TW200900074A - Immune adjuvant - Google Patents

Immune adjuvant Download PDF

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Publication number
TW200900074A
TW200900074A TW096123064A TW96123064A TW200900074A TW 200900074 A TW200900074 A TW 200900074A TW 096123064 A TW096123064 A TW 096123064A TW 96123064 A TW96123064 A TW 96123064A TW 200900074 A TW200900074 A TW 200900074A
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Taiwan
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streptomyces
antigen
tumor
ppd
cells
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TW096123064A
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Chinese (zh)
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TWI339583B (en
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Tadao Ohno
Eiji Uchimura
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Cell Medicine Inc
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to an immune adjuvant, which is capable of bringing in strong immune-stimulating action and having high safety, and which comprises one or two or more immune stimulants respectively carried in two or more different micro-particulate immune stimulant carriers. Moreover, the immune adjuvant comprises (a) inorganic compound such as englobing-sized micro-particulate tertiary calcium phosphate, etc. by means of cells and (b) sediment assemblies by means of coagulation generated by the water-soluble protein and the polysaccharide to be used as the micro-particulate immune stimulant carrier.

Description

200900074 九、發明說明 【發明所屬之技術領域】 本發明係關於免疫佐劑。 【先前技術】 以往常用的大部分的免疫佐劑，係對生體投予後於局 部扮演（1 )使抗原不會快速擴散而保持以水性或油性乳 膠之性狀，緩慢放出抗原而以優異之效率供給與免疫細胞 ，（2 )向引起發炎反應部位集合且活性免疫細胞此2種 角色。近年，已知無論是誘發抗體產生之免疫反應，及誘 發殺手T細胞之細胞性免疫反應，均由抗原呈現細胞擔任 免疫反應的中心，以及做爲抗原呈現細胞的有樹狀細胞， 巨噬細胞以及B細胞。其中以樹狀細胞具有最強的抗原呈 現能力（Dendritic Cells, second edition, ed. Lotze, Μ. T. and Thomson, A. W., Academic Press, San Diego, 2001 ) 。與可有效活化抗原呈現細胞之物質一同投予至生體時， 將該物質用作爲免疫佐劑，使於體液性免疫反應以及細胞 性免疫反應均可以良好的效率誘發•增強對抗原之免疫反 應。 自過去即已知多種免疫佐劑，但以腫瘤治療、轉移及 預防再發等目的，可安全使用於腫瘤免疫療法且低價之免 疫佐劑極少。例如於利用培養樹狀細胞之腫瘤免疫療法， 使用输孔蟲戚血藍蛋白（keyhole limpet hemocianin)做 爲免疫佐劑而加以使用（Geiger, J. D·, et al.，Cancer 200900074200900074 IX. Description of the Invention [Technical Field to Which the Invention Is Ascribed] The present invention relates to an immunological adjuvant. [Prior Art] Most of the immunoadjuvants commonly used in the past are used to locally act on the body (1) to prevent the antigen from diffusing rapidly and to maintain the properties of the aqueous or oily latex, and to slowly release the antigen with excellent efficiency. Supply and immune cells, (2) to the inflammatory reaction site and active immune cells. In recent years, it has been known that both the immune response which induces antibody production and the cellular immune response which induces killer T cells are the center of the immune response by the antigen presenting cells, and the dendritic cells, macrophages which act as antigen presenting cells. And B cells. Among them, dendritic cells have the strongest antigen-presenting ability (Dendritic Cells, second edition, ed. Lotze, Μ. T. and Thomson, A. W., Academic Press, San Diego, 2001). When administered to a living body together with a substance capable of effectively activating an antigen-presenting cell, the substance is used as an immunological adjuvant, so that a humoral immune reaction and a cellular immune reaction can be induced with good efficiency. . A variety of immunological adjuvants have been known since the past, but are safe for tumor immunotherapy and have low-cost immunological adjuvants for the purposes of tumor treatment, metastasis, and prevention of recurrence. For example, using tumor immunotherapy for culturing dendritic cells, using keyhole limpet hemocianin as an immunoadjuvant (Geiger, J. D., et al., Cancer 200900074)

