JP2019206491A - Antibody-drug complex for treatment of breast or gastric cancer - Google Patents
Antibody-drug complex for treatment of breast or gastric cancer Download PDFInfo
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- JP2019206491A JP2019206491A JP2018102760A JP2018102760A JP2019206491A JP 2019206491 A JP2019206491 A JP 2019206491A JP 2018102760 A JP2018102760 A JP 2018102760A JP 2018102760 A JP2018102760 A JP 2018102760A JP 2019206491 A JP2019206491 A JP 2019206491A
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Images
Abstract
Description
本発明は、乳がんまたは胃がんの治療のための抗体-薬物複合体に関する。 The present invention relates to antibody-drug conjugates for the treatment of breast cancer or gastric cancer.
均一な抗体-薬物複合体の作製を行うために、抗体と薬物を結合させるリンカーの開発など様々な手法が試みられている(非特許文献１等を参照)。多くは、リジンアミノ基へのカルボン酸との縮合によるアミド結合形成、あるいは、システインのスルフヒドリル基とマレイミドのマイケル付加反応、ジスルフィド結合形成反応を介しての結合形成により、薬物が抗体側に結合される。抗体は、複数のリジン、システインを含むので、薬物結合の位置と数を制御することができず、不均一になる。また、可変部位のリジンやシステインに薬物が結合すると、抗原への結合能力が低下する。
In order to produce a uniform antibody-drug complex, various techniques such as the development of a linker that binds an antibody and a drug have been attempted (see Non-Patent
アミノ基は、抗体に約９０含まれており、そのうちの３０は、容易に修飾されることが知られている。ヒトIgG1の場合、鎖間ジスルフィド結合は４つあり、その結合を担うシステインは８個存在する。従って、アミノ基やチオール基を介した修飾を行うと、多くの異性体を生じることになる。特に、正電荷を有するリジン側鎖は通常抗体の分子表面に存在するために、疎水性の高い薬物をリジン残基を介して結合させると、薬物動態や体内クリアランスに影響を与え得るため、リジン残基を利用した薬物導入は回避されることが多い。 About 90 amino groups are contained in antibodies, 30 of which are known to be easily modified. In the case of human IgG1, there are four interchain disulfide bonds, and there are eight cysteines responsible for the bonds. Therefore, when modification is performed via an amino group or a thiol group, many isomers are produced. In particular, since lysine side chains with positive charge are usually present on the surface of antibody molecules, binding of highly hydrophobic drugs via lysine residues can affect pharmacokinetics and internal clearance. Drug introduction using residues is often avoided.
また、抗体の一部のアミノ酸残基をアジド基のような官能基をもつ非天然型アミノ酸に置換する方法もあるが、抗体をin vitro protein synthesis により発現させる労力が必要である(非特許文献２等を参照)。 In addition, there is a method of substituting some amino acid residues of an antibody with a non-natural amino acid having a functional group such as an azide group, but it requires labor to express the antibody by in vitro protein synthesis (Non-Patent Documents). (See 2).
また、ある特定のペプチド配列を用いて、sortase A, transglutaminase, formylglycine converting enzyme を用いて部位特異的に薬物を結合することもできる。それぞれ、Leu-Pro-X-Thr-Gly (X = 任意のアミノ酸)、Leu-Leu-Gln-Gly-Ala を認識する。しかしタンパク質に該当する配列が存在しない場合には、付加できない（非特許文献３等を参照）。
Alternatively, a specific peptide sequence can be used to bind a drug site-specifically using sortase A, transglutaminase, formylglycine converting enzyme. Recognizes Leu-Pro-X-Thr-Gly (X = any amino acid) and Leu-Leu-Gln-Gly-Ala, respectively. However, when there is no sequence corresponding to the protein, it cannot be added (see Non-Patent
一方で、抗体はAsn297位に普遍的にN-結合型糖鎖を持つ。N-結合型糖鎖はその複雑な生合成過程から微細構造が少しずつ異なる。抗体医薬において、糖鎖構造が活性に影響を及ぼすことが知られているが(非特許文献４等)、抗体作製時に糖鎖構造を均一にすることは困難である。
On the other hand, the antibody has an N-linked sugar chain universally at the Asn297 position. N-linked sugar chains are slightly different in microstructure due to their complex biosynthetic processes. In antibody drugs, it is known that the sugar chain structure affects the activity (Non-patent
特異的に反応する部位を持つ修飾糖ユニットを糖供与体として、改変したガラクトース転移酵素、シアル酸転移酵素を用いて、糖ユニットをひとつずつ延長する糖転移酵素を用いて、抗体の糖鎖部に薬物を結合させる手法も用いられるが、糖鎖構造が不均一であるために、最終的な抗体-薬物複合体が不均一になったり、複数回の糖鎖伸長反応が必要であったりする問題点がある（非特許文献５等を参照）。また、非ヒト型糖鎖を排除することはできない。また、薬物と特異的に反応可能である修飾基を持つ糖供与体である糖ヌクレオチドの合成は数工程必要であり、全収率は高くなく、中間体などは不安定である。 Using a modified saccharide unit with a site that specifically reacts as a sugar donor, using a modified galactose transferase or sialyltransferase, using a glycosyltransferase that extends the saccharide unit one by one, the sugar chain of the antibody The drug can be bound to the drug, but the sugar chain structure is heterogeneous, resulting in a heterogeneous final antibody-drug complex or multiple sugar chain elongation reactions. There is a problem (see Non-Patent Document 5 etc.). In addition, non-human sugar chains cannot be excluded. In addition, the synthesis of a sugar nucleotide, which is a sugar donor having a modifying group capable of specifically reacting with a drug, requires several steps, the overall yield is not high, and intermediates are unstable.
また、糖鎖の水酸基をNaIO4により酸化開裂して、アルデヒド基を創出し、ヒドラジドと反応させて、位置特異的に反応させることも行われている。しかしながら、糖鎖構造が不均一であることから、結合の数を制御することはできない。また、トリプトファン、システイン、メチオニンなど酸化に不安定な官能基を含むアミノ酸残基に対する影響は無視できない（非特許文献６等を参照）。 In addition, a hydroxyl group of a sugar chain is oxidatively cleaved with NaIO 4 to create an aldehyde group, which is reacted with hydrazide to cause a site-specific reaction. However, since the sugar chain structure is heterogeneous, the number of bonds cannot be controlled. In addition, the influence on amino acid residues containing functional groups unstable to oxidation such as tryptophan, cysteine, and methionine cannot be ignored (see Non-Patent Document 6 and the like).
細胞内の代謝を利用して、官能基修飾したフコースなどを導入し、抗体糖鎖に修飾フコースを導入することが可能である。フコースの官能基を利用して、部位特異的に薬物を結合することが可能である（非特許文献７等を参照）。
It is possible to introduce a modified fucose into an antibody sugar chain by introducing a functional group-modified fucose or the like using intracellular metabolism. It is possible to bind a drug in a site-specific manner using a functional group of fucose (see Non-Patent
上記の問題点を解決するために、抗体にもともと存在する糖鎖をAsnに結合しているN-アセチルグルコサミン１残基のみを残して切断し（糖鎖微細構造、非ヒト型糖鎖は消失する）、一挙に特異的反応を起こすような官能基（例えば、アジド基）を持つ、均一な構造のヒト型糖鎖を改変糖加水分解酵素を用いて転移させる（糖鎖部分が均一となる）。特異的反応をおこす官能基（例えば、アジド基）と反応する官能基を結合した薬物を付加すると、均一構造を持つタンパク質-薬物複合体を作製できる。糖鎖は普遍的なタンパク質修飾であるので、sortaseやtransglutaminaseのような酵素が要求する特定のアミノ酸配列を必要とすることなく、コンジュゲートが可能である。改変糖加水分解酵素を用いてアジド基などの官能基をもつ糖鎖を抗体に導入した例も存在する（非特許文献８等を参照）。 In order to solve the above problems, the sugar chain originally present in the antibody is cleaved leaving only one residue of N-acetylglucosamine bound to Asn (glycan fine structure, non-human sugar chain disappears) ) To transfer a human-type sugar chain with a functional group that causes a specific reaction at once (for example, an azide group) to a uniform structure using a modified sugar hydrolase (the sugar chain part becomes uniform) ). When a drug having a functional group that reacts with a functional group that causes a specific reaction (for example, an azide group) is added, a protein-drug complex having a uniform structure can be prepared. Since sugar chains are universal protein modifications, conjugation is possible without requiring specific amino acid sequences required by enzymes such as sortase and transglutaminase. There is also an example in which a sugar chain having a functional group such as an azide group is introduced into an antibody using a modified sugar hydrolase (see Non-Patent Document 8, etc.).
薬物とリンカーを結合する反応は、アジドとひずんだアルキンとのクリック反応や逆電子要請型Diels-Alder反応の用な生物直交的反応が使用できる。 As the reaction for linking the drug and the linker, a bioorthogonal reaction such as a click reaction between an azide and a distorted alkyne or a reverse electron demand type Diels-Alder reaction can be used.
リンカーとして、カテプシン切断型リンカーについても報告されている（非特許文献９等を参照）。 As a linker, a cathepsin-cleavable linker has also been reported (see Non-Patent Document 9, etc.).
