TW201134480A

TW201134480A - CLUAP1 peptides and vaccines including the same

Info

Publication number: TW201134480A
Application number: TW099135910A
Authority: TW
Inventors: Yusuke Nakamura; Takuya Tsunoda; Ryuji Ohsawa; Sachiko Yoshimura; Tomohisa Watanabe
Original assignee: Oncotherapy Science Inc
Priority date: 2010-04-08
Filing date: 2010-10-21
Publication date: 2011-10-16
Also published as: CA2795423A1; JP2013523083A; SG184468A1; KR20130043632A; WO2011125139A1; MX2012011666A; BR112012025486A2; AU2010350389A1

Abstract

Peptide vaccines against cancer are described herein. In particular, epitope peptides derived from the CLUAP1 gene that elicit CTLs are provided. Antigen-presenting cells and isolated CTLs that target such peptides, as well as methods for inducing the antigen-presenting cell, or CTL are also provided. The present invention further provides pharmaceutical compositions containing as active ingredient peptides derived from CLUAP1 or polynucleotides encoding the peptides as active ingredients. Furthermore, the present invention provides methods for the treatment and/or prophylaxis (i. e., prevention) of cancers (tumors), and/or the prevention of postoperative recurrence thereof, as well as methods for inducing CTLs, methods for inducing anti-tumor immunity, using the peptides derived from CLUAP1, polynucleotides encoding the peptides, or antigen-presenting cells presenting the peptides, or the pharmaceutical compositions of the present invention.

Description

201134480 六、發明說明：【發明所屬之技術領域】本發明係關於生物科學領域，更特別對於癌症治療領域。特別是’本發明係關於新穎之胜肽，其當作癌症疫苗及治療與避免腫瘤之藥物為非常有效。【先前技術】已證實CD8陽性細胞毒殺性T淋巴球辨認來自建造於主要組織相容性抗原複合體（ma jor histocompatibi 1 ity complex, MHC) class I分子上之腫瘤相關抗原 (tumor-associated antigens，TAAs)的抗原決定位胜肽，且之後殺死腫瘤細胞。自從發現黑色素瘤抗原（melanoma antigen，MAGE)家族為腫瘤相關抗原之第一個例子，藉由免疫方法，已發現許多其他腫瘤相關抗原（Boon T，Int J Cancer 1993 May 8, 54(2): 177-80(NPL 1); Boon T & van der Bruggen P, J Exp Med 1996 Mar 1, 183(3): 725-9(NPL 2))’且這些腫瘤相關抗原的一些目前在臨床發展的過程中作為免疫治療標的。能誘導有效且專一之抗腫瘤免疫反應的新腫瘤相關抗原的辨認成為於多種形式癌症中之胜肽疫苗接種策略 (vaccination strategies)之更進一步發展與臨床研究的根據為正進行著（Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20)： 1442-55(NPL 3); Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13): 3134-42(NPL 4); Vissers 201134480 JL et al.，Cancer Res 1 999 Nov 1，59 (2 1 ): 5554-9 (NPL 5); van der Burg SH et al. , J Immuno 1 1 996 May 1，156(9): 3308-14CNPL 6)； Tanaka F et al., Cancer Res 1997 Oct 15, 57(20): 4465-8CNPL 7); Fu j ie T et al. , Int J Cancer 1 999 Jan 1 8, 80 ( 2 ): 1 69-72 (NPL 8); Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66(NPL 9); Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94(NPL 10))。迄今已報導許多使用這些腫瘤相關抗原衍生胜肽的臨床S式驗。不幸地，許多目前癌症疫苗試驗已顯示一低的客觀反應率（objective response rate) (Belli F et al.， J Clin Oncol 2002 Oct 15, 20(20): 4169-80(NPL 11)； Coulie PG et al. , Immunol Rev 2002 Oct, 188: 33-42(NPL 12); Rosenberg SA et al., Nat Med 2004 Sep, 10(9)： 9 0 9-1 5 (NPL 13))。因此’仍然需要新穎之腫瘤相關抗原作為免疫治療標的。為此目的’藉由使用含有23,040個基因之基因體寬度 cDNA微陣列之大腸直腸癌（colorectal cancer)表現輪廊的分析’已鑑定出CLUAP1 ( GenBank獲得編號NM_0 1 5041 或 NM_024793 )，簇蛋白相關蛋白質201134480 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of biological sciences, and more particularly to the field of cancer treatment. In particular, the present invention relates to a novel peptide which is very effective as a cancer vaccine and a drug for treating and avoiding tumors. [Prior Art] It has been confirmed that CD8-positive cytotoxic T lymphocytes are recognized from tumor-associated antigens (tumor-associated antigens) constructed on the main histocombility complex (MHC) class I molecule. The epitope of TAAs) is the peptide, and then the tumor cells are killed. Since the discovery of the melanoma antigen (MAGE) family as the first example of tumor-associated antigens, many other tumor-associated antigens have been discovered by immunological methods (Boon T, Int J Cancer 1993 May 8, 54(2): 177-80 (NPL 1); Boon T & van der Bruggen P, J Exp Med 1996 Mar 1, 183(3): 725-9(NPL 2))' and some of these tumor-associated antigens are currently in clinical development In the process as a target for immunotherapy. The identification of new tumor-associated antigens that induce an effective and specific anti-tumor immune response is a further development of the vaccination strategies in various forms of cancer and the basis for clinical research is ongoing (Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20): 1442-55 (NPL 3); Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13): 3134-42 (NPL 4); Vissers 201134480 JL et Al., Cancer Res 1 999 Nov 1,59 (2 1 ): 5554-9 (NPL 5); van der Burg SH et al. , J Immuno 1 1 996 May 1,156(9): 3308-14CNPL 6) Tanaka F et al., Cancer Res 1997 Oct 15, 57(20): 4465-8CNPL 7); Fu j ie T et al. , Int J Cancer 1 999 Jan 1 8, 80 ( 2 ): 1 69-72 (NPL 8); Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66 (NPL 9); Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387 -94 (NPL 10)). A number of clinical S-tests using these tumor-associated antigen-derived peptides have been reported to date. Unfortunately, many current cancer vaccine trials have shown a low objective response rate (Belli F et al., J Clin Oncol 2002 Oct 15, 20(20): 4169-80 (NPL 11); Coulie PG Et al., Immunol Rev 2002 Oct, 188: 33-42 (NPL 12); Rosenberg SA et al., Nat Med 2004 Sep, 10(9): 9 0 9-1 5 (NPL 13)). Therefore, novel tumor-associated antigens are still required as immunotherapeutic targets. For this purpose 'CLUAP1 (GenBank Accession No. NM_0 1 5041 or NM_024793) has been identified by the analysis of colorectal cancer using a genome-wide cDNA microarray containing 23,040 genes. protein

Kclusterin-associated protein 1)，為在大腸癌中常被轉移活化（transactivated)的基因（Takahashi M et al Oncogene. 2004 Dec 9;23(57): 9289-94(NPL 14))。已觀察到CLUAP1的表現被逐步增加於細胞週期之s相晚期至 G2/M相，回復至於G0/G1相中的基礎程度。此外，藉由siRfu 201134480 之CLUAP1的抑制已顯示導致在轉染腫瘤細胞中之成長遲延。CLUAP1的過度表現也於骨肉瘤（〇ste〇sarc〇ma)、卵巢與肺癌中被觀察到（IshikuraHetal.，IntJOncol. 2007 Feb; 30(2): 461-7(NPL 15))。綜上所述，這些事實建議，在多種癌症中，CLUAP1可為癌症免疫治療的一有效目標。【引用文獻】非專利文獻（Non Patent Literature) [NPL 1]Kclusterin-associated protein 1) is a gene that is often transactivated in colorectal cancer (Takahashi M et al Oncogene. 2004 Dec 9; 23(57): 9289-94 (NPL 14)). It has been observed that the performance of CLUAP1 is gradually increased from the late phase of the s phase to the G2/M phase, returning to the basal level in the G0/G1 phase. Furthermore, inhibition of CLUAP1 by siRfu 201134480 has been shown to result in growth delay in transfected tumor cells. Excessive expression of CLUAP1 was also observed in osteosarcoma (〇ste〇sarc〇ma), ovary and lung cancer (Ishikura Hetal., IntJ Oncol. 2007 Feb; 30(2): 461-7 (NPL 15)). Taken together, these facts suggest that CLUAP1 can be an effective target for cancer immunotherapy in a variety of cancers. [Citations] Non Patent Literature [NPL 1]

Boon T, Int J Cancer 1993 May 8, 54(2): 177-80 [NPL 2]Boon T, Int J Cancer 1993 May 8, 54(2): 177-80 [NPL 2]

Boon T & van der Bruggen P, J Exp Med 1 996 Mar 1, 183(3): 725-9 [NPL 3]Boon T & van der Bruggen P, J Exp Med 1 996 Mar 1, 183(3): 725-9 [NPL 3]

Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20)： 1442-55 [NPL 4]Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20): 1442-55 [NPL 4]

Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13): 3134-42 [NPL 5]Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13): 3134-42 [NPL 5]

Vissers 几 et al·，Cancer Res 1 999 Nov 1，59(21): 5554-9 [NPL 6] van der Burg SH et al., J Immunol 1996 May 1, 6 201134480 156(9): 3308-14 [NPL 7]Vissers et al., Cancer Res 1 999 Nov 1, 59(21): 5554-9 [NPL 6] van der Burg SH et al., J Immunol 1996 May 1, 6 201134480 156(9): 3308-14 [ NPL 7]

Tanaka F et al., Cancer Res 1997 Oct 15, 57(20)： 4465-8 [NPL 8]Tanaka F et al., Cancer Res 1997 Oct 15, 57(20): 4465-8 [NPL 8]

Fujie T et al., Int J Cancer 1 999 Jan 18, 80(2): 169-72 [NPL 9]Fujie T et al., Int J Cancer 1 999 Jan 18, 80(2): 169-72 [NPL 9]

Kikuchi M et al., Int J Cancer 1 999 May 5, 81(3): 459-66 [NPL 10]Kikuchi M et al., Int J Cancer 1 999 May 5, 81(3): 459-66 [NPL 10]

Oiso M et al.， Int J Cancer 1999 May 5， 81(3): 387-94 [NPL 11]Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94 [NPL 11]

Belli F et al., J Clin Oncol 2002 Oct 1 5, 20 (20 ): 4169-80 [NPL 12]Belli F et al., J Clin Oncol 2002 Oct 1 5, 20 (20 ): 4169-80 [NPL 12]

Coulie PG et al. , Immunol Rev 2002 Oct, 1 88: 33-42 [NPL 13]Coulie PG et al., Immunol Rev 2002 Oct, 1 88: 33-42 [NPL 13]

Rosenberg SA et al., Nat Med 2004 Sep, 10(9): 909-15 [NPL 14]Rosenberg SA et al., Nat Med 2004 Sep, 10(9): 909-15 [NPL 14]

Takahashi M et al., Oncogene. 2004 Dec 9;23(57): 9289-94 7 201134480 [NPL 15]Takahashi M et al., Oncogene. 2004 Dec 9;23(57): 9289-94 7 201134480 [NPL 15]

