現在、胃癌の早期スクリーニング検査や診断は主に胃カメラと組織病理学的生検に依存しているが、こうした早期スクリーニング検査や診断に依存した手段では、その特異性、感度、精度及び安全性にある程度の限界があり、普及が困難である。また、様々な干渉要因が存在するほか、臨床での応用効果が満足のいくものとなっていない。結果、胃癌患者の多くには初診の段階ですでに他の臓器への転移が認められ、外科手術や放射線治療、全身化学療法等に頼らざるを得ない。こうした従来の治療或いは救命対策は費用が高騰するだけでなく、個人や社会の経済的負担が大幅に増加するほか、治療の特異性や治療効果が十分でない等の課題がある。概算によれば、末期胃癌患者の５年生存率は一般的に１０％以下となっている。よって、胃癌における特異的且つ高効率の新たな臨床治療方法が緊急に必要とされている。近年、新たな化学治療薬や併用療法（オーダーメイド医療）の実施によって胃癌患者の治療反応性はある程度向上している。しかし、臨床研究についてランダム調査を実施した結果、生存期間中央値１年以上との治療目標は未だ達成されていないことがわかった。 Described in the background knowledge of “Monoclonal antibody MS17-57 that resists ectopic expression on cell surface and its production method and use (Patent Document 1: published and permitted), previously filed by the inventor Rikumei et al. Gastric cancer (Gastric Cancer, GC) is one of the most common digestive system malignancies for humans, and is one of the main causes of tumor-related death. It is the country with the highest incidence of gastric cancer, and in these countries new cases of about 600 to 700,000 cases are newly confirmed every year, accounting for a large number of cases in the world. At present, early screening tests and diagnoses for gastric cancer rely mainly on gastroscope and histopathological biopsies, but hands that depend on such early screening tests and diagnoses There are some limitations in its specificity, sensitivity, accuracy and safety, and it is difficult to disseminate, and there are various interference factors and clinical application effects are not satisfactory. As a result, in many patients with gastric cancer, metastasis to other organs is already recognized at the initial examination, and they have to rely on surgery, radiation therapy, systemic chemotherapy, etc. Such conventional treatment or life-saving measures In addition to soaring costs, the economic burden on individuals and society is greatly increased, and there are problems such as treatment specificity and inadequate treatment effect. The rate is generally less than 10%, so there is an urgent need for new and specific clinical treatment methods for gastric cancer that are both specific and highly efficient. Medical practice) The therapeutic response of patients with gastric cancer has improved to some extent, but as a result of conducting a random survey of clinical studies, it was found that the treatment goal with a median survival of 1 year or more has not yet been achieved.
化学療法や放射線治療は胃癌に対する感度が一般的に低く、且つ、これらに代わる治療プランも存在しない。そのため、ここ数年は胃癌の新たな治療方法や薬物の研究開発が基礎研究及び臨床研究の焦点となっている。２００２年、米国国立癌研究所（ＮＣＩ）の癌専門家らは、腫瘍患者のカスタマイズ治療や再発検査、予後評価等が効果的に実現されるよう、腫瘍の分子分析について定義を提案した。こうした新たな理念を指針として、胃癌の分子標的治療や分子生体機能学的治療が急速に発展しており、腫瘍治療の新たな流れともなっている。 Chemotherapy and radiation therapy are generally less sensitive to gastric cancer, and there is no alternative treatment plan. Therefore, in recent years, research and development of new treatment methods and drugs for gastric cancer have been the focus of basic research and clinical research. In 2002, oncologists at the National Cancer Institute (NCI) proposed a definition for molecular analysis of tumors so that customized treatment, relapse testing, prognosis evaluation, etc. of tumor patients could be effectively realized. With these new ideas as a guide, molecular targeted therapy and molecular biofunctional therapy for gastric cancer are rapidly developing, and it has become a new trend of tumor therapy.
発明者である陸梅生らは、過去に出願した「細胞表面の異所性発現に抵抗するモノクローナル抗体とその生成方法及び使用」（特許文献１。開示・許可済）において、更に、腫瘍マーカーを標的とした治療や生体機能学的治療では、腫瘍細胞の表面に過剰に発現又は機能的に発現する何らかのバイオマーカー分子を標的にすると述べている。１９９０年代以降、様々な分子標的治療薬（抗体薬を含む）が続々と臨床試験における腫瘍治療に取り入れられ、良好な治療効果を示してきた。これらは、大分子のモノクローナル抗体（細胞外に作用する治療抗体薬）と、小分子のチロシンキナーゼ阻害薬（細胞内に作用する化学系薬物）の２種類に大別される。抗体薬は、対応する腫瘍細胞外受容体を高度な特異性をもって識別及び結合可能であり、速やかに細胞の生体機能を活性化する。また、機能の活性化が不可能な場合には、１．抗体−薬物複合（Ａｎｔｉｂｏｄｙ Ｄｒｕｇ Ｃｏｎｊｕｇａｔｉｏｎ，ＡＤＣ）、２．抗体と放射性同位体を結合して実施する特異性放射免疫療法、３．キメラ抗原受容体（Ｃｈｉｍｅｒｉｃ Ａｎｔｉｇｅｎ Ｒｅｃｅｐｔｏｒ，ＣＡＲ）によるＴ細胞免疫療法、といったその他いくつかの抗体療法が存在する。我々は、これらを腫瘍の抗体標的療法（Ａｎｔｉｂｏｄｙ Ｔａｒｇｅｔｉｎｇ Ｔｈｅｒａｐｙ，ＡＴＴ）と抗体生体機能学的療法（Ａｎｔｉｂｏｄｙ Ｂｉｏｆｕｎｃｔｉｏｎａｌ Ｔｈｅｒａｐｙ，ＡＢＴ）としてまとめた。更に、後者の抗体生体機能学的療法（ＡＢＴ）は、抗体と生体機能を連携させるアゴニスト（Ａｇｏｎｉｓｔ）と、抗体と生体機能を競わせるアンタゴニスト（Ａｎｔａｇｏｎｉｓｔ）に分けられる。抗体の生体機能は主な役割として、腫瘍細胞から発生して成長する関連分子を標的とし、腫瘍の特異的殺傷を実現することで、転移を防止したり、腫瘍を成長させる微小環境に干渉したりする。そのため、従来の化学療法や放射線治療、東洋医学・漢方薬と比較して、抗体薬は効果がより明らかであり、効用が長く持続し、且つ副作用も少ない。したがって、腫瘍の抗体生体機能学的療法は腫瘍治療発展の大勢になるものと考えられる。 Rikumei, the inventor of the present invention, further filed a tumor marker in the previously filed "Monoclonal antibody that resists ectopic expression on cell surface and its production method and use" (Patent Document 1: disclosed and permitted). Targeted therapies and biofunctional therapies are said to target any biomarker molecule that is overexpressed or functionally expressed on the surface of tumor cells. Since the 1990's, various molecularly targeted therapeutic agents (including antibody drugs) have been successively incorporated into tumor treatment in clinical trials and have shown good therapeutic effect. These are roughly classified into two types: large molecule monoclonal antibodies (therapeutic antibody drugs acting extracellularly) and small molecule tyrosine kinase inhibitors (chemical drugs acting intracellularly). Antibody drugs can identify and bind corresponding tumor extracellular receptors with high specificity, and rapidly activate cellular biological functions. In addition, when activation of the function is impossible, Antibody-drug conjugate (Antibody Drug Conjugation, ADC); Specific radioimmunotherapy performed by combining an antibody and a radioactive isotope, Several other antibody therapies exist, such as T cell immunotherapy with Chimeric Antigen Receptor (CAR). We summarized these as Tumor Targeting Therapy (ATT) and Antibody Biofunctional Therapy (ABT). Furthermore, the latter antibody biofunctional therapy (ABT) is divided into an agonist (Agonist) that links antibody and biological function, and an antagonist (Antagonist) that competes between antibody and biological function. The biological function of the antibody plays a major role, by targeting related molecules generated and grown from tumor cells, achieving specific killing of the tumor, preventing metastasis or interfering with the microenvironment that causes the tumor to grow. To Therefore, compared with conventional chemotherapies, radiotherapy, and Oriental medicine / Chinese medicine, antibody drugs have more obvious effects, long lasting utility, and less side effects. Therefore, antibody biofunctional therapy of tumors is considered to be the majority of tumor treatment development.
発明者である陸梅生らが過去に出願した「細胞表面の異所性発現に抵抗するモノクローナル抗体とその生成方法及び使用」（特許文献１。開示・許可済）では、胃癌の抗体治療の背景について、更に、抗体薬による胃癌治療がここ数年急速に発展しているとの記載がある。しかし、現在米国ＦＤＡの承認を受けている一部抗体薬については、前臨床毒性試験や各臨床フェーズでの症例報告から、特異性の低さ、標的性の弱さ、実際の臨床治療効果が総体的に十分でないといった点が明らかになっている。これらの薬物には、上皮成長因子受容体（ＥＧＦＲ）に抵抗するセツキシマブ（Ｃｅｔｕｘｉｍａｂ）モノクローナル抗体薬であるアービタックス（Ｅｒｂｉｔｕｘ）、抗上皮成長因子受容体−２（ＥＧＦＲ２）又はＨＥＲ２を標的とするトラスツズマブ（Ｔｒａｓｔｕｚｕｍａｂ）モノクローナル抗体薬であるハーセプチン（Ｈｅｒｃｅｐｔｉｎ）、及び、血管内皮増殖因子（ＶＥＧＦ）を標的とするベバシズマブ（Ｂｅｖａｃｉｚｕｍａｂ）モノクローナル抗体薬であるアバスチン（Ａｖａｓｔｉｎ）等が含まれる。そこで、現在の腫瘍臨床医学における喫緊の必要性に対応すべく、胃癌の生体機能を標的とする高効率のモノクローナル抗体薬を生成することが求められている。学者のＢｒｉｃｈｏｒｙは、世界で初めてプロテオミクスにおけるポリペプチドの合成又は構築方法を、腫瘍関連抗原の鑑定とスクリーニング、及び抗腫瘍抗体分野に取り入れた。人々は、このような構想及び方法から、例えばグルコース調節タンパク質（Ｇｌｕｃｏｓｅ Ｒｅｇｕｌａｔｅｄ Ｐｒｏｔｅｉｎ，ＧＲＰ７８）、熱ショックタンパク質−２７，−６０，−７０等（Ｈｅａｔ Ｓｈｏｃｋ Ｐｒｏｔｅｉｎ，ＨＳＰ−２７，−６０，−７０等）、及びフィブリノペプチド−Ａ（Ｆｉｂｒｉｎ Ｐｅｐｔｉｄｅ−Ａ）など、新たな細胞内及び細胞外の腫瘍マーカータンパク質分子を多数開示してきた。しかし、こうした手段は多くの限界をもたらしてもいる。なぜなら、腫瘍の生体機能学的標的は細胞表面の特異性抗原だけでなく、機能としてタンパク質の空間構造又は天然の立体構造を備えている必要があるからである。このことが、Ｂｒｉｃｈｏｒｙ構想の更なる研究及び応用を大きく制限している。一方、本研究及び発明では、人体が生物学的に自然な状態で、生細胞の免疫及びハイスループットフローサイトメーター（ＦＡＣＳ）による生細胞のスクリーニングを行い、胃癌又はその他腫瘍細胞表面の特異性抗原を鑑定することで、特異的且つ高効率の治療抗体薬を捕捉及び生成する。また、胃癌の分子マーカー鑑定及び胃癌の標的治療用モノクローナル抗体薬の臨床応用に有力なツールを提供する。 The background of antibody therapy for gastric cancer is described in "Monoclonal antibody that resists ectopic expression on the cell surface and its production method and use" (patent document 1; disclosed and permitted) previously filed by the inventor Rikumei, et al. Furthermore, there is a description that the treatment of gastric cancer with antibody drug has been rapidly developed in recent years. However, for certain antibody drugs that are currently approved by the US FDA, low specificity, weak targeting, and actual clinical treatment effects are obtained from preclinical toxicity tests and case reports in each clinical phase. It has become clear that it is not sufficient as a whole. Among these drugs, there is a cetuximab (Cetuximab) monoclonal antibody drug that is resistant to the epidermal growth factor receptor (EGFR), Erbitux, an anti-epidermal growth factor receptor-2 (EGFR2) or trastuzumab (HER2) targeting HER2 Trastuzumab) Herceptin, which is a monoclonal antibody drug, and Avastin, which is a Bevacizumab monoclonal antibody drug that targets vascular endothelial growth factor (VEGF), and the like are included. Therefore, in order to meet the urgent need in current oncology clinics, it is required to generate highly efficient monoclonal antibody drugs targeting the biological functions of gastric cancer. Scholar Brichory is the first in the world to introduce methods for synthesis or construction of polypeptides in proteomics, identification and screening of tumor-related antigens, and in the field of anti-tumor antibodies. From such conception and methods, for example, glucose regulated protein (GRP78), heat shock protein -27, -60, -70 etc. (Heat Shock Protein, HSP -27, -60, -70 etc. A large number of new intracellular and extracellular tumor marker protein molecules, such as Fibrin peptide-A (Fibrin Peptide-A), etc. have been disclosed. However, these measures have also brought many limitations. This is because tumor biofunctional targets need to have not only cell surface specific antigens, but also the protein spatial structure or natural conformation as a function. This greatly limits the further research and application of the Brichory concept. On the other hand, in the present study and invention, when the human body is in a state of natural biology, live cells are screened for live cells by immunization and high-throughput flow cytometer (FACS), and gastric cancer or other tumor cell specific antigens Identify and generate specific and highly efficient therapeutic antibody drugs. It also provides powerful tools for molecular marker identification of gastric cancer and clinical application of monoclonal antibody drugs for targeted treatment of gastric cancer.
