JP2015196665A - Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same - Google Patents
Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same Download PDFInfo
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Abstract
【課題】LR11に特異的に結合し、優れた中和活性を有する新規モノクローナル抗体、及びそれを含有する悪性腫瘍の予防及び／又は治療剤の提供。【解決手段】特定の配列の群から選択される少なくとも１つのアミノ酸配列を相補性決定領域（CDR）として有する抗LR11モノクローナル抗体は、（Ａ）LR11を認識することができ、（Ｂ）LR11のVps10pドメインのアミノ酸配列からなるタンパク質に対する特異的結合能を有し、かつ、（Ｃ）接着性アッセイにおいてヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進される細胞接着性を有意に抑制する、抗LR11モノクローナル抗体。また、前記の抗LR11モノクローナル抗体を含有してなる悪性腫瘍の予防及び／又は治療剤。【選択図】なしThe present invention provides a novel monoclonal antibody that specifically binds to LR11 and has an excellent neutralizing activity, and a preventive and / or therapeutic agent for malignant tumors containing the same. An anti-LR11 monoclonal antibody having, as a complementarity determining region (CDR), at least one amino acid sequence selected from a group of specific sequences can recognize (A) LR11, and (B) LR11 Has a specific binding ability to a protein comprising the amino acid sequence of the Vps10p domain, and (C) significantly suppresses cell adhesion enhanced by a protein comprising the amino acid sequence of the human LR11 Vps10p domain in the adhesion assay; Anti-LR11 monoclonal antibody. In addition, a preventive and / or therapeutic agent for malignant tumors comprising the anti-LR11 monoclonal antibody. [Selection figure] None
Description
本発明は、モノクローナル抗体、特に、LR11の細胞外ドメインであるVps10pドメインによってコードされるエピトープによって細胞表面LR11及び可溶性LR11（以下、「sLR11」という。）に特異的に結合し、多くの好ましい特徴を有するモノクローナル抗体に関する。本発明はまた、細胞表面LR11及びsLR11の持つ生物活性を阻害して中和する活性を持つモノクローナル抗体に関する。本発明はまた、前記モノクローナル抗体を含む医薬組成物、又は各種癌の治療方法における使用のための前記モノクローナル抗体を提供する。本発明はさらに、前記モノクローナル抗体の癌治療用医薬の製造における使用に関する。 The present invention specifically binds to cell surface LR11 and soluble LR11 (hereinafter referred to as “sLR11”) by an epitope encoded by a monoclonal antibody, particularly the Vps10p domain that is the extracellular domain of LR11, and has many preferable features. It relates to a monoclonal antibody having The present invention also relates to a monoclonal antibody having an activity of inhibiting and neutralizing the biological activity of cell surface LR11 and sLR11. The present invention also provides a pharmaceutical composition comprising the monoclonal antibody or the monoclonal antibody for use in a method for treating various cancers. The invention further relates to the use of said monoclonal antibody in the manufacture of a medicament for the treatment of cancer.
LR11（Low-density lipoprotein (LDL) receptor relative with 11 ligand-binding repeats）はLDL受容体ファミリータンパク質であり（特許文献１）、その構造は膜貫通領域を1回含むI型の膜タンパク質で、LDL受容体ファミリーの共通した構造であるリガンド結合領域（clusters of type A ligand-binding repeats又はcomplement-type repeats clusters of complement-type repeats）が11回繰り返している構造を持っている（非特許文献１）。LR11は、ヒト、ウサギ、マウス、ラット、ニワトリ等、種差を超えてそのアミノ酸配列は高く保存され、リガンド結合領域の繰り返し構造のＮ末端側にはVps10p（Vacuolar protein sorting 10 protein）類似ドメインを、Ｃ末端側にはフィブロネクチンtypeIIIドメインといった構造を有し、さらにＣ末端側には膜貫通領域、細胞質尾部Ｃ末端を有している。LR11は神経細胞での発現が高く、ヒトにおいては脳や腎臓での発現が高い。また、細胞表面上だけでなく細胞内のエンドゾームにも分布がみられる。脳組織より同定されたSorLA（sorting protein-related receptor containing LDL receptor class A repeats）とLR11は同一のタンパク質であり、ヒト遺伝子名はSORL1である（非特許文献２）。さらに、細胞表面に存在するLR11は膜貫通領域直上でプロテアーゼTACE（tumor necrosis factor-alpha converting enzyme；TNF-α変換酵素）により切断され、細胞外部分はsLR11としてVps10pを含む画分が細胞外へ放出される。LR11は正常血管では発現がみられないが、動脈硬化巣においては、平滑筋細胞の遊走（cell migration）及び増殖によって形成された血管内膜肥厚部位において特異的に発現が亢進することが知られている（非特許文献３、４）。LR11は膜結合型、放出可溶型ともに、細胞表面上のウロキナーゼ（以下、「uPA」という。）受容体（以下、「uPAR」という。）と結合する（非特許文献５）。uPARはさらに細胞接着分子のインテグリンとも細胞表面上で複合体を形成する。可溶性LR11は、uPA-uPAR系を介したプラスミン・カスケードの亢進により、細胞外マトリックスを分解させるとともに、マトリックスメタロプロテアーゼを活性化させることで、細胞周囲の細胞外マトリックスは分解される。さらに、uPAと結合したuPARはインテグリンを介した細胞内シグナルを誘発するが、放出sLR11はuPARと結合することにより、インテグリン/FAK/Rac1経路が活性化し、細胞骨格の構造を変化させて血管平滑筋細胞の遊走能を亢進させると考えられている（非特許文献６）。 LR11 (Low-density lipoprotein (LDL) receptor relative with 11 ligand-binding repeats) is an LDL receptor family protein (Patent Document 1), and its structure is a type I membrane protein containing a transmembrane region once. The ligand binding region (clusters of type A ligand-binding repeats or complement-type repeats of complement-type repeats), which is a common structure of the receptor family, has a structure that repeats 11 times (Non-patent Document 1). . LR11, such as human, rabbit, mouse, rat, chicken, etc., has a highly conserved amino acid sequence across species differences, and a Vps10p (Vacuolar protein sorting 10 protein) -like domain is present on the N-terminal side of the repeating structure of the ligand binding region. It has a structure such as a fibronectin type III domain on the C-terminal side, and further has a transmembrane region and a cytoplasmic tail C-terminal on the C-terminal side. LR11 is highly expressed in nerve cells, and in humans, it is highly expressed in brain and kidney. Moreover, distribution is seen not only on the cell surface but also in intracellular endosomes. SorLA (sorting protein-related receptor containing LDL receptor class A repeats) identified from brain tissue and LR11 are the same protein, and the human gene name is SORL1 (Non-patent Document 2). Furthermore, LR11 present on the cell surface is cleaved by protease TACE (tumor necrosis factor-alpha converting enzyme) directly above the transmembrane region, and the extracellular portion of the fraction containing Vps10p as sLR11 goes out of the cell. Released. LR11 is not expressed in normal blood vessels, but in arteriosclerotic lesions, it is known that expression is specifically increased at the site of intimal thickening formed by smooth muscle cell migration and proliferation. (Non-Patent Documents 3 and 4). LR11 binds to a urokinase (hereinafter referred to as “uPA”) receptor (hereinafter referred to as “uPAR”) on the cell surface in both membrane-bound and release-soluble forms (Non-patent Document 5). uPAR also forms a complex on the cell surface with the cell adhesion molecule integrin. Soluble LR11 degrades the extracellular matrix by activating the plasmin cascade via the uPA-uPAR system and activates the matrix metalloprotease to degrade the extracellular matrix surrounding the cell. Furthermore, uPAR bound to uPA induces integrin-mediated intracellular signals, but released sLR11 binds to uPAR, thereby activating the integrin / FAK / Rac1 pathway and altering cytoskeletal structure to smooth blood vessels. It is considered to enhance the migration ability of muscle cells (Non-patent Document 6).
また、脳において強く発現しているLR11は神経細胞で細胞内ゴルジ−小胞体ネットワークに局在し神経細胞内で小胞間のタンパク質輸送に関わる（非特許文献７）。LR11はアミロイド前駆体輸送にも関与し、アミロイド前駆タンパク質をゴルジ体中に留め、また、分泌胞に移行したアミロイド前駆タンパク質を再びゴルジ体に戻し、アミロイド前駆タンパク質からアミロイドを生成するβセクレターゼの機能を阻害することで、アミロイドβの産生を抑制することからLR11とアルツハイマー病との関連が報告されている（非特許文献７）。すなわち、アルツハイマー病患者の脳内でのLR11発現減少（非特許文献８、９）や、LR11の遺伝子多型がアルツハイマー病の発症リスクになる可能性が指摘されている（非特許文献１０）。
さらに、髄液中のsLR11濃度はアルツハイマー病で高値となり（非特許文献１１）、アルツハイマー病の発症リスクと関連する患者のLR11遺伝子多型と髄液中のLR11濃度は相関するという報告もある（非特許文献１２−１４）。
LR11, which is strongly expressed in the brain, is localized in the intracellular Golgi-endoplasmic reticulum network in neurons and is involved in protein transport between vesicles in neurons (Non-patent Document 7). LR11 is also involved in amyloid precursor transport, retains the amyloid precursor protein in the Golgi apparatus, returns the amyloid precursor protein transferred to the secretory vesicle to the Golgi apparatus again, and functions of β-secretase to generate amyloid from the amyloid precursor protein By inhibiting the production of amyloid β, the association between LR11 and Alzheimer's disease has been reported (Non-patent Document 7). That is, it has been pointed out that the decrease in LR11 expression in the brain of Alzheimer's disease patients (Non-Patent Documents 8 and 9) and the possibility that an LR11 gene polymorphism is a risk of developing Alzheimer's disease (Non-Patent Document 10).
Furthermore, the sLR11 concentration in the cerebrospinal fluid becomes high in Alzheimer's disease (Non-patent Document 11), and there is a report that the LR11 gene polymorphism in patients associated with the risk of developing Alzheimer's disease correlates with the LR11 concentration in cerebrospinal fluid ( Non-patent literature 12-14).
近年、LR11が血液幹／前駆細胞、すなわち、CD34(+)CD38(-)未分化骨髄幹細胞で高発現する遺伝子の一つであることが見出された（非特許文献１５）。さらに、急性骨髄性白血病（以下、「AML」という。）や急性リンパ性白血病（以下、「ALL」という。）等の急性白血病患者の白血病細胞表面でのLR11の発現が高まることや患者血清中のsLR11濃度が高値になり、化学療法による寛解時にはその値が低下することが報告されている（非特許文献１６）。また、非ホジキンリンパ腫（びまん性大細胞型B細胞性リンパ腫、ろ胞性リンパ腫）においても患者血清でsLR11濃度は高値となり、初発時高値の場合には予後不良となることが報告されている（非特許文献１７）。このようにsLR11は、悪性腫瘍、特に白血病や悪性リンパ腫のような造血器腫瘍疾患で異常高値を示し、重要なバイオマーカーの１つとなり得る（特許文献２）。 Recently, LR11 was found to be one of the genes highly expressed in blood stem / progenitor cells, that is, CD34 (+) CD38 (−) undifferentiated bone marrow stem cells (Non-patent Document 15). Furthermore, the expression of LR11 on the surface of leukemia cells of acute leukemia patients such as acute myeloid leukemia (hereinafter referred to as “AML”) and acute lymphocytic leukemia (hereinafter referred to as “ALL”) is increased, and in the serum of patients. It has been reported that the sLR11 concentration of the drug becomes high, and the value decreases during remission by chemotherapy (Non-patent Document 16). It has also been reported that sLR11 concentration is high in patient serum in non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma), and that the prognosis is poor if the initial high level is high (non Patent Document 17). Thus, sLR11 shows an abnormally high value in malignant tumors, particularly hematopoietic tumor diseases such as leukemia and malignant lymphoma, and can be one of important biomarkers (Patent Document 2).