Ishikura H et al.， Int J Oncol. 2007 Feb; 30(2):461-7 【發明内容】本發明至少部分基於發現新穎的胜肽，其可作為免疫治療之標的。由於腫瘤相關抗原（tumor-associated antigens, TAAs)有時被免疫系統感知為“自身”且因此常不具有免疫抗原性（i mmunogen i c i ty )，所以適合標的的發現極度重要。如上所提到，已確認CLUAP1(由GenBank獲得編號0_015041或關_024793 (例如，序列辨識號：44) 之基因所編碼出的序列辨識號：45 )在包括，但不限於乳癌（breast cancer)、子宮頸癌（cervical cancer)、大腸直腸癌（colorectal cancer)、食道癌（esophageal cancer)、胃癌（gastric cancer)、瀰漫型胃癌（gastric di f fuse-type cancer)、淋巴癌（lymphoma)、神經母細胞瘤（neuroblastoma)、胰臟癌（pancreatic cancer)的癌症中為向上調控。因此’本發明聚焦於CLUAP1為一癌症/腫瘤免疫治療之標的的候選物。本發明更關於具有誘導專一於CLUAP1之細胞毒殺性τ 淋巴球之能力的CLUAP1之基因產物的特定抗原決定位胜狀的確認。詳細如下所討論，使用與HLA (人類白血球組織抗原）-A* 2402或HLA-A* 0201結合之來自CLUAP1的候選胜肽來刺激自健康提供者獲得之周邊血液單核球細胞 201134480 (peripheral blood mononuclear cells, PBMCs)。之後建立細胞毒殺性T淋巴球，其具有抗（against)經各候選胜肽脈衝（pul sed)之HLA-A24或HLA-A2陽性目標細胞的專一細胞毒性。這些結果證明這些胜肽為HLA-A24或HLA-A2限制的抗原決定位胜肽，其可誘導強而專一之抗表現CLUAP1之細胞的免疫反應。此外，結果指出CLUAPI為強效致免疫性且其抗原決定位為癌症/腫瘤免疫治療之有效目標。因此’本發明一目的為提供與HLA抗原結合之經分離的胜肽’特別是包括CLUAP1 (序列辨識號：45 )或其免疫活性片段的那些。這些胜肽被預期具有細胞毒殺性T淋巴球誘發能力且因此可被用於叩誘導細胞毒殺性τ淋巴球或被投予一個體以誘導抗癌症’例如乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌的免疫反應。較佳胜肽為九胜肽或十胜狀’且更佳為具有擇自序列辨識號：2至23與25至43中之胺基酸序列的那些。在這些中，具有擇自序列辨識號·· 3、 4 、 5 、 8 、 9 、 1。、 14 、 15 、 16 、 18 、 23 、 30 、 33 、 34 、 35 、 36與38中之胺基酸序列的胜肽顯示強的細胞毒殺性丁淋巴球誘發能力，且因此為特別被喜愛。本發明胜肽包括其中一、二或多個胺基酸被取代、刪除或加人的那些’只要所產生經修飾之胜肽維持最初之細胞母殺性Τ淋巴球誘發能力。本發明也提供編碼出任何本發明胜肽之經分離的多核㈣。這些多核苦酸可用以誘導或製傷具有細胞毒殺,性τ淋巴球誘發能力之抗原呈現細 201134480 胞’或如本發明胜肽可被投予至一個體以誘導抗癌之免疫反應。當投予一個體時’本發明胜肽較佳被表現於抗原呈現細胞之表面以便誘導將分別之胜肽做為目標之細胞毒殺性 τ淋巴球。因此，本發明一目標為提供誘導細胞毒殺性τ 淋巴球之組合物或物質，此種組合物包括一或多個本發明胜肽或編碼出此類胜肽的多核苷酸。本發明更考慮包括一或多個本發明胜肽或編碼出此類胜肽的多核苷酸之藥學組合物’此組合物被配製用於癌症之治療及/或預防，與其手術後復發的避免’此類癌症包括’但不限於乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌。除了本發明胜肽或多核苷酸外/ 代替本發明胜肽或多核苷酸，本發明藥學組合物或物質可包括呈現任何之本發明胜肽的抗原呈現細胞或外吐小體為活性成分。本發明之胜肽與多核苷酸可誘導於其表面呈現HLa抗原與本發明胜肽之複合物的抗原呈現細胞，例如，藉由將來自一個體之抗原呈現細胞與此胜肽接觸或將編碼出本發明一胜肽的多核苦酸引入抗原呈現細胞。此種抗原呈現細胞具有问的抗目標胜狀之細胞毒殺性τ淋巴球誘發能力且因此提供用途於癌症免疫治療中。因此，本發明考慮誘導具細胞毒殺性τ淋巴球誘發能力之抗原呈現細胞的方法與藉由此方法獲得之抗原呈現細胞兩者。本發明也提供誘導細胞毒殺性T淋巴球的方法，方法 201134480 包括將CD8陽性細胞與表現一或多個本發明胜肽於其表面之抗原呈現細胞或外吐小體共培養之步驟，或引入包括編碼出與本發明胜肽結合之τ細胞受體（T ceu receptQF， TCR)次單元多胜肽之多核苷酸的基因的步驟。藉由此種方法獲得之細胞毒殺性T淋巴球可在癌症之治療與避免中提供用途，癌症之例子包括，但不限於乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌。因此，本發明包括藉由本發明方法獲得之細胞毒殺性Τ淋巴球。本發明另一目標提供於一需要之個體中誘導抗癌症之免疫反應的方法，此方法包含投予一包括CLUApi多胜肽或 —其免疫活性片段、編碼出CLUAP1多胜肽之多核苷酸，與呈現CLUAP1多胜肽之外吐小體或抗原呈現細胞之組合物或物質至此個體的步驟。本發明之應用性擴展至一些關於或起因於CLUAP1過度表現的疾病的任一個，例如癌症，示例之癌整包括’但不限於乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌。更具體地，本發明提供下列事物： [1 ] 一種經分離的胜肽，具有細胞毒殺性τ淋巴球誘發能力’其中該胜肽係由CLUAP1之胺基酸序列或一其免疫活性片段所組成。 [2] [1]之經分離的胜肽，其中該胜肽包括一胺基酸序列’其係擇自由序列辨識號：2至23與25至43所組成之 201134480 群組。 [3] [1]與[2]之任一項所述之匈;八μ 1 κ經分離的胜肽，其中1、 2或數個胺基酸被取代、刪除或加入。 [4] [3]之經分離的胜肽，其中力丹甲在人類白血球組織抗原 -Α24背景下，該胜肽具有下列特徵之—戋兩者· (a) 來自Ν端之第二個胺某酴耿巷酸為經修飾為一胺基酸，其係擇自由苯丙胺酸、酷·胺酸、甲访1 τ硫丁胺酸或色胺酸所組成之群組；以及 ⑻C端胺基酸為，或經修錦為一胺基酸其係擇自由苯丙胺酸、白胺酸、#白胺酸、色胺酸或甲硫丁胺酸所組成之群組。 [5] [3]之經分離的胜肽’其中在人類白血球組織抗原 -Α2背景下，該胜肽具有下列特徵之—或兩者： (a) 來自Ν端之第二個胺基酸為係擇自由白胺酸與甲硫丁胺酸所組成之群組；以及 (b) C端胺基酸為係擇自由纈氨酸與白胺酸所組成之群組。 [6 ][ 1 ]至[5 ]之任一項所述之經分離的胜肽，其中該胜狀為九胜狀或十胜狀。 [7 ] —種經分離之多核苷酸，其編碼出[丨]至[6 ]之任一項所述的該胜肽。 [8 ] —種誘發細胞毒殺性τ淋巴球之組合物，其中該組合物包括[1]至[6]之任一項所述之—或多個該胜肽，或 [7]所述之一或多個該多核苷酸。 12 201134480 [9 ] 一種藥學組合物’用於癌症之治療及/或預防，及 /或其手術後復發的避免，其中該組合物包括[1]至[6]之任一項所述之一或多個該胜肽，或所述之一或多個該多核苷酸。 [1 0 ][ 9 ]之藥學組合物，被配製來用以投予一個體，其人類白血球組織抗原為人類白血球組織抗原-A24或人類白血球組織抗原-A2。 [11] [9]或[10]之藥學組合物，其中該組合物被配製來用於癌症之治療。 [1 2 ] —種誘導具有細胞毒殺性τ淋巴球誘發能力之抗原呈現細胞的方法，其中該方法包括一步驟，其係擇自由下列所組成之群組： (a) pyiro、以或“ 將一抗原呈現細胞與[1]至[6]之任一項所述之該胜肽接觸；以及 (b) 將編碼出[1 ]至[6 ]之任一項所述之該胜肽的一多核苷酸引入一抗原呈現細胞。 [13 ] —種誘導細胞毒殺性τ淋巴球的方法，藉由包括擇自由下列所組成之群組的一步驟的方法： (a) 將CD8陽性T細胞與抗原呈現細胞共培養，抗原呈現細胞表現一人類白血球組織抗原與[1 ]至[6 ]之任一項所述之該胜肽的複合物於其表面上； (b) 將C D 8陽性T細胞與外吐小體共培養，外11土小體表現一人類白血球組織抗原與[丨]至[6 ]之任一項所述之該胜肽的複合物於其表面上；以及 13 201134480 (C)將一包括編碼出一 T細胞受體次單元多胜肽之多核苦酸的基因引入一 Τ細胞，該τ細胞受體次單元多胜月太與[1]至[6]之任一項所述的該胜肽結合。 [14 ] 一種經分離之抗原呈現細胞，其表現一人類白血球組織抗原與[1]至[6]之任一項所述之該胜肽的複合物於其表面上。 [15] [14]之抗原呈現細胞’其藉由[12]之方法來誘導。 [1 6 ] —種經分離之細胞毒殺性τ淋巴球，其以[丨]至 [6]之任一項所述之該胜肽為標的。 [1 7 ] [ 1 6 ]之細胞毒殺性τ淋巴球，其藉由[丨3 ]之方法來誘導。 [18] —種於一個體中誘導一抗癌症之免疫反應的方法，其中該方法包括投予該個體一組合物，該組合物包括 [1]至[6]之任一項所述之一胜肽、一其免疫活性片段或一編碼出該胜肽或該片段多核苷酸。 [19] 一種抗體或其片段，其抗[丨]至[6]之任—項所述之該胜狀_的任一個。 [2〇] —種載體，包括編碼出[n至[6]之任一項所述之該胜肽的一核苷酸序列。 [21] —種宿主細胞，其被以[2]之一表現載體所轉形或轉染。 [22] 一種診斷套組，包括[丨]至[6]之任一項所述之該胜肽、[7]之該核苷酸或[19]之該抗體。 14 201134480 [23 ] [ 1 ]至[6 ]之任一項之經分離的胜肽，其係擇自由序列辨識號：3、4、5、8、9' 10、14、15、16、18、23、 30、33 ' 34、35、36與38所組成之群組。需瞭解的是’本發明前述發明内容與下列詳細敘述兩者為不範之實施例，並不限制本發明或本發明其他替代實施例。除了上述’當以下詳細說明被閱讀並結合伴隨之圖式與貫施例，本發明之其他目的與特徵會變的更完全地明白。然而，可瞭解的是’上面之本發明内容與以下之詳細說明兩者為示範之實施例，並不限制本發明或本發明其他替代實施例。特別當關於一些特定實施例於此敘述之本發明，可以瞭解的是，敘述為本發明之說明且並不建構為本發明之限制。各種修飾與應用可被熟悉此技藝人士想到’而無背離本發明精神與範，如所时請專利範圍所述。同樣地，本發明之其他此内容與下述之特定實施例，藝人士而言可立即明白。此種上述結合伴隨實施例、資料、隨著考慮引入於此之參考文獻楚的。目的、特徵、好處與優點自為清楚的，且對於熟悉此技目的、特徵、好處與優點自圖式與所有要被自其單獨或而描述的所有合理推論為清【實施方式】雖然於本發明實施例之實施 ^ λ ^ , °飞干可使用相似或箄同於在此敘述之那些的任何方寻〃材枓，但是現在敘述較 15 201134480 佳之方法、元件輿奸其、〃 ° ‘，，；而在敘述本發明材料與方法之則，需瞭解的是，★议本發明並不限於敘述於此之特定大小、形狀、尺寸、材料、方法學、步驟等，例如按照慣例實驗法及/或最佳化可將1變町”i更。也需瞭解的是，於此敘述中用之專門用語僅县兔7 &丄疋為了敘述特別之變化形式或實施例，且不傾向限制僅會受限於限於所附上之申請專利範圍的本發明範圍。於本說明書φ担Hi ^及之所有刊物、專利或專利申請於此以其内容被具體弓丨入為i去—虹 1八马參考文獻。然而，於此並沒有被解釋為承認本發明由^^ 1 ± 由於先則發明之效力不被給予先於這些揭露之權力。 I.定義除非特別定義’於此使用屬與本發明之所有技術或科學用語為與熟悉此技藝人士所通常瞭解之意義相同。然而，若發生抵觸，本發明說明書，包括定義將會控制。於此使用之單字“一 ”與“該”意指“至少一，，除非以別的方式明確指出。於此可替換使用之用語“多胜肽”、“胜肽，，與‘‘蛋白質意指胺基酸殘基之一聚合物。此用語適用於胺基酸聚合物’於其中一或多個胺基酸殘基可為經修飾之殘基或非自然發生之殘基’例如對應自然發生胺基酸之人工化學模仿物’與自然發生胺基酸聚合物。於此使用之用語“胺基酸”意指自然發生與合成之胺基酸，及胺基酸類似物與胺基酸模仿物，其與自然發生之 16 201134480 胺基酸起相似作用。胺基酸可為L-胺基酸或D-胺基酸。自然發生胺基酸為基因密碼所編碼的那些與於細胞中在轉譯後被修飾的那些（例如經脯胺酸（hydroxyproΠne)、τ -羧基谷胺酸（gamma-carboxyglutamate)與0-磷絲胺酸 (Ο-phosphoserine))。措辭“胺基酸類似物”意指具有與自然發生胺基酸相同之基礎化學結構（一 <2碳鍵結至一氫、一羧基、一胺基與一 R基）的化合物，但具有一或多個經修飾之R基或經修飾之骨架（例如，同絲胺酸 (homoserine)、降亮胺酸（norieucine)、甲硫胺酸 (methionine)、亞砜（sulf〇xide)、甲基硫氨磺（methi〇nine methylsulfonium))。措辭“胺基酸模仿物，’意指化學化合物其與一般胺基酸具有不同結構，但有相似的功能。可藉由由 IUPAC-IUB Biochemical N〇menclature Commission所建議之其一般所知的三字母符號或一字母符號來指出於此處之胺基酸。於此可替換使用用語“基因”、“多核苷酸”、“核苷酸”與“核酸，’，且除非以別的特別：¾式指ώ，相似於胺基s复其以它們一般被接受的單一字母編碼來指出。此處使用之用語“組合物，’意指包括一產物，其包括於特疋里中特定成分’與任何產物其直接或間接來自於特定量之特定成分的組合。此用語肖“藥學組合物，，相關，意指包括m包括—活性成分與形成載體的惰性成刀’及任何產物其直接或間接來自任兩個或多個成份之組合、複合或聚集’或來自一或多個成分之解離或來自— 17 201134480 或多個成分之反應或相互作用的复 ’、升^ 式0 因 Ή Jr StS- 明内容中，用語“藥學組合物，，音匕，在本發物盘筚學上戍生理上可接… 藉由混合本發明化合视一樂子上及玍理上了接受之載體所如此處所使用之措辭“藥學上、何組合物。予上』接文之載體” 可接受之載體”意指藥學上或生理 4生理上铷铷哲々# 丨Λ,, 接又之材料 '組合物、物質或載劑，包括，但不限於一固體埴右、稀釋劑、賦形劑、溶劑或套膜材料，1 具兄 /、，、自一器官或身體之一部分攜帶或運輸受支配支〆又朱樂效團（scaffolded polypharmacophores)至另-器官或 | ㈡吕次身體之—部分相關。除非以別的方式定義，用語‘‘癌 n 意指過度表現 CLUAPI基因之癌症或腫瘤，其例子田六妁卞巴栝，但不限於乳癌、子宮頸癌、大陽直陽癌、食道癌 '胃I 、猫成、 $逕屈胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、姨臟癌。除非以別的方式定義，於此可替換使用且以別的方式特別指出用語“細胞毒殺性τ 淋巴球、“細胞毒殺性Τ細胞，’肖“CTL”以意指τ淋巴球之次族群，且除非以別的方式指出，意指τ淋巴球之次群組（SUb-group)可辨認非自身細胞（例如，腫瘤/癌症細 I被病毒感染之細胞），且誘導這些細胞死亡。除非特別疋義，用5吾HLA-A24”意指包含次型，例如HLA_A* 2402 之HLA-A24。除非特別定義，用語“HU_A2，，如此處所使用代表性意指次型，例如HLA-A*0201與HLA- A*0206。除非特別定義’於此使用之用語“套組，，被使用於關於4劑與其他材料之組合。與此考慮之套組包括微陣列、晶片、標誌等《並無打算使用語“套組，，限制於試劑及/ 18 201134480 或材料之特定組合。本發明之方法與組合物之範圍提供用途於癌症之治療之内容中，一治療被視為“有效”，若其導致臨床優點，例如於CLUAP1基因之表現中的減少、或於個體中癌症之大小、普遍程度（prevalence)4轉移潛力的減少。當治療為預防性（pr〇phylacticaUy)提供時， “有效”意、指減緩或避免癌症形成，或避免或減輕癌症之臨床症狀。有效性被確認於相關之診斷或治療特定腔瘤形式的任何已知方法。本發明之方法與組合物之範圍提供用途於癌症之“避免/、預防之内谷中，此類用詞為與此交替使用意指任何活性，其減少死亡率之負載或來自疾病之死亡率。避免與預防可發生於“初期、第二期與第三期避免層級”。初期避免與預防避免了疾病之發展，@第二期與第三期層級之避免與預防包括藉由恢復功能與減少疾病相關併發症，以疾病之發展與症狀之浮現及減少已建立之疾病之負向發展的避免與預防為目的。由分 ^或者，治療或避免可包括一廣砣圍之預防疾病治療，其 ^冉以減緩特別疾病之嚴重度為目標，例如減少腫瘤之增殖與轉移。在本發明内容中，癌症 ^ ^ 〈化療及/或預防，或，及/岑其手術後復發的避免包括任何 ^ 下列步驟，例如癌細胞之手術移除、似癌細胞之生長抑制、 .^ w疋哀退或退化、癌絡生之減緩與抑制的誘導、Μ 生,“ 腫•退化與與轉移之減少盘抑制。癌症之有效治療及/戋 ’、抑之個體的預後、減低癌症標 …有癌症 °己於血液中的程度與減緩伴隨 19 201134480 著癌症之可偵測症狀。例如，症狀之減輕或改善構成有效治療及/或預防，其⑽1Q%、2()%、3()%或更加減輕，或穩定疾病。在本發明内容中，用語“抗體，，意指免疫球蛋白與其片段，其專一與選定蛋白質或其片段反應。一抗體可包括人類抗體、靈長類抗體、嵌合抗體（chimeric antib〇dy)、雙專一抗體（bispecific antibody)、人源化抗體、與其他蛋白質或放射標誌融合之抗體，與抗體片段。此外，此處之抗體被使用於最大效用且特別包含完整單株抗體、多株抗體、形成自至少兩個完整抗體之多專一抗體 (multispecific antibody)(例如雙專一抗體）與抗體片段，只要其存在所需生物活性。“抗體”意指所有之種類（例如，IgA、IgD、IgE、IgG 與 IgM)。除非特別疋義，於此使用屬與本發明之所有技術或科學用語為與熟悉此技藝人士所通常瞭解之意義相同。 11.胜肽為了證明來自CLUAPI之胜肽作用如一被細胞毒殺性τ 淋巴球（CTLs)所辨認之抗原，分析來自CLUAP1之胜肽（序列辨識號：45 )以確定是否其為由一般遇到jjLA對偶基因 (&116 16)之111^(人類白血球組織抗原）-入24或八2所限制之抗原決定位（Date Y et al·，Tissue Antigens 47: 93-101, 1996; Kondo A et al. , J Immunol 155: 4307-12, 1 995; Kubo RT et al·，J Immunol 1 52: 391 3-24, 1 994)。 20 201134480 確認來自CLUAP1之HLA-A24結合胜肽的候選物，根據其對HLA-A24之結合親和力。候選胜肽被認為是下列胜肽： CLUAP1-A24-9-244 (序列辨識號：2)、 CLUAP1-A24-9-255 (序列辨識號：3)、 CLUAP1-A24-9-283 (序列辨識號：4)、 CLUAP1-A24-9-31 (序列辨識號·· 5)、 CLUAP1 -A24-9-8 (序列辨識號：6)、 CLUAP1-A24-9-26 (序列辨識號：7)、 CLUAP 1 -A24-9-1 68 (序列辨識號：8)、 CLUAP1-A24-9-64 (序列辨識號：9)、 CLUAP 1-A24-9-216 (序列辨識號：10)， CLUAP1-A24-9-335 (序列辨識號：11)、 CLUAP1-A24-9-86 (序列辨識號：12)、 CLUAP1-A24-9-71 (序列辨識號：13)、 CLUAP1-A24-9-154 (序列辨識號：14)、 CLUAP1-A24-10-91 (序列辨識號：15)、 CLUAP1-A24-1 0-1 04 (序列辨識號：16)、 CLUAP1-A24-10-31 (序列辨識號：17)、 CLUAP1-A24-10-125 (序列辨識號：18)、 CLUAP卜A24-10-17 (序列辨識號：19)、 CLUAP1-A24-1 0-289 (序列辨識號：20)、 CLUAP1-A24-10-258 (序列辨識號：21)與 CLUAP1-A24-1 0-63 (序列辨識號：22) ° 21 201134480 此外’ //7 F/ 藉由以這些胜肽脈衝（載有）之樹犬細胞（dendr i t i c ce 11, DC)刺激Τ細胞後，使用下列胜狀之各個成功建立細胞毒殺性T淋巴球： CLUAP1-A24-9-151-255 (序列辨識號：3)、 CLUAP1-A24-9-1 52-283 (序列辨識號：4)、 CLUAP卜A24-9-1 53-31 (序列辨識號：5)、 CLUAP1-A24-9-156-168 (序列辨識號：8)、 CLUAP卜A24-9-157-64 C 序列辨識號：9)、 CLUAP1-A24-9-158-216 (序列辨識號：10)、 CLUAP卜A24-9-163-154 (序列辨識號：14)、 CLUAP1-A24-1 0-166-91 (序列辨識號：15)、 CLUAP卜A24-10-167-104 (序列辨識號：16)與 CLUAP卜A24-1 0-1 69-125 (序列辨識號：18) 確認來自CLUAP1之HLA-A2結合胜肽的候選物’根據其對HLA-A2之結合親和力。候選胜肽被認為是下列胜狀： CLUAP1-A2-9-34 (序列辨識號： 23)、 CLUAP1-A2-9-95 (序列辨識说· 25)、 CLUAP1-A2-9-85 (序列辨識號： 26)、 CLUAP1-A2-9-290 (序列辨識號 :27)、 CLUAP1-A2-9-265 (序列辨識號 :28)、 CLUAP1-A2-9-297 (序列辨識號 :29)、 CLUAP1-A2-9-99 (序列辨識號： 30)、 CLUAP1-A2-9-188 (序列辨識號 :31)、 22 201134480 CLUAP1-A2-9-96 (序列辨識號：32)、 CLUAP1-A2-10-153 (序列辨識號 :33)、 CLUAP1-A2-10-85 (序列辨識號： 34)、 CLUAP1-A2-10-34 (序列辨識號： 35)、 CLUAP1-A2-10-33 (序列辨識號： 36) ' CLUAP1-A2-10-72 (序列辨識號： 37)、 CLUAP1-A2-10-80 (序列辨識號： 38) ' CLUAP1-A2-10-41 (序列辨識號： 39)、 CLUAP1-A2-10-233 (序列辨識號 :40)、 CLUAP1-A2-10-183 (序列辨識號：41)、 CLUAP1-A2-10-343 (序列辨識號 :42)與 CLUAP1-A2-10-222 (序列辨識號 :43) 之樹突列脞肽此外，//? f/ 藉由以這些胜肽脈衝（載有）細胞（dendritic cel 1，DC)刺激Τ細胞後，使用下之各個成功建立細胞毒殺性T淋巴球： CIjUAP1~A2~9-34 (序列辨識號：23)、 aUAPl~A2-9-99 (序列辨識號：30)、 CLlMPl-A2 —1〇_153 (序列辨識號：33)、 CLUAP卜A2-10 — 85 (序列辨識號：34)、 CLUAPi-A2~i〇 —34 (序列辨識號：35)、 CLUAPi~A2-i〇 —33 (序列辨識號：36)與 CLUAPl-A2~l〇-8〇 (序列辨識號：38) 23 201134480 這些被建立的細胞毒殺性τ淋巴球顯示強而專一之抗經分別之胜狀脈衝之目標細胞的細胞毒殺性τ淋巴球活性。此處這些結果證明CLUAP1為一由細胞毒殺性τ淋巴球所辨認之抗原，且被測試之胜肽為由HLA_A24或HLA_A2限制之CLUAP1的抗原決定位胜肽。由於CLUAP1基因於癌症細胞與組織，包括，但不限於例如乳癌、子宮頸癌。、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌的那些之中被過度表現，且不在大部分正常器官中，所以其代表一良好之免疫治療標的。因此，本發明提供對應於來自CLUApi之細胞毒殺性T淋巴球辨認之抗原決定位的九胜肽（胜肽由九個胺基酸殘基所組成）與十胜肽（胜肽由十個胺基酸殘基所組成）。或者，本發明提供經分離之胜肽，其與HLA抗原結合且誘導細胞毒殺性T淋巴球活性，其中胜肽具有序列辨識號：45之胺基酸序列或為一其免疫活性片段。特別是，本發明九胜肽與十胜肽之較佳實施例包括具有擇自於序列辨識號：3、4、5、8、9、10、U、15、16、18、23、 30、33、34、35、36與38中之胺基酸序列的那些胜肽。通常可使用現今於例如網路可得之軟體程式’例如於 Parker KC et al. , J Immunol 1 994 Jan 1, 152(1)： 163-75 與 Nielsen M et al.’ Pr〇tein Sci 2003; 12: 1007-17Ishikura H et al., Int J Oncol. 2007 Feb; 30(2): 461-7 SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery of novel peptides that are useful as immunotherapeutics. Since tumor-associated antigens (TAAs) are sometimes perceived by the immune system as "self" and therefore often do not have immunogenicity (i mmunogen i c i ty ), the discovery of suitable targets is extremely important. As mentioned above, it has been confirmed that CLUAP1 (sequence identification number encoded by GenBank's gene number 0_015041 or off _024793 (eg, sequence number: 44): 45) includes, but is not limited to, breast cancer. , cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastric di f fuse-type cancer, lymphoma, lymph In cancers of neuroblastoma and pancreatic cancer, it is up-regulated. Thus, the present invention focuses on CLUAP1 as a candidate for cancer/tumor immunotherapy. The present invention further relates to the confirmation of specific epitope determinants of the gene product of CLUAP1 which induces the ability to specifically bind to the cytotoxic tau lymphocytes of CLUAP1. As discussed in detail below, a candidate peptide derived from CLUAP1 in combination with HLA (Human Leukocyte Tissue Antigen)-A* 2402 or HLA-A* 0201 is used to stimulate peripheral blood mononuclear cells obtained from a health provider 201134480 (peripheral blood) Mononuclear cells, PBMCs). A cytotoxic T lymphocyte is then established which has specific cytotoxicity against HLA-A24 or HLA-A2 positive target cells that are pul sed by each candidate peptide. These results demonstrate that these peptides are HLA-A24 or HLA-A2 restricted epitope peptides that induce a strong and specific immune response against cells expressing CLUAP1. Furthermore, the results indicate that CLUAPI is potently immunogenic and its epitope is an effective target for cancer/tumor immunotherapy. Thus, it is an object of the present invention to provide isolated peptides that bind to HLA antigens, particularly those comprising CLUAP1 (SEQ ID NO: 45) or immunologically active fragments thereof. These peptides are expected to have cytotoxic T lymphocyte evokengs and can therefore be used to induce cytotoxic T lymphocytes or to be administered to a body to induce anti-cancer 'eg breast cancer, cervical cancer, colorectal cancer, Immune response in esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, and pancreatic cancer. Preferred peptides are the Nine Peptides or Decoxers' and more preferably those having the amino acid sequence selected from the sequence identification numbers: 2 to 23 and 25 to 43. Among these, there are selected sequence identification numbers ··· 3, 4, 5, 8, 9, and 1. The peptides of the amino acid sequences of 14, 15, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 show strong cytotoxic sputum-inducing ability and are therefore particularly preferred. The peptide of the present invention includes those in which one, two or more amino acids are substituted, deleted or added as long as the resulting modified peptide maintains the initial virulence lymphocyte inducing ability. The invention also provides isolated multinuclei (IV) encoding any of the peptides of the invention. These polynucleic acid can be used to induce or injure an antigen having a cytotoxicity, a stimulating ability of a tau lymphocyte, or a peptide of the present invention, or a peptide which can be administered to a body to induce an anti-cancer immune response. When a subject is administered, the peptide of the present invention is preferably expressed on the surface of the antigen-presenting cell to induce a cytotoxic thymocyte which targets the respective peptide as a target. Accordingly, it is an object of the present invention to provide compositions or materials which induce cytotoxic tau lymphocytes, such compositions comprising one or more of the peptides of the invention or polynucleotides encoding such peptides. The present invention further contemplates pharmaceutical compositions comprising one or more of the peptides of the invention or polynucleotides encoding such peptides. This composition is formulated for the treatment and/or prevention of cancer, and the avoidance of recurrence after surgery 'This type of cancer includes, but is not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. In addition to or in place of the peptide or polynucleotide of the present invention, the pharmaceutical composition or substance of the present invention may comprise an antigen presenting cell or an exosome which exhibits any of the peptides of the present invention as an active ingredient. The peptide and polynucleotide of the present invention can be induced into an antigen-presenting cell whose surface exhibits a complex of the HLa antigen and the peptide of the present invention, for example, by contacting or displaying an antigen-presenting cell from a body with the peptide. The polynucleic acid which is a peptide of the present invention is introduced into an antigen-presenting cell. Such antigens exhibit the ability of the cells to challenge the target-like cytotoxic thymocyte-inducing ability and thus provide utility in cancer immunotherapy. Accordingly, the present invention contemplates both a method of inducing an antigen-presenting cell having a cytotoxic tau lymphocyte-inducing ability and an antigen-presenting cell obtained by the method. The invention also provides a method for inducing cytotoxic T lymphocytes, and the method 201134480 comprises the steps of co-cultivating CD8-positive cells with an antigen-presenting cell or exosome expressing one or more peptides of the invention on the surface thereof, or introducing A step of encoding a gene encoding a polynucleotide of a T cell receptor (T ceu recept QF, TCR) subunit polypeptide in combination with a peptide of the present invention. The cytotoxic T lymphocytes obtained by such methods can be used for the treatment and avoidance of cancer, and examples of cancer include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer. , lymphoma, neuroblastoma, pancreatic cancer. Accordingly, the invention includes a cytotoxic a lymphocyte obtained by the method of the invention. Another object of the present invention is to provide a method for inducing an immune response against cancer in a subject in need thereof, the method comprising administering a polynucleotide comprising a CLUApi polypeptide or an immunologically active fragment thereof, encoding a CLUAP1 polypeptide, And the step of presenting a composition or substance of the CLUAP1 multi-peptide exosome or antigen-presenting cells to the individual. The applicability of the present invention extends to any of the diseases relating to or caused by overexpression of CLUAP1, such as cancer, and exemplary cancer includes, but is not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse type. Gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. More specifically, the present invention provides the following: [1] An isolated peptide having cytotoxic tau lymphocyte-inducing ability, wherein the peptide consists of an amino acid sequence of CLUAP1 or an immunologically active fragment thereof. . [2] The isolated peptide of [1], wherein the peptide comprises an amino acid sequence, which is a group of free radical identification numbers: 2, 23 and 25 to 43 of the 201134480 group. [3] The hungry of the above-mentioned [1] and [2], wherein the 1, 2 or a plurality of amino acids are substituted, deleted or added. [4] [3] The isolated peptide, wherein Lida A has the following characteristics in the context of human leukocyte tissue antigen-Α24—戋 (a) the second amine from the terminal An acid in a lane is modified to be an amino acid, which is selected from the group consisting of free amphetamine, tyrosine, a beta thiobutyric acid or tryptophan; and (8) a C-terminal amino acid. Or, as a group of amino acids, it is a group consisting of free phenylalanine, leucine, #leucine, tryptophan or methionine. [5] [3] The isolated peptide 'wherein the human leukocyte tissue antigen-Α2 background, the peptide has the following characteristics - or both: (a) the second amino acid from the terminal is A group consisting of free leucine and methionine; and (b) a C-terminal amino acid is a group consisting of free proline and leucine. [6] The isolated peptide according to any one of [1] to [5] wherein the winning form is a nine-win or ten-win. [7] An isolated polynucleotide encoding the peptide of any one of [丨] to [6]. [8] A composition for inducing a cytotoxic lymphosphere, wherein the composition comprises any one of [1] to [6], or a plurality of the peptide, or [7] One or more of the polynucleotides. 12 201134480 [9] A pharmaceutical composition for use in the treatment and/or prevention of cancer, and/or its prevention of recurrence after surgery, wherein the composition comprises one of [1] to [6] Or a plurality of the peptide, or the one or more of the polynucleotides. The pharmaceutical composition of [1 0 ] [9] is formulated for administration to a human, and the human leukocyte tissue antigen is human leukocyte antigen-A24 or human leukocyte antigen-A2. [11] The pharmaceutical composition of [9] or [10], wherein the composition is formulated for the treatment of cancer. [1 2 ] A method for inducing an antigen-presenting cell having a cytotoxic lymphotrophy-inducing ability, wherein the method comprises a step of selecting a group consisting of: (a) pyiro, or or An antigen-presenting cell is contacted with the peptide of any one of [1] to [6]; and (b) one of the peptides of any one of [1] to [6] The polynucleotide is introduced into an antigen-presenting cell. [13] A method for inducing a cytotoxic thymocyte, by a method comprising the step of selecting a group consisting of: (a) a CD8-positive T cell Co-culture with antigen-presenting cells, the antigen-presenting cells exhibit a complex of the human leukocyte tissue antigen and the peptide described in any one of [1] to [6] on the surface thereof; (b) CD 8-positive T The cell is co-cultured with the exosome, and the outer 11 soil body exhibits a human leukocyte tissue antigen and a complex of the peptide described in any one of [丨] to [6] on its surface; and 13 201134480 ( C) introducing a gene comprising a polynucleic acid encoding a T cell receptor subunit The tau cell receptor subunit is more than one of the peptides described in any one of [1] to [6]. [14] An isolated antigen-presenting cell which expresses a human leukocyte antigen The complex of the peptide described in any one of [1] to [6] on the surface thereof. [15] The antigen of [14] [14] presents a cell which is induced by the method of [12]. 6] an isolated cytotoxic tau lymphocyte, which is characterized by the peptide described in any one of [丨] to [6]. [1 7 ] [ 1 6 ] cytotoxic τ lymph A sphere, which is induced by the method of [丨3]. [18] A method of inducing an immune response against a cancer in a body, wherein the method comprises administering to the individual a composition comprising the composition [ 1) The peptide according to any one of [6], an immunologically active fragment or a polynucleotide encoding the peptide or the fragment. [19] An antibody or a fragment thereof, which is resistant to [丨] Any one of the winners described in the item [6]. [2〇] A carrier comprising a nucleoside encoding the peptide of any one of [n to [6] Acid sequence. [21] A cell, which is transformed or transfected with one of the expression vectors of [2]. [22] A diagnostic kit comprising the peptide of any one of [丨] to [6], [7] The nucleotide or the antibody of [19]. 14 201134480 [23] The isolated peptide of any one of [1] to [6], which is selected as a free sequence identification number: 3, 4, 5, 8 , 9' 10, 14, 15, 16, 18, 23, 30, 33 '34, 35, 36 and 38 group. It is to be understood that the invention is not intended to be limited to the details of the present invention. Other objects and features of the present invention will become more fully apparent from the description of the appended claims. However, it is to be understood that the invention is not to be construed as limiting the invention or the invention. In particular, the invention as described herein with respect to the specific embodiments thereof is to be understood as illustrative and not restrictive. Various modifications and applications can be made by those skilled in the art without departing from the spirit and scope of the invention, as described in the scope of the claims. As such, other aspects of the invention will be readily apparent to those skilled in the art. Such combinations of the above are accompanied by the examples, the materials, and the references which are incorporated herein by reference. The purpose, the features, the advantages and the advantages of the present invention are obvious, and it is clear that all the reasonable inferences from the schema and all the descriptions are to be Implementation of the inventive embodiment ^ λ ^ , ° fly dry can use any similar or similar to those described herein to find the material, but now describes the method, component, and 〃 ° ' In the description of the materials and methods of the present invention, it is to be understood that the present invention is not limited to the particular size, shape, size, materials, methodology, steps, etc. described herein, such as in conventional practice and / or optimization can be changed to 1 change. I also need to understand that the terminology used in this description is only the county rabbit 7 & 丄疋 in order to describe special variations or examples, and does not tend to limit The scope of the present invention is limited only by the scope of the appended claims. All publications, patents, or patent applications of this specification are hereby incorporated herein in However, this is not to be construed as an admission that the invention is not limited by the scope of the invention. All technical or scientific terms used in the present invention are the same as those commonly understood by those skilled in the art. However, in the event of a conflict, the description of the present invention, including definitions, will be controlled. The words "a" and "the" "meaning" at least one, unless otherwise explicitly stated. As used herein, the terms "polypeptide", "peptide," and "protein" mean a polymer of one of the amino acid residues. This term applies to amino acid polymers in one or more of them. The amino acid residue can be a modified residue or a non-naturally occurring residue 'for example, an artificial chemical mimetic corresponding to a naturally occurring amino acid' and a naturally occurring amino acid polymer. The term "amine group" is used herein. "Acid" means a naturally occurring and synthetic amino acid, and an amino acid analog and an amino acid mimetic, which act similarly to naturally occurring 16 201134480 amino acid. The amino acid may be an L-amino acid. Or D-amino acids. Naturally occurring amino acids are those encoded by the genetic code and those which are modified in the cell after translation (for example, hydroxyproΠne, gamma-carboxyglutamate) And 0-phosphonine. The word "amino acid analog" means having the same basic chemical structure as a naturally occurring amino acid (a <2 carbon bond to a hydrogen, a a compound of a carboxyl group, an amine group and an R group), but having one or a plurality of modified R groups or modified backbones (eg, homoserine, norieucine, methionine, sulfoxide xide, methyl sulphide) Meth 〇nine methylsulfonium. The word "amino acid mimetic," means a chemical compound that has a different structure than a general amino acid but has a similar function. The amino acid herein may be indicated by a three-letter symbol or one-letter symbol as generally known by the IUPAC-IUB Biochemical N〇menclature Commission. The terms "gene", "polynucleotide", "nucleotide" and "nucleic acid," are used interchangeably herein, and unless otherwise specified: the formula is similar to the amine group, which is generally The phrase "composition," as used herein, is meant to include a product comprising a combination of a particular ingredient in a particular product, and any product, directly or indirectly, from a particular amount of a particular component. . The phrase "pharmaceutical composition," is meant to include m including - an inert ingredient forming the active ingredient and forming a carrier, and any product which is directly or indirectly derived from a combination, combination or aggregation of two or more components' or The dissociation from one or more components or from the complex reaction or interaction of - 2011, 2011,344 or multiple components, 0 Ή Ή Ή r Jr StS- ming content, the phrase "pharmaceutical composition, 匕, in The present invention is physiologically achievable by the combination of the present invention and the accepted carrier, as used herein, the phrase "pharmaceutical, what composition." The carrier "acceptable carrier" means pharmaceutically or physiologically physiologically 铷铷铷铷丨Λ , , , , , , , , , , , , , , , 材料材料材料 ' ' ' ' ' ' ' ' ' ' ' ' ' ' 稀释稀释稀释稀释稀释稀释稀释Agent, excipient, solvent or mantle material, 1 brother/,, from one organ or part of the body carrying or transporting the dominating branch and scaffolded polypharmacophores to another organ or | (2) Lvji body It Partially relevant. Unless otherwise defined, the term ''cancer n' refers to a cancer or tumor that overexpresses the CLUAPI gene, examples of which are not limited to breast cancer, cervical cancer, and straight Yang cancer. Esophageal cancer 'stomach I, cat, squamous gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, sputum cancer. Unless otherwise defined, it can be used interchangeably and otherwise specifically stated. "Cytotoxic τ lymphocytes, "cytotoxic sputum cells, 'shaw" CTL" means the subpopulation of τ lymphocytes, and unless otherwise indicated, means subgroups of τ lymphocytes (SUb- Group) identifiable non-self cells (eg, tumor/cancer fine I infected by virus) and induces death of these cells. Unless specifically derogatory, the use of 5 HLA-A24" means subtypes, such as HLA_A* 2402 HLA-A24. Unless specifically defined, the term "HU_A2," as used herein, refers to subtypes, such as HLA-A*0201 and HLA-A*0206. Unless specifically defined as the term "set used herein", it is used in relation to Combination of 4 doses with other materials. Kits contemplated herein include microarrays, wafers, logos, etc., "there is no intended use", and are limited to reagents and / 18 201134480 or specific combinations of materials. The methods and compositions of the present invention provide for use. In the treatment of cancer, a treatment is considered "effective" if it leads to clinical advantages, such as a decrease in the expression of the CLUAP1 gene, or a decrease in the size, prevalence, and metastasis potential of the cancer in the individual. When the treatment is provided as prophylactic (pr〇phylacticaUy), "effective" means slowing or avoiding the formation of cancer, or avoiding or alleviating the clinical symptoms of cancer. The effectiveness is confirmed in the relevant diagnosis or treatment of a specific form of the tumor. Any known method. The scope of the methods and compositions of the present invention provides for use in the "avoidance/prevention" of cancer, and such terms are used interchangeably to mean any activity that reduces the burden of mortality or comes from Mortality of the disease. Avoidance and prevention can occur in the “initial, second and third phase avoidance levels”. Early avoidance and prevention avoids the development of the disease. The avoidance and prevention of the second and third stages includes the restoration of function and the reduction of disease-related complications, the development of the disease and the emergence of symptoms and the reduction of established diseases. The purpose of avoiding and preventing negative development is to achieve. Treatment or avoidance may include a wide-ranging prevention of disease treatment, which aims to reduce the severity of a particular disease, such as reducing tumor proliferation and metastasis. In the context of the present invention, cancer avoidance and/or prevention, and/or prevention of recurrence after surgery include any of the following steps, such as surgical removal of cancer cells, growth inhibition of cancer cells, . w疋疋退退退退退退退退退退退退疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋疋...there is a cancer that has been in the blood and slows down with the detectable symptoms of cancer. For example, the reduction or improvement of symptoms constitutes effective treatment and/or prevention, (10) 1Q%, 2()%, 3() % or more alleviate, or stabilize the disease. In the context of the present invention, the term "antibody," means an immunoglobulin and a fragment thereof, which specifically react with a selected protein or fragment thereof. An antibody can include a human antibody, a primate antibody, a chimeric antibody, a bispecific antibody, a humanized antibody, an antibody fused to other proteins or a radiolabel, and an antibody fragment. Furthermore, the antibodies herein are used for maximum utility and in particular comprise intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and antibody fragments formed from at least two intact antibodies, as long as they There is a desired biological activity. "Antibody" means all species (e.g., IgA, IgD, IgE, IgG, and IgM). Unless otherwise stated, all technical or scientific terms used herein are the same as commonly understood by those skilled in the art. 11. peptides In order to demonstrate the peptide action from CLUAPI, such as an antigen recognized by cytotoxic thymic lymphocytes (CTLs), the peptide derived from CLUAP1 (SEQ ID NO: 45) was analyzed to determine whether it was encountered by The jjLA dual gene (&116 16) 111^ (human leukocyte antigen)-into 24 or VIII restricted antigenic epitopes (Date Y et al., Tissue Antigens 47: 93-101, 1996; Kondo A et Al., J Immunol 155: 4307-12, 1 995; Kubo RT et al., J Immunol 1 52: 391 3-24, 1 994). 20 201134480 A candidate for HLA-A24 binding peptide from CLUAP1 was identified based on its binding affinity for HLA-A24. Candidate peptides are considered to be the following peptides: CLUAP1-A24-9-244 (SEQ ID NO: 2), CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-283 (Sequence Identification No.: 4), CLUAP1-A24-9-31 (sequence identification number·· 5), CLUAP1 -A24-9-8 (sequence identification number: 6), CLUAP1-A24-9-26 (sequence identification number: 7) , CLUAP 1 -A24-9-1 68 (sequence identification number: 8), CLUAP1-A24-9-64 (sequence identification number: 9), CLUAP 1-A24-9-216 (sequence identification number: 10), CLUAP1 -A24-9-335 (sequence identification number: 11), CLUAP1-A24-9-86 (sequence identification number: 12), CLUAP1-A24-9-71 (sequence identification number: 13), CLUAP1-A24-9- 154 (sequence identification number: 14), CLUAP1-A24-10-91 (sequence identification number: 15), CLUAP1-A24-1 0-1 04 (sequence identification number: 16), CLUAP1-A24-10-31 (sequence Identification number: 17), CLUAP1-A24-10-125 (sequence identification number: 18), CLUAP A24-10-17 (sequence identification number: 19), CLUAP1-A24-1 0-289 (sequence identification number: 20) ), CLUAP1-A24-10-258 (sequence identification number: 21) and CLUAP1-A24-1 0-63 (sequence identification number: 22) ° 21 201134480 In addition ' //7 F/ by these peptides After the dog cells (dendr itic ce 11, DC) stimulated the sputum cells, the cytotoxic T lymphocytes were successfully established using each of the following traits: CLUAP1-A24-9-151-255 (SEQ ID NO: 3), CLUAP1-A24-9-1 52-283 (sequence identification number: 4), CLUAP A24-9-1 53-31 (sequence identification number: 5), CLUAP1-A24-9-156-168 (sequence Identification number: 8), CLUAP A24-9-157-64 C Sequence identification number: 9), CLUAP1-A24-9-158-216 (sequence identification number: 10), CLUAP A24-9-163-154 ( Sequence identification number: 14), CLUAP1-A24-1 0-166-91 (sequence identification number: 15), CLUAP A24-10-167-104 (sequence identification number: 16) and CLUAP A24-1 0-1 69-125 (SEQ ID NO: 18) Confirmation of candidate for HLA-A2 binding peptide from CLUAP1 'based on its binding affinity for HLA-A2. Candidate peptides are considered to be the following: CLUAP1-A2-9-34 (SEQ ID NO: 23), CLUAP1-A2-9-95 (Sequence Identification 25), CLUAP1-A2-9-85 (Sequence Identification) No.: 26), CLUAP1-A2-9-290 (sequence identification number: 27), CLUAP1-A2-9-265 (sequence identification number: 28), CLUAP1-A2-9-297 (sequence identification number: 29), CLUAP1-A2-9-99 (sequence identification number: 30), CLUAP1-A2-9-188 (sequence identification number: 31), 22 201134480 CLUAP1-A2-9-96 (sequence identification number: 32), CLUAP1-A2 -10-153 (sequence identification number: 33), CLUAP1-A2-10-85 (sequence identification number: 34), CLUAP1-A2-10-34 (sequence identification number: 35), CLUAP1-A2-10-33 ( Serial identification number: 36) ' CLUAP1-A2-10-72 (sequence identification number: 37), CLUAP1-A2-10-80 (sequence identification number: 38) ' CLUAP1-A2-10-41 (sequence identification number: 39 ), CLUAP1-A2-10-233 (sequence identification number: 40), CLUAP1-A2-10-183 (sequence identification number: 41), CLUAP1-A2-10-343 (sequence identification number: 42) and CLUAP1-A2 -10-222 (SEQ ID NO: 43) of the dendritic peptides, in addition, / / f / by pulsed (loaded) cells with these peptides (dendriti c cel 1,DC) After stimulating sputum cells, the cytotoxic T lymphocytes were successfully established using each of the following: CIjUAP1~A2~9-34 (sequence identification number: 23), aUAP1~A2-9-99 (sequence identification number) :30), CLlMPl-A2 —1〇_153 (sequence identification number: 33), CLUAP A2-10 — 85 (sequence identification number: 34), CLUAPi-A2~i〇—34 (sequence identification number: 35) , CLUAPi~A2-i〇—33 (sequence identification number: 36) and CLUAPl-A2~l〇-8〇 (sequence identification number: 38) 23 201134480 These established cytotoxic τ lymphocytes show strong and specific The cytotoxic thymocyte activity of target cells against the spur pulse. Here, these results demonstrate that CLUAP1 is an antigen recognized by cytotoxic tau lymphocytes, and the peptide to be tested is an epitope of CLUAP1 restricted by HLA_A24 or HLA_A2. Since the CLUAP1 gene is involved in cancer cells and tissues, it includes, but is not limited to, breast cancer, cervical cancer, for example. It is overexpressed in colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, pancreatic cancer, and is not in most normal organs, so it represents a good immunotherapy target. . Accordingly, the present invention provides a nine-peptide (the peptide consists of nine amino acid residues) and a ten-peptide (corresponding to ten amines) corresponding to the epitope of the cytotoxic T lymphocyte recognition from CLUApi. The base acid residue is composed of). Alternatively, the invention provides an isolated peptide that binds to an HLA antigen and induces cytotoxic T lymphocyte activity, wherein the peptide has the amino acid sequence of sequence identification number: 45 or an immunologically active fragment thereof. In particular, preferred embodiments of the nine peptides and ten peptides of the present invention include those having the sequence identification numbers: 3, 4, 5, 8, 9, 10, U, 15, 16, 18, 23, 30, Those peptides of the amino acid sequences of 33, 34, 35, 36 and 38. Software programs available today, for example, over the Internet can be used, for example, Parker KC et al., J Immunol 1 994 Jan 1, 152(1): 163-75 and Nielsen M et al. 'Prötein Sci 2003; 12: 1007-17

