JP6771509B2 - Immunomagnetic composition, its preparation method, its usage method and cancer treatment kit - Google Patents
Immunomagnetic composition, its preparation method, its usage method and cancer treatment kit Download PDFInfo
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- JP6771509B2 JP6771509B2 JP2018088193A JP2018088193A JP6771509B2 JP 6771509 B2 JP6771509 B2 JP 6771509B2 JP 2018088193 A JP2018088193 A JP 2018088193A JP 2018088193 A JP2018088193 A JP 2018088193A JP 6771509 B2 JP6771509 B2 JP 6771509B2
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Description
本発明は、組成物及びその調製方法に関し、特に、特殊の物理形態を特徴とする医薬組成物及びその調製方法に関する。 The present invention relates to a composition and a method for preparing the same, and more particularly to a pharmaceutical composition characterized by a special physical form and a method for preparing the same.
癌は、悪性腫瘍とも言われ、細胞が異常に増殖する状態であり、これらの増殖性細胞が体の他の部分に侵入する可能性があり、細胞分裂や増殖に対する制御機能の異常による疾患である。全世界に渡って、癌に罹患する人がますます増加しており、癌が台湾人の死亡原由の上位１０位にランクインし、２７年連続で死亡原因のトップ１０になっている。 Cancer, also known as malignant tumor, is a condition in which cells proliferate abnormally, and these proliferative cells can invade other parts of the body, resulting in abnormal control over cell division and proliferation. is there. The number of people suffering from cancer is increasing all over the world, and cancer is ranked in the top 10 causes of death among Taiwanese and has been the top 10 causes of death for 27 consecutive years.
癌に対する常規の治療手段としては、手術治療、放射線療法及び化学療法などが含まれる。免疫療法は、上記の治療法以外の癌治療法であり、患者自身の免疫系を活性化させ、腫瘍細胞又は腫瘍抗原物質によって体の特異的細胞免疫及び体液性免疫応答を誘導して、体の抗癌能力を高め、腫瘍の生長、拡散及び再発を阻止することで、腫瘍の摘出又は制御の目的を達成する。免疫チェックポイント（ｉｍｍｕｎｅ ｃｈｅｃｋｐｏｉｎｔ）は、幾つかの免疫療法の中で最も重視されるものであり、２０１５年以来、免疫チェックポイント阻害剤（ｉｍｍｕｎｅ ｃｈｅｃｋｐｏｉｎｔ ｉｎｈｉｂｉｔｏｒ）による５０以上の複合療法が臨床で試験されるようになる。しかしながら、免疫チェックポイント阻害剤は、ヒト免疫系のフィードバック機能をオフにし、細胞傷害性Ｔ細胞（ＣＤ８+ Ｔ細胞）により、癌細胞が攻撃されるほか、皮膚及び胃潰瘍の潰瘍形成等の自己免疫反応も引き起こすことがある。 Regular treatments for cancer include surgical treatment, radiation therapy and chemotherapy. Immunotherapy is a cancer treatment method other than the above-mentioned treatment methods, in which the body activates its own immune system and induces the body's specific cell-mediated immunity and humoral immune response by tumor cells or tumor antigenic substances. Achieve the purpose of tumor resection or control by enhancing the anticancer ability of the tumor and preventing tumor growth, spread and recurrence. Immune checkpoints are the most important of several immunotherapies, and since 2015, more than 50 combined therapies with immune checkpoint inhibitors have been clinically tested. Become so. However, immune checkpoint inhibitors turn off the feedback function of the human immune system, and cytotoxic T cells (CD8 + T cells) attack cancer cells as well as autoimmunity such as ulceration of skin and gastric ulcers. Reactions can also be triggered.
体内の腫瘍に対する特異的な免疫細胞の添加も、癌の治療において非常に有望なリンクであると考えられている。従来の技術の大部分は、患者から腫瘍における免疫細胞を取ってインビトロ培養して、マイクロディメンションのミクロ構造（例えば、マイクロビーズ（ｍｉｃｒｏｂｅａｄ））によって抗原提示細胞（ａｎｔｉｇｅｎ ｐｒｅｓｅｎｔｉｎｇ ｃｅｌｌ；ＡＰＣｓ）を模倣して、Ｔ細胞の増殖や訓練を行い、最後、患者の体に戻って癌細胞を殺す。しかしながら、そのような方法では、時間がかかり、材料をかなり消費し、且つ患者の体内の癌細胞が変異しやすく、再導入された免疫細胞が元々の効果を失う。一方、増殖用のマイクロビーズが大きすぎるため、人の血液循環によって目的領域に入ることができず、インビトロ培養しかで使用できない。また、このような担体は、表面移植抗体又は被覆された活性成分に加えて、通常、担体を形成する材料が賦形剤であり、治療にあまり役立たず、適用時の用量を制限するため、このような素材の固有欠陥となる。 The addition of specific immune cells to tumors in the body is also considered to be a very promising link in the treatment of cancer. Most of the prior art involves taking immune cells in a tumor from a patient and culturing them in vitro to mimic antigen presenting cells (APCs) with microdimensional microstructures (eg, microbeads). It proliferates and trains T cells, and finally returns to the patient's body to kill the cancer cells. However, such a method is time consuming, consumes considerable material, and the cancer cells in the patient's body are susceptible to mutation, and the reintroduced immune cells lose their original effect. On the other hand, the growth microbeads are too large to enter the target area due to human blood circulation and can only be used in in vitro culture. Also, in such carriers, in addition to surface-transplanted antibodies or coated active ingredients, the material forming the carrier is usually an excipient, which is not very therapeutic and limits the dose at the time of application. It becomes an inherent defect of such a material.
これに鑑みて、本発明の一態様は、コア層と、フコイダン（ｆｕｃｏｉｄａｎ）、酸化デキストラン（ｄｅｘｔｒａｎ）及び超常磁性酸化鉄ナノ粒子が疎水性相互作用（ｈｙｄｒｏｐｈｏｂｉｃ ｉｎｔｅｒａｃｔｉｏｎ）によって結合される複合物からなり、コア層を覆うシェル層と、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤である少なくとも１つの抗体を含む外層と、を備え、前記抗体は、シェル層の外にグラフトされ外層を構成する免疫磁気組成物を提供する。 In view of this, one aspect of the invention consists of a core layer and a complex in which fucoidan, dextran oxide and superparamagnetic iron oxide nanoparticles are bound by a hydrophobic interaction. , A shell layer covering the core layer and an outer layer containing at least one antibody that is an immune checkpoint inhibitor and / or a killer T cell proliferating agent, wherein the antibody is grafted out of the shell layer to form an outer layer. To provide an immunomagnetic composition.
前記の免疫磁気組成物によれば、前記免疫磁気組成物は、粒径が８０ｎｍ〜３５０ｎｍにある球体であってもよい。 According to the immunomagnetic composition, the immunomagnetic composition may be a sphere having a particle size of 80 nm to 350 nm.
前記の免疫磁気組成物によれば、前記フコイダンは、ウンデリア・ピンナチフィダ（Ｕｎｄａｒｉａ ｐｉｎｎａｔｉｆｉｄａ）、マクロシスティス・ピリフェラ（Ｍａｃｒｏｃｙｓｔｉｓ ｐｙｒｉｆｅｒａ）又はフーカス・ベシクロサス（Ｆｕｃｕｓ ｖｅｓｉｃｕｌｏｓｕｓ）から抽出されてもよい。 According to immunomagnetic compositions of the said fucoidan, Underia-Pin'nachifida (Undaria pinnatifida), may be extracted from the macro cis infantis-Pirifera (Macrocystis pyrifera) or Fukasu-vesiculosus (Fucus vesiculosus).
前記の免疫磁気組成物によれば、前記酸化デキストランは、アルデヒド基を有してもよい。前記酸化デキストランは、分子量が５ｋＤａ〜２７０ｋＤａにあるデキストランによって調製されてもよい。 According to the immunomagnetic composition, the oxidized dextran may have an aldehyde group. The oxidized dextran may be prepared by dextran having a molecular weight of 5 kDa to 270 kDa.
前記の免疫磁気組成物によれば、前記免疫チェックポイント阻害剤は、ＰＤ−Ｌ１抗体、ＰＤ−１抗体、ＣＴＬＡ−４抗体及びＴＩＭ−３抗体からなる群から選ばれてもよい。前記キラーＴ細胞増殖剤は、ＣＤ３抗体、ＣＤ２８抗体及び４−１ＢＢ抗体からなる群から選ばれてもよい。 According to the immunomagnetic composition, the immune checkpoint inhibitor may be selected from the group consisting of PD-L1 antibody, PD-1 antibody, CTLA-4 antibody and TIM-3 antibody. The killer T cell proliferating agent may be selected from the group consisting of CD3 antibody, CD28 antibody and 4-1BB antibody.
前記の免疫磁気組成物によれば、前記コア層は、活性物質を別に含んでもよい。 According to the immunomagnetic composition, the core layer may separately contain an active substance.
これによって、本発明の免疫磁気組成物は、抗癌活性を有するフコイダン、酸化デキストラン及び超常磁性酸化鉄ナノ粒子を担体として構成され、更に外層が抗体を有し且つコア層で活性物質を覆うナノスケール構造を形成してもよく、そのサイズ及び表面電荷は適切であり、体循環時間を増加させ、腫瘍に浸透し、腫瘍に対するフコイダンの役割を強める。外層の抗体は、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤であってもよく、これによって本発明の免疫磁気組成物が自体材料の抗癌作用に加えて、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤の両方であってもよく、腫瘍微小環境が大幅に改善され、且つ本発明の免疫磁気組成物は、単独の同じ抗体による免疫療法の抗癌効果を大幅に改善し、より少ない抗体用量でより良好な腫瘍抑制能力を達成することができる。また、製造された免疫磁気組成物は、凍結乾燥して粉末状の結晶を形成し、無菌条件下で長時間保存することができ、必要に応じて溶媒で再溶解して用いることができ、簡便で安定した特性を示す。 As a result, the immunomagnetic composition of the present invention is composed of fucoidan having anticancer activity, dextran oxide and superparamagnetic iron oxide nanoparticles as carriers, and the outer layer has an antibody and the core layer covers the active substance. Scale structures may be formed, of which size and surface charge are appropriate, to increase body circulation time, penetrate tumors and enhance the role of fucoidan in tumors. The antibody in the outer layer may be an immune checkpoint inhibitor and / or a killer T cell proliferation agent, whereby the immunomagnetic composition of the present invention can be an immune checkpoint inhibitor and / or an immune checkpoint inhibitor in addition to the anti-cancer effect of its own material. / Or both killer T cell proliferators, the tumor microenvironment is significantly improved, and the immunomagnetic compositions of the present invention significantly improve the anti-cancer effect of immunotherapy with the same antibody alone. Better tumor suppressor capacity can be achieved with lower antibody doses. Further, the produced immunomagnetic composition can be freeze-dried to form powdery crystals, can be stored for a long time under aseptic conditions, and can be redissolved in a solvent and used if necessary. Shows simple and stable characteristics.
本発明の別の態様は、フコイダン及び酸化デキストランを含む水相溶液を提供する工程と、有機溶媒及び超常磁性酸化鉄ナノ粒子を含む油相溶液を提供する工程と、水相溶液と油相溶液を混合し乳濁液を形成するエマルジョン反応を行う工程と、乳濁液における有機溶媒を除去して、磁気フコイダン担体を形成する工程と、磁気フコイダン担体と免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤である少なくとも１つの抗体を混合して、免疫磁気組成物を形成するグラフトを行う工程と、を備える免疫磁気組成物の調製方法を提供することにある。 Another aspect of the present invention is a step of providing an aqueous phase solution containing fucoidan and dextran oxide, a step of providing an oil phase solution containing an organic solvent and hypernormal magnetic iron oxide nanoparticles, and an aqueous phase solution and an oil phase solution. A step of performing an emulsion reaction to form an emulsion by mixing the two, a step of removing an organic solvent in the emulsion to form a magnetic fucoidan carrier, a magnetic fucoidan carrier and an immune checkpoint inhibitor and / or a killer T. It is an object of the present invention to provide a method for preparing an immunomagnetic composition, comprising a step of mixing at least one antibody which is a cell proliferation agent and performing a graft to form an immunomagnetic composition.
前記の免疫磁気組成物の調製方法によれば、前記免疫チェックポイント阻害剤は、ＰＤ−Ｌ１抗体、ＰＤ−１抗体、ＣＴＬＡ−４抗体及びＴＩＭ−３抗体からなる群から選ばれてもよく、前記キラーＴ細胞増殖剤は、ＣＤ３抗体、ＣＤ２８抗体及び４−１ＢＢ抗体からなる群から選ばれてもよい。 According to the method for preparing an immunomagnetic composition, the immune checkpoint inhibitor may be selected from the group consisting of PD-L1 antibody, PD-1 antibody, CTLA-4 antibody and TIM-3 antibody. The killer T cell proliferating agent may be selected from the group consisting of CD3 antibody, CD28 antibody and 4-1BB antibody.
前記の免疫磁気組成物の調製方法によれば、フコイダンと酸化デキストランとの重量比は、１：０．１〜１：４であってもよい。 According to the method for preparing an immunomagnetic composition, the weight ratio of fucoidan to dextran oxide may be 1: 0.1 to 1: 4.
前記の免疫磁気組成物の調製方法によれば、前記酸化デキストランは、アルデヒド基を有してもよく、前記酸化デキストランは、分子量が５ｋＤａ〜２７０ｋＤａにあるデキストランによって調製されてもよい。 According to the method for preparing an immunomagnetic composition, the oxidized dextran may have an aldehyde group, and the oxidized dextran may be prepared by a dextran having a molecular weight of 5 kDa to 270 kDa.
前記の免疫磁気組成物の調製方法によれば、前記有機溶媒は、メタン（ｍｅｔｈａｎｅ）、ジクロロメタン（ｄｉｃｈｌｏｒｏｍｅｔｈａｎｅ）又はクロロホルム（ｃｈｌｏｒｏｆｏｒｍ）であってもよい。 According to the method for preparing an immunomagnetic composition, the organic solvent may be methane (methane), dichloromethane (dichloromethane) or chloroform (chloroform).
これによって、本発明の免疫磁気組成物の調製方法は、他のターゲット担体の複雑な製造プロセスと異なり、構造を安定させるために余分な界面活性剤を使用する必要がなく、材料の入手と生産も非常に簡単である。 Thereby, the method for preparing an immunomagnetic composition of the present invention, unlike the complicated manufacturing process of other target carriers, does not require the use of extra surfactants to stabilize the structure, and the acquisition and production of materials. Is also very easy.