AMLやALL等の急性白血病は、化学療法に対する感受性が高く、抗癌剤によって白血病細胞の数が著しく減少し症状が改善される「寛解」が得られる場合が多い。しかし、一旦寛解状態を得た患者でも再発率が高いことが白血病治療の課題となっている。この白血病における再発のメカニズムは、骨髄内の「ニッチ」と呼ばれる骨髄と骨内膜の境界域に存在する白血病幹細胞が抗癌剤に対して抵抗性があるために生き残り、再び自己複製を行いながら、白血病細胞集団を供給するためと考えられている（非特許文献１８）。白血病幹細胞に抗癌剤抵抗性があるのは、白血病幹細胞がニッチ環境下で特異的に細胞周期を静止させているためであり、抗癌剤は増殖活性の高い癌細胞を標的としていることから、白血病幹細胞が抵抗性を持つと考えられている（非特許文献１８）。このようにニッチと呼ばれる骨内微小環境下で白血病幹細胞が静止期を維持できるのは、白血病幹細胞が周囲の細胞外基質(マトリクス）や間質細胞との接着状態が局在を保っていることが重要と考えられている。一方、最近になって骨髄ニッチ等の低酸素条件下では、造血幹細胞のLR11の発現が亢進され、uPARを介した周囲の細胞外基質や間質細胞との接着を制御していることが報告された（非特許文献１９）。さらに、放射線照射し、抗アシアロGM1抗体を投与した免疫不全マウスに、LR11を高発現しているヒト単球系リンパ腫細胞株U937細胞を移植するAMLモデルにおいて、LR11発現を抑制させたU937細胞(LR11-KD)を移植した場合には、骨髄への浸潤、生着数は減少し、生存延長が認められ、さらにLR11-KD U937細胞はヒト骨髄系間質細胞への接着性も低下していた（非特許文献２０）。以上の結果は、LR11がAMLの病態に重要な修飾因子として関与していることを示唆している。 Acute leukemias such as AML and ALL are highly sensitive to chemotherapy, and anticancer agents often provide a “remission” in which the number of leukemia cells is significantly reduced and symptoms are improved. However, a high recurrence rate even in patients who have achieved remission is a challenge for leukemia treatment. The mechanism of recurrence in this leukemia is that the leukemia stem cells located in the border between the bone marrow and the endosteum, called the “niche” in the bone marrow, survive because they are resistant to anticancer drugs, and self-replicate It is considered to supply a cell population (Non-patent Document 18). Leukemia stem cells are resistant to anticancer drugs because leukemia stem cells specifically arrest the cell cycle in a niche environment, and anticancer drugs target cancer cells with high proliferative activity. It is considered to have resistance (Non-patent Document 18). In this way, leukemia stem cells can maintain a quiescent phase in an intraosseous microenvironment called a niche because leukemia stem cells maintain their local adhesion to the extracellular matrix (matrix) and stromal cells. Is considered important. On the other hand, recently, under hypoxic conditions such as bone marrow niche, LR11 expression of hematopoietic stem cells is enhanced and regulates adhesion to surrounding extracellular matrix and stromal cells via uPAR (Non-patent Document 19). Furthermore, U937 cells with suppressed LR11 expression in an AML model in which human monocyte lymphoma cell line U937 cells highly expressing LR11 were transplanted into immunodeficient mice that had been irradiated and administered anti-asialo GM1 antibody ( When LR11-KD) was transplanted, bone marrow infiltration and engraftment decreased, survival was increased, and LR11-KD U937 cells also had reduced adhesion to human myeloid stromal cells. (Non-Patent Document 20). These results suggest that LR11 is involved as an important modifier in the pathogenesis of AML.
抗LR11抗体は前記疾患の診断マーカーへの利用や治療への応用が示唆されている（特許文献２及び３、非特許文献２１）。また、抗LR11抗体がラット平滑筋細胞に対する接着亢進作用を抑制する効果も知られているが(非特許文献２２)、その作用強度は十分なものではない。 The anti-LR11 antibody has been suggested to be used as a diagnostic marker for the disease and applied to treatment (Patent Documents 2 and 3, Non-Patent Document 21). Moreover, although the effect which anti-LR11 antibody suppresses the adhesion promotion effect | action with respect to a rat smooth muscle cell is also known (nonpatent literature 22), the effect | action intensity | strength is not enough.
本発明の目的は、LR11に特異的に結合し、優れた中和活性を有し、造血器腫瘍、上皮性悪性腫瘍等の悪性腫瘍の予防及び治療効果に優れた新規モノクローナル抗体、及びそれを含有してなる医薬組成物を提供することにある。 An object of the present invention is to provide a novel monoclonal antibody that specifically binds to LR11, has an excellent neutralizing activity, and has an excellent prophylactic and therapeutic effect on malignant tumors such as hematopoietic tumors and epithelial malignant tumors, and It is in providing the pharmaceutical composition which contains.
本発明者らは、既存のLR11を認識するモノクローナル抗体では明確な報告のない、sLR11の持つ生物活性である細胞接着亢進作用や細胞遊走亢進作用を阻害する中和抗体としての作用を有する新規な抗LR11モノクローナル抗体の開発を検討してきたところ、特定の抗原を用いて製造した抗体を、接着性アッセイによりスクリーニングすることにより、細胞接着亢進作用や細胞遊走亢進作用を阻害する中和抗体として優れた作用を有する抗LR11モノクローナル抗体が得られることを見出した。このようにして得られた抗体は、優れた接着抑制作用や遊走抑制作用を有し、本抗体が悪性腫瘍に対して予防及び治療効果を示すことを見出し、本発明を完成するに至った。 The present inventors have not reported clearly with existing monoclonal antibodies recognizing LR11, and have a novel activity that acts as a neutralizing antibody that inhibits the cell adhesion enhancing action and cell migration enhancing action that are biological activities of sLR11. We have been investigating the development of anti-LR11 monoclonal antibodies, and by screening antibodies produced using specific antigens by adhesion assays, they are excellent as neutralizing antibodies that inhibit cell adhesion enhancement and cell migration enhancement. It was found that an anti-LR11 monoclonal antibody having an action can be obtained. The antibody thus obtained has an excellent adhesion inhibitory action and migration inhibitory action, and it has been found that the antibody exhibits preventive and therapeutic effects on malignant tumors, thereby completing the present invention.
即ち、本発明は、LR11のVps10pドメインを抗原として製造された抗体であって、接着性アッセイにおいてヒトLR11のVps10pドメインからなるタンパク質により亢進された細胞接着性を有意に抑制することができる抗LR11モノクローナル抗体、及びその製造方法に関する。
また、本発明は、本発明の抗LR11モノクローナル抗体を含有してなる医薬組成物に関する。
That is, the present invention is an antibody produced using the Vps10p domain of LR11 as an antigen, and can significantly suppress cell adhesion enhanced by a protein comprising the Vps10p domain of human LR11 in an adhesion assay. The present invention relates to a monoclonal antibody and a method for producing the same.
The present invention also relates to a pharmaceutical composition comprising the anti-LR11 monoclonal antibody of the present invention.
さらに、本発明は、本発明の抗LR11モノクローナル抗体の有効量を、悪性腫瘍の患者又は悪性腫瘍の疑いがある患者に投与して、悪性腫瘍を治療する方法、又は悪性腫瘍を予防する方法に関する。
また、本発明は、悪性腫瘍を予防又は治療するための医薬組成物に使用するための抗LR11モノクローナル抗体に関する。
Furthermore, the present invention relates to a method for treating a malignant tumor or a method for preventing a malignant tumor by administering an effective amount of the anti-LR11 monoclonal antibody of the present invention to a patient with a malignant tumor or a patient suspected of having a malignant tumor. .
The present invention also relates to an anti-LR11 monoclonal antibody for use in a pharmaceutical composition for preventing or treating malignant tumors.
より詳細に本発明を説明すれば、以下のとおりである。
（１）配列番号１、２、３、４、５及び６からなる群から選択される少なくとも１つのアミノ酸配列を相補性決定領域（CDR）として有する抗LR11モノクローナル抗体であって、該抗体は、
（Ａ）LR11を認識することができ、
（Ｂ）LR11のVps10pドメインのアミノ酸配列からなるタンパク質に対する特異的結合能を有し、かつ、
（Ｃ）接着性アッセイにおいてヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進される細胞接着性を有意に抑制する、
ことを特徴とする抗LR11モノクローナル抗体。
（２）配列番号１、２、３、４、５及び６のアミノ酸配列を相補性決定領域（CDR）として有する、前記（１）に記載の抗LR11モノクローナル抗体。
The present invention will be described in more detail as follows.
(1) An anti-LR11 monoclonal antibody having, as a complementarity determining region (CDR), at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6,
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
An anti-LR11 monoclonal antibody characterized by that.
(2) The anti-LR11 monoclonal antibody according to (1), which has the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6 as complementarity determining regions (CDRs).
（３）配列番号１、２及び３がＨ鎖の相補性決定領域（CDR）であり、配列番号４、５及び６がＬ鎖の相補性決定領域（CDR）である、前記（１）又は（２）に記載の抗LR11モノクローナル抗体。
（４）Ｈ鎖の可変領域の配列が配列番号７で示されるアミノ酸配列であり、Ｌ鎖の可変領域の配列が配列番号８で示されるアミノ酸配列である、前記（１）から（３）のいずれか一つに記載の抗LR11モノクローナル抗体。
（５）接着性アッセイが、ヒト単球系リンパ腫細胞株U937細胞を用い、ビトロネクチンでコーティングされた容器を用いてのアッセイである、前記（１）から（４）のいずれか一つに記載の抗LR11モノクローナル抗体。
(3) SEQ ID NOs: 1, 2 and 3 are H chain complementarity determining regions (CDR), and SEQ ID NOs: 4, 5 and 6 are L chain complementarity determining regions (CDR), The anti-LR11 monoclonal antibody according to (2).
(4) The sequence of the variable region of the H chain is an amino acid sequence represented by SEQ ID NO: 7, and the sequence of the variable region of the L chain is an amino acid sequence represented by SEQ ID NO: 8, The anti-LR11 monoclonal antibody according to any one of the above.
(5) The adhesion assay is an assay using a human monocyte lymphoma cell line U937 cell and a container coated with vitronectin, according to any one of (1) to (4) above Anti-LR11 monoclonal antibody.
（６）接着性アッセイにおいて、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進された値を１００％としたとき、その値を８０％以下に抑制することができる、前記（１）から（５）のいずれか一つに記載の抗LR11モノクローナル抗体。
（７）接着性アッセイにおける、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質の濃度が１μｇ／ｍＬ〜１００μｇ／ｍＬであり、抗体の濃度が０．１μｇ／ｍＬ〜１００μｇ／ｍＬである、前記（１）から（６）のいずれか一つに記載の抗LR11モノクローナル抗体。
（８）ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質に対する特異的結合能が、少なくとも１μｇ／ｍＬの抗体の濃度において、ヒトソルチリンのVps10pドメインのアミノ酸配列からなるタンパク質からなるタンパク質よりも、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質に強く結合する能力である、前記（１）から（７）のいずれか一つに記載の抗LR11モノクローナル抗体。
（９）さらに、抗体が細胞遊走亢進作用に対する抑制作用を有している、前記（１）から（８）のいずれか一つに記載の抗LR11モノクローナル抗体。
(6) In the adhesion assay, when the value enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 is defined as 100%, the value can be suppressed to 80% or less. The anti-LR11 monoclonal antibody according to any one of 5).