中所敘述的那些，來計算//7 介於各種胜肽與HLA 抗原間之結合親和力。例如，如於概述於，例如，Ufuente EM et al., Current Pharmaceutical Design, 2009, 15, 24 201134480 3209-3220 中之 parker KC et al.，J Immunol 1 994 Jan 1， 1 52( 1 ): 1 63-75, Kuzushima K et a 1. , Blood 2001, 98(6): 1872-81, Larsen MV et a 1. BMC Bioinformatics. 2007 Oct 31; 8: 424，Buus Set al. Tissue Antigens., 62:378-84, 2003, Nielsen M et al., Protein Sci 2003; 1 2: 1 007-1 7 與 Nielsen M et al. PLoS ONE 2007; 2: e796 中所述可測里與HLA抗原之結合親和力。測量親和力之方法欽述於，例如於 Journal of Immunological Methods, 1 995, 1 85: 18卜190 與 Protein Science， 2000， 9: 1838-1846 中。所以使用此種軟體程式可選擇來自CLUAP1的片段，其具有與HLA抗原之南結合親和力。因此藉由此類已知程式本發明包括由來自CLUAP1之任何片段所組成之胜肽，其與hla 結合。此外’此類胜肽可包括CLUAP1之全長胜肽。本發明之九胜肽與十胜肽可於側面具有額外之胺基酸殘基，只要所產生之胜肽維持它們的細胞毒殺性T淋巴球誘發能力。額外之胺基酸殘基可由任何種類之胺基酸所組成’只它們不減少原始胜肽之細胞毒殺性τ淋巴球誘發能力°因此’本發明包含具有對HLA抗原之結合親和力的胜肽’其包括來自CLUAP1之胜肽。此種胜肽，例如小於約個胺基酸’時常小於約20個胺基酸，通常小於約1 5個胺基酸。 —般而言，於一胜肽中一、二或多個胺基酸之修飾，不會影響胜肽的功能’且在一些例子中，甚至增強原始蛋白質所需之功能。事實上，已知經修飾之胜肽（即，當與 25 201134480 原始參考序列比較時，包括胺基酸序列之胜肽’其中一、二或多個胺基酸殘基已被修飾（即，取代、加入、刪除或插入））維持原始胜肽的生物活性（Mark et al.，Proc Natl Acad Sci USA 1 984, 81: 5662-6; Zoller and Smith,Those described in the calculation to calculate the binding affinity between various peptides and HLA antigens. For example, as outlined in, for example, Ufuente EM et al., Current Pharmaceutical Design, 2009, 15, 24 201134480 3209-3220, Parker KC et al., J Immunol 1 994 Jan 1, 1 52( 1 ): 1 63-75, Kuzushima K et a 1. , Blood 2001, 98(6): 1872-81, Larsen MV et a 1. BMC Bioinformatics. 2007 Oct 31; 8: 424, Busus Set al. Tissue Antigens., 62: 378-84, 2003, Nielsen M et al., Protein Sci 2003; 1 2: 1 007-1 7 and Nielsen M et al. PLoS ONE 2007; 2: e796 conjugated affinities with HLA antigens. Methods for measuring affinity are described, for example, in Journal of Immunological Methods, 1 995, 1 85: 18, 190 and Protein Science, 2000, 9: 1838-1846. Therefore, a fragment derived from CLUAP1 having a binding affinity to the south of the HLA antigen can be selected using this software program. Thus, by such known procedures, the invention comprises a peptide consisting of any fragment from CLUAP1 which binds to hla. Further, such peptides may include the full length peptide of CLUAP1. The Nine Peptides and Tokachi peptides of the present invention may have additional amino acid residues on the side as long as the peptides produced maintain their cytotoxic T lymphocyte eliciting ability. The additional amino acid residues may be composed of any kind of amino acid 'only they do not reduce the cellular toxic lymphotropy-inducing ability of the original peptide. Thus 'the present invention encompasses peptides having binding affinity for HLA antigens' It includes the peptide from CLUAP1. Such peptides, e.g., less than about a few amino acids, are often less than about 20 amino acids, typically less than about 15 amino acids. In general, modification of one, two or more amino acids in a peptide does not affect the function of the peptide' and in some instances, even enhances the desired function of the original protein. In fact, it is known that a modified peptide (ie, a peptide comprising an amino acid sequence when compared to the original reference sequence of 25 201134480) wherein one, two or more amino acid residues have been modified (ie, Substituting, adding, deleting or inserting)) maintaining the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1 984, 81: 5662-6; Zoller and Smith,