これによって、本発明の癌治療用キットは、本発明の免疫磁気組成物及び磁場発生装置を含み、磁場発生装置を磁気誘導の補助工具とすることによって、本発明の免疫磁気組成物を患部に蓄積して、局所的な拡大処置の効果を達成し、全身の免疫応答を回避する。本発明の癌治療用キットは、物理的及び生物学的両方の目的の役割を有し、従来の純粋な粋な抗体用量の１％だけを使用するが、より優れた腫瘍抑制能力を示し、半減期を２倍以上高めることができる。 Thereby, the cancer treatment kit of the present invention includes the immunomagnetic composition of the present invention and the magnetic field generator, and by using the magnetic field generator as an auxiliary tool for magnetic induction, the immunomagnetic composition of the present invention is applied to the affected area. Accumulate to achieve the effect of topical dilation treatment and avoid systemic immune response. The cancer treatment kits of the present invention serve both physical and biological purposes, using only 1% of conventional pure and sensible antibody doses, but exhibiting better tumor suppressor capacity. The half-life can be increased more than twice.
本発明の説明は、読者に本開示内容を基本的に理解させるように、本開示内容の簡略化された概要を提供する。この発明の内容は、本開示内容の完全な記述ではなく、また本発明実施例の重要な又は肝心な素子を指摘し、或いは本発明の範囲を限定するものではない。
下記の添付図面の説明は、本発明の上記及び他の目的、特徴、メリット及び実施例をより分かりやすくするためのものである。また、添付ファイル１〜５は、本明細書とは別に、物件提出書にて提出される。
本発明の上記及び他の目的、特徴、メリット及び実施例をより分かりやすくするために、添付図面の説明は下記の通りである。
The description of the present invention provides a simplified overview of the disclosure so that the reader may have a basic understanding of the disclosure. The content of the present invention is not a complete description of the content of the present invention, nor does it point out important or essential elements of the examples of the present invention, or limit the scope of the present invention.
The description of the accompanying drawings below is for the purpose of making the above and other purposes, features, merits and examples of the present invention easier to understand. In addition, Attachments 1 to 5 are submitted in the property submission form separately from this specification.
In order to make the above and other purposes, features, merits and embodiments of the present invention easier to understand, the accompanying drawings are described below.
本明細書の開示内容は、フコイダン及び酸化デキストランを疎水性相互作用によって超常磁性酸化鉄ナノ粒子と結合した後で、また抗体をグラフトしてなる新規の免疫磁気組成物を提出する。製造された免疫磁気組成物は、単独の同じ抗体による免疫療法の抗癌効果を大幅に改善し、より少ない抗体でより良好な腫瘍抑制能力を達成することができる。また、本明細書は、また、本案の免疫磁気組成物及び磁場発生装置を含み、更に本発明の免疫磁気組成物の抗癌効果を向上させることができる新規の癌治療用のキットを開示する。明細書において、乳癌肺転移及び大腸癌のマウス動物モデルによって、本案の免疫磁気組成物及び癌治療用のキットの免疫療法における有効性及びメカニズムを確認する。 The disclosure of this specification provides a novel immunomagnetic composition comprising binding fucoidan and dextran oxide to superparamagnetic iron oxide nanoparticles by hydrophobic interaction and then grafting an antibody. The immunomagnetic composition produced can significantly improve the anti-cancer effect of immunotherapy with the same antibody alone, and achieve better tumor suppressor capacity with fewer antibodies. The present specification also discloses a novel cancer treatment kit that includes the immunomagnetic composition of the present invention and a magnetic field generator, and can further improve the anticancer effect of the immunomagnetic composition of the present invention. .. In the specification, a mouse animal model of breast cancer lung metastasis and colorectal cancer confirms the efficacy and mechanism of the immunomagnetic composition of the present invention and the kit for treating cancer in immunotherapy.
以下、本明細書に用いられる特定用語の説明である。 The following is a description of specific terms used in the present specification.
明細書における前記の「フコイダン（ｆｕｃｏｉｄａｎ）」とは、茶色の海藻の表面における独特のスティックスリップ成分から抽出された水溶性食物繊維である。フコイダンは、フコースが豊富であり、高い生物安全性、抗酸化、抗凝固、抗血栓、抗ウイルス及び抗癌活性を有する天然多糖類である。 The aforementioned "fucoidan" in the specification is a water-soluble dietary fiber extracted from a unique stick-slip component on the surface of brown seaweed. Fucoidan is a natural polysaccharide rich in fucose and having high biosafety, antioxidant, anticoagulant, antithrombotic, antiviral and anticancer activity.
明細書における前記の「デキストラン（ｄｅｘｔｒａｎ）」とは、３Ｄａ〜２０００ｋＤａの範囲の分子量を構成する、複雑で分岐したデキストラン（多くのグルコース分子からなる多糖類）である。デキストランの直鎖部分は、α−１，６グリコシド結合によって互いに連結されたグルコース分子からなり、一方、分枝はα−１，３グリコシド結合に由来する。明細書における前記の「酸化デキストラン（ｏｘｉｄｉｚｅｄ ｄｅｘｔｒａｎ）」は、デキストランに対して表面改質を行い、デキストランにおけるヒドロキシル基をアルデヒド基に酸化して、抗体を更にグラフトすることができる酸化デキストランを得る。 The aforementioned "dextran" in the specification is a complex and branched dextran (polysaccharide consisting of many glucose molecules) having a molecular weight in the range of 3Da to 2000 kDa. The linear portion of dextran consists of glucose molecules linked together by α-1,6 glycosidic bonds, while the branches are derived from α-1,3 glycosidic bonds. The aforementioned "oxidized dextran" in the specification performs surface modification on dextran and oxidizes the hydroxyl group in dextran to an aldehyde group to obtain an oxidized dextran capable of further grafting the antibody.
図１を参照すると、本発明の免疫磁気組成物１００を示す模式図である。免疫磁気組成物１００は、コア層１１０と、シェル層１２０と、外層１３０と、を含む。 With reference to FIG. 1, it is a schematic view showing the immunomagnetic composition 100 of the present invention. The immunomagnetic composition 100 includes a core layer 110, a shell layer 120, and an outer layer 130.
コア層１１０は、サイトカイン又は抗癌薬物のような活性物質を含んでもよい。 The core layer 110 may contain active substances such as cytokines or anti-cancer drugs.
シェル層１２０は、フコイダン、酸化デキストラン及び超常磁性酸化鉄ナノ粒子が疎水性相互作用によって結合される複合物からなり、コア層１１０を覆う。更に、シェル層１２０を構成する複合物に用いられるフコイダンは、ウンデリア・ピンナチフィダ（Ｕｎｄａｒｉａ ｐｉｎｎａｔｉｆｉｄａ）、洋ナシの形嚢大型藻類（Ｍａｃｒｏｃｙｓｔｉｓ ｐｙｒｉｆｅｒａ）又はフーカスベシクロサス（Ｆｕｃｕｓ ｖｅｓｉｃｕｌｏｓｕｓ）から抽出されてもよく、用いられる酸化デキストランがアルデヒド基を有してもよく、且つ分子量が５ｋＤａ〜２７０ｋＤａにあるデキストランによって調製されてもよい。フコイダン、酸化デキストラン及び超常磁性酸化鉄ナノ粒子の間の疎水性相互作用は、乳化作用又はナノ沈殿法等のような方法によって形成されてもよいが、本発明はこれらに限定されない。 The shell layer 120 is composed of a composite in which fucoidan, dextran oxide and superparamagnetic iron oxide nanoparticles are bound by a hydrophobic interaction, and covers the core layer 110. Furthermore, the fucoidan used in the complex constituting the shell layer 120 may be extracted from underia pinnatifida, macrocystis pyrifera or fucus dextran, which is a pear-shaped sac. The dextran oxide used may have an aldehyde group and may be prepared by dextran having a molecular weight of 5 kDa to 270 kDa. Hydrophobic interactions between fucoidan, dextran oxide and superparamagnetic iron oxide nanoparticles may be formed by methods such as emulsification or nanoprecipitation, but the present invention is not limited thereto.
外層１３０は、少なくとも１つの抗体１３１を含み、且つ抗体１３１がシェル層１２０外にグラフトされて外層１３０を構成する。抗体１３１は、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤であってもよい。免疫チェックポイント阻害剤は、ＰＤ−Ｌ１抗体、ＰＤ−１抗体、ＣＴＬＡ−４抗体及びＴＩＭ−３抗体からなる群から選ばれてもよく、キラーＴ細胞増殖剤は、ＣＤ３抗体、ＣＤ２８抗体及び４−１ＢＢ抗体からなる群から選ばれてもよい。 The outer layer 130 contains at least one antibody 131, and the antibody 131 is grafted outside the shell layer 120 to form the outer layer 130. Antibody 131 may be an immune checkpoint inhibitor and / or a killer T cell proliferation agent. The immune checkpoint inhibitor may be selected from the group consisting of PD-L1 antibody, PD-1 antibody, CTLA-4 antibody and TIM-3 antibody, and the killer T cell proliferating agent is CD3 antibody, CD28 antibody and 4 It may be selected from the group consisting of -1BB antibody.
更に、前記免疫磁気組成物１００は、粒径が８０ｎｍ〜３５０ｎｍにある球体であってもよい。また、前記免疫磁気組成物は、中空形状になる。 Further, the immunomagnetic composition 100 may be a sphere having a particle size of 80 nm to 350 nm. In addition, the immunomagnetic composition has a hollow shape.
図２を参照すると、本発明の免疫磁気組成物の調製方法３００を示す工程フロー図である。図２において、免疫磁気組成物の調製方法３００は、工程３１０、工程３２０、工程３３０、工程３４０と工程３５０を含む。 With reference to FIG. 2, it is a process flow chart showing the method 300 for preparing the immunomagnetic composition of the present invention. In FIG. 2, the method 300 for preparing an immunomagnetic composition includes step 310, step 320, step 330, step 340 and step 350.
工程３１０は、フコイダン及び酸化デキストランを含む水相溶液を提供する。用いられるフコイダンは、ウンデリア・ピンナチフィダ（Ｕｎｄａｒｉａ ｐｉｎｎａｔｉｆｉｄａ）、洋ナシの形嚢大型藻類（Ｍａｃｒｏｃｙｓｔｉｓ ｐｙｒｉｆｅｒａ）又はフーカスベシクロサス（Ｆｕｃｕｓ ｖｅｓｉｃｕｌｏｓｕｓ）から抽出されてもよく、用いられる酸化デキストランがアルデヒド基を有してもよく、分子量が５ｋＤａ〜２７０ｋＤａにあるデキストランによって調製されてもよい。フコイダンと酸化デキストランとは、１：０．１〜１：４の重量比で混合される。 Step 310 provides an aqueous phase solution containing fucoidan and dextran oxide. The fucoidan used may be extracted from Undalia pinnatifida, pear-shaped large algae (Macrocystis pyrifera) or Fucus vesiculosus, and the used dextran oxide has an aldehyde group. It may also be prepared by dextran having a molecular weight of 5 kDa to 270 kDa. Fucoidan and dextran oxide are mixed in a weight ratio of 1: 0.1 to 1: 4.
工程３２０は、有機溶媒及び超常磁性酸化鉄ナノ粒子を含む油相溶液を提供する。有機溶媒は、メタン（ｍｅｔｈａｎｅ）、ジクロロメタン（ｄｉｃｈｌｏｒｏｍｅｔｈａｎｅ）又はクロロホルム（ｃｈｌｏｒｏｆｏｒｍ）であってもよい。 Step 320 provides an oil phase solution containing an organic solvent and superparamagnetic iron oxide nanoparticles. The organic solvent may be methane (methane), dichloromethane (dichloromethane) or chloroform (chloroform).
工程３３０は、工程３１０に提供される水相溶液と工程３２０に提供される油相溶液を混合して乳濁液を形成するエマルジョン反応を行う。 Step 330 performs an emulsion reaction in which the aqueous phase solution provided in step 310 and the oil phase solution provided in step 320 are mixed to form an emulsion.
工程３４０において、エマルジョンにおける有機溶媒は、減圧蒸発等の方法によって乳濁液における有機溶媒を除去して、磁気フコイダン担体を形成することができる。 In step 340, the organic solvent in the emulsion can be removed from the emulsion by a method such as evaporation under reduced pressure to form a magnetic fucoidan carrier.
工程３５０は、磁気フコイダン担体と少なくとも１つの抗体をグラフトして、免疫磁気組成物を形成する抗体のグラフトを行う。用いられる抗体は、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤であってもよい。免疫チェックポイント阻害剤は、ＰＤ−Ｌ１抗体、ＰＤ−１抗体、ＣＴＬＡ−４抗体及びＴＩＭ−３抗体からなる群から選ばれてもよく、キラーＴ細胞増殖剤は、ＣＤ３抗体、ＣＤ２８抗体及び４−１ＢＢ抗体からなる群から選ばれてもよい。 In step 350, the magnetic fucoidan carrier is grafted with at least one antibody to graft the antibody to form an immunomagnetic composition. The antibody used may be an immune checkpoint inhibitor and / or a killer T cell proliferator. The immune checkpoint inhibitor may be selected from the group consisting of PD-L1 antibody, PD-1 antibody, CTLA-4 antibody and TIM-3 antibody, and the killer T cell proliferating agent is CD3 antibody, CD28 antibody and 4 It may be selected from the group consisting of -1BB antibody.
これによって、前記の方法で調製された免疫磁気組成物は、後で抗癌剤、例えば、癌細胞増殖抑制剤、癌転移抑制剤、腫瘍免疫反応誘導剤等として使用することができる。上記の方法で調製された免疫磁気組成物は中空形状になり、免疫物質組成物の抗腫瘍効果を高めるためにコア層に活性物質を更に覆ってもよい。 Thereby, the immunomagnetic composition prepared by the above method can be later used as an anticancer agent, for example, a cancer cell growth inhibitor, a cancer metastasis inhibitor, a tumor immune response inducer, or the like. The immunomagnetic composition prepared by the above method has a hollow shape, and the core layer may be further covered with an active substance in order to enhance the antitumor effect of the immune substance composition.