(7) In the adhesion assay, the concentration of the protein comprising the amino acid sequence of the Vps10p domain of human LR11 is 1 μg / mL to 100 μg / mL, and the antibody concentration is 0.1 μg / mL to 100 μg / mL, The anti-LR11 monoclonal antibody according to any one of 1) to (6).
(8) Human LR11 has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of human LR11, compared to a protein consisting of the protein consisting of the amino acid sequence of human sortilin Vps10p domain at a concentration of antibody of at least 1 μg / mL. The anti-LR11 monoclonal antibody according to any one of (1) to (7) above, which has the ability to bind strongly to a protein comprising the amino acid sequence of the Vps10p domain.
(9) The anti-LR11 monoclonal antibody according to any one of (1) to (8), wherein the antibody further has an inhibitory action on a cell migration enhancing action.
（１０）前記（１）から（９）のいずれか一つに記載の抗LR11モノクローナル抗体を含む、悪性腫瘍の予防及び／又は治療剤。
（１１）悪性腫瘍が、造血器腫瘍又は上皮性悪性腫瘍である、前記（１０）に記載の悪性腫瘍の予防及び／又は治療剤。
（１２）造血器腫瘍が、白血病又は悪性リンパ腫である、前記（１１）に記載の悪性腫瘍の予防及び／又は治療剤。
（１３）前記（１）から（９）のいずれか一つに記載の抗LR11モノクローナル抗体を含む、腫瘍細胞の接着抑制及び／又は遊走抑制剤。
（１４）腫瘍細胞が、造血器腫瘍又は上皮性悪性腫瘍である、前記（１３）に記載の腫瘍細胞の接着抑制及び／又は遊走抑制剤。
(10) A preventive and / or therapeutic agent for malignant tumor, comprising the anti-LR11 monoclonal antibody according to any one of (1) to (9).
(11) The preventive and / or therapeutic agent for malignant tumor according to (10), wherein the malignant tumor is a hematopoietic tumor or an epithelial malignant tumor.
(12) The preventive and / or therapeutic agent for malignant tumor according to (11), wherein the hematopoietic tumor is leukemia or malignant lymphoma.
(13) A tumor cell adhesion inhibitor and / or migration inhibitor comprising the anti-LR11 monoclonal antibody according to any one of (1) to (9).
(14) The tumor cell adhesion inhibitory and / or migration inhibitory agent according to (13), wherein the tumor cell is a hematopoietic tumor or an epithelial malignant tumor.
本発明の抗LR11モノクローナル抗体は、特定の抗原を用いて製造されるものであり、細胞接着性アッセイによりスクリーニングすることにより、LR11に対する優れた中和活性を有する。
そして、本発明の抗LR11モノクローナル抗体を用いることにより、造血幹細胞の骨髄への移動（ホーミング）及び生着(定着）を抑制し、優れた抗悪性腫瘍作用、腫瘍細胞の接着抑制作用及び／又は腫瘍細胞の遊走抑制作用を示す。本発明の抗LR11モノクローナル抗体は、悪性腫瘍、特に造血器腫瘍の予防及び／又は治療に有用である。
The anti-LR11 monoclonal antibody of the present invention is produced using a specific antigen and has an excellent neutralizing activity against LR11 by screening by a cell adhesion assay.
And by using the anti-LR11 monoclonal antibody of the present invention, the migration (homing) and engraftment (establishment) of hematopoietic stem cells to the bone marrow is suppressed, and an excellent anti-malignant tumor action, tumor cell adhesion suppressing action and / or It shows tumor cell migration inhibitory action. The anti-LR11 monoclonal antibody of the present invention is useful for the prevention and / or treatment of malignant tumors, particularly hematopoietic tumors.
本明細書において特段の記載が無い限り、本明細書において使用する専門技術用語は、本発明が属する分野の当業者により一般的に理解されている意味の最も広い意味として解釈される。以下に、いくつかの専門技術用語について説明するが、本発明はこれらの説明により限定されるものではない。 Unless otherwise stated herein, technical terms used herein are to be interpreted as having the broadest meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Hereinafter, some technical terms will be described, but the present invention is not limited to these descriptions.
本発明の「モノクローナル抗体」は、一般に、特定のエピトープに対する単一の結合特異性や親和性を有する抗体であり、コーラーらの文献（Kohler, et al, Nature,1975, 256, 495）に記載のハイブリドーマ法；組み換えDNA法（例えば米国特許４，８１６，５６７参照）；クラクソンらの文献（Clackson, et al., Nature, 1991, 352, 624-628）やマークスらの文献（Marks, et al., J. Mol. Biol., 1991, 222, 581-597）に記載のファージ抗体ライブラリーを用いる方法などによって製造することができる。また、本発明の「モノクローナル抗体」の種類は特に制限されず、マウス抗体、ヒト抗体、ラット抗体、ウサギ抗体、ヒツジ抗体、モルモット抗体、ハムスター抗体、トリ抗体等や、ヒトに投与した際に免疫応答を引き起こすことを回避するために、抗体の持つ異種抗原性を低下させることを目的として人為的に改変した遺伝子組換え型抗体、例えば、ヒト化抗体等の何れでもよい。また、これらの抗体は、抗体断片等の低分子化抗体や抗体の修飾物のいずれでもよい。抗体断片としては、Ｆａｂ、Ｆａｂ'、Ｆ（ａｂ'）2、ｓｃＦｖ、ｄｓＦｖ、および可変領域の相補性決定領域（CDR）を含むペプチドなどが挙げられる。 The “monoclonal antibody” of the present invention is generally an antibody having a single binding specificity or affinity for a specific epitope, and is described in Kohler et al. (Kohler, et al, Nature, 1975, 256, 495). Hybridoma method; recombinant DNA method (see, for example, US Pat. No. 4,816,567); Claxson et al. (Clackson, et al., Nature, 1991, 352, 624-628) and Marks et al. (Marks, et al , J. Mol. Biol., 1991, 222, 581-597), and the like. In addition, the type of “monoclonal antibody” of the present invention is not particularly limited, and mouse antibody, human antibody, rat antibody, rabbit antibody, sheep antibody, guinea pig antibody, hamster antibody, avian antibody, etc. In order to avoid inducing a response, any of recombinant antibodies that have been artificially modified for the purpose of reducing the heteroantigenicity of antibodies, such as humanized antibodies, may be used. These antibodies may be either low molecular weight antibodies such as antibody fragments or modified antibodies. Examples of the antibody fragment include Fab, Fab ′, F (ab ′) 2, scFv, dsFv, and a peptide containing a variable region complementarity determining region (CDR).
ヒト抗体については、元来、ヒト体内に天然に存在する抗体を意味するが、遺伝子工学的、細胞工学的、発生工学的な技術の進歩により作製されたヒト抗体ファージライブラリーおよびヒト抗体産生トランスジェニック動物から得られる抗体等も含まれる。ヒト体内に存在する抗体は、例えば、ヒト末梢血リンパ球を単離し、EBウイルス等を感染させ不死化、クローニングすることにより、該抗体を産生するリンパ球を培養でき、培養上清より該抗体を精製することができる。ヒト抗体ファージライブラリーは、ヒトB細胞から調製した抗体遺伝子をファージ遺伝子に挿入することによりFab、scFv等の抗体断片をファージ表面に発現させたライブラリーである。このライブラリーより固定化した抗原に対する結合活性を指標として抗原結合活性を有する抗体断片を発現しているファージを回収することができる。該抗体断片は、更に遺伝子工学的手法によって2本の完全なH鎖および2本の完全なL鎖からなるヒト抗体分子へも変換することができる。ヒト抗体産生トランスジェニック動物は、ヒト抗体遺伝子が細胞内に組み込まれた動物を意味する。具体的には、マウスES細胞へヒト抗体遺伝子を導入し、該ES細胞を他のマウスの初期胚へ移植後、発生させることによりヒト抗体産生トランスジェニック動物を作製することができる。ヒト抗体産生トランスジェニック動物からのヒト抗体の作製方法は、通常のヒト以外の哺乳動物で行われているハイブリドーマ作製方法によりヒト抗体産生ハイブリドーマを得て培養し、培養上清より該抗体を精製することができる。 The term “human antibody” refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody production trans gene produced by the advancement of genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals. The antibody present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing and cloning EB virus and the like, and culturing lymphocytes that produce the antibody. Can be purified. The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. From this library, phages expressing antibody fragments having antigen-binding activity can be recovered using the binding activity to the immobilized antigen as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques. A human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a mouse ES cell, and transplanting the ES cell into an early embryo of another mouse, followed by generation. A human antibody production method from a human antibody-producing transgenic animal is obtained by culturing a human antibody-producing hybridoma obtained by a hybridoma production method performed in a normal non-human mammal, and purifying the antibody from the culture supernatant. be able to.