Res.，61，pp.8 5 1 3 - 8 5 1 9，200 1 )，但該物質極昂貴。亦提 案有將直接活化樹狀細胞之顆粒細胞巨噬細胞集落刺激因 子(granulocyte-macrophage colony stimulating factor : 以下有時會略稱爲「GM-CSF」）等細胞激素類，做爲免疫 佐劑而進行投予之方法，但細胞激素類更爲昂貴。 於製造爲預防感染症之疫苗，做爲安全且低價的免疫 佐劑之氫氧化銘（aluminum hydroxide)、佛氏不完全佐 劑（Freund's imcomplete adjuvant，因該佐劑爲油性而憂 慮其毒性），或使用如蒙他奈（Montanide )等活性不充 分之免疫佐劑。這些物質與使用於動物實驗之佛氏完全佐 劑（Freund's complete adjuvant:以下有時會略稱爲「 FCA」）及 Ribi 佐劑系統（Ribi Adjuvant System)相比 ，雖然毒性較低但免疫佐劑之活性亦弱。 檢測檢核菌感染歷史用之結核菌素，特別結核菌素中 的蛋白成分藉由硫銨沉澱法經純化之結核菌素（以下有時 會略稱爲「PPD」），可極安全地重複對人類進行投予， 且因其價格低廉而於全世界普及使用。本發明之發明者們 ’發現可將PPD用做爲免疫佐劑（PCT/JP00/00692 )。又 ，本發明之發明者們，發現將PPD與可溶性蛋白與黏多 糖藉由凝聚作用形成沉澱物，以物理性方法使其變性再藉 由對腫瘤組織內進行投予，而成爲誘發抗腫瘤免疫反應有 效的免疫佐劑（日本專利第3492671號）。該免疫佐劑與 溶解狀態之PPD相比，具有較高之免疫佐劑活性，且具 有與溶解狀態之PPD相同的極高的安全性。尤其與以 200900074 FCA及內毒素爲主要成分之脂多醣體（以下有時會略稱爲 「LPS J )相比，其免疫佐劑活性較不充分。 末梢血液中流動著可吞噬微粒子狀抗原之未成熟的抗 原呈現細胞。已知添加LPS於生體外（in vitro)培養中 之未成熟的抗原呈現細胞，可增進成熟，使其轉變爲表現 強力的抗原呈現能力。於此過程中被活化的抗原呈現細胞 ，會釋放出GM-CSF、介白素（以下有時會略稱爲「IL」 )、干擾素r (以下有時會略稱爲「IFNg」）等各種細胞 激素。因GM-CSF爲樹狀細胞生存之必須細胞生長因子， 活化後之樹狀細胞可藉由GM - C S F之自體分泌機轉而長期 保持於活化狀態持續地生存。不僅是樹狀細胞，巨噬細胞 亦同樣地分化自造血幹細胞爲相同系統之抗原呈現細胞， 受到免疫刺激時，會與樹狀細胞一樣的產生GM-CSF等細 胞激素。 實際上，藉由將固體化且微粒子化之腫瘤組織做爲抗 原，與GM-CSF等細胞激素一同投予至體內，可更有效率 地誘發對腫瘤細胞之抗腫瘤免疫反應（PCT/JP00/00692 ) 。此事實係來自於GM-CSF本身顯示其爲免疫佐劑同時， 自受到免疫佐劑刺激之抗原呈現細胞所產生之GM - C S F的 量，可得到表示原始免疫佐劑活性之指標。亦即，藉由將 來自人類的抗原呈現細胞所產生之GM-CSF進行定量，不 僅可使用於人體，且可推定於in vitro實驗系中免疫佐劑 之活性。 本發明之發明者們，利用上述方法，呈現了藉由將來 -6 - 200900074 自微生物之可溶性成分〔Mycobacterium bovis Bacillus Calmette-Guerin (以下有時會略稱爲「BCG菌」）等有機 溶劑萃取之可溶性成分〕’固定於做爲免疫刺激物質載體 之固體化組織，可得更強免疫佐劑作用’使用於包含來自 人類末梢血液之抗原呈現細胞之末梢血附著性細胞（ W02003/074079 ) ° 於該免疫佐劑，使用爲免疫刺激物質載體之固體化組 織，係於包含抗原呈現細胞之吞噬細胞中被消化’不殘存 體內會消失之生物分解性材料。尤其適用該免疫佐劑於人 體個體時，未使用主要組織相容性抗原完全一致的本人或 基因一致之同卵性雙胞胎之組織時，免疫刺激物質載體會 對該人體個體產生抗原性，而引起對該免疫刺激物質載體 或其分解之蛋白質片段之抗原抗體反應。因此泛用前述之 免疫佐劑使用爲免疫佐劑時（於免疫佐劑中加入規定之抗 原而誘發對該抗原的免疫反應時），會引起對於免疫刺激 物質載體本身多餘的抗原抗體反應，會引起對該抗原之免 疫反應會埋沒在全體的免疫反應中不易顯現此一問題。 反之，本發明之發明者們，提案1種或2種以上被担 載分子所担載之磷酸鈣微粒子之製造方法（特開2005-1 263 3 5號公報）。該磷酸鈣微粒子係Ca/ P之莫耳比爲 1.3以上之羥基磷灰石。另外，因非晶質磷酸鈣微粒子及 低結晶性磷灰石，不僅可被親水性之蛋白質分子亦可被疏 水性之低分子担載，顯示可得其做爲免疫刺激物質載體（ 特表20 0 2-5 2449 1號公報）。然而，使物理化學性狀完全 200900074 相異之2種以上的免疫刺激物質載體上，担載1種或2種 以上的免疫刺激物質，混合這些物質的免疫佐劑目前並未 獲知。 【發明內容】 本發明之課題係提供安全性優異，且可有效發揮強免 疫佐劑活性之免疫佐劑。 本發明之發明者們，發現藉由將結合菌素製成緩釋性 製劑而可得更高之免疫佐劑活性，及混合白蛋白及肝素藉 由凝聚作用形成沉澱時，和預先混合好的結核菌素蛋白， 發現該結核菌素蛋白會被捲入此沉澱中而形成不溶性微粒 子而成爲緩釋性製劑，以及將該不溶性微粒子與可溶性的 純化結核菌素一同做爲免疫佐劑，投予至做爲抗原的腫瘤 細胞時，可顯示強而有力的腫瘤防禦效果，投予至熱變性 後之體內腫瘤組織時，可誘發強而有力的抗腫瘤免疫反應 (日本專利第3492671號）。 另一方面，顯示可得磷酸鈣微粒子做爲免疫刺激物質 載體（特表2002-524491號公報），另外根據磷酸鈣微粒 子之製造方法（特開2005-126335號公報）所生成之磷酸 鈣微粒子，係Ca/ P之莫耳比爲1.3以上之羥基磷灰石， 含有大量的最大徑未達1/zm之奈米尺寸之微粒子，爲生 體適合性極高且安全之物質。藉由使可被抗原呈現細胞吞 噬尺寸之磷酸鈣微粒子擔載PPD，可製造免疫佐劑。 本發明之發明者們爲解決上述課題進而持續硏究，發 -8- 200900074 現將結核菌素所含之可溶性蛋白及來自微生物之可溶性成 分等使用爲免疫刺激物質，將該免疫刺激物質分別擔載於 微粒子狀之免疫刺激物質載體之（a )無機物，以及（b ) 可溶性蛋白與黏多醣藉由凝聚作用而產生之沉澱物，使用 這些載體的混合物做爲免疫佐劑時，與單獨使用1種免疫 刺激物質載體時相比，可得到相乘性的極強的免疫佐劑作 用。例如，發現混合白蛋白及肝素藉由凝聚作用形成沉澱 時，和結核菌素蛋白混合而得之不溶性微粒子，與含有根 據特開2005- 1 263 3 5號公報所記載之方法而得之Ca/ P之 莫耳比爲1.3以上之羥基磷灰石之磷酸鈣微粒子，使其擔 載PPD後之微粒子之混合物做爲免疫佐劑時，與單獨使 用各種成分時所得之免疫佐劑活性相比，可得遙遙凌駕地 相乘性的高免疫佐劑作用。本發明以這些知識爲基礎而被 完成。 亦即，根據本發明，本發明係包含於2種以上不同之 微粒子狀免疫刺激物質載體分別担載1種或2種以上的免 疫刺激物質之免疫佐劑，提供至少包含該做爲微粒子狀免 疫刺激物質載體之（a )無機物，以及（b )藉由可溶性蛋 白與黏多醣產生凝聚作用之沉澱物之組合之免疫佐劑。 根據本發明所希望之實施方式，2種免疫刺激物質載 體之組合係（a )藉由細胞而爲可被吞噬尺寸之微粒子狀 磷酸鈣，以及（b)藉由可溶性蛋白與黏多醣產生凝聚作 用之沉澱物，提供前述之免疫佐劑。 進而根據本發明所希望之實施方式，前述之（a)之 -9- 200900074 微粒子狀磷酸鈣係最大徑爲以下之磷酸鈣之前述之 免疫佐劑；前述之（a)之微粒子狀磷酸鈣係下記之Ca/ P之莫耳比爲丨·3以上，且含有碳酸根離子d2'爲3〜6 重量％，其結晶性規定爲藉由CuK α射線於X射線繞射光 譜中，以2Θ値26°、32°以及34°爲中心出現寬廣的尖峰値 ，且於33°出現300之米勒指數之肩峰之結晶性以上，且 規定以2 6 °、2 8 · 1 °、2 9 °、3 2 °、3 3 °以及3 4 °爲中心出現尖 峰値或肩峰之外，再加上2 1 1米勒指數與1 1 2米勒指數之 尖峰値係爲分離之結晶性以下之結晶性之微粒子狀磷酸鈣 ，提供前述之免疫佐劑。 進而根據本發明所希望之實施方式，前述（b)可溶 性蛋白係白蛋白之前述之免疫佐劑；前述（b)黏多醣係 肝素之前述之免疫佐劑；前述（b )不溶性物質之可溶性 蛋白與黏多醣產生凝聚作用之沉澱物，經蛋白分子間交聯 劑而被交聯之沉澱物，提供前述之免疫佐劑。 另外，免疫刺激物質係1種或2種以上選自細胞激素 、趨化激素、細胞生長因子以及荷爾蒙所成群之誘發劑之 前述之免疫佐劑；免疫刺激物質係1種或2種以上選自結 核菌素、純化結核菌素（PpD )、來自微生物的可溶性成 分、海藻糖二黴菌酸酯、LPS、脂質A、寡核苷酸、葡聚 醣、鑰孔蟲戚血藍蛋白、胞壁醯二胜肽、貝他定、左旋咪 喽、細胞激素、趨化激素、細胞生長因子以及荷爾蒙所成 群之物質之前述之免疫佐劑：來自微生物的可溶性成分係 i種或2種以上選自微生物之酒精萃取物、丙酮萃取物、 -10- 200900074 吡啶萃取物以及熱水萃取物所成群之萃取物之前述之免疫 佐劑。 自其他觀點，本發明係提供與抗原一同對包含人類之 哺乳類動物之體內進行投予後，而達成誘發對該抗原之全 身性免疫反應之前述之免疫佐劑。 根據本發明所希望之實施方式，本發明係提供抗原係 1種或2種以上選自真菌、放線菌、細菌、病毒、噬菌體 、克次體_、原蟲、真菌成分、放線菌成分、細菌成分、 病毒成分、随菌體成分、立克次體成分、原蟲成分、腫瘤 組織、腫瘤細胞、腫瘤細胞成分、腫瘤抗原蛋白以及腫瘤 抗原胜肽所成群之物質之前述之免疫佐劑。 另外’自其他觀點，本發明係提供將包含人類之哺乳 類動物之腫瘤組織以物理性方法使其變性後，再藉由對該 腫瘤組織進行投予而達成誘發抗腫瘤免疫反應之前述之免 疫佐劑。根據本發明所希望之實施方式，本發明係提供物 理性方法係1種或2種以上選自微波照射法、高週波凝固 法、冷凍凝固法、電燒加熱法、熱水注入法、酒精注入法 、栓塞法、放射線照射法、雷射照射法以及超音波破壞法 所成群之方法之前述之免疫佐劑。進而自其他觀點，本發 明係提供於體外與擔任免疫細胞混合後，藉由對包含人類 之哺乳類動物進行投予，而達成於該動物之生體內誘發全 身性免疫反應之前述之免疫佐劑。 根據本發明所希望之實施方式，本發明係提供擔任免 疫細胞係1種或2種以上選自樹狀細胞、巨噬細胞、b淋 -11 - 200900074 巴球、T淋巴球、自然殺手細胞、自然殺手T細胞以及造 血幹細胞所成群之細胞之前述之免疫佐劑。 進而提供一種包含前述免疫佐劑與抗原之疫苗。 根據本發明所希望之實施方式，本發明係提供抗原係 1種或2種以上選自真菌、放線菌、細菌、病毒、噬菌體 、立克次體、原蟲、或這些微生物成分所成群之抗原’使 用於預防感染症以及/或治療之前述之疫苗；以及提供抗 原係1種或2種以上選自腫瘤組織、腫瘤細胞、腫瘤細胞 成分、腫瘤抗原蛋白以及腫瘤抗原胜肽所成群之抗原，使 用於腫瘤之治療以及/或預防之前述之疫苗。 做爲抗原之例如藉由使用異種動物蛋白質，依據前述 之免疫佐劑於人類之外的哺乳類動物對該抗原可以優異之 效率使抗體產生。因此，根據本發明可提供產生抗體動物 ，及來自該產生抗體動物之產生抗體細胞或抗體基因。 本發明係提供將包含人類之哺乳類動物之腫瘤組織以 物理性方法使其變性後，再藉由對該腫瘤組織進行投予而 達成誘發抗腫瘤免疫反應之包含前述之免疫佐劑之腫瘤疫 苗；於體外與擔任免疫細胞混合後，藉由對包含人類之哺 乳類動物進行投予，而達成於該動物之生體內誘發全身性 免疫反應之包含前述之免疫佐劑之腫瘤疫苗；於體外與擔 任免疫細胞及抗原混合後，藉由對包含人類之哺乳類動物 進行投予，而達成於該動物之生體內誘發抗腫瘤免疫反應 之包含前述之免疫佐劑之腫瘤疫苗。 進而自其他觀點，本發明係提供一種全身性免疫反應 -12- 200900074 之誘發方法，對包含人類之哺乳類動物投予包含前述之免 疫佐劑之步驟之方法；一種誘發抗腫瘤免疫反應之方法， 將包含人類之哺乳類動物之腫瘤組織以物理性方法使其變 性後，包含於該腫瘤組織內投予前述之免疫佐劑之步驟之 方法；以及一種全身性免疫反應之誘發方法，將前述之免 疫佐劑及免疫擔當細胞預先於體外混合後，包含對包含人 類之哺乳類動物之生體內進行投予之步驟之方法。 於本說明書中所使用之用語其意義如下所述。 「免疫刺激物質」係指對包含人類之哺乳類動物進行 投予時，引起以及/或增強體內任何免疫反應之物質，對 包含人類之哺乳類動物之抗原呈現細胞於體外進行添加時 ’可引起以及/或增強該細胞任何活化之物質。因此，包 含對抗原本身具任何免疫刺激作用之免疫刺激物質。 「免疫刺激物質載體」係指目的爲担載前述之免疫刺 激物質之物質。免疫刺激物質其本身完全不具有免疫刺激 作用，或多爲具極低的免疫刺激作用，但載體本身有時也 會具有免疫刺激作用。 「2種以上相異之免疫刺激物質載體」或其類義語， 係指由相異的化學物質或相異的組成物所組成之2種以上 免疫刺激物質載體之外，亦包含由相同的化學物質或組成 物所組成，具相異物理化學性狀之免疫刺激物質載體。例 如’可將含有相同成分且該成分之比例相異之2種組成物 用做爲2種免疫刺激物質載體。或者，其係由相同之化學 物質或組成物所形成之微粒子，但粒子表面的物理化學性 -13- 200900074 狀相異時等，亦包含於此用語中。於本說明書中，並未有 所限制地解釋該用語任何意義，須最廣義地加以解釋。 「免疫佐劑」係指對抗原能夠引起以及/或增強免疫 反應之物質或組成物。將幾乎不具有免疫刺激作用之免疫 刺激物質載體本身稱爲免疫佐劑，但一般大多意指該載體 與任何的免疫刺激物質之組合。另外，也有僅將免疫刺激 物質稱爲免疫佐劑的狀況。本發明之免疫佐劑係指2種以 上之免疫刺激物質載體分別担載1種或2種以上的免疫刺 激物質，必定包含免疫刺激物質載體與免疫刺激物質之組 合。 本發明之免疫佐劑係包含於2種以上不同之微粒子狀 免疫刺激物質載體分別担載1種或2種以上的免疫刺激物 質，該微粒子狀免疫刺激物質載體其特徵爲其中至少1種 爲無機物，另1種爲可溶性蛋白與黏多醣藉由凝聚作用產 生之沉澱物。本發明之免疫佐劑，包含做爲微粒子狀免疫 刺激物質載體之（a )無機物以及（b )可溶性蛋白與黏多 醣藉由凝聚作用產生之沉澱物之組合，於前述（a )以及 (b )各別的微粒子狀免疫剌激物質載體上，担載了相同 或相異的免疫刺激物質。免疫刺激物質可使用1種或2種 以上。例如典型爲可使前述（a )以及（b )微粒子狀免疫 刺激物質載體分別担載相異的免疫刺激物質，或可使前述 (a )以及（b )微粒子狀免疫刺激物質載體分別担載相同 的免疫刺激物質。亦即，前述（a )以及（b )微粒子狀免 疫刺激物質載體中，使其中一個担載2種相異的免疫刺激 -14- 200900074 物質，可於另一個免疫刺激物質載體，使其担載這些免疫 刺激物質中1種或2種免疫刺激物質。 前述（a)以及（b)微粒子狀免疫刺激物質載體，可 含有相異的化學物質或相異的組成物所組成之2種以上微 粒子狀免疫刺激物質載體，或可分別由相同的化學物質或 組成物組成，而爲具相異物理化學性狀之免疫刺激物質載 體之混合物。例如可將前述（b )之沉澱物做爲微粒子狀 免疫刺激物質載體，由相同成分組合組成，且該成分比例 相異之2種組成物之混合物而使用。或亦可使用將前述（ a)之無機物做爲微粒子狀免疫刺激物質載體，由相同無 機物組成之微粒子，粒子表面物理化學性狀相異之微粒子 的混合物。 本發明之免疫佐劑，包含分別担載免疫刺激物質之前 述（a )以及（b )的微粒子狀免疫刺激物質載體之組合， 但可進而含有未担載免疫刺激物質之免疫刺激物質載體。 此時，所使用之免疫刺激物質載體，以選擇其本身具有免 疫刺激作用之免疫刺激物質載體爲佳。另外，本發明之免 疫佐劑，可進而含有未担載於免疫刺激物質載體之免疫刺 激物質。 最佳的實施方式，做爲2種微粒子狀免疫刺激物質載 體之組合係包含（a )藉由細胞而爲可被吞噬尺寸之微粒 子狀磷酸鈣，以及（b)藉由可溶性蛋白與黏多醣產生凝 聚作用之沉澱物之組合。以下，針對該特定的方式加以說 明，但本發明之免疫佐劑並未限定於該特定的方式。前述 -15- 200900074 之（a)藉由細胞而爲可被吞噬尺寸之微粒子狀磷酸鈣， 以及（b)藉由可溶性蛋白與黏多醣產生凝聚作用之沉澱 物之組合並未特別加以限制，可依其成分的種類、比例及 調製方法等任意的加以選擇。另外，前述實施方式之免疫 佐劑可進而混合第3種成分。該第3種成分除抗原外，可 使用與被担載之免疫刺激物質相異之免疫刺激物質等。可 以固體的載體担載抗原，載體亦可利用前述之（a )以及 (b)的微粒子狀免疫刺激物質載體。本發明之免疫佐劑 ，亦可包含未担載有與成分（a)或成分（b)所担載之免 疫刺激物質相同之免疫刺激物質之狀態。 藉由細胞而爲可被吞噬尺寸之微粒子狀磷酸鈣，可使 用例如特開2 005- 1 263 3 5號公報所記載之方法而調製。微 粒子狀磷酸鈣，可使用任意之微粒子狀磷酸鈣。例如根據 業者周知之方法，將市售之微粒子狀磷酸鈣粉末更加進行 粉碎，可選擇出藉由細胞而爲可被吞噬尺寸之微粒子狀磷 酸鈣而加以使用，亦可使用由磷酸溶液以及鈣溶液所製造 者。 亦可使用使磷酸鈣於微粒子狀基材上沉澱或析出，再 於該基材表面形成微粒子狀磷酸鈣薄膜者。此種方式亦包 含微粒子狀磷酸鈣。使用的基材若爲藉由細胞而爲可被吞 噬尺寸之微粒子狀則無特別限定。例如，在中性範圍爲不 溶性蛋白之纖維狀蛋白膠原蛋白及水溶性醣鏈高分子之硫 酸鹽軟骨素，與氫氧化鈣懸浮液及磷酸溶液混合過程中， 與低結晶性的磷灰石一同被沉澱，可製作包含奈米尺寸的 -16- 200900074 微小結晶之膠原蛋白/合成硫酸鹽軟骨素-磷灰石（Rhee， S. H., et al., Biomaterials., 22 ( 21 ) , pp . 2 8 4 3 - 2 8 4 7 , 2001 )，亦可將該微粒子使用做爲基材。但爲調製微粒子 狀磷酸鈣之基材並未限定於這些物質。 並未特別限定磷酸鈣的種類，可爲無水物、無水鹽類 或含水鹽類任一種，亦可爲結晶質或非結晶質任一種。進 而，鈣的一部分或磷酸的一部分亦可被其他原子及原子團 取代。這類型的磷酸鈣具體而言可舉出例如磷灰石（ Cai〇(P04)6(〇H)2 )、缺鈣磷灰石、一部分的金屬離子被 Ca取代之磷灰石、以硫酸根取代一部分磷酸根的磷灰石 、非晶質磷酸鈣、一部分的金屬離子被Ca取代之非晶質 磷酸鈣、八鈣磷酸鹽（Ca8H2(P04)6 · 5H20 )、以及磷酸 三鈣（Ca3(P04)2)、一部分的金屬離子被Ca取代之磷酸 三鈣等，但並非僅限定於這些物質。 以含有碳酸根離子C032·爲1〜15重量％之含碳酸磷 酸鈣爲佳。並未特別限定含碳酸磷酸鈣之種類，可爲無水 物、無水鹽類或含水鹽類任一種，亦可爲結晶質或非結晶 質任一種。含碳酸磷酸鈣可舉出例如含碳酸磷灰石及含碳 酸非結晶性磷酸鈣等。碳酸根離子的佔有位置並未特限定 ，例如可於磷酸位置、羥基位置、其他亦即爲非磷灰石位 置及表面吸附位置。其中最佳者可舉出爲C a/ P之莫耳比 爲1.3以上，且含有碳酸根離子C032·爲3〜6重量％，其 結晶性規定爲藉由CuK α射線於X射線繞射光譜中，以 2Θ値26°、32°以及34°爲中心出現寬廣的尖峰値，且於33° -17- 200900074 出現300之米勒指數之肩峰之結晶性以上，且規定以26° 、28.1°、29°' 32°、33°以及34°爲中心出現尖峰値或肩峰 之外，再加上2 1 1米勒指數與1 1 2米勒指數之尖峰値係爲 分離之結晶性以下之結晶性之微粒子狀磷酸鈣。 藉由細胞而爲可被吞噬微粒子之尺寸，一般以10ym 以下爲佳，最大徑爲1/zm以下特佳。人類細胞本身的大 小通常以球形而言直徑爲20/zm左右，附著之細胞及長 時間未分裂的巨大化細胞其長徑可超過lOOym。吞噬能 力佳之巨噬細胞有時亦會吞噬直徑2〇vm以上的微粒子 。因此，藉由細胞而爲可被吞噬微粒子之尺寸，並未特別 被限制。 使用藉由細胞而爲可被吞噬尺寸之微粒子狀磷酸鈣做 爲免疫刺激物質載體，使其可担載免疫刺激物質之方法， 可使用例如記載於特開2 0 0 5 - 1 2 6 3 3 5號公報之方法，但並 未限定於該方法。於磷酸鈣中，羥基磷灰石微粉末之比表 面積大於l〇〇m2/g時，因具優異之吸附能力，可將羥基磷 灰石微粉末使用爲多種物質之吸附劑。一般認爲羥基磷灰 石微粉末使用於各種化合物，而可得爲調製緩釋性醫藥之 基材，已依循根據吸附脫出之擴散模型，而顯示其緩釋性 之實驗（Burgos, A. E ., et al., B i o m at er i al s ., 23, pp.25 1 9-2526，2002 )。其他，已知低結晶性磷灰石及非 結晶性磷酸鈣等磷酸鈣，係原本爲生體內物質，而可與蛋 白等做爲體內生理活性物質安全的緩釋性醫藥之基材。亦 可使用這些由磷灰石類所形成之微粒子做爲免疫刺激物質 -18- 200900074 載體。 並未特別限定担載於藉由細胞而爲可被吞噬尺寸之微 粒子狀磷酸鈣之免疫刺激物質的種類。如上所述，因羥基 磷灰石微粉末具優異之吸附能力，可使用做爲使其担載免 疫刺激物質之各式各樣的物質。免疫刺激物質係可使用1 種或2種以上選自例如細胞激素、趨化激素、細胞生長因 子以及荷爾蒙誘發劑所成群之物質，例如以使用1種或2 種以上選自結核菌素、PPD、來自微生物的可溶性成分、 海藻糖二黴菌酸酯、LPS、脂質A、寡核苷酸、葡聚醣、 鑰孔蟲戚血藍蛋白、胞壁醯二胜肽、貝他定、左旋咪唑、 細胞激素、趨化激素、細胞生長因子以及荷爾蒙所成群之 物質爲佳。來自微生物的可溶性成分係可使用1種或2種 以上選自微生物之酒精萃取物、丙酮萃取物、吡啶萃取物 以及熱水萃取物所成群之萃取物。另外，細胞激素、趨化 激素、細胞生長因子以及荷爾蒙的誘發劑中，含有細菌的 菌體或細菌的菌體成分。但，磷酸鈣微粒子所担載之免疫 刺激物質並未限定於這些物質。 細胞激素、趨化激素、細胞生長因子或荷爾蒙，若爲 刺激抗原呈現細胞後可誘發其產生細胞激素之物質，無論 任何物質均可使用。包含例如干擾素〇:、干擾素Θ、干擾 素 r、介白素（IL)類（例如 IL-1、IL-2、IL-3、IL-4、 IL-7、IL-11、IL-12、IL-18、IL-27 等）、集落刺激因子 (CSF )(例如 G- CSF、GM-CSF、M-CSF 等）、上皮細 胞成長因子（EGF )、纖維芽細胞成長因子（FGF )(例 -19- 200900074 如FGF-l、FGF-2、FGF-7等）、胰島素、或人類生長荷 爾蒙等。 於調製來自微生物的可溶性成分之步驟中，並未特別 限定於以有機溶劑以及/或熱水等方法萃取可溶性成分， 若爲該業者所周知之方法均可。例如以使用乙醇調製萃取 物爲佳。於調製來自微生物的可溶性成分之步驟中所使用 之微生物，例如可爲細菌、真菌、或放線菌等，以可使用 細菌爲佳。更佳之細菌例示如下。在這些細菌中，可使用 任一種或亦可組合數種而加以使用。 棒狀白喉桿菌（Corynebacterium diphtheriae);假 結核棒桿菌（Corynebacterium pseudotuberculosis);結 膜乾燥症棒狀桿菌（Corynebacterium xerosis);腎棒狀 桿菌（Corynebacterium renale );庫氏柴狀桿菌（ Corynebacterium kutscheri ) ;假棒狀白喉桿菌（Res., 61, pp. 8 5 1 3 - 8 5 1 9,200 1 ), but the material is extremely expensive. Cytokines such as granulocyte-macrophage colony stimulating factor (hereinafter sometimes referred to as "GM-CSF"), which directly activate dendritic cells, are also proposed as immunological adjuvants. The method of administration is carried out, but cytokines are more expensive. For the manufacture of vaccines for the prevention of infectious diseases, as a safe and low-cost immunoadjuvant of aluminum hydroxide, Freund's imcomplete adjuvant, because the adjuvant is oily and worried about its toxicity) Or use an immunological adjuvant such as Montanide that is not sufficiently active. These substances are compared with the Freund's complete adjuvant (hereinafter sometimes referred to as "FCA") and the Ribi Adjuvant System (Ribi Adjuvant System) for animal experiments, although the toxicity is low, but the immunological adjuvant The activity is also weak. Detecting tuberculin in the history of nucleus infection, especially the protein component of tuberculin, which is purified by ammonium sulfate precipitation (hereinafter sometimes referred to as "PPD"), can be extremely safely repeated It is administered to humans and is widely used throughout the world because of its low price. The inventors of the present invention have found that PPD can be used as an immunological adjuvant (PCT/JP00/00692). Further, the inventors of the present invention found that PPD and soluble protein and mucopolysaccharide are precipitated by coagulation, denatured by physical means, and then administered into tumor tissues to induce anti-tumor immunity. A reaction-effective immunoadjuvant (Japanese Patent No. 3492671). The immunoadjuvant has a higher immunoadjuvant activity than the PPD in a dissolved state, and has the same high safety as the PPD in a dissolved state. In particular, compared with the lipopolysaccharide containing 200900074 FCA and endotoxin as the main component (hereinafter sometimes referred to as "LPS J"), the immunoadjuvant activity is insufficient. The peripheral blood flows with phagocytic microparticle antigen. Immature antigen-presenting cells. It is known that the addition of LPS to immature antigen-presenting cells in in vitro culture can enhance maturation and transform it into a strong antigen-presenting ability. The antigen-presenting cells release various cytokines such as GM-CSF, interleukin (hereinafter sometimes referred to as "IL"), and interferon r (hereinafter sometimes referred to as "IFNg"). Since GM-CSF is an essential cell growth factor for dendritic cell survival, the activated dendritic cells can be continuously maintained in an activated state by the autologous secretion mechanism of GM-C S F for a long period of time. Not only dendritic cells, but macrophages are also differentiated from hematopoietic stem cells as antigen-presenting cells of the same system. When immunostimulated, they produce cytokines such as GM-CSF like dendritic cells. In fact, by using solidified and micronized tumor tissue as an antigen, it can be administered to the body together with cytokines such as GM-CSF to induce an anti-tumor immune response to tumor cells more efficiently (PCT/JP00/ 00692 ). This fact is derived from the fact that GM-CSF itself shows that it is an immunoadjuvant and that the amount of GM-C S F produced by the antigen-presenting cells stimulated by the immunoadjuvant provides an index indicating the activity of the original immunoadjuvant. That is, by quantifying the GM-CSF produced by the antigen-presenting cells derived from humans, it can be used not only in the human body but also in the activity of the immunological adjuvant in the in vitro experimental line. The inventors of the present invention have used the above-mentioned method to extract organic solvents such as Mycobacterium bovis Bacillus Calmette-Guerin (hereinafter sometimes referred to as "BCG bacteria") by -6 - 200900074. The soluble component] is immobilized on a solidified tissue that acts as a carrier for the immunostimulatory substance, and a stronger immunoadjuvant effect can be obtained. 'Used in peripheral blood-adherent cells containing antigen-presenting cells derived from human peripheral blood (W02003/074079) ° The immunoadjuvant is a solidified tissue which is an immunostimulating substance carrier, and is digested into a phagocytic cell containing an antigen-presenting cell, and is a biodegradable material which does not remain in the body and disappears. In particular, when the immunoadjuvant is applied to a human individual, when the tissue of the same or homozygous identical twins with the same major histocompatibility antigen is not used, the immunostimulating substance carrier will produce antigenicity to the individual, resulting in The antigen-antibody reaction of the immunostimulatory substance carrier or its decomposed protein fragment. Therefore, when the above-mentioned immunoadjuvant is used as an immunoadjuvant (when an immunological adjuvant is added to an immunoadjuvant to induce an immune response to the antigen), an antigen-antibody reaction to the immunostimulatory substance carrier itself is caused. The immune response to the antigen is buried in the overall immune response and is less likely to manifest this problem. On the other hand, the inventors of the present invention have proposed a method for producing calcium phosphate fine particles supported by one or more kinds of supported molecules (JP-A-2005-1263 35). The calcium phosphate microparticles have a molar ratio of Ca/P of hydroxyapatite of 1.3 or more. In addition, due to the amorphous calcium phosphate microparticles and the low crystalline apatite, not only the hydrophilic protein molecules but also the hydrophobic low molecular molecules can be supported, which can be obtained as an immunostimulating substance carrier (Special Table 20) 0 2-5 2449 Bulletin No. 1). However, one or two or more kinds of immunostimulating substances are supported on a carrier of two or more kinds of immunostimulating substances having different physical and chemical properties, and that the immunoadjuvants in which these substances are mixed are not known at present. SUMMARY OF THE INVENTION An object of the present invention is to provide an immunoadjuvant which is excellent in safety and can effectively exert the activity of a strong immunological adjuvant. The inventors of the present invention have found that a higher immunoadjuvant activity can be obtained by making a combination of bacteriocin into a sustained-release preparation, and a mixed albumin and heparin are formed by agglomeration, and are pre-mixed. The tuberculin protein is found to be involved in the precipitation to form insoluble microparticles into a sustained-release preparation, and the insoluble microparticles are combined with the soluble purified tuberculin as an immunological adjuvant. When it is a tumor cell as an antigen, it exhibits a strong tumor defense effect, and when it is administered to a tumor tissue in thermal degeneration, it can induce a strong antitumor immune response (Japanese Patent No. 3492671). On the other hand, calcium phosphate microparticles which are obtained by the method of producing calcium phosphate microparticles, and calcium phosphate microparticles produced by the method for producing calcium phosphate microparticles (Japanese Laid-Open Patent Publication No. 2005-126335), It is a hydroxyapatite with a molar ratio of Ca/P of 1.3 or more. It contains a large number of fine particles with a maximum diameter of less than 1/zm, which is a highly biocompatible and safe substance. An immunoadjuvant can be produced by supporting PPD with calcium phosphate microparticles which can be engulfed in size by the antigen. In order to solve the above problems, the inventors of the present invention have continued to study the problem, and the soluble protein contained in tuberculin and the soluble component derived from microorganisms are used as immunostimulating substances, and the immunostimulating substances are separately used. (a) an inorganic substance contained in a microparticle-shaped immunostimulating substance carrier, and (b) a precipitate produced by coagulation of a soluble protein and a mucopolysaccharide, and a mixture of these carriers is used as an immunoadjuvant, and is used alone. When the immunostimulating substance carrier is used, a highly immunoadjuvant effect of multiplicity can be obtained. For example, it has been found that mixed albumin and heparin are insoluble fine particles obtained by mixing a tuberculin protein when a precipitate is formed by coagulation, and a Ca/containing method according to the method described in JP-A-2005-263 355. A calcium phosphate microparticle having a molar ratio of P of 1.3 or more, which is a mixture of microparticles after supporting PPD, is used as an immunoadjuvant as compared with the immunoadjuvant activity obtained when each component is used alone. It can be used as a high-immunity adjuvant for the superiority of the multiplication. The present invention has been completed on the basis of this knowledge. That is, according to the present invention, the present invention relates to an immunological adjuvant comprising one or more immunostimulating substances supported on two or more different microparticle-like immunostimulating substance carriers, and at least comprising the microparticle-like immunization An immunological adjuvant that irritates the carrier of (a) an inorganic substance, and (b) a combination of a precipitate of a coagulation effect of a soluble protein and a mucopolysaccharide. According to a preferred embodiment of the invention, the combination of the two immunostimulatory substance carriers (a) is a microparticle-like calcium phosphate of a phagocytic size by the cells, and (b) a coagulation effect by the soluble protein and the mucopolysaccharide The precipitate provides the aforementioned immunological adjuvant. Further, according to a preferred embodiment of the present invention, the above-mentioned (a)-9-200900074 microparticulate calcium phosphate is the above-mentioned immunoadjuvant having the largest diameter of the following calcium phosphate; and the above-mentioned (a) microparticulate calcium phosphate system The molar ratio of Ca/P is 丨·3 or more, and the carbonate ion d2′ is 3 to 6 wt%, and the crystallinity is defined by the CuK α ray in the X-ray diffraction spectrum. A wide peak 値 is present at the center of 26°, 32°, and 34°, and the crystallinity of the shoulder of the 300 Miller index is above 33°, and is specified to be 2 6 °, 2 8 · 1 °, 2 9 °, At 2 2 °, 3 3 ° and 3 4 °, there are spikes or shoulders, and the peaks of the 2 1 1 Miller index and the 1 1 2 Miller index are crystallinity below the crystallinity of separation. The particulate calcium phosphate provides the aforementioned immunological adjuvant. Further, according to a preferred embodiment of the present invention, the (b) soluble protein is an immunoadjuvant of the albumin; the (b) mucopolysaccharide heparin as described above; the (b) soluble protein of the insoluble substance A precipitate which is agglomerated with the mucopolysaccharide and which is crosslinked by a cross-linking agent of the protein molecule, provides the aforementioned immunoadjuvant. Further, the immunostimulating substance is one or more selected from the group consisting of cytokines, chemokines, cell growth factors, and hormone-inducing agents; and the immunostimulating substance is one or more selected. From tuberculin, purified tuberculin (PpD), soluble components from microorganisms, trehalose diguate, LPS, lipid A, oligonucleotide, dextran, keyhole limpet hemocyanin, cell wall The above-mentioned immunoadjuvants of a substance selected from the group consisting of bismuth peptide, betahedine, levorotin, cytokines, chemokines, cell growth factors, and hormones: soluble components derived from microorganisms, or more than two species The aforementioned immunoadjuvant from the extract of the microbial alcohol extract, acetone extract, -10-200900074 pyridine extract and hot water extract. From other viewpoints, the present invention provides an immunoadjuvant as described above which is administered to a mammal comprising a human in combination with an antigen to achieve a full-body immune response to the antigen. According to a preferred embodiment of the present invention, the present invention provides one or more antigenic systems selected from the group consisting of fungi, actinomycetes, bacteria, viruses, bacteriophages, gram-producing bodies, protozoa, fungal components, actinomycetes, bacteria. The aforementioned immunoadjuvant of a substance, a virus component, a bacterial component, a rickettsia component, a protozoal component, a tumor tissue, a tumor cell, a tumor cell component, a tumor antigen protein, and a tumor antigen peptide. In addition, the present invention provides a method for inducing an anti-tumor immune response by administering a tumor tissue containing a human mammalian body by physical means, and then administering the tumor tissue. Agent. According to a preferred embodiment of the present invention, the present invention provides one or more kinds of physical methods selected from the group consisting of microwave irradiation, high-frequency coagulation, freeze coagulation, electro-thermal heating, hot water injection, and alcohol injection. The aforementioned immunoadjuvant of the method of grouping, embolization, radiation irradiation, laser irradiation, and ultrasonic destruction. Further, from another viewpoint, the present invention provides the above-mentioned immunoadjuvant which induces a full-body immune response in a living body of the animal by administering to a human mammal in combination with an immune cell in vitro. According to a preferred embodiment of the present invention, the present invention provides one or more of the immune cell lines selected from the group consisting of dendritic cells, macrophages, b- -11 - 200900074, globules, T lymphocytes, natural killer cells, Natural killer T cells and the aforementioned immunoadjuvants of cells in groups of hematopoietic stem cells. Further provided is a vaccine comprising the aforementioned immunological adjuvant and antigen. According to a preferred embodiment of the present invention, the present invention provides one or more antigenic systems selected from the group consisting of fungi, actinomycetes, bacteria, viruses, bacteriophages, rickettsia, protozoa, or a group of these microbial components. The antigen is used in a vaccine for preventing infection and/or treatment; and one or more antigen systems are selected from the group consisting of tumor tissues, tumor cells, tumor cell components, tumor antigen proteins, and tumor antigen peptides. An antigen, a vaccine as described above for use in the treatment and/or prevention of tumors. As the antigen, for example, by using a heterologous animal protein, the mammal can be produced with an excellent efficiency against the antigen in a mammal other than a human in accordance with the aforementioned immunological adjuvant. Thus, according to the present invention, an antibody-producing animal, and an antibody-producing cell or antibody gene derived from the antibody-producing animal can be provided. The present invention provides a tumor vaccine comprising the above-mentioned immunoadjuvant, which is obtained by physically densifying a tumor tissue of a mammalian human mammal, and then administering the tumor tissue to induce an anti-tumor immune response; A tumor vaccine comprising the aforementioned immunoadjuvant which induces a systemic immune response in a living animal of the animal by administering to a mammal containing a human in vitro, and in vitro, and immunizing After the cells and the antigen are mixed, a mammalian mammal containing the human is administered to achieve a tumor vaccine containing the immunological adjuvant which induces an anti-tumor immune response in the animal. Further, from other viewpoints, the present invention provides a method for inducing a systemic immune response -12-200900074, a method for administering a mammalian mammal comprising a step comprising the aforementioned immunoadjuvant; and a method for inducing an anti-tumor immune response, a method comprising the step of administering the aforementioned immunological adjuvant to the tumor tissue after denaturation of a tumor tissue of a mammalian human mammal, and a method for inducing a systemic immune response, and immunizing the aforementioned The adjuvant and the immunologically-administered cells are mixed in advance in vitro, and include a method of administering a step of administering a human mammalian mammal. The terms used in this specification have the following meanings. "Immuno-stimulating substance" means a substance which causes and/or enhances any immune response in a human mammal when it is administered, and when an antigen-presenting cell containing a human mammal is added in vitro, it may cause and/ Or enhance any activated substance of the cell. Therefore, it contains an immunostimulating substance which has any immunostimulating effect on the antigen itself. The "immunostimulatory substance carrier" means a substance which is intended to carry the aforementioned immunostimulating substance. The immunostimulating substance itself has no immunostimulatory effect at all, or mostly has a very low immunostimulatory effect, but the carrier itself sometimes has an immunostimulating effect. "Two or more different types of immunostimulating substance carriers" or the likes thereof, which means that the same chemical substance is contained in addition to two or more kinds of immunostimulating substance carriers composed of different chemical substances or different compositions. Or an immunostimulatory substance carrier composed of a composition having different physical and chemical properties. For example, two compositions containing the same components and different ratios of the components can be used as carriers for the two immunostimulating substances. Alternatively, it is a microparticle formed of the same chemical substance or composition, but the physicochemical property of the surface of the particle is also included in this term. In this specification, any meaning of the term is not limited and is to be interpreted in the broadest sense. "Immuno adjuvant" means a substance or composition that is capable of causing and/or enhancing an immune response to an antigen. The immunostimulating substance carrier which hardly has an immunostimulating effect is itself called an immunological adjuvant, but generally means a combination of the carrier and any immunostimulating substance. In addition, there are cases where only an immunostimulating substance is referred to as an immunoadjuvant. The immunoadjuvant of the present invention refers to an immunostimulating substance carrier carrying two or more kinds of immunostimulating substances, and a combination of an immunostimulating substance carrier and an immunostimulating substance. The immunoadjuvant of the present invention comprises one or more immunostimulating substances each of which is contained in two or more different microparticle immunostimulating substance carriers, and the microparticle immunostimulating substance carrier is characterized in that at least one of them is an inorganic substance The other is a precipitate produced by coagulation of soluble protein and mucopolysaccharide. The immunoadjuvant of the present invention comprises (a) an inorganic substance as a carrier of the microparticle-like immunostimulating substance and (b) a combination of a soluble protein and a precipitate produced by agglomeration of the mucopolysaccharide, in the aforementioned (a) and (b) The same or different immunostimulating substances are carried on the respective microparticle-like immunostimulating substance carriers. One or two or more kinds of immunostimulating substances can be used. For example, the (a) and (b) microparticle-like immunostimulating substance carriers may each be loaded with a different immunostimulating substance, or the (a) and (b) microparticle-like immunostimulating substance carriers may be respectively carried the same. Immunostimulating substance. That is, in the above-mentioned (a) and (b) microparticle-like immunostimulating substance carriers, one of the two kinds of immunostimulating substances, which are different from each other, can be carried on another immunostimulating substance carrier. One or two immunostimulating substances of these immunostimulating substances. The (a) and (b) microparticle-like immunostimulating substance carriers may contain two or more kinds of microparticle-like immunostimulating substance carriers composed of different chemical substances or different compositions, or may be respectively the same chemical substance or The composition of the composition is a mixture of immunostimulatory substance carriers having different physicochemical properties. For example, the precipitate of the above (b) can be used as a microparticle-like immunostimulating substance carrier, and a mixture of the same components can be used, and a mixture of the two components having different ratios can be used. Alternatively, a mixture of the above-mentioned inorganic substance (a) as a microparticle-like immunostimulating substance carrier, microparticles composed of the same inorganic substance, and fine particles having different physicochemical properties on the surface of the particles may be used. The immunoadjuvant of the present invention comprises a combination of the microparticle-like immunostimulating substance carriers (a) and (b), respectively, which carry the immunostimulating substance, but may further contain an immunostimulating substance carrier which does not carry the immunostimulating substance. In this case, the immunostimulating substance carrier to be used is preferably an immunostimulating substance carrier which has an immunostimulating action itself. Further, the immunoadjuvant of the present invention may further contain an immunostimulating substance which is not carried on the carrier of the immunostimulating substance. In a preferred embodiment, the combination of the two microparticle-like immunostimulating substance carriers comprises (a) microparticle-like calcium phosphate which is phagocytologically sized by cells, and (b) produced by soluble protein and mucopolysaccharide A combination of precipitates of cohesion. Hereinafter, the specific embodiment will be described, but the immunoadjuvant of the present invention is not limited to this specific mode. The combination of (a) the particulate granulated calcium phosphate which can be phagocytized by the cells, and (b) the precipitate which forms a coagulation effect by the soluble protein and the mucopolysaccharide by the above-mentioned -15-200900074 is not particularly limited. It is arbitrarily selected depending on the type, ratio, and preparation method of the components. Further, the immunoadjuvant of the above embodiment may further mix the third component. In addition to the antigen, the third component may be an immunostimulating substance different from the immunostimulating substance to be loaded. The antigen may be carried on a solid carrier, and the carrier may also be a microparticle-like immunostimulating substance carrier of the above (a) and (b). The immunoadjuvant of the present invention may also contain a state in which an immunostimulating substance which is the same as the immunostimulating substance carried on the component (a) or the component (b) is not carried. The particulate calcium phosphate which is devourable by the cells can be prepared by a method described in, for example, JP-A No. 2,005-1,263,5. As the microparticulate calcium phosphate, any of the particulate calcium phosphates can be used. For example, the commercially available micronized calcium phosphate powder is further pulverized according to a method known to those skilled in the art, and a particulate calcium phosphate which can be phagocytized by a cell can be selected and used, and a phosphoric acid solution and a calcium solution can also be used. Manufacturer. It is also possible to use a calcium phosphate precipitated or precipitated on a fine particle substrate to form a fine particle calcium phosphate film on the surface of the substrate. This method also contains particulate calcium phosphate. The substrate to be used is not particularly limited as long as it is a microparticle having a size that can be swallowed by a cell. For example, in the neutral range of fibrous protein collagen of insoluble protein and sulfated chondroitin of water-soluble sugar chain polymer, in combination with low-crystallinity apatite during mixing with calcium hydroxide suspension and phosphoric acid solution It is precipitated to produce collagen-/synthetic sulfate chondroitin-apatite containing nano-sized-16-200900074 (Rhee, SH, et al., Biomaterials., 22 ( 21 ) , pp . 2 8 4 3 - 2 8 4 7 , 2001), the microparticles can also be used as a substrate. However, the substrate for modulating the particulate calcium phosphate is not limited to these materials. The type of calcium phosphate is not particularly limited, and may be either an anhydrous substance, an anhydrous salt or an aqueous salt, and may be either crystalline or amorphous. Further, a part of calcium or a part of phosphoric acid may be substituted by other atoms and radicals. Specific examples of the calcium phosphate of this type include apatite (Cai (P04) 6 (〇H) 2 ), calcium-deficient apatite, a part of metal ions substituted with Ca, apatite, and sulfate. a part of phosphate-substituted apatite, amorphous calcium phosphate, a part of metal ions replaced by Ca-substituted amorphous calcium phosphate, octacalcium phosphate (Ca8H2(P04)6 · 5H20), and tricalcium phosphate (Ca3 ( P04) 2), a part of the metal ions are replaced by Ca, such as tricalcium phosphate, etc., but are not limited to these. It is preferred to contain calcium carbonated phosphate containing 1 to 15% by weight of carbonate ion C032·. The type containing calcium carbonate is not particularly limited and may be any of an anhydrous substance, an anhydrous salt or an aqueous salt, and may be either crystalline or amorphous. Examples of the calcium carbonate-containing phosphate include carbonate-containing apatite and carbonic acid-containing amorphous calcium phosphate. The position of the carbonate ion is not particularly limited, and may be, for example, a phosphoric acid site, a hydroxyl group site, or other non-apatite sites and surface adsorption sites. The best one is that the Mo ratio of C a / P is 1.3 or more, and the carbonate ion C032· is 3 to 6 wt%, and the crystallinity thereof is defined by X-ray diffraction spectrum by CuK α ray. In the middle, 2宽26°, 32°, and 34° are the center of the broad peak 値, and the crystallinity of the shoulder of the 300 Miller index is above 33° -17- 200900074, and is specified to be 26°, 28.1°. , 29 ° ' 32 °, 33 ° and 34 ° centered on the peak or the shoulder, plus the 2 1 1 Miller index and the 1 1 2 Miller index of the peak 値 is the crystal of the separation below the crystal Sexual particulate calcium phosphate. The size of the phagocytose microparticles by the cells is preferably 10 μm or less, and the maximum diameter is preferably 1/zm or less. The size of the human cells themselves is usually about 20/zm in diameter in terms of spheres, and the attached cells and the giant cells that have not been split for a long time may have a long diameter of more than 100 μm. Macrophages with good phagocytosis sometimes also devour microparticles with a diameter of 2 〇vm or more. Therefore, the size of the microparticles that can be phagocytized by the cells is not particularly limited. A method in which a microparticle-like calcium phosphate having a phagocytic size by a cell is used as an immunostimulating substance carrier to carry an immunostimulating substance can be used, for example, as described in JP-A 2 0 0 5 - 1 2 6 3 3 The method of the 5th publication is not limited to this method. In the calcium phosphate, when the specific surface area of the hydroxyapatite fine powder is larger than l〇〇m2/g, the hydroxyapatite fine powder can be used as an adsorbent for various substances because of its excellent adsorption ability. It is generally considered that hydroxyapatite fine powder is used for various compounds, and can be obtained as a substrate for modulating a sustained-release medicine, and has been subjected to an experiment based on a diffusion model derived from adsorption to exhibit sustained release properties (Burgos, A. E., et al., B iom at er i al s ., 23, pp. 25 1 9-2526, 2002). In addition, it is known that calcium phosphate such as low-crystalline apatite and non-crystalline calcium phosphate is a substrate which is originally a living substance and can be used as a sustained-release medicine which is safe as a physiologically active substance in the body. These microparticles formed of apatite can also be used as an immunostimulating substance -18-200900074. The type of immunostimulating substance supported on the microparticulate calcium phosphate which can be phagocytized by the cells is not particularly limited. As described above, since the hydroxyapatite fine powder has an excellent adsorption ability, various materials can be used as the immunostimulating substance. The immunostimulating substance may be one or more selected from the group consisting of, for example, a cytokine, a chemokine, a cell growth factor, and a hormone-inducing agent, for example, one or more selected from the group consisting of tuberculin, PPD, soluble components from microorganisms, trehalose dimycolate, LPS, lipid A, oligonucleotide, dextran, keyhole limpet hemocyanin, cell wall bismuth peptide, betahedine, levamisole It is better for cytokines, chemokines, cell growth factors, and hormones. The soluble component derived from the microorganism may be one or more extracts selected from the group consisting of an alcohol extract of a microorganism, an acetone extract, a pyridine extract, and a hot water extract. Further, among the cytokines, chemokines, cell growth factors, and hormone-inducing agents, bacterial cells or bacterial cells are contained. However, the immunostimulating substance carried by the calcium phosphate microparticles is not limited to these substances. A cytokine, a chemokine, a cell growth factor, or a hormone, which can induce a cytokine-producing substance after stimulating an antigen, can be used regardless of any substance. Including, for example, interferon 〇: interferon Θ, interferon r, interleukin (IL) (eg, IL-1, IL-2, IL-3, IL-4, IL-7, IL-11, IL- 12. IL-18, IL-27, etc., colony stimulating factor (CSF) (eg G-CSF, GM-CSF, M-CSF, etc.), epithelial cell growth factor (EGF), fibroblast growth factor (FGF) (Example -19- 200900074 such as FGF-1, FGF-2, FGF-7, etc.), insulin, or human growth hormone. The step of preparing a soluble component derived from a microorganism is not particularly limited to extraction of a soluble component by an organic solvent or/or a hot water method, and may be a method known to those skilled in the art. For example, it is preferred to use an ethanol to prepare an extract. The microorganism used in the step of preparing the soluble component derived from the microorganism may be, for example, a bacterium, a fungus, or an actinomycete, and the like may be used. More preferred bacteria are exemplified below. Among these bacteria, any one may be used or a combination of several may be used. Corynebacterium diphtheriae; Corynebacterium pseudotuberculosis; Corynebacterium xerosis; Corynebacterium renale; Corynebacterium kutscheri; Diphtheria

