JP2008174466A - Carrier for idiotype antigen and idiotype vaccine using the same - Google Patents

Carrier for idiotype antigen and idiotype vaccine using the same Download PDF

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JP2008174466A
JP2008174466A JP2007007561A JP2007007561A JP2008174466A JP 2008174466 A JP2008174466 A JP 2008174466A JP 2007007561 A JP2007007561 A JP 2007007561A JP 2007007561 A JP2007007561 A JP 2007007561A JP 2008174466 A JP2008174466 A JP 2008174466A
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antigen
idiotype
cells
carrier
crystals
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JP5084012B2 (en
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Hiroshi Tsuya
寛 津谷
Ippei Sakamaki
一平 酒巻
Kunihiro Inai
邦博 稲井
Takanori Ueda
孝典 上田
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Japan Health Sciences Foundation
University of Fukui NUC
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University of Fukui NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method, a reagent (carrier), a vaccine, and the like, each for efficiently making cytotoxic lymphocytes to recognize a cancer-specific antigen in a cancer-specific immunotherapy, especially to provide a method, a reagent (carrier), a vaccine, and the like, each for enabling that the antigen can especially efficiently be taken into antigen-exhibiting cells and simultaneously activate the antigen-exhibiting cells, even when an immunization globulin massively existing in the body is used as a cancer-specific antigen. <P>SOLUTION: An idiotype vaccine comprises a composite material which comprises an idiotype antigen and a carrier comprising urate crystals. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、免疫刺激のための特異的抗原の担体、さらに具体的にはがん細胞を標的とする細胞傷害性T細胞(以下、CTLということがある)を効率的に誘導するための、特異的抗原の担体に関する。本発明はまた、これらの担体を用いた抗原複合体およびそれを含む免疫刺激用組成物(ワクチン)、ならびにワクチンの製造方法等にも関する。   The present invention provides a carrier for specific antigens for immune stimulation, more specifically, for efficiently inducing cytotoxic T cells (hereinafter sometimes referred to as CTL) targeting cancer cells. It relates to a carrier for a specific antigen. The present invention also relates to an antigen complex using these carriers, an immunostimulatory composition (vaccine) containing the same, a method for producing the vaccine, and the like.

近年、腫瘍特異抗原の発見や樹状細胞の利用により腫瘍免疫療法が開発されている。がん抗原タンパク(がんワクチン)を用いた腫瘍特異的細胞免疫は、がんの治療や再発予防に応用することが可能である。腫瘍特異的細胞免疫は、(1)特異的抗原(がん抗原タンパク)の作製、(2)抗原提示細胞への抗原感作、(3)抗原提示細胞によるCTLの教育・増殖の、3段階を経て惹起される。   In recent years, tumor immunotherapy has been developed through the discovery of tumor-specific antigens and the use of dendritic cells. Tumor-specific cellular immunity using cancer antigen protein (cancer vaccine) can be applied to cancer treatment and recurrence prevention. Tumor-specific cellular immunity consists of (1) preparation of specific antigen (cancer antigen protein), (2) antigen sensitization to antigen-presenting cells, and (3) education and proliferation of CTL by antigen-presenting cells. It is evoked through.

がん細胞が特有な抗原を持っている場合、その抗原タンパクは腫瘍細胞内でペプチドに分解され、一部がMHCクラスIと複合体を形成し、がん細胞表面に提示される。末梢血中に存在するCTLは、同細胞表面上のT細胞抗原レセプターを介してがん細胞のMHCクラスI複合体と結合し、がん細胞を非自己と認識した場合には積極的に攻撃する。したがって、クラスI分子上に提示されるようながん特異的抗原をがんワクチンとして使用することによりCTLを効率的に誘導することができる。   When a cancer cell has a unique antigen, the antigen protein is decomposed into a peptide in the tumor cell, a part forms a complex with MHC class I, and is presented on the surface of the cancer cell. CTL present in peripheral blood binds to the MHC class I complex of cancer cells via the T cell antigen receptor on the cell surface, and actively attacks when the cancer cells are recognized as non-self. To do. Therefore, CTL can be efficiently induced by using a cancer-specific antigen as presented on a class I molecule as a cancer vaccine.

がん特異抗原が作製できたら、がん特異的免疫療法の成功のためには、がん特異的抗原をいかに効率的に細胞傷害性リンパ球に認識させるかが課題となる。樹状細胞(以下、DCということがある)は、生体内で最も強力な抗原提示機能を有する細胞であり、自分が取り込んだ抗原をナイーブT細胞に提示し、抗原特異的なCTLの増殖活性を誘導し、特異的免疫を機能させる能力を有する。樹状細胞は、抗原を感作させた後にリンパ球と混合培養すると、腫瘍に特異的なCTLを最も効率的に誘導し感作効率を高めることが知られている。したがって、樹状細胞は、がんワクチンを用いた腫瘍特異的細胞免疫療法の司令塔として働く細胞として利用されている。   Once a cancer-specific antigen has been prepared, the challenge for successful cancer-specific immunotherapy is how to make the cancer-specific antigen recognized by cytotoxic lymphocytes. Dendritic cells (hereinafter sometimes referred to as DC) are cells having the most powerful antigen-presenting function in the living body, presenting the antigens they have taken up to naive T cells, and antigen-specific CTL proliferation activity. And has the ability to function specific immunity. It is known that when dendritic cells are sensitized with an antigen and mixed with lymphocytes, CTLs specific to tumors are induced most efficiently and sensitization efficiency is increased. Therefore, dendritic cells are used as cells that serve as a control tower for tumor-specific cellular immunotherapy using cancer vaccines.

多発性骨髄腫は、白血病や悪性リンパ腫とならぶ造血器悪性腫瘍のひとつで、白血球の一種であるリンパ球から分化・成熟した形質細胞が、がん化して発症する疾患である。形質細胞は、身体に侵入したウイルスや細菌などの異物を排除する作用をもつタンパク質(抗体=免疫グロブリン)を産生する。免疫グロブリンは、可変領域を含むFab領域(ab fraction; Fab)部分と定常領域(constant fraction; Fc)部分とに分かれ、Fabの可変領域は対象抗原に特異的なイディオタイプ抗原である。形質細胞では1種類の免疫グロブリンしか産生されないため、がん化した形質細胞(骨髄腫細胞)の可変領域部分は腫瘍特異抗原として用いることができる。   Multiple myeloma is one of the hematopoietic malignancies similar to leukemia and malignant lymphoma, and is a disease that develops when cancer cells are differentiated and matured from lymphocytes, which are a type of white blood cell. Plasma cells produce a protein (antibody = immunoglobulin) that has the effect of eliminating foreign substances such as viruses and bacteria that have entered the body. An immunoglobulin is divided into a Fab region (ab fraction; Fab) portion including a variable region and a constant region (Fc) portion, and the Fab variable region is an idiotype antigen specific to the target antigen. Since plasma cells produce only one type of immunoglobulin, the variable region portion of cancerous plasma cells (myeloma cells) can be used as a tumor-specific antigen.

既に、本発明者らは、多発性骨髄腫細胞株IM−9の培養上清よりIgG可変領域を切り出してイディオタイプ抗原とし、IM−9細胞とHLA−Aが合致したボランティアの単球より誘導した樹状細胞を感作した後、同じボランティアから得たリンパ球と共培養した実験系において、増殖したリンパ球が骨髄腫細胞株IM−9に対する細胞傷害性を有することを証明した(非特許文献1および2)。すなわち、多発性骨髄腫において、比較的簡単に精製できる免疫グロブリンFab領域を抗原として樹状細胞を感作することにより、腫瘍特異的な細胞傷害性T細胞を効率的に誘導できる可能性があることが証明されている。   Already, the present inventors excised the IgG variable region from the culture supernatant of the multiple myeloma cell line IM-9 to form an idiotype antigen, which was derived from monocytes of volunteers matched with IM-9 cells and HLA-A. In an experimental system co-cultured with lymphocytes obtained from the same volunteer after sensitizing the resulting dendritic cells, it was proved that the proliferated lymphocytes had cytotoxicity against the myeloma cell line IM-9 (non-patented). References 1 and 2). That is, in multiple myeloma, there is a possibility that tumor-specific cytotoxic T cells can be efficiently induced by sensitizing dendritic cells with an immunoglobulin Fab region that can be purified relatively easily as an antigen. It has been proven.