また、前記の方法で調製された免疫磁気組成物は、磁石、三次元磁場磁石又は磁気共鳴スキャナー等の磁場発生装置と組み合わせて、癌を治療するためのキットを構成してもよく、磁場発生装置が発生させた磁場を磁気誘導用の補助工具として使用することによって、本発明の免疫磁気組成物を患部に蓄積させ、局所的な拡大処置の効果を得ることができ、本発明の癌治療用のキットは、従来の純粋な粋な抗体の投与量の１％しか必要としないが、より優れた腫瘍抑制能力を示し、２倍以上の半減期を増加させることができる。 Further, the immunomagnetic composition prepared by the above method may be combined with a magnetic field generator such as a magnet, a three-dimensional magnetic field magnet or a magnetic resonance scanner to form a kit for treating cancer, and the magnetic field generation may be formed. By using the magnetic field generated by the device as an auxiliary tool for magnetic induction, the immunomagnetic composition of the present invention can be accumulated in the affected area, and the effect of local expansion treatment can be obtained, and the cancer treatment of the present invention can be obtained. The kit for the magnet requires only 1% of the dose of conventional pure and stylish antibody, but shows better tumor suppressive ability and can increase the half-life by more than 2 times.
本発明は、本発明の当業者が過度に解読せずに、容易に実施できるように、以下の特定の試験例によって更に説明するが、これらの試験例は本発明の範囲を制限するものではなく、本発明の材料や方法の実施方法を説明するためのものである。
（試験例）
一、本発明の免疫磁気組成物及びその調製方法
１．１ 磁気フコイダン担体の構造及び安定性の分析
The present invention will be further described by the following specific test examples so that those skilled in the art of the present invention can easily carry out the invention without undue decipherment, but these test examples do not limit the scope of the present invention. However, it is for explaining the method of carrying out the material and the method of the present invention.
(Test example)
1. Immunomagnetic composition of the present invention and its preparation method 1.1 Analysis of structure and stability of magnetic fucoidan carrier
本試験例において、抗体がグラフトされていない磁気フコイダン担体を調製して、試験の最適な調製条件で、走査型電子顕微鏡（ｓｃａｎｎｉｎｇ ｅｌｅｃｔｒｏｎ ｍｉｃｒｏｓｃｏｐｙ；ＳＥＭ）及び透過型電子顕微鏡（ｔｒａｎｓｍｉｓｓｉｏｎ ｅｌｅｃｔｒｏｎ ｍｉｃｒｏｓｃｏｐｙ；ＴＥＭ）によって磁気フコイダン担体の形態を観察した。ナノ粒径及び界面電位アナライザー（Ｄｅｌｓａ Ｎａｎｏ Ｃ ｐａｒｔｉｃｌｅ ａｎａｌｙｚｅｒ，ＢＥＣＫＭＡＮ ＣＯＵＬＴＥＲ）によって磁気フコイダン担体のゼータ電位（ｚｅｔａ ｐｏｔｅｎｔｉａｌ）、粒径及び磁気フコイダン担体が二次蒸留（Ｄｏｕｂｌｅ Ｄｉｓｔｉｌｌｅｄ Ｗａｔｅｒ；ＤＤＷ）及びリン酸緩衝溶液（Ｐｈｏｓｐｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅ；ＰＢＳ）における安定性を分析した。 In this test example, a magnetic fucoidan carrier without antibody grafting was prepared, and under the optimum preparation conditions for the test, a scanning electron microscope (SEM) and a transmission electron microscope (TEM) were used. The morphology of the magnetic fucoidan carrier was observed by. The nanoparticle size and interfacial potential analyzer (Delsa Nano C parameter analyzer, BECKMAN COOLTER) allows the zeta potential of the magnetic fucoidan carrier, and the particle size and magnetic fucoidan carrier to be secondary distilled (Double Distilled Water). Stability in solution (Phosphate Buffered Saline; PBS) was analyzed.
磁気フコイダン担体を調製する前に、まず超常磁性酸化鉄ナノ粒子及び酸化デキストランを別々に調製し、後の抗体のグラフトに寄与するように、本試験例においてアルデヒド基を有する酸化デキストランを使用した。超常磁性酸化鉄ナノ粒子（以下、「ＩＯ」で示す）の設計合成は、２００４?~のＳｈｏｕｈｅｎｇ Ｓｕｎチームによって発表される文献（Ｓｈｏｕｈｅｎｇ Ｓｕｎ ｅｔ ａｌ．，Ｍｏｎｏｄｉｓｐｅｒｓｅ ＭＦｅ２Ｏ４（Ｍ＝Ｆｅ，Ｃｏ，Ｍｎ） Ｎａｎｏｐａｒｔｉｃｌｅｓ．Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ａｍｅｒｉｃａｎ Ｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｙ ２００４，１２６（１）：２７３−２７９）を参照し、調製条件として、２ｍｍｏｌのＦｅ（ａｃａｃ）３、１０ｍｍｏｌの１，２−ヘキサデカンジオール、６ｍｍｏｌのオレイン酸及び６ｍｍｏｌのアセトアミドを２０ｍｌのベンジルエーテル２０ｍｌで混合した後で、窒素雰囲気下、１００℃で３０分間還流させた。更に、前記反応物を２００℃で１時間加熱した後で、また２８５℃まで３０分間加熱してＩＯの核形成及び生長を完成した。反応物を室温に冷却した後で、６０００ｒｐｍで遠心分離してＩＯを１０分間回収し、エタノールで３回精製して、ＩＯの調製を完成した。 Before preparing the magnetic fucoidan carrier, superparamagnetic iron oxide nanoparticles and dextran oxide were first prepared separately, and dextran oxide having an aldehyde group was used in this test example so as to contribute to the subsequent grafting of the antibody. The design and synthesis of superparamagnetic iron oxide nanoparticles (hereinafter referred to as “IO”) is described in the literature published by the Shouheng Sun team from 2004? (Shouheng Sun et al., Monodisperse MFe2O4 (M = Fe, Co, Mn)). Refer to Nanoparticles. Journal of the American Chemical Society 2004,126 (1): 273-279), and as preparation conditions, 2 mmol Fe (acac) 3, 10 mmol 1,2-hexadecanediol, 6 mmol oleic acid and 6 mmol. Was mixed with 20 ml of 20 ml of benzyl ether, and then refluxed at 100 ° C. for 30 minutes under a nitrogen atmosphere. Further, after heating the reaction product at 200 ° C. for 1 hour, the reaction product was heated to 285 ° C. for 30 minutes to complete the nucleation and growth of IO. After cooling the reaction to room temperature, the reaction was centrifuged at 6000 rpm to recover IO for 10 minutes and purified 3 times with ethanol to complete the preparation of IO.
アルデヒド基を有する酸化デキストラン（以下「Ｄｅｘ」で示す）の調製方法としては、下記の通りである。デキストラン（分子量：５ｋＤａ〜２７０ｋＤａ）を室温で光を避けて過ヨウ素酸ナトリウム溶液（１０ｍＭ）を含む水性酸化バッファー（０．５−１０ｍｇｍｌ−１、ｐＨ＝５．５）に溶解して３０分間酸化した。Ａｍｉｃｏｎ（分子量：３ｋＤａ）によって修飾したＤｅｘを透析して過ヨウ素酸ナトリウムを除去した。凍結乾燥機（ＦｒｅｅＺｏｎｅ １Ｌ Ｂｅｎｃｈｔｏｐ Ｆｒｅｅｚｅ Ｄｒｙ Ｓｙｓｔｅｍｓ，Ｌａｂｃｏｎｃｏ，ｋａｎｓａｓ）によって前記Ｄｅｘを再散在し、凍結乾燥した。製造されたＤｅｘについては、核磁気共鳴（Ｎｕｃｌｅａｒ Ｍａｇｎｅｔｉｃ Ｒｅｓｏｎａｎｃｅ；ｎｍＲ）によって構造を分析し、比色アルデヒドベースのアッセイキット（ＭＡＫ１４０，ｓｉｇｍａ）を用いてその修飾度を分析した。 The method for preparing dextran oxide having an aldehyde group (hereinafter referred to as "Dex") is as follows. Dextran (molecular weight: 5 kDa to 270 kDa) is dissolved in an aqueous oxidation buffer (0.5-10 mgml-1, pH = 5.5) containing a sodium periodate solution (10 mM) at room temperature to avoid light and oxidized for 30 minutes. did. Dex modified with Amicon (molecular weight: 3 kDa) was dialyzed to remove sodium periodate. The Dex was re-scattered by a freeze-dryer (FreeZone 1L Benchtop Freeze Dry Systems, Labconco, Kansas) and freeze-dried. The structure of the produced Dex was analyzed by Nuclear Magnetic Resonance (nmR), and the degree of modification was analyzed using a colorimetric aldehyde-based assay kit (MAK140, sigma).
本試験例において、磁気フコイダン担体の調製条件としては、下記の通りである。０．５ｍｇ／ｍｌのフコイダン（フーカスベシクロサスから抽出）と０．５ｍｇ／ｍｌのＤｅｘを混合して水相溶液とした。２ｍｇのＩＯを０．２ｍｌのジクロロメタンに溶解して油相溶液とした。前記水相溶液及び油相溶液を混合した後で、ホモジナイザー（Ｄｏｕｂｌｅ Ｅａｇｌｅ Ｅｎｔｅｒｐｒｉｓｅ Ｃｏ，Ｌｔｄ）で１２０Ｗのパワーで５０秒乳化して、乳濁液を得た。ロータリーエバポレーターでジクロロメタンを除去した後で、磁気分離装置（ＭａｇｎｉＳｏｒｔ、ｅＢｉｏｓｃｉｅｎｃｅ）によって製造された磁気フコイダン担体（以下、「ＩＯ＠ＦｕＤｅｘ」で示す）を精製し、ＩＯ＠ＦｕＤｅｘをＤＤＷ又は０．１ＭのＰＢＳ（ｐＨ＝６）で再懸濁して更に抗体のグラフトを行った。
また、本試験例において、別にそれぞれ０．５ｍｇ／ｍｌのフコイダン又は０．５ｍｇ／ｍｌのＤｅｘを水相溶液として、ジクロロメタンに溶解するＩＯを油相溶液として、同じ調製方法によって磁気担体ＩＯ＠Ｆｕ（水相溶液：フコイダン）又はＩＯ＠Ｄｅｘ（水相溶液：アルデヒド基を有する酸化デキストラン）を調製した。調製されたＩＯ＠ＦｕＤｅｘ、ＩＯ＠Ｆｕ及びＩＯ＠Ｄｅｘに対しては、走査型電子顕微鏡及び透過型電子顕微鏡によって形態を分析し、ナノ粒径及び界面電位アナライザーによってゼータ電位と粒径を分析し、超伝導量子干渉磁力計によって磁気分析を分析した。
In this test example, the preparation conditions for the magnetic fucoidan carrier are as follows. 0.5 mg / ml fucoidan (extracted from fucas becyclosus) and 0.5 mg / ml Dex were mixed to prepare an aqueous phase solution. 2 mg of IO was dissolved in 0.2 ml of dichloromethane to prepare an oil phase solution. After mixing the aqueous phase solution and the oil phase solution, they were emulsified with a homogenizer (Double Eagle Enterprise Co, Ltd) at a power of 120 W for 50 seconds to obtain an emulsion. After removing dichloromethane with a rotary evaporator, a magnetic fucoidan carrier (hereinafter referred to as "IO @ FuDex") produced by a magnetic separation device (MagniSort, eBioscience) is purified to obtain IO @ FuDex in DDW or 0.1 M. It was resuspended in PBS (pH = 6) and further grafted with antibodies.
Further, in this test example, 0.5 mg / ml fucoidan or 0.5 mg / ml Dex was used as an aqueous phase solution, and IO dissolved in dichloromethane was used as an oil phase solution, respectively, using the same preparation method as the magnetic carrier IO @ Fu. (Aqueous phase solution: fucoidan) or IO @ Dex (aqueous phase solution: dextran oxide having an aldehyde group) was prepared. For the prepared IO @ FuDex, IO @ Fu and IO @ Dex, the morphology was analyzed by a scanning electron microscope and a transmission electron microscope, and the zeta potential and particle size were analyzed by a nano-particle size and interfacial potential analyzer. , Magnetic analysis was analyzed by a superconducting quantum interference magnetometer.
図３Ａ〜図６Ｃを参照すると、図３Ａ〜図３Ｆは、磁気フコイダン担体を示す構造分析図である。図３Ａは、ＩＯ＠ＦｕＤｅｘの走査電子顕微鏡写真図である。図３Ｂ〜図３Ｆは、ＩＯ＠ＦｕＤｅｘの透過電子顕微鏡写真図である。
図４Ａ〜図４Ｃは、磁気担体ＩＯ＠Ｆｕを示す構造分析図である。図４Ａと図４Ｂは、ＩＯ＠Ｆｕの走査電子顕微鏡写真図である。図４Ｃは、ＩＯ＠Ｆｕの透過電子顕微鏡写真図である。
図５Ａと図５Ｂは、磁気担体ＩＯ＠Ｄｅｘを示す構造分析図である。図５Ａは、ＩＯ＠Ｄｅｘの走査電子顕微鏡写真図である。図５Ｂは、ＩＯ＠Ｄｅｘの透過電子顕微鏡写真図である。
図６Ａ〜図６Ｃは、磁気フコイダン担体の安定性を示す分析結果図である。図６Ａは、ＩＯ＠ＦｕＤｅｘ、ＩＯ＠ＦｕとＩＯ＠Ｄｅｘの流体力学的サイズ分布図である。図６Ｂは、ＩＯ＠ＦｕＤｅｘ、ＩＯ＠ＦｕとＩＯ＠Ｄｅｘのゼータ電位分析図である。図６Ｃは、ＩＯ＠ＦｕＤｅｘとＩＯの磁気分析図である。
With reference to FIGS. 3A to 6C, FIGS. 3A to 3F are structural analysis diagrams showing a magnetic fucoidan carrier. FIG. 3A is a scanning electron micrograph of IO @ FuDex. 3B to 3F are transmission electron micrographs of IO @ FuDex.
4A to 4C are structural analysis diagrams showing the magnetic carrier IO @ Fu. 4A and 4B are scanning electron micrographs of IO @ Fu. FIG. 4C is a transmission electron micrograph of IO @ Fu.
5A and 5B are structural analysis diagrams showing the magnetic carrier IO @ Dex. FIG. 5A is a scanning electron micrograph of IO @ Dex. FIG. 5B is a transmission electron micrograph of IO @ Dex.
6A to 6C are analysis result diagrams showing the stability of the magnetic fucoidan carrier. FIG. 6A is a hydrodynamic size distribution diagram of IO @ FuDex, IO @ Fu and IO @ Dex. FIG. 6B is a zeta potential analysis diagram of IO @ FuDex, IO @ Fu and IO @ Dex. FIG. 6C is a magnetic analysis diagram of IO @ FuDex and IO.