Corynebacterium pseudodiphtheriticum )；馬玫瑰球菌（ Corynebacterium equi )；牛棒狀桿菌（Corynebacterium bovis);短棒狀桿菌（Corynebacterium parvum);萘驅 棒狀桿菌（Corynebacterium paurometabolum);化膿性 棒狀桿菌（Corynebacterium pyogenes);酵素棒狀桿菌 (Corynebacterium enzymicum )；哈其棒狀桿菌（ Corynebacterium hoagii ) ；斜方棒狀桿菌（Corynebacterium pseudodiphtheriticum); Corynebacterium equi; Corynebacterium bovis; Corynebacterium parvum; Corynebacterium paurometabolum; Corynebacterium pyogenes; Corynebacterium enzymicum; Corynebacterium hoagii; Corynebacterium rhizogenes

Corynebacterium striatum ) ；鼠敗血症桿菌（Corynebacterium striatum );

Corynebacterium murisepticum ) ; 腎 棒桿菌 (Corynebacterium murisepticum ) Corynebacterium

Corynebacterium nephridii ) ;福卡棒狀桿菌（ -20- 200900074Corynebacterium nephridii ); Corynebacterium fugues ( -20- 200900074

Corynebacterium p ho c ae ) ; 陰道棒 狀 桿 囷 ( Corynebacterium vaginalis ) ;黃色分 枝 桿 菌 (Corynebacterium p ho c ae ) ; Corynebacterium vaginalis ; Yellow branching bacterium (

Microbacterium flavum);纏繞紅球菌（Corynebacterium fascians);厚皮桿菌（Corynebacterium rathayi);黑穗 桿菌（Corynebacterium agropyri );小麥棒桿菌（ Corynebacterium tritici );伊拉桿菌（Corynebacterium iranicum ) ;腐爛分枝桿菌（Corynebacterium sepedonicum );甜菜桿菌（Corynebacterium beticola ) ;美國冬青桿菌（Corynebacterium ilicis);修密費拉姆 桿菌（Corynebacterium humiferum );修姆立桿菌（ Corynebacterium humuli ):粗厚桿菌（Corynebacterium hypertrophicans ) ;嗜酸桿菌（Corynebacterium ac et o ac i d o p h i 1 u m );阿西費拉姆桿菌（C o r y n e b a c t e r i u m acetophilum ) ；金色桿菌(Corynebacterium aurantiacum ) ;Corynebacterium callunae ；希 拉 朵姆-摩比李斯 ( Corynebacterium citreum-mobilis ) * 乙醇胺桿菌 ( Corynebacterium ethanolaminophilum ) :萎蔫短桿菌 ( Corynebacterium flaccumfaciens ) ♦ 麩胺酸棒桿菌 ( Corynebacterium glutamicum ) ； 力士棒桿菌 ( Corynebacterium herculis ) ； 解 烴棒桿菌 ( Corynebacterium hydrocarboclastus ) :百合棒桿菌 ( Corynebacterium lilium );海水桿 菌 (Corynebacteri um luteum );米德藍諾桿菌（Corynebacterium mediolanum );棲糖蜜棒桿菌（Corynebacterium melas secola ) ； 密 -21 - 200900074 托衣斯桿菌（Corynebacterium mycetoides):努比拉姆 桿菌（Corynebacterium nubilum )；粉紅桿菌（ Corynebacterium roseum ) ；血桿菌(Corynebacterium sanguinis);球形節桿菌（Arthrobacter globiformis); 關節桿菌 （Arthrobacter simplex);腫脹節桿菌 （ Arthrobacter tumescens );檸檬節桿菌（Arthrobacter citreus);乳節桿菌（Arthrobacter terregens);微黃色 節桿菌（Arthrobacter flavescens );脫砂節桿菌（ Arthrobacter duodecadis );藤黃節桿菌（Arthrobacter luteus);海洋節桿菌（Arthrobacter marinus);變異節 桿菌（Arthrobacter variabilis ) ；黏節桿菌（Microbacterium flavum); Corynebacterium fascians; Corynebacterium rathayi; Corynebacterium agropyri; Corynebacterium tritici; Corynebacterium iranicum; Corynebacterium Sepedonicum); Corynebacterium beticola; Corynebacterium ilicis; Corynebacterium humiferum; Corynebacterium humuli: Corynebacterium hypertrophicans; Acidophilus Corynebacterium ac et o ac idophi 1 um ); C orynebacterium acetophilum; Corynebacterium aurantiacum; Corynebacterium callunae; Corynebacterium citreum-mobilis * Corynebacterium Ethylaminophilum ) : Corynebacterium flaccumfaciens ♦ Corynebacterium glutamicum ; Corynebacterium h Erculis); Corynebacterium hydrocarboclastus: Corynebacterium lilium; Corynebacteri um luteum; Corynebacterium mediolanum; Corynebacterium melas secola; 21 - 200900074 Corynebacterium mycetoides: Corynebacterium nubilum; Corynebacterium roseum; Corynebacterium sanguinis; Arthrobacter globiformis; Arthrobacter simplex Arthrobacter tumescens; Arthrobacter citreus; Arthrobacter terregens; Arthrobacter flavescens; Arthrobacter duodecadis; Arthrobacter luteus ); Arthrobacter marinus; Arthrobacter variabilis; Myxobacteria

Arthrobacter viscosus):產多色性節桿菌（Arthrobacter polychromogenes ) ；結合節桿菌（Arthrobacter consociatus ) ;食尼古丁 節桿菌（Arthrobacter nicotinovorus )；亞麻短桿菌（Brevibacterium linens) ;乙酿短桿菌（Brevibacterium acetylicum);紅短桿菌 (Brevibacterium erythro genes ) ;黑利短桿菌（Arthrobacter viscosus): Arthrobacter polychromogenes; Arthrobacter consociatus; Arthrobacter nicotinovorus; Brevibacterium linens; Brevibacterium acetylicum; Brevibacterium erythro genes; Brevibacterium breve

Brevibacterium healii ):脂短桿菌（Brevibacterium lipolyticum );褐短桿菌（Brevibacterium brunneum )； 黄色短桿菌（Brevibacterium fulvum):紫棕短桿菌（ Brevibacterium fuscum)；栗色短桿菌（Brevibacterium helvolum );不動短桿菌（ Brevibacterium immotum ) ； 馬諾比卡短桿菌（Brevibacterium marinopiscum):索西 比巴短桿菌（Brevibacterium sociovivum):斯達歐斯短 -22- 200900074 桿菌（Brevibacterium stationis );海星短桿菌（ Brevibacterium maris);因派耳短桿菌（Brevibacterium imperiale );非特定短桿菌（Brevibacterium incertum ) :因菲力姆短桿菌（Brevibacterium insectiphilium);米 奴其非短桿菌（Brevibacterium minutiferula);庫阿雷短 桿菌（Brevibacterium quale ) ； Brevibacterium tegumenticola ； 產 氨 短 桿 菌 （Brevibacterium f ammoniagenes )；硫擴短桿菌（Brevibacterium sulfureum );撥法米椰短桿菌（Brevibacterium protophormiae); 薩匹迪短桿菌（Brevibacterium saperdae):黃色短桿菌 (Brevibacterium flavum )；因馬歐姆短桿菌（ Brevibacterium immariophilum );乳酸發酵短桿菌（ Brevibacterium lactofermentum ) ；粉紅短桿菌（Brevibacterium healii ): Brevibacterium lipolyticum; Brevibacterium brunneum; Brevibacterium fulvum: Brevibacterium fuscum; Brevibacterium helvolum; Brevibacterium Immotum ); Brevibacterium marinopiscum: Brevibacterium sociovivum: Stadios short-22- 200900074 Brevibacterium stationis; Brevibacterium maris; Brevibacterium imperiale; Brevibacterium incertum: Brevibacterium insectiphilium; Brevibacterium minutiferula; Brevibacterium quale; Brevibacterium Tegumenticola; Brevibacterium f ammoniagenes; Brevibacterium sulfureum; Brevibacterium protophormiae; Brevibacterium saperdae: yellow Tuberculosis (Brevibacterium flavum); Yinmaoumu Brevibacterium (Brevibacterium immariophilum); Brevibacterium (Brevibacterium lactofermentum); Brevibacterium pink (

Brevibacterium roseum):解糖短桿菌（Brevibacterium saccharolyticum.);谷氨酸短桿菌（Brevibacterium I divaricatum );絡乙七槓短桿菌（Brevibacterium leucinophagum ) ； 液化短桿菌 (Brevibacterium liquefaciens);噴頭-阿拉妮卡短桿菌（Brevibacterium pentoso-alanicum )；噴頭-胺基酸性短桿菌 （ Brevibacterium pentoso-aminoacidicum ) ；尿素短桿菌(Brevibacterium roseum): Brevibacterium saccharolyticum.; Brevibacterium I divaricatum; Brevibacterium leucinophagum; Brevibacterium liquefaciens; sprinkler-Alainica Brevibacterium pentoso-alanicum; Brevibacterium pentoso-aminoacidicum; Brevibacterium ureaplasma

Brevibacterium lyticum);短頸短桿菌（Brevibacterium albidum ):檸檬短桿菌（Brevibacterium citreum))； 黃體短桿菌（Brevibacterium luteum);磚紅色短桿菌（ Brevibacterium testaceum ) ;普西拉姆短桿菌（ -23- 200900074Brevibacterium lyticum); Brevibacterium albidum: Brevibacterium citreum; Brevibacterium luteum; Brevibacterium testaceum; Brevibacterium brevis (-23- 200900074)

Brevibacterium pusillum ) ;丙氛酸短桿菌（Brevibacterium pusillum ); Brevibacterium bacterium

Brevibacterium alanicum):產氣短桿菌（Brevibacterium aminogenes );產色短桿菌(Brevibacterium chromogenes );福利哥粒短桿菌（Brevibacterium frigoritolerans); 哈洛雷斯短桿菌（Brevibacterium halotolerans);發酵短 桿菌（Brevibacterium fermentans );氧化短桿菌（ B r e v i b a c t e r i u m ο X y d a n s );乳分枝桿菌（M i c r 〇 b a c t e r i u m lacticum );液化分枝桿菌（Microbacterium liquefaciens );黃色分枝桿菌（Microbacterium flavum ):耐熱分枝 桿菌（Microbacterium thermosphactum);產黃纖維單胞 菌（Cellulomonas Flavigena );酸性纖維單胞菌（ Cellulomonas acidula );歐羅克那纖維單胞菌 （ Cellulomonas aurogena ) ；克巴纖維單胞菌（Brevibacterium alanicum): Brevibacterium aminogenes; Brevibacterium chromogenes; Brevibacterium frigoritolerans; Brevibacterium halotolerans; Brevibacterium fermentans; Brevibacterium ο X ydans; M icr 〇bacterium lacticum; Microbacterium liquefaciens; Microbacterium flavum: Microbacterium thermosphactum; Cellulomonas Flavigena; Cellulomonas acidula; Cellulomonas aurogena; Cellulomonas gingivalis

Cellulomonas galba ):普西拉纖維單胞菌（Cellulomonas pusilla);佐氏庫特氏菌（Kurthia zopfii);變異庫特氏 菌（Kurthia variabilis ):貝索尼庫特氏菌（Kurthia bessonii ) ; 費氏酸桿菌 （Propionibacterium freudenreichii ):丙酸桿菌（P r o p i ο n i b a c t e r i um t h o eni i );嗜酸酸桿菌（Propionibacterium acidi-propionici); 詹氏酸桿菌（Propionibacterium jensenii);貪婪酸桿菌 C Propionibacterium avidum ) ；初油酸桿菌（Cellulomonas galba): Cellulomonas pusilla; Kurthia zopfii; Kurthia variabilis: Kurthia bessonii; Fischer Propionibacterium freudenreichii: Propioni ο nibacteri um tho eni i; Propionibacterium acidi-propionici; Propionibacterium jensenii; Propionibacterium avidum; Oleic acid bacillus

Propionibacterium acnes );第二型初油酸桿菌（ Propionibacterium acnes Type II ) •，嗜淋巴酸桿菌（Propionibacterium acnes ); Type II Phytobacterium bacterium ( Propionibacterium acnes Type II ) •, Lyophilic acidophilus (

Propionibacterium lymphophilum ) ；顆粒酸狀菌（ -24- 200900074Propionibacterium lymphophilum ); granule acid bacteria ( -24- 200900074

Propi onibacterium granulosum );非坎斯歐闻桿菌（ Eucobacterium foedans );酸解歐高桿菌（Eucobacterium alactolyticum ):雷古他歐高桿菌（Eucobacterium rectale);理膜薩歐高桿菌（Eucobacterium limosum); 盧米那姆歐高桿菌（Eucobacterium ruminantium);薩布 姆歐高桿菌（Eucobacterium saburreum);布太歐局桿囷 ( Eucobacterium budayi ) Eucobacterium nitrito genes ) Eucobacterium ventrio sum ) Eucobacterium mutiforme ) Eucobacterium cylindroids ) Eucobacterium moniliforme ) Eucobacterium tortuosum ) ； 產氮歐高桿菌 （ :倍多利歐歐高桿菌（ ；多形歐高桿菌 （ 西多利歐高桿菌 （ :膜米封歐高桿菌（ :圖魯薩歐高桿菌（ ； 纖維歐高桿菌 （Propi onibacterium granulosum ); Eucobacterium foedans; Eucobacterium alactolyticum: Eucobacterium rectale; Eucobacterium limosum; Lumi Eucobacterium ruminantium; Eucobacterium saburreum; Eucobacterium budayi Eucobacterium nitrito genes Eucobacterium ventrio sum Eucobacterium mutiforme ) Eucobacterium ciliindroids Eucobacterium moniliforme ) Eucobacterium tortuosum Nitrogen-producing bacteria (: B. oligobacteria); polymorphic eubacteria (S. euphorbia): : Membrane glutinosa ( : trorus or bacillus) (

Eucobacterium cellul 歐高桿菌）（osolvens);複合歐局 桿菌（Eucobacterium combesii);提奴挪歐商桿囷（ Eucobacterium tenue ):非西卡那歐尚桿菌 （ Eucobacterium fissicatena ) ;連續歐局桿菌(Eucobacterium cellul (osolvens); Eucobacterium combesii; Eucobacterium tenue: Eucobacterium fissicatena;

Eucobacterium contortum ):愛羅歐歐筒桿菌（Eucobacterium contortum): Tyroea

Eucobacterium aerofaciens ) ;乳歐尚桿菌（Eucobacterium aerofaciens );

Eucobacterium lentum);腸內歐高桿菌（Eucobacterium endocarditidis ) ；黑敏歐高桿菌（Eucobacterium helminthoides ) ；假性歐高桿菌（Eucobacterium pseudotortuosum );歐布斯歐高桿菌（Eucobacterium obstii ):紅血球歐高桿菌（Eucobacterium ethylicum ) -25- 200900074 :黑耳威歐高桿菌（Eucobacterium helwigiae);尿素歐 高桿菌（Eucobacterium ureolyticum);巴爾巴歐高桿菌 (Eucobacterium parvum );牛放線菌（Actinomyces bo vis );溶齒放線菌（Actinomyces odontolyticus);人 放線菌 （Actinomyces israelii );內氏放線菌 （ Actinomyces naeslundii );黏性放線菌放線菌 （ Actinomyces viscosus ) ；艾氏方夂線菌(Actinomyces eriksonii);潮濕放線菌（Actinomyces humiferus);竇 放線菌（Actinomyces suis );丙酸蛛菌（Arachnia propionica );雙叉乳桿菌（Bifidobacterium bifidum); 六株雙叉桿菌（Bifidobacterium adolescentis);嬰兒雙 叉桿菌（Bifidobacterium infantis );短雙叉桿菌（ Bifidobacterium breve ) ；龍根菌（Bifidobacterium longum ) ;假長鞭雙叉桿菌（Bifidobacterium pseudolongum ) ;嗜熱雙叉桿菌（Bifidobacterium thermophilum);豬雙叉桿菌（Bifidobacterium suis); 星形雙叉桿菌（Bifidobacterium asteroids);印度雙叉桿 菌（Bifidobacterium indicum );棒狀雙叉桿菌（ Bifidobacterium coryneforme ) ;多形雙叉桿菌（Eucobacterium lentum); Eucobacterium endocarditidis; Eucobacterium helminthoides; Eucobacterium pseudotortuosum; Eucobacterium obstii: Escherichia coli Eucobacterium ethylicum ) -25- 200900074 : Eucobacterium helwigiae; Eucobacterium ureolyticum; Eucobacterium parvum; Actinomyces bo vis; Actinomyces odontolyticus; Actinomyces israelii; Actinomyces naeslundii; Actinomyces viscosus; Actinomyces eriksonii; Actinomyces Humiferus); Actinomyces suis; Arachnia propionica; Bifidobacterium bifidum; six Bifidobacterium adolescentis; Bifidobacterium infantis; Bifidobacterium brev e); Bifidobacterium longum; Bifidobacterium pseudolongum; Bifidobacterium thermophilum; Bifidobacterium suis; Bifidobacterium asteroids Bifidobacterium indicum; Bifidobacterium coryneforme; Bifidobacterium polymorpha

Bacterionema matruchotii ) ;齲齒羅氏菌（Rothia dentocariosa ) ；結核分枝桿菌（Mycobacterium tuberculosis);田鼠分枝桿菌（Mycobacterium microti) ;牛分枝桿菌(Mycobacterium bovis ) ； Mycobacterium bovis BCG ;螺分枝桿菌（Mycobacterium africanum); -26- 200900074 非結核分枝桿菌（Mycobacterium k an sasii);海洋分枝 桿菌（Mycobacterium marinum ):猴分枝桿菌（Bacterionema matruchotii ); Rothia dentocariosa; Mycobacterium tuberculosis; Mycobacterium microti; Mycobacterium bovis; Mycobacterium bovis BCG; Mycobacterium africanum -26- 200900074 Mycobacterium k an sasii; Mycobacterium marinum: Mycobacterium marinum (

Mycobacterium simiae);胃分枝桿菌（Mycobacterium gastri ) ；不產色分枝桿菌（Mycobacterium nonchromogenicum ) ; 土 地分枝桿菌(Mycobacterium terrae );次要分枝桿菌（Mycobacterium triviale ):戈 登分枝桿菌（Mycobacterium gordonae);瘰癧分枝桿菌 (Mycobacterium scrofulaceum ) ;石蠟分枝桿菌（Mycobacterium simiae); Mycobacterium gastri; Mycobacterium nonchromogenicum; Mycobacterium terrae; Mycobacterium triviale: Mycobacterium Gordonae); Mycobacterium scrofulaceum; Mycobacterium parasiticus

Mycobacterium paraffinicum );胞內結核分枝桿菌（ Mycobacterium intracellulare);鳥分枝桿菌副結核亞種 (Mycobacterium avium);蟾分枝桿菌（Mycobacterium xenopi);潰瘍分枝桿菌（Mycobacterium ulcerans);草 分枝桿菌（My cobacterium phlei );母牛分枝桿菌（ Mycobacterium vaccae);迪氏分枝桿菌（Mycobacterium diernhoferi) ; Mycobacterium smegmatis;嗜熱分枝桿菌 (Mycobacterium thamnopheos ) ;龜分枝桿菌（Mycobacterium paraffinicum; Mycobacterium intracellulare; Mycobacterium avium; Mycobacterium xenopi; Mycobacterium ulcerans; Mycobacterium phlei (My cobacterium phlei); Mycobacterium vaccae; Mycobacterium diernhoferi; Mycobacterium smegmatis; Mycobacterium thamnopheos; Mycobacterium marinum (

Mycobacterium flavescens )；分枝桿菌(Mycobacterium fortuitum )；偶然分枝桿菌(Mycobacterium peregrinum );龜分枝桿菌（Mycobacterium chelonei);副結核分 枝桿菌（Mycobacterium paratuberculosis);痲瘋分枝桿 菌（Mycobacterium leprae ):鼠型癞瘋分枝桿菌（ Mycobacterium lepraemurium );檀木弗蘭克氏菌（ Frankia alni);沙棗弗蘭克氏菌（Frankia elaeagni); 迪克弗蘭克氏菌（Frankia discariae);克羅弗蘭克氏菌 -27- 200900074 (Frankia ceanothi ):克拉弗蘭克氏菌（Frankia coriariae );多迪弗蘭克氏菌（Frankia dryadis );普疏 弗蘭克氏菌（Frankia purshiae ):西皮弗蘭克氏菌（ Frankia cercocarpi ):分枝弗蘭克氏菌（Frankia brunchorstii );可迪弗蘭克氏菌（Frankia casuarinae ) ;費氏游動放線菌（Actinoplanes philippinensis):阿美 尼亞游動放線菌（Actinoplanes armeniacus);密蘇里游 動放線菌（Actinoplanes missouriensis ):猶它.游動放線 菌（Actinoplanes utahensis ) ；微白螺孢菌（Mycobacterium flutscens; Mycobacterium fortuitum; Mycobacterium peregrinum; Mycobacterium chelonei; Mycobacterium paratuberculosis; Mycobacterium leprae: Mycobacterium lepraemurium; Frankia alni; Frankia elaeagni; Frankia discariae; Crohn's -27-200900074 (Frankia ceanothi): Frankia coriariae; Frankia dryadis; Frankia purshiae: Frankia cercocarpi: Branched Frankia (Frankia Brunchostisti); Frankia casuarinae; Actinoplanes philippinensis: Actinoplanes armeniacus; Actinoplanes missouriensis: Utah. Actinoplanes utahensis; bacteria(

Spirillospora albida ) ;粉紅鏈孢 囊菌 （Spirillospora albida ); Sphaerotheca fuliginea (

Streptosporangium roseum ) ；禾本鏈孢囊菌（Streptosporangium roseum );

Streptosporangium vulgare );產紫晶鏈孢囊菌（ Streptosporangium amethystogenes);假性禾本鏈孢囊菌 (Streptosporangium pseudo vulgare ) ； 鏈孢囊菌 (Streptosporangium vulgare ); Streptosporangium amethystogenes; Streptosporangium pseudo vulgare; Streptomyces sp.