Nucleic Acids Res 1982， 10: 6487-500;Nucleic Acids Res 1982, 10: 6487-500;

Dalbadie-McFarJand et al. , Proc Natl Acad Sci USA 1 982, 79: 6409-13)。因此，於一實施例中，本發明胜肽具有細胞毒殺性T淋巴球誘發能力與擇自序列辨識號：3、4、5、 8、 9、 10、 14、 15、 16、 18、 23、 30、 33、 34、 35、 36與 38中之胺基酸序列兩者’其中加入、刪除及/或取代一、二甚至更多個胺基酸。熟悉此技藝人士認定改變一單一胺基酸或一小百分比之胺基酸的個別加入、刪除或取代至一胺基酸序列傾向產生保存原始胺基酸支鏈的特性。因此，它們常被意指為“保守取代（conservative substitutions)” 或“保守修飾 (conservative modifications)” ，其中一蛋白質之改變導致一具有類似原始蛋白質之功能的經修飾蛋白質。提供功能相似胺基酸之保守取代表已為本技術領域所熟知。所需保守之胺基酸支鏈的特徵的例子包括，例如疏水胺基酸 (A，I，L, M, F，P，W，Y，V)、親水胺基酸（r，卩，n，C, E, Q, G，H，K’ S，T)與具有下列共同官能基或特徵之支鏈：一脂肪族支鏈（G，A，V，L，丨，p); 一含經基支鏈（s， Τ，Y)，含硫原子支鏈（C，M);含羧酸與胺基支鏈（D，N，E， Q)，含驗支鏈（R，K，H)，以及含芳香族支鏈（h, f，γ，w)。 26 201134480 此外，下列八個知群各包含於本技術領域中被接受為保守取代之胺基酸： 1) 丙胺酸（A)、甘胺酸（G); 2) 天門冬胺酸（D)、麵胺酸（E); 3) 天門冬醯胺（N)、麵胺醯胺（Q); 4) 精胺酸（R)、離胺酸（K); 5) 異白胺酸（I)、白胺酸（L)、曱硫丁胺酸（M)、纈胺酸（V); 6) 苯丙胺酸（F)、酪胺酸（γ)、色胺酸（w); 7) 絲胺酸（S)、蘇胺酸（T);以及 8) 半胱胺酸（C)、甲硫丁胺酸（μ)(參見，例如 Creighton, Proteins 1984)。此種經保守修飾胜肽也被視為本發明之胜肽。然而本發明之胜肽並不限於此’且可包括非保守修飾，只要經修飾之胜肽維持原始胜肽之細胞毒殺性τ淋巴球誘發能力。更進- 一步而言，經修飾之胜肽不排除多形變體 (polymorph ic variant)之細胞毒殺性τ淋巴球誘發的胜肽、種間同質體（interspecies homologues)與 CLUAP1 對偶基因 (all e 1 e s )。為了維持必須之細胞毒殺性τ淋巴球誘發能力，可修飾（插入、加入、刪除及/或取代）一小數 S (例如一、二或數個）或小百分比之胺基酸。此處用語 “ 數個”指 5或更少個胺基酸’例如4個、3個或更少。被修飾之胺: 基酸之百分比較佳為2 0 %或更少，例如，15 % 或更少，且- 甚至更佳為’ 10%或更少或1至5%。 27 201134480 當使用於文中之免疫治療時，本發明之胜肽應被表現於一細胞或外吐小體之表面上，較佳作為一具有HLA抗原之複合物。因此，較佳為選擇胜肽其不止誘導細胞毒殺性 T淋巴球也具有對HLA抗原之高親和力。為達此目的，胜肽可藉由胺基酸殘基之取代、插入及/或加入來修飾以產生具經改善之結合親和力的經修飾之胜肽。除了自然表現之胜肽外，由於已知藉由結合至HLA抗原表現之胜肽序列的規則（J Immunol 1 994，152: 3913; Immunogenetics 1995, 41: 178; J Immunol 1 994，155: 4307)，可將基於此規則之修飾引入本發明之致免疫性胜肽。例如，為了增加HLA-A24結合親和力，可需要以苯丙胺酸、路胺酸、曱硫丁胺酸或色胺酸取代N端的第二個胺基酸，及/或以苯丙胺酸、白胺酸、異白胺酸、色胺酸或甲硫丁胺酸取代位於C端之胺基酸。因此，本發明也包括具有擇自序列辨識號：1至22中之胺基酸序列的胜肽，其中胺基酸序列之N端的第二個胺基酸被苯丙胺酸、酪胺酸、甲硫丁胺酸或色胺酸取代，及/或其中胺基酸序列之C端胺基酸被苯丙胺酸、白胺酸、異白胺酸、色胺酸或曱硫丁胺酸取代。或者’為了增加HLA-A2結合親和力，可需要以白胺酸或曱硫丁胺酸取代N端的第二個胺基酸，及/或以纈胺酸或白胺酸取代位於C端之胺基酸。因此，本發明也包括具有擇自序列辨識號：23與25至43中之胺基酸序列的胜肽，其中所述序列辨識號之胺基酸序列之N端的第二個胺基酸 28 201134480 勢被白胺酸或甲硫丁胺酸取代’及/或其中所述序列辨識號之胺基酸序列之c端胺基酸被纈胺酸或白胺酸或甲硫丁胺酸取代。可將取代引入不止於末端胺基酸，也可於胜肽之潛在 TCR辨認位置。一些研究已證實於一具有胺基酸取代之胜肽可具有等於或比原來更好的功能，例如CAP 1、p53 ¢264- 2 7 2 hDalbadie-McFarJand et al., Proc Natl Acad Sci USA 1 982, 79: 6409-13). Therefore, in one embodiment, the peptide of the present invention has a cytotoxic T lymphocyte evoking ability and a sequence identification number: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, The amino acid sequences of 30, 33, 34, 35, 36 and 38 'in which one, two or even more amino acids are added, deleted and/or substituted. It is well known to those skilled in the art that changing the individual addition, deletion or substitution of a single amino acid or a small percentage of amino acid to an amino acid sequence tends to produce the property of preserving the original amino acid branch. Thus, they are often referred to as "conservative substitutions" or "conservative modifications" in which a change in a protein results in a modified protein having a function similar to that of the original protein. The conservative representation of providing functionally similar amino acids is well known in the art. Examples of characteristics of the desired conservative amino acid branch include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (r, 卩, n) , C, E, Q, G, H, K' S, T) and a branch having the following common functional groups or characteristics: an aliphatic branch (G, A, V, L, 丨, p); Branch group (s, Τ, Y), a branch containing a sulfur atom (C, M); a branch containing a carboxylic acid and an amine group (D, N, E, Q), including a test branch chain (R, K, H), and contains aromatic branches (h, f, γ, w). 26 201134480 In addition, the following eight knowledge groups each contain amino acids that are accepted as conservative substitutions in the art: 1) alanine (A), glycine (G); 2) aspartic acid (D) , face acid (E); 3) aspartame (N), face amine guanamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I) ), leucine (L), thioglycolic acid (M), proline (V); 6) phenylalanine (F), tyrosine (γ), tryptophan (w); 7) silk Amine acid (S), threonine (T); and 8) cysteine (C), methylthiobutyric acid (μ) (see, for example, Creighton, Proteins 1984). Such conservatively modified peptides are also considered to be peptides of the present invention. However, the peptide of the present invention is not limited to this and may include non-conservative modifications, as long as the modified peptide maintains the cytotoxic thymocyte stimulating ability of the original peptide. Further, in one step, the modified peptide does not exclude the polymorphic cytotoxic thymocyte-induced peptide, interspecies homologues and CLUAP1 dual gene (all e 1 Es ). In order to maintain the necessary cellular toxin-like lymphocyte priming ability, a small number S (e.g., one, two or several) or a small percentage of amino acid may be modified (inserted, added, deleted and/or substituted). The term "several" as used herein means 5 or less amino acids' such as 4, 3 or less. The amine to be modified: the percentage of the base acid is preferably 20% or less, for example, 15% or less, and - even more preferably '10% or less or 1 to 5%. 27 201134480 When used in immunotherapy herein, the peptide of the present invention should be expressed on the surface of a cell or exosome, preferably as a complex with an HLA antigen. Therefore, it is preferred to select a peptide which not only induces a cytotoxic T lymphocyte but also has a high affinity for an HLA antigen. To this end, the peptide can be modified by substitution, insertion and/or addition of an amino acid residue to produce a modified peptide having improved binding affinity. In addition to naturally occurring peptides, rules for peptide sequences expressed by binding to HLA antigens are known (J Immunol 1 994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol 1 994, 155: 4307) A modification based on this rule can be introduced into the immunogenic peptide of the present invention. For example, in order to increase the binding affinity of HLA-A24, it may be desirable to replace the N-terminal second amino acid with phenylalanine, lysine, guanidinine or tryptophan, and/or phenylalanine, leucine, Isoamino acid, tryptophan or methylthiobutyric acid replaces the amino acid at the C-terminus. Accordingly, the present invention also encompasses a peptide having an amino acid sequence selected from the sequence identification numbers: 1 to 22, wherein the second amino acid of the N-terminus of the amino acid sequence is amphetamine, tyrosine, methyl sulfide The butyric acid or tryptophan acid is substituted, and/or the C-terminal amino acid of the amino acid sequence is substituted with amphetamine, leucine, isoleucine, tryptophan or guanidinoic acid. Or 'in order to increase the binding affinity of HLA-A2, it may be necessary to replace the N-terminal second amino acid with leucine or thioglycolic acid, and/or replace the amine group at the C-terminus with a proline or leucine acid. acid. Accordingly, the present invention also encompasses a peptide having an amino acid sequence selected from the sequence identification numbers: 23 and 25 to 43, wherein the second amino acid of the N-terminus of the amino acid sequence of the sequence number is 28 201134480 The c-terminal amino acid substituted with leucine or methylthiobutyric acid and/or the amino acid sequence of the sequence number is substituted with lysine or leucine or methionine. Substitutions can be introduced not only at the terminal amino acid, but also at the potential TCR recognition site of the peptide. Some studies have demonstrated that a peptide having an amino acid substitution can have a function equal to or better than the original, such as CAP 1, p53 ¢ 264 - 2 7 2 h.