しかし、免疫グロブリンは患者体内に大量に存在しているため、がん抗原として感作されにくいことが予想される。そのため、免疫グロブリンを抗原として用いた場合であっても、抗原が特異的に抗原提示細胞へ取り込まれ、かつ同時に抗原提示細胞を活性化できるような方法を開発することにより、より効率的に腫瘍特異的細胞免疫が惹起されるようにすることが望まれている。   However, since immunoglobulin is present in a large amount in the patient, it is expected that it is difficult to sensitize as a cancer antigen. Therefore, even when immunoglobulins are used as antigens, tumors can be more efficiently developed by developing a method that allows antigens to be specifically taken into antigen-presenting cells and simultaneously activate antigen-presenting cells. It is desired that specific cellular immunity be elicited.

尿酸ナトリウム(MSU)結晶は、ヒト体内においては体液中に過剰に存在する尿酸が体内で析出することにより形成され、痛風発作と呼ばれる特異的な関節炎症状を引き起こす物質である。MSU結晶は2〜20μmの長針状または桿状の形状を示し、結晶表面は不規則であり、空間の充填が乏しく催炎性が強いとされる表面構造を示す。結晶表面は陰性荷電を持ち、IgGを中心とした免疫グロブリンやリポタンパクの付着が認められ、IgGは陽性に荷電しているFab部分でMSU結晶と結合しているとされている(非特許文献3)。   Sodium urate (MSU) crystals are substances that form in the human body due to precipitation of uric acid that is excessively present in body fluids in the body and cause a specific joint inflammation state called gout attack. The MSU crystal has a long needle shape or a saddle shape of 2 to 20 μm, and the crystal surface is irregular, and has a surface structure in which space filling is poor and the flammability is strong. The surface of the crystal has a negative charge, and adhesion of immunoglobulins and lipoproteins centering on IgG is observed, and IgG is said to be bound to the MSU crystal at the positively charged Fab portion (Non-patent Document) 3).

近年、MSU結晶が局所における免疫反応の重要物質として働いていると考えられるようになってきた。例えば、Shiらは、局所で傷害された細胞から放出された尿酸が内因性の危険信号(danger signal)として樹状細胞に働くこと、および予め形成させたMSU結晶をインビボ注射するとアジュバント活性が見られることを報告した(非特許文献4)。また、Huらは、尿酸濃度に依存して免疫拒絶反応が高まることやMSU結晶の皮下投与により異種移植片の拒絶反応が促進されることを報告した(非特許文献5)。   In recent years, it has come to be considered that MSU crystals work as an important substance of local immune reaction. For example, Shi et al. Show that uric acid released from locally injured cells acts on dendritic cells as an intrinsic danger signal, and that adjuvant activity is seen when pre-formed MSU crystals are injected in vivo. (Non-Patent Document 4). In addition, Hu et al. Reported that immune rejection increased depending on the uric acid concentration and that xenograft rejection was promoted by subcutaneous administration of MSU crystals (Non-patent Document 5).

しかし、MSU結晶を特定の抗原と予め結合させることは記載されていない。
酒巻一平、上田孝典、高澤ゆみえ、岩崎博道、稲井邦博、津谷 寛:「Fabを抗原としたmyeloma cell lineに対するallogenic CTLの誘導」。第65回日本血液学会総会・第45回日本臨床血液学会総会、2003.8. 臨床血液 44(8),874,2003,8。 Sakamaki, I., Inai, K., Takazawa, Y., Tsutani, H., *Ueda, T.:Clonal expansion of TCR V beta repertoires in CD8 and CD4 T cells in response to idiotype protein. American Association for Cancer Research 95th Annual Meeting,Abstract. 45, 294-295, 2004,3. Kozin F. et al., J Lab Clin Med. 1980 Shi et al., Nature 425:516-521 (2003) Hu et al., Cancer Res. 64:5059-5062 (2004)
However, there is no description of pre-binding MSU crystals to specific antigens.
Ippei Sakamaki, Takanori Ueda, Yumie Takasawa, Hiromichi Iwasaki, Kunihiro Inai, Hiroshi Tsuya: “Induction of allogenic CTL against myeloma cell line using Fab as an antigen”. 65th Annual Meeting of the Japanese Society of Hematology, 45th Annual Meeting of the Japanese Society of Clinical Hematology, 2003.8. Clinical Blood 44 (8), 874, 2003, 8. Sakamaki, I., Inai, K., Takazawa, Y., Tsutani, H., * Ueda, T .: Clonal expansion of TCR V beta repertoires in CD8 and CD4 T cells in response to idiotype protein. American Association for Cancer Research 95th Annual Meeting, Abstract. 45, 294-295, 2004, 3. Kozin F. et al., J Lab Clin Med. 1980 Shi et al., Nature 425: 516-521 (2003) Hu et al., Cancer Res. 64: 5059-5062 (2004)

本発明は、がん特異的免疫療法において、効率よくがん特異的抗原を細胞傷害性リンパ球に認識させるための方法および試薬(担体)、ワクチン等を提供することを目的とする。特に、体内に大量に存在している免疫グロブリンをがん特異的抗原として用いた場合であっても抗原が特異的に効率よく抗原提示細胞へ取り込まれ、かつ同時に抗原提示細胞を活性化することを可能にする方法および試薬(担体)、ワクチン等を提供することを目的とする。   An object of the present invention is to provide a method, a reagent (carrier), a vaccine and the like for efficiently recognizing a cancer-specific antigen to cytotoxic lymphocytes in cancer-specific immunotherapy. In particular, even when immunoglobulin present in large amounts in the body is used as a cancer-specific antigen, the antigen is specifically and efficiently taken into the antigen-presenting cell and simultaneously activates the antigen-presenting cell. It is an object to provide a method and a reagent (carrier), a vaccine, and the like that make it possible.

本発明者らは、MSU結晶を抗原担体として用いることにより上記目的が達成されることを見い出し、本発明を完成した。   The present inventors have found that the above object can be achieved by using MSU crystals as an antigen carrier, and have completed the present invention.

すなわち、本発明により、
〔1〕 イディオタイプ抗原と尿酸塩結晶からなる担体とからなる複合体を含むことを特徴とするイディオタイプワクチン;
〔2〕 イディオタイプ抗原が、多発性骨髄腫細胞によって産生される免疫グロブリンまたは可変領域を含むその一部である、前記〔1〕記載のイディオタイプワクチン;
〔3〕 尿酸塩結晶が、針状結晶を超音波処理したものである、前記〔1〕または〔2〕記載のイディオタイプワクチン;
〔4〕 前記〔1〕〜〔3〕のいずれか1項記載のイディオタイプワクチンを製造する方法であって、イディオタイプ抗原と尿酸塩結晶とを一緒にして静置することによりイディオタイプ抗原と尿酸塩結晶との複合体を形成させる工程を含む方法;
〔5〕 前記〔1〕〜〔3〕のいずれか1項記載のイディオタイプワクチンを用いて樹状細胞を刺激し、この樹状細胞を用いて細胞傷害性T細胞を刺激することを特徴とする、特異的細胞傷害性T細胞の免疫応答を促進する方法;
〔6〕 前記〔1〕〜〔3〕のいずれか1項記載のイディオタイプワクチンを製造するための担体であって、尿酸ナトリウム結晶からなる担体、が提供される。
That is, according to the present invention,
[1] An idiotype vaccine comprising a complex comprising an idiotype antigen and a carrier comprising urate crystals;
[2] The idiotype vaccine according to [1], wherein the idiotype antigen is an immunoglobulin produced by multiple myeloma cells or a part thereof including a variable region;
[3] The idiotype vaccine according to the above [1] or [2], wherein the urate crystals are those obtained by ultrasonically treating needle-like crystals;
[4] A method for producing the idiotype vaccine according to any one of [1] to [3] above, wherein the idiotype antigen and urate crystals are allowed to stand together to leave the idiotype antigen and Forming a complex with urate crystals;
[5] A method comprising stimulating dendritic cells using the idiotype vaccine according to any one of [1] to [3], and stimulating cytotoxic T cells using the dendritic cells. A method of promoting an immune response of specific cytotoxic T cells;
[6] A carrier for producing the idiotype vaccine according to any one of [1] to [3] above, which is a carrier made of sodium urate crystals.