ヒト化抗体としては、ヒト型キメラ抗体、ヒト型CDR移植抗体等が挙げられる。ヒト型キメラ抗体は、ヒト以外の動物の抗体重鎖可変領域及び抗体軽鎖可変領域とヒト抗体の重鎖定常領域及びヒト抗体の軽鎖定常領域とからなる抗体を意味する。ヒト以外の動物としては、マウス、ラット、モルモット、ハムスター、ウサギ等ハイブリドーマを作成することが可能であれば、いかなるものも用いることができる。本発明のヒト型キメラ抗体におけるヒト以外の動物はマウスが好ましい。この場合、マウス抗体の重鎖及び軽鎖可変領域をコードするＤＮＡをヒト抗体の重鎖及び軽鎖定常領域をコードするＤＮＡと連結し、これを発現ベクターに組み込んで宿主に導入し産生させることにより得ることができる。ヒト型キメラ抗体の重鎖定常領域としては、ヒト免疫グロブリン（hIg）に属すればいかなるものでもよいが、hIgGクラスのものが好ましく、さらにhIgG1,hIgG2,hIgG3,hIgG4といったサブクラスのいずれも用いることができる。また、ヒト型CDR移植抗体の軽鎖定常領域としては、hIgに属すればいかなるものでもよく、κクラスあるいはλクラスのものを用いることができる。 Examples of humanized antibodies include human chimeric antibodies and human CDR-grafted antibodies. The human chimeric antibody means an antibody comprising an antibody heavy chain variable region and antibody light chain variable region of a non-human animal, a human antibody heavy chain constant region, and a human antibody light chain constant region. Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, guinea pigs, hamsters, rabbits and the like. The non-human animal in the human chimeric antibody of the present invention is preferably a mouse. In this case, the DNA encoding the heavy and light chain variable regions of the mouse antibody is linked to the DNA encoding the heavy and light chain constant regions of the human antibody, and this is incorporated into an expression vector and introduced into the host for production. Can be obtained. The heavy chain constant region of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hIg), but is preferably of the hIgG class, and any of the subclasses of hIgG1, hIgG2, hIgG3, hIgG4 should be used. Can do. The light chain constant region of the human CDR-grafted antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
ヒト型CDR移植抗体は、ヒト以外の動物、例えば、マウス抗体の重鎖可変領域及び軽鎖可変領域の相補性決定領域（CDR）のアミノ酸配列をヒト抗体の重鎖可変領域及び軽鎖可変領域の適切な位置に移植した抗体を意味する。例えばマウス抗体の可変領域のCDR以外のフレームワーク配列と類似のフレームワーク配列を有するヒト化抗体の配列を、例えば、公開されているヒト化抗体のデータベースなどから入手し、選択したヒト化抗体のフレームワーク配列を前記マウス抗体のフレームワーク配列と入れ替えることによって作成できる。さらに、その入れ替えたフレームワーク配列の一部をさらに別のアミノ酸と入れ換えることにより、より親和性の高いヒト化抗体とすることも可能である。具体的には、ヒト以外の動物、例えば、マウス抗体の相補性決定領域（CDR）とヒト抗体の可変領域のフレームワーク領域を連結するように設計したＤＮＡ配列を、末端部にオーバーラップする部分を有するように作製した数個のオリゴヌクレオチドからＰＣＲ法により合成する。得られたＤＮＡを、ヒト抗体定常領域をコードするＤＮＡと連結し、次いで発現ベクターに組み込んで、これを宿主に導入し産生させることにより得られる（ＥＰ２３９４００号公報、国際公開ＷＯ９６／０２５７６号公報など）。ヒト型CDR移植抗体の重鎖定常領域としては、ヒト免疫グロブリン（hIg）に属すればいかなるものでもよいが、hIgGクラスのものが好ましく、さらにhIgG1,hIgG2,hIgG3,hIgG4といったサブクラスのいずれも用いることができる。また、ヒト型CDR移植抗体の軽鎖定常領域としては、hIgに属すればいかなるものでもよく、κクラスあるいはλクラスのものを用いることができる。 The human CDR-grafted antibody is a non-human animal such as a mouse antibody heavy chain variable region and light chain variable region complementarity determining region (CDR) amino acid sequence. Means an antibody transplanted at an appropriate position. For example, a humanized antibody sequence having a framework sequence similar to the framework sequence other than the CDR of the variable region of a mouse antibody is obtained from, for example, a public database of humanized antibodies, and the selected humanized antibody. It can be created by replacing the framework sequence with the framework sequence of the mouse antibody. Furthermore, it is possible to obtain a humanized antibody with higher affinity by replacing a part of the replaced framework sequence with another amino acid. Specifically, non-human animals, for example, a portion that overlaps the terminal portion of a DNA sequence designed to link the complementarity determining region (CDR) of a mouse antibody and the framework region of the variable region of a human antibody It is synthesized by PCR from several oligonucleotides prepared so as to have The obtained DNA is obtained by ligating with a DNA encoding a human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (EP239400, International Publication WO96 / 02576, etc.) ). The heavy chain constant region of the human CDR-grafted antibody may be any as long as it belongs to human immunoglobulin (hIg), but is preferably of the hIgG class, and further, any of the subclasses of hIgG1, hIgG2, hIgG3, hIgG4 are used. be able to. The light chain constant region of the human CDR-grafted antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
相補性決定領域を含むアミノ酸配列は、重鎖又は軽鎖相補性決定領域の少なくとも1領域以上を含んで構成される。複数の相補性決定領域は、直接又は適当なアミノ酸配列リンカーを介して結合することができる。 The amino acid sequence including the complementarity determining region includes at least one region of the heavy chain or light chain complementarity determining region. Multiple complementarity determining regions can be joined directly or via a suitable amino acid sequence linker.
本発明のモノクローナル抗体は、優れた中和活性を有していることを特徴とする。ここで、中和活性とは、当該抗体が特異的に結合するエピトープを含むタンパク質、例えばLR11が有している少なくとも１つの活性を低下させる、又は抑制させることができる活性である。このような中和活性は、in vitro及び／又はin vivoのいずれで確認されるものであってもよい。 The monoclonal antibody of the present invention is characterized by having an excellent neutralizing activity. Here, the neutralizing activity is an activity that can reduce or suppress at least one activity of a protein containing an epitope to which the antibody specifically binds, for example, LR11. Such neutralizing activity may be confirmed either in vitro and / or in vivo.
本発明のモノクローナル抗体は、細胞表面のLR11が有している少なくとも１つの活性、例えば細胞接着性を低下又は抑制することができる。より詳細には、LR11により亢進された細胞接着を、抗体濃度が１０μｇ／ｍＬ、５μｇ／ｍＬ、又は２．５μｇ／ｍＬにおいて、LR11により亢進された値を１００％としたときに、９０％以下、好ましくは８０％以下、６０％以下、又は５０％以下に抑制できる中和活性を有する。 The monoclonal antibody of the present invention can reduce or suppress at least one activity possessed by LR11 on the cell surface, such as cell adhesion. More specifically, cell adhesion enhanced by LR11 is 90% or less when the value enhanced by LR11 is 100% at an antibody concentration of 10 μg / mL, 5 μg / mL, or 2.5 μg / mL. Preferably, it has a neutralizing activity that can be suppressed to 80% or less, 60% or less, or 50% or less.
本発明のモノクローナル抗体は、LR11のVps10pドメインのアミノ酸配列からなるタンパク質を抗原として製造される抗LR11モノクローナル抗体であって、
（Ａ）LR11を認識することができ、
（Ｂ）LR11のVps10pドメインのアミノ酸配列からなるタンパク質に対する特異的結合能を有し、かつ、
（Ｃ）接着性アッセイにおいてヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進される細胞接着性を有意に抑制する、
ことを特徴とする抗LR11モノクローナル抗体である。
The monoclonal antibody of the present invention is an anti-LR11 monoclonal antibody produced using a protein comprising the amino acid sequence of the Vps10p domain of LR11 as an antigen,
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
It is an anti-LR11 monoclonal antibody characterized by this.
LR11のVps10pドメインのアミノ酸配列からなるタンパク質としては、LR11、好ましくはヒトLR11のVps10pドメインからなるタンパク質である。ヒトLR11は、２２１４個のアミノ酸からなるタンパク質であり（Moerwald S., et al., Arterioscler Thromb Vasc Biol., 17(5):996-1002, 1997.等参照）、Vps10pドメインは、ヒトLR11では、Ｎ末端から１番目から７５３番目までの７５３個のアミノ酸で構成される部分である。
本発明における「LR11のVps10pドメインのアミノ酸配列からなるタンパク質」は、取り扱いの簡便性や、入手の容易性から、Myc-タグやHis-タグのような各種のタグを有していてもよい。
The protein comprising the amino acid sequence of the LR11 Vps10p domain is LR11, preferably a protein comprising the Vps10p domain of human LR11. Human LR11 is a protein consisting of 2214 amino acids (see Moerwald S., et al., Arterioscler Thromb Vasc Biol., 17 (5): 996-1002, 1997., etc.), and the Vps10p domain is , A part composed of 753 amino acids from the 1st to the 753rd from the N-terminal.
The “protein consisting of the amino acid sequence of the Vps10p domain of LR11” in the present invention may have various tags such as Myc-tag and His-tag for ease of handling and availability.
また、本発明における「LR11を認識できる」とは、結合能を有しているということであり、抗体濃度が１００００ｎｇ／ｍＬ以下、好ましくは１０００ｎｇ／ｍＬ以下での結合能を有するものである。 In addition, “LR11 can be recognized” in the present invention means that it has binding ability, and it has binding ability when the antibody concentration is 10000 ng / mL or less, preferably 1000 ng / mL or less.
本発明の好ましいモノクローナル抗体としては、３つの重鎖CDRのアミノ酸配列が、それぞれ配列番号１、２、３に記載のアミノ酸配列、及び軽鎖CDRアミノ酸配列が、それぞれ配列番号４、５、６に記載のアミノ酸配列を含有している抗体が挙げられる。この抗体を以下では324-12抗体という。324-12抗体は、ヒトLR11のVps10pドメインを抗原として製造された抗体であり、接着性アッセイによりスクリーニングされた抗体である。 As preferred monoclonal antibodies of the present invention, the amino acid sequences of three heavy chain CDRs are the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, and the light chain CDR amino acid sequences are shown in SEQ ID NOs: 4, 5, and 6, respectively. Antibodies containing the described amino acid sequences are mentioned. This antibody is hereinafter referred to as 324-12 antibody. The 324-12 antibody is an antibody produced using the Vps10p domain of human LR11 as an antigen, and is an antibody screened by an adhesion assay.
接着性アッセイは、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進される細胞接着性をアッセイできるものであれば特に制限はない。アッセイに使用する細胞としては腫瘍細胞が好ましく、例えば、後述する実施例２に記載しているように、ヒト単球系リンパ腫細胞株U937細胞を用い、ビトロネクチンでコーティングされた容器を用いてのアッセイが挙げられる。
本発明のモノクローナル抗体は、このような接着性アッセイにおいて、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進された値を１００％としたときに、抑制することができる抗体であればよく、好ましくは８０％以下、８５％以下、７０％以下、７５％以下、６０％以下、６５％以下、又は５０％以下に抑制するものが挙げられる。接着性アッセイにおける、ヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質の濃度は特に制限はないが、好ましくは０．１μｇ／ｍＬ〜５００μｇ／ｍＬ、０．１μｇ／ｍＬ〜１００μｇ／ｍＬ、１μｇ／ｍＬ〜５００μｇ／ｍＬ、１μｇ／ｍＬ〜１００μｇ／ｍＬ、又は１μｇ／ｍＬ〜５０μｇ／ｍＬが挙げられる。そのときに用いる抗体の濃度は、前記したタンパク質の濃度に応じて適宜調整することができ、好ましくは、０．０１μｇ／ｍＬ〜１００μｇ／ｍＬ、０．０１μｇ／ｍＬ〜５０μｇ／ｍＬ、０．０１μｇ／ｍＬ〜２０μｇ／ｍＬ、０．１μｇ／ｍＬ〜１００μｇ／ｍＬ、０．１μｇ／ｍＬ〜５０μｇ／ｍＬ、又は０．１μｇ／ｍＬ〜２０μｇ／ｍＬが挙げられる。
The adhesion assay is not particularly limited as long as it can assay cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11. Tumor cells are preferred as the cells used in the assay. For example, as described in Example 2 described later, an assay using a human monocyte lymphoma cell line U937 cell and a vessel coated with vitronectin is used. Is mentioned.
The monoclonal antibody of the present invention may be any antibody that can be suppressed when the value enhanced by a protein consisting of the amino acid sequence of the human LR11 Vps10p domain is 100% in such an adhesion assay, Preferred are those that are suppressed to 80% or less, 85% or less, 70% or less, 75% or less, 60% or less, 65% or less, or 50% or less. In the adhesion assay, the concentration of the protein comprising the amino acid sequence of the Vps10p domain of human LR11 is not particularly limited, but preferably 0.1 μg / mL to 500 μg / mL, 0.1 μg / mL to 100 μg / mL, 1 μg / mL. -500 μg / mL, 1 μg / mL-100 μg / mL, or 1 μg / mL-50 μg / mL. The concentration of the antibody used at that time can be appropriately adjusted according to the concentration of the protein described above, and preferably 0.01 μg / mL to 100 μg / mL, 0.01 μg / mL to 50 μg / mL, 0.01 μg / ML to 20 μg / mL, 0.1 μg / mL to 100 μg / mL, 0.1 μg / mL to 50 μg / mL, or 0.1 μg / mL to 20 μg / mL.