ポドカリキシン（Ｐｏｄｏｃａｌｙｘｉｎ，ＰＣ）は、主に糸球体の基底膜における足細胞（Ｐｏｄｏｃｙｔｅ）表面に発現する糖タンパク質である。タンパク質の等電点（ｐＩ）は７未満であり、酸性且つマイナス電荷を有している。ポドカリキシンは、糸球体の足細胞上で濾過隙間の開発を維持する役割を果たす。また、ポドカリキシンは、血小板による凝血、初期糖尿病の発症においても一定の役割を果たす。一方で、ポドカリキシン様タンパク質（Ｐｏｄｏｃａｌｙｘｉｎ−Ｌｉｋｅ ｐｒｏｔｅｉｎ，ＰＯＤＸＬ）はヒト特有のものであり、ＰＯＤＸＬ遺伝子でコードされる唾液ムチンファミリーに属している。ポドカリキシン様タンパク質は、細胞内の構成要素を有するＮａ＋／Ｈ＋交換輸送体（Ｎａ＋／Ｈ＋ ｅｘｃｈａｎｇｅｒ、ＮＨＥ）と複合物を構成するとともに、造血細胞の分化において一定の役割を果たす。ポドカリキシン様タンパク質は血管内皮細胞で発現し、Ｌ−セレクチン（Ｌ−ｓｅｌｅｃｔｉｎ）と結合可能である。また、ＰＯＤＸＬは、異なるアミノ酸配列や異なるポリペプチド断片長さのサブタイプを有し、且つ、異なる組織、細胞及び腫瘍上で発現する。 Podocalyxin (PC) is a glycoprotein mainly expressed on the surface of podocytes in the basement membrane of glomeruli. The isoelectric point (pI) of the protein is less than 7 and has an acidic and negative charge. Podocalyxins play a role in maintaining the development of the filtration gap on glomerular podocytes. Podocalyxin also plays a role in platelet aggregation and in the onset of early diabetes. On the other hand, podocalyxin-like protein (Podocaloxin-Like protein, PODXL) is unique to humans and belongs to the saliva mucin family encoded by PODXL gene. Podocalyxin-like proteins form a complex with Na + / H + exchangers (Na + / H + exchangers, NHE) having intracellular components, and play a role in differentiation of hematopoietic cells. Podocalyxin-like proteins are expressed in vascular endothelial cells and can bind to L-selectin. Also, PODXL has different amino acid sequences and subtypes of different polypeptide fragment lengths and is expressed on different tissues, cells and tumors.
上述した従来技術の欠点に鑑みて、本発明は、従来技術の課題を解決するための胃癌細胞の表面に機能的に発現するポドカリキシン様タンパク質前駆体サブタイプ２（英文略称：ＰＯＤＸＬ−ｖ２、英文正式名称：Ｐｏｄｏｃａｌｙｘｉｎ Ｉｓｏｆｏｒｍ ２ Ｐｒｅｃｕｒｓｏｒ，ＰＯＤＸＬ−ｖ２）に抵抗するモノクローナル抗体とその生成方法及び使用を提供することを目的とする。 SUMMARY OF THE INVENTION In view of the above-mentioned disadvantages of the prior art, the present invention provides a podocalyxin-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells for solving the problems of the prior art. Formal Name: Podocalyxin Isoform 2 Precursor, POD XL-v2) It is an object of the present invention to provide a monoclonal antibody that resists the method and use thereof.
上記の目的及びその他関連の目的を実現すべく、本発明は第一の局面において、胃癌細胞の表面に機能的に発現するポドカリキシン様タンパク質前駆体サブタイプ２（ＰＯＤＸＬ−ｖ２）に対するモノクローナル抗体（ＭＳ１７−３８）であって、当該抗体のＬ鎖可変領域におけるアミノ酸配列がＳＥＱ ＩＤ ＮＯ：２又はその保存的変異体配列であり、当該抗体のＨ鎖可変領域におけるアミノ酸配列がＳＥＱ ＩＤ ＮＯ：４又はその保存的変異体配列であるモノクローナル抗体を提供する。 In order to achieve the above objects and other related objects, the present invention provides, in a first aspect, a monoclonal antibody (MS17) against podocalyxin-like protein precursor subtype 2 (PODXL-v2) functionally expressed on the surface of gastric cancer cells The amino acid sequence in the L chain variable region of the antibody is SEQ ID NO: 2 or a conservative variant sequence thereof, and the amino acid sequence in the H chain variable region of the antibody is SEQ ID NO: 4 or Provided are monoclonal antibodies that are conservative variant sequences thereof.
好ましくは、前記Ｌ鎖可変領域におけるコード配列は、ＳＥＱ ＩＤ ＮＯ：１又はその保存的変異体配列であり、前記Ｈ鎖可変領域におけるコード配列は、ＳＥＱ ＩＤ ＮＯ：３又はその保存的変異体配列である。 Preferably, the coding sequence in the light chain variable region is SEQ ID NO: 1 or a conservative variant sequence thereof, and the coding sequence in the heavy chain variable region is SEQ ID NO: 3 or a conservative variant sequence thereof It is.
好ましくは、前記ＭＳ１７−３８モノクローナル抗体はマウス由来である。 Preferably, said MS17-38 monoclonal antibody is from mouse.
より好ましくは、前記ＭＳ１７−３８モノクローナル抗体は、ＩｇＧ１Ｈ鎖及びκ Ｌ鎖のサブタイプの免疫グロブリンである。 More preferably, said MS17-38 monoclonal antibody is an immunoglobulin of the IgG1 H chain and 及 び L chain subtypes.
本発明で提供するＭＳ１７−３８モノクローナル抗体は、腫瘍細胞の表面に機能的に発現するＰＯＤＸＬ−ｖ２抗原（又は受容体或いはエピトープ）に抵抗（結合又は作用）するか、或いは、部分的にＰＯＤＸＬ抗原に抵抗（結合又は作用）するか、或いは、部分的にＰＯＤＸＬ−ｖ１抗原に抵抗（結合又は作用）するモノクローナル抗体である。 The MS17-38 monoclonal antibody provided by the present invention either resists (binds or acts) PODXL-v2 antigen (or receptor or epitope) functionally expressed on the surface of tumor cells, or partially PODXL antigen A monoclonal antibody that either resists (binds or acts) or partially resists (binds or acts) POD XL-v1 antigen.
更に、本発明は、前記ＭＳ１７−３８モノクローナル抗体の誘導体を提供する。前記誘導体は、前記ＭＳ１７−３８モノクローナル抗体の断片としてもよいし、或いは、前記ＭＳ１７−３８モノクローナル抗体又はＭＳ１７−３８モノクローナル抗体の断片を含む融合タンパク質としてもよい。また、前記モノクローナル抗体の断片は、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ又はｓｃＦｖ等である。 Furthermore, the present invention provides a derivative of the MS17-38 monoclonal antibody. The derivative may be a fragment of the MS17-38 monoclonal antibody, or may be a fusion protein comprising a fragment of the MS17-38 monoclonal antibody or the MS17-38 monoclonal antibody. In addition, fragments of the monoclonal antibody are Fab, Fab ′, F (ab ′) 2 , Fv, scFv or the like.
本発明は第二の局面において、前記ＭＳ１７−３８モノクローナル抗体におけるＨ鎖及び／又はＬ鎖の可変領域或いは全アミノ酸をコードする分離されたＤＮＡ分子を提供する。 The present invention provides, in a second aspect, an isolated DNA molecule encoding the variable region or all amino acids of the H chain and / or L chain in the MS17-38 monoclonal antibody.
本発明は第三の局面において、前記分離されたＤＮＡ分子を含む構造体を提供する。 The present invention provides, in a third aspect, a construct comprising the separated DNA molecule.
好ましくは、前記ＤＮＡベクター発現構造体は、前記分離された抗体のＤＮＡ分子を発現ベクターのマルチクローニングサイトに挿入することで構築される。 Preferably, the DNA vector expression construct is constructed by inserting the DNA molecule of the separated antibody into the multicloning site of the expression vector.
前記発現ベクターは、具体的に、当業者が熟知するこれまで常用されてきた発現ベクターとすればよい。具体的に適用可能な発現ベクターには、ｐＥＴ系発現ベクター、ｐＧＥＸ系発現ベクター、ｐｃＤＮＡ系発現ベクター等が含まれるがこれらに限らない。 Specifically, the expression vector may be a conventionally used expression vector familiar to those skilled in the art. Specific applicable expression vectors include, but are not limited to, pET expression vectors, pGEX expression vectors, pcDNA expression vectors and the like.
本発明は第四の局面において、上記構造体が宿主細胞にトランスフェクションされて構成されるモノクローナル抗体の発現系を提供する。 The present invention provides, in a fourth aspect, a monoclonal antibody expression system constructed by transfecting the above-described construct into a host cell.
発現ベクター（構造体）に適用されて本件特許に記載する抗体を発現するあらゆる細胞が宿主細胞となり得る。例えば、酵母、昆虫、植物等の細胞が可能である。好ましくは、前記宿主細胞は真核細胞であり、抗体を産生しない哺乳動物の宿主細胞系を用いればよい。具体的に使用可能な細胞系には、チャイニーズハムスターの卵巣細胞（ＣＨＯ）、幼少ハムスターの腎細胞（ＢＨＫ、ＡＴＣＣ ＣＣＬ １０）、幼少マウスのセルトリ細胞（Ｓｅｒｔｏｌｉ ｃｅｌｌｓ）、サルの腎細胞（ＣＯＳ細胞）、ＳＶ４０（ＣＯＳ−７、ＡＴＣＣ ＣＲＬ １６５ １）により形質転換したサルの腎臓ＣＶＩ細胞、ヒト胎児腎細胞（ＨＥＫ−２９３）、サルの腎細胞（ＣＶＩ、ＡＴＣＣ ＣＣＬ−７０）、アフリカミドリザルの腎細胞（ＶＥＲＯ−７６、ＡＴＣＣ ＣＲＬ−１５８７）、ヒト子宮頸癌細胞（ＨＥＬＡ、ＡＴＣＣ ＣＣＬ−２）等が含まれるがこれらに限らない。 Any cell which is applied to an expression vector (construct) and which expresses the antibody described in the present patent may be a host cell. For example, cells such as yeast, insects and plants are possible. Preferably, the host cell is a eukaryotic cell, and a mammalian host cell line which does not produce an antibody may be used. Specifically usable cell lines include Chinese hamster ovary cells (CHO), juvenile hamster kidney cells (BHK, ATCC CCL 10), juvenile mouse Sertoli cells (Sertoli cells), monkey kidney cells (COS cells) ), Monkey kidney CVI cells transformed with SV40 (COS-7, ATCC CRL 165 1), human fetal kidney cells (HEK-293), monkey kidney cells (CVI, ATCC CCL-70), African green monkey kidney Cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL-2) and the like, but not limited thereto.