図３Ａの結果から、走査型電子顕微鏡の観察によって、ＩＯ＠ＦｕＤｅｘが球状構造であることが分かり、電子顕微鏡の中空環境によって崩壊の現象があり、それは中空構造であることを示す。
図３Ｂの結果から分かるように、透過型電子顕微鏡によってＩＯ＠ＦｕＤｅｘが崩壊で重なるシェル層による暗いコントラストが観察された。図３Ｃは、シェル層を拡大する透過電子顕微鏡写真図である。数多くのサイズが約５ｎｍの超常磁性酸化鉄ナノ粒子が見られ、それはシェル層の組成の構造であることが証明される。図３Ｄの暗視野観察及び図３Ｅと図３Ｆの要素分布の検出で、再び明らかな中空構造及び酸化鉄が担体に散在している状況が見られる。
From the results of FIG. 3A, it can be seen that IO @ FuDex has a spherical structure by observation with a scanning electron microscope, and there is a phenomenon of collapse due to the hollow environment of the electron microscope, which indicates that it is a hollow structure.
As can be seen from the results of FIG. 3B, a dark contrast was observed by the transmission electron microscope due to the shell layer in which IO @ FuDex collapsed and overlapped. FIG. 3C is a transmission electron micrograph of the shell layer magnified. A large number of superparamagnetic iron oxide nanoparticles with a size of about 5 nm are found, which proves to be the structure of the shell layer composition. The dark-field observation in FIG. 3D and the detection of the element distributions in FIGS. 3E and 3F show that the hollow structure and iron oxide are scattered on the carrier again.
図４Ａ〜図５Ｂの結果から分かるように、ＩＯ＠ＦｕとＩＯ＠ＦｕＤｅｘは同様に均一な構造を有し、ＩＯ＠Ｄｅｘが安定ではなく、蒸発プロセスで凝集しやすくて、広い範囲の粒径分布及びランダムな形態学的外観が観察された。 As can be seen from the results of FIGS. 4A to 5B, IO @ Fu and IO @ FuDex have a similarly uniform structure, IO @ Dex is not stable, easily aggregates in the evaporation process, and has a wide range of particle sizes. Distribution and random morphological appearance were observed.
図６Ａと図６Ｂの結果から分かるように、ＩＯ＠ＦｕＤｅｘは流体中に均一な粒径が現れ、粒径サイズが８０ｎｍ〜３５０ｎｍにあり、且つ平均粒径が１４１．５ｎｍであり、ＩＯ＠Ｆｕの平均粒径（２４１ｎｍ）より小さい。ＩＯ＠ＦｕＤｅｘとＩＯ＠Ｆｕは主体がフコイダンからなり、その中に含まれる硫酸塩によってＩＯ＠ＦｕＤｅｘとＩＯ＠Ｆｕに強い負ゼータ電位を持たせるが、ＩＯ＠ＦｕＤｅｘの主体に別にＤｅｘを有するので、ＩＯ＠ＦｕＤｅｘの負ゼータ電位（−３２．８ｍＶ）がＩＯ＠Ｆｕの負ゼータ電位（−５８．４ｍＶ）よりも低くなるため、ＩＯ＠ＦｕＤｅｘの間の強い反発力がコロイドの安定性と良好な散在性を維持することができる。
図６Ｃの結果から分かるように、超常磁性酸化鉄ナノ粒子（ＩＯ）がかなり高い単位重量あたりの飽和磁化強度があり、ＩＯ＠ＦｕＤｅｘは、超常磁性酸化鉄ナノ粒子、フコイダン及び酸化デキストランを含むので、その数値がやや低減するが、やはり５７．５ｅｍｕ ｇ−１の高数値を保有し、その磁気の強度が複合担体構造の形成によって低減しなく、磁気分離装置によって磁気精製を行うことに寄与して、高収率のＩＯ＠ＦｕＤｅｘを得た。
As can be seen from the results of FIGS. 6A and 6B, IO @ FuDex has a uniform particle size in the fluid, a particle size of 80 nm to 350 nm, and an average particle size of 141.5 nm. It is smaller than the average particle size (241 nm) of. IO @ FuDex and IO @ Fu are mainly composed of fucoidan, and the sulfate contained in them gives IO @ FuDex and IO @ Fu a strong negative zeta potential, but since the main body of IO @ FuDex has a separate Dex. , IO @ FuDex negative zeta potential (-32.8 mV) is lower than IO @ Fu negative zeta potential (-58.4 mV), so strong repulsive force between IO @ FuDex is good for colloidal stability. Can be maintained in a scattered manner.
As can be seen from the results of FIG. 6C, the superparamagnetic iron oxide nanoparticles (IO) have a fairly high saturation magnetization strength per unit weight, and IO @ FuDex contains superparamagnetic iron oxide nanoparticles, fucoidan and dextran oxide. Although the value is slightly reduced, it also has a high value of 57.5 emu g-1, and its magnetic strength is not reduced by the formation of the composite carrier structure, which contributes to magnetic purification by the magnetic separator. A high yield of IO @ FuDex was obtained.
本試験例において、別に異なる分子量の酸化デキストランで磁気フコイダン担体を調製し、それは、図２の工程３１０のように提供される水相溶液に、分子量が５ｋＤａ〜２７０ｋＤａであるデキストランによって酸化デキストランを調製し、また酸化デキストランとフコイダンとを１：０．２の重量比で混合した。残りの工程３２０〜工程３４０の細部については、試験例１．１に記載のものとほぼ同じであるので、ここで説明しない。更に製造された磁気フコイダン担体の粒径サイズをナノ粒径及び界面電位アナライザーによって分析して、その結果を下記の表１に示す。 In this test example, a magnetic fucoidan carrier is separately prepared with dextran oxide having a different molecular weight, and it is prepared by preparing dextran oxide having a molecular weight of 5 kDa to 270 kDa in an aqueous phase solution provided as in step 310 of FIG. Then, dextran oxide and fucoidan were mixed at a weight ratio of 1: 0.2. The details of the remaining steps 320 to 340 are almost the same as those described in Test Example 1.1, and will not be described here. Further, the particle size of the produced magnetic fucoidan carrier was analyzed by a nano-particle size and an interfacial potential analyzer, and the results are shown in Table 1 below.
また、本試験例において、異なるフコイダンと酸化デキストランとの混合比例で磁気フコイダン担体を調製して、図２の工程３１０のように提供された水相溶液に、フコイダンとデキストランとを１：０．１〜１：４の重量比で混合した。残りの工程３２０〜工程３４０の細部については、試験例１．１に記載のものとほぼ同じであるので、ここで説明しない。更に製造された磁気フコイダン担体の粒径サイズをナノ粒径及び界面電位アナライザーによって分析して、その結果を下記の表２に示す。 Further, in this test example, a magnetic fucoidan carrier was prepared in a mixed proportion of different fucoidan and dextran oxide, and fucoidan and dextran were added to the aqueous phase solution provided as shown in step 310 of FIG. The mixture was mixed in a weight ratio of 1 to 1: 4. The details of the remaining steps 320 to 340 are almost the same as those described in Test Example 1.1, and will not be described here. Further, the particle size of the produced magnetic fucoidan carrier was analyzed by a nano-particle size and an interfacial potential analyzer, and the results are shown in Table 2 below.
凍結乾燥されて再溶解した後のＩＯ＠ＦｕＤｅｘが同じ構造を有するかをテストするために、本試験例では、更に調製されたＩＯ＠ＦｕＤｅｘを凍結乾燥機によって凍結乾燥して粉末状の結晶を形成し、また凍結乾燥された結晶を水溶液に再溶解し、透過型電子顕微鏡によってＩＯ＠ＦｕＤｅｘの凍結乾燥前と凍結乾燥された構造を観察した。 In order to test whether IO @ FuDex after being lyophilized and redissolved has the same structure, in this test example, further prepared IO @ FuDex was lyophilized by a lyophilizer to obtain powdery crystals. The formed and lyophilized crystals were redissolved in an aqueous solution, and the pre-lyophilized and lyophilized structures of IO @ FuDex were observed with a transmission electron microscope.
図７Ａと図７Ｂを参照すると、図７Ａは、ＩＯ＠ＦｕＤｅｘの凍結乾燥前の透過電子顕微鏡写真図である。図７Ｂは、ＩＯ＠ＦｕＤｅｘが凍結乾燥されて再溶解した後の透過電子顕微鏡写真図である。結果から分かるように、ＩＯ＠ＦｕＤｅｘの凍結乾燥前の構造は、中空の球状であり、サイズが約８０ｎｍ〜３５０ｎｍにある。凍結乾燥された結晶は、快速に水溶液に再溶解でき、且つ再溶解のＩＯ＠ＦｕＤｅｘ構造が透過型電子顕微鏡で、やはり中空であり、且つサイズが凍結乾燥前の担体と合致する球体となるように保持する。ＩＯ＠ＦｕＤｅｘの安定性が非常に優れたと示す。 Referring to FIGS. 7A and 7B, FIG. 7A is a transmission electron micrograph of IO @ FuDex before lyophilization. FIG. 7B is a transmission electron micrograph after lyophilized and redissolved IO @ FuDex. As can be seen from the results, the structure of IO @ FuDex before lyophilization is a hollow spherical shape with a size of about 80 nm to 350 nm. The lyophilized crystals can be rapidly redissolved in an aqueous solution, and the redissolved IO @ FuDex structure is a transmission electron microscope, which is also hollow and has a size that matches the carrier before freeze-drying. Hold on. It shows that the stability of IO @ FuDex is very good.
（１．２ 実施例１−３の調製）
前記最適なＩＯ＠ＦｕＤｅｘの調製条件で、更に本発明の免疫磁気組成物を調製した。調製されたＩＯ＠ＦｕＤｅｘをそれぞれ異なる抗体を含み且つシアノ水素化ホウ素ナトリウム（５μＭ）を含む緩衝液（０．１Ｍ，ｐＨ＝６）と４℃で抗体のグラフトを４〜６時間行い、Ｄｅｘにおけるアルデヒド基が抗体上の第一級アミンとシッフ塩基を形成し、シアノ水素化ホウ素ナトリウムによって還元アミノ化を行った後で、抗体のグラフトを化学的に安定な状態にした。最後に、製造された免疫磁気組成物を磁気分離装置によって精製した。表３を参照すると、本発明の実施例１−３に用いられる抗体であり、実施例１に用いられる抗体がＣＤ３抗体及びＣＤ２８抗体であり、実施例２に用いられる抗体がＰＤ−Ｌ１抗体であり、実施例３に用いられる抗体がＰＤ−Ｌ１抗体、ＣＤ３抗体及びＣＤ２８抗体であった。
(1.2 Preparation of Examples 1-3)
Further, the immunomagnetic composition of the present invention was prepared under the optimum preparation conditions of IO @ FuDex. The prepared IO @ FuDex was grafted with a buffer solution (0.1 M, pH = 6) containing different antibodies and containing sodium cyanoborohydride (5 μM) at 4 ° C. for 4 to 6 hours in Dex. The aldehyde group formed a Schiff base with the primary amine on the antibody and was reductively aminated with sodium cyanoborohydride, after which the antibody graft was chemically stabilized. Finally, the immunomagnetic composition produced was purified by a magnetic separation device. Referring to Table 3, the antibody used in Examples 1-3 of the present invention, the antibody used in Example 1 is a CD3 antibody and a CD28 antibody, and the antibody used in Example 2 is a PD-L1 antibody. Yes, the antibodies used in Example 3 were PD-L1 antibody, CD3 antibody and CD28 antibody.
調製された実施例は、透過型電子顕微鏡分析の形態、元素分析、及びＸ線光電子分光法（ＸＰＳ）によって実施例３における元素構成を測定した。 In the prepared examples, the elemental composition in Example 3 was measured by the form of transmission electron microscopy, elemental analysis, and X-ray photoelectron spectroscopy (XPS).
図８を参照すると、実施例３の構造分析図である。結果から分かるように、実施例３の構造は、サイズが約８０〜３５０ｎｍの球状の中空構造であった。そして、電子顕微鏡の元素分析によって、実施例３の外層が抗体の存在のため、均一に明らかな窒素信号（Ｎ）が見られた。抗体がグラフトされる前に、担体成分として糖及び酸化鉄だけがあり、窒素を有する材料がないが、抗体がグラフトされた後で、抗体がアミノ基及びペプチド結合を有するので、多量の窒素を有して、本発明の免疫磁性組成物は抗体を有することが実証された。 With reference to FIG. 8, it is a structural analysis diagram of Example 3. As can be seen from the results, the structure of Example 3 was a spherical hollow structure having a size of about 80 to 350 nm. Then, by elemental analysis with an electron microscope, a uniformly clear nitrogen signal (N) was observed due to the presence of the antibody in the outer layer of Example 3. Before the antibody is grafted, there are only sugar and iron oxide as carrier components and no material with nitrogen, but after the antibody is grafted, the antibody has amino groups and peptide bonds, so a large amount of nitrogen is added. It has been demonstrated that the immunomagnetic compositions of the present invention have antibodies.
また図９を参照すると、本発明の実施例３のＸ線光電子分光法分析結果図である。結果から分かるように、ＩＯ＠ＦｕＤｅｘに対して実施例３は結合エネルギーが３９９ｅＶに位置する１本の信号があり、この信号が窒素信号であり、これは実施例３において抗体が担体にうまくグラフトされていることを示す。 Further, referring to FIG. 9, it is an X-ray photoelectron spectroscopy analysis result diagram of Example 3 of the present invention. As can be seen from the results, for IO @ FuDex, Example 3 has a single signal with a binding energy located at 399 eV, which is the nitrogen signal, which in Example 3 the antibody successfully grafted onto the carrier. Indicates that it has been done.
（１．３ 実施例５の調製及び構造確認）
本発明の免疫磁気組成物は、中空形状になるので、更にコア層でサイトカイン又は抗癌薬物等のような活性物質を覆うことができた。本試験例における活性物質は、インターロイキン−２（Ｉｎｔｅｒｌｅｕｋｉｎ−２，ＩＬ−２）を例として、更にＩＬ−２を覆う実施例５を調製した。
(1.3 Preparation of Example 5 and structural confirmation)
Since the immunomagnetic composition of the present invention has a hollow shape, the core layer can further cover an active substance such as a cytokine or an anticancer drug. As the active substance in this test example, Interleukin-2 (Interleukin-2, IL-2) was taken as an example, and Example 5 covering IL-2 was prepared.