本発明における好ましい抗体としては、前記した324-12抗体が挙げられる。324-12抗体は、CDRとして配列番号１〜６に記載したアミノ酸配列を有する。また、324-12抗体は可変領域として配列番号７に記載したＨ鎖のアミノ酸配列、及び配列番号８に記載したＬ鎖のアミノ酸配列を有する。
本発明における他の好ましい抗体としては、配列番号１〜６からなる群から選択される少なくとも１つのアミノ酸配列を含有するモノクローナル抗体である。配列番号１、２又は３で示される配列は重鎖超可変領域の相補性決定領域（CDR：complementarity-determining region）アミノ酸配列であり、配列番号４、５又は６で示される配列は軽鎖超可変領域の相補性決定領域（CDR）アミノ酸配列である。好ましくは、配列番号１、２又は３で示される重鎖CDRアミノ酸配列、及び配列番号４、５又は６で示される軽鎖CDRアミノ酸配列を含む抗体が挙げられる。さらに好ましくは、配列番号１、２及び３で示される重鎖CDRアミノ酸配列を含み、かつ配列番号４、５及び６で示される軽鎖CDRアミノ酸配列を含む抗体が挙げられる。
Preferred antibodies in the present invention include the aforementioned 324-12 antibody. The 324-12 antibody has the amino acid sequence set forth in SEQ ID NOs: 1 to 6 as CDRs. The 324-12 antibody has the amino acid sequence of H chain described in SEQ ID NO: 7 and the amino acid sequence of L chain described in SEQ ID NO: 8 as variable regions.
Another preferable antibody in the present invention is a monoclonal antibody containing at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. The sequence represented by SEQ ID NO: 1, 2 or 3 is the complementarity-determining region (CDR) amino acid sequence of the heavy chain hypervariable region, and the sequence represented by SEQ ID NO: 4, 5 or 6 is a sequence exceeding the light chain It is the complementarity determining region (CDR) amino acid sequence of the variable region. Preferred examples include an antibody comprising a heavy chain CDR amino acid sequence represented by SEQ ID NO: 1, 2 or 3 and a light chain CDR amino acid sequence represented by SEQ ID NO: 4, 5 or 6. More preferably, an antibody comprising a heavy chain CDR amino acid sequence represented by SEQ ID NOs: 1, 2, and 3 and a light chain CDR amino acid sequence represented by SEQ ID NOs: 4, 5, and 6 is exemplified.
本発明のモノクローナル抗体は、優れた中和活性を有しており、LR11の発現、好ましくは過剰発現に起因する各種の疾患の治療剤及び／又は予防剤として、特に悪性腫瘍の治療剤及び／又は予防剤として使用することができる。 The monoclonal antibody of the present invention has excellent neutralizing activity, and is particularly useful as a therapeutic and / or prophylactic agent for various diseases caused by LR11 expression, preferably overexpression. Or it can be used as a preventive agent.
本発明における悪性腫瘍とは、造血器腫瘍、上皮性悪性腫瘍等が挙げられる。ここで造血器腫瘍としては白血病及び悪性リンパ腫等が挙げられる。白血病には急性骨髄性白血病、急性前骨髄球性白血病、成人急性リンパ性白血病、慢性骨髄性白血病等が挙げられ、悪性リンパ腫には非ホジキンリンパ腫等が挙げられる。一方、上皮性悪性腫瘍としては、胃癌、肝臓癌、膵臓癌、肺癌、前立腺癌、膀胱癌、食道癌、乳癌、子宮頸癌、卵巣癌、結腸癌、大腸癌、腎臓癌、胆嚢癌、神経腫瘍（グリオーマ）、悪性黒色腫（メラノーマ）等が挙げられる。 Examples of the malignant tumor in the present invention include hematopoietic tumors and epithelial malignant tumors. Examples of hematopoietic tumors include leukemia and malignant lymphoma. Examples of leukemia include acute myeloid leukemia, acute promyelocytic leukemia, adult acute lymphoblastic leukemia, and chronic myelogenous leukemia. Malignant lymphoma includes non-Hodgkin lymphoma. On the other hand, epithelial malignant tumors include stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, colon cancer, kidney cancer, gallbladder cancer, nerve Tumor (glioma), malignant melanoma (melanoma), etc. are mentioned.
本発明による具体的な治療対象としては、造血器腫瘍においては、白血病のうち、急性白血病がより好ましく、悪性リンパ腫のうち、非ホジキンリンパ腫がより好ましい。また、上皮性悪性腫瘍においては、肝臓癌、膵臓癌、結腸癌、大腸癌、胆嚢癌が好ましい。 As a specific treatment target according to the present invention, among hematopoietic tumors, acute leukemia is more preferable among leukemias, and non-Hodgkin lymphoma is more preferable among malignant lymphomas. In the epithelial malignant tumor, liver cancer, pancreatic cancer, colon cancer, colon cancer, and gallbladder cancer are preferable.
本発明の抗体は、有効成分自体を単独で投与することも可能であるが、通常は薬理学的に許容される一つあるいはそれ以上の担体と一緒に混合し、医薬組成物として投与することができる。本発明の医薬組成物は、経口又は非経口投与に適する医薬用組成物、好ましくは非経口投与に適する医薬組成物とすることができる。非経口投与に適する医薬組成物としては、例えば、静脈内注射剤や筋肉内注射剤などの注射剤、点滴剤、坐剤、吸入剤、点眼剤、点鼻剤、経皮吸収剤、経粘膜吸収剤などを挙げることができるが、これらに限定されることはない。 While it is possible for the antibody of the present invention to be administered alone, the active ingredient itself is usually mixed with one or more pharmacologically acceptable carriers and administered as a pharmaceutical composition. Can do. The pharmaceutical composition of the present invention can be a pharmaceutical composition suitable for oral or parenteral administration, preferably a pharmaceutical composition suitable for parenteral administration. Pharmaceutical compositions suitable for parenteral administration include, for example, injections such as intravenous injections and intramuscular injections, drops, suppositories, inhalants, eye drops, nasal drops, transdermal absorption agents, transmucosal Although an absorbent etc. can be mentioned, it is not limited to these.
上記の医薬組成物は、薬理学的、製剤学的に許容しうる添加物を加えて製造することができる。薬理学的、製剤学的に許容しうる添加物の例としては、例えば、賦形剤、結合剤、増量剤、崩壊剤、界面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、矯味剤、香料、被膜剤、希釈剤などを挙げることができるが、これらに限定されることはない。 The above pharmaceutical composition can be produced by adding pharmacologically and pharmaceutically acceptable additives. Examples of pharmacologically and pharmaceutically acceptable additives include, for example, excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, Examples include, but are not limited to, flavoring agents, fragrances, coating agents, and diluents.
本発明の医薬の投与量は特に限定されず、疾患の種類、予防又は治療の目的、有効成分の種類などに応じて適宜選択することができ、さらに患者の体重や年齢、症状、投与経路など通常考慮すべき種々の要因に応じて、適宜増減することができる。例えば、静脈内投与の場合には成人一日あたり有効成分の重量として０．１ｍｇ〜１０ｇ程度の範囲で用いることができるが、投与量は当業者に適宜選択可能であり、上記の範囲に限定されることはない。成人一日あたり有効成分の重量として好ましくは、約０．１ｍｇ〜１０００ｍｇ、より好ましくは約０．５ｍｇ〜５００ｍｇの用量で静脈内投与することができる。また、投与は一日１回又は数回に分けて行うことができる。 The dosage of the pharmaceutical agent of the present invention is not particularly limited, and can be appropriately selected according to the type of disease, the purpose of prevention or treatment, the type of active ingredient, etc., and the patient's weight and age, symptoms, administration route, etc. It can be increased or decreased as appropriate according to various factors that should be normally considered. For example, in the case of intravenous administration, the weight of the active ingredient per day for an adult can be used in the range of about 0.1 mg to 10 g, but the dosage can be appropriately selected by those skilled in the art and is limited to the above range. It will never be done. Preferably, the weight of the active ingredient per adult can be intravenously administered at a dose of about 0.1 mg to 1000 mg, more preferably about 0.5 mg to 500 mg. Moreover, administration can be performed once a day or divided into several times.
抗LR11モノクローナル抗体の作製や単離・精製には、公知のモノクローナル抗体の作製方法、例えば、浜窪隆雄（2012）「新機能抗体開発ハンドブック（エヌ・ティー・エス）」又は大海忍（1994）「新版抗ペプチド抗体実験プロトコール（秀潤社）」等に従い作製、単離・精製することができる。
抗LR11モノクローナル抗体は、常法に従い、作製したハイブリドーマを培養して、培養上清から分離する方法、ハイブリドーマをこれと適合性のある哺乳類動物に投与して、腹水内に抗体を産生させる方法により製造できる。抗体は、必要に応じてそれをより精製して使用することができる。抗体を精製、単離する方法としては、従来公知の方法、例えば、硫酸アンモニウム沈殿法などの塩析、ゲルろ過法、イオン交換法、プロテインＡ又はＧなどのカラムなどや免疫抗原を固定化したカラムによるアフィニティー精製法などがある。
For the production, isolation and purification of anti-LR11 monoclonal antibodies, known monoclonal antibody production methods, for example, Takao Hamakubo (2012) “New Functional Antibody Development Handbook (NTS)” or Shinobu Oumi (1994) It can be prepared, isolated and purified according to "New Edition Anti-Peptide Antibody Experiment Protocol (Shyujunsha)".
The anti-LR11 monoclonal antibody is obtained by culturing the prepared hybridoma according to a conventional method and separating it from the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and producing the antibody in ascites. Can be manufactured. The antibody can be used after further purification as necessary. As a method for purifying and isolating antibodies, conventionally known methods, for example, salting out such as ammonium sulfate precipitation, gel filtration, ion exchange, columns such as protein A or G, and columns on which immune antigens are immobilized There is an affinity purification method.
（モノクローナル抗体作製）
LR11に対するモノクローナル抗体は、具体的に例えば次のようにして作製することができる。抗体の作製に用いられる免疫抗原としては、LR11タンパク質、又はその一部断片（ペプチド）等を用いることができるが、LR11のＮ末端側のVps10pドメイン部分が好ましい。生体組織、細胞由来の試料から精製することによって得てもよいし、組換えタンパク質や合成ペプチドを用いてもよい。免疫する動物としては、例えばウサギ、マウス、ラットなどが挙げられるが、モノクローナル抗体作製にはマウスを好ましく用いることができる。免疫方法は、公知の手法に従って行われる。例えば、免疫抗原を腹腔内注入、静脈注入、皮下注射などの投与方法によって、抗体産生が可能な部位にそれ自体単独であるいは担体、希釈剤、あるいは抗体産生能を高めるために、完全フロイントアジュバントや不完全フロイントアジュバントなどの補液と混合、エマルジョン化させて投与してもよい。投与は、通常2〜6週毎に1回ずつ、計2〜10回程度行われる。
(Monoclonal antibody production)
Specifically, a monoclonal antibody against LR11 can be prepared, for example, as follows. As an immunizing antigen used for the production of an antibody, LR11 protein or a partial fragment (peptide) thereof can be used, and the Vps10p domain portion on the N-terminal side of LR11 is preferable. You may obtain by refine | purifying from the sample derived from a biological tissue and a cell, and you may use a recombinant protein and a synthetic peptide. Examples of animals to be immunized include rabbits, mice, and rats, but mice can be preferably used for the production of monoclonal antibodies. The immunization method is performed according to a known method. For example, in order to increase the ability to produce an antibody by itself, or a carrier, diluent, or antibody at a site where antibody production is possible by administration methods such as intraperitoneal injection, intravenous injection, and subcutaneous injection, complete Freund's adjuvant or You may mix and emulsify with fluids such as incomplete Freund's adjuvant. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total.