本発明では、効果的な腫瘍生細胞のハイスループットスクリーニング方法を構築する。ヒトの胃癌細胞系生細胞を混合してマウスを免疫するとともに、高い免疫反応を示したマウスの脾臓を選択してマウスのＳＰ２／０細胞系と融合することで抗体ハイブリドーマを産生する。そして、抗胃癌細胞系ハイブリドーマ抗体に対しハイスループット生細胞スクリーニングを行うとともに、健常人の新鮮な末梢血単核細胞（ＰＢＭＣ）に対し対照スクリーニングを行うことで、このハイブリドーマ株からなる胃癌細胞に対し高い特異的反応を示すモノクローナル抗体ＭＳ１７−３８を取得する。更に、本発明は発現ベクターを構築すべく、モノクローナル抗体を培養する細胞株からスクリーニングにより目的の抗体の遺伝子コード配列を取得する。発現系による発現で抗体は活性を再構築し、ＭＳ１７−３８モノクローナル抗体が得られる。 In the present invention, a method for high-throughput screening of effective tumor live cells is constructed. Human gastric cancer cell lines are mixed to immunize mice, and spleens of mice showing a high immune response are selected and fused with the mouse SP2 / 0 cell line to produce antibody hybridomas. Then, high-throughput live cell screening is performed on anti-stomach cancer cell line hybridoma antibodies, and control screening is performed on fresh peripheral blood mononuclear cells (PBMCs) of healthy individuals, thereby treating gastric cancer cells consisting of this hybridoma line. The monoclonal antibody MS17-38 showing highly specific reaction is obtained. Furthermore, in the present invention, in order to construct an expression vector, the gene coding sequence of the target antibody is obtained by screening from cell lines in which monoclonal antibodies are cultured. The antibody reconstitutes the activity upon expression by the expression system to obtain the MS17-38 monoclonal antibody.
前記ＭＳ１７−３８モノクローナル抗体は、次の手順でスクリーニングすることで得られる。即ち、４種類の混合胃癌細胞株ＳＧＣ７９０１、ＢＧＣ８２３、ＭＫＮ２８、ＭＫＮ４５の生細胞を用いて複数部位でマウスを免疫し、免疫したマウスの脾臓細胞とマウスの骨髄腫細胞を融合する。そして、上記４種類の胃癌生細胞の表面に結合可能な抗原を分泌可能だが健常人の末梢血単核細胞とは反応しないハイブリドーマ株をスクリーニングする。このハイブリドーマ株をサブクローニングしてから、培養されたハイブリドーマの上清液を取得して、親和及び精製すれば前記モノクローナル抗体ＭＳ１７−３８が得られる。本発明の一実施例において、前記４種類の混合胃癌細胞株ＳＧＣ７９０１、ＢＧＣ８２３、ＭＫＮ２８、ＭＫＮ４５の生細胞は等しい比率で混合することが好ましい。また、前記マウスの骨髄腫細胞は、ＳＰ２／０マウスの骨髄腫細胞である。具体的な融合方法としてはＰＥＧ化学融合法を用い、ハイブリドーマの培養上清液を免疫アフィニティークロマトグラフィーで精製する。 The MS17-38 monoclonal antibody can be obtained by screening according to the following procedure. Specifically, live cells of four types of mixed gastric cancer cell lines SGC7901, BGC823, MKN28, and MKN45 are used to immunize mice at multiple sites, and spleen cells of the immunized mice and mouse myeloma cells are fused. Then, a hybridoma strain capable of secreting an antigen capable of binding to the surface of the above four types of gastric cancer living cells but not reacting with peripheral blood mononuclear cells of healthy individuals is screened. After this hybridoma strain is subcloned, the supernatant fluid of the cultured hybridoma is obtained, and affinity and purification are performed to obtain the monoclonal antibody MS17-38. In one embodiment of the present invention, it is preferable that the living cells of the four mixed gastric cancer cell lines SGC7901, BGC823, MKN28 and MKN45 be mixed at an equal ratio. Also, the mouse myeloma cells are myeloma cells of SP2 / 0 mice. As a specific fusion method, the culture supernatant of the hybridoma is purified by immunoaffinity chromatography using PEG chemical fusion method.
本発明は第五の局面において、前記ＭＳ１７−３８モノクローナル抗体の生成方法であって、前記抗体の発現に適した条件で、前記モノクローナル抗体の発現系を培養することで前記モノクローナル抗体を発現し、前記モノクローナル抗体を精製及び分離するステップを含む方法を提供する。 The present invention provides, in a fifth aspect, a method for producing the MS17-38 monoclonal antibody, wherein the monoclonal antibody is expressed by culturing the expression system of the monoclonal antibody under conditions suitable for expression of the antibody, There is provided a method comprising the steps of purifying and separating said monoclonal antibody.
本発明の抗体をコードする核酸配列を取得した後、以下の方法で目的の抗体を生産可能である。例えば、目的の抗体をコードする核酸を含むベクターを宿主細胞に直接組み込み、適切な条件で細胞を培養することでコードされた抗体の発現を誘導する。本発明で使用する発現ベクター及び宿主細胞はいずれも従来技術であり、商業ルートから直接取得可能である。培養に使用する１０％ＦＢＳのＤＭＥＭ培地もまた各種の一般的な哺乳細胞培地である。当業者であれば、適用するＤＭＥＭ培地を経験的に選択し、宿主細胞の成長に適した条件で培養することが可能である。宿主細胞が適切な細胞密度まで成長すると、適切な方法（例えば、温度変換又は化学誘導）で誘導及び選択したプロモーターを用い、細胞を更に一定期間培養する。上記方法における組み換えポリペプチドは、細胞内又は細胞膜上に発現可能であるか、細胞外に分泌される。本発明で述べるモノクローナル抗体を取得すれば、その物理的特性、化学的特性及びその他の特性を利用して、各種分離方法により前記モノクローナル抗体を分離及び精製可能となる。これらの方法は、当業者が熟知するものである。また、これらの方法の例としては、一般的な復元処理、タンパク質沈殿剤処理（塩析方法）、遠心、浸透菌破砕、超処理、超遠心、分子ふるいクロマトグラフィー（ゲルろ過）、吸着クロマトグラフィー、イオン交換クロマトグラフィー、高速液体クロマトグラフィー（ＨＰＬＣ）及びその他各種の液体クロマトグラフィー技術及びこれらの組み合わせを含むが上記に限らない。 After the nucleic acid sequence encoding the antibody of the present invention is obtained, the target antibody can be produced by the following method. For example, a vector containing a nucleic acid encoding an antibody of interest is directly incorporated into a host cell, and culturing of the cell under appropriate conditions induces expression of the encoded antibody. The expression vectors and host cells used in the present invention are both conventional techniques and can be obtained directly from commercial routes. DMEM medium of 10% FBS used for culture is also various common mammalian cell culture medium. Those skilled in the art can empirically select the DMEM medium to be applied, and culture it under conditions suitable for the growth of host cells. Once the host cells are grown to the appropriate cell density, the cells are cultured for an additional period of time using promoters selected and selected by appropriate methods (eg, temperature conversion or chemical induction). The recombinant polypeptide in the above method can be expressed intracellularly or on a cell membrane, or secreted extracellularly. Once the monoclonal antibody described in the present invention is obtained, it becomes possible to separate and purify the monoclonal antibody by various separation methods using its physical properties, chemical properties and other properties. These methods are familiar to those skilled in the art. In addition, examples of these methods include general refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic bacterial disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography Including, but not limited to, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations thereof.
本発明は第六の局面において、前記ＭＳ１７−３８モノクローナル抗体の、腫瘍治療薬の生成又はスクリーニングにおける使用、或いは、腫瘍診断薬の生成における使用を提供する。 The present invention provides, in a sixth aspect, the use of the MS17-38 monoclonal antibody in the production or screening of a tumor therapeutic or in the production of a tumor diagnostic.
前記腫瘍治療薬とは、具体的に、腫瘍細胞の表面に機能的に発現するＰＯＤＸＬ−ｖ２を抗原とし、前記抗原ＰＯＤＸＬ−ｖ２に結合又は作用することで癌の治療及び原発癌の転移予防を行う薬物のことをいう。前記抗原ＰＯＤＸＬ−ｖ２に対する結合又は作用には、具体的に、ＰＯＤＸＬ−ｖ２抗原の免疫抑制、又は、特異性モノクローナル抗体により腫瘍の抗原を識別し、腫瘍治療薬を指向的に腫瘍部位に集中させることで腫瘍細胞を選択的に殺傷すること等が含まれるが、これらに限らない。 Specifically, PODXL-v2 functionally expressed on the surface of a tumor cell is used as an antigen, and the tumor therapeutic agent specifically binds to or acts on the antigen PODXL-v2 to treat cancer and prevent metastasis of primary cancer. It refers to the drug to be done. Specifically, for the binding or action to the antigen PODXL-v2, the tumor antigen is identified by immunosuppression of POD XL-v2 antigen or a specific monoclonal antibody, and a tumor therapeutic drug is directed to focus on the tumor site. Selectively kills tumor cells, but is not limited thereto.
したがって、腫瘍診断薬とは、具体的に、腫瘍細胞の作用標的であるＰＯＤＸＬ−ｖ２に対し、ＰＯＤＸＬ−ｖ２をバイオマーカーとして、鑑別診断、病理診断、早期診断、映像診断及び予後診断等を行うための診断用試薬をいう。 Therefore, a tumor diagnostic agent specifically performs differential diagnosis, pathological diagnosis, early diagnosis, video diagnosis, prognosis diagnosis, etc. using PODXL-v2 as a biomarker for PODXL-v2, which is an action target of tumor cells. For diagnostic reagents.
好ましくは、前記腫瘍は胃癌である。 Preferably, the tumor is gastric cancer.
より好ましくは、前記胃癌は、胃扁平上皮癌、胃腺癌、胃小細胞癌、胃腺扁平上皮癌、胃カルチノイド又は胃・十二指腸癌である。 More preferably, the gastric cancer is gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric adenosquamous cell carcinoma, gastric carcinoid or gastric / duodenal carcinoma.
本発明は第七の局面において、治療有効量の前記ＭＳ１７−３８モノクローナル抗体又はその免疫複合体を含む医薬組成物を提供する。 The present invention provides, in a seventh aspect, a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned MS17-38 monoclonal antibody or an immunoconjugate thereof.
前記免疫複合体は、前記モノクローナル抗体又はその断片及び薬物、毒素、サイトカイン、放射性核種、酵素又はその他の診断用試薬等を組み合わせてなる複合体を含むがこれらに限らない。 The immune complex includes, but is not limited to, a complex formed by combining the monoclonal antibody or a fragment thereof and a drug, a toxin, a cytokine, a radionuclide, an enzyme or another diagnostic reagent and the like.
前記医薬組成物とは、腫瘍細胞の表面に機能的に発現するＰＯＤＸＬ−ｖ２を抗原とし、前記抗原に結合又は作用することで癌の治療及び／又は原発癌の転移予防を行うものをいう。 The above-mentioned pharmaceutical composition refers to one that uses PODXL-v2 functionally expressed on the surface of a tumor cell as an antigen and binds to or acts on the antigen to treat cancer and / or prevent metastasis of primary cancer.
本発明のＭＳ１７−３８モノクローナル抗体は、当該分野における既知のあらゆる方式で医薬組成物を調合して使用すればよい。このような組成物は、前記モノクローナル抗体を活性成分とし、１又は複数の薬学的に受け入れ可能なベクター又は賦形剤が加えられる。前記ベクター又は賦形剤は、前記モノクローナル抗体と親和性のあるものとすべきである。 The MS17-38 monoclonal antibody of the present invention may be used to formulate the pharmaceutical composition in any manner known in the art. Such compositions comprise the monoclonal antibody as an active ingredient, to which one or more pharmaceutically acceptable vectors or excipients are added. The vector or excipient should be compatible with the monoclonal antibody.
前記医薬組成物は胃癌の治療に用いられ、好ましくは、胃扁平上皮癌、胃腺癌、胃小細胞癌、胃腺扁平上皮癌、胃カルチノイド又は胃・十二指腸癌の１又は複数の組み合わせの治療に用いられる。前記医薬組成物を対象体内の腫瘍の予防又は治療に用いる場合には、有効投与量の前記医薬組成物を対象に投与すればよい。 The pharmaceutical composition is used for the treatment of gastric cancer, preferably for the treatment of one or more combinations of gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric adenosquamous cell carcinoma, gastric carcinoid or gastric / duodenal carcinoma Be When the pharmaceutical composition is used for the prevention or treatment of a tumor in a subject, an effective dose of the pharmaceutical composition may be administered to the subject.
本発明は第八の局面において、治療有効量の前記ＭＳ１７−３８モノクローナル抗体又はその免疫複合体を含む診断用試薬キットを提供する。 The present invention provides, in an eighth aspect, a diagnostic reagent kit comprising a therapeutically effective amount of the MS17-38 monoclonal antibody or an immunoconjugate thereof.
したがって、診断用試薬キットとは、腫瘍細胞の作用標的であるＰＯＤＸＬ−ｖ２に対し、ＰＯＤＸＬ−ｖ２をバイオマーカーとして、鑑別診断、病理診断、早期診断、映像診断及び予後診断等を行うものをいう。 Therefore, a diagnostic reagent kit refers to one that performs differential diagnosis, pathological diagnosis, early diagnosis, video diagnosis, prognosis diagnosis, etc. using PODXL-v2 as a biomarker for PODXL-v2 that is an action target of tumor cells. .