本発明のイディオタイプ抗原用担体は、樹状細胞にイディオタイプ抗原を効率的に取り込ませることができる。さらに、本発明のイディオタイプ抗原用担体を使用することにより、抗原の取り込み促進と同時に樹状細胞を活性化することができる。すなわち本発明の担体は、抗原取り込み能の優れた未熟樹状細胞に抗原を取り込ませると同時に、従来使用されてきた各種の成熟化物質を使用することなしに抗原提示能の優れた成熟樹状細胞への成熟化を行なうことができるので、非常に有利である。その結果、本発明のイディオタイプ抗原用担体を用いたイディオタイプワクチンは非常に有効性が高く、腫瘍特異的免疫療法が成功する可能性が高くなると期待される。また、本発明のイディオタイプ抗原用担体は生体内に存在しうる物質に基づいているため、予測できない副作用を引き起こす危険がなく、安全性が高い。したがって、本発明の担体に腫瘍由来のイディオタイプ抗原を結合させた複合体は、がんワクチン(抗がん剤)として使用することができる。   The idiotypic antigen carrier of the present invention can efficiently take up an idiotype antigen into dendritic cells. Further, by using the idiotype antigen carrier of the present invention, dendritic cells can be activated simultaneously with the promotion of antigen uptake. That is, the carrier of the present invention allows immature dendritic cells with excellent antigen uptake ability to take up antigens, and at the same time, mature dendrites with excellent antigen presentation ability without using various conventionally used mature substances. This is very advantageous because it allows maturation into cells. As a result, the idiotype vaccine using the idiotype antigen carrier of the present invention is very effective and is expected to increase the possibility of successful tumor-specific immunotherapy. Further, since the idiotype antigen carrier of the present invention is based on a substance that can exist in the living body, there is no risk of causing unpredictable side effects, and the safety is high. Therefore, a complex obtained by binding a tumor-derived idiotype antigen to the carrier of the present invention can be used as a cancer vaccine (anticancer agent).

本発明の担体は、尿酸塩結晶からなる。尿酸塩結晶としては各種の形状のものが知られているが、どのような形状のものであってもよく、また、結晶は、公知のいずれの方法で作製してもよい。一般的には、水に難溶性の尿酸をアルカリや加熱により溶解して尿酸を含む溶液を用意し、静置して冷却することにより結晶を析出させる。   The carrier of the present invention consists of urate crystals. Urate crystals are known in various shapes, but any shape may be used, and crystals may be produced by any known method. In general, a solution containing uric acid is prepared by dissolving uric acid that is hardly soluble in water by alkali or heating, and crystals are precipitated by standing and cooling.

結晶のサイズは小さい方が細胞による取り込みのために有利である。例えば、結晶は、貪食されやすいように樹状細胞より小さいサイズ、例えば直径10μm以下のサイズであることが好ましい。細かい結晶を得るためには、いったん製造した結晶を超音波処理等によって破砕してもよい。この場合、破砕のしやすさの点からは、針状結晶のみまたは針状結晶を主に含む結晶を使用することが好ましい。   Smaller crystal sizes are advantageous for cellular uptake. For example, the crystal is preferably smaller in size than the dendritic cell so as to be easily phagocytosed, for example, having a diameter of 10 μm or less. In order to obtain fine crystals, once produced crystals may be crushed by ultrasonic treatment or the like. In this case, from the viewpoint of easy crushing, it is preferable to use only acicular crystals or crystals mainly containing acicular crystals.

尿酸塩として特に好ましいのは尿酸ナトリウム(MSU)および尿酸水素ナトリウムであり、最も好ましいのは尿酸一ナトリウム一水和物である。痛風結節内など生体内の尿酸塩結晶は尿酸水素ナトリウムであり、さらに結晶水1分子を含み、尿酸一ナトリウム一水和物が形成される(非特許文献3)。MSU結晶は、ピロリン酸カルシウム結晶やヒドロキシアパタイトとともに結晶誘発性関節炎の原因物質であり、催炎性が最も強い結晶である。また、生体への安全性、作製の容易性等の点からも、担体として最も好ましい。   Particularly preferred as urate are sodium urate (MSU) and sodium hydrogen urate, and most preferred is monosodium urate monohydrate. The urate salt in vivo such as in gout nodules is sodium hydrogen urate, and further contains 1 molecule of crystal water to form monosodium urate monohydrate (Non-patent Document 3). MSU crystals, together with calcium pyrophosphate crystals and hydroxyapatite, are causative substances for crystal-induced arthritis, and are the crystals with the strongest inflammatory effect. In addition, it is most preferable as a carrier from the viewpoints of safety to living bodies and ease of production.

また、本発明の担体は、タンパクとの結合能および樹状細胞の感作能等の機能を保持する範囲内で、適宜修飾または改変されていてもよい。   In addition, the carrier of the present invention may be appropriately modified or modified as long as it retains functions such as protein binding ability and dendritic cell sensitization ability.

尿酸塩結晶は、陰性に荷電していることが知られている。したがって、本発明の担体は、陽性に荷電している免疫グロブリンFab部分と電気的に結合するという物理的特性を有する。本発明のイディオタイプワクチンは、このようにして結合した本発明の尿酸塩結晶からなる担体とイディオタイプ抗原とからなる複合体を含む。本発明のイディオタイプワクチンに含まれるイディオタイプ抗原としては、少なくとも可変領域を含むタンパクまたはポリペプチドであればよく、例えば免疫グロブリン全体、または可変領域を含む部分、例えばFab、F(ab’)、軽鎖、重鎖、および重鎖のFab部分が挙げられる。ワクチン用として、免疫グロブリンは、IgGが好ましいが、場合によってはそれ以外の免疫グロブリン等またはそれらに由来する部分であってもよい。このような抗体の調製および抗体フラグメントの調製は当業者には充分公知である。 Urate crystals are known to be negatively charged. Thus, the carrier of the present invention has the physical property of electrically binding to a positively charged immunoglobulin Fab moiety. The idiotype vaccine of the present invention comprises a complex composed of the carrier comprising the urate crystals of the present invention and the idiotype antigen bound in this manner. The idiotype antigen contained in the idiotype vaccine of the present invention may be any protein or polypeptide containing at least a variable region, for example, whole immunoglobulin or a portion containing a variable region, such as Fab, F (ab ′) 2. , Light chain, heavy chain, and the Fab portion of the heavy chain. For vaccine use, IgG is preferably IgG, but in some cases, other immunoglobulin or the like or a portion derived therefrom may be used. The preparation of such antibodies and antibody fragments are well known to those skilled in the art.

本発明のイディオタイプワクチンは、多発性骨髄腫を代表とするモノクローナルタンパクを産生する腫瘍などに対して治療目的で適用しうる。したがって、イディオタイプ抗原は、多発性骨髄腫等の患者の血液等から得ることができる。   The idiotype vaccine of the present invention can be applied for therapeutic purposes to tumors that produce monoclonal proteins typified by multiple myeloma. Therefore, an idiotype antigen can be obtained from blood of a patient such as multiple myeloma.