Her-2/neu U 6 9 - 3 7 7 )或 gplOO ( 2 0 9 - 2 1 7) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002) Feb 1;168(3):1338-47., S. 0. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and S. 〇. Dionne et al. Cancer Immunology,Her-2/neu U 6 9 - 3 7 7 ) or gplOO ( 2 0 9 - 2 1 7) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al. J Immunol. (2002 Feb 1;168(3):1338-47., S. 0. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and S. 〇. Dionne et al. Cancer Immunology,

Immunotherapy (2004) 53，307-314^ 本發明也考慮一、一個或數個胺基酸也可被加至本發明胜肽之N及/或c端。本發明也包括具有高HLA抗原結合親和力且維持細胞毒殺性T淋巴球誘發能力之此種經修飾的胜肽。然而’當胜狀序列與—具有不同功能之内生或外生蛋 $質之胺基酸序列的一部份相同時，可能誘導副作用，例如自體免疫疾病或抗特定物質之過敏症候群。因此，較佳為使用可知之貝料庫執行同源搜尋以避免胜肽之胺基酸序列符合其他蛋白質之疋扣基酸序列的情況。當與目標胜肽比較時不只存在具有—忐 4兩個胺基酸不同之胜肽的同源搜砰變得清楚時，為了辦Λ ^Θ加其與HLA抗原之結合親和力，及/ 或增加其細胞毒殺性夂& 1淋巴球誘發能力而不具副作用之任何危險，可修飾目標胺基酸。 29 201134480 雖然如上述之具有對HLA抗原高結合親和力的胜肽被預期為高效能’但根據作為指示之高親和表現而被選擇之候選胜肽’更進一步被測試細胞毒殺性τ淋巴球誘發能力的表現。此處措辭“細胞毒殺性T淋巴球誘發能力”意指當表現於抗原呈現細胞時’胜肽誘導細胞毒殺性T淋巴球的能力。此外，“細胞毒殺性τ淋巴球誘發能力”包括胜肽誘導細胞毒殺性τ淋巴球活化、細胞毒殺性τ淋巴球增殖、促進細胞毒殺性τ淋巴球分解目標細胞與增加細胞毒殺性T淋巴球IFN- 7產生的能力。藉由誘導攜帶人類MHC抗原之抗原呈現細胞（例如B-淋巴球、巨嗟細胞與樹突細胞）或更專一地來自人類周邊血液單核細胞之樹突細胞，並在以胜肽刺激之後與CD8陽性細胞混合，且之後測量由抗目標細胞之細胞毒殺性T淋巴球產生並釋放之IFN- r來達成細胞毒殺性T淋巴球誘發能力的確定。如此反應系統，可使用已被產生來表現人類 HLA之基因轉殖動物（例如，於BenMohamed L，KrishnanR, Longraate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61(8): 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependent on MHC (HLA) class II restricted T(H) response中的描述）^例如可以51Cr放射標示目標細胞，且可從自目標細胞釋放出的放射活性計算細胞毒殺活性。或者在攜帶經固定之胜肽之抗原呈現細胞存在下，藉由測 30 201134480 量由細胞毒殺性τ淋巴球產生並釋放的IFN-r，且使用抗 IFNi單株抗體來使於培養基上之抑制區可見來分析細胞毒殺性T淋巴球誘發能力。由於如上述測試胜肽之細胞毒殺性τ淋巴球誘發能力’發現擇自由序列辨識號：3、4、5、8、9、1〇、14、15、 ^ 18、23、30、33、34、35、36與38所指出的胺基酸序列中之九胜肽或十胜肽顯示特別高之細胞毒殺性τ淋巴球誘發能力與對HLA抗原之高結合親和力。因此這些胜肽為本發明之較佳實施例。此外，同源分析之結果顯示那些胜肽不與來自任何其他已知人類基因產物之胜肽有顯著之同源性。因此當用於免疫治療時，降低了未知或不需要之免疫反應提升的可能性。因此，也來自此態樣，這些胜肽提供在癌症病患中引起抗CLUAP1免疫反應的用途。因此本發明之胜肽較佳為具有擇自序列辨識號：3、4、5、8、9、10、14、15、16、18、 23、30、33、34、35、36與38之胺基酸序列的胜肽。除了上述修飾之外’本發明胜肽也可連接其他胜肽，只要所產生之經連接的胜肽維持原始胜肽之細胞毒殺性τ 淋巴球誘發能力。適合胜肽的例子包括：本發明胜肽或來自其他腫瘤相關抗原之細胞毒殺性Τ淋巴球誘導胜肽。適合之胜肽内（inter-peptide)連結器為被本技術領域所熟知與包括，例如 AAY (P. M. Daftarian et al·，J Trans Med 2007, 5:26) ^ AAA > NKRK (R. P. M. Sutmuller et al., J Immunol. 2000，165: 7308-731 5)或 K (S. Ota el; al 31 201134480Immunotherapy (2004) 53, 307-314^ The present invention also contemplates that one or more amino acids may also be added to the N and/or c terminus of the peptide of the present invention. The present invention also encompasses such modified peptides having high HLA antigen binding affinity and maintaining cytotoxic T lymphocyte eliciting ability. However, when the winning sequence is identical to a portion of the amino acid sequence of the endogenous or exogenous egg having different functions, side effects such as autoimmune diseases or anti-specific substances may be induced. Therefore, it is preferred to perform a homologous search using a known shell library to avoid the case where the amino acid sequence of the peptide conforms to the thiol acid sequence of other proteins. When compared with the target peptide, there is not only a homologous search with a peptide having a different amino acid, but also a binding affinity to the HLA antigen, and/or an increase. The cytotoxic sputum & 1 lymphocyte-inducing ability without any risk of side effects can modify the target amino acid. 29 201134480 Although a peptide having high binding affinity for HLA antigen as described above is expected to be highly potent 'but a candidate peptide selected based on high affinity performance as an indication' is further tested for cytotoxic titoblast induction ability Performance. The phrase "cytotoxic T lymphocyte-inducing ability" as used herein means the ability of the peptide to induce a cytotoxic T lymphocyte when expressed as an antigen presenting cell. In addition, "cytotoxic tau lymphocytic evoked ability" includes peptide-induced cytotoxic tau lymphocyte activation, cytotoxic tau lymphocyte proliferation, promotion of cytotoxic tau lymphocyte decomposition target cells and increased cytotoxic T lymphocytes The ability of IFN-7 to produce. Presenting cells (eg, B-lymphocytes, giant sputum cells, and dendritic cells) or more specifically dendritic cells derived from human peripheral blood mononuclear cells by inducing antigens carrying human MHC antigens, and after stimulation with peptides The CD8-positive cells were mixed, and then the IFN-r produced and released by the cytotoxic T lymphocytes of the target cells was measured to determine the cytotoxic T lymphocyte-inducing ability. Such a reaction system can use genetically modified animals that have been produced to express human HLA (for example, in Ben Mohamed L, Krishnan R, Longraate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61 (8) ): 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependent on MHC (HLA) class II restricted T(H) response))^ For example, the target cell can be radiolabeled with 51Cr, and the cytotoxic activity can be calculated from the radioactivity released from the target cell. Or in the presence of antigen-presenting cells carrying the immobilized peptide, the IFN-r produced and released by the cytotoxic tau lymphocytes is measured by 30 201134480, and the anti-IFNi monoclonal antibody is used to inhibit the medium. The area can be seen to analyze the cytotoxic T lymphocyte evoked ability. Due to the cytotoxicity of the cytotoxicity of the cell as described above, the discovery of the free sequence identification number: 3, 4, 5, 8, 9, 1 , 14, 15, ^ 18, 23, 30, 33, 34 The nine peptides or the ten peptides in the amino acid sequence indicated by 35, 36 and 38 show particularly high cytotoxic tau lymphocyte inducing ability and high binding affinity to HLA antigen. These peptides are therefore preferred embodiments of the invention. Furthermore, the results of the homology analysis showed that those peptides did not have significant homology to peptides derived from any other known human gene product. Therefore, when used in immunotherapy, the possibility of an unknown or unwanted immune response elevation is reduced. Therefore, also from this aspect, these peptides provide the use of an anti-CLUAP1 immune response in cancer patients. Therefore, the peptide of the present invention preferably has the following sequence identification numbers: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38 The peptide of the amino acid sequence. In addition to the above modifications, the peptide of the present invention may also be linked to other peptides as long as the resulting peptide is maintained to maintain the cytotoxic thymocyte inducing ability of the original peptide. Examples of suitable peptides include: the peptide of the present invention or a cytotoxic a lymphocyte-inducing peptide derived from other tumor-associated antigens. Suitable inter-peptide linkers are well known and included in the art, for example, AAY (PM Daftarian et al., J Trans Med 2007, 5:26) ^ AAA > NKRK (RPM Sutmuller et al , J Immunol. 2000, 165: 7308-731 5) or K (S. Ota el; al 31 201134480

Can Res. 62, 1471-1476, K. S. Kawamura et al., JCan Res. 62, 1471-1476, K. S. Kawamura et al., J

Immunol. 2002， 168: 5709-5715)。例如’也可實質同時使用非CLUAP1腫瘤相關抗原胜肽以增加經由HL A class I及/或class II之免疫反應。其已相當確認，癌症細胞可表現多於一個腫瘤相關基因。因此，其為在對於熟悉此技藝人士例行實驗之範圍中以確認是否一特定個體表現額外腫瘤相關基因，且之後包括來自此類基因之表現產物的HLA class I及/或class II結合胜肽於根據本發明之CLUAP1組合物或疫苗中。 HLA class I與HLA class II結合胜肽之例子對於熟悉此技藝人士而言為已知（例如，參見C〇ui ie，stem Cel Is 13:393-403，1 995 )，且可以一如此處所揭露之那些的類似方式被使用於發明中。因此，使用分子生物之標準程序，熟悉此技藝人士可立即製備包括一或多個CLUAP1胜肽與一或多個非CLUAP1胜狀的多胜肽，或編碼出此類多胜肽的核酸。上述此類連結胜肽於此處意指為“多面體 (poly tope)即’兩個或多個潛在免疫原性（immunogeni c) 或免疫反應刺激胜肽的群組’胜肽可互相連接以多種排列 (例如，連成一串或部分重疊）。多面體（或編碼出多面體的核酸）可以一標準免疫步驟被投予，例如至動物，以測試多面體於刺激、增強及/或誘導一免疫反應之功效。胜狀可被直接連接或經由使用位於側面之序列以形成多面體，且多面體為疫苗之用途為本技術領域所熟知（參 32 201134480 見，Thomson et al·， Proc. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., NatureImmunol. 2002, 168: 5709-5715). For example, a non-CLUAP1 tumor associated antigen peptide can also be used substantially simultaneously to increase the immune response via HL A class I and/or class II. It has been fairly confirmed that cancer cells can exhibit more than one tumor-associated gene. Thus, it is within the scope of routine experimentation for those skilled in the art to determine whether a particular individual exhibits additional tumor-associated genes, and then includes HLA class I and/or class II binding peptides from the performance products of such genes. In a CLUAP1 composition or vaccine according to the invention. Examples of HLA class I binding to HLA class II peptides are known to those skilled in the art (see, for example, C〇ui ie, Stem Cel Is 13: 393-403, 995) and can be as disclosed herein. A similar way of those is used in the invention. Thus, using standard procedures for molecular biology, one skilled in the art can immediately prepare a multi-peptide comprising one or more CLUAP1 peptides and one or more non-CLUAP1 traits, or a nucleic acid encoding such a multi-peptide. The above-mentioned linked peptides are herein referred to as "poly tope" or "two or more potential immunogenic (immunogeni c) or immunoreactive stimulating peptides. The peptides can be linked to each other in a variety of ways. Arrangement (eg, in a series or partial overlap). Polyhedra (or nucleic acids encoding polyhedra) can be administered in a standard immunization step, such as to an animal, to test the efficacy of a polyhedron in stimulating, enhancing, and/or inducing an immune response. The winning form can be directly linked or via the use of a sequence located on the side to form a polyhedron, and the use of a polyhedron for a vaccine is well known in the art (see 32 201134480, see Thomson et al., Proc. Natl. Acad. Sci USA 92). (13): 5845-5849, 1995; Gilbert et al., Nature

Biotechnol. 15(12):1280-1284, 1997; Thomson et al.， J Immunol. 157(2):822-826, 1996； Tarn et al., J Exp. Med. 1 71 ( 1 ):299-306, 1 990 )。製備並含有抗原決定位之不同數目與組合的多面體並為了藉由細胞毒殺性T淋巴球的辨認與為了於增加免疫反應中之功效將其進行測試。本發明之胜肽可被連接至其他物質，只要所產生之連結維持原始胜肽之必不可少之細胞毒殺性T淋巴球誘發能力。適合之物質的例子包括，胜肽 '脂質、糖與糖鏈、乙醯基，天然與合成之聚合物等。本發明胜肽可包含修飾，例如醣基化、支鏈氧化及/或磷酸化，其提供修飾不損壞原始胜肽之生物活性。此修飾可被執行以授予額外之功能（例如目標功能與傳送功能）或穩定多胜肽。例如，為了增加多胜肽之穩定度，本技術領域已知引入D_胺基酸、胺基酸模仿物或非天然胺基酸；此内容也適合本發明之多胜肽。可以一些方法分析一多胜肽的穩定度。例如，可使用肽酶與多種生物培養基’例如人類血漿與血清來測試穩定度（參見，例如 Verhoef et al., Eur j Drug _ Pharmacokin 1986， 11: 291-302)。、此外如上所提到，在藉由_、二或數個胺基酸殘基取代刪除或加人之經修飾的胜肽中，可筛選或選擇盘原 :胜肽：較具有相同或較高之活性的那些。因此本發明也 β ’、4選或選擇與原始相較具有相@或較高之活性的經 33 201134480 修飾胜肽的方法 a :將至少一加入，說月之方法包括下列步驟：個本發明胜肽之胺基酸殘基取代、刪除或 b :確定胜肽的活性，與 c:選擇與原始相較具有相同或較高之活性的胜肽。此處，要被分析之活性可包括MHC結合活性、抗原呈現細胞或細胞毒殺性T淋P社噠旅处丄… 淋巴球誘發能力與細胞毒性活性。此處’本發明之胜狀_ "όΓ ϋ 、+，4 « λί 肱乜可被描述為CLUAP1胜肽，，或 “CLUAP1多胜肽”。 III. CLUAP1胜肽之製備使用熟知之技術可製備本發明之胜狀。例如，使用重組DNA技術或化學合成可以合成方法地製備胜狀。本發明胜肽可單獨合成或為包括兩或多個胜肽之較長多胜肽。之後可分離此胜肽’即純化或分離以使其實質上無其他自然發生之宿主細胞蛋白質與其片段或任何其他化學物質。本發明胜肽可包含修飾，例如醣基化、支鏈氧化或磷酸化，其提供修飾不損壞原始胜肽之生物活性。其他修飾包括可用來’例如增加胜肽之血清半衰期之D_胺基酸或其他胺基酸模仿物的合併。藉由根據經選擇之胺基酸序列的化學合成可獲得本發明之胜肽。適合此合成之一般胜肽合成方法的例子包括： (i)胜狀合成（peptide Synthesis) Interscience， New York, 1966； 34 201134480 (ii)蛋白質（The Proteins), Vol. 2， Academic Press, New York, 1976; (iii) 胜肽合成（Peptide Synthesis)(in Japanese), Maruzen Co. , 1975; (iv) 胜肽合成之基礎與實驗（Basics and Experiment of Peptide Synthesis) (in Japanese), Maruzen Co., 1985; (v) 藥學的發展（Development of Pharmaceuticals) (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hi rokawa, 1991; (vi) W099/67288 ;以及 (vii) Barany G. & Merrifield R.B., Peptides Vol. 2， Solid Phase Peptide Synthesis, Academic Press, New York， 1980， 100-118。或者’藉由適應任何已知產生胜肽之基因工程方法可獲得本發明之胜肽（例如，Morrison J，J Bacteriology 1977, 132: 349-51； Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983， 101: 347-62)。例如，首先製備一適合之載體’其懷有一多核苷酸其編碼出目標胜肽於一可表達的形式中（例如，調控序列之下游相當於啟動子序列）’並將載體轉殖進入適合之宿主細胞。此種載體或宿主細胞也由本發明所提供。之後培養宿主細胞以產生感興趣之胜狀。使用一 a W加轉譯系統可μ 產生胜肽。 35 201134480 IV ·多核苦酸本發明也提供-多核苦酸，其編碼出任何本發明上述之胜肽。這些包括來自自然發生之⑽以基因（GenBankBiotechnol. 15(12): 1280-1284, 1997; Thomson et al., J Immunol. 157(2): 822-826, 1996; Tarn et al., J Exp. Med. 1 71 (1):299- 306, 1 990). Different numbers and combinations of polymorphs of epitopes were prepared and included for testing by cytotoxic T lymphocytes and for increasing efficacy in immune responses. The peptide of the present invention can be linked to other substances as long as the resulting linkage maintains the essential cytotoxic T lymphocyte eliciting ability of the original peptide. Examples of suitable materials include peptides, lipids, sugars and sugar chains, ethyl sulfonyl groups, natural and synthetic polymers, and the like. The peptide of the present invention may comprise modifications, such as glycosylation, branched oxidation, and/or phosphorylation, which provide for modification without damaging the biological activity of the original peptide. This modification can be performed to grant additional functionality (e. g., target function and delivery function) or to stabilize the multi-peptide. For example, in order to increase the stability of a multi-peptide, it is known in the art to introduce a D-amino acid, an amino acid mimetic or an unnatural amino acid; this content is also suitable for the multi-peptide of the present invention. The stability of a multi-peptide can be analyzed in some ways. For example, peptidase can be used to test for stability with various biological media such as human plasma and serum (see, for example, Verhoef et al., Eur j Drug _ Pharmacokin 1986, 11: 291-302). Further, as mentioned above, in the substitution of the deleted or added modified peptide by _, two or several amino acid residues, the disc original: peptide: more identical or similar Those with high activity. Therefore, the present invention also β ', 4 selects or selects the method 33 of the 2011 201180 modified peptide which has a phase @ or higher activity compared with the original one: at least one is added, and the method of the month includes the following steps: The amino acid residue of the peptide is substituted, deleted or b: the activity of the peptide is determined, and c: the peptide having the same or higher activity than the original is selected. Here, the activity to be analyzed may include MHC binding activity, antigen presenting cells or cytotoxic T lymphocytes, lymphocyte inducing ability and cytotoxic activity. Here, the winner of the present invention _ "όΓ ϋ , +, 4 « λί 肱乜 can be described as CLUAP1 peptide, or "CLUAP1 multi-peptide". III. Preparation of CLUAP1 peptides The advantages of the present invention can be prepared using well-known techniques. For example, a victory can be prepared synthetically using recombinant DNA techniques or chemical synthesis. The peptide of the present invention can be synthesized alone or as a longer multi-peptide comprising two or more peptides. The peptide can then be isolated' purified or isolated such that it is substantially free of other naturally occurring host cell proteins and fragments thereof or any other chemical. The peptide of the present invention may comprise modifications, such as glycosylation, branched oxidation or phosphorylation, which provide for modification without damaging the biological activity of the original peptide. Other modifications include the combination of D_amino acids or other amino acid mimetics that can be used, e.g., to increase the serum half-life of the peptide. The peptide of the present invention can be obtained by chemical synthesis according to the selected amino acid sequence. Examples of general peptide synthesis methods suitable for this synthesis include: (i) peptide synthesis Interscience, New York, 1966; 34 201134480 (ii) Protein (The Proteins), Vol. 2, Academic Press, New York , 1976; (iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975; (iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co. , 1985; (v) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hi rokawa, 1991; (vi) W099/67288; and (vii) Barany G. & Merrifield RB, Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-118. Alternatively, the peptide of the present invention can be obtained by adapting to any genetic engineering method known to produce a peptide (for example, Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds Wu et al.) 1983, 101: 347-62). For example, a suitable vector is first prepared which has a polynucleotide which encodes the target peptide in an expressible form (eg, downstream of the regulatory sequence corresponding to the promoter sequence) and transfers the vector into a suitable vector. Host cell. Such vectors or host cells are also provided by the present invention. The host cells are then cultured to produce a victory of interest. A peptide can be generated using a w plus translation system. 35 201134480 IV. Polynucleic acid The present invention also provides a polynucleic acid which encodes any of the above-described peptides of the present invention. These include genes from natural (10) genes (GenBank)