本試験例において、実施例５の調製条件は下記の通りである。０．５ｍｇ／ｍｌのフコイダン、０．５ｍｇ／ｍｌのＤｅｘ及び５０μｇのＩＬ−２を混合して水相溶液とした。２ｍｇのＩＯを０．２ｍｌのジクロロメタンに溶解して油相溶液とした。前記水相溶液と油相溶液を混合した後で、ホモジナイザーで１２０Ｗのパワーで５０秒乳化して、乳濁液を得た。ロータリーエバポレーターでジクロロメタンを除去した後で、製造された実施例５を磁気分離装置によって精製した。調製された実施例については、走査型電子顕微鏡によって形態を分析し、ナノ粒径及び界面電位アナライザーによってその粒径を分析した。 In this test example, the preparation conditions of Example 5 are as follows. 0.5 mg / ml fucoidan, 0.5 mg / ml Dex and 50 μg IL-2 were mixed to prepare an aqueous phase solution. 2 mg of IO was dissolved in 0.2 ml of dichloromethane to prepare an oil phase solution. After mixing the aqueous phase solution and the oil phase solution, they were emulsified with a homogenizer at a power of 120 W for 50 seconds to obtain an emulsion. After removing dichloromethane with a rotary evaporator, the manufactured Example 5 was purified by a magnetic separation device. For the prepared examples, the morphology was analyzed by a scanning electron microscope, and the particle size was analyzed by a nanoparticle size and an interfacial potential analyzer.
図１０Ａと図１０Ｂを参照すると、実施例５を示す構造分析図である。
図１０Ａは、実施例５の走査電子顕微鏡写真図である。図１０Ｂは、実施例５の流体力学的サイズ分布図である。結果から分かるように、実施例５のＩＬ−２のカバー率は９９．９３％まで達する。ＩＬ−２がコア層を満たして、実施例５の平均粒径サイズが純粋な担体の１６１．２ｎｍから３５６．２ｎｍに増加し、本発明の免疫磁気組成物が更にコア層で活性物質を覆うことが証明され、例えばドキソルビシン薬物（Ｄｏｘｏｒｕｂｉｃｉｎ）、ドセタキセル薬物（Ｄｏｃｅｔａｘｅｌ又はＰａｃｌｉｔａｘｅｌ）及びアスタキサンチン（Ａｓｔａｘａｎｔｈｉｎ；ＡＳＴＸ）等の活性物質も、同じ方式によって本発明の免疫磁気組成物に覆われることができる。
With reference to FIGS. 10A and 10B, it is a structural analysis diagram showing Example 5.
FIG. 10A is a scanning electron micrograph of Example 5. FIG. 10B is a hydrodynamic size distribution map of Example 5. As can be seen from the results, the coverage of IL-2 in Example 5 reaches 99.93%. IL-2 fills the core layer, the average particle size of Example 5 increases from 161.2 nm of the pure carrier to 356.2 nm, and the immunomagnetic composition of the invention further covers the active material with the core layer. It has been demonstrated that active substances such as, for example, doxorubicin, docetaxel or paclitaxel and astaxanthin (ASTX) can also be covered with the immunomagnetic composition of the invention by the same method.
（１．４ 本発明の免疫磁気組成物の目的能力の分析）
本試験例において、本発明の免疫磁気組成物がＴ細胞免疫力の低下を逆転してマウスの腫瘍退縮を促進することができるかを検証するために、調製された実施例１−３に対して目的能力分析及び細胞結合能力分析を行った。試験的に、実験モデルとして、転移能力、浸潤性及びＰＤ−Ｌ１発現を有するトリ陰性乳癌細胞株（４Ｔ１）を選択した。蛍光顕微鏡下での観察を容易にするために、蛍光顕微鏡分析群の実施例１−３でそれぞれ量子ドットに接続した。
(1.4 Analysis of Objective Capability of Immunomagnetic Composition of the Present Invention)
In this test example, with respect to Examples 1-3 prepared in order to verify whether the immunomagnetic composition of the present invention can reverse the decrease in T cell immunity and promote tumor regression in mice. The purpose and ability analysis and cell binding ability analysis were performed. Experimentally, a trinegative breast cancer cell line (4T1) with metastatic potential, invasiveness and PD-L1 expression was selected as an experimental model. In order to facilitate observation under a fluorescence microscope, each quantum dot was connected in Example 1-3 of the fluorescence microscope analysis group.
目的効率を評価するために、試験的に、実施例１−３及び４Ｔ１細胞株（４×１０５）を、ウシ血清アルブミン（Ｂｏｖｉｎｅ Ｓｅｒｕｍ Ａｌｂｕｍｉｎ；ＢＳＡ）を基礎とする培養液の中で、４℃で３０分間培養し、次いでフローサイトメトリー（Ｎｏｖｏｃｙｔｅ Ｆｌｏｗ Ｃｙｔｏｍｅｔｅｒ；ＡＣＥＡ Ｂｉｏｓｃｉｅｎｃｅｓ）によって分析した。細胞結合能力を評価するために、量子ドットに接続したＩＯ＠ＦｕＤｅｘ及び実施例２をそれぞれ４Ｔ１細胞株と１、４、１２と２４時間培養し、またフローサイトメトリー又は蛍光顕微鏡（Ｃａｒｌ Ｚｅｉｓｓ；Ｔｈｏｒｎｗｏｏｄ）によって分析した。 To assess the efficiency of interest, on a trial basis, Examples 1-3 and 4T1 cell lines (4 × 105) were placed in a culture medium based on bovine serum albumin (BSA) at 4 ° C. The cells were cultured for 30 minutes and then analyzed by flow cytometry (Novocate Flow Cytometer; ACEA Biosciences). To assess cell binding capacity, quantum dot-connected IO @ FuDex and Example 2 were cultured with a 4T1 cell line for 1, 4, 12 and 24 hours, respectively, and flow cytometry or fluorescence microscopy (Carl Zeiss; Hornwood). ).
図１１Ａ〜図１１Ｆを参照すると、本発明の免疫磁気組成物の目的能力と細胞結合能力の分析結果図である。
図１１Ａは、グラフト濃度が１．４μｇ／ｍｌ（低濃度；Ｌ）、７μｇ／ｍｌ（中濃度；Ｍ）と３５μｇ／ｍｌ（高濃度；Ｈ）であるＰＤ−Ｌ１抗体の実施例２の体外の目的能力のテスト結果である。図１１Ｂは、実施例２とＩｇＧがグラフトされたＩＯ＠ＦｕＤｅｘの体外の目的能力のテスト結果である。図１１Ｃは、ＩＯ＠ＦｕＤｅｘが第１、４、１２と２４時間に４Ｔ１細胞株との結合能力の分析結果図である。図１１Ｄは、実施例２が第１、４、１２と２４時間に４Ｔ１細胞株との結合能力の分析結果図である。図１１Ｅは、グラフト濃度が１．４μｇ／ｍｌ（低濃度；Ｌ）、７μｇ／ｍｌ（中濃度；Ｍ）と３５μｇ／ｍｌ（高濃度；Ｈ）であるＣＤ３抗体／ＣＤ２８抗体の実施例３の体外の目的能力のテスト結果である。図１１Ｆは、実施例２と実施例３が第１と２４時間に４Ｔ１細胞株との結合能力の分析結果図である。
With reference to FIGS. 11A to 11F, it is an analysis result diagram of the objective ability and the cell binding ability of the immunomagnetic composition of the present invention.
FIG. 11A shows the in vitro PD-L1 antibody Example 2 having graft concentrations of 1.4 μg / ml (low concentration; L), 7 μg / ml (medium concentration; M) and 35 μg / ml (high concentration; H). It is a test result of the purpose ability of. FIG. 11B shows the test results of the in vitro target ability of IO @ FuDex grafted with Example 2 and IgG. FIG. 11C is an analysis result diagram of the binding ability of IO @ FuDex to the 4T1 cell line at the first, fourth, twelfth and 24 hours. FIG. 11D is an analysis result diagram of the binding ability of Example 2 to the 4T1 cell line at the first, fourth, twelfth and 24 hours. FIG. 11E shows Example 3 of the CD3 / CD28 antibody having graft concentrations of 1.4 μg / ml (low concentration; L), 7 μg / ml (medium concentration; M) and 35 μg / ml (high concentration; H). It is a test result of the purpose ability outside the body. FIG. 11F is an analysis result diagram of the binding ability of Example 2 and Example 3 to the 4T1 cell line in the first and 24 hours.
図１１Ａの結果から分かるように、１時間培養した後で、実施例２（Ｈ）の群が一番高い平均蛍光強度（ｍｅｄｉａｎ ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎｄｅｘ；ＭＤＩ）を有し実施例２が４Ｔ１細胞株（ＰＤ−Ｌ１を発見する）と結合する量がその表面抗体密度と緊密な関係があるので、後の試験実施例２にグラフトされるＰＤ−Ｌ１濃度が高濃度（３５μｇ／ｍｌ）であった。
図１１Ｂの結果から分かるように、ＩｇＧが接続されるＩＯ＠ＦｕＤｅｘにＭＤＩ変位が観察されなく、実施例２が４Ｔ１細胞株に対する親和性はＩＯ＠ＦｕＤｅｘとの非特異的相互作用ではなく、ＰＤ−Ｌ１抗体に由来することを示す。
As can be seen from the results of FIG. 11A, after culturing for 1 hour, the group of Example 2 (H) had the highest average fluorescence intensity (MDI), and Example 2 had a 4T1 cell line (PD). Since the amount that binds to (discovering -L1) is closely related to its surface antibody density, the PD-L1 concentration grafted in Test Example 2 later was high (35 μg / ml).
As can be seen from the results of FIG. 11B, no MDI displacement was observed in IO @ FuDex to which IgG was connected, and in Example 2, the affinity for the 4T1 cell line was not a non-specific interaction with IO @ FuDex, but PD. It is shown that it is derived from the -L1 antibody.
図１１Ｃ図と図１１Ｄの結果から分かるように、ＩＯ＠ＦｕＤｅｘと細胞結合の量が時間依存性を有し、培養後の第１２時間後に明らかな細胞取り込みがあった。対照的に、実施例２において培養後の第１時間に細胞結合の現象があり、その後、ＭＤＩが緩い変位になり、培養時間の増加につれて、実施例２が細胞との結合量が向上することを示し、この結果から分かるように、ＰＤ−Ｌ１抗体をＩＯ＠ＦｕＤｅｘにグラフトした後で、元のＩＯ＠ＦｕＤｅｘ及び４Ｔ１細胞株の結合能力が変わった。 As can be seen from the results of FIGS. 11C and 11D, the amount of IO @ FuDex and cell binding was time-dependent, and there was clear cell uptake 12 hours after culturing. In contrast, in Example 2, there is a phenomenon of cell binding in the first hour after culturing, after which the MDI becomes loosely displaced, and as the culturing time increases, the amount of binding to cells in Example 2 increases. As can be seen from this result, the binding ability of the original IO @ FuDex and 4T1 cell lines changed after grafting the PD-L1 antibody to IO @ FuDex.
本発明の免疫磁気組成物を同時に免疫チェックポイント阻害剤とキラーＴ細胞増殖剤にするために、実施例３は、同時にＰＤ−Ｌ１抗体、ＣＤ３抗体及びＣＤ２８抗体をＩＯ＠ＦｕＤｅｘにグラフトしてなる免疫磁気組成物であった。図１１Ｅを参照すると、異なる濃度のＣＤ３抗体／ＣＤ２８抗体をグラフトする実施例３の目的能力分析であった。
図１１Ｅの結果から分かるように、１時間のインビトロ培養後、実施例３（Ｈ）の群が一番高いＭＤＩを有し、実施例３（Ｈ）群がＣＤ８+ Ｔ細胞に対して強い親和性を有することを示す。図１１Ｆの結果から分かるように、実施例３が４Ｔ１細胞株との間に実施例２と類似する結合力を有し、ＩＯ＠ＦｕＤｅｘに複数の抗体がグラフトされては４Ｔ１細胞株に対する親和性を影響しないことを示す。
In order to simultaneously make the immunomagnetic composition of the present invention an immune checkpoint inhibitor and a killer T cell proliferation agent, Example 3 comprises grafting PD-L1 antibody, CD3 antibody and CD28 antibody into IO @ FuDex at the same time. It was an immunomagnetic composition. With reference to FIG. 11E, it was a purpose-ability analysis of Example 3 in which different concentrations of CD3 antibody / CD28 antibody were grafted.
As can be seen from the results of FIG. 11E, after 1 hour of in vitro culture, the group of Example 3 (H) had the highest MDI, and the group of Example 3 (H) had a strong affinity for CD8 + T cells. Indicates that it has sex. As can be seen from the results of FIG. 11F, Example 3 has a binding force similar to that of Example 2 with the 4T1 cell line, and has an affinity for the 4T1 cell line when multiple antibodies are grafted on IO @ FuDex. Indicates that it does not affect.
図１２Ａ〜図１２Ｄを参照すると、実施例３がＣＤ８+ Ｔ細胞との結合能力の分析結果図である。試験的に、量子ドットが接続される実施例３をＣＤ８+ Ｔ細胞と３０分間培養し、Ａｌｅｘａ Ｆｌｕｏｒ ４８８ ＰｈａｌｌｏｉｄｉｎとＤＡＰＩによってＣＤ８+ Ｔ細胞に対して固定染色を行い、共役焦点顕微鏡によって実施例３がＣＤ８+ Ｔ細胞の細胞内部位に位置することを確認した。
図１２Ａは、細胞核の位置を示し、図１２Ｂは、実施例３の位置を示し、図１２Ｃは、明視野下の顕微鏡写真図である。図１２Ｄは、合併後の顕微鏡写真図であり、スケールバーは、５μＭの長さを示す。結果から分かるように、ＣＤ８+ Ｔ細胞で複数の実施例３が見られ、実施例３が確実にＣＤ８+ Ｔ細胞と結合できることが証明された。
With reference to FIGS. 12A to 12D, Example 3 is an analysis result diagram of the binding ability to CD8 + T cells. On a trial basis, the Example 3 in which the quantum dots are connected by culturing CD8 + T cells and 30 minutes, subjected to fixed stained for CD8 + T cells by Alexa Fluor 488 Phalloidin and DAPI, Example 3 by conjugate focal microscopy Was confirmed to be located at the intracellular site of CD8 + T cells.
12A shows the position of the cell nucleus, FIG. 12B shows the position of Example 3, and FIG. 12C is a photomicrograph under a bright field of view. FIG. 12D is a photomicrograph after the merger, with scale bars showing a length of 5 μM. As can be seen from the results, CD8 + T cells several of Example 3 was observed in proved to be Example 3 can be combined reliably CD8 + T cells.
（二、免疫磁気組成物の用途）
（２．１ 本発明の免疫磁気組成物による癌治療の治療効果）
本試験例は、乳癌肺転移及び大腸癌のマウスモデルで更に本発明の免疫磁気組成物が癌治療の効能を有するか、そして磁気誘導によって患部に蓄積されるかをテストした。
(Second, use of immunomagnetic composition)
(2.1 Therapeutic effect of cancer treatment by the immunomagnetic composition of the present invention)
This test example is a mouse model of breast cancer lung metastasis and colorectal cancer, and further tested whether the immunomagnetic composition of the present invention has a cancer therapeutic effect and whether it is accumulated in the affected area by magnetic induction.