好ましくは、前記診断用試薬キットは、ＭＳ１７−３８モノクローナル抗体のマーカーを更に含む。 Preferably, the diagnostic reagent kit further comprises a marker for MS17-38 monoclonal antibody.
前記ＭＳ１７−３８モノクローナル抗体のマーカーとＭＳ１７−３８モノクローナル抗体との結合に適用可能なマーカーの種類には、蛍光マーカー、放射性マーカー、酵素標識マーカー、化学発光性マーカーのうち１又は複数の組み合わせが含まれるがこれらに限らない。 Types of markers applicable to the binding of the MS17-38 monoclonal antibody marker to the MS17-38 monoclonal antibody include one or more combinations of a fluorescent marker, a radioactive marker, an enzyme labeling marker, and a chemiluminescent marker. But not limited to.
試薬キットの検出原理に応じて、前記試薬キットは検出に必要な１又は複数の試薬を含んでもよい。また、必要に応じて、前記試薬キットには、容器、対照物（陰性又は陽性）、緩衝剤、助剤等を含んでもよい。 Depending on the detection principle of the reagent kit, said reagent kit may comprise one or more reagents necessary for the detection. In addition, if necessary, the reagent kit may contain a container, a control (negative or positive), a buffer, an auxiliary agent, and the like.
本発明は、更に、逆スクリーニングにより胃癌細胞表面の抗原を鑑定し、新たなバイオマーカー抗原を取得する抗胃癌モノクローナル抗体を生成して取得する。具体的に、本発明は、腫瘍バイオマーカーを発見及び鑑定するとともに、当該マーカー抗原（胃癌細胞表面における天然立体構造のＰＯＤＸＬ−ｖ２抗原）に対する特異性ＭＳ１７−３８モノクローナル抗体を生成する一方、当該特異性抗体に基づいて、対応する特異性胃癌分子マーカーである細胞膜発現タンパク質ＰＯＤＸＬ−ｖ２をスクリーニングにより鑑定する。 The present invention also identifies antigens on the surface of gastric cancer cells by reverse screening, and generates and acquires anti-gastric cancer monoclonal antibodies that acquire new biomarker antigens. Specifically, the present invention discovers and identifies a tumor biomarker, and while producing a specific MS17-38 monoclonal antibody against the marker antigen (PODXL-v2 antigen in a natural configuration on the surface of a gastric cancer cell), Based on the sex antibodies, cell membrane expressed protein PODXL-v2, which is a corresponding specific gastric cancer molecular marker, is identified by screening.
本発明において、抗体に結合される腫瘍細胞表面の発現抗原を抗体のハイスループットスクリーニング（ＨＴＳ）によって逆鑑定するとの戦略は、胃癌細胞表面の分子マーカーに対する特異性モノクローナル抗体を生成するとともに、スクリーニングにより新たな胃癌分子マーカーを鑑定可能とすることを目的としている。モノクローナル抗体は、プロテオミクス研究にとって良好なツールである。従来のモノクローナル抗体生成過程では抗体をハイブリダイゼーションする方法を用いており、通常は分子マーカーによる免疫は用いず、融合後に大量のプレーティングを行うＨＴＳ方法を用いることも少なかった。そのため、抗細胞抗原天然エピトープ抗体が取得される確率が低かった（従来の抗体スクリーニング生成方法では、腫瘍生細胞表面の天然立体構造の抗原を識別するような抗体を得ることは難しかった）。これに対し、本発明では、「ショットガン法」（“Ｓｈｏｔ−Ｇｕｎ”ｍｅｔｈｏｄ）に類似する方法で生細胞を免疫し、抗体のハイブリダイゼーション技術とフローサイトメトリーハイスループット検出を組み合わせている。腫瘍生細胞でマウスを免疫し、独特の抗体ハイブリダイゼーションによる高融合手法で融合及び大量のプレーティング（毎回５０〜８０枚）を行い、生細胞に対する蛍光フローサイトメトリー（ＦＡＣＳ）−ＨＴＳを組み合わせて検出及びスクリーニングする方法で、細胞表面に立体構造エピトープ（Ｃｏｎｆｏｒｍａｔｉｏｎａｌ Ｅｐｉｔｏｐｅ）を備える分子マーカーに対するモノクローナル抗体を生細胞レベルで直接スクリーニングするとともに、再検査により抗体ハイブリドーマのサブクローンを鑑定する。そして、最後に、特異的且つ高親和性のモノクローナル抗体として、胃癌細胞表面の天然立体構造ＰＯＤＸＬ−ｖ２抗原に対するＭＳ１７−３８特異性抗体を選択する。また、ＭＳ１７−３８抗体を鑑定するとともに、ＭＳ１７−３８抗体及び胃癌について臨床病理学的なパラメーターの相関分析を行った後、ウェスタンブロッティング、免疫沈降法及びタンパク質質量分析によって、生成されたＭＳ１７−３８抗体が特異的に結合する標的抗原タンパク質を迅速に鑑定する。こうして、ＰＯＤＸＬ−ｖ２という新たな胃癌細胞表面の分子マーカーがスクリーニングにより発見された。 In the present invention, a strategy to reversely identify antigens expressed on the surface of tumor cells bound to antibodies by high-throughput screening (HTS) of antibodies is to generate specific monoclonal antibodies against molecular markers on the surface of gastric cancer cells as well as screening. The purpose is to make it possible to identify new gastric cancer molecular markers. Monoclonal antibodies are good tools for proteomics research. In the conventional monoclonal antibody production process, a method of hybridizing antibodies is used, and usually, immunization with a molecular marker is not used, and it is also rare to use an HTS method in which a large amount of plating is performed after fusion. Therefore, the probability of obtaining anti-cell antigen natural epitope antibodies was low (it was difficult to obtain an antibody that can identify an antigen of a natural conformation on the surface of a living tumor cell by the conventional antibody screening method). On the other hand, in the present invention, living cells are immunized by a method similar to the "Shot-Gun" method, and antibody hybridization technology is combined with flow cytometry high throughput detection. Immunize mice with live tumor cells, perform fusion and mass plating (50 to 80 each time) in a high fusion method with unique antibody hybridization, and combine fluorescence flow cytometry (FACS) -HTS on live cells In the detection and screening method, monoclonal antibodies against molecular markers having conformational epitopes on the cell surface are directly screened at live cell level, and subclones of antibody hybridomas are identified by re-examination. Finally, as a specific and high affinity monoclonal antibody, an MS17-38 specific antibody against the native three-dimensional structure PODXL-v2 antigen on the stomach cancer cell surface is selected. In addition, after identifying the MS17-38 antibody and performing correlation analysis of clinicopathological parameters for the MS17-38 antibody and gastric cancer, MS17-38 produced by Western blotting, immunoprecipitation and protein mass spectrometry. Rapid identification of target antigen proteins to which antibodies specifically bind. Thus, a new gastric cancer cell surface molecular marker named PODXL-v2 was discovered by screening.
本発明のＭＳ１７−３８モノクローナル抗体は、複合物全体の分子量が１３５ｋＤａであり、腫瘍細胞表面に自然に発現するとともに空間立体構造を有するポドカリキシン様タンパク質前駆体サブタイプ２（ＰＯＤＸＬ−ｖ２）の分子に対しては高い特異性をもって反応する一方、ＰＯＤＸＬ−ｖ２単体の分子量が約５１ｋＤａである線形体分子のタンパク質に対しては反応しない。本発明における特殊なモノクローナル抗体のハイブリドーマスクリーニング設計では、発現するモノクローナル抗体がＭＳ１７−３８と称されるモノクローナル抗体のハイブリドーマ株から分泌されて産生する。また、マウスのモノクローナル抗体のサブタイプはＩｇＧ１Ｈ鎖及びκＬ鎖に属している。 The MS17-38 monoclonal antibody of the present invention is a molecule of podocalyxin-like protein precursor subtype 2 (PODXL-v2), which has a molecular weight of 135 kDa as a whole complex and is naturally expressed on the surface of tumor cells and has spatial conformation. While it reacts with high specificity, it does not react to the protein of the linear body molecule whose molecular weight of PODXL-v2 alone is about 51 kDa. In the hybridoma screening design of a specific monoclonal antibody in the present invention, an expressed monoclonal antibody is secreted and produced from a hybridoma strain of a monoclonal antibody designated as MS17-38. In addition, mouse monoclonal antibody subtypes belong to IgG1 H chain and κ L chain.
本発明では、生成により取得した胃癌混合細胞株に対する特異性モノクローナル抗体と胃癌等の消化器腫瘍について臨床病理学的パラメーターの関連分析を実施した。そして、腫瘍細胞表面のＰＯＤＸＬ−ｖ２分子に対するＭＳ１７−３８モノクローナル抗体の作用及び機能を分析したところ、ＭＳ１７−３８モノクローナル抗体がヒト胃癌細胞の表面のＰＯＤＸＬ抗原に対して産生されたものであり、特異性及び機能的生体反応を誘導可能なことから、胃癌等の消化器腫瘍を特異的に識別して標的治療することが可能であるとともに、消化器腫瘍の診断及び映像にも応用可能であることを発見した。本発明は、胃癌及び消化器固形腫瘍細胞の表面の天然立体構造を持つ抗原に対する特異性抗体をハイスループットで生成可能な全く新しい方法を構築した。また、取得されるＭＳ１７−３８モノクローナル抗体はプロテオミクス研究のツールとなり、胃癌等の消化器固形腫瘍細胞の表面における分子標的マーカーを逆スクリーニングにより鑑定可能である。前記モノクローナル抗体は、キメラ化又はヒト化することで、更にヒトの胃癌治療用バイオ薬剤を開発可能となる。 In the present invention, association analysis of clinicopathological parameters was performed for specific monoclonal antibodies against gastric cancer mixed cell line obtained by generation and gastrointestinal tumors such as gastric cancer. Then, when the action and function of MS17-38 monoclonal antibody against PODXL-v2 molecules on the surface of tumor cells were analyzed, MS17-38 monoclonal antibody was produced against PODXL antigen on the surface of human gastric cancer cells and was specific It is possible to specifically identify and target digestive tract tumors such as gastric cancer because it can induce sexual and functional biological responses, and it can also be applied to diagnosis and imaging of digestive tract tumors Found. The present invention has constructed an entirely new method capable of generating a high-throughput specific antibody against an antigen having a natural three-dimensional structure on the surface of gastric cancer and gastrointestinal solid tumor cells. In addition, the MS17-38 monoclonal antibody obtained is a tool for proteomics research, and molecular target markers on the surface of digestive organ solid tumor cells such as gastric cancer can be identified by reverse screening. The above-mentioned monoclonal antibody can be further chimerized or humanized to develop a biopharmaceutical for human gastric cancer treatment.
以下に、特定の具体的実施例を挙げて本発明の実施形態につき説明する。なお、当業者であれば本明細書に開示の内容から本発明のその他の利点及び効果を容易に理解可能である。更に、本発明はその他の異なる具体的実施形態によっても実施又は応用可能である。また、本明細書の各詳細事項については、視点及び応用の違いに応じて、本発明の精神を逸脱しないことを前提に各種の補足又は変更が可能である。 Hereinafter, embodiments of the present invention will be described with reference to specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed herein. Furthermore, the present invention can be practiced or applied according to other different specific embodiments. In addition, with respect to each detailed matter in the present specification, various additions or changes can be made on the premise that the spirit of the present invention is not deviated, depending on the difference in viewpoint and application.
本発明の具体的実施形態について更に記載する前に、本発明による保護の範囲は後述する特定の具体的実施方案に限らないと解釈すべきである。また、本発明の実施例で使用される用語は特定の具体的実施方案を記載するためのものであって、本発明による保護の範囲を制限するものではないと解釈すべきである。本発明の明細書及び請求の範囲では、特に明記しない限り、「１つ」「一の」及び「この」といった単数形には複数形が含まれる。 Before further describing specific embodiments of the present invention, it should be construed that the scope of protection according to the present invention is not limited to the specific embodiments described below. It is also to be understood that the terminology used in the examples of the present invention is for the purpose of describing a particular specific implementation and does not limit the scope of protection according to the present invention. In the specification and claims of the present invention, the singular forms "one", "one" and "this" include plural forms unless otherwise stated.