本発明の担体と、イディオタイプ抗原とを結合させることによりそれらの複合体が得られる。担体と抗原との結合は、適当量の両者を混合して静置するだけで容易に起こすことができる。例えば、尿酸塩結晶:Fabを重量比0.1:1〜4:1程度でリン酸緩衝生理食塩水中で混合し、4℃〜室温程度で1〜48時間静置することにより複合体が形成される。特定の条件下での最適な複合体形成条件は後述する方法などによって適宜決定することができる。   These complexes can be obtained by binding the carrier of the present invention to the idiotype antigen. The binding between the carrier and the antigen can be easily caused only by mixing a suitable amount of both and allowing to stand. For example, a complex is formed by mixing urate crystals: Fab in a phosphate buffered saline in a weight ratio of about 0.1: 1 to 4: 1, and allowing to stand at 4 ° C. to room temperature for 1 to 48 hours. Is done. Optimum complex formation conditions under specific conditions can be appropriately determined by the method described below.

本発明のイディオタイプワクチンは、患者に直接投与することができる。投与経路としては、特に制限はないが、非経口経路が好ましく、一般的には皮内投与であり、リンパ節近傍の皮内に投与することが好ましい。したがって、本発明のイディオタイプワクチンは、イディオタイプ抗原と本発明の担体との複合体を含むほか、製造過程で使用されるリン酸水素ナトリウム、リン酸二水素ナトリウム、塩化ナトリウム、水酸化ナトリウム、塩酸などの緩衝剤とpH調整用試薬が添加されていてもよく、さらに投与経路や剤型等によって必要に応じてベンジルアルコールなどの無痛化剤、乳糖水和物、D−ソルビトール、L−グルタミン、ゼラチンなどの安定剤、フェノール、ホルマリンなどの不活化剤など製剤分野で公知の成分を含んでいてもよい。また、他の抗がん剤等と組み合わせてもよい。   The idiotype vaccine of the present invention can be administered directly to a patient. The administration route is not particularly limited, but a parenteral route is preferable, generally intradermal administration, and preferably intradermally in the vicinity of lymph nodes. Therefore, the idiotype vaccine of the present invention includes a complex of the idiotype antigen and the carrier of the present invention, as well as sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide, A buffering agent such as hydrochloric acid and a pH adjusting reagent may be added, and a soothing agent such as benzyl alcohol, lactose hydrate, D-sorbitol, L-glutamine as necessary depending on the administration route and dosage form. Ingredients known in the pharmaceutical field such as stabilizers such as gelatin and inactivators such as phenol and formalin may be included. Moreover, you may combine with another anticancer agent etc.

投与量は、患者の状態、投与経路、投薬スケジュール等によって変動するが、一般的には、抗原量として1.0〜20.0μg/kg体重程度、担体量として2.0〜40.0μg/kg体重程度(抗原と担体との複合体の量として3.0〜60.0μg/kg体重)である。このような尿酸塩結晶の投与量は、ごく少量のため痛風患者の皮膚局所でみられる痛風結節のMSU結晶量の2万分の1〜1000分の1程度に相当し、肉眼的に認識できる痛風結節を作ることもなく、そもそも皮膚ではみることはない痛風発作も当然惹起されることはないと考えられる。また例えすべて溶解し血液中内に移行したとしても血清尿酸値を0.0025〜0.05mg/dl上昇させる程度であり、担体の投与が尿酸塩結晶の組織への沈着を促進するとは考えられない。したがって、尿酸塩結晶による副作用の可能性はほとんどないと考えられる。   The dose varies depending on the patient's condition, administration route, dosing schedule, etc., but generally, the antigen amount is about 1.0 to 20.0 μg / kg body weight, and the carrier amount is 2.0 to 40.0 μg / kg. It is about kg body weight (3.0 to 60.0 μg / kg body weight as the amount of the complex of antigen and carrier). Since the dosage of such urate crystals is very small, it corresponds to about 20,000 to 1/1000 of the amount of MSU crystals in gout nodules found in the skin of gout patients, and gout that can be recognized visually. Gout attacks that do not make nodules and are not seen in the skin in the first place are not likely to be triggered. Moreover, even if all dissolved and moved into the blood, the serum uric acid level is increased by 0.0025 to 0.05 mg / dl, and it is considered that administration of the carrier promotes deposition of urate crystals in the tissue. Absent. Therefore, it is considered that there is almost no possibility of side effects due to urate crystals.

また、本発明のイディオタイプワクチンは、直接投与せずに、体外で本発明のイディオタイプワクチンで樹状細胞を感作することにより、がん細胞を標的とする樹状細胞を産生したり、さらにはこの樹状細胞を用いて特異的CTLを誘導したりするために使用することができる。このような樹状細胞またはCTLを患者に与えることによっても腫瘍の治療を行なうことができる。   Moreover, the idiotype vaccine of the present invention produces dendritic cells targeting cancer cells by sensitizing dendritic cells with the idiotype vaccine of the present invention outside the body without direct administration, Further, the dendritic cells can be used to induce specific CTLs. Tumor treatment can also be performed by providing such a dendritic cell or CTL to a patient.

1.尿酸ナトリウム(MSU)結晶担体の作製(1)
尿酸ナトリウム結晶を、MaCartyらの方法(Lancet 2:682, 1962)に基づいて以下のようにして作製した。
1. Preparation of sodium urate (MSU) crystal carrier (1)
Sodium urate crystals were prepared as follows based on the method of MaCarty et al. (Lancet 2: 682, 1962).

尿酸(ナカライテスク(株)、京都)336mgに蒸留水40mLおよび水酸化ナトリウムを0.080mg加えて攪拌した。さらに蒸留水を加えて80mLとし、加熱沸騰させて尿酸を完全に溶解させた。加熱を終了し、再び溶液量を80mLに調整し(尿酸濃度420mg/dL)、自然冷却下で45℃となったところでろ過滅菌した(商品名「ステリカップ‐GPフィルターユニット」、日本ミリポア(株)、東京)。ろ過液を速やかに1.5mL容の滅菌済みマイクロチューブに分注し、4℃で5日間以上保存して、針状結晶の生成を最大限に促した。この間、ウリカーゼ・ペルオキシダーゼ法を用いて溶液中の尿酸濃度の減少を経時的にモニタリングした。その結果を図1に示す。   To 336 mg of uric acid (Nacalai Tesque, Kyoto), 40 mL of distilled water and 0.080 mg of sodium hydroxide were added and stirred. Distilled water was further added to make 80 mL, and the mixture was heated to boiling to completely dissolve uric acid. After heating, the solution volume was adjusted again to 80 mL (uric acid concentration 420 mg / dL), and sterilized by filtration when the temperature reached 45 ° C. under natural cooling (trade name “Sterilcup-GP Filter Unit”, Nippon Millipore Corporation ),Tokyo). The filtrate was quickly dispensed into 1.5 mL sterilized microtubes and stored at 4 ° C. for over 5 days to maximize the formation of needle crystals. During this time, the decrease in uric acid concentration in the solution was monitored over time using the uricase peroxidase method. The result is shown in FIG.

生成した尿酸ナトリウム(MSU)結晶を、樹状細胞による貪食を容易にするために、イディオタイプ抗原と結合させる直前に10μm以下になるように破砕した。閉式超音波細胞破砕装置(バイオラプターUCW200TM、東湘電気(株)、横浜)を用い、200Wにて30秒間処理を1回とし、計4回断続して無菌的に上記低温保存しMSU結晶が析出したマイクロチューブを処理し、マイクロチューブ内のMSU結晶を細かくした。   The generated sodium urate (MSU) crystals were crushed to 10 μm or less immediately before binding to idiotype antigens to facilitate phagocytosis by dendritic cells. Using a closed ultrasonic cell crusher (Bioraptor UCW200TM, Tojo Electric Co., Ltd., Yokohama), treatment at 200W is performed once for 30 seconds. The deposited microtube was processed to make the MSU crystal in the microtube fine.

