JP2016169182A - Magnetic antibodies - Google Patents
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Abstract
Description
本発明は、医薬等に使用される抗体であって、標的抗原（例えば、タンパク質またはペプチド）の複数の異なるエピトープに対する結合能を実質的に減じられることなく、リンカーを介して、抗体に磁性を発現させるためのエッセンシャルポーションが当該抗体に結合された磁性抗体に関するものである。 The present invention relates to an antibody used in medicine or the like, wherein the antibody can be magnetized via a linker without substantially reducing the binding ability of a target antigen (eg, protein or peptide) to a plurality of different epitopes. The essential portion for expression relates to a magnetic antibody bound to the antibody.
従来から、抗体を用いた疾患の治療が行われており、その一つとしてがんの抗体治療が知られている。がんに対する抗体治療は、現在では、血液疾患や固形腫瘍の治療のための重要な治療法の一つとなっている。腫瘍細胞では、細胞表面抗原の過剰発現や変異が見られ、正常組織にはない特異的抗原が腫瘍細胞に出現することが多い。そこで、腫瘍細胞の表面抗原を標的とした抗体治療が検討されている。この検討において、抗体を用いて、表面抗原、受容体機能、そして、免疫系に変化を生じさせたり、特異的薬剤を抗体に結合させたものを、特異的抗原を発現する組織に対する標的薬として用いる等の応用が試みられている。 Conventionally, treatment of diseases using antibodies has been performed, and one of them is known as antibody treatment for cancer. Antibody therapy against cancer has now become one of the important therapies for the treatment of blood diseases and solid tumors. In tumor cells, cell surface antigens are overexpressed and mutated, and specific antigens not found in normal tissues often appear in tumor cells. Thus, antibody therapy targeting tumor cell surface antigens has been studied. In this study, antibodies that cause changes in the surface antigen, receptor function, and immune system, or that have specific drugs bound to antibodies, are targeted drugs for tissues that express specific antigens. Application such as use has been attempted.
抗体治療では、標的となる抗原をどのように選択するか、抗体と抗原との親和性は十分か、抗体の標的を何にするか(例えば、腫瘍細胞の抗原、細胞内シグナル伝達、Ｔ細胞活性化などの免疫機能等)、抗体の薬物動態特性はどうか等、抗体治療の効果を発揮させる上での様々な影響因子があるため、これら各種の因子による影響を改善しつつ効果的な治療を達成しなければならない。 In antibody therapy, how to select the target antigen, whether the antibody has sufficient affinity for the antigen, what is the target of the antibody (eg, tumor cell antigen, intracellular signaling, T cell There are various influencing factors in exerting the effects of antibody therapy, such as how immune functions such as activation) and the pharmacokinetic properties of antibodies, so effective treatment while improving the effects of these various factors Must be achieved.
この種の抗体医薬として、例えば、特表２０１１−５３０５３６号公報に記載の抗膵癌抗体がある。また、本願出願人は、金属サレン錯体に、ジスルフィド結合、エーテル結合、エステル結合、アミド結合の少なくとも一つを備える結合領域を介して、抗体を結合させ得る金属サレン錯体化合物を提案している（国際公開第２０１１／１２５３３１号公報）。 As this type of antibody drug, for example, there is an anti-pancreatic cancer antibody described in JP-T-2011-530536. Further, the applicant of the present application has proposed a metal-salen complex compound capable of binding an antibody to a metal-salen complex via a binding region having at least one of a disulfide bond, an ether bond, an ester bond, and an amide bond ( International Publication No. 2011/125331).
従来の抗体治療では、既述のとおり、抗体医薬の薬物動態特性の改良を図ることも実行されてはいるものの、抗体医薬は体内代謝による影響を受けやすいため、抗体医薬の投与量は他の種類の薬剤に比較して多いものとなっていた。即ち、現在、医薬品として承認されている抗体医薬の大部分は、その投与量が１日当たり数ｍｇ〜数１００ｍｇと非常に多く、かつ高価なものが大半である。抗体医薬品以外の生物医薬品の多くが、１日当たり数十μｇ〜１ｍｇの投薬量であるのと比較すると、抗体医薬品の一日当たりの投与量はその約１０倍から１０００倍にもなっていた。 In conventional antibody therapy, as described above, improvement of the pharmacokinetic properties of antibody drugs has also been implemented, but antibody drugs are easily affected by metabolism in the body. It was more than the kind of drugs. That is, most of antibody drugs currently approved as pharmaceuticals have a very high dose of several mg to several hundred mg per day, and most of them are expensive. Compared with the dosage of several tens of micrograms to 1 mg per day for many biopharmaceuticals other than antibody pharmaceuticals, the daily dosage of antibody pharmaceuticals was about 10 to 1000 times that amount.