実施例が数値範囲を提示する場合、本発明においては特に説明がない限り、各数値範囲の２つの端点及び２つの端点間の任意の数値をいずれも使用可能であると解釈すべきである。また、特に定義されない限り、本発明で使用されるあらゆる技術用語及び科学用語は、当業者が一般的に理解する意味と同義である。実施例で使用される具体的方法、デバイス、材料のほかに、当業者が把握する従来技術及び本発明の記載に基づいて、本発明の実施例における上記の方法、デバイス、材料と類似又は同等の従来技術における任意の方法、デバイス及び材料を用いて本発明を実現してもよい。 Where the examples present numerical ranges, it is to be understood that in the present invention any two endpoints of each numerical range and any numerical value between the two endpoints may be used, unless specifically stated otherwise. Also, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, and materials used in the examples, the above-described methods, devices, and materials in the examples of the present invention are similar or equivalent based on the description of the prior art and the present invention understood by those skilled in the art. The invention may be practiced using any of the methods, devices and materials in the prior art of
また、別途説明がない限り、本発明で開示される実験方法、検出方法、生成方法には、当業者にとって一般的な分子生物学、生物化学、クロマチン構造及び分析、分析化学、細胞培養、組換えＤＮＡ技術及び関連分野の一般的技術を採用可能である。これら技術については、既存の文献に十分に説明されている。具体的には、以下の文献等を参照すればよい。
Ｓａｍｂｒｏｏｋ 他，ＭＯＬＥＣＵＬＡＲ ＣＬＯＮＩＮＧ：Ａ ＬＡＢＯＲＡＴＯＲＹ ＭＡＮＵＡＬ，Ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，１９８９ ａｎｄ Ｔｈｉｒｄ ｅｄｉｔｉｏｎ，２００１
Ａｕｓｕｂｅｌ 他，ＣＵＲＲＥＮＴ ＰＲＯＴＯＣＯＬＳ ＩＮ ＭＯＬＥＣＵＬＡＲ ＢＩＯＬＯＧＹ，Ｊｏｈｎ Ｗｉｌｅｙ＆Ｓｏｎｓ，Ｎｅｗ Ｙｏｒｋ，１９８７ ａｎｄ ｐｅｒｉｏｄｉｃ ｕｐｄａｔｅｓ
ｔｈｅ ｓｅｒｉｅｓ ＭＥＴＨＯＤＳ ＩＮ ＥＮＺＹＭＯＬＯＧＹ，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｓａｎ Ｄｉｅｇｏ
Ｗｏｌｆｆｅ，ＣＨＲＯＭＡＴＩＮ ＳＴＲＵＣＴＵＲＥ ＡＮＤ ＦＵＮＣＴＩＯＮ，Ｔｈｉｒｄ ｅｄｉｔｉｏｎ，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｓａｎ Ｄｉｅｇｏ，１９９８
ＭＥＴＨＯＤＳ ＩＮ ＥＮＺＹＭＯＬＯＧＹ，Ｖｏｌ．３０４，Ｃｈｒｏｍａｔｉｎ（Ｐ．Ｍ．Ｗａｓｓａｒｍａｎ ａｎｄ Ａ．Ｐ．Ｗｏｌｆｆｅ，ｅｄｓ．），Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｓａｎ Ｄｉｅｇｏ，１９９９
ＭＥＴＨＯＤＳ ＩＮ ＭＯＬＥＣＵＬＡＲ ＢＩＯＬＯＧＹと、Ｖｏｌ．１１９，Ｃｈｒｏｍａｔｉｎ Ｐｒｏｔｏｃｏｌｓ（Ｐ．Ｂ．Ｂｅｃｋｅｒ，ｅｄ．）Ｈｕｍａｎａ Ｐｒｅｓｓ，Ｔｏｔｏｗａ，１９９９
In addition, unless otherwise stated, experimental methods, detection methods, and production methods disclosed in the present invention include molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, cell culture, which are common to those skilled in the art General techniques of recombinant DNA technology and related fields can be employed. These techniques are fully described in the existing literature. Specifically, the following documents may be referred to.
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates
the series METHODS IN ENZYMOLOGY, Academic Press, San Diego
Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998
METHODS IN EN ZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999
METHODS IN MOLECULAR BIOLOGY and Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999
実施例１
４種類の等しい比率で混合した４株の胃癌細胞（ＢＧＣ−８２３、ＭＫＮ−２８、ＭＫＮ−４５及びＳＧＣ−７９０１。いずれも中国科学院上海細胞生物学研究所製）を用い、１０％ＦＢＳを含有するＤＭＥＭ培地及び５％ＣＯ２、３７℃環境で培養した後、収集した生細胞をＰＢＳ緩衝液内で混合して免疫原とした。続いて、これをＡ／Ｊ−ＪＡＸマウス（南京大学実験動物モデルセンターより購入。マウスは米国ジャクソン研究所由来）の背部皮下及び尾静脈に注射して免疫した。なお、毎回マウス１匹につき各部位３〜５百万の細胞（０．１ｍｌ）を注射した。免疫は隔週で実施された。３回目の免疫から１週間後にマウスの血清を抽出し、フローサイトメトリーハイスループットシステム（ＦＡＣＳ−ＨＴＳ）によって、血清と４種類の混合胃癌細胞株との反応状況を検出した（具体的には実施例３を参照）。また、健常人ボランティアのＰＢＭＣを対照細胞とした（健常人ボランティアの末梢血からフィコール液で末梢血単核細胞（Ｐｅｒｉｐｈｅｒａｌ Ｂｌｏｏｄ Ｍｏｎｏｎｕｃｌｅａｒ Ｃｅｌｌｓ，ＰＢＭＣ）を分離し、蛍光フローサイトメトリーハイスループットスクリーニング（ＦＡＣＳ−ＨＴＳ）における健常人の細胞抗原として対照スクリーニングした）。そして、血清の力価が高く、且つ対照細胞ＰＢＭＣとの交叉反応レベルが低いマウス（同じ希釈比率で、血清と胃癌細胞及び対照細胞との平均蛍光強度値の差＞５００）を選択し、融合前に免疫を強化した。
Example 1
Using 4 strains of gastric cancer cells (BGC-823, MKN-28, MKN-45 and SGC-7901, all of which are made by Shanghai Cell Biology Research Institute of Chinese Academy of Sciences) mixed in 4 equal proportions, containing 10% FBS after culturing in DMEM medium and 5% CO 2, 37 ℃ environment, the collected live cells were used as an immunogen is mixed with PBS buffer solution. Subsequently, the mice were immunized by injecting them into the back subcutaneous and tail veins of A / J-JAX mice (purchased from Nanjing University Experimental Animal Model Center, mice from Jackson Laboratory, USA). Each mouse was injected with 3 to 5 million cells (0.1 ml) at each site. Immunization was performed every other week. One week after the third immunization, mouse serum was extracted, and the reaction situation between the serum and the four mixed gastric cancer cell lines was detected by a flow cytometry high throughput system (FACS-HTS) (specifically, it was carried out See example 3). In addition, PBMCs of healthy volunteers are used as control cells (Peripheral Blood Mononuclear Cells (PBMCs) are separated from peripheral blood of healthy volunteers with Ficoll solution, and fluorescent flow cytometry high-throughput screening (FACS- Control screened as cell antigen of healthy human in HTS). Then select and fuse mice with high serum titers and low levels of cross-reactivity with control cell PBMCs (difference in mean fluorescence intensity values between serum and gastric cancer cells and control cells> 500 at the same dilution ratio) Before strengthening the immunity.
そして、免疫を強化したマウスの脾臓を抽出し、無血清のＤＭＥＭ培養液を用いて免疫応答性の高かった１号マウスの脾臓細胞を単一細胞懸濁液とした。５０％ＰＥＧ（ｐＨ７．４）条件下で脾臓細胞とＳＰ２／０マウスの骨髄腫細胞を融合し、融合後に大量プレーティング（５０プレート）してから、抗体ハイブリドーマのクローンが形成されるまでＨＡＴ選択培地で１０日間培養した。次に、大量培養した前記４種類の胃癌細胞株をスクリーニング対象として収集及び混合するとともに、健常人の末梢血ＰＢＭＣ細胞をスクリーニング用の対照細胞として分離した。２群の細胞（第１群は４種類の胃癌細胞を等しい比率で混合したもの、第２群は健常人の末梢血ＰＢＭＣ細胞）はいずれも氷上で予め冷却した１．５％ＢＳＡ／ＰＢＳ系ブロッキング溶液中でそれぞれ再懸濁し、５１枚の９６ウェルＵ型プレート内に均一に（各ウェルにつき約１０万〜２０万細胞）分配した（合計１０２枚。５１枚目のプレート２枚については、それぞれ陽性（免疫後のマウス血清且つ段階希釈）及び陰性（正常なマウス血清、段階希釈及びＨＡＴ選択培養液）の対照プレートとした）。 Then, the spleens of the mice in which the immunity was enhanced were extracted, and the spleen cells of highly immunoresponsive No. 1 mouse were used as a single cell suspension using a serum-free DMEM culture solution. Fusion of spleen cells and myeloma cells from SP2 / 0 mice under 50% PEG (pH 7.4) conditions, mass plating (50 plates) after fusion, and then HAT selection until antibody hybridoma clones are formed It culture | cultivated in the culture medium for 10 days. Next, the four types of gastric cancer cell lines cultured in large amounts were collected and mixed as screening targets, and peripheral blood PBMC cells of healthy individuals were separated as control cells for screening. 1.5% BSA / PBS system pre-chilled on ice for 2 groups of cells (group 1 mixed 4 stomach cancer cells in equal proportions, group 2 peripheral blood PBMC cells of healthy people) on ice Each was resuspended in blocking solution and evenly distributed (approximately 100,000 to 200,000 cells per well) in 51 96-well U-shaped plates (total 102, for two 51 plates): Control plates were positive (mouse serum and serial dilution after immunization) and negative (normal mouse serum, serial dilution and HAT selection medium) respectively.
続いて、５０枚の抗体ハイブリドーマ融合プレート（９６ウェルプレート）の上清液（初期モノクローナル抗体である第１抗体に相当）を毎回７０μＬ／ウェルずつ対応するスクリーニングプレート及び対照プレートにそれぞれ移し、振とう混合した。そして、氷浴反応させ、ブロッキング溶液で洗浄した後に、１００μＬ／ウェルのＦＩＴＣ蛍光標識したヤギ抗マウスを加えて細胞蛍光染色実験を実施し反応させた。これによる蛍光フローサイトメトリーハイスループットスクリーニング（ＦＡＣＳ−ＨＴＳ）では、まずブランク対照ウェルとアイソタイプ対照ウェルの細胞でＦＡＣＳのパラメーターを調節し、バックグラウンドとした。そして、２群の９６ウェルＵ型プレートにおける各ウェルの細胞サンプルについて一つずつＦＡＣＳ検出を実施し、下記２つの条件を満たしたものを陽性細胞ウェルとした。
（１）４株の胃癌細胞の表面抗原との間に結合反応がある（即ち、アイソタイプ対照の信号ピークと比較して、サンプルの信号ピークのずれ幅が対数値１つ分よりも大きい）。
（２）ＰＢＭＣ細胞との間に結合反応がない（即ち、アイソタイプ対照の信号ピークと比較して、サンプルの信号ピークのずれ幅が５〜１０％未満）。
このようにして選択及び採取された独特な結合を示すハイブリドーマのハイスループットスクリーニング細胞は、選択培地や一般的な１０％ＦＢＳのＤＭＥＭ培地、ハイブリドーマのサブクローニング及び複数回に渡る抗体ハイブリドーマの培養上清検出を経たものである。ＭＳ１７−３８モノクローナル抗体に対応するハイブリドーマの上清は、親和及び精製を経て０．２μｍ膜で濾過し、４℃で無菌保存した。或いは、５０％グリセリンを添加して−２０℃で長期保存した。
Subsequently, the supernatant (corresponding to the first antibody, which is the initial monoclonal antibody) of 50 antibody hybridoma fusion plates (96-well plate) is transferred to the corresponding screening plate and control plate each time 70 μL / well, and shaken. Mixed. After ice bath reaction and washing with a blocking solution, 100 μL / well of FITC fluorescently labeled goat anti-mouse was added to carry out a cytofluorescent staining experiment and make a reaction. For fluorescence flow cytometry high-throughput screening (FACS-HTS) with this, the FACS parameters were first adjusted in the blank control wells and in the cells of the isotype control wells to provide a background. Then, FACS detection was performed on cell samples of each well in two groups of 96-well U-type plates, and those satisfying the following two conditions were defined as positive cell wells.
(1) There is a binding reaction between the surface antigens of four types of gastric cancer cells (ie, the shift width of the signal peak of the sample is larger than one logarithmic value compared to the signal peak of the isotype control).
(2) There is no binding reaction with PBMC cells (i.e., the shift width of the signal peak of the sample is less than 5-10% as compared to the signal peak of the isotype control).