Streptosporangium nondiastaticum ) ; 鍵 抱囊菌 (Streptosporangium nondiastaticum ) ;

Streptosporangium longisporum );綠灰鏈孢囊菌（ Streptosporangium viridogriseum ) ； 鏈 孢囊菌 (Streptosporangium longisporum ); Streptosporangium viridogriseum; Streptomyces (

Streptosporangium album ) ； 鍵 孢囊菌 (Streptosporangium album ) ;

Streptosporangium albidum ) ； 鍵 孢囊菌 (Streptosporangium albidum ) ;

Streptosporangium viridialbum );紅色鍵抱囊菌（ Streptosporangium rubrum ) ； ( Amorphosphorangium auranticolor ) ；小瓶菌(Ampullariella regularis ) ；小 瓶菌（Ampullariella campanulata ) ；小瓶菌（Streptosporangium viridialbum); Streptosporangium rubrum; (Amorphosphorangium auranticolor); Ampullariella regularis; Ampullariella campanulata;

Ampullariella lobata)；小并瓦菌(Ampullariella digitata -28- 200900074 );水生角質菌（Pilimelia terevasa):水生角質菌（ Pilimelia anulata );游動單孢菌（Planomonospora paronto spora ) ；游動單孢菌（Planomonospora venezuelensis );長鞭游動單孢菌（Planobispora longispora);粉紅游動單孢菌（Planobispora rosea); 孢囊菌（Dactylosporangium aurantiacum);孢囊菌（ Dactylosporangium thailandense ):結合孢囊菌（ D ermatophilus congolensis ) ;昏暗地嗜皮菌（Ampullariella lobata); Ampullariella digitata -28- 200900074; Pilimelia terevasa: Pilimelia anulata; Planomonospora paronto spora; Actinomonospora Planomonospora venezuelensis ); Planobispora longispora; Planobispora rosea; Dactylosporangium aurantiacum; Dactylosporangium thailandense: combined with cystice (D ermatophilus Congolensis ); dimly secreted bacterium (

Geodermatophilus obscurus ) ;皮痕諾卡氏菌（Nocardia farcinica ):諾卡氏菌（Nocardia otitidus-caviarum); 巴西諾卡氏菌（Nocardiabrasiliensis);星形諾卡氏菌（ Nocardia asteroids );耳道炎諾卡氏菌（Nocardia transvalensis);諾卡氏菌（Nocardia formicae);腸諾 卡氏菌（Nocardia coeliaca):產多色性諾卡氏菌（ Nocardia polychromogenes );石鱲諾卡氏菌（Nocardia paraffinae);諾卡氏菌（Nocardia petroleophila);諾卡 氏菌（N 〇 c ar d i a s atur ne a );黑石諾卡氏菌（Nocardia kuroishii );諾卡氏菌（Nocardia rugosa);粉紅色諾卡 氏菌（Nocardia rhodnii);諾卡氏菌（Nocardia vaecinii );渾濁諾卡氏菌（Nocardia minima);黑色諾卡氏菌 (Nocardia blackwellii );結合諾卡氏菌（Nocardia convoluta);細胞諾卡氏菌（Nocardia cellulans);諾卡 氏菌（Nocardia lutea);球形諾卡氏菌（Nocardia globerula);深紅色諾卡氏菌（Nocardia rubropertincta -29- 200900074 );核諾卡氏菌（Nocardia corallina);橙色諾卡氏菌（ Nocardia salmonicolor);紅色諾卡氏菌（Nocardia rubra ):諾卡氏菌（Nocardia opaca ):釣諾卡氏菌 （ N 〇 c a r d i a c a 1 c a r e a );諾卡氏菌（N 〇 c a r d i a r e s t r i c t a ); 紅血球諾卡氏菌（Nocardia erythropolis);海洋諾卡氏 菌（Nocardia marina):諾卡氏菌（Nocardia atlantica ) :產諾卡氏菌（Nocardia aerocolonigenes);金接黃諾卡 氏菌（Nocardia aurantia);幫卡諾卡氏菌（Nocardia butanica);諾卡氏菌（Nocardia dassonvillei);諾卡氏 菌（Nocardia histidans);馬杜拉諾卡氏菌（Nocardia madurae ):諾卡氏菌（Nocardia neoopaca);皮耳哥諾 卡氏菌（Nocardia pellegrino);白樂杰馬杜拉諾卡氏菌 (Nocardia pelletieri ) :諾卡氏菌（N o c ar d i a sylvodorifera);特氏諾卡氏菌（Nocardia turbata);白 色諾卡氏菌 （Nocardia tenuis );變異諾卡氏菌 （ Nocardia variabilis);嗜熱假諾卡氏菌（Pseudonocardia thermophila);酸棗假諾卡氏菌（Pseudonocardia spinosa );白長鏈黴菌（Streptomyces albolongus);變異鏈黴 菌（Streptomyces viridaris ) ;白色鏈黴菌（Geodermatophilus obscurus); Nocardia farcinica: Nocardia otitidus-caviarum; Nocardia brasiliensis; Nocardia asteroids; ear canal Nocardia transvalensis; Nocardia formicae; Nocardia coeliaca: Nocardia polychromogenes; Nocardia paraffinae Nocardia petroleophila; N 〇c ar dias atur ne a ; Nocardia kuroishii; Nocardia rugosa; Nocardia pinkis Nocardia vaecinii; Nocardia minima; Nocardia blackwellii; Nocardia convoluta; Nocardia Nocardia cellulans); Nocardia lutea; Nocardia globerula; Nocardia rubropertincta -29-200900074; Nocardia corallina; orange Nocardia salmonicolor; Nocardia rubra: Nocardia opaca: N 〇cardiaca 1 carea; N 〇cardiarestricta Nocardia erythropolis; Nocardia marina: Nocardia atlantica: Nocardia aerocolonigenes; Nocardia aurantia ); Nocardia butanica; Nocardia dassonvillei; Nocardia histidans; Nocardia madurae: Nocardia neoopaca ); Nocardia pellegrino; Nocardia pelletieri: Noco dia sylvodorifera; Nocardia turbata; Nocardia tenuis; Nocardia variabilis; Pseudonocardia thermophila; Pseudonocardia spinosa; Streptomyces (Streptomyces albolongus); variant Streptomyces (Streptomyces viridaris); Streptomyces albus (

Streptomyces albo-niger):白孢鏈黴菌（Streptomyces albosporeus )；鏈黴菌(Streptomyces albovinaceus )； 鏈黴菌（Streptomyces aureocirculatus );鏈黴菌（ Streptomyces baarnensis ) ;鏈黴菌（Streptomyces clavifer);鏈黴菌（Streptomyces galtieri);撥比鏈黴 -30- 200900074 菌（Streptomyces bobili );鏈黴菌（Streptomyces longispororuber )；鏈黴菌(Streptomyces longisporus ) ;鏈黴菌（Streptomyces herbeus );鏈黴菌（ Streptomyces albofaciens );白色鏈黴菌（Streptomyces albus );白色鏈黴菌亞種（Streptomyces albus subsp · bruneomycini ):白色鏈黴菌亞種（Streptomyces albus subsp. pathocidicus ) i 鏈徽菌(Streptomyces almquistii );嗜氣鏈黴菌（S t r e p t o m y c e s a m i η o p h i 1 u s );可可鏈黴 菌(Streptomyces cacaoi ) ;鏈黴菌（Streptomyces chrestomyceticus ) ； 紙球 鏈徽菌 （Streptomyces flocculus);灰色鏈黴菌（Streptomyces gibsonii);鏈 黴菌（Streptomyces herbescens ):引朵鏈黴菌（ Streptomyces iodoformicus ) ;鍵黴菌（Streptomyces ochraceiscleroticus );使君子鏈黴菌（Streptomyces rangoon);龜裂鏈黴菌（Streptomyces rimosus);龜裂 鏈黴菌亞種(Streptomyces rimosus subsp. paromomycinus ):龜裂鏈黴菌亞種（Streptomyces rimosus subsp. pseudo verticillatus ) ; 鏈黴菌 （ Streptomyces spiroverticillatus );鏈徽菌（Streptomyces subflavus ) :變換鏈黴菌（Streptomyces varsoviensis);鏈黴菌（ Streptomyces xantholiticus );白色鏈黴菌亞種（ Streptomyces albus subsp. fungatus ):產氫鏈黴菌（ Streptomyces hydrogenans )；凡登鏈黴菌(Streptomyces vendargus )；產色鏈黴菌(Streptomyces achromo genes ) 200900074 :星狀鏈黴菌（Streptomyces antibioticus);貝氏鏈黴菌 (Streptomyces bikiniensis );可可鏈黴菌亞種（ Streptomyces cacaoi subsp. asoensis ) ； 鏈黴菌 (Streptomyces albo-niger): Streptomyces albosporeus; Streptomyces albovinaceus; Streptomyces aureocirculatus; Streptomyces baarnensis; Streptomyces clavifer; Streptomyces galtieri; Strepttomyces bobili; Streptomyces longispororuber; Streptomyces longisporus; Streptomyces herbeus; Streptomyces albofaciens; Streptomyces albus; Streptomyces albus subsp · bruneomycini: Streptomyces albus subsp. pathocidicus i Streptomyces almquistii; Streptomycesami η ophi 1 us; Streptomyces cocoa (Streptomyces cacaoi); Streptomyces chrestomyceticus; Streptomyces flocculus; Streptomyces gibsonii; Streptomyces herbescens: Streptomyces Iodoformicus); Streptomyces ochraceiscleroticus; Streptomyces rangoon; Streptomyces rimosus; Streptomyces rimosus subsp. paromomycinus: Streptomyces Rimosus subsp. pseudo verticillatus ); Streptomyces spiroverticillatus; Streptomyces subflavus: Streptomyces varsoviensis; Streptomyces xantholiticus; Streptomyces albus subsp. fungatus: Streptomyces hydrogenans; Streptomyces vendargus; Streptomyces achromo genes 200900074: Streptomyces antibioticus; Streptomyces bikiniensis; Streptomyces cocoa Subspecies (Streptomyces cacaoi subsp. asoensis); Streptomyces (

Streptomyces cinereoruber );鏈黴菌亞種（Streptomyces cinereoruber subsp. fructofermentans ) ;鍵黴菌亞種（Streptomyces cinereoruber); Streptomyces cinereoruber subsp. fructofermentans; subsp.

Streptomyces cylindrosporus subsp. piceus ) ;鏈黴菌(Streptomyces cylindrosporus subsp. piceus ); Streptomyces (

Streptomyces ederensis ) ; 鏈黴菌 （Streptomyces ful vo violaceus ) ;黃色鏈黴菌（Streptomyces fulvoviridis);均得鏈黴菌（Streptomyces gardneri); 球形鏈黴菌（Streptomyces globosus);腸炎鏈黴菌（ Streptomyces griseorubiginosus );鏈黴菌(Streptomyces herbaricolor )；英氏鏈黴菌(Streptomyces indigoferus ) ;印氏鏈黴菌（Streptomy ces litmocidini ) •，那氏鏈黴菌 (Streptomyces narbonensis ) ;鍵徵菌（Streptomyces nashvillensis ) ;鍵徽菌（Streptomyces noboritoensis ) ;鏈黴菌（Streptomyces phaeopurpureus );鏈黴菌（ Streptomyces purpeofuscus ) ;鏈徵菌（Streptomyces showdoensis ) ;鏈黴菌（Streptomyces tanashiensis ); 紫色鏈黴菌（Streptomyces violaceorectus );鏈黴菌（ Streptomyces zaomy ceticus ) ;阿布拉鍵徵菌（Streptomyces ederensis ); Streptomyces ful vo violaceus; Streptomyces fulvoviridis; Streptomyces gardneri; Streptomyces globosus; Streptomyces griseorubiginosus; Streptomyces Herbaricolor ); Streptomyces indigoferus; Streptomyces ces litmocidini • Streptomyces narbonensis; Streptomyces nashvillensis; Streptomyces noboritoensis; Streptomyces (Streptomyces phaeopurpureus); Streptomyces purpeofuscus; Streptomyces showdoensis; Streptomyces tanashiensis; Streptomyces violaceorectus; Streptomyces zaomy ceticus;

Streptomyces aburaviensis );青花鍵徵菌(Streptomyces caeruleus )；小串鍵黴菌(Streptomyces catenul ae )；金 羊毛鍵徽菌亞種（S t r e p t o m y c e s c h r y s o m a 11 u s s u b s p · fumigatus ) ;鏈黴菌(treptomyces x a n t h o c i d i c u s );產 -32- 200900074 色鏈黴菌亞種（S t r e p t 〇 m y c e s a c h r 〇 m 〇 g e n e s s u b s p · rubradiris );鏈黴菌（Streptomyces anandii );鏈黴菌 (Streptomyces aurantiogriseus ):撥比鏈黴菌亞種（ Streptomyces bobili subsp. sporificans ) ;鏈黴菌（Streptomyces aburaviensis ); Streptomyces caeruleus; Streptomyces catenul ae; S treptomyceschrysoma 11 ussubsp fumigatus; treptomyces xanthocidicus; producing -32- 200900074 Strepept 〇mycesachr 〇m 〇genessubsp · rubradiris; Streptomyces anandii; Streptomyces aurantiogriseus: Streptomyces bobili subsp. sporificans; Streptomyces

Streptomyces cinerochromogenes ) ;卷鬚鏈黴菌（Streptomyces cinerochromogenes ); Streptomyces faecalis

Streptomyces cirratus ) ;山丘鏈黴菌（Streptomyces collinus )；泛溫鏈黴菌(Streptomyces eurythermus )； 鮮黃鏈黴菌（Streptomyces galbus);加利鏈黴菌（ Streptomyces galilaeus )；力tl 利鏈黴菌（Streptomyces griseoruber ) ；鏈黴菌(Streptomyces griseosporeus ) ； 氫鏈黴菌亞種(Streptomyces hygr oscopicus sub sp. ossamyceticus ) ;庫氏鏈徽菌(Streptomyces kurssano vii ) ;藤黃灰鏈黴菌（Streptomyces luteogriseus ) ； massasporeus ( Streptomyces massasporeus ) ;奇異鏈徽 菌(Streptomyces mirabilis ) ;鍵徽菌（Streptomyces multispiralis );耐氏鏈黴菌(Streptomyces naganishii ) ;內亞氏鏈黴菌（Streptomyces neyagawaensis);諾亞氏 鏈黴菌（Streptomyces nojiriensis);橄欖色鏈黴菌（ Streptomyces olivochromogenes ) ; 鏈黴菌 （Streptomyces cirratus; Streptomyces collinus; Streptomyces eurythermus; Streptomyces galbus; Streptomyces galilaeus; Streptomyces griseoruber; Streptomyces griseosporeus; Streptomyces hygr oscopicus sub sp. ossamyceticus; Streptomyces kurssano vii; Streptomyces luteogriseus; massasporeus ( Streptomyces massasporeus ); singular Streptomyces mirabilis; Streptomyces multispiralis; Streptomyces naganishii; Streptomyces neyagawaensis; Streptomyces nojiriensis; Streptomyces olivaceus Streptomyces olivochromogenes ); Streptomyces (

Streptomyces phaeofaciens ) ;鏈黴菌（Streptomyces pulveraceus);枝鏈黴菌（Streptomyces rameus);抗黴 素鏈黴菌（Streptomyces resistomycificus );香豆鏈黴菌 (Streptomyces rishiriensis ) ;熱紫鏈黴菌（Streptomyces phaeofaciens); Streptomyces pulveraceus; Streptomyces rameus; Streptomyces resistomycificus; Streptomyces rishiriensis; Streptomyces thermophilus

Streptomyces thermo violaceus ) ;鏈黴菌(Streptomyces -33- 200900074 violaceochromogenes ) ; 鏈黴菌 （ Streptomyces afghaniensis);鏈黴菌（Streptomyces arenae);鏈黴菌 (Streptomyces attrocyaneus ) :色褐鏈黴菌（ Streptomyces chromofuscus ) ;鍵徽菌（Streptomyces durhamensis ):疾黎鏈黴菌（Streptomyces echinatus ) ;鏈黴菌（Streptomyces filipinensis ):鑲邊鏈黴菌（ Streptomyces fimbriatus );灰色產色鏈黴菌（ Streptomyces griseochromogenes )；印阿卡鏈黴菌（ Streptomyces iakyrus ) ；鏈黴菌(Streptomyces lucensis );鏈黴菌（Streptomyces malachitofuscus );鏈黴菌（ Streptomyces malachitorectus ) ;叢毛鏈黴菌（Streptomyces thermo violaceus ); Streptomyces -33- 200900074 violaceochromogenes ; Streptomyces afghaniensis; Streptomyces arenae; Streptomyces attrocyaneus: Streptomyces chromofuscus; Streptomyces durhamensis ): Streptomyces echinatus; Streptomyces filipinensis: Streptomyces fimbriatus; Streptomyces griseochromogenes; Streptomyces iakyrus; Streptomyces lucensis; Streptomyces malachitofuscus; Streptomyces malachitorectus; Streptomyces faecalis

Streptomyces pilosus ) ; 鍵徽菌 （ Streptomyces albidofuscus ) ；白淺灰鏈黴菌（Streptomyces albogriseolus ) ;生二素鍵徽菌（Streptomyces ambofaciens ) ；鏈黴菌(Streptomyces anthocyanicus ) ； 鏈黴菌（Streptomyces antimycoticus );碳青鏈黴菌（ Streptomyces argenteolus ) ; 鏈黴菌 （Streptomyces atratus ) ;金色鍵黴菌（Streptomyces aureofaciens )； 鏈黴菌（Streptomyces avellaneus);紅莓鏈黴菌（ Streptomyces caesius ):肉膜鏈徵菌（Streptomyces carnosus);鍵黴菌（Streptomyces chibaensis);鏈黴菌 (Streptomyces coelescens )；鏈黴菌亞種(Streptomyces coelicolor subsp· achrous);鏈黴菌亞種（Streptomyces c o e 1 i c o 1 o r subsp. coelicofers ) ;鍵徵菌亞種（ -34- 200900074Streptomyces pilosus ); Streptomyces albidofuscus; Streptomyces albogriseolus; Streptomyces ambofaciens; Streptomyces anthocyanicus; Streptomyces antimycoticus; carbon blue chain Streptomyces argenteolus; Streptomyces atratus; Streptomyces aureofaciens; Streptomyces avellaneus; Streptomyces caesius: Streptomyces carnosus; Streptomyces chibaensis); Streptomyces coelescens; Streptomyces coelicolor subsp. achrous; Streptomyces coe 1 ico 1 or subsp. coelicofers; Bacterial subspecies (-34- 200900074)

Streptomyces coelicolor sub sp. coelicolatus )；鏈黴菌亞 種(Streptomyces coelicolor subsp. coelicovarians )；柯 基鏈黴菌（Streptomyces corchorusii )鏈黴菌；（ Streptomyces cyanogenus ):殺粉酶鏈黴菌亞種（ Streptomyces diastaticus subsp. ardesiacus ) ；殿粉酶產 色鏈黴菌(Streptomyces d i a s t at o c hr o m o g e ne s subsp. bracus);塗鏈黴菌（Streptomyces endus);鏈黴菌（ Streptomyces erumpens ) ;灰橙鏈黴菌（Streptomyces griseoaurantiacus );灰褐類群鏈黴菌（S t r e p t o m y c e s griseofuscus ) ；鏈黴菌(Streptomyces griseolosuffuscus );鏈黴菌（Streptomyces griseoluteus ):灰色鏈黴菌 亞種(Streptomyces griseus subsp. difficilis )；濕鏈黴 菌（Streptomyces humidus);吸水鍵徽菌（Streptomyces hygroscopicus );吸水鏈黴菌亞種（Streptomyces hygroscopicus subsp. angustmy ceticus ) ；吸水鏈黴菌亞 種(Streptomyces hygroscopicus subsp. decoyicus) ；吸 水鏈黴菌亞種 (Streptomyces hy groscopius subsp. glebosus);黎巴嫩鏈黴菌（Streptomyces libani);黎巴 嫩鏈黴菌亞種（Streptomyces libani subsp. rufus);變錯 青鏈黴菌（Streptomyces lividans ):盧氏鏈黴菌（ Streptomyces lusitanus ) ；利迪鏈徵菌(Streptomyces lydicus ) ; 長川 鏈黴菌 （ Streptomyces me 1 ano spo ro faciens ) ; 鍵徵菌 （ Streptomyces misionensis );鏈徽菌（Streptomyces murinus ):鏈黴 -35- 200900074 菌（Streptomyces mutabilis ):產黑鏈黴菌（ Streptomyces nigrescens);多結鏈黴菌（Streptomyces nodosus);黑胡桃鏈黴菌（Streptomyces nogal at er)+; 鏈黴菌（Streptomyces olivaceiscleroticus);橄欖綠鏈黴 菌（Streptomyces olivaceoviridis) •，橄檀色鏈黴菌（ Streptomyces olivaceus );小小鏈黴菌（Streptomyces parvullus);平板鏈黴菌（Streptomyces platensis);普 卡鏈黴菌（Streptomyces plicatus );鏈黴菌（ Streptomyces poonensis ) ; 鍵徽菌 （Streptomyces psammoticus ) •’ 鍵徽菌 ( Streptomyces purpurogeneiscleroticus ) ; 鏈黴菌 （Streptomyces recifenis);鏈黴菌（Streptomyces rochei);鏈黴菌（ Streptomyces rokugoensis ) ;鍵黴菌 （Streptomyces roseodiastaticus );魯地鏈黴菌亞種（Streptomyces rutgersensis subsp. castelarense ) ;佐山鍵黴菌（Streptomyces coelicolor subsp. coelicolatus; Streptomyces coelicolor subsp. coelicovarians; Streptomyces corchorusii Streptomyces; Streptomyces cyanogenus: Streptomyces diastaticus subsp. ardesiacus Streptomyces diast at oc hr omoge ne s subsp. bracus; Streptomyces endus; Streptomyces erumpens; Streptomyces griseoaurantiacus; Streptomyces griseoaurantiacus; Streptomyces griseofuscus ); Streptomyces griseolosuffuscus; Streptomyces griseoluteus: Streptomyces griseus subsp. difficilis; Streptomyces humidus; Streptomyces hygroscopicus; Streptomyces hygroscopicus subsp. angustmy ceticus; Streptomyces hygroscopicus subsp. decoyicus; Streptomyces hy groscopius subsp. Glebosus); Streptomyces libani; Streptomyces libani subsp. rufus; Streptomyces lividans: Streptomyces lusitanus; Streptomyces Lydicus ); Streptomyces me 1 ano spo ro faciens; Streptomyces misionensis; Streptomyces murinus: Streptomyces mutabilis: Streptomyces mutabilis Streptomyces nigrescens); Streptomyces nodosus; Streptomyces nogal at er+; Streptomyces olivaceiscleroticus; Streptomyces olivaceoviridis • Streptomyces olivaceus Streptomyces parvullus; Streptomyces platensis; Streptomyces plicatus; Streptomyces poonensis; Streptomyces psammoticus • Streptomyces purpurogeneiscleroticus Streptomyces (Streptomyces recifenis); Streptomyces rochei; Streptomyces rokugoensis; Streptomyces roseodiastaticus; Streptomyces rutgersensis subsp. castelarense;

Streptomyces sayamaensis ) ;鏈黴菌（Streptomyces sendaiensis );鏈黴菌（Streptomyces sioyaensis );唐 德鏈黴菌（Streptomyces tendae );熱普通鏈黴菌（ Streptomyces thermo vulgaris ) :三色鍵黴菌 （ Streptomyces tricolor ) ; 鏈黴菌 （Streptomyces tubercidicus )；鏈黴菌(Streptomyces tumemacerans )； 鏈黴菌 （Streptomyces vastus );紫邊鏈黴菌 （ Streptomyces v i o 1 ac e o 1 atu s );三七根際鏈黴菌（Streptomyces sayamaensis); Streptomyces sendaiensis; Streptomyces sioyaensis; Streptomyces tendae; Streptomyces thermo vulgaris: Streptomyces tricolor; Streptomyces tubercidicus Streptomyces tumemacerans; Streptomyces vastus; Streptomyces vio 1 ac eo 1 atu s; Streptomyces vio ac eo 1 atu s

Streptomyces violaceus-niger );紫紅鏈黴菌（ -36- 200900074Streptomyces violaceus-niger ); Streptomyces rhodochrous ( -36- 200900074

Streptomy ce s violaceus-ruber ) :生綠鏈黴菌（ Streptomyces viridifaciens ) ;鍵徵菌（Streptomyces atroolivaceus ) ;鏈黴菌（Streptomyces cyanocolor ) ； 禾粟鏈黴菌（Streptomyces graminofaciens);鏈黴菌（Streptomy ce s violaceus-ruber ): Streptomyces viridifaciens; Streptomyces atroolivaceus; Streptomyces cyanocolor; Streptomyces graminofaciens; Streptomyces (Streptomyces viridifaciens)

Streptomyces griseoplanus ) ;鏈黴菌(Streptomyces alb aduncus )；鏈黴菌(Streptomyces albo spinus );小白 色鏈黴菌（Streptomyces albulus );鏈黴菌（ Streptomy ce s althioticus ) ;阿拉伯鏈黴菌（Streptomyces griseoplanus; Streptomyces alb aduncus; Streptomyces albo spinus; Streptomyces albulus; Streptomy ce s althioticus; Streptomyces alpina (