Accession No. NMJ)1 5041 或關_〇24793 (例如，序列辨識號：44))的核苦酸序列與具有其之保守修飾之核苦酸序列的那些。此處措辭“保守修飾之核苷酸序列，，指序列其編碼出相同或實質上相同之胺基酸序列。由於基因密碼的退化，一大份之功能相同之核酸編碼出任何已知蛋白質。例如，密碼GCA、GCC、GCG與GC"編碼出胺基酸丙胺酸。因此，於藉由一密碼具體指定丙胺酸之每個位置，可改變密碼《為任何上述不會改變編碼出《胜肽的對應密碼。此核酸變化為“沈默變化（silent variati〇n)” ，其為保守修飾變化的一種。此處編碼出一胜肽之每個核酸序列也描述核酸之每種可能的沈默變化。熟悉此技藝人士明白於-核酸中各密碼（除了 AUG，其原本為甲硫胺酸之唯一密碼、與TGG其原本為色胺酸之唯一密碼）可被修飾以產生一功能相同分子。因此編碼出一胜肽之核酸的各沈默變化係為於各所揭露之序列中被暗示性描述。本發明之多核苷酸可由DNA、RNA或其衍生物所組成。如本技術領域所熟知，DNA分子由鹼基所組成，例如自然發生之鹼基a、τ、 C與G，而T於RNA中為U所取代。熟悉此技藝人士可瞭解非自然發生驗基也被包含於本發明中。本發明之多核苷酸可編碼出本發明之多個胜肽，具有 36 201134480 s /、有’丨於中間之胺基酸序列。例如介於中間之胺基酸序列可提供多核苦酸或經轉譯之胜肽—裂解位（例如酵素辨 < 序列）。更進—步而言，多核苷酸可包括任何額外之序列至編碼出本發明胜肽之編碼序列。例如，多核苦酸可為一重組多核苷酸，其包括胜肽表現所需之調控序列，或可為一具有標誌基因與此類之表現載體（質體）。一般而。可製備此重組多核苷酸，藉由經由使用一般重組技術，例如聚合酶與内切酶之多核苷酸操作。可使用重組與化學合成技術兩者以產生本發明之多核苷酸。例如，藉由插進入一適合之載體可產生一多核苷酸，當轉染進入一勝任細胞時，其可被表現。或者，使用PCR 技術或表現於適合的宿主可將一多核苷酸放大（參見，例如 Sambrook et al.， Molecular Cloning: A LaboratoryAccession No. NMJ) 1 5041 or off _ 〇 24793 (eg, Sequence ID: 44)) of the nucleotide sequence and those with a conservatively modified nucleotide sequence. The phrase "conservatively modified nucleotide sequence" refers to a sequence which encodes the same or substantially the same amino acid sequence. Due to the degeneration of the gene code, a large number of functionally identical nucleic acids encode any known protein. For example, the codes GCA, GCC, GCG, and GC" encode amino acid alanine. Therefore, by specifying a position of alanine by a password, the password can be changed. "For any of the above, the peptide is not changed." Corresponding code. This nucleic acid change is "silent variati", which is one of the conservative modification changes. Each nucleic acid sequence encoding a peptide here also describes every possible silent change in the nucleic acid. Those skilled in the art will appreciate that the various codes in the nucleic acid (except AUG, which is originally the unique code for methionine, and the unique code for TMG which is originally tryptophan) can be modified to produce a functionally identical molecule. Each silent change in the nucleic acid of a peptide is implicitly described in each of the disclosed sequences. The polynucleotide of the present invention may be composed of DNA, RNA or a derivative thereof. As is well known in the art, DNA molecules are composed of bases such as naturally occurring bases a, τ, C and G, and T is replaced by U in RNA. Those skilled in the art will be aware of non-natural occurrence tests. The bases are also included in the present invention. The polynucleotide of the present invention encodes a plurality of peptides of the present invention having a ratio of 36 201134480 s /, having an amino acid sequence in the middle. For example, an intermediate amino group The acid sequence may provide a polynucleic acid or a transduced peptide-cleavage site (e.g., an enzyme recognition < sequence). Further, the polynucleotide may include any additional sequences to encode the peptide of the present invention. For example, the polynucleic acid may be a recombinant polynucleotide comprising a regulatory sequence required for the performance of the peptide, or may be a vector (plastid) having a marker gene and such a gene. Recombinant polynucleotides are manipulated by using polynucleotides using general recombinant techniques, such as polymerases and endonucleases. Both recombinant and chemical synthesis techniques can be used to produce polynucleotides of the invention. For example, by insertion Suitable The vector can produce a polynucleotide which can be expressed when transfected into a competent cell. Alternatively, a polynucleotide can be amplified using PCR techniques or in a suitable host (see, for example, Sambrook et Al., Molecular Cloning: A Laboratory

Manual, Cold Spring Harbor Laboratory, New York, 1989) °或者，使用固態技術如於BeaucageSL&IyerRP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984，3: 801-5中所敘述，可合成多核苷酸。 V·外吐小體（exosomes) 本發明進一步地提供稱為外吐小體的胞間囊泡 (intracel lular vesicles)，其呈現形成於本發明之胜肽與人類白血球抗原表面之間的複合物。利用例如Japanese Patent Application Kohyo Publications Nos. He i 1 1 -5 1 0507與W099/03499所詳述的方法以及從接受治療和 37 201134480 戈預防之病人所#的抗原表現 ^ |備外吐小體。本發月之外吐J、體可如疫苗般地接種之方弋似於本發明的胜肽包含在複合物中的人類白血球抗原形式必須血需要户療和/或預防之個體的人類白血球抗原形式相符。例如，於日本族群中，HLA-A24與HLA_A2,特別是hu_a*24〇2斑 HU-A*〇2〇mA_A*〇2()6為普遍且因此對於日本人病患的治療而言為適合的。高度表現於日本人與高加索人之中的A24型或A2型之使用有助於獲得有效的結果，且次型a * 2402與^ 0201與a* 0206也提供用途。一般在臨床上，需接受治療之病患的人類白血球抗原形式係進行預先的研究，這可適當地選擇對此抗原具有高度結合親合力的胜肽或經由抗原表現具有細胞毒性T淋巴細胞誘發性的胜狀。此外’為了獲得具有高度結合親合力與細胞毒性τ淋巴細胞誘發性兩者的胜肽’可以天然產生之CLUAP1部分胜肽的胺基酸序列為基礎，然後進行1、2或數個胺基酸的取代、插入、刪除及/或添加。當對本發明外吐小體使用A24型HLA抗原時，具有擇自序列辨識號：1至22中之序列的胜肽提供用途。或者，當對本發明外吐小體使用A2型HLA抗原時，具有擇自序列辨識號：23與25至43中之序列的胜肽提供用途。 VI.抗原呈現細胞Manual, Cold Spring Harbor Laboratory, New York, 1989) ° Alternatively, using solid state techniques as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5 , can synthesize polynucleotides. V. Exosomes The present invention further provides intercel vesicles called exocytosis, which exhibit a complex formed between the peptide of the present invention and the surface of human leukocyte antigen. . The method described in, for example, Japanese Patent Application Kohyo Publications Nos. He i 1 1 - 5 1 0507 and WO99/03499, and the antigen expression from the patient who received the treatment and 37 201134480 戈 prevent the external body. The human leukocyte antigen of the individual who needs to be treated and/or prevented by blood in the human leukocyte antigen form contained in the complex is not included in the present invention. The form matches. For example, in the Japanese ethnic group, HLA-A24 and HLA_A2, especially hu_a*24〇2 spot HU-A*〇2〇mA_A*〇2()6 are common and therefore suitable for the treatment of Japanese patients. of. The use of A24 or A2, which is highly representative of Japanese and Caucasians, helps to obtain effective results, and subtypes a* 2402 and ^0201 and a* 0206 also provide use. Generally, clinically, the human leukocyte antigen form of the patient to be treated is subjected to a prior study, which can appropriately select a peptide having a high binding affinity for the antigen or exhibiting cytotoxic T lymphocyte-induced property via the antigen. Victory. In addition, 'in order to obtain a peptide with both high binding affinity and cytotoxic tau lymphocyte inducibility' can be based on the amino acid sequence of the naturally occurring CLUAP1 partial peptide, and then 1, 2 or several amino acids are carried out. Replace, insert, delete and/or add. When the A24 type HLA antigen is used for the exocytosis of the present invention, the peptide having the sequence selected from the sequence identification numbers: 1 to 22 provides a use. Alternatively, when an A2 type HLA antigen is used for the exosome of the present invention, a peptide having a sequence selected from the sequence identification numbers: 23 and 25 to 43 provides use. VI. Antigen presenting cells

本發明也提供抗經分離之原呈現細胞，其表現以HLA 38 201134480 抗原與本發明胜肽形成的複合物於其表面◦抗原呈現細胞可來自受到治療及/或避免之病患，且藉由其本身或與包括本發明之胜肽、外吐小體或細胞毒殺性τ淋巴球之其他藥物結合可被投予如一疫苗。抗原呈現細胞並不限於特定種類之細胞，且包括樹突細胞、蘭格罕細胞（Langerhans ce 11 )、巨嗜細胞、B細胞與活化之T細胞，已知其表現蛋白質（proteinaceous)抗原於其細胞表面以被淋巴球所辨認。由於樹突細胞為一典型抗原呈現細胞，其於抗原呈現細胞中具最強之細胞毒殺性T淋巴球誘導作用，樹突細胞供給使用如本發明之抗原呈現細胞。例如，藉由誘導來自周邊血液單核細胞之樹突細胞與之後//? F/ ίΓσ、以F/ FO或7·/7 W叩以本發明胜肽接觸（刺激）其可獲得一抗原呈現細胞。當本發明之胜肽投予至一個體’於個體身體内誘導表現本發明胜肽之抗原呈現細胞。措辭“誘導抗原呈現細胞”包括以本發明之胜肽或編碼出本發明胜肽之核苷酸接觸（刺激）一細胞，以表現形成於HLA抗原與本發明胜肽之間的複合物於細胞表面。因此，藉由在將本發明胜肽投予至一個體後，自此個體收集抗原呈現細胞可獲得本發明之抗原呈現細胞。或者，藉由將自個體收集之抗原呈現細胞與本發明胜肽接觸可獲得本發明之抗原呈現細胞。可將本發明之抗原呈現細胞投予至一個體以誘導於個體中之抗癌免疫反應’藉由其本身或與包括本發明之胜肽、外吐小體或細胞毒殺性Τ淋巴球之其他藥物結合。例 39 201134480 如，^ F/ 投予可包括步驟： a :自一第一個體收集抗原呈現細胞， b .以胜肽接觸步驟a之抗原呈現細胞，以及 c .將步驟b之抗原呈現細胞投予一第二個體。第一個體與第二個體可為相同個體或可為不同個體。或者，根據本發明，提供本發明胜肽於製造一誘導抗原呈現細胞之藥學組合物的用途。此外，本發明提供製造誘導抗原呈現細胞之藥學組合物的方法或製程。更進一步，本發明也提供用於誘導抗原呈現細胞之本發明胜肽。自步驟 b獲付之抗原呈現細胞可一為疫苗，用以治療及/或預防癌症，例如乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、騰臟癌。根據本發明一方面，本發明之抗原呈現細胞具高程度細胞毒殺性T淋巴球誘發能力。在用語“高程度細胞毒殺性T淋巴球誘發能力”中，高程度相對於藉由抗原呈現細胞沒有與胜狀接觸或與無法誘導細胞毒殺性T淋巴球之胜肽接觸的程度。藉由包括k 將編碼出本發明胜肽之 ^核苷Si轉移至抗原呈現細胞的步驟的方法與上述之方法’可製備此種具高程度細胞毒殺性T淋巴球誘發能力之抗原呈現細胞。此經引入之基因可為DNA $ RNA形式。引入方法的例子包括，並無特別限制，可使用各種於此領域一般被執行的方法，例如可使用脂質體轉染 (lipofecti on)、電穿孔法（electroporaUon)或碳酸妈方法更特別地’可執行其如Cancer Res 1996，56: 5672-7; 40 201134480 * J Immunol 1998， 161: 5607-13; J Exp Med 1996， 184: 465-72; Published Japanese Translation 〇f International Publication No. 2000-509281 中所述。藉由轉移基因進入抗原呈現細胞，基因遭遇轉錄、轉譯與此類於細胞中’且之後所獲得之蛋白質藉由MHC C1 ass I 或C1 ass 11處理’並經由一呈現途徑進行以與呈現胜肽。 VII.細胞毒殺性T淋巴球抗任何本發明胜肽誘導之細胞毒殺性T淋巴球增強加叩以癌症細胞為標的之免疫反應，且因此就其本身而言，可使用為疫苗以相似於胜肽之方式。因此本發明提供經分離之細胞毒殺性T淋巴球其藉由任何本發明之胜肽專一地被誘導或活化。可獲得此種細胞毒殺性τ淋巴球，藉由（1)將本發明胜肽投予至一個體；或（2)將來自個體之抗原呈現細胞與CD8陽性細胞或周邊血液單核淋巴球與本發明之胜肽h κ/π接觸（刺激）或（3)將CD8陽性細胞或周邊血液單核淋巴球與表現HU抗原與胜肽之複合物：其表面上之抗原呈現細胞或外吐小體接觸或（4)引入勺括編碼出與與本發明胜肽結合之了細胞受體夕^ 入平儿夕胜肽的多核皆酸的基因。藉由上述方法可製備此類抗么或外吐小體’且⑷之方法之詳細被敘述於下$ 、、、田胞細胞受體（TCR)”的段落中。 Π· Τ 本發明細胞毒殺性Τ淋巴球可來避免之m藉由其身或與包括本發明之胜^ /或 4芍了調 41 201134480 節作用之外吐小體的其他藥物結合可被投予。所獲得之細胞毋殺n τ淋巴球起專一抗目標細胞的作用，#目標細胞其表現本發明胜肽，例如用於誘導之相同胜肽。目標細胞可為’’’田I其内生性表現，例如癌細胞，或被以 CLUAP1基因轉殖之細胞；且由於藉由胜肽刺激表現本發明胜肽於細胞表面之細胞，也可做為經活化之細胞毒殺性τ 淋巴球攻擊的目標。 νπ I. τ細胞受體（TCR) 本發明也提供一組合物其包括由編碼出可形成Τ細胞受體之次單位之多胜肽的核酸，與其使用方法。Τ細胞受體之次單位具有能力形成τ細胞受體，其授與專一性至抗腫瘤細胞的Τ細胞，腫瘤細胞表現CLUAP1。藉由使用本技術領域所知的方法可確認作為細胞毒殺性Τ淋巴球之τ細胞受體次單元之α -與沒-支鏈的核酸，而細胞毒殺性τ淋巴球以一或多個本發明之胜肽所誘導W02007/032255與 Morgan et al·，J Immunol, 171，3288 (2003)。例如，喜好以聚合酶鏈鎖反應方法來分析T細胞受體次單元。用於分析之聚合酶鍵鎖反應引子可為，例如5’ -R引子 (5， -gtctaccaggcattcgcttcat-3’ ）為 5’ 端引子（序列辨識號： 46 ) 與 3-TRa-C 引子 (5，-tcagctggaccacagccgcagcgt-3’ )專一於 T 細胞受體alpha鏈C區（序列辨識號：47) 、3-TRb-Cl引子 (5，-tcagaaatcctttc1;cttgac-3’ ）專一於 T 細胞受體 42 201134480 beta鏈Cl區（序列辨識號：48 )或3-TRbeta-C2引子（5，- ctagcctctggaatcctttctctt-3’ ）專一於 T 細胞受體 beta 鏈C2區（序列辨識號：49 )為3’ 端引子，但不限於其。引出之τ細胞受體可以高親合力結合表現CLUAP1胜肽之目標細胞’且視需要FO與ίΓΟ居中有效殺死表現 CLUAP1之目標細胞。編碼出Τ細胞受體次單位的核酸序列可合併進入適合之載體’例如反轉錄病毒載體。這些載體為本技術領域所熟知。通常包含其之核酸或載體可被轉移至一 Τ細胞，例如一來自一病患之Τ'細胞。有用地，本發明提供一現成 (off-the-shelf)的組合物允許快速修飾病人所擁有之τ 細胞（或其他哺乳動物之那些）以快速簡單產生具有優秀之癌症細胞殺死特性的經修飾T細胞。特定之T細胞受體可專一地辨認本發明之一胜肽與 HLA分子之複合物，當T細胞受體於τ細胞表面時給予τ 細胞抗目標細胞之專一活性。藉由任何已知方法可確認上述複合物之專一辨認’其較佳例子包括使用HLA分子與本發明胜肽之HLA多聚體染色（muitimer staining)分析，與 ELISP0T分析。藉由執行ELISp〇T分析，其可確認表現τ 細胞受體於細胞表面上之τ細胞藉由τ細胞受體辨認一細胞，且訊息傳送於細胞内。當複合物存在於τ細胞表面時藉由已知方法也可執行上述複合物可給予一 τ細胞細胞毒性活性的確認。較佳方法包括，例如，抗HLA陽性目標細胞之細胞毋性活性測定，例如鉻（chr〇m丨um)釋放分析。 43 201134480 本發明也提供細胞毒殺性τ淋巴球，其藉由以編碼出在HLA-24存在下與例如序列辨識號：3、4、5、r η 0、y、1 〇、 14、15、16與18之CLlJAP1胜肽結合，與以編碼出在h以1 存在下與序列辨識號：23、30、33、34、35、36與38之胜肽結合的T細胞受體次單位多胜肽的核酸轉導來製備。經轉導之細胞毒殺性T淋巴球可//? 叩自引導至癌症、屈胞，且可藉由熟知的培養方法4 叩擴張（例=The present invention also provides an anti-isolated pro-presented cell which exhibits a complex formed by the HLA 38 201134480 antigen and the peptide of the present invention on its surface. The antigen-presenting cells can be derived from a patient being treated and/or avoided, and by It may be administered as a vaccine by itself or in combination with other drugs including the peptide of the present invention, exosome or cytotoxic lymphocytes. Antigen presenting cells are not limited to a particular type of cell, and include dendritic cells, Langerhans cells (Langerhans ce 11 ), macrophages, B cells, and activated T cells, which are known to represent proteinaceous antigens. The cell surface is recognized by the lymphocytes. Since the dendritic cells are a typical antigen-presenting cell, they have the strongest cytotoxic T lymphocyte-inducing action in the antigen-presenting cells, and the dendritic cells are supplied using the antigen-presenting cells as the present invention. For example, an antigen can be obtained by inducing dendritic cells derived from peripheral blood mononuclear cells and then contacting (stimulating) the peptide with the present invention at F/FO or 7·/7 W叩. cell. When the peptide of the present invention is administered to an individual, an antigen presenting cells expressing the peptide of the present invention is induced in the body of the individual. The phrase "inducing an antigen presenting cell" includes contacting (stimulating) a cell with a peptide of the present invention or a nucleotide encoding the peptide of the present invention to express a complex formed between the HLA antigen and the peptide of the present invention. surface. Therefore, the antigen-presenting cells of the present invention can be obtained by collecting antigen-presenting cells from the individual after administering the peptide of the present invention to one body. Alternatively, the antigen-presenting cells of the present invention can be obtained by contacting antigen-presenting cells collected from an individual with the peptide of the present invention. The antigen presenting cells of the present invention can be administered to a body to induce an anti-cancer immune response in an individual 'by itself or with other peptides including the peptides of the present invention, exosome or cytotoxic lymphocytes Drug combination. Example 39 201134480 For example, ^F/ administration can include the steps of: a: collecting antigen presenting cells from a first individual, b. presenting cells with the peptide contacting step a of the peptide, and c. presenting the antigen of step b to the cell Give a second individual. The first individual and the second individual may be the same individual or may be different individuals. Alternatively, according to the present invention, there is provided the use of a peptide of the present invention for the manufacture of a pharmaceutical composition for inducing antigen presenting cells. Furthermore, the invention provides a method or process for making a pharmaceutical composition that induces antigen presenting cells. Still further, the present invention also provides a peptide of the present invention for inducing antigen-presenting cells. The antigen-presenting cells obtained from step b can be used as a vaccine for treating and/or preventing cancer, such as breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma. Dirty cancer. According to one aspect of the invention, the antigen presenting cells of the invention have a high degree of cytotoxic T lymphocyte evokengsing ability. In the phrase "high degree of cytotoxic T lymphocyte evoking ability", the degree of contact with the peptide by the antigen is not as high as that of the peptide which is incapable of inducing cytotoxic T lymphocytes. Such antigen-presenting cells having a high degree of cytotoxic T lymphocyte-inducing ability can be prepared by a method comprising the step of transferring k-nucleoside Si encoding the peptide of the present invention to an antigen-presenting cell and the above method. The introduced gene can be in the form of DNA $ RNA. Examples of the introduction method include, without particular limitation, various methods generally performed in the field can be used, for example, lipofecti on, electroporaUon or carbonated mother method can be used more particularly Executing it as in Cancer Res 1996, 56: 5672-7; 40 201134480 * J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Japanese Translation 〇f International Publication No. 2000-509281 Said. By transferring a gene into an antigen-presenting cell, the gene undergoes transcription, translation and such a protein in the cell 'and the protein obtained afterwards is treated by MHC C1 ass I or C1 ass 11' and via a presentation pathway to present the peptide . VII. Cytotoxic T lymphocytes against any of the peptide-inducible cytotoxic T lymphocytes of the present invention enhances the immune response to cancer cells, and thus, as such, can be used as a vaccine to resemble a victory The way of peptides. The invention therefore provides isolated cytotoxic T lymphocytes which are specifically induced or activated by any of the peptides of the invention. Such a cytotoxic tau lymphocyte can be obtained by (1) administering the peptide of the present invention to a single body; or (2) antigen-presenting cells from an individual and CD8-positive cells or peripheral blood mononuclear lymphocytes The peptide of the present invention is h κ/π contact (stimulation) or (3) a CD8-positive cell or a peripheral blood mononuclear lymphocyte and a complex which expresses a HU antigen and a peptide: the antigen on the surface thereof presents a cell or a small exocytosis The body contact or (4) introduces a gene encoding a polynuclear acid that binds to a cell receptor that binds to the peptide of the present invention. The method of preparing such an anti- or exosome body by the above method and the method of (4) is described in detail in the paragraph below, the cell cell receptor (TCR). Π· Τ The cell of the present invention is poisoned. The sputum lymphocytes can be prevented from being administered by their body or in combination with other drugs including the sputum of the present invention. Killing n τ lymphocytes acts as a specific anti-target cell, #target cells which express the peptide of the present invention, for example, for the same peptide to be induced. The target cell may be '''I's endogenous performance, such as cancer cells, Or cells that have been transfected with the CLUAP1 gene; and because the peptides of the present invention are expressed on the cell surface by stimulation of the peptide, they can also be used as targets for activated cytotoxic T lymphocytes. νπ I. Receptor (TCR) The present invention also provides a composition comprising a nucleic acid encoding a multi-peptide which can form a subunit of a purine cell receptor, and a method of using the same. The subunit of the purine cell receptor has the ability to form a tau cell. Body, specificity to resistance The sputum cells of the tumor cells, the tumor cells exhibit CLUAP 1. The α-and non-branched nucleic acids of the tau cell receptor subunit of the cytotoxic sputum lymphocytes can be confirmed by using methods known in the art, and the cells are Toxic tau lymphocytes are induced by one or more peptides of the invention W02007/032255 and Morgan et al, J Immunol, 171, 3288 (2003). For example, prefer to analyze T cells by polymerase chain reaction method. Receptor subunit. The polymerase keying reaction primer used for analysis can be, for example, a 5'-R primer (5, -gtctaccaggcattcgcttcat-3') is a 5' end primer (SEQ ID NO: 46) and 3-TRa- The C primer (5,-tcagctggaccacagccgcagcgt-3') is specific to the T cell receptor alpha chain C region (SEQ ID NO: 47), and the 3-TRb-Cl primer (5, -tcagaaatcctttc1; cttgac-3') is specific to T cells. Receptor 42 201134480 beta chain Cl region (SEQ ID NO: 48) or 3-TRbeta-C2 primer (5, - ctagcctctggaatcctttctctt-3') specific to T cell receptor beta chain C2 region (SEQ ID NO: 49) is 3 'end primer, but not limited to it. The receptor can bind to the target cell expressing CLUAP1 peptide with high affinity' and effectively kill the target cell expressing CLUAP1 by FO and ΓΟ. The nucleic acid sequence encoding the Τ cell receptor subunit can be incorporated into a suitable vector' Retroviral vector. These vectors are well known in the art. A nucleic acid or vector containing the same can be transferred to a cell, such as a cell from a patient. Usefully, the present invention provides an off-the-shelf composition that allows for rapid modification of tau cells (or those of other mammals) possessed by a patient to rapidly and easily produce modified cells with excellent cancer cell killing properties. T cells. The specific T cell receptor can specifically recognize the complex of one peptide of the present invention and the HLA molecule, and the specific activity of the tau cell against the target cell when the T cell receptor is on the surface of the tau cell. The specific recognition of the above complex can be confirmed by any known method. Preferred examples thereof include HLA multimer staining analysis using HLA molecules and the present peptide, and ELISP0T analysis. By performing the ELISp〇T analysis, it was confirmed that the tau cells expressing the tau cell receptor on the cell surface recognize a cell by the tau cell receptor, and the message is transmitted into the cell. When the complex is present on the surface of the tau cell, the above complex can also be administered to confirm the cytotoxic activity of a tau cell by a known method. Preferred methods include, for example, assays for cellular activity of anti-HLA positive target cells, such as chromium (chr〇m丨um) release assays. 43 201134480 The present invention also provides cytotoxic tau lymphocytes by encoding in the presence of HLA-24 with, for example, sequence identification numbers: 3, 4, 5, r η 0, y, 1 〇, 14, 15, 16 and 18 of the CL1JAP1 peptide binds to the T cell receptor subunit with a peptide encoding the sequence identification number: 23, 30, 33, 34, 35, 36 and 38 in the presence of h in the presence of 1 The peptide is prepared by nucleic acid transduction. Transduced cytotoxic T lymphocytes can be self-directed to cancer, stagnation, and can be expanded by well-known culture methods (example =