The high-throughput screening of hybridomas exhibiting unique binding thus selected and collected can be performed by using selective medium, general 10% FBS DMEM medium, hybridoma subcloning and detection of culture supernatants of antibody hybridomas over multiple rounds. Have passed. Hybridoma supernatants corresponding to the MS17-38 monoclonal antibody were affinity and purified, filtered through a 0.2 μm membrane and stored sterile at 4 ° C. Alternatively, 50% glycerin was added and stored at -20 ° C for a long time.
実施例２
ＱＩＡＧＥＮ社（米国カリフォルニア州バレンシア）のＲＮｅａｓｙ試薬キットを用いてＭＳ１７−３８モノクローナル抗体のハイブリドーマ株からトータルＲＮＡを抽出し、インビトロジェン社（米国ニューヨーク州グランドアイランド）のＳｕｐｅｒＳｃｒｉｐｔ ＩＩＩ Ｆｉｒｓｔ−Ｓｔｒａｎｄ試薬キットを用いてｍＲＮＡをＭＳ１７−３８モノクローナル抗体のｃＤＮＡライブラリに逆転写した。次に、ドイツＰｒｏｇｅｎ Ｂｉｏｔｅｃｈｎｉｋ社の「Ｍｏｕｓｅ ＩｇＧ Ｌｉｂｒａｒｙ Ｐｒｉｍｅｒ Ｓｅｔ」（Ｆ２０１０）試薬キットに含まれる２３個のプライマー及び実験方法を用い、２１個の特定のプライマー対によるＰＣＲ反応を実施した（λＬ鎖の反応は含まない）。そして、発生した特異的なＬ鎖・Ｈ鎖の産物について、ＤＮＡシークエンシング、アミノ酸ポリペプチド配列の翻訳、及びＣＤＲｓ（抗原決定基領域）とＦＷ（足場領域）の識別を実施した。具体的な結果を以下に示す。
Example 2
Total RNA is extracted from the hybridoma strain of MS17-38 monoclonal antibody using RNeasy reagent kit from QIAGEN (Valencia, California, USA), using SuperScript III First-Strand reagent kit from Invitrogen (Grand Island, NY, USA) The mRNA was reverse transcribed into a cDNA library of MS17-38 monoclonal antibody. Next, using the 23 primers and experimental method included in the “Mouse IgG Library Primer Set” (F2010) reagent kit from Progen Biotechnik, Germany, PCR reactions with 21 specific primer pairs were carried out (on the λ L chain) Reaction not included). Then, DNA sequencing, translation of amino acid polypeptide sequence, and discrimination between CDRs (antigenic determinant region) and FW (scaffold region) were carried out for the generated specific light chain and heavy chain products. Specific results are shown below.
ＭＳ１７−３８モノクローナル抗体のＬ鎖可変領域におけるコード配列は、ＳＥＱ ＩＤ ＮＯ：１のようになった。
The coding sequence in the light chain variable region of the MS17-38 monoclonal antibody was as in SEQ ID NO: 1.
ＭＳ１７−３８モノクローナル抗体のＬ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：２のようになった。
The amino acid sequence in the light chain variable region of the MS17-38 monoclonal antibody was as SEQ ID NO: 2.
ＭＳ１７−３８モノクローナル抗体のＨ鎖可変領域におけるコード配列は、ＳＥＱ ＩＤ ＮＯ：３のようになった。
The coding sequence in the heavy chain variable region of the MS17-38 monoclonal antibody was as in SEQ ID NO: 3.
ＭＳ１７−３８モノクローナル抗体のＨ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：４のようになった。
The amino acid sequence in the heavy chain variable region of the MS17-38 monoclonal antibody was as SEQ ID NO: 4.
コード配列であるＳＥＱ ＩＤ ＮＯ：１及びＳＥＱ ＩＤ ＮＯ：３は、それぞれアミノ酸配列であるＳＥＱ ＩＤ ＮＯ：２及びＳＥＱ ＩＤ ＮＯ：４に対応している。アミノ酸配列における下線部はＣＤＲ領域の位置を示しており、ＣＤＲ１、ＣＤＲ２、ＣＤＲ３の順で配列されている。また、これらの間の領域が足場タンパク質配列（ＦＷ）である。 The coding sequences SEQ ID NO: 1 and SEQ ID NO: 3 correspond to the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The underlined portion in the amino acid sequence indicates the position of the CDR region, and is arranged in the order of CDR1, CDR2 and CDR3. Also, the region between them is the scaffold protein sequence (FW).
実施例３
ＭＳ１７−３８モノクローナル抗体全長における真核発現ベクターの構築及びその発現細胞株の作製：
Example 3
Construction of eukaryotic expression vector in full-length MS17-38 monoclonal antibody and preparation of its expression cell line:
ＰＣＲ反応によって上記Ｌ鎖可変領域及びＨ鎖可変領域のＤＮＡのコード配列を増幅し、抗体のＨ鎖可変領域とＬ鎖可変領域の遺伝子の両端に適切な酵素切断点を組み込んだ。ＰＣＲ増幅後に、Ｌ鎖可変領域の遺伝子及びＨ鎖可変領域の遺伝子のＰＣＲ産物をアガロースゲル電気泳動で回収し精製した。Ｌ鎖及びＨ鎖の増幅産物はそれぞれ対応する制限酵素に加え、酵素切断産物をＤＮＡ回収・精製試薬キットにより精製した。ＰＣＲにより取得したＨ鎖（ＶＨ）及びＬ鎖（ＶＬ）の産物をヒトＩｇＧ１ＣＨ１及びＣＬを含有する中間ベクター（ｐＧＥＭ−Ｔ）とライゲーションすることで中間ベクターｐＧＥＭ−Ｔ−Ｈ及びｐＧＥＭ−Ｔ−Ｌをそれぞれ取得してから、取得したＶＬ＋ＣＬ及びＶＨ＋ＣＨ１遺伝子をベクターｐｃＤＮＡ３．１に組み替えた。ライゲーション産物はＤＨ５α大腸菌に形質転換し、５０μｇ／ｍｌのカルベニシリンを含有する２ＹＴ寒天培地に塗布した。取得した陽性クローンは５０μｇ／ｍｌのカルベニシリンを含有する２ＹＴ液体培地で培養し、インビトロジェン社によるシークエンシング検証の後に、Ｐｌａｓｍｉｄ Ｍａｘｉ Ｐｒｅｐａｒａｔｉｏｎ Ｋｉｔを用いて陽性クローンのプラスミドを抽出した。 The coding sequences of the light chain variable region and heavy chain variable region DNA were amplified by PCR reaction, and appropriate enzyme cleavage points were incorporated at both ends of the antibody heavy chain variable region and light chain variable region genes. After PCR amplification, PCR products of the light chain variable region gene and the heavy chain variable region gene were recovered and purified by agarose gel electrophoresis. The amplification products of the L chain and H chain were added to the corresponding restriction enzymes, respectively, and the enzyme cleavage products were purified by the DNA recovery and purification reagent kit. Intermediate vectors pGEM-T-H and pGEM-T-L are obtained by ligating products of H chain (VH) and L chain (VL) obtained by PCR with an intermediate vector (pGEM-T) containing human IgG1 CH1 and CL. Were obtained, and the obtained VL + CL and VH + CH1 genes were recombined into vector pcDNA3.1. The ligation product was transformed into DH5α E. coli and coated on 2YT agar containing 50 μg / ml carbenicillin. The obtained positive clones were cultured in 2YT liquid medium containing 50 μg / ml carbenicillin, and after verification of sequencing by Invitrogen, plasmids of positive clones were extracted using a Plasmid Maxi Preparation Kit.
続いて、インビトロジェン社のＮｅｏｎシステムを用い、線形化したプラスミドＤＮＡをＣＨＯ細胞にトランスフェクションした。トランスフェクションしたＣＨＯ細胞は希釈クローニング培養し、５０マイクロモル（μｍｏｌ）のメチオニンスルホキシミン（ｍｅｔｈｉｏｎｉｎｅ ｓｕｌｆｏｘｉｍｉｎｅ，ＭＳＸ）を含有する９４１１３培地（Ｉｒｖｉｎｅ Ｓｃｉｅｎｔｉｆｉｃ社製）でスクリーニングすることで、モノクローナル抗体発現細胞系を取得した。 Subsequently, linearized plasmid DNA was transfected into CHO cells using the Invitrogen Neon system. The transfected CHO cells are subjected to dilution cloning culture, and screened with 94113 medium (manufactured by Irvine Scientific) containing 50 micromoles (μmol) of methionine sulfoximine (MSX) to obtain a monoclonal antibody-expressing cell line. I got
取得したモノクローナル細胞系は、５０μｍｏｌのメチオニンスルホキシミンを含有する９４１１３培地で振とうフラスコ培養し、生細胞の密度が３０％未満となってから細胞培養液の上清を収集した。そして、Ｐｒｏｔｅｉｎ Ａアフィニティークロマトグラフィーカラムを用い、細胞培養上清から目的の抗体を分離精製した。 The obtained monoclonal cell line was cultured in shake flask in 94113 medium containing 50 μmol of methionine sulfoximine, and the cell culture medium supernatant was collected when the density of living cells became less than 30%. Then, using the Protein A affinity chromatography column, the target antibody was separated and purified from the cell culture supernatant.
次に、モノクローナル細胞からＲＮＡを抽出して目的遺伝子の検出を行い、取得したモノクローナル細胞の目的遺伝子のコピー数を確認して、前記ＭＳ１７−３８モノクローナル抗体であることを検証した。また、抗体タンパク質のＮ末端シークエンシングを実施した結果、抗体ハイブリドーマ株から検出される抗体アミノ酸と配列と一致していた。 Next, RNA was extracted from the monoclonal cells to detect the target gene, and the copy number of the target gene of the obtained monoclonal cell was confirmed to verify that it was the MS17-38 monoclonal antibody. Moreover, as a result of performing N-terminal sequencing of the antibody protein, it was in agreement with the antibody amino acid and the sequence detected from the antibody hybridoma strain.
実施例４
９６ウェル「Ｕ」型プレート上で、１％ＢＳＡ／ＰＢＳを用いて各ウェルにつき平均約２０万個の異なる胃癌細胞（胃癌のＳＧＣ７９０１及びＢＧＣ８２３細胞株と、対照として健常人ＰＢＭＣ）／１００μＬとなるよう配合し、Ｕ型プレートに加えた。そして、胃癌生細胞で免疫した血清（実施例１で取得した免疫後のマウス血清）をそれぞれ５倍力価で段階希釈した後、各ウェルに１００マイクロリットル（μＬ）ずつ加えて均一に混合し、氷上又は４℃下で２０分間反応させた。そして、２回洗浄してから１：３３３で希釈したヤギ抗マウスＩｇＧＦｃ−ＦＩＴＣを１００μＬ／ウェル加え、４℃で反応及び洗浄した後、ＢＤ社のＬＳＲ−ＩＩ蛍光フローサイトメーター・ＨＴＳ機で各ウェルの平均蛍光強度値（ＭＦＩ）を読み取った。
Example 4
On a 96-well "U" -shaped plate, average about 200,000 different gastric cancer cells (SGC7901 and BGC823 cell lines for gastric cancer and healthy human PBMC as a control) / 100 μL per well using 1% BSA / PBS Formulated and added to a U-shaped plate. Then, after serially diluting each serum immunized with live gastric cancer cells (immunized mouse serum obtained in Example 1) with 5-fold titer, add 100 microliters (μL) to each well and mix uniformly The reaction was carried out for 20 minutes on ice or at 4 ° C. Then, after washing twice, add 100 μL / well of goat anti-mouse IgG Fc-FITC diluted 1: 333 and react and wash at 4 ° C, then use LSR-II fluorescence flow cytometer and HTS machine from BD. The mean fluorescence intensity value (MFI) of the wells was read.
その結果、免疫応答性の高かった１号マウスの血清を胃癌細胞と結合させた場合の力価反応は、健常人ＰＢＭＣと結合させた場合の力価よりも明らかに高かった。そこで、当該マウスの脾臓細胞をＭＳ１７−３８モノクローナル抗体のハイブリドーマ産生のための融合実験に使用した（図１は、４種類の混合胃癌細胞株で免疫したマウスの血清と、健常人ＰＢＭＣ、胃癌ＳＧＣ７９０１及びＢＧＣ８２３細胞株との力価反応をＦＡＣＳにより検出したものである）。 As a result, the titer response when the serum of highly immunoresponsive mouse No. 1 was combined with gastric cancer cells was clearly higher than the titer when combined with healthy human PBMC. Therefore, spleen cells of the mouse were used in fusion experiments for producing hybridomas of MS17-38 monoclonal antibody (FIG. 1 shows sera of mice immunized with four types of mixed gastric cancer cell lines, PBMC of healthy subjects, gastric cancer SGC7901. And the titer reaction with the BGC 823 cell line was detected by FACS).