Streptomyces arabicus ) ；鏈黴菌亞種(Streptomyces atroolivaceus subsp. mutomycini );拿肯氏鏈黴菌（ Streptomyces canus);恰塔努加鏈黴菌（Streptomyces chattano o g ens i s ) ；鏈黴菌(Streptomyces chlorobiens ) ;鏈黴菌（Streptomyces cuspidosporus );鏈黴菌（Streptomyces arabicus); Streptomyces atroolivaceus subsp. mutomycini; Streptomyces canus; Streptomyces chattano og ens is; Streptomyces chlorobiens; Streptomyces Streptomyces cuspidosporus); Streptomyces (

Streptomyces gancidicus);卡特利鍵徽菌（Streptomyces griseoflavus ) ;鏈黴菌（Streptomyces griseoincarnatus );灰略紅鏈黴菌（Streptomyces griseorubens );鏈黴 菌（Streptomyces macrosporeus );孔雀石鏈黴菌（ Streptomyces malachiticus ) ;鏈黴菌（Streptomyces matensis );諾個思鏈黴菌（Strepomyces noursei );鏈 黴菌（Streptomyces olivoviridis );假淺灰鏈黴菌（ Streptomyces pseudogriseolus ) ;赤色鏈球菌（Streptomyces gancidicus); Streptomyces griseoflavus; Streptomyces griseoincarnatus; Streptomyces griseorubens; Streptomyces macrosporeus; Streptomyces malachiticus; Streptomyces Streptomyces matensis ); Strepomyces noursei; Streptomyces olivoviridis; Streptomyces pseudogriseolus; Streptococcus erythropolis

Streptomyces rubiginosus)；稀疏鏈徵菌（Streptomyces sparsogenes ) ；鏈黴菌 C Streptomyces viridiviolaceus ) ;鏈黴菌（Streptomyces virido-diastaticus );充裸鏈黴 -37- 200900074 菌（Streptomyces calvus );鏈黴菌（Streptomyces cyanoalbus)；鏈黴菌（Streptomyces finlayi);淺黃鏈 黴菌（Streptomyces flaveolus ):噴泉鏈黴菌（ Streptomyces gey siriensis ) ;鏈黴菌（Streptomyces herbiferis);角蛋白鏈黴菌（Streptomyces pactum);鏈 黴菌（Streptomyces akitaensis );阿希氏鏈黴菌（ Streptomyces akiyoshiensis ) ;鏈黴菌（Streptomyces alanosinicus );淺白鍵黴菌亞種（Streptomyces albidus subsp. invertens );白產色鏈黴菌（Streptomyces albochromogenes );圏卷產色鍵徽菌（Streptomyces ansochromogenes);圈卷產色鏈黴菌亞種（Streptomyces ansochromogenes subsp. pallens );鏈黴親和素菌（ Streptomyces a v i d ini i ) ; 鏈黴菌 （Streptomyces carcinomycicus ) ; 鏈黴菌 （ Streptomyces castaneglobisporus ) ；鏈黴菌(Streptomyces castaneus ) ;鏈黴菌（Streptomyces cyanoflavus );鏈黴菌（ Streptomyces djakartensis );產紅色鏈黴菌亞種（ Streptomyces erythrochromogenes subsp. narutoensis ) ； 產色鍵黴菌（Streptomy ces glomerochromogenes );鏈黴 菌(Streptomyces grisinus ) ;鏈黴菌（Streptomyces haranomachiensis )；鍵黴菌(Streptomyces hy gro staticus );鏈黴菌（Streptomyces insulatus );產鏈黴菌（ Streptomyces inversochromogenes ) ; 鍵徵菌 （Streptomyces rubiginosus); Streptomyces sparsogenes; Streptomyces viridiviolaceus; Streptomyces virido-diastaticus; Streptomyces calvus; Streptomyces cyanoalbus; Streptomyces cyanoalbus; Streptomyces finlayi; Streptomyces flaveolus: Streptomyces gey siriensis; Streptomyces herbiferis; Streptomyces pactum; Streptomyces akitaensis; Axi Streptomyces akiyoshiensis; Streptomyces alanosinicus; Streptomyces albidus subsp. invertens; Streptomyces albochromogenes; Streptomyces ansochromogenes Streptomyces ansochromogenes subsp. pallens; Streptomyces avid ini i; Streptomyces carcinomycicus; Streptomyces castaneglobisporus; Streptomyces (Streptomyces castaneus); Streptomyces cyanoflavus; Streptomyces djakartensis; Streptomyces erythrochromogenes subsp. narutoensis; Streptomy ces glomerochromogenes; Streptomyces grisinus; Streptomyces haranomachiensis; Streptomyces hy gro staticus; Streptomyces insulatus; Streptomyces inversochromogenes;

Streptomyces kitazuwaensi s ) ;馬力鍵徽菌（ -38- 200900074Streptomyces kitazuwaensi s ); horsepower key bacteria ( -38- 200900074

Streptomyces mariensi s ) ; 鏈徽菌 （Streptomyces minutiscleroticus ) ;鍵黴菌（Streptomyces mitakaensis );鏈黴菌（Streptomyces nigrogriseolus);尼格鏈黴菌 (Streptomyces ogaensis );配達鍵黴菌（Streptomyces piedadensis );藤黃灰鏈黴菌（Streptomyces regensis )Streptomyces mariensi s ); Streptomyces minutiscleroticus; Streptomyces mitakaensis; Streptomyces nigrogriseolus; Streptomyces ogaensis; Streptomyces piedadensis; Streptomyces lividans Streptomyces regensis )

;魯伕鏈黴菌（Streptomyces robefuscus);魯博鏈黴菌 (Streptomyces robeus );魯布思鏈黴菌（Streptomyces robustrus ) ;玫瑰淺灰鏈黴菌（Streptomyces roseogriseolus ) ;玫瑰灰鏈徽菌（Streptomyces ro seogriseus );撒哈鍵徽菌(Streptomyces sahachiroi ) :西諾鏈黴菌（Streptomyces senoensis);田無鏈黴菌亞 種(Streptomyces tanashiensis sub sp. cephalomyceticus ) ;嗜熱硝基鍵徽菌（Streptomyces thermonitrifi cans)； 熱紫鏈黴菌亞種（S t r e p t o m y c e s t h e rm o v i o 1 a c e u s s u b s p · apingens);微帆鏈黴菌（Streptomyces viridoniger); 微若鏈黴菌（Streptomyces werraensis);灰色鏈黴菌（ Streptomyces alboflavus );桿狀鏈徽菌（Streptomyces bacillaris);卡渥鍵徽菌（Streptomyces cavourensis); 鏈黴菌（Streptomyces cyaneofuscatus );暗黃鏈黴菌（ Streptomyces fulvissimus ) ;傑斯托鏈黴菌（Streptomyces robefuscus; Streptomyces robeus; Streptomyces robustrus; Streptomyces roseogriseolus; Streptomyces ro seogriseus; Streptomyces sahachiroi: Streptomyces senoensis; Streptomyces tanashiensis sub sp. cephalomyceticus; Streptomyces thermonitrifi cans; Streptomyces thermophilus Subspecies (S treptomycesthe rm ovio 1 aceussubsp · apingens); Streptomyces viridoniger; Streptomyces werraensis; Streptomyces alboflavus; Streptomyces bacillaris; Streptomyces cavourensis; Streptomyces cyaneofuscatus; Streptomyces fulvissimus; Streptomyces genus

Streptomyces griseobrunneus ) ;密執安鏈黴菌（Streptomyces griseobrunneus); Streptomyces militaris (

Streptomyces michiganensis ) ;處西馬鏈黴菌（Streptomyces michiganensis ); Streptomyces sinensis (

Streptomyces tsusimaensis ) ;黃色菌素鏈黴菌（Streptomyces tsusimaensis ); yellow streptomyces streptomyces (

Streptomyces xanthochromogenus ) ; 白黃鏈黴菌（ -39- 200900074Streptomyces xanthochromogenus ); Streptomyces fulvum ( -39- 200900074)

Streptomyces albidoflavus ) ;鏈黴菌（Streptomyces alboviridis );安第鏈黴菌（Streptomyces anulatus )； 栗褐鏈黴菌（Streptomyces badius):加州鏈黴菌（ Streptomyces californicus );肯氏鍵黴菌(Streptomyces canescens ) ；纖維黃色鏈黴菌（Streptomyces celluloflavus);纖維鏈黴菌（Streptomyces cellulosae) ;強怕瓦第鏈_黴菌（Streptomyces champavatii );金羊毛 鏈黴菌（Streptomyces chrysomallus);檸檬螢光鏈黴菌 (Streptomyces citreofluorescens );天藍色鏈黴菌（ Streptomyces coelicolor);苦膽鏈黴菌（Streptomyces felleus );非米卡鏈徽菌（Streptomyces fimicarius)； 佛州鏈黴菌（Streptomyces floridae):螢光鏈黴菌（ Streptomyces fluorescens);球抱鍵徽菌（Streptomyces globisporus );球孢鏈黴菌亞種（Streptomyces globisporus subsp. caucasicus )球孢鏈黴菌亞種（ treptomyces globisporus subsp. flavofuscus) ；球孢鏈黴 菌亞種(Streptomyces globisporus subsp. vulgaris ) ；谷 氏鏈黴菌（Streptomyces gougerotii):格西鏈黴菌（ Streptomyces griseinus );白邊鏈黴菌（Streptomyce s griseoloalbus )；灰色鏈黴菌(Streptomyces griseus)； 灰色鏈黴菌亞種（Streptomyces griseus subsp. alpha); 灰色鏈黴菌亞種（Streptomyces griseus subsp. cretosus) ;灰色鏈黴菌亞種（Streptomyce s griseus subsp. solvifaciens);中間型鏈黴菌（Streptomyces intermedius -40- 200900074 );卡麥鏈黴菌（Streptomyces kanamyceticus);制酵母 鏈黴菌（Streptomyces levoris);淚泥鏈黴菌（ Streptomyces limosus )；林帕鏈黴菌（Streptomyces lipmanii);細黃鍵黴菌（Streptomyces microflavus); 芳香鏈黴菌（S t r e p t o m y c e s o d o r i f e r );巴微斯鏈黴菌（ Streptomyces parvus ) :多色鍵黴菌（Streptomyces plurico lorescens ):液化性肺炎鏈黴菌（Streptomyces pneumonicus ):白環鏈黴菌（Streptomyces praecox )； 子鏈黴菌（Streptomyces puniceus);棉子糖鏈黴菌（ Streptomyces raffinosus ):魯地鏈黴菌（Streptomyces rutgersensis ) ;桑氏鏈黴菌（Streptomyces sampsonii ) ;西唐氏鏈黴菌（S t r e p t o m y c e s s e t ο n i i );西登鏈黴菌（ Streptomyces sindenensi s ) :榆樹心材鏈黴菌（ Streptomyces sulphureus ) ；維摩鏈黴菌(Streptomyces willmorei)；夏威夷鏈黴菌（Streptomyces hawaiiensis ) ;阿帕鏈黴菌（Streptomyces albohelvatus);鏈黴菌（ Streptomyces aurigineus ) ;雀鏈黴菌（Streptomyces canarius);淡紫色鏈黴菌（Streptomyces chryseus); 鏈黴菌（Streptomyces flavidovirens ):黑瓦鏈黴菌（ Streptomyces helvaticus);淡紫灰鏈黴菌（Streptomyces longisporoflavus )；香豆鏈黴菌（Streptomyces niveus ) ;鏈黴菌（Streptomyces paucidiastaticus ):球形鏈黴菌 (Streptomyces spheroides );哈黴素鏈黴菌（ Streptomyces pimprina ) ；卡胞鏈黴菌(Streptomyces -41 - 200900074 capoamus )；朱糸工鏈黴菌(Streptomyces cinnabarinus ) ;水玉鏈黴菌（Streptomyces crystallinus);鏈黴菌（ Streptomyces flavotricini);哥博鏈黴菌（Streptomyces gobitricini ):林肯鍵黴菌（Streptomyces lincolnensis ) ;產黑鏈黴菌（Streptomyces melanogenes );暗色產色 鍵黴菌（Streptomyces phaeochromogenes )；暗色產色鏈 黴菌亞種 (Streptomyces phaeochromogenes subsp. chloromy ceticus ) ；假性鏈黴菌（Streptomyces pseudovenezuelae ) ；粉紅鏈黴菌（Streptomyces roseoviridis )；重葛鍵黴菌(Streptomyces spectabillis ) :孢鏈黴菌（Streptomyces subrutilus);粗集胞鏈黴菌 (Streptomyces umbrinus ):委內瑞拉鍊黴菌（ Streptomyces venezuelae ) ; 鍵徽菌 （Streptomyces xanthophaeus ) ; 鏈黴菌 （ Streptomyces aur e o mo no p o d i a 1 e s );鏈黴菌（Streptomyces exfoliatus );介殼鏈黴菌（Streptomyces Hlamentosus );鏈黴菌 (Streptomyces prunicolor);玫瑰鏈黴菌（Streptomyces roseofulvus );淺玫瑰鏈黴菌（Streptomyces roseolus ) ;降玫瑰鏈黴菌（Streptomyces roseoporus);路比鏈黴 菌（Streptomyces rubiginosohe 丨 volus);得米提煉黴菌（ Streptomyces termitum);肉桂地鏈黴菌（Streptomyces cinnamonensis );哥倫比亞鍵徽菌（Streptomyces colombiensis ) ；勾戌吉鏈黴菌（Streptomyces goshikiensis);卡特那鏈黴菌（Streptomyces katrae); -42- 200900074 縮狀鏈黴菌（Streptomyces lavendofoliae);薰衣草鏈黴 菌（Streptomyces lavendulae );薰衣草鏈黴菌亞種（ Streptomyces lavendulae subsp· avireus);薰衣草鏈黴菌 亞種(Streptomyces lavendulae subsp. brasilicus ) ；薰衣 草鏈徽菌亞種（Streptomyces lavendulae subsp. grasserius )；薰衣草色鏈黴菌（Streptomyces lavendulcolor);牛肝菌鏈徽菌（Streptomyces luridus) :環內酰胺鏈黴菌（Streptomyces orchidaceus);瑞擾思 鏈黴菌(Streptomyces racemochromogenes )；冰河鏈黴 菌（Streptomyces syringae ) ;毒三素鏈黴菌（Streptomyces albidoflavus); Streptomyces alboviridis; Streptomyces anulatus; Streptomyces badius: Streptomyces californicus; Streptomyces canescens; Streptomyces candida (Streptomyces celluloflavus); Streptomyces cellulosae; Streptomyces champavatii; Streptomyces chrysomallus; Streptomyces citreofluorescens; Streptomyces Coelicolor); Streptomyces felleus; Streptomyces fimicarius; Streptomyces floridae: Streptomyces fluorescens; Streptomyces globisporus; Streptomyces globisporus subsp. caucasicus (Treptomyces globisporus subsp. flavofuscus); Streptomyces globisporus subsp. vulgaris; Streptomus Streptomyces Yces gougerotii): Streptomyces griseinus; Streptomyce s griseoloalbus; Streptomyces griseus; Streptomyces griseus subsp. alpha; Streptomyces griseus subsp. Streptomyces griseus subsp. cretosus); Streptomyce s griseus subsp. solvifaciens; Streptomyces intermedius (40-200900074); Streptomyces kanamyceticus; Streptomyces levoris Streptomyces limosus; Streptomyces lipmanii; Streptomyces microflavus; Streptomyces odorifer; Streptomyces parvus: multicolor bonds Streptomyces plurico lorescens: Streptomyces pneumonicus: Streptomyces praecox; Streptomyces puniceus; Streptomyces raffinosus: Streptomyces Rutg Ersensis ); Streptomyces sampsonii; Streptomyceset ο nii; Streptomyces sindenensi s: Streptomyces sulphureus; Streptomyces willmorei Streptomyces hawaiiensis; Streptomyces albohelvatus; Streptomyces aurigineus; Streptomyces canarius; Streptomyces chryseus; Streptomyces flavidovirens: black Streptomyces helvaticus; Streptomyces longisporoflavus; Streptomyces niveus; Streptomyces paucidiastaticus: Streptomyces spheroides; Streptomyces pimprina Streptomyces -41 - 200900074 capoamus; Streptomyces cinnabarinus; Streptomyces crystallinus; Streptomyces flavotricini; (Streptomyces gobitricini): Streptomyces lincolnensis; Streptomyces melanogenes; Streptomyces phaeochromogenes; Streptomyces phaeochromogenes subsp. chloromy ceticus; Streptomyces pseudovenezuelae; Streptomyces roseoviridis; Streptomyces spectabillis: Streptomyces subrutilus; Streptomyces umbrinus: Streptomyces venezuelae; Streptomyces xanthophaeus; Streptomyces aur eo mo no podia 1 es; Streptomyces exfoliatus; Streptomyces Hlamentosus; Streptomyces prunicolor; Streptomyces roseofulvus Streptomyces roseolus; Streptomyces roseoporus; Streptomyces rubiginosohe 丨volus; Streptomy Ces termitum); Streptomyces cinnamonensis; Streptomyces colombiensis; Streptomyces goshikiensis; Streptomyces katrae; -42- 200900074 Streptomyces variabilis Streptomyces lavendofoliae); Streptomyces lavendulae; Streptomyces lavendulae subsp. avireus; Streptomyces lavendulae subsp. brasilicus; Lavender streptomyces subsp. (Streptomyces lavendulae subsp. grasserius ); Streptomyces lavendulcolor; Streptomyces luridus: Streptomyces orchidaceus; Streptomyces racemochromogenes; Streptomyces syringae; Streptomyces fuliginea

Streptomyces toxytricini ) ;度瑞斯鏈黴菌（Streptomyces toxytricini ); Streptomyces genus

Streptomyces tuirus ):酒紅鏈黴菌（Streptomyces vinaceus);維吉尼亞鏈黴菌（Streptomyces virginiae) ;賴特瑞鏈黴菌（Streptomyces lateritus);福氏鏈黴菌 (Streptomyces flavo variabilis ) ;姜氏鏈黴菌（Streptomyces tuirus): Streptomyces vinaceus; Streptomyces virginiae; Streptomyces lateritus; Streptomyces flavo variabilis; Streptomyces gallisepticum

Streptomyces j anthinus ) ;紫鏈黴菌（Streptomyces purpurascens ) ；糸工粉鏈黴菌(Streptomyces roseospinus );玫瑰紫鏈黴菌（Streptomyces roseoviolaceus);紫 色鏈黴菌（Streptomyces violaceus);紫色鏈黴菌亞種（ Streptomyces violaceus subsp. confinus);紫色鍵徵菌亞 種（Streptomyces violaceus subsp. vicinus);澳拉錬徵 菌（Streptomyces violarus );澳羅鍊黴菌（Streptomyces violatus)；橫村鍵黴菌(Streptomyces yokosukanensis ) ;白孢鏈黴菌（Streptomyces albosporeus);橙色鏈黴菌 -43- 200900074 (Streptomyces aurantiacus );黃直絲鏈黴菌（ Streptomyces aureoverticillatus );阿魯尼鏈黴菌（ Streptomyces aurini ) ；淡黃鏈黴菌(Streptomyces cremeus );田鼠鏈黴菌（Streptomyces daghestanicus ) :弗氏鏈黴菌（Streptomyces fradiae):脆弱鏈黴菌（ Streptomyces fragilis )；富馬鏈黴菌（Streptomyces fumanus ) ; 葛 氏 鏈 黴 菌 ( Streptom.yces glomeroaurantiacus ):淺灰綠鏈黴菌（Streptomyces griseoviridis )；最氏鏈黴菌（Streptomyces niveoruber ) ;蒽環鏈黴菌（Streptomyces peucetius);費氏鏈黴菌（ Streptomyces phaeoviridis);羅世鏈黴菌（Streptomyces roseiscleroticus ) ;玫瑰黃鏈黴菌（Streptomyces roseoflavus ) ;玫瑰絲鏈黴菌（Streptomyces roseolilacinus);魯博-山南鏈黴菌（Streptomyces rubo-cyaneus);公牛鏈黴菌（Streptomyces tauricus);文-卓 氏鏈黴菌（Streptomyces vinaceus-drappus );密羅氏鏈 黴菌（Streptomyces virocidus);紅黴素鏈黴菌（ S t r e p t 〇 m y c e s e r y t h r a e u s );菊螢光鏈黴菌（S t r e p t o m y c e s luteofluorescens ) ;產紅鏈徵菌（Streptomyces erythrogriseus);環絲鏈黴菌（Streptomyces garyphalus );拉文督鏈黴菌（S t r e p t o m y c e s 1 a v e n d u 1 a r e c t u s );長 崎煉黴菌（Streptomyces nagasakiensis);紅白黴素鏈黴 菌（Streptomyces rubrolavendulae);肉桂地鏈黴菌（ Streptomyces cinnamonensis ) ; 阿薩 鏈黴菌 （ -44- 200900074Streptomyces j anthinus ); Streptomyces purpurascens; Streptomyces roseospinus; Streptomyces roseoviolaceus; Streptomyces violaceus; Streptomyces violaceus subsp. confinus ); Streptomyces violaceus subsp. vicinus; Streptomyces violarus; Streptomyces violatus; Streptomyces yokosukanensis; Streptomyces Albosporeus); Streptomyces aurantiacus; Streptomyces aureoveriatus; Streptomyces aurini; Streptomyces cremeus; Streptomyces daghestanicus: Eph. Streptomyces fradiae: Streptomyces fragilis; Streptomyces fumanus; Streptom.yces glomeroaurantiacus: Streptomyces griseoviridis Streptomyces niveoruber; Streptomyces peucetius; Streptomyces phaeoviridis; Streptomyces roseiscleroticus; Streptomyces roseoflavus; Streptomyces roseosum Streptomyces roseolilacinus); Streptomyces rubo-cyaneus; Streptomyces tauricus; Streptomyces vinaceus-drappus; Streptomyces virocidus; erythromycin Strept 〇myceserythraeus; Streptomyces luteofluorescens; Streptomyces erythrogriseus; Streptomyces garyphalus; Streptomyces 1 avendu 1 Arectus ); Streptomyces nagasakiensis; Streptomyces rubrolavendulae; Streptomyces cinnamonensis; Streptomyces argenatus ( -44- 200900074)

Streptomyces ashchabadicus );多產色性鏈黴菌（ Streptomyces ρ ο 1 y c hr o m o g en e s ) :前昭鏈黴菌 （ Streptomyces amakusaensis ) ;鏈黴菌（Streptomyces caelestis);阿祖魯鏈黴菌（Streptomyces azureus);貝 耳司鏈黴菌（Streptomyces bellus ):查氏鏈黴菌（ Streptomyces chartreusis ) ；可利如鏈黴菌（Streptomyces ashchabadicus); Streptomyces ρ ο 1 yc hr omog en es : Streptomyces amakusaensis; Streptomyces caelestis; Streptomyces azureus; Bel Streptomyces bellus: Streptomyces chartreusis; such as Streptomyces

Streptomyces coeliatus);可利得鍵黴菌（Streptomyces coerulatus ):可利得鏈黴菌亞種（Streptomyces coerulatus subsp. amylolyticus ):可利如伕鏈黴菌（ Streptomyces c o e r u 1 e o fu s c us ):天蘭淡紅鏈黴菌（ treptomyces c o e r u 1 e o r ub i du s ) ;天藍色鏈黴素（Streptomyces coeliatus); Streptomyces coerulatus: Streptomyces coerulatus subsp. amylolyticus: Streptomyces coeru 1 eo fu sc us: Streptomyces coleoptera (treptomyces) Coeru 1 eor ub i du s ) ; sky blue streptomycin (

Streptomyces coerulescens ) ;可拉可鏈黴菌（Streptomyces coerulescens ); Streptomyces cole

Streptomyces curacoi);賽阿尼思鏈黴菌（Streptomyces cyaneus);賽阿龍鏈黴菌（Streptomyces cyanoglomerus );印多鏈黴菌（Streptomyces indigocolor);瓜鏈黴菌 (Streptomyces lanatus );拉魯鏈黴菌（Streptomyces lazureus);瓦雷鏈黴菌（Streptomyces valynus);綠色 產色鏈黴菌（Streptomyces viridochromogenes);淡青鏈 黴菌（Streptomyces glaucescens );布蘭氏鏈黴菌（ Streptomyces blensis);可拉鏈黴菌亞種（Streptomyces coerulatus subsp. anaseuli);可拉鍵徵菌（Streptomyces coeruleoro seus ) ;立枯病鏈黴菌（Streptomyces 4 ipomoeae );使拼歐鍵徽菌（Streptomyces spinosus); 格利歐鏈黴菌（Streptomyces griseomycini);灰草黃鏈 -45- 200900074 黴菌（Streptomyces griseostramineus );帕氏鏈黴菌（ Streptomyces prasinosporus ) ;迦鈉氏鏈黴菌（Streptomyces curacoi); Streptomyces cyaneus; Streptomyces cyanoglomerus; Streptomyces indigocolor; Streptomyces lanatus; Streptomyces lazureus Streptomyces valynus; Streptomyces viridochromogenes; Streptomyces glaucescens; Streptomyces blensis; Streptomyces coerulatus subsp. anaseuli Streptomyces coeruleoro seus; Streptomyces 4 ipomoeae; Streptomyces spinosus; Streptomyces griseomycini; gray grass yellow chain-45- 200900074 Streptomyces griseostramineus; Streptomyces prasinosporus; Streptomyces glabrata

Streptomyces ghanaensis ) ;鏈黴菌（Streptomyces hirsutus);帕拉新鏈黴菌（Streptomyces prasinus);綠 孢鏈黴菌（Streptomyces viridosporus);阿卡挖鏈黴菌 (Streptomyces acrimycini)；斑貝鏈黴菌（Streptomyces b a m b e r g i e n s i s ) ;帕拉鏈黴菌（Streptomyces prasinopilosus);霍頓鏈黴菌（Streptomyces horton); 瑞氏鏈黴菌（Streptomyces rectiviolaceus);利拉鏈黴菌 (Streptomyces lilacinofulvus ) ;錦葵鏈黴菌（Streptomyces ghanaensis; Streptomyces hirsutus; Streptomyces prasinus; Streptomyces viridosporus; Streptomyces acrimycini; Streptomyces bambergiensis; Streptomyces prasinopilosus; Streptomyces horton; Streptomyces rectiviolaceus; Streptomyces lilacinofulvus; Streptomyces lilac (