この操作における破砕の進行状況を顕微鏡観察した(倍率100倍)。結果を図2に示す。30秒間の破砕(パネルD)で、きれいな断片化が行なわれたが、60秒間(パネルE)では断片サイズが30秒間より大きそうなものも含まれていたため、安定的に破砕が完了する120秒間(30秒オン、30秒オフのサイクルを4回;パネルF)を標準的に使用する破砕条件とした。   The progress of crushing in this operation was observed with a microscope (magnification 100 times). The results are shown in FIG. Clean fragmentation was performed in 30 seconds of crushing (Panel D), but in 60 seconds (Panel E), the fragment size was likely to be larger than 30 seconds. The crushing conditions were typically used for 2 seconds (4 cycles of 30 seconds on, 30 seconds off; panel F).

破砕後の溶液を7,000×gでフラッシングした後、沈降させた結晶を0.1Mリン酸緩衝食塩水(pH7.0)で洗浄して、最終的に同液に沈降させて、イディオタイプ抗原用担体とした。   After flushing the crushed solution at 7,000 × g, the precipitated crystals were washed with 0.1M phosphate buffered saline (pH 7.0) and finally settled in the same solution. An antigen carrier was used.

2.MSU結晶担体の作製(2)
0.1Mリン酸緩衝食塩水(pH7.4)80mLに尿酸100mgを加えて加熱沸騰させて尿酸を完全に溶解させた。加熱を終了し、再び溶液量を80mLに調整し、自然冷却下で45℃となったところでろ過滅菌した(ステリカップ-GP フィルターユニット、日本ミリポア(株)、東京)。ろ過液を速やかに1.5mLの滅菌済みエッペンチューブに分注し、4℃で5日間以上保存して、針状結晶の生成を最大限に促した。
2. Preparation of MSU crystal support (2)
100 mg of uric acid was added to 80 mL of 0.1 M phosphate buffered saline (pH 7.4) and heated to boiling to completely dissolve uric acid. The heating was terminated, the amount of the solution was adjusted again to 80 mL, and the solution was sterilized by filtration when the temperature reached 45 ° C. under natural cooling (Stericup-GP filter unit, Nippon Millipore Corporation, Tokyo). The filtrate was quickly dispensed into 1.5 mL sterilized Eppendorf tubes and stored at 4 ° C. for over 5 days to maximize the formation of needle crystals.

その後は上記と同様にしてイディオタイプ抗原用担体を調製した。   Thereafter, an idiotype antigen carrier was prepared in the same manner as described above.

3.イディオタイプ抗原の作製
IgG型多発性骨髄腫の患者血清0.5〜1.0mL(IgG 40mg相当)を採取し、0.1Mリン酸緩衝食塩水(pH7.4)で希釈して、同液にて予め充足させてあるProtein Gカラム(商品名「Hitrap protein G 1mL」、アマシャム ファルマシア バイオテク(株)、東京)に注入し、吸着させた。さらに0.1Mリン酸緩衝食塩水(pH7.0)を注入して非吸着成分をカラムより洗い出した。1M グリシン−塩酸緩衝液(pH2.7)3mLを注入して、吸着していたIgGを溶出させ、溶出液に1Mトリス−塩酸緩衝液(pH9.0)を加えてpHを7.4程度に調整した。
3. Preparation of idiotype antigen 0.5-1.0 mL of IgG multiple myeloma patient serum (equivalent to 40 mg IgG) was collected and diluted with 0.1 M phosphate buffered saline (pH 7.4). Were injected onto a Protein G column (trade name “Hitrap protein G 1 mL”, Amersham Pharmacia Biotech Co., Ltd., Tokyo) previously adsorbed and adsorbed. Further, 0.1M phosphate buffered saline (pH 7.0) was injected to wash out non-adsorbed components from the column. 3 mL of 1M glycine-hydrochloric acid buffer (pH 2.7) was injected to elute the adsorbed IgG, and 1M Tris-hydrochloric acid buffer (pH 9.0) was added to the eluate to bring the pH to about 7.4. It was adjusted.

溶出させたIgGを、0.12mg/mLシステインおよび2mM EDTAナトリウム(pH7.4)混合液中で0.25mg/mLパパイン(MERCK社、Darmstadt, Germany)を用いて、37℃にて12時間孵置することにより、IgGをFab部分とFc部とに切断した。その後、10.8mMヨードアセタミドで反応を停止した。   The eluted IgG was digested with 0.25 mg / mL papain (MERCK, Darmstadt, Germany) in a mixture of 0.12 mg / mL cysteine and 2 mM sodium EDTA (pH 7.4) at 37 ° C. for 12 hours. The IgG was cleaved into the Fab part and the Fc part. The reaction was then stopped with 10.8 mM iodoacetamide.

プロテインAカラム(商品名「Hitrap protein A 5mL」、アマシャム ファルマシア バイオテク(株)、東京)に注入してFc部分および消化されていないIgGを吸着し、さらに0.1Mリン酸緩衝食塩水(pH7.4)を注入して非吸着成分であるFab部分を採取した。次に、遠心濃縮(商品名「アミコンウルトラ‐4 遠心式フィルターユニット」、日本ミリポア(株)、東京)した後、0.1Mリン酸緩衝食塩水(pH7.4)を透析液として透析膜(ミリポア0.025μM VSWP 25mm、日本ミリポア(株)、東京)を用いたDrop Dialysis法にて90分間透析し、さらに滅菌済シリンジ加圧式フィルターユニット(商品名「マイレクス−GV」、0.22μm、PVDF、日本ミリポア(株)、東京)を通してろ過滅菌し、イディオタイプ抗原液とした。   It is injected into a protein A column (trade name “Hitrap protein A 5 mL”, Amersham Pharmacia Biotech Co., Ltd., Tokyo) to adsorb the Fc portion and undigested IgG, and further 0.1 M phosphate buffered saline (pH 7. 4) was injected to collect the Fab portion which is a non-adsorbing component. Next, after centrifugal concentration (trade name “Amicon Ultra-4 Centrifugal Filter Unit”, Nippon Millipore Co., Ltd., Tokyo), 0.1M phosphate buffered saline (pH 7.4) was used as a dialysis solution (dialysis membrane ( Dialyzed for 90 minutes by Drop Dialysis method using Millipore 0.025μM VSWP 25mm, Nippon Millipore Co., Ltd., Tokyo), and further sterilized syringe pressure type filter unit (trade name “Mirex-GV”, 0.22μm, PVDF And sterilized by filtration through Nippon Millipore Corporation, Tokyo) to obtain an idiotype antigen solution.

4.複合体の形成(1)
一定量のイディオタイプ抗原(Fab)を含む溶液を用いて、MSU結晶担体量と吸着したFabタンパク量との関係を調べた。
4). Formation of complex (1)
Using a solution containing a certain amount of idiotype antigen (Fab), the relationship between the amount of MSU crystal carrier and the amount of adsorbed Fab protein was examined.

上記1で作製したMSU結晶担体0〜360μgが入ったマイクロチューブに、上記3で作製したイディオタイプ抗原(Fab)100μgを含有する0.1Mリン酸緩衝生理食塩水100μLを加え、4℃にて24時間静置した。この溶液を7,000×gでフラッシングした後に、上清のタンパク濃度をピロガロールレッド法(商品名「マイクロTP−テスト」、和光純薬(株)、東京)で測定した。   100 μL of 0.1 M phosphate buffered saline containing 100 μg of the idiotype antigen (Fab) prepared in 3 above is added to the microtube containing 0 to 360 μg of the MSU crystal carrier prepared in 1 above at 4 ° C. It was allowed to stand for 24 hours. After flushing this solution at 7,000 × g, the protein concentration of the supernatant was measured by the pyrogallol red method (trade name “Micro TP-Test”, Wako Pure Chemical Industries, Ltd., Tokyo).