本発明は、抗体の体内における薬物動態を改善して、抗体による生物活性や治療効果を向上することを目的とする。本発明の他の目的は、抗体医薬の投与量を削減することにある。本発明のさらに他の目的は、体内代謝の影響を受け難い改良された抗体を提供することにある。本発明のさらに他の目的は、体内における薬物動態を制御可能な改良抗体を提供することにある。本発明のさらに他の目的は、特定の器官、組織、部位、細胞、酵素等の対象領域に向けて誘導可能な改良抗体を提供することにある。本発明のさらに他の目的は、対象領域に磁場によって誘導可能な改良抗体を提供することにある。本発明のさらに他の目的は、少ない投与量で生物活性を発揮し得る改良抗体を提供することにある。本発明のさらに他の目的は、体内での動態が改良されても、抗体の標的抗原との親和性に実質的に影響を及ぼさない抗体を提供することにある。 An object of the present invention is to improve the pharmacokinetics of an antibody in the body and improve the biological activity and therapeutic effect of the antibody. Another object of the present invention is to reduce the dose of the antibody drug. It is still another object of the present invention to provide an improved antibody that is less susceptible to body metabolism. Still another object of the present invention is to provide an improved antibody capable of controlling pharmacokinetics in the body. Still another object of the present invention is to provide an improved antibody that can be directed to a target region such as a specific organ, tissue, site, cell, enzyme or the like. Still another object of the present invention is to provide an improved antibody that can be induced in a target region by a magnetic field. Still another object of the present invention is to provide an improved antibody capable of exerting biological activity with a small dose. It is still another object of the present invention to provide an antibody that does not substantially affect the affinity of the antibody with a target antigen even if the kinetics in the body is improved.
前記目的を解決するために、本発明者が鋭意検討したところ、抗原−抗体反応に関係する生体内機能性タンパク（抗原・抗体）の結合部位に実質的に影響がないように金属錯体からなる強磁性体が結合できたことを見出した。この知見に基づいた、第１の発明は、抗原の一つの又は複数のエピトープに対する結合能を実質的に減じられることなく、リンカーを介して、抗体に磁性を発現させるためのエッセンシャルポーションが結合された磁性抗体であることを特徴とするものである。 In order to solve the above-mentioned object, the present inventor has intensively studied, and consists of a metal complex so that the binding site of an in vivo functional protein (antigen / antibody) related to antigen-antibody reaction is not substantially affected. We found that ferromagnets could be coupled. Based on this finding, the first invention is such that an essential portion for causing an antibody to express magnetism is bound via a linker without substantially reducing the ability to bind one or more epitopes of an antigen. A magnetic antibody.
同様に、第２の発明は、抗体にリンカーを介して磁性金属サレン錯体化合物等の磁性金属錯体が結合された磁性抗体であって、個体に投与された後でも、体外からの磁場によって対象領域に誘導され、当該領域において蓄積されながら、前記磁性金属錯体によって実質的に影響されることなく、前記対象領域の目的抗原との抗原抗体反応が達成される磁性抗体である。 Similarly, the second invention is a magnetic antibody in which a magnetic metal complex such as a magnetic metal salen complex compound is bound to the antibody via a linker, and even after administration to an individual, a magnetic field from outside the body causes a target region. It is a magnetic antibody in which an antigen-antibody reaction with the target antigen in the target region is achieved without being substantially affected by the magnetic metal complex while being accumulated in the region and accumulated in the region.
本発明によれば、対象領域に磁場によって誘導可能な改良抗体を提供することができる。 According to the present invention, an improved antibody that can be induced in a target region by a magnetic field can be provided.
抗体に磁性を発現させるためのエッセンシャルポーション（有効部位）の好適な形態が磁性金属サレン錯体化合物等の磁性金属錯体である。磁性金属錯体としては、例えば、体外から磁場によって誘導可能な程度の磁性を持つものであれば特に限定されない。磁性金属錯体としては、強磁性を持った金属サレン錯体が好適である。磁性金属サレン錯体化合物としては、出願人によって、後述のとおり提案され、自身で磁性を有する金属サレン錯体、その誘導体、金属サレン錯体の多量体、金属サレン錯体と医薬分子の結合体等の関連形態を含む。金属サレン錯体の磁性は、他の化合物、例えば、鉄化合物のキャリアの援助を必要としない。金属サレン錯体は、（Ｎ，Ｎ，Ｏ，Ｏ）を４座配位子として金属に配位させたものであり、例えば、種々の金属サレン錯体誘導体の主骨格として、下記式のものを列挙することができる。