Streptomyces mauvecolor ) ；維歐連鏈黴菌（Streptomyces mauvecolor ); Violet Streptomyces (

Streptomyces violans );淺紫色鏈黴菌（Streptomyces violascens ) ;巴達西輪絲菌（Streptoverticillium baldaccii );非瑞輪絲菌（Streptoverticillium fervens) ;魯本輪絲菌（Streptoverticillium rubrochlorinum);輪 絲菌（Streptoverticillium biverticillatum);阿魯本輪絲 菌（Stre_ptoverticillium aureoversales);潘他丁 輪絲菌 (Streptoverticillium pentaticum )；玫瑰輪絲菌（ Streptoverticillium roseoverticillatum )；魯本微輪絲菌 (Streptoverticillium rubro verticillatum )；荷魯斯輪絲 菌（Streptoverticillium hiroshimense):沙孟斯輪絲菌（ Streptoverticillium salmoni s )；魯頭氏微輪絲菌（ Streptoverticillium luteoverticillatum )；撤檀網狀輪絲 菌（Streptoverticillium olivoreticuli);瓦克青輪絲菌（ -46- 200900074 %Streptomyces violans ); Streptomyces violascens; Streptoverticillium baldaccii; Streptoverticillium fervens; Streptoverticillium rubrochlorinum; Streptoverticillium biverticillatum ); Stre_ptoverticillium aureoversales; Streptoverticillium pentaticum; Streptoverticillium roseoverticillatum; Streptoverticillium rubro verticillatum; Horus filament Streptoverticillium hiroshimense: Streptoverticillium salmoni s; Streptoverticillium luteoverticillatum; Streptoverticillium olivoreticuli; 46- 200900074 %

Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium waksmanii ) ； griseocarneum ) cinnamoneum ) hachij o ens e ) ； ardum ) ； 阿 abikoense ) ； albireticuli ) ； eurocidicum ) ； kishiwadense ) mashuense ) o 1 ivovertici 11 atum ) o r i η o c i ) parvisporogenes ) kentuckense ) album ) ； distallicum ) ehimense ) ； flavopersicum ) griseoverticillatum ) netropsis ) rectiverticillatum ) s ept atum ) mobaraense ) blastmyceticum ) 葛斯氏輪絲菌（ ;肉桂鏈輪絲菌（ :哈邱輪絲菌（ 阿當姆輪絲菌（ 孫鏈輪絲菌 （ 阿巴瑞輪絲菌 （ 一優西底輪絲菌（ 其西瓦第輪絲菌（ ;馬術森輪絲菌（ 橄欖網狀輪絲菌（ ;歐輪輪絲菌（ 帕維斯輪絲菌 （ ;坎圖輪絲菌（ 阿普姆輪絲菌（ 迪斯輪絲菌 （ 因門斯輪絲菌（ 香氛輪絲菌 （ 灰色鏈輪絲菌（ ;紡錘輪絲菌（ 瑞提氏輪絲菌 （ 絲狀輪絲菌（ 茂原輪鏈絲菌 （ 不來氏輪絲菌（ ;薰衣草輪絲菌（ -47- 200900074Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium Streptoverticillium waksmanii); griseocarneum) cinnamoneum) hachij o ens e); ardum); A abikoense); albireticuli); eurocidicum); Kishiwadense ) mashuense ) o 1 ivovertici 11 atum ) ori η oci ) parvisporogenes ) kentuckense ) album ) ; farlicum ) ehimense ) ; flavopersicum ) griseoverticillatum ) netropsis ) rectiverticillatum ) s ept atum ) mobaraense ) blastmyceticum ) Spirulina cinnamensis (H. sylvestris) (A. serrata (A. serrata) A. serrata (the spirulina of S. cerevisiae;; Phytophthora serrata (the genus of the genus Phytophthora (the genus Phytophthora (the genus Paclitaxus) (A. mutans (Drosophila fuliginea (H. meliloti) (Spirulina sphaeroides (Spirulina serrata); Helicobacter pneumoniae (filamentous filaments (filamentous filaments) Bacteria (M. serrata (H. leucocephala); lavender rot fungi ( -47- 200900074

Streptoverticillium lavenduligriseum ) ；薄青輪糸糸菌(Streptoverticillium lavenduligriseum );

Streptoverticillium lilacinum );克什米爾倫絲菌（ Streptoverticillium kashmir en se ) ； 硫輪 糸糸菌 (Streptoverticillium lilacinum ); Streptoverticillium kashmir en se ;

Streptoverticillium thioluteum ) ；游魚抱菌(Sporichthya f polymorpha )；灰質小耳孢囊菌（Microellobosporia cinerea);紫質小耳孢囊菌（Microellobosporia violacea );黃質小耳孢囊菌（Microellobosporia flavea);葛莉 塞小耳孢囊菌（Microellobosporia grisea);青銅小耳孢 囊菌（Micromonospora chalcea):嗜鹽小耳孢囊菌（ M i c r 〇 m ο η 〇 s ρ 〇 r a halophytica );海洋小耳孢囊菌（ Micromonospora carbonacea );那拉氏小耳孢囊菌（ Micromonospora narashinoensis )；每拉諾小耳抱囊菌(Streptoverticium thioluteum ); Sporichthya f polymorpha; Microellobosporia cinerea; Microellobosporia violacea; Microellobosporia flavea; Microellobosporia grisea; Micromonospora chalcea: M icr 〇m ο η 〇s ρ 〇ra halophytica; Micromonospora carbonacea; Micromonospora narashinoensis; each sacral ear

Micromonospora melanosporea ) ；棘孢小耳孢囊菌（Micromonospora melanosporea ); Microspora oxysporum (

Micromonospora echinospora );絳紅色小耳抱囊菌（ Micromonospora purpurea )；產紫質色性小耳孢囊菌（ Micromonospora purpureochromogenes ) ;雙色小耳抱囊 菌（Micromonospora bicolor); 天藍色小耳孢囊菌（ Micromonospora coerulea ) ;抱器小耳抱囊菌（Micromonospora echinospora ); Micromonospora purpurea; Micromonospora purpureochromogenes; Micromonospora bicolor; Micromonospora coerulea; Holding a small ear

Micromonospora globosa ) ；緬甸小耳孢囊菌（Micromonospora globosa); Burmese echinococcosis (

Micromonospora elongata ) •’ 普羅挖小耳孢囊菌（Micromonospora elongata ) •’

Micromonosora parva ) ； 粗毛小耳孢囊菌 (Micromonosora parva ) ;

Micromonospora gallica ) ；安同小耳孢囊菌(Micromonospora gallica );

Micromonospora acetoformici ) ；丙酸小耳孢囊菌（Micromonospora acetoformici ); Microcystis acnes (

Micromonospora propionici ):普通高溫放線菌（ -48- 200900074Micromonospora propionici ): Ordinary high temperature actinomycetes ( -48- 200900074

Thermoactinomyces vulgaris )；糖高溫放線菌（ Thermoactinomyces sacchari);底邱頭米卡高溫放線菌（ Actinobifida dichotomica )；阿爾巴雙歧放線菌（ Actinobifida alba );產色雙岐放線菌（Actinobifida chromogena );珊湖筒溫單抱菌（Thermomonspora curvata);綠色局溫單抱菌（Thermomonospora viridis ) ;玫瑰小雙孢菌（Microbispora rosea);薔薇小雙孢菌 (Microbispora aerata);產紫晶小雙孢菌（Microbispora amethysto genes );小雙孢小雙孢菌（Microbispora bispora);產色性小雙孢菌（Microbispora chromogenes );玫瑰小雙孢菌亞種（Microbispora diast at ica):普羅 挖小雙孢菌（Microbispora parva);高溫小雙孢菌（ Microbispora thermodiastati ca )；高溫小雙孢菌亞種（ Microbispora thermorosea ) ；短鍵小多抱菌（Thermoactinomyces vulgaris ); Thermoactinomyces sacchari; Actinobifida dichotomica; Actinobifida alba; Actinobifida chromogena; Thermomonspora curvata; Thermomonospora viridis; Microbispora rosea; Microbispora aerata; Microbispora Amethysto genes ); Microbispora bispora; Microbispora chromogenes; Microbispora diast at ica: Microbispora Parva); Microbispora thermodiastati ca; Microbispora thermorosea;

Micropolyspora brevicatena );安及羅小多孢菌（ Micropolyspora angiospora ) ；凯氏小多孢菌（Micropolyspora brevicatena ); Micropolyspora angiospora; Microsporum kj.

Micropolyspora caesia);乾草小多孢菌（Micropolyspora faeni);直桿小多孢菌（Micropolyspora rectivirgula); 魯氏小多抱菌（Micropolyspora rubrobrunea);高溫小多 孢菌（Micropolyspora thermovirida);衛瑞丁掛小多孢 菌(Micropolyspora viridinigra ) 〇 於藉由細胞而爲可被吞噬尺寸之微粒子狀磷酸鈣上， 可使其與免疫刺激物質一同或是分別担載抗原。使用爲該 目的之抗原若爲微粒子狀磷酸鈣可担載者，並無特別限定 -49- 200900074 其種類。例如可將具水溶性且溶解度極高之牛血清白蛋白 做爲抗原而担載（特開2005-126335號公報）。與前述來 自微生物的可溶性成分相同，亦可將疏水性物質做爲抗原 而担載。抗原係以1種或2種以上選自真菌、放線菌、細 菌、病毒、噬菌體、立克次體、原蟲、這些微生物的成分 、原蟲成分、腫瘤組織、腫瘤細胞、腫瘤細胞成分、腫瘤 抗原蛋白以及腫瘤抗原胜肽所成群之物質爲佳，但並非限 定於這些物質。 可溶性蛋白與黏多醣產生凝聚作用而形成微粒子狀沉 澱物之調製方法，詳細記載於例如美國專利第5,75 9,5 82 號說明書中，相關業者可容易地調製上述之沉澱物。例如 針對（i )可溶性蛋白與白蛋白，（ii )黏多醣與肝素，以 及（iii )使用 PPD做爲免疫刺激次質，詳細敘述於日本 專利第349267 1號說明書中，以使用該方法爲最佳，但並 未限定於該方法。可溶性蛋白與黏多醣之組合並未限定於 前述之組合。免疫刺激物質可使用例如包含於結核菌素中 之可溶性蛋白，或可以相關業者所周知之方法，使用部分 或全部自結核菌素所調製之可溶性蛋白。藉由將前述之（ i ) 、（ ii )及（iii ) 3種成分進行攪拌，使成分（i )及 成分（ii)產生凝聚作用而形成沉澱，此時可提供使成分 (iii)被捲入該沉澱中而一同沉澱之沉澱物。可溶性蛋白 與黏多醣產生凝聚作用而形成之沉澱物，以調製成進行投 予方式簡便且爲可通過注射針筒大小之微粒子狀爲佳。該 微粒子之粒徑一般以1 # m以下爲佳。將沉澱物調製成微 -50- 200900074 粒子狀之方法，以相關業者所周知之方法爲佳，並未限定 於特定之方法。 並未特別限定包含於免疫刺激物質中之可溶性蛋白（ i)與可溶性蛋白之比例，（i)可溶性蛋白與（ϋ)黏多 醣若在可引起凝聚作用之範圍，任何比例均可。試舉一例 使用ρΗ2·5之2.5%人類血清白蛋白溶液爲可溶性蛋白時 ，黏多醣使用5mg/ml之市售肝素溶液，相對於600 # 1之 黏多醣，溶解2.5yg之PPD後，持續攪拌該混合物並滴 下人類血清白蛋白溶液，形成凝聚（coacervation)所致之 微粒子。將該懸濁液進行離心，定量其上清液中之蛋白質 含量。以選擇混合後之蛋白質爲捲入了大部分微粒子之容 量比者爲佳。更無遑多言成分（i )及成分（iii )比例及 凝聚作用條件等相關業者可適宜地選擇。所得之沉澱物中 所含微粒子之粒徑一般爲l//m左右，但並無限定於特定 之尺寸。 所得之微粒子狀沉澱係做爲担載免疫刺激物質之載體 ’可直接使用爲本發明之佐劑的1種成分，且可因應需要 將沉澱以蒸餾水加以洗淨。另外，爲使沉澱安定化，可藉 由使蛋白分子間交聯劑進行作用而形成交聯，形成不溶性 之微粒子。並未特別限定蛋白分子間交聯劑之種類，可以 相關業者所周知之交聯劑使用周知之方法。例如將20 mg/ml之1-乙基-3-(3-二甲基胺基丙基）碳二亞胺製成水 溶液，加入同時以旋渦混合器混合，使其最終濃度成爲 0.8〜1.5 mg/ml’其後，置於室溫下15分鐘，沉澱中之蛋 -51 - 200900074 白質分子間可形成充分安定之交聯。其中，交聯之形成並 未特別限定於前述之特定條件或特定之蛋白分子間交聯劑 。進行如前述步驟後所得之微粒子狀沉澱，以經交聯處理 之微粒子狀沉澱爲佳，即便以水洗淨亦不會溶解’極適於 本發明佐劑之成分使用。 根據最適之實施方式，預先混合具抗原性之可溶性蛋 白與具免疫佐劑活性之結核菌素可溶性蛋白，取代上述之 (iii )而使用該混合物，進行如上述之步驟亦可得沉澱物 。該沉澱物係担載免疫刺激物質與抗原之載體，可使用爲 本發明之佐劑的1種成分。 於可溶性蛋白與黏多醣產生凝聚作用而形成之沉澱物 中，可使其與免疫刺激物質一同或是與其他物質進行担載 抗原。抗原可使用細胞激素、趨化激素、細胞生長因子以 及荷爾蒙之誘發劑。另外，可使用1種或2種以上選自結 核菌素、PPD、來自微生物的可溶性成分、海藻糖二黴菌 酸酯、LPS、脂質A、寡核苷酸、葡聚醣、鑰孔蟲戚血藍 蛋白、胞壁醯二胜肽、貝他定、左旋咪唑、細胞激素、趨 化激素、細胞生長因子以及荷爾蒙所成群之物質做爲抗原 。另外可使用1種或2種以上微生物之酒精萃取物、丙酮 萃取物、吡啶萃取物以及熱水萃取物所成群之萃取物做爲 抗原。微生物以細菌爲佳，且該萃取物與可溶性蛋白結合 者更佳。微生物可使用示於上述者。其中於担載可溶性蛋 白與黏多醣產生凝聚作用而形成之沉澱物之抗原，並非限 定於上述特定之例。 -52- 200900074 亦可使可溶性蛋白與黏多醣產生凝聚作用而形成之沉 澱物，與其他不溶性蛋白分子及抗原，藉由蛋白分子間交 聯劑而進行交聯，將担載免疫刺激物質之載體調製成不溶 性之微粒子型態。並未特別限定蛋白分子間交聯劑種類， 可以相關業者所周知之交聯劑使用周知之方法。例如可使 用1-乙基-3- (3-二甲基胺基丙基）碳二亞胺等。 本發明免疫佐劑之免疫佐劑免疫活性，根據具體說明 於本發明說明書中之方法，以及於下所說明之方法，相關 業者可容易地進行測定。已知THP-1細胞株係來自人類 之巨噬細胞系列之建立細胞株，於培養中以卩11〇6〇112-Myristate 1 3 - Acetate (以下略稱爲「PM A」）使其作用誘 發進行分化後，顯示其具有吞噬能力（Kurosaka，K.，et al.，J. Immunol., 161，pp.6245-6249，1 998 )，分化爲抗原 呈現細胞（Hu，P. Q·, et al.，J. Immunol.，172, pp.1595- 1601,2004) 。 另外 ，將該 細胞以 IFNg 進 行前處 理時， 可增強對T細胞之抗原呈現能力（8^11，8.〗.，61&1.，1· Immunol·，150，pp_2869-84，1993)。因此，藉由使 THP-1 細胞株進行分化，再定量所產生的GM-CSF之量，可不必 使用含人類抗原呈現細胞之來自週邊血之附著性細胞，而 測定人類抗原呈現細胞之活化程度。因THP-1細胞株係 建立細胞株，並無人類個體之差異，可確保品質安定之實 驗材料。 本發明之免疫佐劑與抗原混合後，可藉由對包含人類 之哺乳類動物之體內進行投予而誘發對該抗原的全身性免 -53- 200900074 疫反應，可與一般所使用之免疫佐劑以相同方式而加以使 用。所使用之抗原，可使用被投予個體內原本所不存在之 生物或物質，更具體而言係可使用1種或2種以上選自真 菌、放線菌、細菌、病毒、噬菌體、立克次體、原蟲、這 些微生物的成分、原蟲成分、腫瘤組織、腫瘤細胞、腫瘤 細胞成分、腫瘤抗原蛋白以及腫瘤抗原胜肽所成群之物質 ，但並非限定於這些物質，若其爲可用作抗原之物質，使 用任何一種物質均可。將抗原用作獨立製劑，對包含人類 之哺乳類動物之體內進行投予，將本發明之免疫佐劑對各 別之動物之體內進行投予，可誘發對該抗原的全身性免疫 反應。或如先前所說明，可使爲本發明免疫佐劑之成分2 種以上之免疫刺激物質載體任1個或2種以上，進行担載 抗原，進而於該狀況下，以其他方法進行抗原投予亦可。 抗原爲真菌、放線菌、細菌、病毒、噬菌體、立克次 體、原蟲以及/或這些微生物的成分時，藉由投予本發明 之免疫佐劑及抗原，可進行感染症之預防以及/或治療爲 目的之疫苗療法。另外抗原爲腫瘤組織、腫瘤細胞、腫瘤 細胞成分、腫瘤抗原蛋白以及/或腫瘤抗原胜肽時，可藉 由一同投予本發明之免疫佐劑及抗原，而進行感染症之預 防以及/或治療爲目的之疫苗療法。例如，藉由將自患者 所分離之不活化腫瘤細胞，與本發明之免疫佐劑混合而對 患者進行投予，可誘發於患者生體內對該腫瘤細胞之腫瘤 免疫反應。其中，本發明之混合免疫佐劑製劑之使用方法 ，並未限定於上述之實施方式，可使用任何一種一般使用 -54- 200900074 免疫佐劑之方式。並未限定本發明免疫佐劑之投予量及投 予方法，一般而言，可使用注射（皮下注射等）非經口投 予之方式充分引起免疫反應而進行投予。 進而，將於患者體內之腫瘤組織以物理性方法使其變 性後，藉由對該組織投予本發明之免疫佐劑，而可誘發對 殘存於患者體內之該腫瘤組織之抗腫瘤免疫反應。並未特 別限定腫瘤組織之物理性變性之方法，可採用例如微波照 射法、高週波凝固法、冷凍凝固法、電燒加熱法、熱水注 入法、酒精注入法、栓塞法、放射線照射法、雷射照射法 以及超音波破壞法等，其中物理性變性之方法並未限制於 這些方法，若爲可誘發腫瘤組織內之腫瘤細胞壞死之方法 ，使用任何1種均可。亦可適宜地組合2種以上的物理性 的方法。 例如，藉由微波照射法將腫瘤組織加熱凝固，對該凝 固組織內投予本發明之免疫佐劑時，可誘發對該腫瘤組織 內部及其週邊所殘存之腫瘤細胞之腫瘤免疫反應。投予本 發明之免疫佐劑時，以同時投予抗結核菌素爲佳。其中， 本發明之免疫佐劑之投予方法並未限定於上述之實施方式 ，對集合聚集於該變性的腫瘤組織之抗原呈現細胞，本發 明之免疫佐劑可針對變性腫瘤組織中所包含之腫瘤抗原， 或本發明之免疫佐劑可提供抗原呈現細胞直接刺激之環境 ，可採用任何一種方法。 預先於體外混合本發明之免疫佐劑與免疫擔任細胞再 對患者體內進行投予，可刺激體內之免疫反應。該擔任免 -55- 200900074 疫細胞可使用樹狀細胞、B淋巴球、T淋巴球、自然殺手 細胞、自然殺手Τ細胞以及/或造血幹細胞等，但並非限 定於這些細胞。 自其他觀點，根據本發明可提供含本發明之免疫佐劑 及抗原爲有效成分之疫苗。投予該疫苗時，可使用相關業 者所周知之方法，可同時混合擔任免疫細胞。抗原可藉由 使用引起病毒或病毒成分之感染症之來自感染源之物質， 不僅對未感染者可預防感染，亦可對罹患該感染症之患者 進行治療。另外，亦可藉由將包含1種或2種以上選自自 患者所分離之腫瘤組織、腫瘤細胞、腫瘤細胞成分、腫瘤 抗原蛋白以及腫瘤抗原胜肽之生體試樣，以及前述之免疫 佐劑之疫苗，對該提供腫瘤之患者進行投予，而治療腫瘤 〇 進而，抗原爲例如異種動物蛋白時，藉由與前述之免 疫佐劑一同對人類以外之動物進行投予，而可使宿主動物 有效率地產生對該抗原之抗體。藉由該方法可容易獲得生 產抗體動物，亦可容易獲得來自該生產抗體動物之生產抗 體細胞及抗體基因。抗原若爲對宿主具抗原性者則可使用 任一種物質，並未限定於這些物質。 【實施方式】 實施例 以下藉由實施例更具體地說明本發明，但本發明之範 圍並未限定於下述之實施例 -56- 200900074 例1 :使2種免疫刺激物質載體担載〗種免疫刺激物質之 免疫佐劑之抗原呈現細胞刺激效果 將以PMA使THP-1細胞株分化後之細胞做爲抗原呈 現細胞’使2種免疫刺激物質載體担載PPD，於培養條件 下添加於抗原呈現細胞後時，測定GM-CSF之產生量。 A ·方法以及材料 1 .調製磷酸鈣（以下略記爲CaP )微粒子核液 量取相當於2mol之碳酸鈣CaC03 ( Karushido股份有 限公司製’純度99.99%)置入高純度氧化鋁匣缽，使用 箱形爐以1 〇〇〇°C，燒製3小時，得進行脫碳酸之高純度 氧化鈣CaO，再將其置入燒杯中。使空氣中之水分與CaO 進行反應，爲極力抑制其再進而與二氧化碳反應，以保鮮 膜封住燒杯之上方，置於做爲反應槽之內容量爲10L之聚 乙烯容器中。使該容器中充滿氮氣，其後取下保鮮膜，再 滴下超純水於燒杯中。此時，爲避免過熱聚乙烯容器中亦 充滿超純水。吸收完反應熱時燒杯中之反應物亦全部被超 純水沖洗而移入至聚乙烯容器中。此處所使用之超純水總 量，以符合使自CaO量所計算出之濃度爲〇.5m〇l/L之量 。藉此獲得氫氧化鈣Ca(ΟH)2懸浮液。將該懸浮液使用攪 拌機（EYELA 公司製》MAZELA Z- 1 000 )，以約 500rpm 攪拌同時，再在其中加入2L之調製爲〇.6mol/L之磷酸 H2P〇4(國產化學股份有限公司製，濃度85%)，以不超 -57- 200900074 過25mL/min之速度滴下。完成滴下後繼續攪拌24 而成爲CaP核液。 2.調製CaP微粒子液，CaP-PPD微粒子液 2 · 1調製磷酸鈣過飽和溶液與C aP微粒子液 將林格氏液（扶桑藥品工業股份有限公司製）以 mL，Klinisalz B (小林製藥工業股份有限公司製 1.24 mL，Bifil專用碳酸氫鈉補充液（清水製藥製 0.4 8 mL之比例混合，再調製爲10 mL。於該磷酸銳 溶液9 mL中，添加1 mL之乙醇，於該混合液每10 加入1滴CaP微粒子液再放置一晚。將該懸浮液進朽 ，將所得之沉澱以注射用之生理食鹽水充分洗淨，再 於1 mL之注射用生理食鹽水。將其做爲C aP微粒子 2.2調製CaP-PPD微粒子液 將林格氏液以 8.28 mL，Klinisalz B以 1.24 Bifil專用碳酸氫鈉補充液以0.48 mL之比例混合， 製爲10mL。於該磷酸鈣飽和溶液9 mL中，添0_5, 1 〇 μ g之結核菌素標準品（日本BCG製造股份有陆 ，一般診斷用純化結核菌素（PPD ))，並加以溶解 該溶液9 mL中，添加1 mL之乙醇，於該混合液每： ，加入1滴C aP微粒子液再放置一晚。將該懸浮液連 心，將所得之沉澱以注射用之生理食鹽水充分洗淨， 浮於1 mL之注射用生理食鹽水。所得之CaP-PPD横 小時 8.28 )以 ;)以 5飽和 mL， Γ離心 f懸浮 液。 mL， 再調 :g至 公司 。於 0 m L :行離 再懸 :粒子 -58- 200900074 液懸浮液，相對應調製所使用之PPD量紀錄CaP-PPD爲 (0.5/z g/mL )及 CaP-PPD 爲（10// g/mL )。另外，這些 溶液添加於培養液（含10%牛胎兒血清之RPMI1 640培養 基）後，其最終濃度爲其1/5時，紀錄CaP-PPD爲（0.1 fi g/mL )及 CaP-PPD 爲（2·0/ζ g/mL)。 2.3調製PPD液 將〇.25μ g之結核菌素標準品（日本BCG製造股份 有限公司，一般診斷用純化結核菌素（PPD ))溶解於 0.5 mL之培養液中，做爲PPD液。 2.4 調製PPD担載凝聚作用沉澱微粒子（以下略稱作 TuMP)液 1) 將25%之人類血清白蛋白（1^八，8&\{6犷八11)\11113(：， Baxter公司製），以滅菌水稀釋至2 · 5 %，再以4N之H C1 調整pH至2.5。Micropolyspora caesia); Micropolyspora faeni; Micropolyspora rectivirgula; Micropolyspora rubrobrunea; Micropolyspora thermovirida; Wei Ruiding Micropolyspora viridinigra is a microparticle-like calcium phosphate that can be phagocytized by cells, and can be loaded with an antigen together with an immunostimulating substance. The antigen to be used for this purpose is not particularly limited as long as it is a particulate calcium phosphate. -49-200900074. For example, bovine serum albumin which is water-soluble and has a high solubility can be carried as an antigen (Japanese Laid-Open Patent Publication No. 2005-126335). The hydrophobic substance may be carried as an antigen in the same manner as the soluble component derived from the microorganism described above. The antigen system is one or more selected from the group consisting of fungi, actinomycetes, bacteria, viruses, bacteriophages, rickettsia, protozoa, components of these microorganisms, protozoal components, tumor tissues, tumor cells, tumor cell components, tumors. The antigenic protein and the tumor antigen peptide are preferably a group of substances, but are not limited to these substances. A method for preparing a microparticle-like precipitate by agglomeration of a soluble protein with a mucopolysaccharide is described in detail in, for example, U.S. Patent No. 5,75,5,528, the entire disclosure of which is incorporated herein by reference. For example, (i) soluble protein and albumin, (ii) mucopolysaccharide and heparin, and (iii) use of PPD as an immunostimulatory sub-mass, as described in detail in Japanese Patent No. 3492671, to use this method as the most Good, but not limited to this method. The combination of soluble protein and mucopolysaccharide is not limited to the combination described above. The immunostimulating substance may be, for example, a soluble protein contained in tuberculin, or a soluble protein prepared by some or all of the tuberculin by a method well known to those skilled in the art. By stirring the above three components (i), (ii) and (iii), the component (i) and the component (ii) are agglomerated to form a precipitate, and at this time, the component (iii) can be supplied. A precipitate which precipitated together in the precipitate. The precipitate formed by the agglomeration of the soluble protein and the mucopolysaccharide is preferably prepared in a manner suitable for administration and is preferably in the form of a microparticle having a syringe size. The particle diameter of the fine particles is generally preferably 1 # m or less. The method of preparing the precipitate into a micro-50-200900074 particle form is preferably a method known to a person skilled in the art, and is not limited to a specific method. The ratio of the soluble protein (i) to the soluble protein contained in the immunostimulating substance is not particularly limited, and (i) the soluble protein and the (ϋ) mucopolysaccharide may be in any range which can cause agglomeration. When using a 2.5% human serum albumin solution of ρΗ2·5 as a soluble protein, the mucopolysaccharide uses a commercially available heparin solution of 5 mg/ml, and dissolves 2.5 dyg of PPD after continuous dissolution with respect to 600 g of the mucopolysaccharide. The mixture is dripped with a human serum albumin solution to form microparticles caused by coacervation. The suspension was centrifuged to quantify the protein content in the supernatant. It is preferred to select the mixed protein as the volume of most of the particles involved. More specific ingredients such as the ratio of component (i) and component (iii) and coacervation conditions can be appropriately selected. The particle size of the fine particles contained in the obtained precipitate is generally about 1/m, but is not limited to a specific size. The obtained microparticle precipitate is used as a carrier for supporting an immunostimulating substance. One component of the adjuvant of the present invention can be used as it is, and the precipitate can be washed with distilled water as needed. Further, in order to stabilize the precipitation, cross-linking can be formed by causing a protein intermolecular crosslinking agent to act to form insoluble fine particles. The type of the intermolecular crosslinking agent of the protein is not particularly limited, and a known method can be used by a known crosslinking agent. For example, 20 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is made into an aqueous solution, and simultaneously mixed with a vortex mixer to have a final concentration of 0.8 to 1.5 mg. /ml' Thereafter, at room temperature for 15 minutes, the egg in the precipitate -51 - 200900074 can form a well-stabilized crosslink between the white matter molecules. Here, the formation of the cross-linking is not particularly limited to the specific conditions described above or a specific interprotein crosslinking agent. The fine particle-like precipitate obtained as described above is preferably precipitated by microparticulation of a cross-linking treatment, and even if it is washed with water, it does not dissolve, and it is used as a component which is extremely suitable for the adjuvant of the present invention. According to the most preferred embodiment, the antigenic soluble protein and the immunologically adjuvanted tuberculin soluble protein are mixed in advance, and the mixture is used instead of the above (iii), and a precipitate can be obtained by the above steps. This precipitate is a carrier for supporting an immunostimulating substance and an antigen, and one component which is an adjuvant of the present invention can be used. In the precipitate formed by the coagulation of soluble protein and mucopolysaccharide, it can be carried together with an immunostimulating substance or with other substances. The antigen can use cytokines, chemokines, cell growth factors, and hormone inducers. Further, one type or two or more types selected from the group consisting of tuberculin, PPD, soluble components derived from microorganisms, trehalose dipic acid ester, LPS, lipid A, oligonucleotide, dextran, and keyhole worm blood can be used. A substance composed of cyanoprotein, cell wall bismuth peptide, betahidine, levamisole, cytokines, chemokines, cell growth factors, and hormones is used as an antigen. Further, an extract of one or more kinds of microbial alcohol extract, acetone extract, pyridine extract, and hot water extract may be used as an antigen. The microorganism is preferably bacteria, and the extract is better combined with the soluble protein. Microorganisms can be used as indicated above. The antigen in which the precipitate formed by the co-aggregation of the soluble protein and the mucopolysaccharide is not limited to the above specific examples. -52- 200900074 A precipitate formed by agglomeration of soluble protein and mucopolysaccharide, and other insoluble protein molecules and antigens, which are crosslinked by a cross-linking agent of an intermolecular molecule to carry an immunostimulating substance. Modulated into insoluble microparticle type. The type of the intermolecular crosslinking agent of the protein is not particularly limited, and a known method can be used for a crosslinking agent known to a person skilled in the art. For example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or the like can be used. The immunoadjuvant immunological activity of the immunoadjuvant of the present invention can be easily determined by a person skilled in the art according to the method described in the specification of the present invention and the method described below. It is known that the THP-1 cell line is derived from a cell line of human macrophage series, and is induced by 卩11〇6〇112-Myristate 1 3 - Acetate (hereinafter abbreviated as "PM A") in culture. After differentiation, it was shown to have phagocytic ability (Kurosaka, K., et al., J. Immunol., 161, pp. 6245-6249, 1 998), and differentiated into antigen-presenting cells (Hu, P. Q., et Al., J. Immunol., 172, pp. 1595-1601, 2004). In addition, when the cells are pretreated with IFNg, the antigen presentation ability to T cells can be enhanced (8^11, 8.〗., 61 & 1,. Immunol, 150, pp_2869-84, 1993). Therefore, by diluting the THP-1 cell line and quantifying the amount of GM-CSF produced, it is possible to determine the degree of activation of the human antigen-presenting cells without using adherent cells derived from peripheral blood containing human antigen-presenting cells. . Since the THP-1 cell line establishes a cell line, there is no difference in human individuals, and the test material for ensuring quality stability can be ensured. After the immunoadjuvant of the present invention is mixed with an antigen, the systemic immunity of the antigen can be induced by administration to a mammal containing a human mammal, and the immunological adjuvant generally used can be used. Use it in the same way. The antigen to be used may be a living organism or a substance which is not originally present in the individual, and more specifically, one or more selected from the group consisting of a fungus, an actinomycete, a bacterium, a virus, a phage, and a rickett. a substance, a protozoan, a component of these microorganisms, a protozoal component, a tumor tissue, a tumor cell, a tumor cell component, a tumor antigen protein, and a tumor antigen peptide, but is not limited to these substances, if they are available As the substance of the antigen, any substance can be used. The antigen is used as a separate preparation, administered to a mammal containing a human, and the immunological adjuvant of the present invention is administered to each animal to induce a systemic immune response to the antigen. Or, as described above, one or two or more kinds of immunostimulating substance carriers which are components of the immunoadjuvant of the present invention may be used to carry an antigen, and in this case, antigen administration may be carried out by other methods. Also. When the antigen is a fungus, an actinomycete, a bacterium, a virus, a bacteriophage, a rickettsia, a protozoan, and/or a component of these microorganisms, the immunological adjuvant and the antigen of the present invention can be administered to prevent infection and/or Or vaccine therapy for the purpose of treatment. When the antigen is a tumor tissue, a tumor cell, a tumor cell component, a tumor antigen protein, and/or a tumor antigen peptide, the immunological adjuvant and the antigen of the present invention can be administered together to prevent and/or treat the infectious disease. Vaccine therapy for the purpose. For example, administration of a patient by injecting an inactivated tumor cell isolated from a patient with the immunoadjuvant of the present invention induces a tumor immune response to the tumor cell in the patient's body. Here, the method of using the mixed immunoadjuvant preparation of the present invention is not limited to the above embodiment, and any of the methods of using the adjuvant of -54-200900074 in general can be used. The administration amount and administration method of the immunoadjuvant of the present invention are not limited, and in general, administration can be carried out by sufficiently injecting an immunological reaction by injection (subcutaneous injection or the like). Further, after the tumor tissue in the patient is physically deformed, the anti-tumor immune response to the tumor tissue remaining in the patient can be induced by administering the immunological adjuvant of the present invention to the tissue. The method of physically denaturation of the tumor tissue is not particularly limited, and for example, microwave irradiation, high-frequency coagulation, freeze coagulation, electro-thermal heating, hot water injection, alcohol injection, embolization, radiation irradiation, The laser irradiation method, the ultrasonic destruction method, and the like, wherein the method of physical denaturation is not limited to these methods, and any method can be used as a method for inducing tumor cell necrosis in tumor tissues. Two or more physical methods may be combined as appropriate. For example, when the tumor tissue is heated and coagulated by microwave irradiation, and the immunoadjuvant of the present invention is administered to the coagulated tissue, a tumor immune response to tumor cells remaining in and around the tumor tissue can be induced. When the immunological adjuvant of the present invention is administered, it is preferred to simultaneously administer anti-tuberculosis. The method for administering the immunoadjuvant of the present invention is not limited to the above embodiment, and the antigen-presenting cells which are aggregated in the denatured tumor tissue, the immunoadjuvant of the present invention may be included in the degenerative tumor tissue. The tumor antigen, or the immunoadjuvant of the present invention, can provide an environment in which the antigen exhibits direct stimulation of the cells, and any method can be employed. The immunological adjuvant of the present invention is mixed with the immune-preserving cells in advance and administered to the patient in advance to stimulate the immune response in the body. The use of dendritic cells, B lymphocytes, T lymphocytes, natural killer cells, natural killer cells, and/or hematopoietic stem cells may be used for the disease-free cells, but is not limited to these cells. From other viewpoints, according to the present invention, a vaccine comprising the immunological adjuvant of the present invention and an antigen as an active ingredient can be provided. When the vaccine is administered, it can be mixed as an immune cell by a method known to a person skilled in the art. The antigen can be treated by a substance derived from an infection source which causes an infection of a virus or a viral component, not only for an uninfected person, but also for a patient suffering from the infection. In addition, a biological sample containing one or more selected from the group consisting of tumor tissues, tumor cells, tumor cell components, tumor antigen proteins, and tumor antigen peptides isolated from a patient, and the aforementioned immunological test may also be used. a vaccine for administering a tumor to a patient who provides a tumor, and in addition, when the antigen is, for example, a heterologous animal protein, the host can be administered to an animal other than human by the immunological adjuvant described above. Animals efficiently produce antibodies to the antigen. The antibody-producing animal can be easily obtained by this method, and the production of the antibody-resistant antibody gene and the antibody gene from the antibody-producing animal can be easily obtained. The antigen may be any substance if it is antigenic to the host, and is not limited to these. EXAMPLES Hereinafter, the present invention will be specifically described by way of Examples, but the scope of the present invention is not limited to the following Examples -56-200900074 Example 1: Two kinds of immunostimulating substance carriers are carried The antigen of the immune adjuvant of the immunostimulating substance exhibits a cell-stimulating effect, and the cells differentiated by the THP-1 cell line are used as antigen-presenting cells by PMA. The two immunostimulating substance carriers are loaded with PPD and added to the antigen under culture conditions. When the cells were present, the amount of GM-CSF produced was measured. A. Method and material 1. The calcium phosphate (hereinafter abbreviated as CaP) fine particle nucleus is adjusted to take 2 mol of calcium carbonate CaC03 (purity: 99.99% manufactured by Karushido Co., Ltd.) and placed in a high-purity alumina crucible. The furnace was fired at 1 〇〇〇 ° C for 3 hours to obtain decarbonated high-purity calcium oxide CaO, which was then placed in a beaker. The water in the air is reacted with CaO to suppress it and react with carbon dioxide as much as possible, and the fresh film is sealed on the upper side of the beaker, and placed in a polyethylene container having a content of 10 L as a reaction tank. The vessel was filled with nitrogen gas, after which the plastic wrap was removed, and ultrapure water was dropped into the beaker. At this time, in order to avoid overheating, the polyethylene container is also filled with ultrapure water. The reactants in the beaker were also washed with ultrapure water and absorbed into a polyethylene container. The total amount of ultrapure water used herein is such that the concentration calculated from the amount of CaO is 〇.5 m〇l/L. Thereby, a calcium hydroxide Ca(ΟH) 2 suspension was obtained. The suspension was stirred at about 500 rpm using a stirrer (MAZELA Z-1 000, manufactured by EYELA Co., Ltd.), and 2 L of phosphoric acid H2P〇4 (manufactured by National Chemical Co., Ltd.) was prepared. The concentration was 85%), and it was dripped at a rate of not more than -57-200900074 at 25 mL/min. After the completion of the dropping, stirring 24 was continued to become a CaP nucleus. 2. Modulation of CaP microparticles, CaP-PPD microparticles 2 · 1 Preparation of calcium phosphate supersaturated solution and C aP microparticles solution Ringer's solution (made by Fuso Pharmaceutical Co., Ltd.) in mL, Klinisalz B (Kobayashi Pharmaceutical Industry Co., Ltd. The company made 1.24 mL, Bifil special sodium bicarbonate replenishing solution (mixed in 0.4 8 mL of Qingshui Pharmaceutical Co., Ltd., and then prepared to 10 mL. In 9 mL of the phosphoric acid sharp solution, add 1 mL of ethanol, in the mixture every 10 One drop of CaP microparticle solution was added and left for one night. The suspension was immersed, and the resulting pellet was washed thoroughly with physiological saline for injection, and then injected with physiological saline solution of 1 mL. This was used as C aP. The microparticles 2.2 modulate the CaP-PPD microparticles solution. The Ringer's solution is mixed with 8.28 mL of Klinisalz B and the 1.24 Bifil sodium bicarbonate supplement solution at a ratio of 0.48 mL to prepare 10 mL. In the calcium phosphate saturated solution 9 mL, 0_5, 1 gμ g of tuberculin standard (Japan BCG manufacturing shares have land, general diagnostic use of purified tuberculin (PPD)), and dissolved 9 mL of this solution, add 1 mL of ethanol, in the mix Liquid per: , plus One drop of the C aP microparticle solution was left for one night. The suspension was placed in a heart, and the resulting pellet was thoroughly washed with physiological saline for injection, and floated in 1 mL of physiological saline for injection. The obtained CaP-PPD was horizontally obtained. Hour 8.28) to ;) Centrifuge the suspension with 5 saturated mL, Γ. mL, then adjust: g to the company. At 0 m L : row resuspension: particle -58- 200900074 liquid suspension, the amount of PPD used for the corresponding preparation is recorded as CaP-PPD (0.5/zg/mL) and CaP-PPD is (10//g/ mL). In addition, when these solutions were added to the culture solution (RPMI1 640 medium containing 10% fetal bovine serum), the final concentration was 1/5, and the CaP-PPD was recorded as (0.1 fi g/mL) and CaP-PPD was ( 2·0/ζ g/mL). 2.3 Preparation of PPD solution The 结核.25 μg tuberculin standard (Japan BCG Manufacturing Co., Ltd., general diagnostic purified tuberculin (PPD)) was dissolved in 0.5 mL of the culture solution as a PPD solution. 2.4 Modulation of PPD-loaded coacervation Precipitated microparticles (hereinafter abbreviated as TuMP) solution 1) 25% human serum albumin (1^8, 8&\{6犷811)\11113 (:, Baxter) Dilute to 2 · 5 % with sterile water, then adjust the pH to 2.5 with 4 N of H C1.