モノクローナル抗体の作製に際しては、LR11抗原を免疫された動物、例えばマウスからLR11に対するモノクローナル抗体の産生が認められた個体を選択し、脾臓又はリンパ節を採取し、それらに含まれる抗体産生細胞を骨髄腫細胞（ミエローマ）と融合させることにより、抗LR11モノクローナル抗体産生ハイブリドーマを調製することができる。細胞融合操作は公知の手法に従って行われる。ポリエチレングリコール（PEG）を融合促進剤として使用したり、電気パルスによって融合してもよいが、好ましくはPEGが用いられる。骨髄腫細胞としては、例えばNS-1,P3U1,SP2/0,AP-1などが挙げられるが、P3U1などが好ましく用いられる。
抗LR11抗体産生ハイブリドーマのスクリーニングには種々の方法が使用できるが、例えばLR11又はその部分ペプチドなどを吸着させた固相（例、マイクロプレート）にハイブリドーマ培養上清を添加し、次に、酵素などで標識した抗免疫グロブリン抗体（細胞融合に用いられる細胞がマウスの場合、抗マウス免疫グロブリン抗体が用いられる。）を加え、固相に結合した抗LR11モノクローナル抗体を検出する方法などが挙げられる。抗LR11モノクローナル抗体産生ハイブリドーマのスクリーニング、培養は通常HAT（ヒポキサンチン、アミノプテリン、チミジン）を添加して、１０〜２０％牛胎児血清を含む細胞培養用培地（例、RPMI1640）で行われる。
抗LR11モノクローナル抗体の分離精製は、通常のポリクローナル抗体の分離精製と同様に免疫グロブリンの分離精製法（例、塩析法、イオン交換体（例、DEAE）による吸脱着法、ゲルろ過法、抗原結合固相あるいはプロテインＡあるいはプロテインＧなどのアフィニティー精製法など）に従って行われる。以上のようにして、ハイブリドーマ細胞を培養した培養上清やハイブリドーマと適合した動物の体内で増殖させた後の体液から抗体を精製することによって、本発明の抗体を製造することができる。
When preparing monoclonal antibodies, select animals that have been immunized with LR11 antigen, such as mice that have been confirmed to produce monoclonal antibodies against LR11, collect spleen or lymph nodes, and use them to produce antibody-producing cells in bone marrow. An anti-LR11 monoclonal antibody-producing hybridoma can be prepared by fusing with a tumor cell (myeloma). The cell fusion operation is performed according to a known method. Polyethylene glycol (PEG) may be used as a fusion accelerator or may be fused by electric pulse, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., and P3U1 is preferably used.
Various methods can be used for screening anti-LR11 antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) adsorbed with LR11 or a partial peptide thereof, and then an enzyme, etc. And a method of detecting an anti-LR11 monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with (in which the mouse used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used). Screening and culture of anti-LR11 monoclonal antibody-producing hybridomas are usually carried out in a cell culture medium (eg, RPMI1640) containing 10-20% fetal calf serum with addition of HAT (hypoxanthine, aminopterin, thymidine).
Separation and purification of anti-LR11 monoclonal antibody is the same as separation and purification of ordinary polyclonal antibodies, such as immunoglobulin separation and purification methods (eg, salting out method, adsorption / desorption method using ion exchanger (eg, DEAE), gel filtration method, antigen A binding solid phase or an affinity purification method such as protein A or protein G). As described above, the antibody of the present invention can be produced by purifying the antibody from the culture supernatant obtained by culturing the hybridoma cells or the body fluid after growing in the body of an animal compatible with the hybridoma.
以下、実施例を挙げて本発明を詳細に説明するが、本発明の範囲はこれらの実施例により限定されるものではない。
本発明の明細書において、アミノ酸等を略号で表示する場合、IUPAC-IUB Commission on Biochemical Nomenclatureによる略号あるいは当該分野における慣用略号に基づくものであり、その例を下記する。
PBS：リン酸緩衝生理食塩水（Phosphate buffered saline）
FCS：ウシ胎児血清（Fetal calf serum）
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples.
In the specification of the present invention, amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below.
PBS: Phosphate buffered saline
FCS: Fetal calf serum
モノクローナル抗体324-12の製造
（１）ヒト可溶性LR11(hsLR11)発現ベクターの構築
hsLR11発現ベクターの構築は、クローニングしたhsLR11をpcDNA3.1A-MycHis（V800-20, Life Technologies社）のマルチクローニングサイトに制限酵素EcoRI, PmeIを用いて、常法により挿入した。このhsLR11発現ベクターをpcDNA3.1-hsLR11-MycHis Aとした。
（２）ヒトVps10p(hVps10p)発現ベクターの構築
hVps10pの発現ベクターの構築は、前記（１）で作製した発現プラスミドpcDNA3.1-hsLR11-MycHis Aを鋳型とし、配列番号９及び１０で示されるプライマーを用い、ヒトLR11のVps10pドメインの塩基配列内にあるNheI認識部位からVps10pのＣ末部分までをPCR法で増幅した。
Production of monoclonal antibody 324-12 (1) Construction of human soluble LR11 (hsLR11) expression vector
The hsLR11 expression vector was constructed by inserting the cloned hsLR11 into the multicloning site of pcDNA3.1A-MycHis (V800-20, Life Technologies) using restriction enzymes EcoRI and PmeI by a conventional method. This hsLR11 expression vector was designated as pcDNA3.1-hsLR11-MycHis A.
(2) Construction of human Vps10p (hVps10p) expression vector
The hVps10p expression vector was constructed using the expression plasmid pcDNA3.1-hsLR11-MycHis A prepared in (1) above as a template, and using the primers shown in SEQ ID NOs: 9 and 10, within the nucleotide sequence of the human LR11 Vps10p domain. From the NheI recognition site to the C-terminal part of Vps10p was amplified by PCR.
Forward primer: 5’- CCGTGGCTAGCAAGACAAACGTG-3’ （配列番号９）
Reverse primer: 5’- CGATCTAGAGGGACAGGGGACCAGCTCT-3’ （配列番号１０）
Forward primerの下線がNheI認識部位、Reverse primerの下線がXbaI認識部位
Forward primer: 5'- CCGTG GCTAGC AAGACAAACGTG-3 '(SEQ ID NO: 9)
Reverse primer: 5'- CGA TCTAGA GGGACAGGGGACCAGCTCT-3 '(SEQ ID NO: 10)
Forward primer underline is NheI recognition site, Reverse primer underline is XbaI recognition site
得られた増幅産物を、さらに制限酵素NheI及びXbaIを用い処理して精製した。このようにして得られた増幅産物は、Vps10pドメイン中の制限酵素Nhelサイトから、Vps10pドメインのＣ末端の制限酵素Xbalサイトまでのアミノ酸配列をコードする断片である。
一方、hsLR11発現ベクターであるpcDNA3.1-hsLR11-MycHis Aは、LR11の細胞外部分の３’末端側に制限酵素Xbalサイトを有しており、制限酵素Nhel及び制限酵素Xbalで処理することにより、ヒト可溶性LR11(hsLR11)のVps10pドメイン中の制限酵素NhelサイトからＣ末端部分を切り取ることができる。
hsLR11発現ベクターpcDNA3.1-hsLR11-MycHis Aを、制限酵素NheI及びXbaI処理し、次いで、前記で得られた増幅産物とをAlkaline phosphataseで処理したpcDNA3.1-hsLR11-MycHis A に接合した。得られた発現ベクターは、Myc-タグ及びHis-タグを有しているので、Ｃ末端にMyc-タグ及びHis-タグが付加されたhVps10pの発現ベクターであり、pcDNA3.1/myc-His(+)-hsVps10pとした。
The obtained amplification product was further purified by treatment with restriction enzymes NheI and XbaI. The amplification product thus obtained is a fragment encoding the amino acid sequence from the restriction enzyme Nhel site in the Vps10p domain to the restriction enzyme Xbal site at the C-terminal of the Vps10p domain.
On the other hand, pcDNA3.1-hsLR11-MycHis A, which is an hsLR11 expression vector, has a restriction enzyme Xbal site on the 3 ′ end side of the extracellular portion of LR11 and is treated with restriction enzyme Nhel and restriction enzyme Xbal. The C-terminal portion can be excised from the restriction enzyme Nhel site in the Vps10p domain of human soluble LR11 (hsLR11).
The hsLR11 expression vector pcDNA3.1-hsLR11-MycHis A was treated with restriction enzymes NheI and XbaI, and then the amplified product obtained above was conjugated to pcDNA3.1-hsLR11-MycHis A treated with Alkaline phosphatase. Since the obtained expression vector has a Myc-tag and a His-tag, it is an expression vector of hVps10p with a Myc-tag and a His-tag added to the C-terminus, and pcDNA3.1 / myc-His ( +)-hsVps10p.
（３）ヒトVps10pタンパク質の調製
前記（２）で得られた発現ベクターpcDNA3.1/myc-His(+)-hsVps10pを以下のようにしてFreeStyle 293-F細胞にトランスフェクションしてヒトLR11 Vps10pタンパク質を発現させた。
ベクターDNA 37.5μgは無血清培地OptiPro SFM（Life Technologies社）を加え600μLとし、トランスフェクション試薬FreeStyle MAX Reagent（Life Technologies社）37.5μLとOptiPro SFM 562.5μLの混合液（total 600μL）と穏やかに混合し、室温で10分間インキュベーション後に125mL三角フラスコのFreeStyle 293-F細胞（1×106cells /mL, 30mL）に添加して4日間の旋回培養（8%CO2, 125rpm, 37℃）を行った。回収した培養上清を限外ろ過（Amicon Ultra, 分画分子量30,000, Merck Millipore社）で約10倍に濃縮後、コバルトカラム用のEquilibration buffer（Clontech社）で希釈後、Equilibration bufferで平衡化したコバルトカラム (1mL, Clontech社）に添加した。10% Elution buffer（Clontech社）でカラムを洗浄し、60% Elution bufferで溶出した。溶出画分はさらに限外ろ過で濃縮しPD-10カラム（GEヘルスケア社）によりPBSにbuffer交換して、精製hVps10pタンパク質を得た。
(3) Preparation of human Vps10p protein The human LR11 Vps10p protein was prepared by transfecting the expression vector pcDNA3.1 / myc-His (+)-hsVps10p obtained in (2) above into FreeStyle 293-F cells as follows. Was expressed.
Add 37.5 μg of vector DNA to 600 μL of serum-free medium OptiPro SFM (Life Technologies), and gently mix with 37.5 μL of the transfection reagent FreeStyle MAX Reagent (Life Technologies) and 562.5 μL of OptiPro SFM (total 600 μL). After 10 minutes incubation at room temperature, it was added to FreeStyle 293-F cells (1 × 10 6 cells / mL, 30 mL) in a 125 mL Erlenmeyer flask and swirled for 4 days (8% CO 2 , 125 rpm, 37 ° C.) . The collected culture supernatant was concentrated approximately 10 times by ultrafiltration (Amicon Ultra, molecular weight cut off 30,000, Merck Millipore), diluted with equilibration buffer for cobalt column (Clontech), and equilibrated with equilibration buffer. Added to a cobalt column (1 mL, Clontech). The column was washed with 10% Elution buffer (Clontech) and eluted with 60% Elution buffer. The eluted fraction was further concentrated by ultrafiltration, and the buffer was exchanged with PBS using a PD-10 column (GE Healthcare) to obtain purified hVps10p protein.