化１の化合物と化２の化合物の違いは、後者は、前者（単量体）が電子供与体である酸素等の介在体を介して結合した多量体（２量体）であるという点である。金属サレン錯体化合物が単量体構造をとるか、多量体構造をとるかは、製造工程、例えば、金属キレート構造を生成させる際に使用するハロゲン化金属のハロゲンの価数、或いは、ハロゲン化金属が水和物であるか否か等によって制御可能である。例えば、２ハロゲン化金属の水和塩（例：FeCl2・4H2O）によって、金属サレン錯体化合物は金属錯体部分の単量体構造（化１）をとり、３ハロゲン化金属（例：FeCl3）によって、金属サレン錯体は金属錯体部分の多量体構造（化２）をとる。金属サレン錯体化合物のうち、多量体構造は単量体構造に比較してより高いレベルの磁性を有していることが発明者によって確認されている。金属サレン錯体化合物が強磁性を有することは、本願発明者に初めて見出された（例えば、国際公開第２０１０／０５８２８０号公報）。金属サレン錯体化合物は、上記のものの他、側鎖の水素が官能基、医薬分子等で置換された誘導体の形態をとることは勿論可能である。また、主骨格が、上記とは異なる、特開２０１３−２８５４３号公報に記載の金属錯体化合物でもよい。本発明の金属錯体化合物とは、（Ｎ，Ｎ，Ｏ，Ｏ）を４座配位子として金属に配位させてものであり、サレンはその一例である。 The difference between the compound of chemical formula 1 and the compound of chemical formula 2 is that the latter is a multimer (dimer) in which the former (monomer) is bonded through an intermediate such as oxygen as an electron donor. is there. Whether the metal-salen complex compound takes a monomer structure or a multimeric structure depends on the manufacturing process, for example, the valence of the halogen of the metal halide used in forming the metal chelate structure, or the metal halide It can be controlled by whether or not is a hydrate. For example, a metal-salen complex compound takes a monomer structure (Chemical formula 1) of a metal complex portion by a hydrated salt of a metal dihalide (eg FeCl 2 .4H 2 O), and a metal trihalide (eg FeCl 2 ). According to 3 ), the metal-salen complex takes a multimeric structure of the metal complex part (Chemical formula 2). Among the metal salen complex compounds, the inventors have confirmed that the multimeric structure has a higher level of magnetism than the monomer structure. It has been found for the first time by the present inventors that the metal-salen complex compound has ferromagnetism (for example, International Publication No. 2010/058280). In addition to the above, the metal-salen complex compound can naturally take the form of a derivative in which the hydrogen in the side chain is substituted with a functional group, a pharmaceutical molecule, or the like. Further, the main skeleton may be a metal complex compound described in JP2013-28543A, which is different from the above. The metal complex compound of the present invention is one in which (N, N, O, O) is coordinated to a metal as a tetradentate ligand, and salen is one example.
Ｍは金属サレン錯体の中心金属であり、例えば、Fe、Cr、Mn、Co、Ni、Mo、Ru、Rh、Pd、W、Re、0s、Ir、Pt、Nd、Sm、Eu、又は、Gdである。金属サレン錯体の多くのものは、自身で強磁性を有している。強磁性を有する金属サレン錯体、及び、その誘導体を本願発明では磁性金属サレン錯体化合物と呼んでいる。金属サレン錯体の誘導体には、化１、化２の側鎖の水素が他の官能基で置換されたものが含まれる。 M is the central metal of the metal-salen complex, for example, Fe, Cr, Mn, Co, Ni, Mo, Ru, Rh, Pd, W, Re, 0s, Ir, Pt, Nd, Sm, Eu, or Gd It is. Many of the metal salen complexes themselves have ferromagnetism. In the present invention, the metal-salen complex having ferromagnetism and its derivative are called magnetic metal-salen complex compounds. Derivatives of metal salen complexes include those in which the hydrogen in the side chain of Chemical Formula 1 and Chemical Formula 2 is substituted with another functional group.
磁性金属錯体化合物に結合可能な抗体は、特に、限定されるものではないが、ガン等特定の器官、組織の領域に局在する疾患に関係する抗原を標的とするものであることが本発明の効果を発揮する上で好適である。本発明の抗体は、好適には、リンカー等の結合手段を介して磁性金属錯体化合物に結合されることによって、抗体は、磁性抗体として改良されたものになる。本発明に係る改良された抗体は、抗体が磁性金属錯体化合物に結合されても、磁性金属錯体化合物に由来する強磁性を維持する。改良された磁性抗体は、この維持された強磁性によって、人間や動物等の個体に注射、輸液等の全身投与、又は、塗布、噴霧、動注等の局所投与等投与形態の違いに拘わらず、投与の際あるいはその後、外部磁場によって、ガン等の疾患によって影響された、組織、器官等の目的領域に、特異的に、即ち、磁性を持たない化合物からは区別されて誘導される。 The antibody capable of binding to the magnetic metal complex compound is not particularly limited, but is intended to target an antigen related to a disease localized in a specific organ or tissue region such as cancer. It is suitable for exhibiting the above effect. The antibody of the present invention is preferably improved as a magnetic antibody by being bound to a magnetic metal complex compound via a binding means such as a linker. The improved antibody according to the present invention maintains the ferromagnetism derived from the magnetic metal complex compound even when the antibody is bound to the magnetic metal complex compound. Due to this maintained ferromagnetism, the improved magnetic antibody can be administered to individuals such as humans and animals regardless of the administration mode such as systemic administration such as injection, infusion, or local administration such as coating, spraying, and arterial injection. At the time of administration or thereafter, it is induced by an external magnetic field specifically in a target region such as a tissue or an organ affected by a disease such as cancer, that is, separately from a non-magnetic compound.