2) 預先將該2.5 %之人類血清白蛋白溶液與肝素溶液 (Novo Heparin 注 1000，1000 單位/mL，約 7.69mg/mL 以下’ AVENTISPHAMA股份有限公司）以各種比例進行 混合’決定肝素溶液之最適合混合比。最適合混合比，以 初始最適當之混合比製作微粒，再以2，5 0 Orpm ( 1 3 0 0 g ) 進行離心15分鐘，其上清液之蛋白質含量以蛋白質分析 套組1 (日本Bio-Rad Laboratories，東京）進行定量，以 混合後之蛋白質之9 9 · 9 %以上均包含於微粒中爲條件。 -59- 200900074 3 )將PPD懸浮於注射用之肝素溶液，以漩渦混合機 混合同時，以預先決定之混合比例滴下2.5%之人類血清 白蛋白溶液。 4) 以4,300rpm( 1300g)進行離心15分鐘，以蒸餾 水清洗2次，再懸浮於蒸餾水中。2) Pre-mix the 2.5% human serum albumin solution with heparin solution (Novo Heparin 1000, 1000 units/mL, about 7.69 mg/mL or less 'AVENTISPHAMA Co., Ltd.) in various ratios to determine the highest heparin solution. Suitable for mixing ratios. The most suitable mixing ratio is to prepare the microparticles at the initial optimum mixing ratio, and then centrifuge at 2,500 rpm (130 g) for 15 minutes, and the protein content of the supernatant is analyzed by protein analysis kit 1 (Japan Bio -Rad Laboratories, Tokyo) Quantitatively, such that more than 99.9% of the mixed protein is contained in the microparticles. -59- 200900074 3) The PPD was suspended in the heparin solution for injection, and mixed with a vortex mixer, and a 2.5% human serum albumin solution was dropped at a predetermined mixing ratio. 4) Centrifuge at 4,300 rpm (1300 g) for 15 minutes, wash twice with distilled water, and resuspend in distilled water.

5) 將1-乙基-3- (3-二甲基胺基丙基）碳二亞胺溶液 (EDC，SIGMA公司製，已溶解於水中濃度爲20 mg/mL 者）以渦混合機混.合同時加入，使其最終濃度爲0.8〜 1.5mg/mL，之後，靜置於室溫下15分鐘。 6) 添加等量0.1M之Glycine水溶液，靜置於室溫下 1 5分鐘。 7) 以4,30 Orpm ( 1 3 00 g)進行離心15分鐘，以蒸餾 水清洗3次’再使PPD量爲0.5 // g/mL之濃度懸浮於生 理食鹽水中。 將該溶液做爲TuMP液。 2.5調製LPS液 將脂多醣體（以下稱作LPS) ( Sigma-Aldrich Japan 股份有限公司’東京）溶解於不含鈣鎂之磷酸鹽緩衝液（ 以下稱作PBS(-)) (SIGMA公司製），調製爲lmg/mL 2.6 THP-1細胞株之分化誘發培養及GM-CSF產生試驗 將人類巨噬細胞細胞株THP -1以離心方式回收，使 -60- 200900074 用PBS(-)清洗2次’於培養液（含ι〇%牛胎兒血清之 RPMI 1 640培養基）中調整細胞數爲400萬個/mL，再添 加進PMA溶液（SIGMA公司製，已將PMA溶解於DMSO 中濃度爲1.62mM者）使其最終濃度爲1.6 " Μ，於24孔 盤上，以每孔播入0.5 mL進行培養4天。培養後，使用 PBS清洗2次’更換新的培養液，再進行培養丨夜。之後 ’更新爲各種刺激用培養液0.5 mL。調製此0.5mL培養 液時，加入1/5容量之CaP微粒子液，CaP-PPD微粒子液 。另外，亦將PPD溶解於培養液中使其濃度爲〇.5 β g/mL，而添加1/5容量。TuMP液（2.5 // g/mL )添加 1/25容量。LPS液則以使其如表1所載濃度以培養液稀釋 而添加。其各別之最終濃度記載於表1。 2.7 GM-CSF之測定試樣 更新各種刺激用培養液0 · 5mL後，再進行培養24小 時，其後回收上清液做爲GM-CSF之測定用試樣。5) A solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, manufactured by SIGMA, dissolved in water at a concentration of 20 mg/mL) mixed with a vortex mixer At the time of contract, it was added to a final concentration of 0.8 to 1.5 mg/mL, and then left to stand at room temperature for 15 minutes. 6) Add an equal amount of 0.1 M aqueous Glycine solution and let stand at room temperature for 15 minutes. 7) Centrifuge at 4,30 Orpm (130 g) for 15 minutes, rinse 3 times with distilled water, and suspend the PPD in a concentration of 0.5 // g/mL in physiological saline. This solution was used as a TuMP solution. 2.5 Preparation of LPS Solution Lipopolysaccharide (hereinafter referred to as LPS) (Sigma-Aldrich Japan Co., Ltd. 'Tokyo) was dissolved in phosphate buffer containing no calcium and magnesium (hereinafter referred to as PBS(-)) (manufactured by SIGMA) , differentiation-induced culture and GM-CSF production assay of 1 mg/mL 2.6 THP-1 cell line. Human macrophage cell line THP-1 was recovered by centrifugation, and -60-200900074 was washed twice with PBS(-). 'Adjust the number of cells in the culture solution (RPMI 1 640 medium containing 〇%% fetal serum) to 4 million cells/mL, and then add the PMA solution (made by SIGMA), the PMA was dissolved in DMSO at a concentration of 1.62 mM. The final concentration was 1.6 " Μ, and cultured for 4 days on a 24-well plate with 0.5 mL per well. After the culture, the cells were washed twice with PBS to replace the new culture solution, and the culture was further incubated overnight. After that, it was updated to 0.5 mL of various stimulating culture solutions. When this 0.5 mL culture solution was prepared, 1/5 volume of CaP fine particle liquid and CaP-PPD fine particle liquid were added. Further, PPD was also dissolved in the culture solution to have a concentration of 〇.5 β g/mL, and a capacity of 1/5 was added. Add 1/25 capacity to TuMP solution (2.5 // g/mL). The LPS solution was added by diluting it with the culture solution at the concentration shown in Table 1. The respective final concentrations are shown in Table 1. 2.7 Measurement sample of GM-CSF After regenerating each culture medium for stimulation, 0·5 mL, the culture was further carried out for 24 hours, and then the supernatant was collected as a measurement sample for GM-CSF.

3.定量 GM-CSF 使用人類 GM-CSF定量用套組（Human GM-CSF ELISA System Biotrak, Amersham LIFE SCIENCE 公司製 )，根據套組使用說明書而使用。GM-CSF之定量以 duplicate方式進行。再以微量滴定盤偵測器（Biotrakll， Amersham Biosciences 公司製），於 30 分鐘內測定 45 Onm之吸光度，根據檢量線而計算出GM-CSF之値。 -61 - 200900074 B ·結果 結果示於表1。表中之簡稱Cap係磷酸鈣微粒子： PPD係純化結核菌素（最終濃度記載於括號中）；TuMP 係PPD担載凝聚作用沉澱微粒子（所担載之PPD之最終 濃度記載於括號中）；CaP-PPD係PPD担載磷酸鈣微粒 子（所担載之PPD之最終濃度記載於括號中）；LPS爲 脂多醣體（最終濃度記載於括號中）。於本實驗系中所產 生之GM-CSF之量係相對於所添加之LPS濃度，顯示具 定量的用量反應性。因此本實驗係具優異之定量性。分化 爲抗原呈現細胞之THP-1細胞，以PPD、TuMP以及2者 刺激時，僅產生少量的 GM-CSF。相對地，使用 CaP-PPD0.1//g/mL濃度時可使所產生之GM-CSF爲未刺激時 之3倍，2.0 # g/mL之濃度時可使GM-CSF爲未刺激時之 13倍。進而於CaP-PPD追加PPD、TuMP時，較僅分別單 純添力[]CaP-PPD、PPD、TuMP時，可使其產生更多之 GM-CSF。類似這種超越相加性的效果亦可見於CaP-PPD 之濃度爲0.1、0.4、2.0// g/mL時，遂確認CaP-PPD之濃 度愈高會表現愈多，而具有相乘性之效果。 -62- 200900074 表13. Quantification GM-CSF was used according to the kit instruction manual using a human GM-CSF quantitative kit (Human GM-CSF ELISA System Biotrak, manufactured by Amersham LIFE SCIENCE). The quantification of GM-CSF was carried out in duplicate. Further, a microtiter plate detector (Biotrakll, Amersham Biosciences) was used to measure the absorbance at 45 Onm in 30 minutes, and the GM-CSF was calculated based on the calibration curve. -61 - 200900074 B · Results The results are shown in Table 1. Abbreviated as Cap calcium phosphate microparticles in the table: PPD-based purified tuberculin (final concentration is shown in parentheses); TuMP-based PPD-loaded coacervation precipitated microparticles (the final concentration of the supported PPD is shown in parentheses); CaP - PPD-based PPD carries calcium phosphate microparticles (the final concentration of the PPD carried is shown in parentheses); LPS is a lipopolysaccharide (final concentration is shown in parentheses). The amount of GM-CSF produced in this experimental line showed a quantitative amount of reactivity with respect to the added LPS concentration. Therefore, this experiment has excellent quantitative properties. THP-1 cells differentiated into antigen-presenting cells, when stimulated with PPD, TuMP, and 2, produced only a small amount of GM-CSF. In contrast, when using a concentration of CaP-PPD0.1//g/mL, the GM-CSF produced can be 3 times that of unstimulated, and when the concentration of 2.0 #g/mL can make GM-CSF unstimulated. 13 times. Further, when PPD or TuMP is added to CaP-PPD, more GM-CSF can be produced than when purely adding [] CaP-PPD, PPD, or TuMP, respectively. Similar effects of this super-additiveness can also be seen when the concentration of CaP-PPD is 0.1, 0.4, 2.0//g/mL, and the higher the concentration of CaP-PPD, the more performance, and the multiplicity. effect. -62- 200900074 Table 1