本発明は、抗HER2抗体と抗がん剤の抗体-薬物複合体を含む乳がんまたは胃がん治療剤の提供を目的とする。 An object of the present invention is to provide a therapeutic agent for breast cancer or stomach cancer containing an antibody-drug complex of an anti-HER2 antibody and an anticancer agent.
本発明者らは、HER2を発現しているがんの治療剤として、抗HER2抗体と抗がん剤である薬物を含む抗体-薬物複合体（ADC)の治療効果を向上させる方法について鋭意検討を行った。in vitro でのHER2を発現するがん細胞に対する効果の検討において、抗体に薬物を糖鎖とカテプシン切断型リンカーを介して結合させたADCが薬物単独と同等の殺細胞効果を奏するという予測できない結果が得られた。また、糖鎖を介して薬物を付加することにより、均一なADCが得られることを見出した。これらの結果より、抗HER2抗体に薬物を糖鎖およびカテプシン切断型リンカーを介して付加したADCが乳がんまたは胃がん、特に乳がん治療に有効であることを見出し本発明を完成させるに至った。 As a therapeutic agent for cancer expressing HER2, the present inventors have intensively studied a method for improving the therapeutic effect of an antibody-drug complex (ADC) containing an anti-HER2 antibody and an anticancer drug. Went. In the study of in vitro effects on HER2-expressing cancer cells, unpredictable results indicate that ADCs that bind antibodies to drugs via sugar chains and cathepsin-cleavable linkers have the same cytocidal effect as drugs alone was gotten. It was also found that a uniform ADC can be obtained by adding a drug via a sugar chain. From these results, it was found that an ADC obtained by adding a drug to an anti-HER2 antibody via a sugar chain and a cathepsin-cleavable linker is effective in treating breast cancer or stomach cancer, particularly breast cancer, and has completed the present invention.
すなわち、本発明は以下のとおりである。
[１] 抗HER2抗体に抗がん剤がカテプシン切断型リンカーであるバリン-シトルリン(Val-Cit)ペプチドリンカーおよび糖鎖を介して付加されており、細胞中でカテプシンによりリンカーが切断され、抗がん剤が放出される、抗体-薬物複合体。
[２] 抗がん剤がアウリスタチンである、[１]の抗体-薬物複合体。
[３] アウリスタチンが、monomethyl auristatin E (MMAE)、monomethyl auristatin D (MMAD)およびmonomethyl auristatin F (MMAF)からなる群から選択される、[２]の抗体-薬物複合体。
[４] 抗HER2抗体が均一糖鎖構造を有する糖鎖改変した抗体である、[１]〜[３]のいずれかの抗体-薬物複合体。
[５] 抗体がトラスツズマブである、[１]〜[４]のいずれかの抗体-薬物複合体。
[６] [１]〜[５]のいずれかの抗体-薬物複合体を含む、乳がんまたは胃がん治療剤。
[７] 他の抗がん剤と併用される、[６]の乳がんまたは胃がん治療剤。
That is, the present invention is as follows.
[1] An anticancer agent is added to the anti-HER2 antibody via a cathepsin-cleavable linker, valine-citrulline (Val-Cit) peptide linker and a sugar chain, and the linker is cleaved by cathepsin in the cell. An antibody-drug complex from which a cancer drug is released.
[2] The antibody-drug conjugate according to [1], wherein the anticancer agent is auristatin.
[3] The antibody-drug complex according to [2], wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), and monomethyl auristatin F (MMAF).
[4] The antibody-drug complex according to any one of [1] to [3], wherein the anti-HER2 antibody is a sugar chain-modified antibody having a uniform sugar chain structure.
[5] The antibody-drug complex according to any one of [1] to [4], wherein the antibody is trastuzumab.
[6] A therapeutic agent for breast cancer or stomach cancer comprising the antibody-drug complex of any one of [1] to [5].
[7] The breast or stomach cancer therapeutic agent according to [6], which is used in combination with another anticancer agent.
抗HER2抗体に薬物を糖鎖およびカテプシン切断型リンカーを介して付加したADCは、HER2を発現するがんの中でも、乳がんまたは胃がんに対して顕著な抗がん効果を示す。 An ADC obtained by adding a drug to an anti-HER2 antibody via a sugar chain and a cathepsin-cleavable linker exhibits a remarkable anticancer effect against breast cancer or stomach cancer among cancers expressing HER2.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明は、がん治療のための抗体-薬物複合体（ADC: Antibody-drug conjugate)である。本発明のADCにおいて、抗体は抗HER2抗体であり、薬物は抗がん剤である。抗がん剤は、チューブリン合成阻害剤（抗がん性植物アルカロイド）、トポイソメラーゼ阻害剤、核酸合成の阻害剤であるアルキル化剤、代謝拮抗剤、抗生物質抗癌剤、ホルモン製剤、白金製剤、非特異的抗悪性腫瘍剤等の公知の抗がん剤の他新規に開発される抗がん剤を含む。具体的には、例えば、アウリスタチン、硫酸ビンクリスチン、硫酸ビンブラスチン、硫酸ビンデシン、ドセタキセル水和物、パクリタキセル、酒石酸ビノレルビン、メイタンシノイド等のチューブリン合成阻害剤（抗がん性植物アルカロイド）；カンプトテシン、トポテカン、エトポシド、塩酸イリノテカン、塩酸ノギテカン等のトポイソメラーゼ阻害薬；シクロフォスファミド、イフォスファミド、メルファラン、チオテバ、ブスルファン、カルボコン、ダカルバジン、塩酸ニムスチン、ラニムスチン等のアルキル化剤；メトトレキサート、メルカプトプリン、6-メルカプトプリンリボシド、フルオロウラシル(5-FU)、テガフール、テガフールウラシル、カルモフール、ドキシフルリジン、シタラビンオクホスファート、ヒドロキシカルバミド、シタラビン、塩酸ゲムシタビン、リン酸フルダラビン、エノシタビン、ロイコボリン等の代謝拮抗剤；塩酸ドキソルビシン、塩酸イダルビシン、塩酸エピルビシン、塩酸ピラルビシン、塩酸ダウノルビシン、塩酸アクラルビシン、マイトマイシンC、アクチノマイシンD、ブレオマイシン、硫酸ペプロマイシン、ネオカルチノスタチン、ジノスタチンスチマラマー等の抗がん性抗生物質；リン酸エストラムスチンタトリウム、フルタミド、ビカルタミド、酢酸ゴセレリン、酢酸ニュープロレリン、クエン酸タモキシフェン、塩酸フォドロゾール水和物、アナストロゾール、メピチオスタン、エピチオスタノール、酢酸メドロキシプロゲステロン等のホルモン製剤；シスプラチン、カルボプラチン、ネダプラチン等の白金製剤；クレスチン、レンアチン、シゾフィラン、ウベニメクス等の非特異的抗悪性腫瘍剤；塩酸ミトキサントロン、塩酸プロカルバジン、ペントスタチン、ソブゾキサン、トレチノイン、L-アスパラギナーゼ、アセグラトン、ミトタン、ポルフィマーナトリウム等が挙げられる。 The present invention is an antibody-drug conjugate (ADC) for cancer treatment. In the ADC of the present invention, the antibody is an anti-HER2 antibody and the drug is an anticancer agent. Anticancer agents include tubulin synthesis inhibitors (anticancer plant alkaloids), topoisomerase inhibitors, nucleic acid synthesis inhibitors alkylating agents, antimetabolites, antibiotic anticancer agents, hormone preparations, platinum preparations, non In addition to known anticancer agents such as specific antineoplastic agents, newly developed anticancer agents are included. Specifically, for example, tubulin synthesis inhibitors (anticancer plant alkaloids) such as auristatin, vincristine sulfate, vinblastine sulfate, vindesine sulfate, docetaxel hydrate, paclitaxel, vinorelbine tartrate, maytansinoid; camptothecin, Topoisomerase inhibitors such as topotecan, etoposide, irinotecan hydrochloride, nogitecan hydrochloride; Mercaptopurine riboside, fluorouracil (5-FU), tegafur, tegafur uracil, carmofur, doxyfluridine, cytarabine ocphosphate, hydroxycarbami , Cytarabine, gemcitabine hydrochloride, fludarabine phosphate, enocitabine, leucovorin, and other antimetabolites; doxorubicin hydrochloride, idarubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, daunorubicin hydrochloride, aclarubicin hydrochloride, mitomycin C, actinomycin D, bleomycin, peomycin Anticancer antibiotics such as cartinostatin and dinostatin stimamarer; estramustine phosphate, flutamide, bicalutamide, goserelin acetate, neuprorelin acetate, tamoxifen citrate, fodozole hydrochloride hydrate, anastrozole Hormone preparations such as mepithiostan, epithiostanol and medroxyprogesterone acetate; platinum preparations such as cisplatin, carboplatin and nedaplatin; Achin, schizophyllan, nonspecific antineoplastic agents such as ubenimex; mitoxantrone hydrochloride, procarbazine hydrochloride, pentostatin, sobuzoxane, tretinoin, L- asparaginase, aceglatone, mitotane, include porfimer sodium and the like.