本願発明者は、抗原−抗体反応に関係する生体内機能性タンパク（抗原・抗体）の結合部位に実質的に影響がないように強磁性体を結合できること見出している。即ち、抗体に磁性金属錯体化合物が結合しても、抗体と抗原との特異点な結合に影響がないことを、後述のとおり、磁性錯体化合物をＩｇＧに結合させた磁性抗体が、プロテインＡと結合できることによって確認している。ＩｇＧのＦｃ領域は貪食細胞のＦｃレセプターに結合する。ＦｃレセプターはＩｇＧのＣＨ２のドメインに結合する。ＩｇＧのＣＨ２とＣＨ３間で黄色ブドウ球菌のようなグラム陽性細菌の細胞壁の成分であるプロテインＡと結合する。即ち、磁性金属錯体化合物−ＩｇＧがプロテインＡと結合できるということは、磁性ＩｇＧと抗原（Ｆｃレセプター）との結合親和性に影響がないことを証明している。このことは、金属錯体化合物が結合可能なあらゆる種類の抗体や抗原に対しても同様なこととして理解されるべきである。結局、磁性錯体化合物は抗体の抗原識別領域に影響がない部位で抗体に結合することができるということである。換言すれば、抗体に、金属錯体化合物以外の他成分が結合すると、抗体の抗原認識能力に影響を与えるおそれあるが、磁性金属錯体化合物と結合しても標的抗原との結合性能が阻害されないような抗体が、本発明の磁性抗体の要素として好適である。今までの説明によって、抗原に磁性金属錯体を結合させても、目的抗体（例えば、生体内の特定抗体）との結合親和性に影響がないことも十分に論理付けることが可能である。このような抗原として、プロテインＡの他、例えば、さらに幅広い抗体に結合する抗原である、Ｇ群レンサ球菌由来のプロテインＧもある。 The inventor of the present application has found that a ferromagnetic substance can be bound so as not to substantially affect the binding site of an in vivo functional protein (antigen / antibody) related to the antigen-antibody reaction. That is, even if the magnetic metal complex compound is bound to the antibody, there is no effect on the specific point binding between the antibody and the antigen. As described later, the magnetic antibody in which the magnetic complex compound is bound to IgG is It is confirmed by being able to combine. The Fc region of IgG binds to the phagocytic Fc receptor. The Fc receptor binds to the CH2 domain of IgG. Between IgG CH2 and CH3, it binds to protein A, which is a component of the cell wall of Gram-positive bacteria such as S. aureus. That is, the fact that magnetic metal complex compound-IgG can bind to protein A proves that the binding affinity between magnetic IgG and antigen (Fc receptor) is not affected. This should be understood as the same for all kinds of antibodies and antigens to which the metal complex compound can bind. Eventually, the magnetic complex compound can bind to the antibody at a site that does not affect the antigen recognition region of the antibody. In other words, when other components other than the metal complex compound are bound to the antibody, it may affect the antigen recognition ability of the antibody. However, even if it binds to the magnetic metal complex compound, the binding ability to the target antigen is not inhibited. Are suitable as elements of the magnetic antibodies of the present invention. According to the explanation so far, it is possible to reason enough that binding of a magnetic metal complex to an antigen does not affect the binding affinity with a target antibody (for example, a specific antibody in a living body). In addition to protein A, for example, there is protein G derived from group G streptococci, which is an antigen that binds to a wider range of antibodies.
本発明に適用可能な抗体としては、既述の要求される属性を維持する限りにおいて、特定の抗体に制限されるべきではない。後述のウサギのＩｇＧは好適な抗体の一例である。その他、例えば、腫瘍壊死因子（ＴＮＦ−α：Tumor Necrosis Factor α）に対する抗体も本発明に適用可能な抗体の一例である。 The antibody applicable to the present invention should not be limited to a specific antibody as long as the required attributes described above are maintained. Rabbit IgG, described below, is an example of a suitable antibody. In addition, for example, an antibody against tumor necrosis factor (TNF-α) is an example of an antibody applicable to the present invention.
本発明に適用可能な抗体のタイプは特定のものに限られない。例えば、モノクローナル抗体、ポリクローナル抗体、Ｆａｂ抗体、一本鎖抗体等種々のタイプの抗体を利用することができる。 The type of antibody applicable to the present invention is not limited to a specific type. For example, various types of antibodies such as a monoclonal antibody, a polyclonal antibody, a Fab antibody, and a single chain antibody can be used.
本発明のリンカーは、抗体に磁性を付与するためのエッセンシャルポーションと抗体との接合を実現し、エッセンシャルポーションの強磁性、抗体の抗原との結合、抗体の抗原認識機能に不利な影響を及ぼさない限り、特に限定されず、公知のリンカーを使用することができる。 The linker of the present invention realizes the bonding of an essential portion for imparting magnetism to an antibody and the antibody, and does not adversely affect the ferromagnetism of the essential portion, the binding of the antibody to the antigen, and the antigen recognition function of the antibody. As long as it is not particularly limited, a known linker can be used.
リンカーとしての架橋剤は、ジスルフィド結合、エーテル結合、エステル結合、又は、アミド結合等を有する結合領域を形成して、エッセンシャルポーションと抗体の夫々に直接或いは側鎖、又は官能基を介して結合することによって、エッセンシャルポーションと抗体とを連結する。後述のテレフタル酸の他、Ｎ−ヒドロキシコハク酸イミドは、リンカーの一連である。 The cross-linking agent as a linker forms a binding region having a disulfide bond, an ether bond, an ester bond, an amide bond or the like, and binds to the essential portion and the antibody directly or through a side chain or a functional group. As a result, the essential portion and the antibody are linked. In addition to terephthalic acid described below, N-hydroxysuccinimide is a series of linkers.