（２．１．１ 本発明の免疫磁気組成物による乳癌肺転移の治療効果）
本試験例は、生物発光酵素を安定に発現するｌｕｃｉｆｅｒａｓｅ遺伝子を持つ４Ｔ１−Ｌｕｃ腫瘍マウスを乳癌肺転移の動物モデル（中国医薬大学付属病院分子医療センターによって提供される）として、マーカー１２５Ｉを含む実施例１−３の製剤を右大腿静脈を介して４Ｔ１−Ｌｕｃ腫瘍マウスに投与し、試験的に、別に実施例４（本発明の癌治療用のキット）があり、実施例４においてマーカー１２５Ｉを含む実施例３の製剤を投与し、円状表面の磁石（直径０．５ｃｍ，０．５Ｔｅｓｌａ）を合わせて磁気誘導を行った。続いて、単一光子放出コンピュータ断層撮影法（Ｓｉｎｇｌｅ Ｐｈｏｔｏｎ Ｅｍｉｓｓｉｏｎ Ｃｏｍｐｕｔｅｄ Ｔｏｍｏｇｒａｐｈｙ；ＳＰＥＣＴ）によって実施例３及び実施例４の腫瘍組織における動的蓄積をモニターした。
(2.1.1 Therapeutic effect of the immunomagnetic composition of the present invention on breast cancer lung metastasis)
In this test example, a 4T1-Luc tumor mouse having a luciferase gene that stably expresses bioluminescent enzyme was used as an animal model of breast cancer lung metastasis (provided by the Molecular Medical Center of China Pharmaceutical University Hospital), and included the marker 125I. The preparation of Example 1-3 was administered to 4T1-Luc tumor mice via the right femoral vein, and there is another Example 4 (kit for cancer treatment of the present invention) on a trial basis, and the marker 125I was used in Example 4. The preparation of Example 3 including the above was administered, and magnetic induction was performed by aligning a magnet (diameter 0.5 cm, 0.5 Tesla) with a circular surface. Subsequently, dynamic accumulation in the tumor tissues of Examples 3 and 4 was monitored by single photon emission computed tomography (SPECT).
図１３Ａ〜図１３Ｃを参照すると、本発明の癌治療用のキットが集中的に腫瘍に蓄積する分析結果図である。
図１３Ａは、１２５Ｉが標識される実施例３又は実施例４が４Ｔ１−Ｌｕｃ腫瘍マウスで第０、４、６、１２と２４時間にある時間活性曲線であり、ｎ＝４である。図１３Ｂは、４Ｔ１−Ｌｕｃ腫瘍マウスが１２５Ｉが標識される実施例３又は実施例４が投与された後の第２４時間の全身単一光子放出コンピュータ断層撮影法図である。図１３Ｃは、４Ｔ１−Ｌｕｃ腫瘍マウスが１２５Ｉが標識される実施例３又は実施例４が投与された後の、第２４時間の生体分布定量図であり、ｎ＝４であり、＊は統計的に明らかな差を示す（ｐ＜０．０５）。
With reference to FIGS. 13A to 13C, it is an analysis result diagram in which the kit for cancer treatment of the present invention intensively accumulates in a tumor.
FIG. 13A is a time activity curve in Example 3 or Example 4 labeled 125I at 0, 4, 6, 12 and 24 hours in 4T1-Luc tumor mice, n = 4. FIG. 13B is a 24 hour systemic single photon emission computed tomography diagram after administration of Example 3 or Example 4 in which 4T1-Luc tumor mice are labeled with 125I. FIG. 13C is a biological distribution quantitative chart at 24 hours after administration of Example 3 or Example 4 in which 4T1-Luc tumor mice are labeled with 125I, n = 4, and * is statistical. Shows a clear difference in (p <0.05).
図１３Ａの結果から分かるように、実施例３の腫瘍における蓄積は、投与後の第６時間に組織１ｇあたりに注射される用量の最大濃度（５．０８％ＩＤ／ｇ）に達した。対照的に、実施例４は、磁気誘導によって腫瘍の用量投与の領域性を促進し、実施例４が２４時間内に腫瘍で持続に蓄積することが見られ、第２４時間に、実施例４が腫瘍における蓄積量は実施例３の３．６倍となった。
図１３Ｂの結果から分かるように、実施例３又は実施例４を投与した後の２４時間後に、実施例３及び実施例４の間の放射性同位体は異なる生体分布パターンを有した。
図１３Ｃの生体分布定量図から分かるように、実施例３及び実施例４において、主に腫瘍、肝臓、脾臓及び膀胱に蓄積するが、筋肉、脳、心臓、肺、胃、腎臓、結腸と血液に蓄積されにくい（５％ＩＤ／ｇよりも小さい）。実施例３は実施例４と比べて、実施例４が腫瘍における蓄積を向上させることだけでなく、著しく肝臓と脾臓におけるその全身蓄積を低減して、実施例４が効果的に作用部位に集中し得ることを示す。
As can be seen from the results of FIG. 13A, the accumulation in the tumor of Example 3 reached the maximum concentration (5.08% ID / g) of the dose injected per 1 g of tissue 6 hours after administration. In contrast, Example 4 promoted the regionality of dose administration of the tumor by magnetic induction, and Example 4 was found to accumulate persistently in the tumor within 24 hours, and at 24 hours, Example 4 However, the amount accumulated in the tumor was 3.6 times that of Example 3.
As can be seen from the results of FIG. 13B, 24 hours after administration of Example 3 or Example 4, the radioisotopes between Examples 3 and 4 had different biodistribution patterns.
As can be seen from the biological distribution quantitative diagram of FIG. 13C, in Examples 3 and 4, the accumulation mainly in the tumor, liver, spleen and bladder, but in muscle, brain, heart, lung, stomach, kidney, colon and blood. Hard to accumulate in (smaller than 5% ID / g). In Example 3, as compared to Example 4, Example 4 not only improves the accumulation in the tumor, but also significantly reduces its systemic accumulation in the liver and spleen, and Example 4 effectively concentrates on the site of action. Show that it can be done.
本試験例は、更にそれぞれ右大腿静脈を介してＩｇＧ（対照組）、ＩＯ＠ＦｕＤｅｘ、実施例１−４を４Ｔ１−Ｌｕｃ腫瘍マウスに投与し、投与形態として腫瘍接種の８日後、４日ごとに連続して３回（ｑ４ｄｘ３）投与した。図１３Ｄ及び添付ファイル１を参照すると、添付ファイル１は、図１３Ｄのカラー図面である。４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４を投与した後の４週のヘマトキシリン−エオシン染色がプロシアンブルー染色に合わせる結果図である。スケールバーは５０μＭの長さを示す。図１３Ｄの結果から分かるように、他の群と比べて、実施例４は腫瘍組織において散在したプロシアンブルー染色結果が観察された。 In this test example, IgG (control group), IO @ FuDex, and Example 1-4 were further administered to 4T1-Luc tumor mice via the right femoral vein, respectively, and the administration form was 8 days after tumor inoculation and every 4 days. Was administered three times in succession (q4dx3). With reference to FIG. 13D and Attachment 1, Attachment 1 is a color drawing of FIG. 13D. It is a result figure that hematoxylin-eosin staining at 4 weeks after administration of Example 1-4 to 4T1-Luc tumor mice is matched with Procyan blue staining. The scale bar indicates a length of 50 μM. As can be seen from the results in FIG. 13D, compared with the other groups, Example 4 observed scattered Procyan blue staining results in the tumor tissue.
本発明の免疫磁気組成物及び癌治療用のキットが４Ｔ１−Ｌｕｃ腫瘍マウスに対して治療効果を有することを確認するために、本試験例では、更に右大腿静脈からＩｇＧ（対照組）、ＩＯ＠ＦｕＤｅｘ、実施例１−４を４Ｔ１−Ｌｕｃ腫瘍マウスに投与し、投与形態として腫瘍接種の８日後、４日ごとに連続して３回（ｑ４ｄｘ３）投与した。腫瘍体積をデジタルキャリパー（ｍｉｔｕｔｏｙｏ）を用いて２〜３日ごとにモニターし、腫瘍体積を以下の式Ｉに基づいて計算した。 In order to confirm that the immunomagnetic composition of the present invention and the kit for treating cancer have a therapeutic effect on 4T1-Luc tumor mice, in this test example, IgG (control group) and IO from the right femoral vein are further added. @ FuDex, Examples 1-4 were administered to 4T1-Luc tumor mice, and as the administration form, 8 days after tumor inoculation, 3 times (q4dx3) were continuously administered every 4 days. Tumor volume was monitored every 2-3 days using a digital caliper (mitutoyo) and tumor volume was calculated based on Formula I below.
非侵襲的生体内分子イメージングシステム（Ｎｏｎ Ｉｎｖａｓｉｏｎ Ｉｎ Ｖｉｖｏ Ｉｍａｇｉｎｇ Ｓｙｓｔｅｍ，ＩＶＩＳ；Ｘｅｎｏｇｅｎ）によって生物発光評価を行った。４Ｔ１−Ｌｕｃ腫瘍マウスの生存率は、Ｋａｐｌａｎ−Ｍｅｉｅｒ法によって分析した。 Bioluminescence evaluation was performed by a non-invasive in vivo molecular imaging system (Non Invasion In Vivo Imaging System, IVIS; Xenogen). Survival rates of 4T1-Luc tumor mice were analyzed by the Kaplan-Meier method.
図１４Ａ〜図１４Ｅ及び添付ファイル２を参照すると、本発明の免疫磁気組成物及び癌治療用のキットが乳癌肺転移マウスモデルで癌細胞増殖と癌転移を阻害する分析結果図である。
図１４Ａは、４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４が投与された後の２４時間の非侵襲的生体内分子イメージングシステムスキャン結果図である。添付ファイル２は、図１４Ａのカラー図面である。図１４Ｂは、４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４が投与された後の腫瘍体積統計図である。図１４Ｃは、４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４が投与された後の生存曲線である。図１４Ｄは、４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４が投与された後の腫瘍と肺臓の写真図である。図１４Ｅは、４Ｔ１−Ｌｕｃ腫瘍マウスに実施例１−４が投与された後の肺転移の統計図である。
With reference to FIGS. 14A-14E and Attachment 2, it is an analysis result diagram in which the immunomagnetic composition of the present invention and a kit for treating cancer inhibit cancer cell proliferation and cancer metastasis in a mouse model of breast cancer lung metastasis.
FIG. 14A is a 24-hour non-invasive in-vivo molecular imaging system scan result diagram after administration of Example 1-4 to 4T1-Luc tumor mice. Attachment 2 is a color drawing of FIG. 14A. FIG. 14B is a tumor volume statistical diagram after administration of Example 1-4 to 4T1-Luc tumor mice. FIG. 14C is a survival curve after administration of Examples 1-4 to 4T1-Luc tumor mice. FIG. 14D is a photograph of the tumor and lung after administration of Example 1-4 to 4T1-Luc tumor mice. FIG. 14E is a statistical diagram of lung metastasis after administration of Example 1-4 to 4T1-Luc tumor mice.
図１４Ａと図１４Ｂの結果から分かるように、対照組と比べて、ＩＯ＠ＦｕＤｅｘは、腫瘍生長を阻害できるが、統計的に明らかな差異がなかった。実施例１−４は、抗腫瘍効果を有する以外、且つ統計的に明らかな差異があり（＊はｐ＜０．０５を示し、＊＊はｐ＜０．０１を示す）、特に、実施例４は、３０日以内に腫瘍の増殖をほとんど阻害した。
図１４Ｃの結果から分かるように、対照組、ＩＯ＠ＦｕＤｅｘ、実施例１、実施例２、実施例３及び実施例４を投与した４Ｔ１−Ｌｕｃ腫瘍マウスの生存期間の中央値は、それぞれ２４日、３４日、３４日、４３天と４４日であった。実施例４を投与した４Ｔ１−Ｌｕｃ腫瘍マウスの生存期間の中央値は、著しく６３日まで延長した。実施例４におけるＰＤ−Ｌ１抗体の投与量は、普通の純粋な抗体投与量の１％しかないが、より優れた腫瘍抑制能力を示して、半減期が２倍以上に増加した。
また、図１４Ｄの結果から分かるように、実施例１−４は何れも抗腫瘍転移能力を有した。図１４Ｄにおいて、他の群と比べて、実施例３及び実施例４の投与は著しく肺腫瘍転移を阻害できる。
別に図１４Ｅの結果から分かるように、対照組の４Ｔ１−Ｌｕｃ腫瘍マウスの肺に２０の結節を超える肺転移を有し、実施例４が投与される４Ｔ１−Ｌｕｃ腫瘍マウスの肺に平均して５つの結節未満の肺転移が発見された。従って、上記の結果から分かるように、本発明の免疫磁気組成物及び癌治療用のキットは、明らかな阻害腫瘍と抗腫瘍転移作用を有した。対照組と比べて、ＩＯ＠ＦｕＤｅｘは、腫瘍の転移を著しく減少させなかった。本発明の免疫磁気組成物及び癌治療用のキットの有する抗腫瘍転移作用は、フコイダンの阻害作用に加え、腫瘍微小環境の動的応答に関連することを推測した。
As can be seen from the results of FIGS. 14A and 14B, IO @ FuDex was able to inhibit tumor growth as compared to the control group, but there was no statistically clear difference. Examples 1-4 have a statistically clear difference other than having an antitumor effect (* indicates p <0.05, ** indicates p <0.01), and in particular, Examples. 4 almost inhibited the growth of the tumor within 30 days.
As can be seen from the results in FIG. 14C, the median survival time of 4T1-Luc tumor mice treated with the control group, IO @ FuDex, Example 1, Example 2, Example 3 and Example 4 was 24 days, respectively. , 34th, 34th, 43rd and 44th. The median survival time of 4T1-Luc tumor mice treated with Example 4 was significantly extended to 63 days. The dose of PD-L1 antibody in Example 4 was only 1% of the normal pure antibody dose, but showed better tumor suppressor capacity and the half-life was more than doubled.
Moreover, as can be seen from the results of FIG. 14D, all of Examples 1-4 had antitumor metastasis ability. In FIG. 14D, administration of Example 3 and Example 4 can significantly inhibit lung tumor metastasis as compared to the other groups.