この中でも、チューブリン合成阻害剤であり、非常に強い抗腫瘍剤であるauristatin（アウリスタチン）が挙げられる。アウリスタチンには、monomethyl auristatin E (MMAE、モノメチルアウリスタチンE)、monomethyl auristatin D (MMAD、モノメチルアウリスタチンD)、monomethyl auristatin F (MMAF、モノメチルアウリスタチンF)等が含まれる。この中でも、monomethyl auristatin E (MMAE、モノメチルアウリスタチンE)が好ましい。 Among them, aristatin (auristatin), which is a tubulin synthesis inhibitor and is a very strong antitumor agent, can be mentioned. Examples of auristatin include monomethyl auristatin E (MMAE, monomethyl auristatin E), monomethyl auristatin D (MMAD, monomethyl auristatin D), monomethyl auristatin F (MMAF, monomethyl auristatin F) and the like. Among these, monomethyl auristatin E (MMAE, monomethyl auristatin E) is preferable.
本発明のADCは、乳がんおよび胃がんの治療に用いられる。
HER2は、細胞表面に存在する、EGFR(epidermal growth factor receptor)ファミリーに属する約185kDaの受容体型チロシンキナーゼであり、上皮成長因子受容体（EGFR)に類似した構造を有している。HER2は、乳がんや胃がんにおいて過剰に発現することがあることが知られている。本発明のADCに用いられる抗HER2抗体は、リコンビナント抗HER2モノクローナル抗体であり、ヒトの定常領域を有するキメラ抗体、ヒト抗体のCDR（相補性決定領域）をヒト以外の動物の抗HER2抗体のCDRで移植したヒト化抗体（CDR移植抗体）、ヒト由来の抗体遺伝子の発現産物であるヒト抗体が含まれるが、ヒト化抗体やヒト抗体が好ましい。完全ヒトモノクローナル抗体は、ヒト免疫グロブリン重鎖および軽鎖遺伝子座の大部分が遺伝子導入されたマウスを免疫することにより作製することができる。抗体のクラスは好ましくはIgG1である。好ましい抗HER2抗体として、ヒト化IgG1抗体であるトラスツズマブ（Trastuzumab）（ハーセプチン（登録商標））が挙げられる。
The ADC of the present invention is used for the treatment of breast cancer and gastric cancer.
HER2 is an approximately 185 kDa receptor tyrosine kinase belonging to the EGFR (epidermal growth factor receptor) family that exists on the cell surface, and has a structure similar to that of epidermal growth factor receptor (EGFR). HER2 is known to be overexpressed in breast cancer and gastric cancer. The anti-HER2 antibody used in the ADC of the present invention is a recombinant anti-HER2 monoclonal antibody, a chimeric antibody having a human constant region, a CDR of a human antibody (complementarity determining region), and a CDR of an anti-HER2 antibody of a non-human animal. And human antibodies that are expression products of human-derived antibody genes are preferred, but humanized antibodies and human antibodies are preferred. Fully human monoclonal antibodies can be made by immunizing mice in which most of the human immunoglobulin heavy and light chain loci have been transfected. The antibody class is preferably IgG1. A preferred anti-HER2 antibody is humanized IgG1 antibody Trastuzumab (Herceptin®).
本発明のADCにおいては、薬物は抗体のアミノ基やシステイン基を介して抗体に付加しているのではなくて、抗体の定常領域に結合している糖鎖を介して付加している。 In the ADC of the present invention, the drug is not added to the antibody via the amino group or cysteine group of the antibody, but is added via a sugar chain bound to the constant region of the antibody.
構造が均一なADCの作製は、活性薬物活性、薬物動態を制御するために必須であり、レギュラトリー・サイエンスからもその重要性が指摘され、ADC開発の中心的議題である。ADCは、薬物（イメージング剤、RI、siRNA等も含む）を抗体に結合させ、体内でがん等の病変部位に選択的に送達させ、薬物を病変部位のみにて放出する（イメージング剤の場合には放出させない）ことにより、効果の有効発現と副作用の低減を狙うDDS（Drug delivery system）技術のひとつである。 Preparation of ADC with uniform structure is essential for controlling active drug activity and pharmacokinetics, and its importance is pointed out by regulatory science, and it is a central agenda for ADC development. ADC binds a drug (including imaging agents, RI, siRNA, etc.) to an antibody, selectively delivers it to a lesion site such as cancer in the body, and releases the drug only at the lesion site (in the case of an imaging agent) This is one of the DDS (Drug Delivery System) technologies aiming at effective expression and reduction of side effects.
一方、抗体に代表されるタンパク質には、不均一な構造の糖鎖が付加しており、糖鎖構造により生理活性も異なることが知られている。例えば、ポテリジオ(登録商標）（フコース除去型抗CCR4抗体）にみられるように、糖鎖構造を改変することにより、ADCC活性が100倍に上昇した例も知られている。また、抗体作製にあたりCHO細胞等を使用すると、抗体に非ヒト型糖鎖が付加され、その抗原性からアナフィラキシーショックを起こすことがある。 On the other hand, it is known that sugar chains having a heterogeneous structure are added to proteins typified by antibodies, and the physiological activity varies depending on the sugar chain structure. For example, as seen in Poterigio (registered trademark) (fucose-removed anti-CCR4 antibody), an example in which ADCC activity is increased 100-fold by modifying the sugar chain structure is also known. In addition, when CHO cells or the like are used for antibody production, a non-human sugar chain is added to the antibody, and anaphylactic shock may occur due to its antigenicity.
これら２つの問題点を解決するために、抗体が本来持っている糖鎖を切断、再構成することにより、糖鎖構造を制御しつつ、糖鎖を介して、構造均一であるヒト型糖鎖を持つ抗体-薬物複合体を作製することができる。この方法により、in vitroにおけるタンパク質合成などの分子生物学的手法を使用することなく、薬物を抗体に位置特異的に結合させ、薬物結合数を制御することが可能になる。 In order to solve these two problems, the sugar chain originally possessed by the antibody is cleaved and reconstructed to control the sugar chain structure, and the human-type sugar chain having a uniform structure via the sugar chain. An antibody-drug complex having can be prepared. This method makes it possible to bind a drug to an antibody in a position-specific manner and control the number of drug bindings without using molecular biological techniques such as in vitro protein synthesis.
本発明のADCにおいては、糖鎖構造が均一となるように、薬物を糖鎖を介して抗体に付加してある。 In the ADC of the present invention, a drug is added to an antibody via a sugar chain so that the sugar chain structure is uniform.
本発明のADCの作製方法を以下に例示する。
（１） 元々糖鎖構造が不均一な抗HER2抗体の糖鎖を切断する。糖鎖の切断は、エンド-β-N-アセチルグルコサミニダーゼ（EndoS）を用いて行うことができる。EndoSにより、抗体のFc領域のCHドメインに位置する297番目のアスパラギン（Asn)に結合しているN型複合型糖鎖の還元末端のN-アセチルグルコサミン（GlcNAc)１残基を残して切断される。還元末端のN-アセチルグルコサミンには、フコースが結合していても、結合していなくてもよい。この後、クロマトグラフィーを用いて、糖鎖が切断された抗体を精製すればよい。クロマトグラフィーとしては、ゲル濾過クロマトグラフィー、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水性クロマトグラフィー等が挙げられる。
A method for manufacturing the ADC of the present invention is illustrated below.
(1) Cleave the sugar chain of an anti-HER2 antibody that originally has a non-uniform sugar chain structure. The sugar chain can be cleaved using endo-β-N-acetylglucosaminidase (EndoS). EndoS is cleaved leaving 1 residue of N-acetylglucosamine (GlcNAc) at the reducing end of the N-type complex sugar chain bound to the 297th asparagine (Asn) located in the CH domain of the Fc region of the antibody. The N-acetylglucosamine at the reducing end may or may not be bound to fucose. Thereafter, the antibody having the sugar chain cleaved may be purified using chromatography. Examples of the chromatography include gel filtration chromatography, affinity chromatography, ion exchange chromatography, hydrophobic chromatography and the like.
（２） 次いで、糖鎖を切断した抗体に、還元末端にオキサゾリン化されたGlcNAcを有し、反対側の末端にアジド基（N3)を付加した均一なオキサゾリン糖鎖(アジドオキサゾリン糖鎖）をドナーとして、アクセプターである（１）で得られた糖鎖を切断した抗体のN-アセチルグルコサミン（GlcNAc)残基に糖鎖を結合させる。この反応においては、抗体のN-アセチルグルコサミン（GlcNAc)残基の４位にドナーのオキサゾリン環が反応し、キトビオース構造を形成し結合する。この際、例えば、糖鎖切断活性を抑制し、糖鎖転移活性を向上させたEndoSのD233Q変異体を用いればよい。この工程により抗体へ均一な糖鎖を付加させることができる。この後、クロマトグラフィーにより糖鎖が結合させた抗体を精製すればよい。このようにして糖鎖を結合させた構造は、均一糖鎖構造を有する糖鎖改変した抗体である。 (2) Next, a homogenous oxazoline sugar chain (azidooxazoline sugar chain) having a GlcNAc oxazolineated at the reducing end and an azido group (N 3 ) added to the opposite end of the antibody whose sugar chain has been cleaved As a donor, the sugar chain is bound to the N-acetylglucosamine (GlcNAc) residue of the antibody obtained by cleaving the sugar chain obtained in the acceptor (1). In this reaction, the donor oxazoline ring reacts with the 4-position of the N-acetylglucosamine (GlcNAc) residue of the antibody to form a chitobiose structure and bind thereto. In this case, for example, the EndoS D233Q mutant with suppressed sugar chain cleavage activity and improved sugar chain transfer activity may be used. By this step, a uniform sugar chain can be added to the antibody. Thereafter, the antibody to which the sugar chain is bound may be purified by chromatography. The structure in which the sugar chains are bound in this manner is a sugar chain-modified antibody having a uniform sugar chain structure.