下記化３は磁性抗体の第１の例（磁性金属サレン錯体と抗体との結合体）であり、下記化４は磁性抗体の第２の例（磁性金属サレン錯体の２量体と抗体との結合体）である。
化３に示す構造においては、ａ５、ａ６の２箇所で抗体が磁性金属サレン錯体に結合し得る。化４に示す構造においては、ａ1、ａ2、ａ3、ａ4の４箇所で抗体が磁性金属サレン錯体に結合し得る。したがって、磁性金属サレン錯体（２量体）の方が磁性金属サレン錯体（単量体）よりも、抗体を多く結合できる分、抗原に対する効果が向上される。ａ1、ａ2、ａ3、ａ4、ａ５、ａ６の夫々について、これらが抗体でない場合には、例えば、水素、又は、水素を置換可能な公知の原子又は官能基である。
なお、化３、化４の構造において、
In the structures of Chemical Formula 3 and Chemical Formula 4,
本発明の磁性抗体は、例えば、特定疾患治療用の抗体医薬として利用される。磁性抗体を含む抗体医薬は、公知のとおり、注射、或いは、輸液用の製剤として提供される。 The magnetic antibody of the present invention is used, for example, as an antibody drug for treating a specific disease. As is known, an antibody drug containing a magnetic antibody is provided as a preparation for injection or infusion.
磁性抗体は、外部磁場によって、体内における目的の領域に誘導されることが可能になるために、磁性抗体の体内動態が、抗体単独の場合（外部磁場による誘導ができない）に比べて、より高い生物活性を示すことができるようになる。さらに、磁性抗体が体内の目的領域に誘導できることによって、非磁性抗体よりも投与量が少なくて済むことになる。磁性抗体では、外部磁場によって、目的領域に到達するまでの所要時間が短縮され、その結果、体内代謝の影響を少なくできることにより、磁性抗体の生物活性の半減期を延長することができる。 Since magnetic antibodies can be induced to a target region in the body by an external magnetic field, the pharmacokinetics of magnetic antibodies is higher than that of the antibody alone (cannot be induced by an external magnetic field). Be able to show biological activity. Furthermore, since the magnetic antibody can be directed to the target area in the body, the dose can be reduced as compared with the non-magnetic antibody. In magnetic antibodies, the external magnetic field shortens the time required to reach the target region, and as a result, the influence of metabolism in the body can be reduced, thereby extending the half-life of the biological activity of the magnetic antibody.
化合物１：１７ｇ、０．１０ｍｏｌ、無水酢酸（acetic anhydride）：２００ｍｌ、H２SO４：少々、を室温で１時間攪拌させた。得られた溶液を２ｌの氷水中に入れて０．５時間混ぜ、加水分解を行った。次に、得られた溶液をフィルターにかけ、大気中で乾燥させたところ白い粉末状のものが得られた。酢酸エチルを含む溶液を使ってその粉末を再結晶化させたところ、２４ｇ（収率７６％）の白い結晶（化合物２）を得ることができた。化合物２：２４ｇ、７７ｍｍｏｌと、メタノール；５００ｍｌに１０％のパラジウムを担持したカーボン：２．４ｇの混合物を一晩、１．５気圧の水素還元雰囲気で還元した。終了後、フィルターでろ過したところ茶色油状の化合物３：２１ｇを合成できた。 Compound 1: 17 g, 0.10 mol, acetic anhydride: 200 ml, H 2 SO 4 : a little was allowed to stir at room temperature for 1 hour. The resulting solution was placed in 2 l of ice water and mixed for 0.5 hour to effect hydrolysis. Next, when the obtained solution was filtered and dried in the air, a white powder was obtained. When the powder was recrystallized using a solution containing ethyl acetate, 24 g (yield 76%) of white crystals (compound 2) could be obtained. Compound 2: A mixture of 24 g, 77 mmol and methanol; carbon loaded with 10% palladium in 500 ml was reduced overnight in a hydrogen reducing atmosphere at 1.5 atm. After completion, the mixture was filtered with a filter to synthesize brown oily compound 3:21 g.
無水エタノール；４００ｍｌの中に化合物６：１０ｇ、４２ｍｍｏｌを入れ、加熱しながら還流させ、無水エタノール：２０ｍｌにエチレンジアミン：１．３ｇ、２１ｍｍｏｌを０．５時間攪拌しながら数滴加えた。そして、その混合溶液を氷の容器に入れて冷却し１５分間かき混ぜた。その後、２００ｍｌのエタノールで洗浄し、フィルターをかけ、真空で乾燥させたところ、３．５ｇ（収率９３．９％）の化合物７（黄色）を合成できた。 Absolute ethanol; Compound 6: 10 g, 42 mmol was placed in 400 ml, refluxed with heating, and ethylenediamine: 1.3 g, 21 mmol was added to absolute ethanol: 20 ml with stirring for a few hours with a few drops. Then, the mixed solution was cooled in an ice container and stirred for 15 minutes. Then, it was washed with 200 ml of ethanol, filtered, and dried in vacuum. As a result, 3.5 g (yield 93.9%) of compound 7 (yellow) could be synthesized.