Separately, as can be seen from the results of FIG. 14E, the lungs of the 4T1-Luc tumor mice of the control group had lung metastases exceeding 20 nodules, and the lungs of the 4T1-Luc tumor mice to which Example 4 was administered were averaged. Less than 5 nodules of lung metastases were found. Therefore, as can be seen from the above results, the immunomagnetic composition of the present invention and the kit for treating cancer had a clear inhibitory tumor and antitumor metastatic effect. Compared to the control group, IO @ FuDex did not significantly reduce tumor metastasis. It was speculated that the antitumor metastatic effect of the immunomagnetic composition of the present invention and the kit for treating cancer is related to the dynamic response of the tumor microenvironment in addition to the inhibitory effect of fucoidan.
（２．１．２ 本発明の免疫磁気組成物による大腸癌の治療効果）
本試験例は、生物発光酵素を安定に発現するｌｕｃｉｆｅｒａｓｅ遺伝子を持つＣＴ−２６細胞株を大腸癌の動物モデル（中国医薬大学付属病院分子医療センターによって提供される）として、ＩｇＧ（対照組）、ＩＯ＠ＦｕＤｅｘ及び実施例３を右大腿静脈を介してＣＴ−２６腫瘍マウスに投与し、試験的に、別に実施例３が投与され円状表面の磁石（直径０．５ｃｍ，０．５Ｔｅｓｌａ）を合わせて磁気誘導を行う実施例４（本発明の癌治療用のキット）、及びＩＯ＠ＦｕＤｅｘが投与され円状表面の磁石を合わせて磁気誘導を行う比較例１があった。投与形態として、腫瘍接種の８日後、４日ごとに連続して３回（ｑ４ｄｘ３）投与した。腫瘍体積をデジタルキャリパーを用いて２〜３日ごとにモニターし、腫瘍体積を前記の式Ｉに基づいて計算し、非侵襲的生体内分子イメージングシステムによって生物発光評価を行った。ＣＴ−２６腫瘍マウスの生存率は、Ｋａｐｌａｎ−Ｍｅｉｅｒ法によって分析した。
(2.1.2 Therapeutic effect of the immunomagnetic composition of the present invention on colorectal cancer)
In this test example, a CT-26 cell line having a luciferase gene that stably expresses bioluminescent enzyme was used as an animal model of colorectal cancer (provided by the Molecular Medical Center of the Chinese Pharmaceutical University Hospital), and IgG (control group), IO @ FuDex and Example 3 were administered to CT-26 tumor mice via the right femoral vein, and Example 3 was separately administered on a trial basis to magnetize a circular surface (0.5 cm in diameter, 0.5 Tesla). There was Example 4 (kit for cancer treatment of the present invention) in which magnetic induction was performed together, and Comparative Example 1 in which IO @ FuDex was administered and magnets on a circular surface were combined to perform magnetic induction. As an administration form, 8 days after tumor inoculation, 3 times (q4dx3) were continuously administered every 4 days. Tumor volume was monitored every 2-3 days using a digital caliper, tumor volume was calculated based on Formula I above, and bioluminescence evaluation was performed by a non-invasive in vivo molecular imaging system. Survival of CT-26 tumor mice was analyzed by the Kaplan-Meier method.
図１５Ａ〜図１５Ｃ及び添付ファイル３を参照すると、本発明の免疫磁気組成物及び癌治療用のキットが大腸癌マウスモデルで癌細胞増殖を阻害する分析結果図である。
図１５Ａは、ＣＴ−２６腫瘍マウスに実施例３−４が投与された後の２４時間の非侵襲的生体内分子イメージングシステムスキャン結果図である。添付ファイル３は、図１５Ａのカラー図面であり。図１５Ｂは、ＣＴ−２６腫瘍マウスに実施例３−４が投与された後の腫瘍体積統計図である。図１５Ｃは、ＣＴ−２６腫瘍マウスに実施例３−４が投与された後の生存曲線である。
With reference to FIGS. 15A-15C and Attachment 3, it is an analysis result diagram in which the immunomagnetic composition of the present invention and a kit for treating cancer inhibit cancer cell growth in a mouse model of colorectal cancer.
FIG. 15A is a 24-hour non-invasive in-vivo molecular imaging system scan result diagram after administration of Example 3-4 to CT-26 tumor mice. Attachment 3 is a color drawing of FIG. 15A. FIG. 15B is a tumor volume statistical diagram after administration of Example 3-4 to CT-26 tumor mice. FIG. 15C is a survival curve after administration of Example 3-4 to CT-26 tumor mice.
図１５Ａと図１５Ｂの結果から分かるように、対照組と比べて、ＩＯ＠ＦｕＤｅｘは、腫瘍生長を阻害できるが、統計的に明らかな差異がなかった。ＩＯ＠ＦｕＤｅｘを投与し磁気誘導を合わせる比較例１は、ＩＯ＠ＦｕＤｅｘの治療効果を向上させることができた。本発明の実施例３−４は、抗腫瘍効果を有する以外、且つ統計的に明らかな差異があり（＊はｐ＜０．０５を示し、＊＊はｐ＜０．０１を示す）、特に、実施例４は、３０日以内に腫瘍の増殖をほとんど阻害した。
図１５Ｃの結果から分かるように、実施例３及び実施例４を投与すると、著しくマウスの生存時間を延長して、本発明の免疫磁気組成物が腫瘍生長と転移を阻害する効果があることが再び証明された。
As can be seen from the results of FIGS. 15A and 15B, IO @ FuDex was able to inhibit tumor growth as compared to the control group, but there was no statistically clear difference. In Comparative Example 1 in which IO @ FuDex was administered and the magnetic induction was combined, the therapeutic effect of IO @ FuDex could be improved. Examples 3-4 of the present invention have a statistically clear difference other than having an antitumor effect (* indicates p <0.05, ** indicates p <0.01), and in particular. Example 4 almost inhibited the growth of the tumor within 30 days.
As can be seen from the results of FIG. 15C, administration of Example 3 and Example 4 significantly prolongs the survival time of mice, and the immunomagnetic composition of the present invention has an effect of inhibiting tumor growth and metastasis. Proven again.
（２．２ 本発明の免疫磁気組成物を投与した後の免疫作用の分析）
本試験例は、更に４Ｔ１腫瘍モデルで、本発明の免疫磁気組成物及び癌治療用のキットの腫瘍内の微環境における免疫作用を検討した。初期腫瘍（１０日）から晩期（３０日）の４Ｔ１−Ｌｕｃ腫瘍マウスの腫瘍、血液、腹水及び脾臓におけるリンパ球の数の変化を実験的にモニターした。
(2.2 Analysis of immune action after administration of the immunomagnetic composition of the present invention)
In this test example, a 4T1 tumor model was used to examine the immune action of the immunomagnetic composition of the present invention and the kit for treating cancer in the microenvironment within the tumor. Changes in the number of lymphocytes in tumors, blood, ascites and spleen of 4T1-Luc tumor mice from early tumor (10 days) to late (30 days) were experimentally monitored.
図１６Ａ〜図１６Ｉを参照すると、本発明の免疫磁気組成物及び癌治療用のキットを投与した後の腫瘍微小環境における腫瘍浸潤リンパ球数変化とサイトカイン含有量変化の分析結果図である。
図１６Ａと図１６Ｂの結果から分かるように、実施例１−４が投与される群、特に、実施例４が投与される群（ｐ＜０．０１）は、著しく抗腫瘍リンパ球細胞、例えば、ＣＤ８+ Ｔ細胞とＣＤ４+ Ｔ細胞の数を向上させることができた。
図１６Ｃと図１６Ｄの結果から分かるように、実施例１−４が投与される群は、著しく前腫瘍抗腫瘍リンパ球細胞、例えばＣＤ４+ＣＤ２２５+Ｆｏｘｐ３+Ｔｒｅｇ（調節性Ｔ細胞）とＣＤ１１ｂ+ＣＤ２０６+Ｆ４／８０+ＴＡＭ（腫瘍関連マクロファージ）の数を低減させることができ、特に、実施例４が投与される群は、大幅に調節性Ｔ細胞（Ｔｒｅｇ）数（ｐ＜０．０１）を低減させることができた。
図１６Ｅ〜図１６Ｇにおいて、異なる群の４Ｔ１−Ｌｕｃ腫瘍マウス血清を回収し、ＴＡＭによって分泌されたＴＮＦ−α、ＶＥＧＦとＴＧＦ−βの含有量を分析し、実施例１−４が投与される群、特に、実施例４が投与される群（ｐ＜０．０１）は、著しくＴＮＦ−α、ＶＥＧＦとＴＧＦ−β等の前炎症性サイトカインの含有量を低減させることができた。
図１６Ｈと図１６Ｉにおいて、細胞内グランザイムＢ（ＧｒＢ+）とＫｉ６７の発現レベルによってＣＤ８+ Ｔ細胞の活性化程度を評価して、実施例１−４が投与される群、特に、実施例４が投与される群（ｐ＜０．０１）は、ＣＤ８+腫瘍浸潤リンパ球細胞の機能を効果的に活性化することができた。
With reference to FIGS. 16A to 16I, it is an analysis result diagram of the change in the number of tumor-infiltrating lymphocytes and the change in the cytokine content in the tumor microenvironment after administration of the immunomagnetic composition of the present invention and the kit for treating cancer.
As can be seen from the results of FIGS. 16A and 16B, the group to which Examples 1-4 are administered, particularly the group to which Example 4 is administered (p <0.01), are markedly antitumor lymphocyte cells, eg, , CD8 + T cells and CD4 + T cells could be improved.
As can be seen from the results of FIGS. 16C and 16D, the group to which Example 1-4 was administered was significantly pretumor antitumor lymphocyte cells such as CD4 + CD225 + Foxp3 + Treg (regulatory T cells) and CD11b +. The number of CD206 + F4 / 80 + TAM (tumor-related macrophages) can be reduced, especially in the group to which Example 4 is administered, the number of regulatory T cells (Tregs) (p <0.01). Was able to be reduced.
In FIGS. 16E-16G, different groups of 4T1-Luc tumor mouse sera are collected, the content of TNF-α, VEGF and TGF-β secreted by TAM is analyzed and Example 1-4 is administered. The group, in particular the group to which Example 4 was administered (p <0.01), was able to significantly reduce the content of pre-inflammatory cytokines such as TNF-α, VEGF and TGF-β.
In FIGS. 16H and 16I, the degree of activation of CD8 + T cells was evaluated based on the expression levels of intracellular granzyme B (GrB + ) and Ki67, and the group to which Examples 1-4 were administered, particularly Example 4. The group to which was administered (p <0.01) was able to effectively activate the function of CD8 + tumor-infiltrating lymphocyte cells.
前記試験例で本発明の免疫磁気組成物及び癌治療用のキットを投与すると腫瘍微小環境を変えることができると確認した後で、本試験例で更に本発明の免疫磁気組成物及び癌治療用のキットの腫瘍に対する特異的免疫応答と全身作用の変化を検討した。試験的に、異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスの腫瘍、血清、脾臓を回収し、ＩＮＦ−γ +ＣＤ４４+ Ｔ細胞とＣＤ８+ＣＤ３+ Ｔ細胞の変化をモニターして、ＴＵＮＥＬテストによって異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスの皮膚組織のアポトーシス状況を観察した。 After confirming that the tumor microenvironment can be changed by administering the immunomagnetic composition of the present invention and the kit for cancer treatment in the above test example, the immunomagnetic composition of the present invention and the kit for cancer treatment are further described in the present test example. The kit's specific immune response to tumors and changes in systemic effects were investigated. On a trial basis, tumors, serum, and spleen of 4T1-Luc tumor mice in different groups were collected, and changes in INF- γ + CD44 + T cells and CD8 + CD3 + T cells were monitored, and different groups were tested by the TUNEL test. The apoptosis status of the skin tissue of 4T1-Luc tumor mice was observed.
図１７Ａと図１７Ｂを参照すると、本発明の免疫磁気組成物及び癌治療用のキットの反応部位の分析結果図である。図１７Ａは、異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスのＩＮＦ−γ +ＣＤ４４+ Ｔ細胞の変化結果図である。図１７Ｂは、異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスのＣＤ８+ＣＤ３+ Ｔ細胞の変化結果図である。 With reference to FIGS. 17A and 17B, it is an analysis result diagram of the reaction site of the immunomagnetic composition of the present invention and the kit for treating cancer. FIG. 17A is a change result diagram of INF- γ + CD44 + T cells of 4T1-Luc tumor mice in different groups. FIG. 17B is a change result diagram of CD8 + CD3 + T cells of 4T1-Luc tumor mice in different groups.
図１７Ａの結果から分かるように、ＩＮＦ−γ +ＣＤ４４+ Ｔ細胞は実施例１−４が投与された後で増殖の数が向上し、特に、実施例４が投与される群は高く増殖した。図１７Ｂの結果から分かるように、実施例１−３が投与された後で、ＣＤ８+ＣＤ３+ Ｔ細胞が血清と脾臓で何れも増殖するが、実施例４が投与される群と実施例３が投与される群と比べて、磁気誘導によってＣＤ８+ＣＤ３+ Ｔ細胞が血清と脾臓における増殖を低減させた。 As can be seen from the results of FIG. 17A, the number of proliferation of INF- γ + CD44 + T cells increased after the administration of Examples 1-4, and in particular, the group to which Example 4 was administered proliferated highly. .. As can be seen from the results of FIG. 17B, after the administration of Example 1-3, both CD8 + CD3 + T cells proliferate in the serum and spleen, but the group to which Example 4 is administered and Example 3 CD8 + CD3 + T cells reduced proliferation in serum and spleen by magnetic induction compared to the group receiving.
皮膚の毒性によるかゆみや白斑が免疫チェックポイント阻害剤治療における副作用の一つであり、免疫関連の有害事象がしばしば長期間の場合に起こるので、本試験例では、腫瘍が接種された4週間後で、４Ｔ１−Ｌｕｃ腫瘍マウスの皮膚組織に対してＴＵＮＥＬテストを行って、浸潤しているＴ細胞が免疫応答を誘発し皮膚組織損傷を引き起こすかを評価した。 Itching and white spots due to skin toxicity are one of the side effects of immune checkpoint inhibitor treatment, and immune-related adverse events often occur over a long period of time. Therefore, in this study example, 4 weeks after tumor infiltration. Then, a TUNEL test was performed on the skin tissue of 4T1-Luc tumor mice to evaluate whether the infiltrating T cells induce an immune response and cause skin tissue damage.