抗体に結合させる均一な構造の糖鎖は目的に応じて適宜選択されるが、還元末端のN-アセチルグルコサミン（GlcNAc)を含め、ジアセチルキトビオース(GlcNAc-GlcNAc)にマンノース（Man)のオリゴマーが結合した構造を有する高マンノース型糖鎖、ジアセチルキトビオースにMan並びにGlcNAc、ガラクトース（Gal)、シアル酸（Neu5Ac)の少なくとも１つが結合した複合（コンプレックス）型糖鎖、ジアセチルキトビオースに高マンノース型と複合型が混成した糖鎖構造を有する混成（ハイブリッド）型糖鎖が挙げられ、シアル酸の有無、コアフコースの有無、分岐鎖の有無等により種々の構造の糖鎖がある。 The sugar chain with a uniform structure to be bound to the antibody is appropriately selected according to the purpose, but includes N-acetylglucosamine (GlcNAc) at the reducing end and oligomers of mannose (Man) in diacetylchitobiose (GlcNAc-GlcNAc). A high mannose sugar chain with a structure in which diacetylchitobiose is linked to diacetylchitobiose, a complex (complex) sugar chain in which at least one of Man and GlcNAc, galactose (Gal), and sialic acid (Neu5Ac) is bound Examples include hybrid (hybrid) type sugar chains having a sugar chain structure in which a high mannose type and a complex type are mixed, and there are sugar chains of various structures depending on the presence or absence of sialic acid, the presence or absence of core fucose, the presence or absence of branched chains, and the like.
代表的な糖鎖として、図１に示す構造を有する糖鎖が挙げられる。図１に示す糖鎖の還元末端のN-アセチルグルコサミン（GlcNAc)には、フコースが結合していてもよい。 A typical sugar chain includes a sugar chain having the structure shown in FIG. Fucose may be bound to N-acetylglucosamine (GlcNAc) at the reducing end of the sugar chain shown in FIG.
（３） 次いで、抗体に結合させたアジド基を有する糖鎖にmonomethyl auristatin E (MMAE)の薬物をリンカーを介して結合させる。この際、アジド基を有する糖鎖のアジド基にリンカーと結合させた薬物を結合させればよい。リンカーはカテプシン等のシステインプロテアーゼ酵素により切断されるリンカー、すなわちカテプシン切断型リンカーを用いればよい。このようなリンカーとして、バリンとシトルリンを結合させたバリン-シトルリン(Val-Cit)ペプチドリンカーが挙げられる。アジド基とリンカーと結合させた薬物との結合は、リンカーと薬物の結合体のリンカー側にWSCDI等の環状アルキンをクリック反応により結合させればよい。この後、クロマトグラフィーにより生成物を精製すればよい。 (3) Next, a monomethyl auristatin E (MMAE) drug is bound to a sugar chain having an azide group bound to an antibody via a linker. At this time, a drug bonded with a linker may be bound to the azide group of the sugar chain having an azide group. The linker may be a linker that is cleaved by a cysteine protease enzyme such as cathepsin, that is, a cathepsin-cleavable linker. Examples of such a linker include a valine-citrulline (Val-Cit) peptide linker obtained by binding valine and citrulline. The azide group and the drug bonded to the linker may be bonded to the linker side of the linker / drug conjugate by a click reaction of a cyclic alkyne such as WSCDI. Thereafter, the product may be purified by chromatography.
図２に本発明のADCの作製方法の概略を示す。
このようにして得られたADCは、抗HER2抗体に薬物がカテプシン切断型リンカーを介して付加されている。
FIG. 2 shows an outline of a method for manufacturing the ADC of the present invention.
In the ADC thus obtained, a drug is added to the anti-HER2 antibody via a cathepsin-cleavable linker.
本発明のADCは、薬物を糖鎖改変抗体の特定の部位の糖鎖に結合させるため、均一な構造を有している。また、抗体に結合させる糖鎖の分岐鎖の数に応じて、１分子の抗体に対して、２〜８分子の薬物を結合させることができる。例えば、図1に示す糖鎖を結合させた場合、１分子の抗体に対して、４分子の薬物が結合する（DAR:Drug Antibody ratio = 4)。 The ADC of the present invention has a uniform structure for binding a drug to a sugar chain at a specific site of a sugar chain-modified antibody. Further, 2 to 8 molecules of drug can be bound to one molecule of antibody depending on the number of sugar chain branches to be bound to the antibody. For example, when the sugar chain shown in FIG. 1 is bound, four molecules of drug bind to one molecule of antibody (DAR: Drug Antibody ratio = 4).
本発明のADCは、その塩、又はそれらの水和物も包含する。
ADCを被験体に投与した場合、ADCは乳がんおよび胃がん細胞に発現しているHER2に結合し、ADCとHER2の複合体が細胞内に取り込まれる。がん細胞中に存在するカテプシンによりカテプシン切断型リンカーが切断され、細胞内でMMAEが放出される。その後、MMAEの作用でがん細胞が死滅する。
The ADC of the present invention includes a salt thereof or a hydrate thereof.
When ADC is administered to a subject, ADC binds to HER2 expressed in breast and gastric cancer cells, and the ADC-HER2 complex is taken up into the cell. Cathepsin-cleavable linkers are cleaved by cathepsins present in cancer cells, and MMAE is released in the cells. After that, cancer cells are killed by the action of MMAE.
本発明のADCは、乳がんまたは胃がんの治療に有用であり、特に乳がん治療に有用である。本発明のADCはカテプシン切断型リンカーを有しており、細胞中でのADCからの薬物の放出は細胞内のカテプシンの作用により行われる。カテプシンはがん細胞で発現が向上しているが、特に乳がんで発現が向上している可能性があり、乳がん細胞中で薬物が効率的に放出される可能性がある。 The ADC of the present invention is useful for the treatment of breast cancer or stomach cancer, and particularly useful for the treatment of breast cancer. The ADC of the present invention has a cathepsin-cleavable linker, and the release of the drug from the ADC in the cell is performed by the action of the intracellular cathepsin. Cathepsin has improved expression in cancer cells, but expression may be improved particularly in breast cancer, and the drug may be efficiently released in breast cancer cells.
ADCが抗がん効果を発揮するためには、ADCががん細胞に到達し取り込まれる工程とがん細胞中で薬物を放出する工程があり、ADCの抗がん効果はこの２つの工程の効率性を考慮する必要がある。従来は、これら２つの工程の効率性を評価することができず、ADCの設計において、抗体、薬物、リンカーの組合せを適切に決め、ADCを設計し、さらに効果が高いがん種を特定することはできなかった。本発明においては、HER2の発現が高いがん（乳がん、胃がん）を対象として、抗体として抗HER抗体を用い、薬物としてMMAEを用いて、リンカーとして、カテプシン切断型リンカーを用いることにより、乳がんに特に効果の高いADCを作製することができた。 In order for ADC to exert its anticancer effect, there are a process in which ADC reaches and is taken up by cancer cells, and a process in which drugs are released in the cancer cells. It is necessary to consider efficiency. Conventionally, the efficiency of these two processes could not be evaluated. In designing ADCs, the combination of antibodies, drugs, and linkers is appropriately determined, ADCs are designed, and cancer types that are more effective are identified. I couldn't. In the present invention, for cancer (breast cancer, gastric cancer) with high expression of HER2, anti-HER antibody is used as an antibody, MMAE is used as a drug, and a cathepsin-cleavable linker is used as a linker. A particularly effective ADC could be fabricated.
本発明は、上記ADCを有効成分として含む抗乳がん剤および抗胃がん剤も包含する。
本発明のADCを含む抗乳がん剤および抗胃がん剤の投与形態は限定されず、経口、非経口等により投与することができる。非経口投与として、静脈内、皮下、筋肉内、腹腔内注射等が挙げられる。製剤の投与形態としては、経口液剤、錠剤、カプセル剤、丸剤、散剤等が挙げられる。カプセル剤、錠剤、散剤、顆粒剤などは、乳糖、ブドウ糖、ショ糖、マンニトールなどの賦形剤；デンプン、アルギン酸ナトリウムなどの崩壊剤；ステアリン酸マグネシウム、タルクなどの滑沢剤；ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチンなどの結合剤；脂肪酸エステルなどの界面活性剤；グリセリンなどの可塑剤などを添加剤として用いて製造できる。
The present invention also includes an anti-breast cancer agent and an anti-gastric cancer agent containing the ADC as an active ingredient.
The dosage form of the anti-breast cancer agent and anti-gastric cancer agent containing the ADC of the present invention is not limited, and can be administered orally, parenterally or the like. Parenteral administration includes intravenous, subcutaneous, intramuscular, intraperitoneal injection and the like. Examples of the dosage form of the preparation include oral solutions, tablets, capsules, pills, powders and the like. For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin; a surfactant such as fatty acid ester; and a plasticizer such as glycerin can be used as additives.