化合物７の構造を1ＨＮＭＲによって配位子の骨格の確認、質量分析によって化合物７の分子量の確認を行った。化合物７の1ＨＮＭＲの分析結果（使用機種名Bruker 300MHz/54mm UltraShield）を図２に示す。測定方法は、次の通りである。 The structure of Compound 7 was confirmed by 1 HNMR for the skeleton of the ligand and by mass spectrometry for the molecular weight of Compound 7. FIG. 2 shows the 1 HNMR analysis result of Compound 7 (model name used: Bruker 300 MHz / 54 mm UltraShield). The measuring method is as follows.
サンプル作成について、ＮＭＲ試料管（直径５ｍｍ）にサンプル（約２ｍｇ）を入れ、ピペットを用いて重溶媒d6-DMSO ０．６ ｍＬを加え、振り混ぜてサンプルを溶かす。次に、ＮＭＲ 試料管を専用のスピニングフォルダーに差し込む。差し込む長さは，専用のゲージで調整する。スピニングフォルダーの上部を持って、超伝導磁石(ＳＣＭ)上部に差し込む。最初にスピニングフォルダーがＳＣＭに浮いた状態で、マニュアル通りで操作し、スピニングフォルダーをＳＣＭ内部に入れることで1ＨＮＭＲスペクトルを得た。 For sample preparation, put a sample (about 2 mg) into an NMR sample tube (diameter 5 mm), add 0.6 mL of heavy solvent d6-DMSO using a pipette, and shake to dissolve the sample. Next, the NMR sample tube is inserted into a dedicated spinning folder. Adjust the length of insertion with a special gauge. Hold the upper part of the spinning folder and insert it into the upper part of the superconducting magnet (SCM). First, the spinning folder was floated on the SCM, and the operation was performed according to the manual, and the spinning folder was placed inside the SCM to obtain a 1 HNMR spectrum.
化合物７の質量分析結果（使用機種名Agilent G1956B）を図３に示す。測定方法は次の通りである。
移動相： 酢酸アンモニウム溶液０．１％：アセトニトリル（３０：７０）
カラム： C18 (３０ ｍｍ× ２．１ｍｍ， ３．５ μｍ)；流速 ０．４ ｍＬ／ｍｉｎ
質量分析の測定条件：
印加電圧 ７０ Ｖ
質量範囲 ７０．００ ― ２０００．００
乾燥気流量 ７ Ｌ／ｍｉｎ
噴霧室圧力 ２５ ｐｓｉｇ
乾燥気体温度 ３００ ℃
毛細管電圧 ３５００ Ｖ
FIG. 3 shows the mass analysis result of Compound 7 (model name used: Agilent G1956B). The measuring method is as follows.
Mobile phase: Ammonium acetate solution 0.1%: Acetonitrile (30:70)
Column: C18 (30 mm × 2.1 mm, 3.5 μm); flow rate 0.4 mL / min
Measurement conditions for mass spectrometry:
Applied voltage 70 V
Mass range 70.00-2000.00
Dry air flow rate 7 L / min
Spray chamber pressure 25 psig
Drying gas temperature 300 ° C
Capillary voltage 3500 V
サンプル処理は、適量のサンプルを少量のアセトニトリルで溶かし、約１００μｇ／ｍＬまで希釈して測定用サンプル溶液とする。測定用サンプル溶液１０μＬをHPLC-MASSに注入し、ＭＡＳＳスペクトルを記録した。その結果、質量分析の値で理論値ｍ／Ｚ ５９４．３５に対して、実測値 ｍ／Ｚ ５９５．３という測定値が得られたため、化合物７が既述の構造式で示されることを確認できた。 In sample processing, an appropriate amount of sample is dissolved in a small amount of acetonitrile, and diluted to about 100 μg / mL to obtain a sample solution for measurement. 10 μL of the sample solution for measurement was injected into HPLC-MASS, and a MASS spectrum was recorded. As a result, a measured value of measured value m / Z 595.3 was obtained with respect to the theoretical value m / Z 594.35 in terms of mass spectrometry, and it was confirmed that compound 7 was represented by the structural formula described above. did it.