結果を図3に示す。添加MSU結晶担体量が多いほど緩衝液中のタンパク量は少なくなり、MSU結晶担体がFabタンパクを吸着していることが推測された。また、MSU結晶担体100μgではおよそ50μgのFabタンパクが吸着していることが計算より推定されるため、0.1Mリン酸緩衝生理食塩水100μL中では、100μgのMSU結晶担体に対して倍量のFabタンパクを加えることにより、MSU結晶担体が50μg以上のFabタンパクの吸着を確保できると考えられた。   The results are shown in FIG. It was estimated that the amount of protein in the buffer solution decreased as the amount of added MSU crystal carrier increased, and the MSU crystal carrier adsorbed Fab protein. Moreover, since it is estimated from calculation that about 50 μg of Fab protein is adsorbed with 100 μg of MSU crystal carrier, in 100 μL of 0.1 M phosphate buffered saline, the amount is doubled with respect to 100 μg of MSU crystal carrier. It was considered that by adding Fab protein, the MSU crystal carrier can secure the adsorption of Fab protein of 50 μg or more.

5.複合体の形成(2)
結合の様子を確認するために、上記1と同様に作製したイディオタイプ抗原用担体混濁液400μ1(MSU結晶1.4mg相当;但し、30秒間の超音波処理を行ったもの)を、マウスFITC標識IgG・F(ab’)(ダコ・ジャパン、京都)100μLと、あるいは等量の上記3の方法でマウスFITC標識IgG・F(ab’)より作製したマウスFITC標識IgG・Fabと混合し、4℃で1時間静置した。希塩酸液(pH6.5〜7.0)で3回洗浄し、光学顕微鏡下でMSU結晶の透過像を得た後で、励起波長488nmによる蛍光顕微鏡像を観察した。
5. Formation of complex (2)
In order to confirm the state of binding, 400 μ1 of the idiotype antigen carrier suspension prepared in the same manner as in 1 above (corresponding to 1.4 mg of MSU crystal; but subjected to ultrasonic treatment for 30 seconds) was labeled with the mouse FITC label. 100 μL of IgG • F (ab ′) 2 (Dako Japan, Kyoto), or mixed with mouse FITC-labeled IgG • Fab prepared from mouse FITC-labeled IgG • F (ab ′) 2 by the same method described in 3 above. It left still at 4 degreeC for 1 hour. After washing three times with dilute hydrochloric acid solution (pH 6.5 to 7.0) and obtaining a transmission image of the MSU crystal under an optical microscope, a fluorescence microscope image with an excitation wavelength of 488 nm was observed.

無添加のMSU結晶は輪郭のみが識別できるのみで蛍光顕微鏡下で無色透明であった(結果は示さず)。一方、マウスFITC標識IgG・FabまたはマウスFITC標識IgG・F(ab’)を混合したMSU結晶は、蛍光検鏡下で緑色に輝いて見えた。図4、パネルA、BにMSU結晶担体とマウスFITC標識IgG・Fabとの結合を、またパネルE、FにマウスFITC標識IgG・F(ab’)との結合の結果を示す。パネルA、Eは光学顕微鏡像、パネルB、Fは蛍光顕微鏡像と光学顕微鏡像との結合像である。さらに、あらかじめMSU結晶を牛胎児血清と混合した後にMSU結晶担体とマウスFITC標識IgG・Fabと混合すると、蛍光顕微鏡下での緑色の輝きは減弱した。 The additive-free MSU crystal was colorless and transparent under a fluorescence microscope with only the outline being distinguishable (results not shown). On the other hand, MSU crystals mixed with mouse FITC-labeled IgG • Fab or mouse FITC-labeled IgG • F (ab ′) 2 appeared to shine green under a fluorescence microscope. In FIG. 4, panels A and B show the results of binding of the MSU crystal carrier and mouse FITC-labeled IgG • Fab, and panels E and F show the results of binding of mouse FITC-labeled IgG • F (ab ′) 2 . Panels A and E are optical microscope images, and panels B and F are combined images of a fluorescence microscope image and an optical microscope image. Further, when the MSU crystal was previously mixed with fetal calf serum and then mixed with the MSU crystal carrier and mouse FITC-labeled IgG • Fab, the green glow under the fluorescence microscope was attenuated.

6.単球よりの未熟樹状細胞の作製
健常人ドナーよりヘパリン採血し、「RosetteSep Human Monocyte Enrichment Cocktail」(商品名;StemCell Technologies Inc, Vancouver, Canada)を用いて単球を取り出した。この単球(末梢血単球)を、GM−CSF(PEPRO TECH INC, Rocky Hill, NJ)(100ng/mL)、IL−4(PEPRO TECH EC, London, UK)(100ng/mL)を加えた10%AB血清含有RPMI1640培養液にて37℃、5%COで6日間培養し、未熟樹状細胞を作製した。
6). Preparation of immature dendritic cells from monocytes Heparin blood was collected from a healthy donor, and monocytes were removed using “RosetteSep Human Monocyte Enrichment Cocktail” (trade name; StemCell Technologies Inc, Vancouver, Canada). GM-CSF (PEPRO TECH INC, Rocky Hill, NJ) (100 ng / mL) and IL-4 (PEPRO TECH EC, London, UK) (100 ng / mL) were added to these monocytes (peripheral blood monocytes). Immature dendritic cells were prepared by culturing in RPMI 1640 medium containing 10% AB serum at 37 ° C. and 5% CO 2 for 6 days.

7.尿酸塩結晶による樹状細胞の成熟化
20μgの上記3で作製したイディオタイプ抗原(Fab)と20μgの上記1で作製したMSU結晶担体とを4℃、3時間静置して複合体を形成させた後、未熟樹状細胞(1×10個/ウェル)と100μLの10%FBS含有RPMI1640培養液中で48時間培養した。同様に、未熟樹状細胞のみを48時間培養した。これらの樹状細胞について、成熟化の指標であるCD83の発現強度を、PE標識した抗CD83抗体(PE COULTER;Coulter-Immunotec, Krefeld, Germany)を用いてフローサイトメトリー(EPICS)(Coulter-Immunotec, Krefeld, Germany)にてsingle color条件にて測定した。
7). Maturation of dendritic cells by urate crystals 20 μg of the idiotype antigen (Fab) prepared in 3 above and 20 μg of the MSU crystal carrier prepared in 1 above were allowed to stand at 4 ° C. for 3 hours to form a complex. Thereafter, the cells were cultured with immature dendritic cells (1 × 10 5 cells / well) in 100 μL of RPMI1640 culture medium containing 10% FBS for 48 hours. Similarly, only immature dendritic cells were cultured for 48 hours. For these dendritic cells, the expression intensity of CD83, which is an index of maturation, was measured by flow cytometry (EPICS) (Coulter-Immunotec) using PE-labeled anti-CD83 antibody (PE COULTER; Coulter-Immunotec, Krefeld, Germany). , Krefeld, Germany) under single color conditions.

結果を図5に示す。あらかじめ尿酸塩結晶担体と抗原との複合体と共培養した樹状細胞のCD83発現強度(パネルB)は、樹状細胞のみで培養したもののCD83発現強度(パネルA)よりも明らかに高かった。したがって、尿酸塩結晶担体と抗原との複合体は未熟樹状細胞の成熟を促進することが確認された。   The results are shown in FIG. The CD83 expression intensity (panel B) of the dendritic cells co-cultured with the complex of the urate crystal carrier and the antigen in advance (panel B) was clearly higher than the CD83 expression intensity (panel A) of those cultured only with dendritic cells. Therefore, it was confirmed that the complex of the urate crystal carrier and the antigen promotes the maturation of immature dendritic cells.

樹状細胞は、未熟な状態では抗原の取り込みには優れているが、抗原提示能、T細胞への刺激能については成熟樹状細胞の方が優れていると考えられている。樹状細胞の成熟化を促すために、TNF−α、IL−6といったサイトカインやプロスタグランディンE2、またピシバニール等の使用が報告されているが、尿酸塩結晶担体と抗原との複合体を用いることで、抗原タンパクの取り込みと同時に樹状細胞を成熟化させることができ、また上記のような成熟化物質を使用することなしに抗原提示能を上昇させることができることがわかった。   Although dendritic cells are excellent in antigen uptake when immature, mature dendritic cells are considered to be superior in antigen-presenting ability and ability to stimulate T cells. In order to promote maturation of dendritic cells, the use of cytokines such as TNF-α and IL-6, prostaglandin E2, and picibanil has been reported, but a complex of urate crystal carrier and antigen is used. Thus, it was found that dendritic cells can be matured at the same time as antigen protein uptake, and that the ability to present antigen can be increased without the use of a maturation substance as described above.