乳剤及びシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖などの糖類；ポリエチレングリコール、プロピレングリコールなどのグリコール類；ごま油、オリーブ油、大豆油などの油類；ｐ−ヒドロキシ安息香酸エステル類などの防腐剤；ストロベリーフレーバー、ペパーミントなどのフレーバー類などを添加剤として用いて製造できる。 Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; p-hydroxybenzoic acid Preservatives such as esters; flavors such as strawberry flavor and peppermint can be used as additives.
注射剤は、水、ショ糖、ソルビトール、キシロース、トレハロース、果糖などの糖類；マンニトール、キシリトール、ソルビトールなどの糖アルコール；リン酸緩衝液、クエン酸緩衝液、グルタミン酸緩衝液などの緩衝液；脂肪酸エステルなどの界面活性剤などを添加剤として用いることができる。 Injections include sugars such as water, sucrose, sorbitol, xylose, trehalose, and fructose; sugar alcohols such as mannitol, xylitol, and sorbitol; buffers such as phosphate buffer, citrate buffer, and glutamate buffer; fatty acid esters A surfactant such as can be used as an additive.
また、抗HER2抗体により乳がんまたは胃がん細胞を標的として、乳がんまたは胃がん細胞に効率的に到達するので、全身投与してもよいが、がん組織に局所的に投与してもよい。 In addition, since the anti-HER2 antibody targets breast cancer or stomach cancer cells and efficiently reaches the breast cancer or stomach cancer cells, it may be administered systemically or locally to cancer tissue.
治療に用いるのに必要な本発明のADCの投与量は、投与する患者の年齢、性別、重篤度等により変えることができるが、最終的には担当医師が決めることができる。例えば、ADCを１回あたり0.05〜10mg/kg体重、好ましくは0.1〜２mg/kg体重で投与すればよい。また、MMAE等の薬物当量で、１回当たり0.001nM〜1000nM投与してもよい。 The dosage of the ADC of the present invention necessary for treatment can be changed depending on the age, sex, severity, etc. of the patient to be administered, but can ultimately be decided by the doctor in charge. For example, ADC may be administered at 0.05 to 10 mg / kg body weight, preferably 0.1 to 2 mg / kg body weight per time. Moreover, 0.001 nM to 1000 nM may be administered at a time with a drug equivalent such as MMAE.
所定の投与量は１回の投与で与えてもよいし、１日当たり２回、３回、４回またはそれ以上の分割投与とし、１日から数年にわたって、適当な間隔で投与してもよい。 The prescribed dose may be given as a single dose, or divided into two, three, four or more divided doses per day, and may be administered at appropriate intervals over a period of one day to several years. .
本発明のADCを含む抗乳がん剤または抗胃がん剤は、他の抗がん剤や他の療法と併用することもできる。他の療法として、例えば、放射線治療、ホルモン療法、外科手術等が挙げられる。 The anti-breast cancer agent or anti-gastric cancer agent containing ADC of the present invention can be used in combination with other anti-cancer agents and other therapies. Examples of other therapies include radiation therapy, hormonal therapy, and surgery.
本発明を以下の実施例によって具体的に説明するが、本発明はこれらの実施例によって限定されるものではない。 The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
[実施例１] 抗体-薬物複合体（ADC)の作製
抗体として、抗HER2抗体(トラスツズマブ：Trastuzumab）を使用した。トラスツズマブは、乳がん、胃がんに対する抗体医薬である。糖鎖部分の合成は以下のように行った。
[Example 1] Preparation of antibody-drug complex (ADC) An anti-HER2 antibody (Trastuzumab) was used as an antibody. Trastuzumab is an antibody drug against breast cancer and stomach cancer. The synthesis of the sugar chain portion was performed as follows.
１． 薬物結合部位を有し、安定な構造を有する糖供与体の合成
DMF（333μL）中ジシアリルオクタサッカライド（30mg、0.0148mmol）、2-（2-（2-（2-アジドエトキシ）エトキシ）エトキシ）エタン-1-アミン（DMF中4M、37μL、0.148mmol）およびDIPEA（26μL、0.148mmol）の懸濁液に、PyBOP（77mg、0.148mmol）を4℃で加え、混合物を40℃で8時間撹拌した。分析用HPLCは、0％ MeCNを含むC8逆相カラム（Mightysil RP-8 GP、4.6×250mm、関東化学社）を用いて2分間、0.1％トリフルオロ酢酸水溶液中0〜100％MeCNの直線勾配で40分間行った（室温、流速1mL/分、214nm、280nmでの検出）。8時間後、反応混合物をゲル濾過カラムクロマトグラフィー（G-25、GE Healthcare Life Sciences、溶出液：H2O）により精製した。次いで、分取HPLC（Mightysil RP-18 GP、20×250mm、関東化学）により、所望の生成物（アジド基を有する糖鎖）（20.4mg、収率57％）を得た。
1H-NMR (D2O) d 5.18 (m, 1H), 5.10 (s, 1H), 4.91 (s, 1H), 4.57-4.55 (m, 2H), 4.42-4.49 (m, 2H), 4.41-4.29 (m, 2H), 4.23 (s, 1H), 4.16 (s, 1H), 4.08 (s, 1H), 2.69-2.65 (m, 2H), 2.02 (s, 15H), 2.00 (s, 9H), 1.87-1.78 (t, 2H); 13C-NMR (D2O) d 175.1, 174.8, 174.7, 169.2, 103.8, 103.8, 99.7, 99.5, 81.0, 80.9, 80.6, 80.4, 76.5, 74.5, 76.3, 74.5, 73.7, 72.6, 74.5, 73.7, 72.6, 72.3, 72.2, 71.2, 70.8, 69.8, 69.6, 69.3, 69.3, 68.6, 68.4, 67.9, 67.5, 67.4, 67.1, 63.1, 60.3, 60.1, 54.8, 51.8, 50.3, 39.1, 38.9, 38.4, 38.4, 38.2, 38.0, 37.8, 37.6, 22.6, 22.2, 22.1; MS calcd for [C92H152N13O61+Na+] 2443, found 2433.
1. Synthesis of sugar donors with drug binding sites and stable structures
Disialyl octasaccharide (30 mg, 0.0148 mmol), 2- (2- (2- (2-azidoethoxy) ethoxy) ethoxy) ethan-1-amine (4M in DMF, 37 μL, 0.148 mmol) in DMF (333 μL) and To a suspension of DIPEA (26 μL, 0.148 mmol), PyBOP (77 mg, 0.148 mmol) was added at 4 ° C. and the mixture was stirred at 40 ° C. for 8 hours. Analytical HPLC is a linear gradient of 0-100% MeCN in 0.1% aqueous trifluoroacetic acid using a C8 reverse phase column (Mightysil RP-8 GP, 4.6 x 250 mm, Kanto Chemical) containing 0% MeCN for 2 minutes. For 40 minutes (room temperature, flow
1 H-NMR (D 2 O) d 5.18 (m, 1H), 5.10 (s, 1H), 4.91 (s, 1H), 4.57-4.55 (m, 2H), 4.42-4.49 (m, 2H), 4.41 -4.29 (m, 2H), 4.23 (s, 1H), 4.16 (s, 1H), 4.08 (s, 1H), 2.69-2.65 (m, 2H), 2.02 (s, 15H), 2.00 (s, 9H ), 1.87-1.78 (t, 2H); 13 C-NMR (D 2 O) d 175.1, 174.8, 174.7, 169.2, 103.8, 103.8, 99.7, 99.5, 81.0, 80.9, 80.6, 80.4, 76.5, 74.5, 76.3 , 74.5, 73.7, 72.6, 74.5, 73.7, 72.6, 72.3, 72.2, 71.2, 70.8, 69.8, 69.6, 69.3, 69.3, 68.6, 68.4, 67.9, 67.5, 67.4, 67.1, 63.1, 60.3, 60.1, 54.8, 51.8 , 50.3, 39.1, 38.9, 38.4, 38.4, 38.2, 38.0, 37.8, 37.6, 22.6, 22.2, 22.1; MS calcd for [C 92 H 152 N 13 O 61 + Na + ] 2443, found 2433.
オキサゾリンの調製
D2O（590μL）中のラクトール（5.0mg、0.00207mmol）およびトリエチルアミン（20.2μL、0.145mmol）の溶液に、塩化2-クロロ-1,3-ジメチル-1H-ベンズイミダゾール-3-イウム（CDMBI、20.2 0.0930mmol）を4℃で添加し、2時間撹拌した。反応の進行を1H NMRにより分析し、オキサゾリンの形成を確認した。次いで、ゲル濾過カラムクロマトグラフィー（G-25、GE Healthcare Life Sciences）、溶出液：0.1％トリエチルアミン水溶液）により精製した。凍結乾燥後、所望の生成物（アジドオキサゾリン糖鎖）が74％の収率で得られた。分析用および分取用HPLCは、C18逆相カラム（YMC-Triart C18; YMC Co.、Inc. 4.6×150mm）を用いて0％MeCNで2分間、0.1％トリエチルアミン水溶液中15〜37.5％MeCNの直線勾配で45分間行った（室温、流速0.7mL /分、214nm、254nmでの検出）。
1H-NMR d 6.06 (d, J = 7.3 Hz, 1H), 5.09 (s, 1H), 4.92 (s, 1H), 4.57-4.55 (m, 2H), 4.41-4.39 (m, 2H), 4.34 (m, 1H), 4.15-4.12 (m, 4H), 2.68-2.65 (m, 2H), 2.03 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.80 (t, J = 13.2 Hz, 2H); 13C-NMR (D2O) d 181.6, 175.1, 174.8, 169.2, 168.7, 103.8, 101.5, 99.5, 81.0, 80.6, 78.1, 76.5, 76.1, 74.5, 73.6, 72.6, 72.2, 71.2, 71.0, 70.8, 70.4, 69.8, 69.7, 69.4, 69.3, 68.6, 68.5, 67.9, 67.4, 67.1, 63.2, 62.8, 61.8, 60.3, 54.7, 51.9, 50.3, 49.0, 38.9, 38.4, 23.4, 22.6, 22.0, 20.2, 13.1, 12.5.