実施例５
親和及び精製したＭＳ１７−３８モノクローナル抗体に対し実施例４の説明で述べたＵ型ウェルプレートによる細胞染色方法を適用し、４種類の免疫用胃癌細胞株を結合染色反応させてＬＳＲ−ＩＩ ＦＡＣＳメーターでＭＦＩを読み取った。なお、その他の実験手順については実施例４と同様とした。
Example 5
The cell staining method with U-well plate described in the description of Example 4 is applied to the affinity and purified MS17-38 monoclonal antibody, and four types of gastric cancer cell lines for immunization are subjected to a binding staining reaction to obtain an LSR-II FACS meter. Read the MFI. The other experimental procedures were the same as in Example 4.
結果、ＭＳ１７−３８モノクローナル抗体と３種類の免疫用胃癌細胞株とを異なるレベルでそれぞれ結合反応させてＦＡＣＳにより検出したところ、ＭＳ１７−３８モノクローナル抗体はＭＫＮ−４５胃癌細胞株に対する反応が最も高いことがわかった。これに対し、ＳＧＣ−７９０１細胞及びＢＧＣ−８２３細胞に対する反応はやや低く、健常人ＰＢＭＣの応答性については結合反応がみられなかった（図２は、ＭＳ１７−３８モノクローナル抗体と、３種類の免疫用胃癌細胞株及び健常人ＰＢＭＣ細胞とを異なるレベルでそれぞれ結合反応させた場合をＦＡＣＳにより検出したものである。このうち、図２（Ａ）は、ＭＳ１７−３８モノクローナル抗体及び対照抗体（アイソタイプが同じ無関係のマウスのモノクローナル抗体。ＩｇＧ１Ｈ鎖、ＫａｐｐａＬ鎖。後述する対照抗体はいずれも同一の対照モノクローナル抗体）と、ＳＧＣ−７９０１及びＢＧＣ−８２３胃癌細胞株との結合反応を示している。また、図２（Ｂ）は、ＭＳ１７−３８モノクローナル抗体及び対照抗体と、ＭＫＮ−４５、ＢＧＣ−８２３及び健常人の末梢血ＰＢＭＣ細胞とをそれぞれ結合反応させた場合を示している）。 As a result, when MS17-38 monoclonal antibody and 3 kinds of gastric cancer cell lines for immunity were made to bind at different levels respectively and detected by FACS, MS17-38 monoclonal antibody had the highest reaction to MKN-45 gastric cancer cell line I understand. On the other hand, the response to SGC-7901 cells and BGC-823 cells was rather low, and no binding reaction was found for the responsiveness of healthy human PBMC (FIG. 2 shows MS17-38 monoclonal antibody and three types of immunity). 2 (A) shows the MS17-38 monoclonal antibody and the control antibody (isotype were different from each other). The same irrelevant mouse monoclonal antibody, IgG1 H chain, Kappa L chain (the control antibody described later is the same control monoclonal antibody) shows the binding reaction of SGC-7901 and BGC-823 gastric cancer cell lines. FIG. 2 (B) shows that MS17-38 monoclonal antibody and control antibody, MKN- 5, BGC-823 and normal human peripheral blood PBMC cells and the shows when combined react respectively).
実施例６
親和及び精製したＭＳ１７−３８モノクローナル抗体に対し実施例４の説明で述べたＵ型ウェルプレートによる細胞染色方法を適用し、健常人ＰＢＭＣ、胃癌細胞株ＭＫＮ−２８、ＧＥＳ−１、ＡＧＳ−Ｎを結合染色反応させてＬＳＲ−ＩＩ ＦＡＣＳメーターでＦＩＴＣ蛍光のＭＦＩ値を読み取った。
Example 6
The cell staining method with U-well plate described in the description of Example 4 is applied to the affinity and purified MS17-38 monoclonal antibody, and healthy human PBMC, gastric cancer cell lines MKN-28, GES-1, AGS-N The combined staining reaction was performed, and the MFI value of FITC fluorescence was read on an LSR-II FACS meter.
結果、ＭＳ１７−３８モノクローナル抗体はＧＥＳ−１及びＡＧＳ細胞株の双方に対し高い結合反応を有するが、健常人ＰＢＭＣには結合反応がみられないことがわかった。また、アイソタイプ対照である無関係のモノクローナル抗体については、いずれも結合反応のない陰性対照であった（図３及び図４は、ＭＳ１７−３８モノクローナル抗体と、２種類の胃癌細胞株及び２つの対照細胞株とを異なるレベルでそれぞれ結合反応させた場合をＦＡＣＳにより検出したものである。このうち、図３では、ＭＳ１７−３８モノクローナル抗体及び対照抗体と、ＭＫＮ−２８及びＡＧＳ−Ｎ細胞株との反応を示している。また、図４では、ＭＳ１７−３８モノクローナル抗体及び対照抗体と、健常人の末梢血ＰＢＭＣ細胞及びＧＥＳ−１（胎児の胃粘膜上皮形質転換細胞）細胞株とをそれぞれ結合反応させた場合を示している）。 As a result, it was found that the MS17-38 monoclonal antibody had high binding responses to both GES-1 and AGS cell lines, but no binding response was seen in healthy human PBMC. In addition, all irrelevant monoclonal antibodies, which are isotype controls, were negative controls without binding reaction (FIGS. 3 and 4 show MS17-38 monoclonal antibody, two gastric cancer cell lines and two control cells. When the binding reaction was carried out at different levels with the strain, they were detected by FACS.In FIG. 3, among the reaction of MS17-38 monoclonal antibody and control antibody with MKN-28 and AGS-N cell lines Also, in Fig. 4, MS17-38 monoclonal antibody and control antibody were respectively reacted with peripheral blood PBMC cells of healthy individuals and GES-1 (fetal gastric epithelial cell transformed cells) cell lines. Show the case).
実施例７
親和及び精製したＭＳ１７−３８モノクローナル抗体を、それぞれ胃癌細胞株ＢＧＣ８２３及び胃癌細胞株ＭＫＮ−４５の細胞膜抽出タンパク質（事前に米国Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製のＩｍｍｕｎｌｏｎ−ＩＩ ９６−ウェルＥＬＩＳＡ反応プレートに充填）と酵素結合反応をさせ（例えばＥＬＩＳＡ等）、ＥＬＩＳＡプレートリーダーにおいて４５０−ｎｍの光学密度でＯＤ値を読み取って作図した。
Example 7
Affinity and purified MS17-38 monoclonal antibody and enzyme extract from gastric cancer cell line BGC 823 and gastric cancer cell line MKN-45 (previously filled in Immunlon-II 96-well ELISA reaction plate manufactured by Fisher Scientific, USA) and enzyme Binding reactions were allowed to occur (e.g. ELISA etc) and OD values were read and plotted at an optical density of 450-nm in an ELISA plate reader.
ＥＬＩＳＡの結果より、ＭＳ１７−３８モノクローナル抗体と胃癌ＢＧＣ８２３及び胃癌ＭＫＮ−４５の膜抽出タンパク質との間には結合反応が認められなかった。このことは、ＭＳ１７−３８モノクローナル抗体がＥＬＩＳＡプレートに付着して分解された結合標的タンパク質とは反応し得ないことを意味している。つまり、ＭＳ１７−３８モノクローナル抗体と抗原の立体構造エピトープとの特異な反応が示された。これは、後述する抗体の共免疫沈降法における特殊な操作条件のための準備的実験となった（ＭＳ１７−３８モノクローナル抗体と、胃癌ＢＧＣ８２３（図５参照）及び胃癌ＭＫＮ−４５（図６参照）の細胞膜抽出タンパク質とを結合させたＥＬＩＳＡ検出反応を参照）。 From the results of the ELISA, no binding reaction was observed between the MS17-38 monoclonal antibody and the membrane extract proteins of gastric cancer BGC823 and gastric cancer MKN-45. This means that the MS17-38 monoclonal antibody can not react with the bound target protein attached to the ELISA plate and degraded. That is, a specific reaction between the MS17-38 monoclonal antibody and a conformational epitope of the antigen was shown. This has become a preliminary experiment for the special operating conditions in the co-immunoprecipitation method of antibodies described later (MS17-38 monoclonal antibody, gastric cancer BGC823 (see FIG. 5) and gastric cancer MKN-45 (see FIG. 6) (See ELISA detection reaction coupled to cell membrane extract protein of
実施例８
胃粘膜形質転換細胞ＧＥＳ−１及び胃癌細胞ＢＧＣ８２３、ＭＫＮ４５をサイトスピン（Ｃｙｔｏｓｐｉｎ）してからＭＳ１７−３８モノクローナル抗体と結合し、カタラーゼ染色反応させた結果、標的タンパク質がいずれも細胞膜表面に分布していることがわかった（拡大倍率は各４０ｘ）（免疫組織化学法）。また、胃粘膜形質転換細胞ＧＥＳ−１についてはサイトスピン（Ｃｙｔｏｓｐｉｎ）後に、培養上清液を親和及び精製して取得したＭＳ１７−３８モノクローナル抗体と結合した。これをカタラーゼ染色反応させた結果、モノクローナル抗体が細胞膜表面の標的タンパク質に結合可能なことがわかった。
Example 8
Gastric mucosa-transformed cells GES-1 and gastric cancer cells BGC823 and MKN45 were cytospin (Cytospin), then combined with MS17-38 monoclonal antibody and catalase stained. As a result, all target proteins were distributed on the cell membrane surface (Magnification: 40x each) (immunohistochemistry). In addition, with respect to gastric mucosa transformed cell GES-1, after culture cells were subjected to cytospin (Cytospin), the culture supernatant fluid was bound to the MS17-38 monoclonal antibody obtained by affinity and purification. As a result of catalase staining reaction, it was found that the monoclonal antibody can bind to a target protein on the cell membrane surface.
免疫組織化学法の検出結果から、ＭＳ１７−３８モノクローナル抗体が胃粘膜形質転換細胞ＧＥＳ−１及び胃癌細胞ＢＧＣ８２３、ＭＫＮ４５の細胞膜表面に結合可能なことが示されたが、このことは、ＭＳ１７−３８モノクローナル抗体が標的タンパク質に照準を合わせていることの証拠でもある（図７は、ＭＳ１７−３８モノクローナル抗体及びアイソタイプが同じ無関係なモノクローナル抗体の、Ａ．胃粘膜形質転換細胞ＧＥＳ−１、Ｂ．胃癌細胞ＭＫＮ−４５、Ｃ．胃癌細胞ＢＧＣ−８２３における免疫組織化学反応を検出したものである）。 The results of detection by immunohistochemistry showed that the MS17-38 monoclonal antibody was able to bind to the cell membrane surface of gastric mucosa transformed cell GES-1 and gastric cancer cells BGC823 and MKN45, which means that MS17-38 It is also evidence that the monoclonal antibody is targeting the target protein (FIG. 7 shows A. gastric mucosa transformed cell GES-1, B. gastric cancer, of MS17-38 monoclonal antibody and unrelated isotype identical monoclonal antibody). Immunohistochemical reaction in cells MKN-45, C. gastric cancer cells BGC-823 is detected.
実施例９
胃癌細胞株ＢＧＣ８２３及びＭＫＮ４５の細胞膜抽出タンパク質をＭＳ１７−３８モノクローナル抗体との親和カラム（特に、結合するＭＳ１７−３８モノクローナル抗体との親和精製カラム）で精製及び共免疫沈降（ＩＰ）した後、産物を分子量の異なる分子マーカーサンプルとともにそれぞれサンプル投入して電気泳動（ＳＤＳ−ＰＡＧＥ）を実施した。電気泳動後にＳＤＳ−ＰＡＧＥゲルをブロモフェノールブルー染色した結果、ＭＳ１７−３８モノクローナル抗体により精製された胃癌ＭＫＮ４５及びＢＧＣ８２３の細胞膜抽出タンパク質は抗原純度がたいへん高いことがわかった。また、抗原ストリップゲルをそれぞれ切り出すことで、更に抗原の質量分析を実施したいとの要求にも対応可能であった（図８は、ＭＳ１７−３８モノクローナル抗体によって胃癌ＢＧＣ８２及びＭＫＮ４５の細胞膜抽出標的タンパク質に対し実施したＳＤＳ−ＰＡＧＥゲルの精製ストリップを示す）。
Example 9
After purification and co-immunoprecipitation (IP) of cell membrane-extracted proteins of gastric cancer cell lines BGC 823 and MKN 45 on an affinity column with MS 17-38 monoclonal antibody (in particular, affinity purification column with MS 17-38 monoclonal antibody to be bound) Electrophoresis (SDS-PAGE) was performed by loading each sample together with molecular marker samples of different molecular weights. As a result of performing bromophenol blue staining of SDS-PAGE gel after electrophoresis, it was found that cell membrane extract proteins of gastric cancer MKN45 and BGC823 purified by MS17-38 monoclonal antibody had very high antigen purity. In addition, it was possible to cope with the need to further analyze the mass of the antigen by cutting out each of the antigen strip gels (FIG. 8 shows the target proteins of cell membranes of gastric cancer BGC 82 and MKN 45 by the MS 17-38 monoclonal antibody. The purification strip of the SDS-PAGE gel carried out is shown.