8.自己T細胞増殖試験
上記6におけるのと同様に、20μgの上記3で作製した患者由来IgGのFabと20μgのMSU結晶担体とを4℃、3時間静置後、1×10個の未熟樹状細胞と48時間共培養した。また、Fabタンパクのみ、尿酸塩結晶のみ、のいずれかを添加し、または刺激なしで、未熟樹状細胞と48時間培養した。このうちのそれぞれ1×10個の樹状細胞に対して1×10個のT細胞を混合し、100μL/ウェルで5日間培養した。
8). Autologous T cell proliferation test As in 6 above, 20 μg of the patient-derived IgG Fab prepared in 3 above and 20 μg of the MSU crystal carrier were allowed to stand at 4 ° C. for 3 hours, and then 1 × 10 5 immature trees. Co-cultured with dendritic cells for 48 hours. In addition, the cells were cultured with immature dendritic cells for 48 hours without adding any of Fab protein alone or urate crystals alone. The respective mixing 1 × 10 5 cells of T cells to 1 × 10 4 cells dendritic cells of, were cultured for 5 days at 100 [mu] L / well.

これら樹状細胞を冷PBSにて2回洗浄後、同じ健常人ドナーより「RosetteSep Human T cell Enrichment Cocktail」(商品名;StemCell Technologies Inc, Vancouver, Canada)を用いて採取したT細胞(1×10/mL CD3陽性細胞>95%)とともに、T細胞:樹状細胞の細胞数の比が10:1となるようにして(すなわち1×10個の樹状細胞に対して1×10個のT細胞)、最終的に100μLとなるように96ウェル平底プレートにて10%AB血清含有AIM−V培養液(SIGMA)で5日間培養した。T細胞の増殖は、「CellTiter 96 Non-Radioactive Cell Proliferation Assay」(商品名;Promega, WI, USA)を用いて測定した。増殖比率(Stimulation Index;SI)を、樹状細胞と共培養したT細胞の各ウェルの測定値/樹状細胞で刺激しないT細胞の測定値として計算した。各データは3人の健常人の結果の平均±標準偏差で示した。 These dendritic cells were washed twice with cold PBS and then collected from the same healthy donor using “RosetteSep Human T cell Enrichment Cocktail” (trade name; StemCell Technologies Inc, Vancouver, Canada) (1 × 10 6 / mL CD3 positive cells> 95%) and a T cell: dendritic cell number ratio of 10: 1 (ie 1 × 10 5 for 1 × 10 4 dendritic cells). In a 96-well flat-bottom plate, the cells were cultured in a 10% AB serum-containing AIM-V medium (SIGMA) for 5 days so that the final volume was 100 μL. The proliferation of T cells was measured using “CellTiter 96 Non-Radioactive Cell Proliferation Assay” (trade name: Promega, WI, USA). The growth ratio (Stimulation Index; SI) was calculated as the measured value of each well of T cells co-cultured with dendritic cells / measured value of T cells not stimulated with dendritic cells. Each data is shown as the mean ± standard deviation of the results of 3 healthy subjects.

結果を、図6に示す。尿酸塩結晶担体とFabタンパクとの複合体と共培養した樹状細胞で刺激したT細胞の増殖(カラムA;SI=2.44±0.15)は、Fabのみと共培養した樹状細胞で刺激したT細胞の増殖(カラムC;SI=1.73±0.24)に比して優位に高かった。尿酸塩結晶のみと共培養した樹状細胞で刺激したT細胞の増殖(カラムB;SI=2.27±0.23)は、樹状細胞のみで刺激したT細胞の増殖(カラムD;SI=1.29±0.13)に比して優位に高かった。   The results are shown in FIG. Proliferation of T cells stimulated with dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein (column A; SI = 2.44 ± 0.15) was obtained by dendritic cells co-cultured with Fab alone It was significantly higher than the proliferation of T cells stimulated with (column C; SI = 1.73 ± 0.24). Proliferation of T cells stimulated with dendritic cells co-cultured only with urate crystals (column B; SI = 2.27 ± 0.23) is the proliferation of T cells stimulated only with dendritic cells (column D; SI = 1.29 ± 0.13).

9.細胞傷害性試験
HLA−A2陽性の健常人ドナーより「RosetteSep Human Monocyte Enrichment Cocktail」(StemCell Technologies Inc)を用いて単球を取り出し、GM−CSF(100ng/mL)、IL−4(100ng/mL)を加えた10%AB血清含有RPMI1640培養液にて37℃、5%COで6日間培養し、未熟樹状細胞を作製した。
9. Cytotoxicity test Monocytes were extracted from healthy donors positive for HLA-A2 using “RosetteSep Human Monocyte Enrichment Cocktail” (StemCell Technologies Inc), GM-CSF (100 ng / mL), IL-4 (100 ng / mL) Was cultured for 6 days at 37 ° C. and 5% CO 2 in a 10% AB serum-containing RPMI 1640 culture solution to which immature dendritic cells were prepared.

HLA−A2陽性のIgG産生骨髄腫細胞株IM−9の培養上清より上記3と同様にして同株細胞が分泌した単クローン性IgGを精製した後、Fab領域を切り出してイディオタイプ抗原(Fabタンパク)を作製した。このFabタンパクと上記1または2で作製した尿酸塩結晶担体とを結合させてから48時間共培養した樹状細胞(A)、共培養の際に用いるFBS(ウシ血清)と尿酸結晶担体とを結合させてからFabと共に共培養した樹状細胞(Fabと尿酸結晶担体とを結合させずにDCと共培養するモデル)(B)、Fabのみと48時間共培養した樹状細胞(C)、樹状細胞のみ(D)を用いて、同じドナーより採取したT細胞を上記7と同様にして刺激した。7日間培養後、再度同様に樹状細胞にて刺激し、さらに7日間培養後、「MACS」(商品名;Miltenyi Biotec, Bergisch Glanbach, Germany)を使用してCD8陽性細胞を取り出した。CD8陽性細胞:腫瘍細胞を5:1として(すなわち1×10個の腫瘍細胞に対してCD8陽性細胞を1×10個)混合して100μL中で4時間培養後、「CytoTox 96」(登録商標)Non-Radioactive Cytotoxicity Assay(Promega, WI, USA)にてIM−9細胞に対するT細胞の細胞傷害性を測定した。 Monoclonal IgG secreted by the same cell line was purified from the culture supernatant of HLA-A2-positive IgG-producing myeloma cell line IM-9 in the same manner as above 3, and then the Fab region was excised and idiotype antigen (Fab Protein). Dendritic cells (A) co-cultured for 48 hours after binding the Fab protein and the urate crystal carrier prepared in 1 or 2 above, FBS (bovine serum) used in the co-culture, and urate crystal carrier Dendritic cells that have been bound and co-cultured with Fab (a model in which Fab is not co-cultured with DC without binding uric acid crystal carrier) (B), dendritic cells that have been co-cultured with Fab alone for 48 hours (C), Using only dendritic cells (D), T cells collected from the same donor were stimulated in the same manner as described above. After culturing for 7 days, the cells were similarly stimulated with dendritic cells. After further culturing for 7 days, CD8 positive cells were taken out using “MACS” (trade name; Miltenyi Biotec, Bergisch Glanbach, Germany). CD8 positive cells: 5: 1 tumor cells (ie, 1 × 10 4 tumor cells to 1 × 10 5 CD8 positive cells) were mixed and cultured in 100 μL for 4 hours, then “CytoTox 96” ( Cytotoxicity of T cells against IM-9 cells was measured with a registered trademark Non-Radioactive Cytotoxicity Assay (Promega, WI, USA).