No 免疫刺激物質 產生GM-CSF (ng/ml) 1 未刺激 1.7 2 PPD ( 0.1 // g/mL) 2.9 3 TuMP ( 0.1 /z g/mL) 2.5 4 PPD ( 0.1 /z g/mL ) +TuMP (0.1// g/mL ) 2.8 5 CaP-PPD ( 0.1 # g/mL) 5.1 6 CaP-PPD ( 0.4/z g/mL) 6.4 7 CaP-PPD ( 2.2 β g/mL) 22.2 8 CaP-PPD ( 0.1 μ g/mL) +PPD ( 0.1 β g/mL) TuMP (0.1 /Z g/mL) 12.4 9 CaP-PPD ( 0.4 β g/mL ) +PPD ( 0.1 // g/mL ) TuMP (0.1 # g/mL ) 14.3 10 CaP-PPD ( 2.0 μ g/mL ) +PPD ( 0.1 β g/mL ) TuMP (0.1// g/mL ) 67.7 11 LPS ( lOng/mL) 34.1 12 LPS ( lOOng/mL) 65.2 13 LPS ( lOOOng/mL) 131.3 例2 :將2種免疫刺激物質各自載持於其他免疫刺激物質 載體之免疫佐劑之抗原提示細胞刺激效果 A ·方法以及材料 試藥之調製、THP-1細胞株之分化誘發培養及GM-CSF之產生試驗、GM-CSF之測定試樣、以及GM-CSF之 定量係根據例1。其他試藥之調製係如下所述。 1 ·調製担載BCG菌萃取物之磷酸鈣微粒子（以下略記爲 CaP-Bx )液 -63- 200900074 1)將乾燥BCG疫苗安瓶1瓶（日本BCG製造股份 有限公司，12mg裝），以110°C滅菌5分鐘，加入lmL 乙醇，攪拌6小時以上，再採取其懸浮液加入另一瓶乾燥 之BCG製劑中，再充分攪拌，之後，以eppendorf型微量 高速離心機以12,00 Orpm離心5分鐘，回收該上清液，以 其爲來自BCG菌體之乙醇可溶性成分溶液（以下稱作 BCG萃取液）。 2 )將林格氏液以 8.28 mL，Klinisalz B 液以 1.24 mL ，Bifil專用碳酸氫鈉補充液以0.48 mL之比例混合，再 調製至1 〇 m L。於該磷酸耗飽和溶液9 m L中，添加1 mL 之BCG萃取液，於該混合液每10 mL，加入1滴CaP微 粒子液再放置一晚。將該懸浮液進行離心，將所得之沉澱 以注射用之生理食鹽水充分洗淨，再懸浮於1 mL之注射 用生理食鹽水。將其做爲CaP-Bx液。於產生GM-CSF試 驗中，添加1 /5容量於刺激用培養液。 B ·結果 結果示於圖1。No.10、11以及12其棒狀柱狀圖上之 縱線表示標準偏差。圖中之簡稱CaP係磷酸鈣微粒子； PPD係純化結核菌素（最終濃度爲0.1〆g/mL ) ; TuMP、 PPD担載凝聚作用沉澱微粒子（所担載之PPD之最終濃 度爲0.1/zg/mL) ; CaP-Bx、BCG菌萃取物担載磷酸鈣微 粒子（BCG菌萃取物之製作方法及最終濃度係參照上述 A 1 · 1 ) ; LP S爲脂多醣體（最終濃度記載於括號中）。圖 -64 - 200900074 中試樣之免疫刺激物質示於下表2。 表2No Immune stimulating substance produces GM-CSF (ng/ml) 1 Unstimulated 1.7 2 PPD ( 0.1 // g/mL) 2.9 3 TuMP ( 0.1 /zg/mL) 2.5 4 PPD ( 0.1 /zg/mL ) +TuMP ( 0.1// g/mL ) 2.8 5 CaP-PPD ( 0.1 # g/mL) 5.1 6 CaP-PPD ( 0.4/zg/mL) 6.4 7 CaP-PPD ( 2.2 β g/mL) 22.2 8 CaP-PPD ( 0.1 μ g/mL) +PPD ( 0.1 β g/mL) TuMP (0.1 /Z g/mL) 12.4 9 CaP-PPD ( 0.4 β g/mL ) +PPD ( 0.1 // g/mL ) TuMP (0.1 # g /mL ) 14.3 10 CaP-PPD ( 2.0 μ g/mL ) + PPD ( 0.1 β g / mL ) TuMP (0.1 / / g / mL ) 67.7 11 LPS ( lOng / mL ) 34.1 12 LPS ( lOOng / mL ) 65.2 13 LPS (100Ong/mL) 131.3 Example 2: Antigens of the immunoadjuvants of the two immunostimulatory substances carried by the other immunostimulating substance carriers, the cell stimulating effect A · Method and preparation of the material reagent, THP-1 cells The differentiation induction culture of the strain, the production test of GM-CSF, the measurement sample of GM-CSF, and the quantitative measurement of GM-CSF are based on Example 1. The modulation of other reagents is as follows. 1·Preparation of calcium phosphate microparticles (hereinafter abbreviated as CaP-Bx) loaded with BCG bacteria extract-63-200900074 1) 1 bottle of dried BCG vaccine ampoules (Japan BCG Manufacturing Co., Ltd., 12mg), to 110 Sterilize for 5 minutes at °C, add 1mL of ethanol, stir for more than 6 hours, then add the suspension to another bottle of dried BCG preparation, stir well, then centrifuge at 12,00 Orpm in an eppendorf type micro-speed centrifuge. The supernatant was recovered as a solution of the ethanol soluble component from BCG cells (hereinafter referred to as BCG extract). 2) Ringer's solution was mixed with 8.28 mL, Klinisalz B solution at 1.24 mL, Bifil-specific sodium bicarbonate supplement solution at a ratio of 0.48 mL, and then adjusted to 1 〇 m L. To 9 m L of the phosphoric acid-saturated solution, 1 mL of BCG extract was added, and 1 drop of CaP microparticle liquid was added to the mixture for 10 mL, and then left overnight. The suspension was centrifuged, and the resulting precipitate was thoroughly washed with physiological saline for injection, and resuspended in 1 mL of physiological saline for injection. Use it as a CaP-Bx solution. In the GM-CSF test, 1/5 volume was added to the stimulation medium. B · Results The results are shown in Figure 1. No. 10, 11 and 12, the vertical line on the bar histogram indicates the standard deviation. In the figure, it is abbreviated as CaP calcium phosphate microparticles; PPD is purified by tuberculin (final concentration is 0.1〆g/mL); TuMP and PPD are supported by coacervation precipitation microparticles (the final concentration of PPD supported is 0.1/zg/ mL); CaP-Bx, BCG extracts loaded with calcium phosphate microparticles (the preparation method and final concentration of BCG extracts refer to the above A 1 · 1); LP S is lipopolysaccharide (final concentration is shown in brackets) . The immunostimulatory substances of the samples in Figures -64 - 200900074 are shown in Table 2 below. Table 2

No. 免疫刺激物質 1 未刺激 2 CaP 3 TuMP 4 PPD 5 PPD+TuMP 6 CaP + TuMP 7 CaP+PPD 8 CaP + PPD + TuMP 9 CaP-Bx 10 CaP-Bx + TuMP 11 CaP-Bx+PPD 12 CaP-Bx + PPD + TuMP 13 LPS ( lOng/mL) 14 LPS ( lOOng/mL) 15 LPS ( lOOOng/mL) 未發現做爲担載載體之C aP微粒子具有抗原呈現細胞 之刺激作用。TuMP、PPD與例1相同，僅具極弱之刺激 作用，混合CaP、TuMP、PPD時其刺激作用亦弱，最大 之產生 GM-CSF 量亦僅 12.4ng/mL(No.5) 。CaP-Bx 則爲 -65- 200900074 128.7 ng/mL (No.9)即便追加PPD亦幾乎無變化，追加 TuMP時可觀察到清楚的相乘性的效果（No · 1 0及Νο · 1 2 ) 例3 :使1種免疫刺激物質載體担載2種免疫刺激物質， 使其他免疫刺激物質載體担載1種免疫刺激物質之免疫佐 劑之抗原呈現細胞刺激效果 A.方法以及材料 試藥之調製、THP-1細胞株之分化誘發培養及GM-CSF之產生試驗、GM-CSF之測定試樣、以及GM-CSF之 定量係根據例1。其他試藥之調製係如下所述。 1.調製担載PPD-BCG菌萃取物之磷酸鈣微粒子（以下略 記爲 CaP-PPD-Bx)液 1 )將林格氏液以 8.28 mL，Klinisalz B 液以 1.24 mL ，Bifil專用碳酸氫鈉補充液以0.48 mL之比例混合，再 調製至10mL。於該磷酸鈣飽和溶液9 mL中，溶解5 # g 之PPD。於該溶液9mL中添加1 mL之BCG萃取液，於 該混合液每1 〇 mL，加入1滴CaP微粒子液再放置一晚。 將該懸浮液進行離心，將所得之沉澱以注射用之生理食鹽 水充分洗淨，再懸浮於1 mL之注射用生理食鹽水。將其 做爲CaP-PPD -Bx液。於產生GM-CSF試驗中，添加1/5 容量於刺激用培養液。 -66- 200900074 B.結果 結果示於表3。表中之簡稱CaP係磷酸鈣微粒子； PPD係純化結核菌素（最終濃度爲〇 .丨# g/mL ) ; TuMP ' PPD担載凝聚作用沉澱微粒子（所担載之PPD之最終濃 度爲0.1 y g/mL ) ; CaP-PPD、PPD担載磷酸鈣微粒子（ 所担載之PPD最終濃度爲1 .〇 " g/mL ) •，同時担載CaP- f PPD-Bx、PPD與BCG菌萃取物之磷酸鈣微粒子（製作方 k 法及最終濃度係參照上述A 1 . 1。所担載之P P D最終濃度 爲1.0# g/mL) ; LPS爲脂多醣體（最終濃度記載於括號 中）。表3中No.8係確認例1之結果的再現性，於CaP-PPD中追力口 PPD、TuMP時，因較僅分別單純添加CaP-PPD、PPD、TuMP時，可使其產生更多之GM-CSF，判斷 其具有良好的再現性。亦發現自該試樣除去PPD液之 No.7同樣具有相乘性的效果。其中亦發現與於CaP-PPD-( Bx中添加TuMP時相同，具極強的相乘性效果。 -67- 200900074 表3No. Immunostimulatory substance 1 Not stimulated 2 CaP 3 TuMP 4 PPD 5 PPD+TuMP 6 CaP + TuMP 7 CaP+PPD 8 CaP + PPD + TuMP 9 CaP-Bx 10 CaP-Bx + TuMP 11 CaP-Bx+PPD 12 CaP -Bx + PPD + TuMP 13 LPS (10 ng/mL) 14 LPS (100 ng/mL) 15 LPS (100Ong/mL) It has not been found that the C aP microparticles as a supporting carrier have an antigen-presenting cell stimulating effect. TuMP and PPD were the same as in Example 1, and had only a very weak stimulating effect. The stimulating effect was also weak when CaP, TuMP and PPD were mixed, and the maximum amount of GM-CSF produced was only 12.4 ng/mL (No. 5). CaP-Bx is -65-200900074 128.7 ng/mL (No.9) Even if PPD is added, there is almost no change, and the effect of clear multiplicity can be observed when TuMP is added (No · 1 0 and Νο · 1 2 ) Example 3: One type of immunostimulating substance carrier is loaded with two kinds of immunostimulating substances, and the antigen of the immunological adjuvant in which the other immunostimulating substance carrier carries one kind of immunostimulating substance exhibits a cell stimulating effect A. Method and modulation of material reagent The differentiation-inducing culture of THP-1 cell strain, the production test of GM-CSF, the measurement sample of GM-CSF, and the quantitation of GM-CSF are according to Example 1. The modulation of other reagents is as follows. 1. Preparation of calcium phosphate microparticles (hereinafter abbreviated as CaP-PPD-Bx) loaded with PPD-BCG extract 1) Ringer's solution was 8.28 mL, Klinisalz B solution was 1.24 mL, supplemented with Bifil-specific sodium bicarbonate The solution was mixed at a ratio of 0.48 mL and then adjusted to 10 mL. 5 g of PPD was dissolved in 9 mL of the saturated calcium phosphate solution. 1 mL of the BCG extract was added to 9 mL of the solution, and 1 drop of CaP fine particle solution was added to the mixture for 1 night, and then left overnight. The suspension was centrifuged, and the resulting precipitate was sufficiently washed with physiological saline for injection and resuspended in 1 mL of physiological saline for injection. This was used as a CaP-PPD-Bx solution. In the GM-CSF test, a 1/5 capacity was added to the stimulation medium. -66- 200900074 B. Results The results are shown in Table 3. The table is abbreviated as CaP calcium phosphate microparticles; PPD is purified tuberculin (final concentration is 〇.丨# g/mL); TuMP ' PPD supports coagulation precipitation microparticles (the final concentration of PPD supported is 0.1 yg) /mL) ; CaP-PPD, PPD carrying calcium phosphate microparticles (the final concentration of PPD carried is 1. 〇 " g / mL) •, while carrying CaP- f PPD-Bx, PPD and BCG extract The calcium phosphate microparticles (the method of the k method and the final concentration are referred to the above A 1.1. The final concentration of the PPD carried is 1.0 # g/mL); the LPS is a lipopolysaccharide (the final concentration is shown in parentheses). In Table 3, No. 8 confirms the reproducibility of the results of Example 1. When the PDP and TuMP are searched for in the CaP-PPD, it is possible to generate more when only CaP-PPD, PPD, and TuMP are simply added. The GM-CSF is judged to have good reproducibility. It was also found that No. 7 in which the PPD liquid was removed from the sample also had the effect of multiplicative. It is also found to have the same strong multiplicative effect as CaP-PPD- (the same as when adding TuMP in Bx. -67- 200900074 Table 3

No 免疫刺激物質 產生GM-CSF (ng/ml) 1 未刺激 1.0 2 PPD 0.3 3 TuMP 0.6 4 PPD+TuMP 1.2 5 CaP-PPD 11.4 6 CaP-PPD+PPD 12.0 7 CaP-PPD + TuMP 20.6 8 CaP-PPD + PPD+TuMP 19.1 9 CaP-PPD-Bx 187.5 10 CaP-PPD-Bx+PPD 23 1.9 11 CaP-PPD-Bx + TuMP 263.0 12 CaP-PPD-Bx + PPD + TuMP 268.3 13 LPS ( lOng/mL) 12.4 14 LPS ( 1 OOng/mL ) 26.0 15 LPS ( lOOOng/mL) 50.9 使用本發明之免疫佐劑時，可使抗原呈現細胞釋放出 與使用已知爲具有強力免疫刺激作用之LPS刺激抗原呈 現細胞時，產生相同程度或超越之大量的GM-CSF。因而 GM-CSF可做爲自體內分泌細胞生長因子，作用於抗原呈 現細胞之強力化性化與維持其狀態。故藉由將本發明之免 疫佐劑與抗原一同對包含人類之哺乳類動物之生體內進行 投予，而可誘發依賴活化之抗原呈現細胞對該抗原之體液 性免疫反應以及/或細胞性免疫反應，而可進行對感染症 及腫瘤極有效之疫苗療法。 -68- 200900074 【圖式簡單說明】 〔圖1〕係表示使2種免疫刺激物質分別担載於相異 的2種免疫刺激物質載體之本發明免疫佐劑之抗原呈現細 胞刺激效果之圖。將磷酸鈣（以下略稱爲「CaP」）做爲載 體使其担載做爲免疫刺激物質之BCG菌萃取物。另外， 使用凝聚作甩沉澱微粒子做爲載體使其担載PPD後之 TuMP。 -69-No Immune stimulating substance produces GM-CSF (ng/ml) 1 Unstimulated 1.0 2 PPD 0.3 3 TuMP 0.6 4 PPD+TuMP 1.2 5 CaP-PPD 11.4 6 CaP-PPD+PPD 12.0 7 CaP-PPD + TuMP 20.6 8 CaP- PPD + PPD+TuMP 19.1 9 CaP-PPD-Bx 187.5 10 CaP-PPD-Bx+PPD 23 1.9 11 CaP-PPD-Bx + TuMP 263.0 12 CaP-PPD-Bx + PPD + TuMP 268.3 13 LPS (10Ong/mL) 12.4 14 LPS (100 ng/mL) 26.0 15 LPS (100Ong/mL) 50.9 When the immunoadjuvant of the present invention is used, the antigen-presenting cells can be released and expressed using an LPS-stimulated antigen known to have potent immunostimulatory effects. At the same time, a large amount of GM-CSF is produced to the same extent or beyond. Therefore, GM-CSF can be used as a cell growth factor secreted from the body, and acts on the antigen presenting cells to maintain the state and maintain its state. Therefore, by administering the immunoadjuvant of the present invention together with an antigen to a human mammalian mammal, the antigen-dependent immune antigen can be induced to exhibit a humoral immune response and/or a cellular immune response to the antigen. It is a highly effective vaccine therapy for infectious diseases and tumors. -68-200900074 [Brief Description of the Drawings] Fig. 1 is a view showing the effect of the antigen-stimulated effect of the antigen of the immunoadjuvant of the present invention in which two kinds of immunostimulating substances are respectively carried on two different immunostimulating substance carriers. Calcium phosphate (hereinafter abbreviated as "CaP") is used as a carrier to carry a BCG extract as an immunostimulating substance. In addition, TuMP was obtained by coagulating the precipitated microparticles as a carrier to carry PPD. -69-

Claims (1)

200900074 十、申請專利範圍 1. 一種免疫佐劑，其係包含於2種以上不同之微粒子 狀免疫刺激物質載體分別担載1種或2種以上的免疫刺激 物質之免疫佐劑，且做爲該微粒子狀免疫刺激物質載體至 少包含（a)藉由細胞而爲可被吞噬尺寸之微粒子狀磷酸 鈣，以及（b)藉由可溶性蛋白與黏多醣產生凝聚作用之 沉澱物之組合，該（a )之微粒子狀磷酸鈣係Ca/ P之莫 耳比爲1.3以上，且含有碳酸根離子（：032_爲1〜15重量 %，其結晶性規定爲藉由CuK α射線於X射線繞射光譜中 ，以2Θ値26°、32°以及34°爲中心出現寬廣的尖峰値，且 於3 3 °出現3 0 0之米勒指數之肩峰之結晶性以上，且規定 以26°、2 8. 1°、29°、3 2°、33°以及3 4°爲中心出現尖峰値 或肩峰之外，再加上211米勒指數與112米勒指數之尖峰 値係爲分離之結晶性以下之結晶性之微粒子狀磷酸鈣。 2. 如申請專利範圍第1項之免疫佐劑，其中該（a ) 之微粒子狀磷酸鈣係最大徑爲以下之磷酸鈣。 3. 如申請專利範圍第1或2項之免疫佐劑，其中該（ b)之可溶性蛋白係白蛋白。 4. 如申請專利範圍第1〜3項中任一項之免疫佐劑， 其中該（b)之黏多醣係肝素。 5. 如申請專利範圍第1〜4項中任一項之免疫佐劑， 其係由該（b)之不溶性物質之可溶性蛋白與黏多醣產生 凝聚作用之沉澱物’經蛋白分子間交聯劑而被交聯之沉澱 物。 -70- 200900074 6 .如申請專利範圍第1〜5項中任一項之免疫佐劑’ 其中免疫刺激物質係1種或2種以上選自細胞激素、趨化 激素、細胞生長因子以及荷爾蒙所成群之誘發劑。 7.如申請專利範圍第1〜6項中任一項之免疫佐劑’ 其中免疫刺激物質係1種或2種以上選自結核菌素、純化 結核菌素、來自微生物的可溶性成分、海藻糖二黴菌酸酯 、脂多醣類、脂質A、寡核苷酸、葡聚醣、鑰孔蟲戚血藍 蛋白、胞壁醯二胜肽、貝他定（bestatin )、左旋咪唑、 細胞激素、趨化激素、細胞生長因子以及荷爾蒙所成群之 物質。 8 .如申請專利範圍第7項之免疫佐劑，其中來自微生 物的可溶性成分係1種或2種以上選自微生物之酒精萃取 物、丙酮萃取物、吡啶萃取物以及熱水萃取物所成群之萃 取物。 9.如申請專利範圍第7或8項之免疫佐劑，其中微生 物係細菌。 1 0·如申請專利範圍第1〜9項中任一項之免疫佐劑， 其係與抗原一同對包含人類之哺乳類動物之體內進行投予 後，而達成誘發對該抗原之全身性免疫反應。 1 1 ·如申請專利範圍第1 0項之免疫佐劑，其中該抗原 係1種或2種以上選自真菌、放線菌、細菌、病毒、噬菌 體、克次體、原蟲、真菌成分、放線菌成分、細菌成分 、病毒成分、噬菌體成分、立克次體成分、原蟲成分、腫 瘤組織、腫瘤細胞、腫瘤細胞成分、腫瘤抗原蛋白以及腫 -71 - 200900074 瘤抗原胜肽所成群之物質。 1 2 .如申請專利範圍第1〜9項中任一項 其係將包含人類之哺乳類動物之腫瘤組織以 其變性後，再藉由對該腫瘤組織進行投予而 瘤免疫反應。 1 3 .如申請專利範圍第1 2項之免疫佐劑 性方法係1種或2種以上選自微波照射法、 、冷凍凝固法、電燒加熱法、熱水注入法、 栓塞法、放射線照射法、雷射照射法以及超 成群之方法。 1 4.如申請專利範圍第1〜9項中任一項 其係於體外與擔任免疫細胞混合後，藉由對 乳類動物進行投予，而達成於該動物之生體 免疫反應。 1 5 ·如申請專利範圍第1 4項之免疫佐劑 疫細胞係1種或2種以上選自樹狀細胞、巨 巴球、T淋巴球、自然殺手細胞、自然殺手 血幹細胞所成群之細胞。 16.—種疫苗，其係包含如申請專利範匱 任一項之免疫佐劑與抗原。 之免疫佐劑， 物理性方法使 達成誘發抗腫 ，其中該物理 高週波凝固法 酒精注入法、 音波破壞法所 之免疫佐劑， 包含人類之哺 內誘發全身性 ，其中擔任免 噬細胞、B淋 T細胞以及造 第1〜9項中 -72-200900074 X. Patent application scope 1. An immunological adjuvant comprising an immunological adjuvant containing one or more immunostimulating substances in two or more different microparticle immunostimulating substance carriers, and The microparticulate immunostimulating substance carrier comprises at least (a) a particulate calcium phosphate which can be phagocytized by a cell, and (b) a combination of a precipitate which is agglomerated by a soluble protein and a mucopolysaccharide, (a) The particulate calcium phosphate Ca/P has a molar ratio of 1.3 or more and contains carbonate ions (: 032_ is 1 to 15% by weight, and its crystallinity is defined by CuK α ray in the X-ray diffraction spectrum. , a wide peak 値 at the center of 2Θ値26°, 32°, and 34°, and the crystallinity of the shoulder of the 300-mile Miller index appears above 3 3 °, and is specified to be 26°, 2 8. 1 °, 29°, 3 2°, 33°, and 3 4° are centered with spikes or shoulders, plus the 211 Miller index and the 112 Miller index peaks are crystalline crystals below the separation. Microparticle calcium phosphate. 2. If the scope of patent application An immunoadjuvant of the item 1, wherein the microparticle-like calcium phosphate of the (a) has a maximum diameter of calcium phosphate. 3. The immunological adjuvant according to claim 1 or 2, wherein the soluble protein of the (b) 4. The immunological adjuvant according to any one of claims 1 to 3, wherein the mucopolysaccharide of (b) is heparin. 5. If any one of claims 1 to 4 of the patent application scope An immunoadjuvant which is a precipitate which is crosslinked by a protein-intermolecular cross-linking agent by a soluble protein of the insoluble matter of (b) and a coagulating polysaccharide-aggregating agent. -70-200900074 6 . The immunological adjuvant of any one of the first to fifth aspects of the invention, wherein the immunostimulating substance is one or more selected from the group consisting of cytokines, chemokines, cell growth factors, and hormones. The immunoadjuvant of any one of claims 1 to 6 wherein the immunostimulating substance is one or more selected from the group consisting of tuberculin, purified tuberculin, soluble component derived from microorganisms, and trehalose Mycophenolate, lipopolysaccharide, lipid A, oligo Nucleotide, dextran, keyhole limpet hemocyanin, cell wall bismuth peptide, bestatin, levamisole, cytokines, chemokines, cell growth factors, and hormones 8. The immunological adjuvant according to claim 7, wherein the soluble component derived from the microorganism is one or more selected from the group consisting of an alcohol extract of a microorganism, an acetone extract, a pyridine extract, and a hot water extract. 9. The extract of the group. 9. The immunological adjuvant according to claim 7 or 8, wherein the microorganism is a bacterium. The immunological adjuvant according to any one of claims 1 to 9 of the patent application, The antigen is administered together to the body of a human mammal, and a systemic immune response to the antigen is induced. 1 1 · The immunological adjuvant according to claim 10, wherein the antigen is one or more selected from the group consisting of a fungus, an actinomycete, a bacterium, a virus, a phage, a gramin, a protozoa, a fungal component, and a line. Bacterial components, bacterial components, viral components, phage components, rickettsia components, protozoal components, tumor tissues, tumor cells, tumor cell components, tumor antigen proteins, and tumor-derived antigens . 1 2 . According to any one of claims 1 to 9 of the patent application, the tumor tissue containing a mammalian mammal is denatured, and then the tumor immune response is administered by administering the tumor tissue. 1 3 . The immunoadjuvant method according to claim 12 of the patent application is one or more selected from the group consisting of microwave irradiation, freeze coagulation, electrothermal heating, hot water injection, embolization, and radiation irradiation. Method, laser irradiation method and super cluster method. 1 4. According to any one of claims 1 to 9 of the patent application, which is mixed with an immune cell in vitro, the mammalian animal is subjected to a biological immune response by administration to a mammal. 1 5 · If the immune adjuvant cytopathic cell line of claim 14 of the patent scope is one or more selected from the group consisting of dendritic cells, giant palms, T lymphocytes, natural killer cells, and natural killer blood stem cells cell. 16. A vaccine comprising an immunological adjuvant and an antigen as claimed in any of the patent applications. The immune adjuvant, the physical method is used to achieve the induction of anti-tumor, wherein the physical high-frequency coagulation method, the alcohol injection method, and the ultrasonic adjuvant of the sonication method, including human feeding, induces systemicity, wherein the activizing cells, B Throwing T cells and making items 1 to 9 in -72-
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