Preparation of oxazoline
To a solution of lactol (5.0 mg, 0.00207 mmol) and triethylamine (20.2 μL, 0.145 mmol) in D 2 O (590 μL) was added 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI). 20.2 0.0930 mmol) at 4 ° C. and stirred for 2 hours. The progress of the reaction was analyzed by 1 H NMR to confirm the formation of oxazoline. Subsequently, it was purified by gel filtration column chromatography (G-25, GE Healthcare Life Sciences), eluent: 0.1% triethylamine aqueous solution). After lyophilization, the desired product (azidooxazoline sugar chain) was obtained in 74% yield. Analytical and preparative HPLC were performed using a C18 reverse phase column (YMC-Triart C18; YMC Co., Inc. 4.6 × 150 mm) for 2 minutes at 0% MeCN, 15-37.5% MeCN in 0.1% aqueous triethylamine. A linear gradient was performed for 45 minutes (room temperature, flow rate 0.7 mL / min, detection at 214 nm, 254 nm).
1 H-NMR d 6.06 (d, J = 7.3 Hz, 1H), 5.09 (s, 1H), 4.92 (s, 1H), 4.57-4.55 (m, 2H), 4.41-4.39 (m, 2H), 4.34 (m, 1H), 4.15-4.12 (m, 4H), 2.68-2.65 (m, 2H), 2.03 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H ), 1.80 (t, J = 13.2 Hz, 2H); 13 C-NMR (D 2 O) d 181.6, 175.1, 174.8, 169.2, 168.7, 103.8, 101.5, 99.5, 81.0, 80.6, 78.1, 76.5, 76.1, 74.5, 73.6, 72.6, 72.2, 71.2, 71.0, 70.8, 70.4, 69.8, 69.7, 69.4, 69.3, 68.6, 68.5, 67.9, 67.4, 67.1, 63.2, 62.8, 61.8, 60.3, 54.7, 51.9, 50.3, 49.0, 38.9, 38.4, 23.4, 22.6, 22.0, 20.2, 13.1, 12.5.
２． 糖鎖除去と糖転移酵素による抗体への転移
（１）トラスツズマブの糖鎖の除去
50mMリン酸緩衝液(pH 7.5)3mLにトラスツズマブ製剤を60 mg溶解し、そこに2 mg/mLのEndoS (E.coliより精製, His tag付き)50 mMリン酸緩衝液(pH 7.5)溶液30μLを添加し、37℃でインキュベーションを16時間行った。16時間後、トラスツズマブの糖鎖の切断をUPLC (AQUITY UPLC H-Class, Waters)で確認した。分析条件を以下に示した(分析法（１）)。表１に移動相Ａと移動相Ｂの濃度勾配の詳細を示す。
2. Sugar chain removal and transfer to antibody by glycosyltransferase (1) Removal of sugar chain of trastuzumab
Dissolve 60 mg of trastuzumab preparation in 3 mL of 50 mM phosphate buffer (pH 7.5), and add 30 mg of 2 mg / mL EndoS (purified from E. coli, with His tag) 50 mM phosphate buffer (pH 7.5) solution And incubated at 37 ° C. for 16 hours. After 16 hours, the sugar chain cleavage of trastuzumab was confirmed by UPLC (AQUITY UPLC H-Class, Waters). The analysis conditions are shown below (Analysis method (1)). Table 1 shows the details of the concentration gradient of mobile phase A and mobile phase B.
LCシステム: AQUITY UPLC H-Class
カラム: AQUITY UPLC Glycoprotein Amide 300 Å(2.1 × 150 mm)
サンプラー温度: 8℃
カラム温度: 80℃
流速: 0.4 mL/分
検出: 蛍光、Ex : 280 nm、Em : 320 nm
移動相A : 0.1% TFA・0.3%HFIP（水中）
移動相B : 0.1%TFA・0.3%HFIP（アセトニトリル中）
LC system: AQUITY UPLC H-Class
Column: AQUITY UPLC Glycoprotein Amide 300 Å (2.1 x 150 mm)
Sampler temperature: 8 ℃
Column temperature: 80 ℃
Flow rate: 0.4 mL / min Detection: Fluorescence, Ex: 280 nm, Em: 320 nm
Mobile phase A: 0.1% TFA, 0.3% HFIP (underwater)
Mobile phase B: 0.1% TFA, 0.3% HFIP (in acetonitrile)
回収したフラクションをAmicon 10K (Amicon Ultra 10K, Merck)で限外ろ過し濃縮した(3000 rpm, 4℃)。次の糖鎖付加反応の反応緩衝液(50 mMリン酸緩衝液(pH 6.5))に置換するため、Amicon10Kに残る濃縮液の体積の10倍量の50 mMリン酸緩衝液(pH 6.5)を加え、限外ろ過による濃縮を行った。緩衝液の置換では置換緩衝液の添加→濃縮の操作を計3回行った。濃縮操作後、糖鎖が除去されたトラスツズマブの溶液濃度を測定するため、280 nmにおける紫外可視吸光度測定を行った。 The collected fraction was ultrafiltered with Amicon 10K (Amicon Ultra 10K, Merck) and concentrated (3000 rpm, 4 ° C.). In order to replace the reaction buffer for the next glycosylation reaction (50 mM phosphate buffer (pH 6.5)), 50 mM phosphate buffer (pH 6.5), 10 times the volume of the concentrate remaining in Amicon10K, was used. In addition, concentration by ultrafiltration was performed. In the replacement of the buffer solution, the operation of adding the replacement buffer solution → concentration was performed three times. After concentration, in order to measure the solution concentration of trastuzumab from which sugar chains were removed, UV-visible absorbance measurement at 280 nm was performed.
（２）トラスツズマブへのアジドオキサゾリン糖鎖の付加
アジドオキサゾリン糖鎖を50 mMリン酸緩衝液(pH 8.0)に溶解し、0.1 mg/mLとした(オキサゾリン骨格は酸性条件により損壊するため、反応溶液である50 mMリン酸緩衝液(pH 6.5)ではなく、pH 8.0の緩衝液によりストックソリューション化した)。（１）で精製したトラスツズマブ0.1 mgを50 mMリン酸緩衝液(pH 6.5)で容量を40μLとし、15.0 mg/mLのEndoS(D233Q) 50 mMリン酸緩衝液(pH 6.5)を2.34μL添加した。この反応溶液にアジドオキサゾリン糖鎖のストックソリューション0.1 mg/μLを0.75μL添加し、30℃でインキュベーションを行った。15分毎にUPLC(分析法（１）)により反応解析を行い、糖鎖が除去されたトラスツズマブとオキサゾリンが一つ付加したトラスツズマブのピークが消失した段階でアジドオキサゾリン糖鎖の添加を止め、（１）と同様の手順で精製・濃縮を行った。最後の緩衝液置換では次のクリック反応で使用する50 mMリン酸緩衝液(pH 8.0)を使用し、（１）と同様の操作を行った。抗体濃度は分析法（１）と同様の操作により算出した。
(2) Addition of azidooxazoline sugar chain to trastuzumab Dissolve the azidooxazoline sugar chain in 50 mM phosphate buffer (pH 8.0) to 0.1 mg / mL (reaction solution because the oxazoline skeleton is damaged by acidic conditions) The stock solution was made with pH 8.0 buffer instead of 50 mM phosphate buffer (pH 6.5). The volume of trastuzumab 0.1 mg purified in (1) was adjusted to 40 μL with 50 mM phosphate buffer (pH 6.5), and 2.34 μL of 15.0 mg / mL EndoS (D233Q) 50 mM phosphate buffer (pH 6.5) was added. . To this reaction solution, 0.75 μL of 0.1 mg / μL stock solution of azidooxazoline sugar chain was added and incubated at 30 ° C. Analyze the reaction every 15 minutes by UPLC (analysis method (1)), and stop adding the azidooxazoline sugar chain when the peak of trastuzumab from which sugar chain was removed and trastuzumab added with one oxazoline disappeared ( Purification and concentration were performed in the same procedure as in 1). In the final buffer replacement, 50 mM phosphate buffer (pH 8.0) used in the next click reaction was used, and the same operation as in (1) was performed. The antibody concentration was calculated by the same operation as in analysis method (1).