結果を図7に示す。尿酸塩結晶担体とFabタンパクとの複合体と共培養した樹状細胞によって誘導したCD8陽性細胞のIM−9に対する細胞傷害性は、他に比して有意に高かった。特に尿酸結晶担体とFabを結合させずに共培養したものに比して尿酸塩結晶担体とFabタンパクとの複合体と共培養した樹状細胞によって誘導したCD8陽性細胞のIM−9に対する細胞傷害性は有意に高く、抗原タンパクと尿酸結晶をあらかじめ結合させておくことがCTLを効率的に誘導するのに必要であることが示された。したがって、本発明のイディオタイプワクチンは、腫瘍細胞を標的とするCTLを効率的に誘導することが判明した。   The results are shown in FIG. The cytotoxicity to IM-9 of CD8 positive cells induced by dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein was significantly higher than others. In particular, cytotoxicity to IM-9 of CD8 positive cells induced by dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein compared to those co-cultured without binding uric acid crystal carrier and Fab The sex was significantly higher, and it was shown that the antigen protein and uric acid crystals were previously bound to efficiently induce CTL. Therefore, it was found that the idiotype vaccine of the present invention efficiently induces CTL targeting tumor cells.

図1は、溶液からのMSU結晶の経時的な生成を示す図である。縦軸は溶液中の尿酸濃度(mg/dL)、横軸は時間(hr)を表す。FIG. 1 shows the generation of MSU crystals from solution over time. The vertical axis represents the uric acid concentration (mg / dL) in the solution, and the horizontal axis represents time (hr). 図2は、MSU結晶の破砕を示す写真(光学顕微鏡、倍率100倍)である。パネルAは処理前、パネルB〜Fはそれぞれパネルの左下に示した時間、超音波処理を行なったサンプルの顕微鏡像である。FIG. 2 is a photograph (optical microscope, 100 × magnification) showing the crushing of MSU crystals. Panel A is a microscopic image of a sample that has been subjected to ultrasonic treatment for the time indicated at the lower left of the panel before panels A to B. 図3は、一定量のイディオタイプ抗原(Fab)を含む溶液を用いて、MSU結晶担体量と吸着したFabタンパク量との関係(MSU結晶添加によるFab溶液中におけるタンパク濃度の変化)を示す図である。FIG. 3 is a graph showing the relationship between the amount of MSU crystal carrier and the amount of adsorbed Fab protein (change in protein concentration in Fab solution due to addition of MSU crystal) using a solution containing a certain amount of idiotype antigen (Fab). It is. 図4は、MSU結晶とFabとの結合を示す写真である。パネルA、C、Eは光学顕微鏡像、パネルB、D、Fは蛍光顕微鏡像と光学顕微鏡像との結合像である(倍率100倍)。パネルA、BはMSU結晶担体とマウスFITC標識IgG・Fabとの結合を、パネルC、DはあらかじめMSU結晶を牛胎児血清と混合した後にMSU結晶担体とマウスFITC標識IgG・Fabとを結合させた結果を、パネルE、FはMSU結晶担体とマウスFITC標識IgG・F(ab’)とを結合させた結果を、それぞれ観察したもの。各パネルの右下のバーは50μm。FIG. 4 is a photograph showing the binding of MSU crystal and Fab. Panels A, C, and E are optical microscope images, and panels B, D, and F are combined images of a fluorescence microscope image and an optical microscope image (magnification 100 times). Panels A and B show the binding between the MSU crystal carrier and mouse FITC-labeled IgG / Fab. Panels C and D show the MSU crystal carrier and mouse FITC-labeled IgG / Fab bound after mixing the MSU crystal with fetal bovine serum in advance. Panels E and F show the results of binding the MSU crystal carrier and mouse FITC-labeled IgG · F (ab ′) 2 , respectively. The lower right bar of each panel is 50 μm. 図5は、MSU結晶による樹状細胞の成熟を示す図である。左パネル(A)は対照(MSU結晶の添加なし)、右パネルはMSU結晶添加(B)の結果(フローサイトメーターで検出した樹状細胞のCD83の発現量)である。FIG. 5 is a diagram showing maturation of dendritic cells by MSU crystals. The left panel (A) is the control (no MSU crystal added), and the right panel is the result of MSU crystal addition (B) (dendritic cell CD83 expression level detected by flow cytometer). 図6は、本発明のイディオタイプワクチンを取り込ませた樹状細胞の自己リンパ球増殖刺激作用を示す図である。A=本発明のイディオタイプワクチン(MSU結晶担体とFabとの複合体)、B=MSU結晶担体のみ、C=Fabのみ、D=添加物なし(樹状細胞のみ)で、それぞれ培養した樹状細胞を用いてT細胞を誘導したもの。(N=3)FIG. 6 is a diagram showing the autologous lymphocyte proliferation stimulating action of dendritic cells incorporating the idiotype vaccine of the present invention. A = idiotype vaccine of the present invention (complex of MSU crystal carrier and Fab), B = MSU crystal carrier only, C = Fab only, D = no additives (dendritic cells only) Induced T cells using cells. (N = 3) 図7は、本発明のイディオタイプワクチンで誘導されたCTLの骨髄性株細胞に対する細胞傷害性を示す図である。A=本発明のイディオタイプワクチン、B=MSU結晶担体をあらかじめFBSでブロックしたものとFabの混合物(FabとMSU結晶担体とを結合させずにDCと共培養するモデル)、C=Fabのみ、D=添加物なしで、それぞれ培養した樹状細胞を用いてT細胞を誘導したもの。(N=3)FIG. 7 is a graph showing cytotoxicity of CTL induced by the idiotype vaccine of the present invention against myeloid cell lines. A = Idiotype vaccine of the present invention, B = MSU crystal carrier previously blocked with FBS and Fab mixture (model co-cultured with DC without binding Fab and MSU crystal carrier), C = Fab only, D = Induced T cells using dendritic cells cultured without additives. (N = 3)

Claims (6)

イディオタイプ抗原と尿酸塩結晶からなる担体とからなる複合体を含むことを特徴とするイディオタイプワクチン。   An idiotype vaccine comprising a complex comprising an idiotype antigen and a carrier comprising urate crystals. イディオタイプ抗原が、多発性骨髄腫細胞によって産生される免疫グロブリンまたは可変領域を含むその一部である、請求項1記載のイディオタイプワクチン。   The idiotype vaccine of claim 1, wherein the idiotype antigen is an immunoglobulin produced by multiple myeloma cells or a part thereof comprising a variable region. 尿酸塩結晶が、針状結晶を超音波処理したものである、請求項1または2記載のイディオタイプワクチン。   The idiotype vaccine according to claim 1 or 2, wherein the urate crystals are those obtained by sonicating acicular crystals. 請求項1〜3のいずれか1項記載のイディオタイプワクチンを製造する方法であって、イディオタイプ抗原と尿酸塩結晶とを一緒にして静置することによりイディオタイプ抗原と尿酸塩結晶との複合体を形成させる工程を含む方法。   A method for producing the idiotype vaccine according to any one of claims 1 to 3, wherein the idiotype antigen and the urate crystals are combined by allowing the idiotype antigen and the urate crystals to stand together. A method comprising the step of forming a body. 請求項1〜3のいずれか1項記載のイディオタイプワクチンを用いて樹状細胞を刺激し、この樹状細胞を用いて細胞傷害性T細胞を刺激することを特徴とする、特異的細胞傷害性T細胞の免疫応答を促進する方法。   A specific cytotoxicity characterized by stimulating dendritic cells using the idiotype vaccine according to any one of claims 1 to 3 and stimulating cytotoxic T cells using the dendritic cells. A method of promoting an immune response of sex T cells. 請求項1〜3のいずれか1項記載のイディオタイプワクチンを製造するための担体であって、尿酸ナトリウム結晶からなる担体。   A carrier for producing the idiotype vaccine according to any one of claims 1 to 3, wherein the carrier comprises sodium urate crystals.
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WO2004100984A1 (en) * 2003-05-13 2004-11-25 The University Of Massachusetts Endogenous adjuvant molecules and uses thereof
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