Kawakami et al. , J Immunol., 142, 3452-3461 (ig89)} 〇本發明細胞毒殺性T淋巴球也可用來形成一致免疫組合物，其於一需要治療或保護之病患中治療或避免癌症為有效（W02006/031221 )。避免與預防包括任何活性，其減少死亡率之負載或來自疾病之死亡率。避免與預防可發生於“初期、第二期與第一期避免層級”。初期避免與預防避免了疾病之發展，而第-期與第三期層級之避免與預防包括藉由恢復功能與減少疾病相關併發症，以疾病之發展與症狀之浮現及減少已建立之疾病之負向發展的避免與預防為目的。或者，治療或避免包括一廣範圍之預防疾病治療，纟以減緩特別疾病之嚴重度為目標，例如減少腫瘤之增殖與轉移、減少血管新生。癌症之治療及/或預防，或，及/或其手術後復發的避免〇括任何下列步驟，例如癌細胞之手術移除、似癌細胞 =生長抑制、腫瘤之衰退或退化、癌發生之減緩與抑制的 έ導腫瘤退化與血管新生抑制的誘導。癌症之有效治療 44 201134480 及/或預防減少致死率與改善具有瓜屁之個體的預後、減低癌症輮記於血液中的程度與減緩牛隨者癌症之可偵測症狀。例如，症狀之減輕或改善構成有效治療及/或預防，其包括_、2。％、30%或更加減輕，或穩定疾病。、 ΪΧ.藥學物質或組合物由於與正常組織相較，CLUAP1表現於癌症中特別被提尚’癌症的例子包括’但不限於乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、騰臟癌，本發明之胜狀或編碼出此類胜肽之多核皆酸可用於癌症之治療及/或預肖，及/或避免其手術後之復發。因此’本發明提供一藥學物質或組合物用來治療及/ 或避免其手術後之復發，此類組合物包括_或多個本發明胜肽或多料酸作為活性成分。或者，本發明之胜肽可表現於任何前述外吐小體或細胞表面，例如抗原呈現細胞，以用來作為藥學物質或組合物。此外，上述以本發明任何胜肽為標的之細胞毒殺性T淋巴球也可用來作為本發明藥學：質或組合物之活性成分。本發明之藥學物質與組合物也提仏使用如疫田。在本發明内文中措辭“疫苗，，（也指-致免疫組合物）意指一物質，其藉由接種至動物具有誘導抗腫瘤免疫力。本發明之藥學物質或組合物可用於治療及/或避免癌症，及/或其手術後復發的避免於一個體或病患中，個體或病患包括人類與任何其他偶動物，#包括，但不限於小 45 201134480 ’綿羊、山羊、諸、牛、一商業上重要動物或被鼠、大鼠、天竺鼠、兔子、猶、狗馬、猴子、狒拂與黑猩猩，特別是馴養了的動物。在另一實施例中，症或腫瘤之藥學組合物其擇自：本發明也在製造用以治療或避免癌或物質中提供一活性成分的使用， (a) 本發明胜肽； (b) 於一可表現之形式， Λ 編碼出如此處揭露之此種胜肽的核酸； (c) 表現本發明一胜肽於复^ ^ ^ ^ ^ ^ ^ ^ 丹衣面上之抗原呈現細胞或外吐小體；以及 (d) 本發明之細胞毒殺性τ淋巴球。或者，本發明更提供—用用以治療或避免癌症或腫瘤的活性成分擇自： (a) 本發明胜肽； (b) 於一可表現之形式，編碼出如此處揭露之此種胜肽的核酸； (c) 表現本發明-胜肽於其表面上之抗原呈現細胞或外吐小體；以及 (d) 本發明之細胞毒殺性τ淋巴球。或者，本發明更提供-製造用以治療或避免癌症或腫瘤之藥學組合物或物質的方法或製程，其中方法或製程包括將-藥學上或生理上可接受之載體與一活性成分一起配製的步驟，活性成分擇自： 46 201134480 (a) 本發明胜肽； (b) 於一可表現之形式，編碼出如此處揭路之此種胜肽的核酸； (c) 表現本發明—胜狀於1矣二胜肽於其表面上之抗原呈現細胞或外吐小體；以及 (d) 本發明之細胞毒殺性τ淋巴球，為活性成分。在另貫施例中，本發明也提供—製造用以治療或避免癌症或腫瘤之藥學組合物或物質的方法或製程，盆中方法或製程包括將一藥學上戋生次玍理上可接受之載體與一活性成分一起混合的步驟’其中活性成分擇自： (a) 本發明胜肽； (b) 於一可表現之形式，編碼出如此處揭露之此種胜肽的核酸； (c) 表現本發明—胜狀於其表面上之抗原呈現細胞或外吐小體；以及 (d) 本發明之細胞毒殺性τ淋巴球。根據本發明’已發現具有擇自序列辨識號：2至2 2中之胺基鲅序列的胜肽為hla_A24限制之抗原決定位胜肽或候選物，且也已發現具有序列辨識號：23與25至43之胺基鲅序列的胜肽為HLA_A2限制之抗原決定位胜肽或候選物，其可誘導強而專一之免疫反應。因此包括任一具有序列辨識號· 3、4、5、8、9、10、14、15、16、18 與 23、 30、33、34、35、％、38之胺基酸序列之胜肽的本發明藥 47 201134480 學物質或組合物特別適合投予HU抗原為分別HLA — 心與 HLA A2之個冑相同的東西提供至包含編碼出任何這些胜肽之多核皆酸（即，本發明之多核苷酸）㈣學物質絲合物。由本發明藥于物質或組合物治療之癌症不限於且包括其中關於CLUAP1 (例如，為過度表現）之任何之癌症，包括’例如乳癌、子宮頸癌…大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌。本發明藥學物質或組合物可包括除了上述活性成分外’具有誘導細胞毒殺性τ淋巴球抗似癌細胞之能力的其他胜肽、編碼出此其他胜肽之其他多核苷酸、其他表現此其他胜肽之細胞或此類。於此，具有誘導細胞毒殺性τ淋巴球抗似癌細胞之能力的其他胜肽由癌症專一抗原所例示 (例如，經定義之腫瘤相關抗原），但不限於此。若需要，本發明之藥學物質或組合物可視需要包括其他治療物質為活性成分，只要此物質不抑制活性成分之抗腫瘤功效，活性成分例如任何本發明胜肽。例如，配方可包括抗發炎物質或組合物、止痛劑、化學治療與其類似。除了其他治 :物質於藥劑其本身中’也可將本發明之藥劑與一或多個〃他藥學物質或組合物相繼或同時投予。藥劑與藥學物質 $組合物的量依照，例如使用何種藥學物質或組合物、要》台療之疾病與投藥的計畫與方式。 ^應瞭解的是，除了此處特別提及之成分外，本發明之 '于物質或組合物可包括本技術領域一般之其他物質或組 48 201134480 合物，其具有關於討論中之配中，本發明之缠& 己方形式。在本發明一實施例二本：月之樂學物質或組合物可被包含於製造之商品* 套組，其包含對於要被治療之 /、 yk m ^ 、扃例如癌症的病理情況有用之材枓。製造之商品可藥與铷哲—人匕括八有一標織之任何本發明與試管。容的合盗包括瓶、小瓶（V1al) °° 乂成自各種材料，例如玻璃或塑膠。於容器上之標籤需指出物質或組合物為用來治療或避免疾病之或多個情況。標籤也可指出投藥指示等。除了上述容器外’套组包括本發明藥學物質或組合物可視需要更進一步包括一第二容器，其儲藏一藥學上可接受之稀釋液。其可更包括商業或使用者觀點需要之其他材料’包括其他緩衝溶液、稀釋液、濾器、針、注射器與具有使用說明之包裝插入物。藥學組合物若需要可被呈現於包（pack)或一分配器，其可包含含有活性成分之一或多單位劑量形式。包裝可例如包括金屬或塑膠羯，例如一泡棉箱（blister pack)。包或分配器可伴隨著投藥指示。 (1 )藥學物質或組合物包含胜肽作為活性成分可直接投予本發明胜肽為一藥學物質或組合物，若需要的話，其已被一般配方方法所配製。在之後的例子，除了本發明胜肽外、若適合可包括載體、賦形劑與原始做為藥物使用之此類而無特別限制。上述載體的例子為滅菌水生理食鹽水、磷酸緩衝溶液與培養液體（culture nuid) 與此類。更進一步而言，若必須，藥學物質或組合物可含 49 201134480 t疋劑、懸液劑、防腐劑、界面活性劑與此類。本發明之藥學物質或組合物可用來抗癌目的。可將本發明之胜肽製備為一組合，其由兩或更多個本發明之胜肽所組成’以j/2 h叩誘導細胞毒殺性T淋巴球。胜肽組合可以雞尾酒形式執行或可使用標準技術彼此結合。例如，胜肽可被化學連接或表現如一單—融合多胜肽序列’其可具有一或數個胺基酸為連接器（例如，Lysine linker： K. S. Kawamura et al. J. Immunol. 2002, 168: 5709 5715)。結合之胜肽可為相同或不同。藉由投予本發明之胜肽，藉由HLA抗原，高密度呈現胜肽於抗原呈現細胞上’之後對形成於呈現胜肽與HLA抗原之間的複合物專一反應之細胞毒殺性ΐ淋巴球被誘導。或者，抗原呈現細胞（例如，樹突細胞）自一個體移出且之後以本發明胜肽刺激以獲得表現任何本發明之胜肽於其細胞表面之抗原呈現細胞。將這些抗原呈現細胞再投予至該個體以於該個體中誘導細胞毒殺性Τ淋巴球，且因此可增加朝向腫瘤相關内皮的侵犯。驭組合物，其包含療及/或發明之胜肽為活性成分，也可包含一已知為有效建立細免疫力之佐劑。或者藥學組合物可與其他活性成分一起技予，或以配製成細粒被投予。佐劑指一任何化合物、 f或組合物，當與具有免疫活性之蛋白質一起投予（或次）時，其增強抗蛋白質之免疫反應。於此考慮之佐劑 i 括於文獻（Clin Microbiol Rev 1 994，7: 277 89)中 50 201134480 描述的那些。適合之佐劑的例子包括，但不限於磷酸鋁、氮氧化紹、明濛、霍亂毒素、沙門氏菌毒素、佛氏不完全佐劑（Incomplete Freund，s adjuvant, IFA)、佛氏完全佐劑（Complete Freund’s adjuvant, CFA)、 ISCOMatrix、 GM_CSF、CpG、0/W乳劑與其類似物。此外’於微脂體（1 i posome )配方與細粒配方中，胜肽連結至幾個微米直徑之小珠，且於配方中，可便利地使用連結至胜肽之脂質。在本發明另一實施例中，本發明胜肽也可以一藥學上可接受之鹽類被投予。鹽類之較佳例子包括具有驗金屬之鹽、具金屬之鹽、具有機鹼之鹽、具有機酸之鹽與具無機酸之鹽。如此處所使用“藥學上可接受之鹽類意指維持化合物生物有效性與特性及獲得自與無機酸或鹼，例如鹽酸、氫溴酸、硫酸、硝酸、磷酸、曱基磺酸（methanesulfonic acid)、乙基磺酸（ethanesulfonic acid)、對曱苯磺酸（p — toluenesulf〇nic aCid) ' 水楊酸 (salicylic acid)與其類似物反應的那些鹽。在一些實施例中，本發明之藥學物質或組合物可更包括一成分其啟動細胞毒殺性T淋巴球。已定義脂質為可如以叩啟動抗病毒抗原之細胞毒殺性T淋巴球的物質或組合物。例如’可將棕橺酸殘基黏附至離胺酸殘基之ε _與以_ 胺基，且之後連結至本發明之一胜肽。之後脂質胜肽可被直接投予於微胞或顆粒中、併入微脂體或乳化於一佐劑中。如脂質啟動細胞毒殺性Τ淋巴球反應之另一例子，$ 脂蛋白’例如三軟脂酸-S甘油半胱氨酰—絲氨酰基絲 51 201134480 氨酸（tripalmitoyl-S-giyCerylCySterny_sery卜serine) 可使用來啟動細胞毒殺性T淋巴球，當共價附加至一合適之胜肽（參見，例如 Deres et al.，Nature 1 989，342: 56 卜 4)。投藥之方法可為口服、皮膚内、皮下、靜脈内注射或此類’以及全身投藥或局部投藥至標的位置的鄰近區域。可執行單次投樂或藉由多次投藥追加。本發明之胜肽劑量可適合地調整根據要治療之疾病、病患年紀、體重、投藥方法、與此類，且本發明之胜肽劑量一般為〇. 〇(n mg至 1 0 0 0 mg ’例如0. 1 mg至10 mg，且可於數天至數個月投藥认。熟悉此技藝人士可適合地選擇一合適的劑量。 (2)藥學物質或組合物包含多核苦酸為活性成分本發明之藥學物質或組合物也可包含編碼出此處揭露之胜狀的核酸於一可表達之形式中。此處措辭“於一可表達之形式中意指多核普酸，當引入一細胞，//? 會Kawakami et al., J Immunol., 142, 3452-3461 (ig89)} The cytotoxic T lymphocytes of the present invention can also be used to form a consensus immunological composition that is treated or avoided in a patient in need of treatment or protection. Cancer is effective (W02006/031221). Avoidance and prevention include any activity that reduces the burden of mortality or mortality from disease. Avoidance and prevention can occur in the “initial, second and first phase avoidance levels”. Early avoidance and prevention avoids the development of the disease, and the avoidance and prevention of the first and third stages includes the restoration of function and the reduction of disease-related complications, the development of the disease and the emergence of symptoms and the reduction of established diseases. The purpose of avoiding and preventing negative development is to achieve. Alternatively, treatment or avoidance includes a wide range of disease prevention treatments, with the goal of slowing the severity of specific diseases, such as reducing tumor proliferation and metastasis, and reducing angiogenesis. The treatment and/or prevention of cancer, or/and its recurrence after surgery, includes any of the following steps, such as surgical removal of cancer cells, cancer-like growth inhibition, tumor regression or degeneration, and slowing of cancer Induction of Tumor Degeneration with Inhibition and Induction of Angiogenesis Inhibition Effective treatment of cancer 44 201134480 and / or to prevent reduction in mortality and improve the prognosis of individuals with melons, reduce the extent of cancer in the blood and slow down the detectable symptoms of bovine cancer. For example, a reduction or improvement in symptoms constitutes an effective treatment and/or prevention, including _, 2. %, 30% or less, or stabilize the disease.药学.Pharmaceutical substance or composition CLUAP1 is particularly recommended in cancer as compared with normal tissues. Examples of cancer include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse type. Gastric cancer, lymphoma, neuroblastoma, sputum cancer, the victory of the present invention or the polynucleic acid encoding such a peptide can be used for the treatment and/or pre-surgery of cancer, and/or to avoid recurrence after surgery . Thus, the present invention provides a pharmaceutical substance or composition for treating and/or avoiding recurrence after surgery, such compositions comprising - or a plurality of peptides or polyacids of the present invention as active ingredients. Alternatively, the peptide of the present invention may be expressed on any of the aforementioned exosome or cell surface, such as antigen presenting cells, for use as a pharmaceutical substance or composition. Furthermore, the above-mentioned cytotoxic T lymphocytes which are subject to any of the peptides of the present invention can also be used as the active ingredient of the medicinal substance or composition of the present invention. The pharmaceutical substances and compositions of the present invention are also useful for use as a field. The phrase "vaccine," (also referred to as an immunogenic composition), is used in the context of the present invention to mean a substance which induces anti-tumor immunity by inoculation into an animal. The pharmaceutical substance or composition of the present invention can be used for treatment and/or Or avoiding cancer, and/or its recurrence after surgery is avoided in a single body or patient, including individuals and any other even animals, #include, but not limited to small 45 201134480 'Sheep, goat, mutton, cattle a commercially important animal or a rat, a rat, a guinea pig, a rabbit, a juvenile, a dog, a monkey, a donkey and a chimpanzee, especially a domesticated animal. In another embodiment, the pharmaceutical composition of the disease or tumor The invention is also: The invention also provides for the use of an active ingredient in the treatment or avoidance of cancer or a substance, (a) a peptide of the invention; (b) in a form that can be expressed, Λ encoded as disclosed herein a nucleic acid of such a peptide; (c) an antigen presenting cell or exosome expressing a peptide of the present invention on a complex surface; and (d) a cytotoxicity of the present invention τ lymphocytes. Or, this Further provided - the active ingredient for treating or avoiding cancer or tumor is selected from: (a) a peptide of the invention; (b) a nucleic acid encoding such a peptide as disclosed herein in a form that can be expressed; (c) an antigen presenting cell or exocytosis of the present invention - a peptide on its surface; and (d) a cytotoxic tau lymphocyte of the invention. Alternatively, the invention provides - manufacturing for treatment or avoidance A method or process for a pharmaceutical composition or substance of a cancer or a method, wherein the method or process comprises the step of formulating a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from: 46 201134480 (a) Inventing the peptide; (b) in a form that can express the nucleic acid of such a peptide as disclosed herein; (c) exhibiting the present invention - the antigen present on the surface of the peptide a cell or an exocytosis; and (d) a cytotoxic tau lymphocyte of the invention as an active ingredient. In a further embodiment, the invention also provides a pharmaceutical composition for the treatment or avoidance of cancer or tumor Or material method or system The method or process in the pot comprises the step of mixing a pharmaceutically acceptable carrier with an active ingredient, wherein the active ingredient is selected from: (a) a peptide of the invention; (b) a form of expression encoding a nucleic acid of such a peptide as disclosed herein; (c) exhibiting the present invention - an antigen presenting cell or exosome on its surface; and (d) cytotoxicity of the present invention τ lymphocytes. According to the present invention, it has been found that a peptide having an aminopurine sequence selected from the sequence identification numbers: 2 to 2 2 is an epitope-determining peptide or candidate restricted by hla_A24, and has also been found to have sequence identification. The peptides of the amino group 23 sequence of 23 and 25 to 43 are HLA_A2 restricted epitope peptides or candidates which induce a strong and specific immune response. Therefore, any peptide having the amino acid sequence of sequence number 3, 4, 5, 8, 9, 10, 14, 15, 16, 18 and 23, 30, 33, 34, 35, %, 38 is included. The present invention 47 201134480 The substance or composition is particularly suitable for administration of HU antigen to HLA - the same thing as HLA A2, to the polynuclear acid containing any of these peptides (ie, the present invention) Polynucleotide) (4) Material substance silk compound. The cancer treated by the agent of the present invention in a substance or composition is not limited to and includes any cancer in which CLUAP1 (for example, is overexpressed), including 'e.g. breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse type Gastric cancer, lymphoma, neuroblastoma, pancreatic cancer. The pharmaceutical substance or composition of the present invention may include, in addition to the above-mentioned active ingredients, other peptides having the ability to induce cytotoxic lymphocytes against cancer-like cells, other polynucleotides encoding the other peptides, and others. The cell of the peptide or the like. Here, other peptides having the ability to induce cytotoxic cytotoxicity against cancer-like cells are exemplified by cancer-specific antigens (e.g., defined tumor-associated antigens), but are not limited thereto. If desired, the pharmaceutical substance or composition of the present invention may optionally include other therapeutic substances as active ingredients, as long as the substance does not inhibit the antitumor efficacy of the active ingredient, such as any of the peptides of the present invention. For example, the formulation may include an anti-inflammatory substance or composition, an analgesic, chemotherapeutic and the like. The agent of the present invention can be administered sequentially or simultaneously with one or more pharmaceutically acceptable agents or compositions, among other things, in the agent itself. The amount of the agent and the pharmaceutical substance is in accordance with, for example, the pharmaceutical substance or composition used, the disease and the plan and method of administration. It is to be understood that the "substance or composition of the present invention", in addition to the ingredients specifically mentioned herein, may include other substances or groups 48 201134480 in the art, which have a discussion in the discussion, The wrap & own form of the invention. In a second embodiment of the present invention, the monthly music material or composition may be included in the manufactured article* kit, which contains materials useful for the pathological condition of the yk m ^ , 扃 such as cancer to be treated. Hey. The manufactured goods are medicinal and 铷哲-人匕八八有一有一有一之任何任何任何任何任何任何任何任何任何任何任何The thief of the capsule includes bottles and vials (V1al) °° from various materials such as glass or plastic. The label on the container indicates that the substance or composition is used to treat or avoid the disease or multiple conditions. The label can also indicate the indication of administration. In addition to the above containers, the kit comprising the pharmaceutical material or composition of the present invention may further comprise, if desired, a second container for storing a pharmaceutically acceptable diluent. It may include other materials that are required by a commercial or user's opinion' including other buffer solutions, diluents, filters, needles, syringes, and package inserts with instructions for use. The pharmaceutical composition may be presented as a pack or a dispenser, if desired, which may contain one or more unit dosage forms containing the active ingredient. The package may, for example, comprise a metal or plastic file, such as a blister pack. The pack or dispenser can be accompanied by a medication indication. (1) A pharmaceutical substance or composition comprising a peptide as an active ingredient The peptide of the present invention can be directly administered as a pharmaceutical substance or composition, and if necessary, it has been formulated by a general formulation method. In the following examples, there are no particular limitations other than the peptide of the present invention, if appropriate, including carriers, excipients, and the original use as a drug. Examples of such carriers are sterile water physiological saline, phosphate buffer solution and culture nuid and the like. Further, if necessary, the pharmaceutical substance or composition may contain 49 201134480 t tanning agent, suspension, preservative, surfactant, and the like. The pharmaceutical substance or composition of the present invention can be used for anti-cancer purposes. The peptide of the present invention can be prepared as a combination consisting of two or more peptides of the present invention to induce cytotoxic T lymphocytes in j/2 h叩. The peptide combination can be performed in the form of a cocktail or can be combined with each other using standard techniques. For example, a peptide can be chemically linked or behave as a single-fused multipeptide sequence which can have one or several amino acids as a linker (eg, Lysine linker: KS Kawamura et al. J. Immunol. 2002, 168 : 5709 5715). The peptides to be combined may be the same or different. By administering the peptide of the present invention, the HLA antigen, the high density exhibits the peptide on the antigen presenting cells, and then the cytotoxic lymphocytes which are formed in response to the complex between the peptide and the HLA antigen. Induced. Alternatively, the antigen-presenting cells (e.g., dendritic cells) are removed from one body and then stimulated with the peptide of the present invention to obtain antigen-presenting cells exhibiting any of the peptides of the present invention on the cell surface thereof. These antigen-presenting cells are re-administered to the individual to induce cytotoxic sputum lymphocytes in the individual, and thus may increase invasion toward the tumor-associated endothelium. A bismuth composition comprising the therapeutic and/or inventive peptide as an active ingredient may also comprise an adjuvant known to be effective in establishing a fine immunity. Alternatively, the pharmaceutical composition may be administered together with other active ingredients or formulated as fine granules. An adjuvant refers to any compound, f or composition that, when administered (or in times) with an immunologically active protein, enhances an anti-protein immune response. The adjuvants considered herein are those described in the literature (Clin Microbiol Rev 1 994, 7: 277 89) 50 201134480. Examples of suitable adjuvants include, but are not limited to, aluminum phosphate, oxynitride, montmorillon, cholera toxin, salmonella toxin, Incomplete Freund (s adjuvant, IFA), complete Freund's adjuvant (Complete) Freund's adjuvant, CFA), ISCOMatrix, GM_CSF, CpG, 0/W emulsions and their analogs. Further, in the formulation of the liposome and the fine particle formulation, the peptide is linked to beads of several micrometer diameter, and in the formulation, the lipid linked to the peptide can be conveniently used. In another embodiment of the invention, the peptide of the invention may also be administered as a pharmaceutically acceptable salt. Preferred examples of the salt include a salt having a metal test, a salt having a metal, a salt having an organic base, a salt having an organic acid, and a salt having an inorganic acid. As used herein, "pharmaceutically acceptable salts" means maintaining the biological effectiveness and properties of the compound and obtaining it from inorganic acids or bases such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid. , ethanesulfonic acid, p-toluenesulfoxime a Cid, a salt of salicylic acid and its analogs. In some embodiments, the pharmaceutical material of the invention Or the composition may further comprise a component which initiates a cytotoxic T lymphocyte. The defined lipid is a substance or composition which can initiate a cytotoxic T lymphocyte of an antiviral antigen, such as 'a palm citrate residue The base is attached to the ε- and _ amine groups of the amino acid residue, and is then linked to one of the peptides of the present invention. The lipid peptide can then be directly administered into the micelles or granules, incorporated into the liposome or emulsified. In an adjuvant, another example of a lipid-inducing cytotoxic lymphocyte reaction, $lipoproteins such as tristearic acid-S glycerol cysteinyl-serine filament 51 201134480 lysine (tripalmitoyl-S -giyCer ylCySterny_seryb serine) can be used to initiate cytotoxic T lymphocytes when covalently attached to a suitable peptide (see, eg, Deres et al., Nature 1 989, 342: 56 Bu 4). Oral, intradermal, subcutaneous, intravenous or such 'and adjacent to systemic administration or topical administration to the vicinity of the target position. Single shots can be performed or added by multiple administrations. The peptide dose of the present invention can suitably be The dosage according to the disease to be treated, the age of the patient, the body weight, the method of administration, and the like, and the dosage of the peptide of the present invention is generally 〇. 〇 (n mg to 1 000 mg 'eg, 0.1 mg to 10 mg And can be administered in a few days to several months. Those skilled in the art can suitably select a suitable dosage. (2) The pharmaceutical substance or composition comprises polynucleic acid as the active ingredient. The pharmaceutical substance or composition of the present invention is also A nucleic acid encoding a winning form disclosed herein may be included in an expressible form. The phrase "in an expressible form, means a multi-nucleotide, when introduced into a cell, //"