通常のメタノール（昭和化学製メタノール、純度９９．５％以上）；５０ｍｌ中に化合物７：８．２ｇ、１６ｍｍｏｌ、トリエチルアミン：２２ｍｌ、１６０ｍｍｏｌを入れ、１０ｍｌのメタノールの中に、FeCl３）：２．７ｇ、１６ｍｍｏｌ（なお、鉄サレン以外の金属サレン、例えば、Mnサレンの場合は、MnCl３、Crサレンの場合は、CrCl３を使用する。）を加えた溶液を窒素雰囲気下で混合した。次いで、室温窒素雰囲気で１時間混合したところ茶色の化合物が得られた。その後、真空中或いはマグネシウムを使う等して十分に水を乾燥或いはマグネシウムに吸着除去させた。得られた化合物は、４００ｍｌのジクロロメタンで希釈し、塩性溶液で２回洗浄し、Na２S0４で乾燥させ、真空中で乾燥させたところ２量体の金属サレン錯体化合物８(化２)が得られた。 Normal methanol (Showa Chemical methanol, purity 99.5% or higher); Compound 7: 8.2 g, 16 mmol, triethylamine: 22 ml, 160 mmol in 50 ml; FeCl 3 ) in 10 ml of methanol: 2. 7 g, 16 mmol (Note that other than iron salen metal-salen, for example, in the case of Mn-salen, if the MnCl 3, Cr salen uses CrCl 3.) the solution was added and mixed under a nitrogen atmosphere. Subsequently, when mixed for 1 hour in a nitrogen atmosphere at room temperature, a brown compound was obtained. Thereafter, water was sufficiently dried or adsorbed and removed by magnesium in vacuum or using magnesium. The obtained compound was diluted with 400 ml of dichloromethane, washed twice with a salt solution, dried with Na 2 SO 4 , and dried in vacuum. As a result, dimeric metal-salen complex compound 8 (Chemical Formula 2) was gotten.
次いで、化合物９：２０ｇ、０．１ｍｍｏｌとシグマアルドリッチ社製ウサギ血清由来のＩｇＧ抗体（製品名Ｉ５００６）：１５０ｍｇとを精製水１ｍＬで３０分攪拌した。その後、リン酸緩衝液１５０ｍｌｍＬ、ｐＨ＝７．０１で撹拌した。そして、攪拌から６時間後に化合物１０を得た。 Next, Compound 9: 20 g, 0.1 mmol and Sigma Aldrich rabbit serum-derived IgG antibody (product name I5006): 150 mg were stirred with 1 mL of purified water for 30 minutes. Thereafter, the mixture was stirred at 150 ml of phosphate buffer, pH = 7.01. And compound 10 was obtained 6 hours after stirring.
次に、化合物１０が強磁性であることは、精製水を入れたシャーレに化合物１０の粒子を投入し、シャーレの下から化合物１０の粒子が磁石で誘導できたことによって確認した。また、Quantum Design MPMS７を用いて、化合物１０について磁場―磁化曲線を測定したところ、−２６８℃から３７℃まで磁場印加とともに磁化が上昇し、外部からの磁場誘導でドラッグデリバリーが可能であることが示唆される結果が得られた。 Next, it was confirmed that the compound 10 was ferromagnetic by introducing particles of the compound 10 into a petri dish containing purified water, and that the particles of the compound 10 could be induced by a magnet from under the petri dish. In addition, when the magnetic field-magnetization curve of Compound 10 was measured using Quantum Design MPMS7, the magnetization increased with application of the magnetic field from −268 ° C. to 37 ° C., and drug delivery was possible by external magnetic field induction. Suggested results were obtained.
次に、化合物１０が抗体の性質を失っていないことの確認を、化合物１０とプロテインＡと反応性を試験することによって行った。
プロテインＡ：Protein A ELISA キット（コスモ・バイオ株式会社製）
プロテインＡによる生成に用いた試料：
カラム IgG Purification Kit-A (コスモバイオ＃AP01)
抗体 Normal Rabbit IgG（シグマ・アルドリッチ社製）
磁性化合物 化合物８
磁性抗体 化合物１０
（磁性Rabbit IgG抗体（０．８ｍｇ／２００μＬ（生理食塩水）の５０μＬをプロテインＡの精製に使用）
Next, confirmation that Compound 10 did not lose antibody properties was performed by testing the reactivity of Compound 10 and Protein A.
Protein A: Protein A ELISA kit (manufactured by Cosmo Bio)
Samples used for protein A production:
Column IgG Purification Kit-A (Cosmo Bio # AP01)
Antibody Normal Rabbit IgG (Sigma-Aldrich)
Magnetic compound Compound 8
Magnetic antibody Compound 10
(Magnetic Rabbit IgG antibody (50 μL of 0.8 mg / 200 μL (saline) is used for protein A purification)
プロテインＡによる精製方法は以下のとおりである。なお、詳細については、コスモバイオ#AP01記載の実験手順書（http://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/DMT_/AP01.20060929.pdf）を参照されたい。
（１）化合物１０の精製に使用するカラムが詰まると精製できなくなってしまうので、軽く遠心をして、溶解している上清50μLを精製した。
（２）（１）のサンプル５０μＬとwash buffer ５０μＬを混合した。
（３）プロテインＡカートリッジに（２）を入れた(２ｍｉｎ)。
（４）（３）のカートリッジを８０００Ｇで遠心分離した（３０秒）。
（５）（４）にwash buffer ２００μＬ
を加えた。
（６）再度、８０００Ｇで遠心分離した（３０秒）。
（７）新しいチューブにcatching buffer ６０μＬを入れ、これに（６）のカートリッジの内容物を移した。
（８）（７）のチューブにElution buffer ７０μＬを加え、磁性抗体をcatching buffer に溶出させた。
The purification method using protein A is as follows. For details, refer to the experimental procedure document (http://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/DMT_/AP01.20060929.pdf) described in Cosmobio # AP01.