３．薬物の設計と合成と結合
Monomethyl auristatin E (MMAE)は、ADCに用いられる強力な活性を持つ抗がん剤である。細胞内において抗体から放出可能であるように、カテプシン切断型のリンカーを用いた。アジド基と選択的に反応可能であるひずみのかかったアルキンを反応させた。
反応式を化学式１に示す。
3. Drug design, synthesis and binding
Monomethyl auristatin E (MMAE) is a potent anticancer agent used for ADC. A cathepsin-cleavable linker was used so that it could be released from the antibody intracellularly. A strained alkyne that can selectively react with an azide group was reacted.
The reaction formula is shown in
Ｂの合成
DMF（4.7mL）中のFmoc-Val-Cit-PABC-MMAE（0.34g）の溶液に、Et2NH（1.2mL）を添加した。1時間後、混合物を減圧下で濃縮し、残渣をLH20（CHCl3：MeOH 1：1）で精製し、0.24gのH-Val-Cit-PABC-MMAEを得た。CH2Cl2（10mL）中のH-Val-Cit-PABC-MMAE（0.234mmol）およびFmoc-PEG11-CO2H（0.19g、0.234mmol）の溶液に、WSCDI（67mg、0.351mmol）、HOBt（47mg、0.351mmol）およびi-Pr2NEt（0.13mL、0.702mmol）を4℃で添加した。一晩経過後、混合物をLH20（CHCl3：MeOH 1：1）を用いて精製して、0.31gの化合物Bを得た。
Synthesis of B
To a solution of Fmoc-Val-Cit-PABC-MMAE (0.34 g) in DMF (4.7 mL) was added Et 2 NH (1.2 mL). After 1 hour, the mixture was concentrated under reduced pressure and the residue was purified with LH20 (CHCl 3 : MeOH 1: 1) to give 0.24 g of H-Val-Cit-PABC-MMAE. To a solution of H-Val-Cit-PABC-MMAE (0.234 mmol) and Fmoc-PEG 11 -CO 2 H (0.19 g, 0.234 mmol) in CH 2 Cl 2 (10 mL), WSCDI (67 mg, 0.351 mmol), HOBt (47 mg, 0.351 mmol) and i-Pr 2 NEt (0.13 mL, 0.702 mmol) were added at 4 ° C. After overnight, the mixture was purified using LH20 (CHCl 3 : MeOH 1: 1) to give 0.31 g of compound B.
Ｃの合成
DMF（1.5mL）中のFmoc-PEG11-Val-Cit-PABC-MMAE（0.31g、0.160mmol）の溶液に、Et2NH（0.3mL）を添加した。1時間後、混合物を減圧下で濃縮し、残渣をLH20（CHCl3：MeOH 1：1）で精製して、0.27gのH-PEG11-Val-Cit-PABC-MMAEを得た。CH2Cl2（10mL）中のH-PEG11-Val-Cit-PABC-MMAE（0.27g）およびDBCO酸（58mg、0.234mmol）の溶液に、WSCDI（46mg、0.240mmol）、HOBt 、0.240mmol）およびi-Pr2NEt（83μL、0480mmol）を4℃で添加した。一晩経過後、混合物をLH20（CHCl3：MeOH 1：1）を用いて精製して、0.29gの化合物Cを得た。
Synthesis of C
To a solution of Fmoc-PEG 11 -Val-Cit-PABC-MMAE (0.31 g, 0.160 mmol) in DMF (1.5 mL) was added Et 2 NH (0.3 mL). After 1 hour, the mixture was concentrated under reduced pressure and the residue was purified with LH20 (CHCl 3 : MeOH 1: 1) to give 0.27 g of H-PEG 11 -Val-Cit-PABC-MMAE. To a solution of H-PEG 11 -Val-Cit-PABC-MMAE (0.27 g) and DBCO acid (58 mg, 0.234 mmol) in CH 2 Cl 2 (10 mL) was added WSCDI (46 mg, 0.240 mmol), HOBt, 0.240 mmol. ) And i-Pr 2 NEt (83 μL, 0480 mmol) were added at 4 ° C. After overnight, the mixture was purified using LH20 (CHCl 3 : MeOH 1: 1) to give 0.29 g of compound C.
薬物部位MMAE-PEG12の導入によるADCの作製
精製したアジドオキサゾリン糖鎖が付加されたトラスツズマブを最終濃度が1.78 mg/mLになるように50 mMリン酸緩衝液(pH 8.0)に溶解し、25.3 mg/mLのMMAE-PEG12 (上記構造) DMSO溶液を20当量分添加した。MMAE-PEG12の水溶性が低いため、反応溶液体積の30%になるようにDMSOを更に添加し、25℃でインキュベーションを16時間行った。16時間後、反応溶液をmilliQで3倍希釈し、希釈液を上記２の分析法（１）と同様の手順で精製・濃縮を行った。その後、上記２の分析法（１）と同様の操作により50 mMリン酸緩衝液(pH 7.5)に置換し保存した。抗体濃度は分析法（１）と同様の操作により算出した。
Preparation of ADC by introduction of drug site MMAE-PEG12 Dissolve the purified azidooxazoline sugar chain trastuzumab in 50 mM phosphate buffer (pH 8.0) to a final concentration of 1.78 mg / mL, 25.3 mg 20 equivalents of / mL MMAE-PEG12 (structure above) DMSO solution was added. Since MMAE-PEG 12 has low water solubility, DMSO was further added so that the volume of the reaction solution was 30%, and incubation was performed at 25 ° C. for 16 hours. After 16 hours, the reaction solution was diluted 3-fold with milliQ, and the diluted solution was purified and concentrated in the same procedure as in the above analysis method (1). Then, it replaced with 50 mM phosphate buffer (pH 7.5) by the same operation as analysis method (1) of said 2 and preserve | saved. The antibody concentration was calculated by the same operation as in analysis method (1).
構造の解析
構造はAspN（エンドプロテイナーゼ）やイムノグロブリン分解酵素により作製したADC を分解し、質量分析により構造と均一性を確認した。
Analysis of the structure The structure was decomposed by ADC with AspN (endoproteinase) or immunoglobulin degrading enzyme, and the structure and homogeneity were confirmed by mass spectrometry.
[実施例２] ADCの活性測定
HER2を発現している細胞（MKN-45（胃がん（腺がん）細胞）、MCF-7（乳がん細胞）、N-87（胃がん（管状腺がん）細胞）、OE-19（食道がん、胃がん細胞）およびSK-BR-3（乳がん細胞）)に対する作製したトラスツズマブとMMAEのADCとトラスツズマブの結合を比較した。MKN-45はHER2の発現が低い胃がん細胞、N-87、OE-19はHER2の発現が高い胃がん細胞、MCF-7はHER2の発現が低い乳がん細胞、SK-BR-3はHER2の発現が低い乳がん細胞である。
[Example 2] ADC activity measurement
HER2-expressing cells (MKN-45 (gastric cancer (adenocarcinoma) cells)), MCF-7 (breast cancer cells), N-87 (gastric cancer (tubular adenocarcinoma) cells), OE-19 (esophageal cancer) , Gastric cancer cells) and SK-BR-3 (breast cancer cells)) were compared for the binding of trastuzumab and MMAE ADC to trastuzumab. MKN-45 is a gastric cancer cell with low HER2 expression, N-87 and OE-19 are gastric cancer cells with high HER2 expression, MCF-7 is a breast cancer cell with low HER2 expression, and SK-BR-3 has HER2 expression Low breast cancer cells.
また、トラスツズマブとMMAEのADCの殺細胞効果をトラスツズマブ単独およびMMAE単独と比較した。 We also compared the cytocidal effects of trastuzumab and MMAE ADC with trastuzumab alone and MMAE alone.
１．方法
細胞培養
SK-BR-3、MCF-7およびNCI-N87 (N87)細胞はAmerican Type Culture Collection (ATCC)から購入した。OE-19細胞は、European Collection of Cell Culture (ECACC)から入手した。MKN-45細胞はJapanese Collection of Research Bioresources (JCRB)から入手した。OE-19およびNCI-N87細胞はRoswell Park Memorial Institute 培地 (RPMI) 1640により維持した。MKN-45およびMCF-7細胞はEagle's minimal essential medium (EMEM)により維持した。SK-BR-3細胞はMcCoy’s 5A培地により維持した。全ての培地は10％ウシ胎仔血清(FBS)と1％の抗生物質を添加し、5％CO2下37℃にて培養した。
1. Cell culture
SK-BR-3, MCF-7 and NCI-N87 (N87) cells were purchased from American Type Culture Collection (ATCC). OE-19 cells were obtained from the European Collection of Cell Culture (ECACC). MKN-45 cells were obtained from the Japanese Collection of Research Bioresources (JCRB). OE-19 and NCI-N87 cells were maintained with Roswell Park Memorial Institute medium (RPMI) 1640. MKN-45 and MCF-7 cells were maintained by Eagle's minimal essential medium (EMEM). SK-BR-3 cells were maintained in McCoy's 5A medium. All media were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and cultured at 37 ° C. under 5% CO 2 .
in vitro 細胞障害アッセイ
細胞を96穴プレート(Corning, USA)に5000個/ウェルとなるように分注し、10％FBSを含む至適培地の中で24時間プレインキュベーションした。プレインキュベーション後、各細胞に、MMAE、トラスツズマブあるいは、ADCを0.50 nM MMAE 等量となるように（トラスツズマブはADCとタンパク質量が等しくなるように）各濃度で添加し、72時間培養した。培養後、細胞の生存率をCell Counting Kit-8 (Dojindo Molecular Technologies, Japan)により評価した。相対的な細胞生存率は、各ウェルの吸光度を、各プレートの薬剤を含まない培地で処理したウェルの平均吸光度で割ることにより算出した。
In Vitro Cytotoxicity Assay Cells were dispensed into 96-well plates (Corning, USA) at 5000 cells / well and preincubated for 24 hours in an optimal medium containing 10% FBS. After pre-incubation, MMAE, trastuzumab, or ADC was added to each cell at various concentrations (so that trastuzumab had the same amount of protein as ADC) and cultured for 72 hours. After the culture, the cell viability was evaluated by Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan). Relative cell viability was calculated by dividing the absorbance of each well by the average absorbance of wells treated with medium without drug on each plate.