実施例１０
実施例９で述べたように、間接免疫沈降法では、ＭＳ１７−３８モノクローナル抗体との親和カラムによって、結合する抗原を胃癌細胞（ＭＫＮ４５及びＢＧＣ８２３）の膜抽出タンパク質から精製し、抗体のＦｃ末端がＰｒｏｔｅｉｎ−Ａの磁気ビーズに特異的に結合して非特異的吸着タンパクが洗い流された後、ＳＤＳ−ＰＡＧＥにサンプルを投入し、緩衝液を加えて加熱解離した。一方、直接免疫沈降法では、ＭＳ１７−３８モノクローナル抗体を、米国インビトロジェン社（米国ニューヨーク州グランドアイランド）製の活性化したＤｙｎａｂｅａｄｓ磁気ビーズに直接連結し、それ以降の反応手順は間接免疫沈降法と同様とした。実践より、ＭＳ１７−３８モノクローナル抗体の免疫沈降法としては、直接法でなければ特異的且つ明瞭な標的ストリップを得られないことが証明された（図８参照）。その後、複数回の高感度質量分析によって、ＭＳ１７−３８モノクローナル抗体に対応する標的がＰＯＤＸＬ−ｖ２タンパク質であることが特定された（図９は、ＭＳ１７−３８モノクローナル抗体によって胃癌ＢＧＣ８２及びＭＫＮ４５の細胞膜抽出標的タンパク質に対し実施した３度の質量分析の結果を示す）。
Example 10
As described in Example 9, in the indirect immunoprecipitation method, the binding antigen is purified from a membrane extract of gastric cancer cells (MKN45 and BGC823) by affinity column with MS17-38 monoclonal antibody, and the Fc end of the antibody is After binding specifically to Protein-A magnetic beads and washing away nonspecific adsorbed proteins, samples were loaded on SDS-PAGE, buffer was added, and heat dissociation was performed. On the other hand, in direct immunoprecipitation, the MS17-38 monoclonal antibody is directly linked to activated Dynabeads magnetic beads manufactured by Invitrogen (Grand Island, NY, USA), and the subsequent reaction procedure is the same as indirect immunoprecipitation. And From practice, it has been proved that specific and clear target strips can not be obtained only by direct methods for immunoprecipitation of MS17-38 monoclonal antibody (see FIG. 8). After that, the target corresponding to the MS17-38 monoclonal antibody was identified as PODXL-v2 protein by multiple times of high sensitivity mass spectrometry (FIG. 9: Cell membrane extraction of gastric cancer BGC82 and MKN45 by the MS17-38 monoclonal antibody The results of three mass analyzes performed on the target protein are shown).
実施例１１
ＮＣＢＩの大規模データベースのデータより、ＰＯＤＸＬのいくつかのサブタイプのうち最長のアミノ酸配列における８−ｍｅｒのアミノ酸と６−ｍｅｒのアミノ酸を重複させ、マイクロマトリックスチップに積層した。次に、ＭＳ１７−３８モノクローナル抗体に蛍光標識したヤギ抗体を加えて間接的なハイブリダイゼーションを実施したところ、アミノ酸配列のうち２２１〜２２４と４７３〜４７７の２つセグメントにおいて強い蛍光結合強度が認められた。即ち、ＭＳ１７−３８モノクローナル抗体は、異性体ＰＯＤＸＬ−ｖ２と結合することが示された（図１０は、マイクロマトリックスチップに積載した６−ｍｅｒ及び８−ｍｅｒのアミノ酸をＭＳ１７−３８モノクローナル抗体により分析したものである）。ＭＳ１７−３８モノクローナル抗体が異性体ＰＯＤＸＬ−ｖ２と特異的に結合したのは、ＰＯＤＸＬ−ｖ２のみが２つの空間立体構造の結合座位間に配列の欠損を有していたからである。このような２つの立体構造の座位が形成されるにはフォールディングが必要であり、ＰＯＤＸＬとＰＯＤＸＬ−ｖ１にはＭＳ１７−３８モノクローナル抗体とＰＯＤＸＬ−ｖ２との結合のような立体構造座位は形成され得ない（図１１は、ＰＯＤＸＬ、ＰＯＤＸＬ−ｖ１、ＰＯＤＸＬ−ｖ２のアミノ酸配列における比較及びこれらの違いと、ＰＯＤＸＬ−ｖ２における空間立体構造の座位のうちＭＳ１７−３８モノクローナル抗体が具体的に結合する２つの座位を示している）。
Example 11
From data of a large-scale database of NCBI, 8-mer amino acids and 6-mer amino acids in the longest amino acid sequence among several subtypes of PODXL were overlapped and stacked on a micro matrix chip. Next, when fluorescently labeled goat antibody was added to MS17-38 monoclonal antibody and indirect hybridization was performed, strong fluorescence binding strength was observed in two segments of 221 to 224 and 473 to 477 in the amino acid sequence. The That is, the MS17-38 monoclonal antibody was shown to bind to the isomer PODXL-v2 (FIG. 10, analysis of the 6-mer and 8-mer amino acids loaded on the micromatrix chip with the MS17-38 monoclonal antibody) That). The MS17-38 monoclonal antibody specifically bound to the isomer PODXL-v2 because only PODXL-v2 had a sequence defect between the binding loci of the two spatial conformations. Such two conformational loci require folding to form, and PODXL and PODXL-v1 may form conformational loci such as the binding of the MS17-38 monoclonal antibody to PODXL-v2. (FIG. 11 shows the comparison of the amino acid sequences of PODXL, PODXL-v1 and PODXL-v2 and their differences, and the two-dimensional binding of MS17-38 monoclonal antibody among the loci of spatial conformation in PODXL-v2 Show sitting position).
実施例１２
ＭＫＮ−４５細胞の培養では、Ａｍｂｉｏｎ Ｌｉｆｅｔｅｃｈ社（現サーモフィッシャー・サイエンティフィック社）から購入した各２５ｎＭ（ナノモル）の最終濃度のＰＯＤＸＬ−ｖ２（品番ｓ１０７６９）、ＦＡＭ１２０Ｂ、Ｓｅｃ１６Ａ、ＳＭＡＲＣＣ１及びブランク対照ｓｉＲＮＡをそれぞれ添加した。そして、細胞に対しｓｉＲＮＡをトランスフェクションすることで、最終的に各ｓｉＲＮＡの標的タンパク質の発現が抑制された。次に、処理済みの細胞タンパク質を抽出し、それぞれＳＤＳ−ＰＡＧＥにおいて各タンパク質ストリップを分離した。そして、ＭＳ１７−３８モノクローナル抗体とβ−Ａｃｔｉｎ抗体（ウェスタンブロットの対照）を用いて特異的に反応させたところ、ＰＯＤＸＬ−ｖ２とｓｉＲＮＡで処理したＭＫＮ−４５の細胞抽出タンパク質のみにおいて、ＭＳ１７−３８モノクローナル抗体と結合可能なＰＯＤＸＬ−ｖ２ストリップベルトが消失した。即ち、別の観点からもＭＳ１７−３８モノクローナル抗体がＰＯＤＸＬ−ｖ２と特異的に反応することが証明された（図１２は、ＭＫＮ４５細胞膜上でのＰＯＤＸＬ−ｖ２の発現をＰＯＤＸＬ−ｖ２とｓｉＲＮＡが干渉することをウェスタンブロット試験により示したものである）。
Example 12
For the culture of MKN-45 cells, PODXL-v2 (part number 10769), FAM120B, Sec16A, SMARCC1 and blank control siRNA at a final concentration of 25 nM (nanomolar) each purchased from Ambion Lifetech (now Thermo Fisher Scientific). Were each added. Then, by transfecting the cells with siRNA, the expression of the target protein of each siRNA was finally suppressed. The treated cell proteins were then extracted and each protein strip separated in SDS-PAGE. Then, when specifically reacted using the MS17-38 monoclonal antibody and the β-Actin antibody (control of the Western blot), only in the cell extract protein of MKN-45 treated with PODXL-v2 and siRNA, MS17-38 The POD XL-v2 strip belt capable of binding to the monoclonal antibody disappeared. That is, it was proved that the MS17-38 monoclonal antibody specifically reacts with PODXL-v2 also from another point of view (FIG. 12 shows that PODXL-v2 and siRNA interfere with the expression of PODXL-v2 on MKN45 cell membrane) Western blot test)).
以上述べたように、本発明は従来技術における様々な欠点を解消するものであり、高度な産業上の利用価値を有する。 As mentioned above, the present invention solves various drawbacks in the prior art, and has a high industrial value.
上記の実施例は本発明の原理と効果を例示的に説明するものにすぎず、本発明を制限するものではない。本技術を熟知する者であれば、本発明の精神及び範囲を逸脱しないことを前提に、上記の実施例を補足又は変形可能である。したがって、当業者が本発明で開示した精神及び技術的思想から逸脱することなく遂行するあらゆる等価の補足又は変形もまた本発明の請求の範囲に含まれる。 The above embodiments are merely illustrative of the principles and advantages of the present invention and are not intended to limit the present invention. Those skilled in the art can supplement or modify the above-described embodiments, provided that they do not depart from the spirit and scope of the present invention. Accordingly, all equivalent supplements or modifications which one of ordinary skill in the art performs without departing from the spirit and technical concept disclosed in the present invention are also included in the claims of the present invention.
Claims (13)
当該抗体のＬ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：２又はその保存的変異体配列であり、当該抗体のＨ鎖可変領域におけるアミノ酸配列は、ＳＥＱ ＩＤ ＮＯ：４又はその保存的変異体配列であるモノクローナル抗体。 A monoclonal antibody to podocalyxin-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells,
The amino acid sequence in the L chain variable region of the antibody is SEQ ID NO: 2 or a conservative variant sequence thereof, and the amino acid sequence in the H chain variable region of the antibody is SEQ ID NO: 4 or a conservative variant thereof A monoclonal antibody that is a sequence.
前記抗体の発現に適した条件で、前記モノクローナル抗体の発現系を培養することで前記モノクローナル抗体を発現し、前記モノクローナル抗体を精製及び分離するステップを含む方法。 A method for producing the monoclonal antibody according to any one of claims 1 to 4, wherein
A method comprising the steps of: expressing the monoclonal antibody by culturing an expression system of the monoclonal antibody under conditions suitable for expression of the antibody; and purifying and separating the monoclonal antibody.
A diagnostic reagent kit comprising a monoclonal antibody against podocalyxin-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells or an immunocomplex thereof.
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| CN201511029117.3A CN105504060B (en) | 2015-12-31 | 2015-12-31 | A kind of monoclonal antibody and its preparation method and application of the sufficient calyx sample amyloid protein precursor hypotype 2 of anti-gastric cancer cell surface functional expression |
| PCT/CN2016/110695 WO2017114204A1 (en) | 2015-12-31 | 2016-12-19 | Monoclonal antibody of anti-podocalyxin-like protein precursor subtype 2 and preparation method and use thereof |
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| CN108101990B (en) * | 2017-11-28 | 2020-11-03 | 浙江大学 | Monoclonal antibody for blocking human Tim-3 function and coding gene and application thereof |
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| WO2011004837A1 (en) * | 2009-07-08 | 2011-01-13 | 株式会社ＡＣＴＧｅｎ | Antibody having anti-cancer activity |
| JP2014525584A (en) * | 2011-08-31 | 2014-09-29 | オンコサイト コーポレーション | Methods and compositions for the treatment and diagnosis of cancer |
| WO2016129660A1 (en) * | 2015-02-12 | 2016-08-18 | 国立大学法人東北大学 | Cancer cell-specific anti-podocalyxin antibody and method for producing same |
| WO2017054089A1 (en) * | 2015-10-01 | 2017-04-06 | The Centre For Drug Research And Development | Anti-podocalyxin antibodies and methods of using the same |
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| JP2014525584A (en) * | 2011-08-31 | 2014-09-29 | オンコサイト コーポレーション | Methods and compositions for the treatment and diagnosis of cancer |
| WO2016129660A1 (en) * | 2015-02-12 | 2016-08-18 | 国立大学法人東北大学 | Cancer cell-specific anti-podocalyxin antibody and method for producing same |
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