（４）ハイブリドーマの作製
免疫抗原として、前記（３）で調製したヒトLR11の一部アミノ酸配列[1-753]を有するタンパク質(hVps10p)を用いて、この免疫抗原0.1mgを完全フロイントアジュバンドと混和乳化し、6週齢の雌BALB/Cマウスの皮下に2週間間隔で2回投与後、2回目の免疫の3日後に脾臓及びリンパ節を摘出した。摘出した脾臓及びリンパ節から得られた細胞と骨髄腫細胞P3U1とを混合し、ポリエチレングリコール存在下にて細胞融合させた。融合細胞はHAT培地に懸濁し、限外希釈法で96穴培養プレートに分注した。これを5％CO2インキュベーター中で37℃にて培養し、ハイブリドーマの生育してきたウェルの培養上清について、次に示すELISA（Enzyme-Linked Immuno Sorbent Assay）法にしたがって、ヒトLR11のVps10pドメイン（hVps10p)に対する結合性でスクリーニングし、陽性ウェルを24穴培養プレートまで培養させ、培養上清を再度、ELISAと細胞接着アッセイでスクリーニングし、陽性ウェルを限界希釈法でクローニングしてハイブリドーマ株(324-12)を樹立した。
(4) Preparation of hybridoma Using the protein (hVps10p) having a partial amino acid sequence [1-753] of human LR11 prepared in (3) above as an immunizing antigen, 0.1 mg of this immunizing antigen was completely Freund's adjuvant. The mixture was emulsified and administered twice subcutaneously in 6-week-old female BALB / C mice at 2-week intervals, and the spleen and lymph nodes were removed 3 days after the second immunization. Cells obtained from the removed spleen and lymph nodes were mixed with myeloma cells P3U1, and the cells were fused in the presence of polyethylene glycol. The fused cells were suspended in HAT medium and dispensed into 96-well culture plates by the limiting dilution method. This was cultured at 37 ° C. in a 5% CO 2 incubator, and the culture supernatant of the well in which the hybridoma had grown was cultured according to the following ELISA (Enzyme-Linked Immuno Sorbent Assay) method. hVps10p) was screened, positive wells were grown to 24-well culture plates, culture supernatants were screened again by ELISA and cell adhesion assay, positive wells were cloned by limiting dilution, and hybridoma strains (324- 12) was established.
（５）モノクローナル抗体の調製
2週間前にプリスタン0.5mLを腹腔内に注射しておいた12週齢の雌BALB/Cマウスに、上記で得られたハイブリドーマを細胞数0.5×106個の量で腹腔内に投与した。約14日後に腹水を採取し、遠心分離して上清を得た。該上清を等量のPBSと混和後、0.45μmのフィルター濾過した。濾液をPBSで平衡化したAb-Capcher ExTraカラム（プロテノバ社製）に通して抗体をカラムに吸着させた後、PBSで十分にカラムを洗浄した後、0.1Mクエン酸緩衝液(pH3.0)でカラムより溶出させ、抗LR11モノクローナル抗体（324-12抗体）を精製した。
(5) Preparation of monoclonal antibody
The hybridoma obtained above was intraperitoneally administered in an amount of 0.5 × 10 6 cells to 12-week-old female BALB / C mice that had been injected intraperitoneally with 0.5 mL of pristane two weeks ago. About 14 days later, ascites was collected and centrifuged to obtain a supernatant. The supernatant was mixed with an equal volume of PBS, and filtered through a 0.45 μm filter. The filtrate was passed through an Ab-Capcher ExTra column (manufactured by Protenova) equilibrated with PBS, and the antibody was adsorbed to the column. After thoroughly washing the column with PBS, 0.1 M citrate buffer (pH 3.0) And purified from the column with an anti-LR11 monoclonal antibody (324-12 antibody).
（６）アミノ酸配列の解析
得られた抗LR11モノクローナル抗体の可変領域及び超可変領域のアミノ酸配列を常法にしたがって解析した。その結果を配列番号１〜８に示す。
(6) Analysis of amino acid sequence The amino acid sequences of the variable region and the hypervariable region of the obtained anti-LR11 monoclonal antibody were analyzed according to a conventional method. The results are shown in SEQ ID NOs: 1-8.
可溶性LR11の生物活性に対する中和活性の測定
（１）可溶性LR11の細胞接着亢進作用に対する抑制作用
工程１：可溶性LR11（sLR11）の調製
アフリカミドリザル腎臓由来の細胞株COS7細胞にヒトLR11の全長cDNAをコードした発現プラスミド（RC214154、Origene社）を、Effectene（Qiagen社）を用いたリポフェクション法によって一過的にトランスフェクションした後、37℃でインキュベーションした。16時間後に無血清培地に交換後、さらに37℃で24時間培養してCOS7細胞表面上にLR11を発現させた。LR11の細胞外ドメインは、sLR11として培養上清中に遊離されることから、細胞培養上清を回収し、限外ろ過（Amicon Ultra, 分画分子量100,000、Merck Millipore社）で約100倍に濃縮し、sLR11を得た。
Measurement of neutralizing activity on the biological activity of soluble LR11 (1) Inhibitory action on the enhancement of cell adhesion of soluble LR11 Step 1: Preparation of soluble LR11 (sLR11) Full-length cDNA of human LR11 was introduced into cell line COS7 derived from African green monkey kidney The encoded expression plasmid (RC214154, Origene) was transiently transfected by the lipofection method using Effectene (Qiagen) and then incubated at 37 ° C. After 16 hours, the serum-free medium was replaced, and further cultured at 37 ° C. for 24 hours to express LR11 on the surface of COS7 cells. Since the extracellular domain of LR11 is released into the culture supernatant as sLR11, the cell culture supernatant is collected and concentrated approximately 100 times by ultrafiltration (Amicon Ultra, molecular weight cut off 100,000, Merck Millipore) And obtained sLR11.
工程２：可溶性LR11の細胞接着亢進作用に対する抑制作用
細胞外基質のヒトビトロネクチン（ベクトン・ディッキンソン アンド カンパニー社、以下、「BD社」という。）をPBSで25μg/mLの濃度に調製した溶液を96ウェル黒色プレート（BD社）に50μL/ウェルずつ分注後、37℃で1時間静置してコーティングした後、PBSで洗浄し、1%ウシ血清アルブミン（BSA）を50μL/ウェルずつ分注後、除去して風乾した。ヒト単球系細胞株U937細胞は無血清のRPMI1640培地で1晩培養後、1μMのCalcein-AMを含む0.1%BSA-ハンクス平衡塩溶液（HBSS）に懸濁して、37℃で30分間インキュベーションしてCalcein-AMを細胞内に取り込ませて細胞を蛍光標識した。その後、無血清培地に3×106cells /mLとなるように細胞を懸濁し、正常マウスIgG抗体又はマウス抗LR11モノクローナル抗体（324-12抗体）を終濃度2.5μg/mLとなるように添加し、その直後にsLR11(COS7培養上清）を培地の1/100量添加し、37℃で3時間反応させた。なお、比較として、各抗体及びsLR11を未添加のウェル、sLR11のみを添加したウェルも設定した。次に、ヒトビトロネクチンをコーティングしたプレートにsLR11及び抗体を含むU937細胞懸濁液を分注し(3×105cells/100μL/ウェル)、低速遠心(1800rpm, 2分間)の後、37℃で20分間静置した。PBSで3回ウェルを洗浄した後、蛍光プレートリーダー（PerkinElmer社）でプレートに結合した細胞の蛍光強度（RFU)を測定した。各実験はすべてN=3で行った。この結果を図１に示す。
Step 2: Inhibitory action of soluble LR11 on cell adhesion enhancing action A solution of an extracellular matrix human vitronectin (Becton Dickinson and Company, hereinafter referred to as “BD”) prepared in PBS to a concentration of 25 μg / mL is 96. Dispense 50 μL / well into a well black plate (BD), leave it at 37 ° C for 1 hour to coat, wash with PBS, and dispense 1% bovine serum albumin (BSA) at 50 μL / well. Removed and air dried. Human monocytic cell line U937 cells are cultured overnight in serum-free RPMI1640 medium, then suspended in 0.1% BSA-Hanks balanced salt solution (HBSS) containing 1 μM Calcein-AM, and incubated at 37 ° C for 30 minutes Then, Calcein-AM was incorporated into the cells and the cells were fluorescently labeled. Then, suspend cells in serum-free medium to 3 × 10 6 cells / mL, and add normal mouse IgG antibody or mouse anti-LR11 monoclonal antibody (324-12 antibody) to a final concentration of 2.5 μg / mL Immediately after that, 1/100 volume of sLR11 (COS7 culture supernatant) was added and reacted at 37 ° C. for 3 hours. For comparison, a well to which each antibody and sLR11 were not added and a well to which only sLR11 was added were also set. Next, dispense U937 cell suspension containing sLR11 and antibody on a plate coated with human vitronectin (3 × 10 5 cells / 100 μL / well), spin at low speed (1800 rpm, 2 minutes), then at 37 ° C. Let stand for 20 minutes. After washing the wells three times with PBS, the fluorescence intensity (RFU) of the cells bound to the plate was measured with a fluorescence plate reader (PerkinElmer). All experiments were performed at N = 3. The result is shown in FIG.
可溶性LR11の細胞遊走亢進作用に対する抑制作用
工程１：部分精製ヒトsLR11タンパク質（アミノ酸配列1-2110のＣ末端にMyc-tag及びHis-tagを付加した部分タンパク質、以下、「hsLR11」という。）の調製
実施例１の（１）で得られたhsLR11発現ベクターpcDNA3.1-hsLR11-MycHis AをFreeStyle 293-F細胞（Life Technologies社）にトランスフェクションしてhsLR11タンパク質を発現させた。トランスフェクション試薬としては、Targefect-293FS reagent（Targetingsystems社）を使用し、トランスフェクション後、3日間の旋回培養（8%CO2, 125rpm, 37℃）を行った。遠心により回収したsLR11発現細胞はLysis buffer（20mM Tris-HCl, pH7.5-1%NP-40-150mM NaCl）に懸濁して可溶化した後、PBSで平衡化した抗His-tag抗体カラム（株式会社医学生物学研究所）に添加し、PBSで洗浄後、酸性条件下（pH3.0）で溶出し、直ちに0.1M Tris-HCl, pH7.5で中和した。溶出分画は引き続き抗Myc-tag抗体カラム（株式会社医学生物学研究所）に添加し、PBSで洗浄後、酸性条件下（pH3.0）で溶出後、直ちに中和し、部分精製hsLR11タンパク質を得た。
Inhibitory action of soluble LR11 on cell migration enhancing step 1: Partially purified human sLR11 protein (partial protein with Myc-tag and His-tag added to C-terminal of amino acid sequence 1-2110, hereinafter referred to as “hsLR11”) Preparation The hsLR11 expression vector pcDNA3.1-hsLR11-MycHis A obtained in (1) of Example 1 was transfected into FreeStyle 293-F cells (Life Technologies) to express the hsLR11 protein. As a transfection reagent, Targefect-293FS reagent (Targetingsystems) was used, and after the transfection, swirling culture (8% CO 2 , 125 rpm, 37 ° C.) was performed for 3 days. The sLR11-expressing cells collected by centrifugation were suspended in Lysis buffer (20 mM Tris-HCl, pH 7.5-1% NP-40-150 mM NaCl), solubilized, and then anti-His-tag antibody column equilibrated with PBS ( And then washed with PBS, eluted under acidic conditions (pH 3.0), and immediately neutralized with 0.1 M Tris-HCl, pH 7.5. The eluted fraction is then added to an anti-Myc-tag antibody column (Medical and Biological Laboratories), washed with PBS, eluted under acidic conditions (pH 3.0), and immediately neutralized, partially purified hsLR11 protein Got.