添付ファイル４、図１７Ｃと図１７Ｄを参照すると、本発明の免疫磁気組成物及び癌治療用のキットのＴＵＮＥＬテスト結果図である。図１７Ｃは、ＴＵＮＥＬテストによる異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスの皮膚組織アポトーシス指数の統計結果図である。アポトーシス指数は、ＴＵＮＥＬ+アポトーシス核の比例を細胞核総数（ランダムに選択された微小範囲）で割ったものとして、２因子の変動性の分析とＮｅｗｍａｎ−Ｋｅｕｌｓ事後比較試験を用いて異なる群の間の差異を評価した。図１７Ｄは、ＴＵＮＥＬテストによる異なる群の４Ｔ１−Ｌｕｃ腫瘍マウスの蛍光顕微鏡写真図であり、スケールバーは、５０μＭの長さを示す。添付ファイル４は、図１７Ｄのカラー図面である。 With reference to Attachment 4, FIGS. 17C and 17D, it is a TUNEL test result diagram of the immunomagnetic composition of the present invention and the kit for treating cancer. FIG. 17C is a statistical result diagram of the skin tissue apoptosis index of 4T1-Luc tumor mice in different groups by the TUNEL test. The apoptotic index is the proportion of TUNEL + apoptotic nuclei divided by the total number of cell nuclei (a small range randomly selected) between different groups using a two-factor variability analysis and a Newman-Keuls ex-post comparison study. The difference was evaluated. FIG. 17D is a fluorescence micrograph of 4T1-Luc tumor mice from different groups by TUNEL test, with scale bars showing a length of 50 μM. Attachment 4 is a color drawing of FIG. 17D.
図１７Ｃと図１７Ｄの結果から分かるように、実施例１−３が投与された後で、ＴＵＮＥＬで標識される（緑色）アポトーシス細胞が増加し、対照群と比べて実施例３が投与される群のアポトーシス指数が３．３倍増加した。しかしながら、実施例４が投与される群は実施例３が投与される群と比べて、磁気誘導は効果的に皮膚組織におけるアポトーシス細胞の数を低減させることができた。 As can be seen from the results of FIGS. 17C and 17D, after the administration of Examples 1-3, the number of TUNEL-labeled (green) apoptotic cells increased, and Example 3 was administered as compared to the control group. The apoptosis index of the group increased by 3.3 times. However, in the group to which Example 4 was administered, magnetic induction was able to effectively reduce the number of apoptotic cells in the skin tissue as compared with the group to which Example 3 was administered.
（２．３ 本発明の免疫磁気組成物の免疫関連の有害事象試験）
本試験例において更に本発明の免疫磁気組成物又は癌治療用のキットを投与した４Ｔ１腫瘍モデルで、試験的に実施例３又は実施例４を４Ｔ１腫瘍マウスに投与し、腫瘍が接種された４週後、４Ｔ１腫瘍マウスに対して免疫関連有害事象（Ｉｍｍｕｎｅ−ｒｅｌａｔｅｄ ａｄｖｅｒｓｅ ｅｖｅｎｔｓ；ｉｒＡＥｓ）をテストして、本発明の免疫磁気組成物及び癌治療用のキットの安全性を分析した。
(2.3 Immunity-related adverse event test of the immunomagnetic composition of the present invention)
In this test example, in a 4T1 tumor model further administered with the immunomagnetic composition of the present invention or a kit for treating cancer, Example 3 or Example 4 was experimentally administered to 4T1 tumor mice, and the tumor was inoculated 4 After a week, 4T1 tumor mice were tested for immune-related advanced events (irAEs) to analyze the safety of the immunomagnetic compositions of the invention and kits for the treatment of cancer.
本試験例は、実施例３及び実施例４によって治療された４週後のマウスのＣＤ４+ Ｔ細胞及びＣＤ８+ Ｔ細胞の主要臓器への浸潤程度を観察して副作用の影響を判断した。図１８Ａ〜図１８Ｅを参照すると、本発明の免疫磁気組成物及び癌治療用のキットを投与した後のマウスＣＤ４+ Ｔ細胞とＣＤ８+ Ｔ細胞の浸潤程度の分析図である。
図１８Ａは、肝臓の分析結果図である。図１８Ｂは、肺臓の分析結果図である。図１８Ｃは、脾臓の分析結果図である。図１８Ｄは、腎臓の分析結果図である。図１８Ｅは、大腸の分析結果図である。結果から分かるように、実施例３は肝臓、肺臓、脾臓、腎臓と大腸へのＴ細胞浸潤程度が対照組よりも低く、実施例４は磁石の吸引作用によって、更に周りへの蓄積（肝臓、肺臓、脾臓、腎臓と大腸）を著しく低減させることができ、更にＴ細胞の浸潤を低減させた。
In this test example, the effect of side effects was determined by observing the degree of infiltration of CD4 + T cells and CD8 + T cells of mice treated by Example 3 and Example 4 into major organs after 4 weeks. 18A-18E are analytical diagrams of the degree of infiltration of mouse CD4 + T cells and CD8 + T cells after administration of the immunomagnetic composition of the present invention and the kit for treating cancer.
FIG. 18A is an analysis result diagram of the liver. FIG. 18B is an analysis result diagram of the lungs. FIG. 18C is an analysis result diagram of the spleen. FIG. 18D is an analysis result diagram of the kidney. FIG. 18E is an analysis result diagram of the large intestine. As can be seen from the results, Example 3 has a lower degree of T cell infiltration into the liver, lungs, spleen, kidneys and large intestine than the control group, and Example 4 further accumulates around (liver,) due to the attractive action of the magnet. The liver, spleen, kidneys and large intestine) could be significantly reduced, and the infiltration of T cells was further reduced.
本試験例において更に血液生化分析によって、肝機能指標（ＡＳＴとＡＬＴ）、腎機能指標（クレアチニン）及び血糖値を分析して副作用の影響を判断する。図１９Ａ〜図１９Ｄを参照すると、本発明の免疫磁気組成物及び癌治療用のキットを投与した後のマウス血液生化学分析の結果図である。
図１９Ａは、ＡＳＴ値分析図である。図１９Ｂは、ＡＬＴ値分析図である。図１９Ｃは、クレアチニン値分析図である。図１９Ｄは、血糖値分析図である。図面から分かるように、実施例３又は実施例４を投与した治療群と対照組の生化値がやはり正常値の範囲に保持され、本発明の免疫磁気組成物及び癌治療用のキットはある程度の安全性を有することを示す。
In this test example, liver function indexes (AST and ALT), renal function indexes (creatinine) and blood glucose levels are further analyzed by blood biochemistry analysis to determine the effects of side effects. 19A to 19D are the results of mouse blood biochemical analysis after administration of the immunomagnetic composition of the present invention and the kit for treating cancer.
FIG. 19A is an AST value analysis diagram. FIG. 19B is an ALT value analysis diagram. FIG. 19C is a creatinine level analysis diagram. FIG. 19D is a blood glucose level analysis diagram. As can be seen from the drawings, the biochemical values of the treatment group and the control group to which Example 3 or Example 4 was administered are also maintained in the normal range, and the immunomagnetic composition of the present invention and the kit for cancer treatment have a certain degree. Shows that it has safety.
また、腫瘍が接種された４週後のマウスに対して病理学的切片の分析を行い、図２０及添付ファイル５を参照すると、実施例３及び実施例４を投与した後のマウスの病理学的切片の画像である。添付ファイル５は、図５Ａのカラー図面である。結果から分かるように、対照組と比べて、実施例３又は実施例４を投与した治療群は、肝臓、肺臓、脾臓、腎臓及び大腸において明らかな組織損傷がなく、本発明の免疫磁気組成物及び癌治療用のキットはある程度の安全性を有することを示す。 In addition, pathological sections were analyzed for mice 4 weeks after inoculation with the tumor, and referring to FIG. 20 and Attachment 5, the pathology of the mice after administration of Examples 3 and 4 It is an image of a target section. Attachment 5 is a color drawing of FIG. 5A. As can be seen from the results, the treatment group to which Example 3 or Example 4 was administered had no obvious tissue damage in the liver, lungs, spleen, kidneys and large intestine as compared to the control group, and the immunomagnetic composition of the present invention. And the kits for cancer treatment are shown to have some degree of safety.
要するに、本発明の免疫磁気組成物は、調製方法の製造プロセスが簡単であり、抗癌活性を有するフコイダンを担体の構成として、超常磁性酸化鉄ナノ粒子を組み合わせて、外層が抗体をグラフトすることができ活性物質をコア層に覆うことができる免疫磁気組成物を形成して、調製された免疫磁気組成物は、ナノスケール構造であり、そのサイズが適切で腫瘍に浸透して、腫瘍に対するフコイダンの役割を強めることができた。外層の抗体は、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤であってもよく、これによって本発明の免疫磁気組成物が自体材料の抗癌作用に加えて、免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤の両方であってもよく、腫瘍微小環境が大幅に改善され、且つ本発明の免疫磁気組成物は、単独の同じ抗体による免疫療法の抗癌効果を大幅に改善し、より少ない抗体用量でより良好な腫瘍抑制能力を達成することができた。また、製造された免疫磁気組成物は、凍結乾燥して粉末状の結晶を形成し、無菌条件下で長時間保存することができ、必要に応じて溶媒で再溶解して用いることができ、簡便で安定した特性を示す。 In short, in the immunomagnetic composition of the present invention, the manufacturing process of the preparation method is simple, fucoidan having anticancer activity is used as a carrier, and superparamagnetic iron oxide nanoparticles are combined, and the outer layer grafts the antibody. The immunomagnetic composition prepared by forming an immunomagnetic composition capable of covering the core layer with an active substance can have a nanoscale structure, is of an appropriate size and penetrates the tumor, and fucoidan against the tumor. I was able to strengthen the role of. The antibody in the outer layer may be an immune checkpoint inhibitor and / or a killer T cell proliferation agent, whereby the immunomagnetic composition of the present invention can be an immune checkpoint inhibitor and / or an immune checkpoint inhibitor in addition to the anti-cancer effect of its own material. / Or both killer T cell proliferators, the tumor microenvironment is significantly improved, and the immunomagnetic compositions of the present invention significantly improve the anti-cancer effect of immunotherapy with the same antibody alone. Better tumor suppressor capacity could be achieved with lower antibody doses. Further, the produced immunomagnetic composition can be freeze-dried to form powdery crystals, can be stored for a long time under aseptic conditions, and can be redissolved in a solvent and used if necessary. Shows simple and stable characteristics.
本発明の癌治療用キットは、本発明の免疫磁気組成物及び磁場発生装置を含み、磁場発生装置によって磁場を発生させ、磁気誘導の補助工具として、本発明の免疫磁気組成物を患部に蓄積して、腫瘍で免疫細胞の増殖が強く、且つ全身の免疫応答を低下させることができ、本発明の免疫磁気組成物の抗癌効果を更に高めることができる。前記試験のデータから確認されるように、局所に対する治療能力を有し、本発明の癌治療用キットは、同時に物理的及び生物学的目的作用を有するので、免疫療法及び化学療法の組み合わせ、又は免疫療法の化合物治療に寄与し、過剰な免疫反応による重篤な副作用を避ける。 The cancer treatment kit of the present invention includes the immunomagnetic composition of the present invention and a magnetic field generator, generates a magnetic field by the magnetic field generator, and accumulates the immunomagnetic composition of the present invention in an affected area as an auxiliary tool for magnetic induction. Thus, the proliferation of immune cells in the tumor is strong, the systemic immune response can be reduced, and the anticancer effect of the immunomagnetic composition of the present invention can be further enhanced. As confirmed from the data of the test, the kit for treating cancer of the present invention has a therapeutic ability locally and has physical and biological objective effects at the same time, so that it is a combination of immunotherapy and chemotherapy, or Contributes to compound therapy of immunotherapy and avoids serious side effects due to excessive immune response.
本発明の実施形態を前述の通りに開示したが、これは、本発明を限定するものではなく、当業者であれば、本発明の精神と範囲から逸脱しない限り、多様の変更や修飾を加えてもよく、したがって、本発明の保護範囲は、特許請求の範囲で指定した内容を基準とするものである。 Although the embodiments of the present invention have been disclosed as described above, this does not limit the present invention, and those skilled in the art can make various changes and modifications as long as they do not deviate from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is based on the content specified in the claims.
１００ 免疫磁気組成物
１１０ コア層
１２０ シェル層
１３０ 外層
１３１ 抗体
３００ 免疫磁気組成物の調製方法
３１０、３２０、３３０、３４０、３５０ 工程
100 Immunomagnetic composition 110 Core layer 120 Shell layer 130 Outer layer 131 Antibody 300 Preparation method of immunomagnetic composition 310, 320, 330, 340, 350 steps
Claims (16)
フコイダン、酸化デキストラン及び複数の超常磁性酸化鉄ナノ粒子が疎水性相互作用によって結合される複合物からなり、前記コア層を覆うシェル層と、
免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤である少なくとも１つの抗体を含む外層と、
を備え、
前記抗体は、前記シェル層の外にグラフトされ前記外層を構成する免疫磁気組成物。 With the core layer
A shell layer comprising fucoidan, dextran oxide and a plurality of superparamagnetic iron oxide nanoparticles bonded by hydrophobic interaction, and a shell layer covering the core layer.
An outer layer containing at least one antibody that is an immune checkpoint inhibitor and / or a killer T cell proliferative agent.
With
The antibody is an immunomagnetic composition that is grafted on the outside of the shell layer to form the outer layer.
有機溶媒及び超常磁性酸化鉄ナノ粒子を含む油相溶液を提供する工程と、
前記水相溶液及び前記油相溶液を混合し乳濁液を形成するエマルジョン反応を行う工程と、
前記乳濁液における前記有機溶媒を除去して、磁気フコイダン担体を形成する工程と、
前記磁気フコイダン担体と免疫チェックポイント阻害剤及び／又はキラーＴ細胞増殖剤である少なくとも１つの抗体を混合して、免疫磁気組成物を形成するグラフトを行う工程と、
を備える免疫磁気組成物の調製方法。 A step of providing an aqueous phase solution containing fucoidan and dextran oxide,
A step of providing an oil phase solution containing an organic solvent and superparamagnetic iron oxide nanoparticles,
A step of performing an emulsion reaction in which the aqueous phase solution and the oil phase solution are mixed to form an emulsion, and
A step of removing the organic solvent in the emulsion to form a magnetic fucoidan carrier, and
A step of mixing the magnetic fucoidan carrier with at least one antibody which is an immune checkpoint inhibitor and / or a killer T cell proliferating agent to perform a graft to form an immunomagnetic composition.
A method for preparing an immunomagnetic composition comprising.
磁場発生装置と、
を含む癌治療用キット。 The immunomagnetic composition according to any one of claims 1 to 8, and the immunomagnetic composition.
Magnetic field generator and
Cancer treatment kit including.
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