(1) Since the column used for the purification of compound 10 becomes clogged, it cannot be purified, and thus light centrifugation was performed to purify 50 μL of the dissolved supernatant.
(2) 50 μL of the sample of (1) and 50 μL of wash buffer were mixed.
(3) (2) was placed in a protein A cartridge (2 min).
(4) The cartridge of (3) was centrifuged at 8000 G (30 seconds).
(5) Wash buffer 200 μL in (4)
Was added.
(6) Centrifugation was again performed at 8000 G (30 seconds).
(7) 60 μL of the catching buffer was placed in a new tube, and the contents of the cartridge in (6) were transferred to this.
(8) 70 μL of Elution buffer was added to the tube of (7), and the magnetic antibody was eluted in the catching buffer.
化合物１０とプロテインＡとの結合を、次のとおり電気泳動法によるバンドによって確認した。図１の左側のバンドは、カラムに充填したたんぱく質であるプロテインＡの電気泳動法によるバンドである。中央の領域はカラム洗浄液のみの電気泳動法による結果を示す領域である。図１から明らかなように、中央の領域には、分子量の大きなタンパク質は含まれてなく、バンドのようなものを確認できなかった。右側の領域は、プロテインＡを充填したカラムに磁性抗体（化合物１０）をカラム洗浄液とともに溶出した後のバンドである。プロテインＡと磁性抗体（化合物１０）とが抗体―抗原反応によって結合しため、バンドが１本になったことが確認された。もし、磁性抗体（化合物１０）の抗体の抗原認識部位が金属サレン化合物によって影響を受け、抗体がプロテインＡと結合できない場合には、バンドが２本観察されるはずである。このことより、磁性抗体（化合物１０）は抗体―抗原反応をプロテインＡと間で起こすことから、磁性抗体の抗体部分は、抗体としての活性を失っていないことが確認できた。 The binding between compound 10 and protein A was confirmed by a band obtained by electrophoresis as follows. The left band in FIG. 1 is a band obtained by electrophoresis of protein A, which is a protein packed in a column. The central area is an area showing the result of electrophoresis using only the column cleaning solution. As is clear from FIG. 1, a protein having a large molecular weight was not included in the central region, and a band-like object could not be confirmed. The region on the right side is a band after elution of the magnetic antibody (compound 10) together with the column washing solution on a column filled with protein A. It was confirmed that protein A and the magnetic antibody (compound 10) were bound by an antibody-antigen reaction, so that one band was formed. If the antigen recognition site of the antibody of the magnetic antibody (compound 10) is affected by the metal salen compound and the antibody cannot bind to protein A, two bands should be observed. From this, since the magnetic antibody (compound 10) caused an antibody-antigen reaction with protein A, it was confirmed that the antibody portion of the magnetic antibody did not lose its activity as an antibody.
既述の磁性抗体を、例えば、抗ガン剤などの治療薬として用いる場合には、磁性抗体を投与（全身又は局所）後、体外からの磁場によって磁性抗体を目的の組織、器官に誘導する。例えば、磁性抗体の適量を生理食塩水に溶解したものが静脈注射されればよい。また、磁性抗体を目的抗原に対する検査薬として使用する場合には、磁性抗体を全身投与後、ＭＲＩによって磁性抗体の造影像を確認すればよい。
When the magnetic antibody described above is used as a therapeutic agent such as an anticancer agent, for example, the magnetic antibody is induced (systemic or local), and then the magnetic antibody is induced to the target tissue or organ by a magnetic field from outside the body. For example, a solution obtained by dissolving an appropriate amount of a magnetic antibody in physiological saline may be injected intravenously. In addition, when a magnetic antibody is used as a test agent for a target antigen, a magnetic antibody contrast image may be confirmed by MRI after systemic administration of the magnetic antibody.
Claims (7)
個体に投与された後でも、体外からの磁場によって目的領域に誘導され、
当該領域において蓄積されながら、前記磁性金属錯体化合物によって実質的に影響されることなく、前記目的領域の標的抗原との抗原抗体反応が実現される磁性抗体。 A magnetic antibody in which a magnetic metal complex compound is bound to an antibody via a linker,
Even after being administered to an individual, it is guided to the target area by a magnetic field from outside the body,
A magnetic antibody that realizes an antigen-antibody reaction with a target antigen in the target region without being substantially affected by the magnetic metal complex compound while being accumulated in the region.
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| JP2009287949A (en) * | 2008-05-27 | 2009-12-10 | Yoshihiro Ishikawa | Antibody or antigen quantifying method |
| WO2011125331A1 (en) * | 2010-04-06 | 2011-10-13 | 株式会社Ihi | Metal salen complex derivative and process for production thereof |
| JP2012176905A (en) * | 2011-02-25 | 2012-09-13 | Ihi Corp | Metal-salen complex compound |
| JP2014210742A (en) * | 2013-04-19 | 2014-11-13 | 株式会社Ihi | Persistent magnetic anticancer agent |
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| WO2011125331A1 (en) * | 2010-04-06 | 2011-10-13 | 株式会社Ihi | Metal salen complex derivative and process for production thereof |
| JP2012176905A (en) * | 2011-02-25 | 2012-09-13 | Ihi Corp | Metal-salen complex compound |
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