工程２：可溶性LR11の細胞遊走亢進作用に対する抑制作用
U937細胞の細胞遊走の実験は上部チャンバー底に8μmのポアサイズの穴の開いたメンブレンが付いている24ウェルのトランスウェル（コーニング社）を用いて行った。U937細胞は無血清培地で1晩培養後、1μMのCalcein-AMを含む0.1%BSA-ハンクス平衡塩溶液（HBSS）に懸濁して、37℃で30分間インキュベーションしてCalcein-AMを細胞内に取り込ませて、U937細胞を蛍光標識した。無血清培地に1×106cells/mLとなるように蛍光標識したU937細胞を懸濁し、正常マウスIgG抗体又はマウス抗LR11モノクローナル抗体（324-12抗体）と部分精製hsLR11を混合した（抗体終濃度130ng/mL 、hsLR11終濃度500ng/mL）。なお、比較として、各抗体及びsLR11を未添加のウェル、sLR11のみを添加したウェルも設定した。次に、トランスウェルの下部チャンバーに10%ウシ胎児血清（FCS）を含むRPMI1640培地を分注し（500μL/ウェル）、上部チャンバーには上記の部分精製hsLR11及び抗体を含むU937細胞懸濁液を分注し（1.5×106cells/150μL/ウェル）、37℃で4時間反応させた。上部チャンバーから下部チャンバーに遊走(移動）した細胞数は、図２に見られるように細胞の蛍光強度で示した。上部チャンバーを取り除いた後に、下部チャンバーの培地を1.5mLチューブに回収し、遠心（1500rpm, 5min）後、沈殿した細胞を100μLのPBSに懸濁して96ウェル黒色プレート（BD社）に分注し、蛍光プレートリーダー（PerkinElmer社）で細胞の蛍光強度を測定した。各実験はすべてN=3で行った。
Step 2: Inhibitory effect of soluble LR11 on cell migration enhancing effect
Experiments on cell migration of U937 cells were performed using a 24-well transwell (Corning) with a membrane with an 8 μm pore hole at the bottom of the upper chamber. U937 cells are cultured overnight in serum-free medium, suspended in 0.1% BSA-Hanks balanced salt solution (HBSS) containing 1 μM Calcein-AM, and incubated at 37 ° C for 30 minutes to allow Calcein-AM to enter the cells. Upon incorporation, U937 cells were fluorescently labeled. Suspend U937 cells fluorescently labeled to 1 × 10 6 cells / mL in serum-free medium, and mix normal mouse IgG antibody or mouse anti-LR11 monoclonal antibody (324-12 antibody) with partially purified hsLR11 (antibody end) Concentration 130 ng / mL, hsLR11 final concentration 500 ng / mL). For comparison, a well to which each antibody and sLR11 were not added and a well to which only sLR11 was added were also set. Next, RPMI1640 medium containing 10% fetal calf serum (FCS) is dispensed into the lower chamber of the transwell (500 μL / well), and the U937 cell suspension containing the above partially purified hsLR11 and antibody is added to the upper chamber. Aliquots (1.5 × 10 6 cells / 150 μL / well) were allowed to react at 37 ° C. for 4 hours. The number of cells that migrated (moved) from the upper chamber to the lower chamber was indicated by the fluorescence intensity of the cells as seen in FIG. After removing the upper chamber, the medium in the lower chamber is collected in a 1.5 mL tube. After centrifugation (1500 rpm, 5 min), the precipitated cells are suspended in 100 μL of PBS and dispensed into a 96-well black plate (BD). The fluorescence intensity of the cells was measured with a fluorescence plate reader (PerkinElmer). All experiments were performed at N = 3.
＜マウス抗LR11モノクローナル抗体のLR11特異性の検討＞
工程１：ヒトLR11 Vps10pタンパク質の調製
実施例１の（２）で調製したヒトLR11 Vps10p の発現ベクターpcDNA3.1/myc-His(+)-hsVps10pを用いて、実施例１の（３）に記載の方法で精製hVps10pタンパク質を得た。
工程２： LR11及びSortilin（ソルチリン）のVps10pタンパク質とのELISA
LR11はVps10pドメインタンパク質ファミリーに属することから、マウス抗LR11モノクローナル抗体のヒトLR11に対する特異性は、同じVps10pドメインのタンパク質ファミリーに属するヒトSortilin Vps10pタンパク質とヒトLR11 Vps10pタンパク質に対するELISAの反応性で確認した。ELISAは以下のように行った。96ウェルELISAプレート（Nunc社）に0.05M炭酸-重炭酸緩衝液（pH9.6）で2μg/mLに調製したヒトSortilinのVps10pタンパク質（Se78-Asn755, R＆D社, 3154ST）又は精製hVps10pタンパク質を50μL/wellずつ分注し、コーティング（25℃、2時間）した後、0.05%Tween20を含むPBS（PBS-T）で3回プレートを洗浄した後、25%に希釈したブロックエース（DSファーマバイオメディカル社）をウェルに添加してブロッキングを行った（25℃,1時間）。PBS-Tで1回洗浄後、PBS-Tで調製したマウス抗LR11モノクローナル抗体の希釈系列溶液（4000ng/mLより公比４で希釈）を分注し（50μL/well）、2時間反応させた（25℃）。PBS-Tで3回洗浄後、PBS-Tで0.3μg/mLに調製したペルオキシダーゼ標識ロバ抗マウスIgG抗体（Merck Millipore社）をウェルに添加（50μL/well）して1時間反応させた（25℃）。次に、ペルオキシダーゼ基質TMB（3, 3', 5, 5'-tetramethylbenzidine）を添加（50μL/well）して発色反応を行い、１M硫酸（10μL/well）で反応を止めた後に450nmの吸光度を測定した。その結果、マウス抗LR11モノクローナル抗体は、Vps10pドメインタンパク質ファミリーのヒト Sortilin Vps10pタンパク質とは全く反応がみられなかった。一方、hVps10pタンパク質に対しては反応性がみられ、マウス抗LR11モノクローナル抗体のヒトLR11に対する特異性が確認された。この結果を図３に示す。
<Examination of LR11 specificity of mouse anti-LR11 monoclonal antibody>
Step 1: Preparation of human LR11 Vps10p protein As described in (3) of Example 1 using the expression vector pcDNA3.1 / myc-His (+)-hsVps10p of human LR11 Vps10p prepared in (2) of Example 1. In this way, purified hVps10p protein was obtained.
Step 2: ELISA of LR11 and Sortilin with Vps10p protein
Since LR11 belongs to the Vps10p domain protein family, the specificity of the mouse anti-LR11 monoclonal antibody for human LR11 was confirmed by ELISA reactivity to human Sortinin Vps10p protein and human LR11 Vps10p protein belonging to the same Vps10p domain protein family. ELISA was performed as follows. 50 μL of human Sortilin Vps10p protein (Se78-Asn755, R & D, 3154ST) or purified hVps10p protein prepared at 2 μg / mL in 96-well ELISA plate (Nunc) with 0.05 M carbonate-bicarbonate buffer (pH 9.6) After aliquoting and coating each well (25 ° C, 2 hours), the plate was washed 3 times with PBS containing 0.05% Tween20 (PBS-T), and then Block Ace diluted to 25% (DS Pharma Biomedical) Were added to the wells for blocking (25 ° C., 1 hour). After washing once with PBS-T, a diluted series solution of mouse anti-LR11 monoclonal antibody prepared with PBS-T (diluted with a common ratio of 4 from 4000 ng / mL) was dispensed (50 μL / well) and reacted for 2 hours. (25 ° C). After washing 3 times with PBS-T, peroxidase-labeled donkey anti-mouse IgG antibody (Merck Millipore) prepared to 0.3 μg / mL with PBS-T was added to the well (50 μL / well) and allowed to react for 1 hour (25 ° C). Next, the peroxidase substrate TMB (3,3 ', 5,5'-tetramethylbenzidine) was added (50 μL / well) to perform a color reaction, and after stopping the reaction with 1 M sulfuric acid (10 μL / well), the absorbance at 450 nm was measured. It was measured. As a result, the mouse anti-LR11 monoclonal antibody did not react at all with the human Sortilin Vps10p protein of the Vps10p domain protein family. On the other hand, reactivity was observed for the hVps10p protein, confirming the specificity of the mouse anti-LR11 monoclonal antibody for human LR11. The result is shown in FIG.
本発明は、優れた中和活性を有する新規な抗LR11モノクローナル抗体を提供するものであり、本発明の抗LR11モノクローナル抗体は、LR11又はその断片タンパク質が関与する疾患、例えば悪性腫瘍の治療及び／又は予防のための医薬として有用であり、有用な医薬を提供するものであり、産業上の利用可能性を有している。 The present invention provides a novel anti-LR11 monoclonal antibody having excellent neutralizing activity, and the anti-LR11 monoclonal antibody of the present invention is used for the treatment of diseases involving LR11 or a fragment protein thereof, for example, malignant tumor treatment and / or Or it is useful as a medicine for prevention, provides a useful medicine, and has industrial applicability.
配列番号 １： 抗体324-12のＨ鎖のCDR１
配列番号 ２： 抗体324-12のＨ鎖のCDR２
配列番号 ３： 抗体324-12のＨ鎖のCDR３
配列番号 ４： 抗体324-12のＬ鎖のCDR１
配列番号 ５： 抗体324-12のＬ鎖のCDR２
配列番号 ６： 抗体324-12のＬ鎖のCDR３
配列番号 ７： 抗体324-12のＨ鎖の可変領域の全長
配列番号 ８： 抗体324-12のＬ鎖の可変領域の全長
配列番号 ９： Vsp10p フォーワードプライマー配列
配列番号１０： Vsp10p リバースプライマー配列
SEQ ID NO: 1: CDR1 of H chain of antibody 324-12
SEQ ID NO: 2: CDR2 of H chain of antibody 324-12
SEQ ID NO: 3: CDR3 of H chain of antibody 324-12
SEQ ID NO: 4: CDR1 of L chain of antibody 324-12
SEQ ID NO: 5: CDR2 of L chain of antibody 324-12
SEQ ID NO: 6: CDR3 of L chain of antibody 324-12
SEQ ID NO: 7: Full length of variable region of H chain of antibody 324-12 SEQ ID NO: 8: Full length of variable region of L chain of antibody 324-12 SEQ ID NO: 9: Vsp10p forward primer SEQ ID NO: 10: Vsp10p reverse primer sequence
Claims (14)
（Ａ）LR11を認識することができ、
（Ｂ）LR11のVps10pドメインのアミノ酸配列からなるタンパク質に対する特異的結合能を有し、かつ、
（Ｃ）接着性アッセイにおいてヒトLR11のVps10pドメインのアミノ酸配列からなるタンパク質により亢進される細胞接着性を有意に抑制する、
ことを特徴とする抗LR11モノクローナル抗体。 An anti-LR11 monoclonal antibody having, as a complementarity determining region (CDR), at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6;
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
An anti-LR11 monoclonal antibody characterized by that.
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| JP2014075821A JP2015196665A (en) | 2014-04-01 | 2014-04-01 | Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same |
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| JP2015196665A true JP2015196665A (en) | 2015-11-09 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE112016004473T5 (en) | 2015-10-02 | 2018-06-14 | Denso Corporation | Heating management device for a vehicle |
| AU2018236819A1 (en) * | 2017-12-01 | 2019-06-20 | Turun Yliopisto | Treatment of HER2-dependent cancers by SORL1 inhibition |
-
2014
- 2014-04-01 JP JP2014075821A patent/JP2015196665A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE112016004473T5 (en) | 2015-10-02 | 2018-06-14 | Denso Corporation | Heating management device for a vehicle |
| AU2018236819A1 (en) * | 2017-12-01 | 2019-06-20 | Turun Yliopisto | Treatment of HER2-dependent cancers by SORL1 inhibition |
| AU2018236819B2 (en) * | 2017-12-01 | 2020-01-23 | Turun Yliopisto | Treatment of HER2-dependent cancers by SORL1 inhibition |
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