フローサイトメトリー
トラスツズマブまたはADCのHER2高発現細胞と低発現細胞への結合能についてはフローサイトメトリーにより以下のように評価した。細胞を、30mMクエン酸三ナトリウム二水和物および270mM塩化カリウムを含有するリン酸緩衝生理食塩水（PBS）中において、37℃で30分間処理し剥離させた。次いで、細胞を、2mMのEDTAおよび0.5％のウシ血清アルブミンを含むPBSでブロッキングした。トラスツズマブおよびADCを10μg/mLで添加し、細胞を4℃で1時間インキュベートし、続いて10μg/mLのGoat anti human-Alexa Fluor 647（Life Technologies、USA）と共に4℃で1時間インキュベートした。試料は、Guava easyCyte TM（Millipore、USA）を用いて分析した。データ解析はFlowJo 7.6.5 (FLOWJO, USA)によって実施した。
Flow cytometry The ability of trastuzumab or ADC to bind to cells expressing high and low HER2 was evaluated by flow cytometry as follows. Cells were detached by treatment for 30 minutes at 37 ° C. in phosphate buffered saline (PBS) containing 30 mM trisodium citrate dihydrate and 270 mM potassium chloride. Cells were then blocked with PBS containing 2 mM EDTA and 0.5% bovine serum albumin. Trastuzumab and ADC were added at 10 μg / mL and cells were incubated for 1 hour at 4 ° C., followed by 10 μg / mL Goat anti human-Alexa Fluor 647 (Life Technologies, USA) for 1 hour at 4 ° C. Samples were analyzed using Guava easyCyte ™ (Millipore, USA). Data analysis was performed with FlowJo 7.6.5 (FLOWJO, USA).
２．結果
結合能の確認
薬物の結合が抗体の抗原への結合に影響を及ぼさないことを確認するためにHER2低発現と高発現細胞それぞれへの抗体またはADCの結合をフローサイトメトリーと用いて評価した。
2. Results Confirmation of binding ability To confirm that drug binding does not affect antibody antigen binding, antibody or ADC binding to HER2 low and high expression cells, respectively, was assessed using flow cytometry. .
結果を図３に示す。図３中「Trastuzumab-ADC」は、トラスツズマブとMMAEのADCを、「Trastuzumab」はトラスツズマブ単独を、「2ndary only」はGoat anti human-Alexa Fluor 647のみの結果を示す。 The results are shown in FIG. In FIG. 3, “Trastuzumab-ADC” shows the results of trastuzumab and MMAE, “Trastuzumab” shows the results of trastuzumab alone, and “2ndary only” shows the results of Goat anti human-Alexa Fluor 647 only.
すべてのHER2低発現と高発現細胞それぞれにおいてトラスツズマブとADCは同様の結合能を示した。これらの結果は、糖鎖への薬物の付加が抗原への結合に影響しないことを示している。 Trastuzumab and ADC showed similar binding ability in all HER2 low and high expressing cells, respectively. These results indicate that the addition of the drug to the sugar chain does not affect the binding to the antigen.
in vitro細胞障害アッセイ
細胞のHER2発現とADCの細胞障害効果が正の相関があるかを評価するために、トラスツズマブとADCとMMAEのHER2低発現細胞と高発現細胞それぞれに対しての細胞障害効果をin vitroで測定した。
In vitro cytotoxicity assay To assess whether there is a positive correlation between cellular HER2 expression and ADC cytotoxicity, cytotoxic effects of trastuzumab, ADC and MMAE on HER2 low and high expression cells, respectively Was measured in vitro.
図４〜６に結果を示す。図４はNKN-45（HER2低発現）およびN-87（HER2高発現）に対する殺細胞効果を、図５はMKN-45（HER2低発現）およびOE-19（HER2高発現）に対する殺細胞効果を、図６はMCF-7（HER2低発現）およびSK-BR-3（HER2高発現）に対する殺細胞効果を示す。「MMAE」はMMAE単独の結果を、「Trastuzumab」はトラスツズマブ単独の結果を、「Trastuzumab-ADC」はトラスツズマブとMMAEのADCの結果を示す。縦軸はMMAE濃度のときの細胞生存率を１とした場合の相対的な細胞生存率を示し、横軸はMMAEの量（nM 当量)を示す。 The results are shown in FIGS. Figure 4 shows cell killing effect on NKN-45 (HER2 low expression) and N-87 (HER2 high expression), and Figure 5 shows cell killing effect on MKN-45 (HER2 low expression) and OE-19 (HER2 high expression). FIG. 6 shows the cell killing effect on MCF-7 (HER2 low expression) and SK-BR-3 (HER2 high expression). “MMAE” shows the results of MMAE alone, “Trastuzumab” shows the results of trastuzumab alone, and “Trastuzumab-ADC” shows the results of ADC of trastuzumab and MMAE. The vertical axis shows the relative cell viability when the cell viability at the MMAE concentration is 1, and the horizontal axis shows the amount of MMAE (nM equivalent).
HER2発現が低い乳がん細胞であるMCF-7や胃がん細胞のMKN-45は抗体トラスツズマブに対しても、ADCに対してもHER2抗原が少ないため効果を示さなかった。HER2発現が高い乳がん細胞SK-BR-3や胃がん細胞OE-19やN87細胞に対しては、トラスツズマブ単独による弱い細胞障害活性は、観察されたが、このアッセイ系では、効果が弱いため、IC50は決定できなかった（20μg/mL IgGまで）。一方で、HER2高発現細胞のADC への曝露は、細胞増殖の用量依存的な減少を引き起こし、SK-BR-3、OE-19およびN87に対するIC50値は、それぞれ、0.3, 0.9, および、1.8 nM MMAE 当量 (MMAE eq.)であった。
これらの結果は、ADCの障害活性がHER2発現に依存するものであることを示している。
MCF-7, a breast cancer cell with low HER2 expression, and MKN-45, a gastric cancer cell, showed no effect on the antibody trastuzumab and ADC because of the low HER2 antigen. Weaker cytotoxic activity with trastuzumab alone was observed against breast cancer cells SK-BR-3 and gastric cancer cells OE-19 and N87 cells with high HER2 expression. Could not be determined (up to 20 μg / mL IgG). On the other hand, exposure of HER2 high-expressing cells to ADC causes a dose-dependent decrease in cell proliferation, and IC50 values for SK-BR-3, OE-19 and N87 are 0.3, 0.9 and 1.8, respectively. nM MMAE equivalent (MMAE eq.).
These results indicate that the dysfunctional activity of ADC is dependent on HER2 expression.
乳がん細胞であるSK-BR-3と胃がん細胞であるN-87はどちらもHER2を高発現している（図３）。しかしながら、図４および図６を比較すると、SK-BR-3においては、ADCはMMAE単独と同程度の殺細胞効果を示すが、N-87においては、ADCの効果はMMAE単独の効果よりも小さい。本発明のADCは、細胞に到達し取り込まれるステップ、細胞に取り込まれた後に薬物が放出されるステップの２つのステップを経て抗がん効果を発揮するので、２つのステップの効率がいずれも高い必要がある。図３の結果は、ADCはSK-BR-3およびN-87のいずれにも高い効率で結合することを示している。また、カテプシンは細胞内に通常存在する分解酵素であり、普遍的に作用する。従って、本発明のADCは乳がんまたは胃がんの治療に特に有用である。 Both breast cancer cells SK-BR-3 and gastric cancer cells N-87 highly express HER2 (FIG. 3). However, comparing FIG. 4 and FIG. 6, in SK-BR-3, ADC shows a cell killing effect similar to that of MMAE alone, but in N-87, the effect of ADC is higher than that of MMAE alone. small. The ADC of the present invention exerts an anti-cancer effect through two steps of reaching and taking up the cell and releasing the drug after being taken up by the cell, so the efficiency of both steps is high. There is a need. The results in FIG. 3 indicate that the ADC binds to both SK-BR-3 and N-87 with high efficiency. Cathepsin is a degrading enzyme usually present in cells and acts universally. Therefore, the ADC of the present invention is particularly useful for the treatment of breast cancer or stomach cancer.
抗HER2抗体に抗がん剤を糖鎖およびカテプシン切断型リンカーを介して付加したADCは、乳がんまたは胃がん治療に利用することができる。 An ADC obtained by adding an anticancer agent to an anti-HER2 antibody via a sugar chain and a cathepsin-cleavable linker can be used for the treatment of breast cancer or stomach cancer.
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