被表現成一誘導抗腫瘤免疫力之多胜肽。在一代表實施例中’感興趣之多核苷酸的核酸序列包括對於表現多核苷酸而言必須之調控要素。可裝配多核苷酸以達到穩定插入目標細胞之基因體（參見，例如敘述同源重組盒式载體 (cassette vector)的 Thomas KR & Capecchi MR，Cell 1 987, 51: 503-12。也參見，例如 Wolff et al.，Science 1 990， 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739，118; 5,736,524; 5,679,647;與 WO 52 201134480 98/04720 )。舰輸送技術的例子包括“裸D·”、經促進 (buPiVacaine、聚合物、胜肽居中之）之輸送、陽離子脂質複合物與顆粒居中之（“基因搶”）或壓力居中之傳送 (參見’例如 U.s. Patent No. 5,922,687 )。本發明之胜肽也可藉由病毒或細菌載體來表現。表現載體的例子包括減弱病毒宿主，例如牛痘或禽痘。此方法包括使用牛痘病毒，例如為一載體以表現編碼胜肽之核苷酸序列。#由引入一宿主，此重組之牛痘病毒表現致免疫胜肽且因此引起一免疫反應。於免疫步驟中為有效之牛痘載體與方法敘述於，例如u_s. Patent N〇. 4,722,848。另一載體包括 BCG (Baci lie Calmette Guerin)。BCG 載體敘述於 Stover et al.，Nature 1 991, 35 1: 456-60 中。對於治療投藥或免疫有用之其他多種載體，例如腺與腺病毋相關之載體、反轉錄病毒載體、傷寒沙門氏菌 (Salmonella typhi)載體、經解毒之炭疽毒素載體與其類似為明顯的。參見，例如Shata et al.，Mol Med Today 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al.， In Vivo 2000， 14: 571_85。輸送多核苷酸進入一病患可為直接，於其例子中，病患直接暴露於一攜帶多核苷酸之載體，或為間接，於其例子中’細胞首先//? f/ 以感興趣之多核苷酸轉形，之後將細胞轉殖進入病患。此兩方法分別為已知，為與ez p/fo基因治療。基因治療之方法之大體回顧，參見 Goldspiel et al., Clinical Pharmacy 1993，\ 2 . 488-505 53 201134480It is expressed as a multi-peptide that induces anti-tumor immunity. The nucleic acid sequence of the polynucleotide of interest in a representative embodiment includes regulatory elements necessary for the expression of the polynucleotide. Polynucleotides can be assembled to achieve stable insertion into the genome of the target cell (see, for example, Thomas KR & Capecchi MR, Cell 1 987, 51: 503-12, which describes a homologous recombination cassette vector. See also For example, Wolff et al., Science 1 990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 52 201134480 98/04720). Examples of ship transport technologies include "naked D.", transport of promoted (buPiVacaine, polymer, peptide centered), cationic lipid complexes and particles centered ("gene grab") or pressure-centered delivery (see ' For example, Us Patent No. 5,922,687). The peptide of the present invention can also be expressed by a viral or bacterial vector. Examples of performance vectors include attenuating viral hosts such as vaccinia or fowl pox. This method involves the use of a vaccinia virus, such as a vector to represent the nucleotide sequence encoding the peptide. By introducing a host, the recombinant vaccinia virus exhibits an immunogenic peptide and thus causes an immune response. The vaccinia carrier and method which are effective in the immunization step are described, for example, in U_s. Patent N〇. 4,722,848. Another vector includes BCG (Baci lie Calmette Guerin). The BCG vector is described in Stover et al., Nature 1 991, 35 1: 456-60. A variety of other vectors useful for therapeutic administration or immunization, such as vectors associated with glandular and adenosis, retroviral vectors, Salmonella typhi vectors, detoxified anthrax vectors, and the like are apparent. See, for example, Shata et al., Mol Med Today 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571_85. Delivery of a polynucleotide into a patient can be direct, in which case the patient is directly exposed to a vector carrying the polynucleotide, or indirectly, in the example of which the cell is first//?f/ is of interest The polynucleotide is transformed and the cells are then transferred into the patient. These two methods are known separately for treatment with the ez p/fo gene. For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 1993, \ 2 . 488-505 53 201134480

Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan,Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan,

Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in Biotechnology 1993，11(5): 155-215)。於重組DMA技術中一般熟知的方法其也可用於本發明’例如，欽述於編者A u s u b e 1 e t a 1., Current Protocols in Molecular Biology, John Wiley & Sons’ NY, 1993; and Kr i eger, Gene Transfer andScience 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in Biotechnology 1993, 11(5): 155-215). Methods well known in the art of recombinant DMA can also be used in the present invention', for example, in the editor of Ausube 1 eta 1., Current Protocols in Molecular Biology, John Wiley & Sons' NY, 1993; and Kr i eger, Gene Transfer and

Expression, A Laboratory Manual, Stockton Press, NY, 1 990。投藥之方法可為口服、皮膚内、皮下、靜脈内注射或此類，以及全身投藥或局部投藥至標的位置的鄰近區域提供使用。可執行單次投藥或藉由多次投藥追加。於適合載體中或於以編碼出本發明之胜肽的多核苷酸轉形之細胞中的多核苷酸的劑量可適合地調整，根據要治療之疾病、病患年紀、體重、投藥方法、與此類，且本發明之胜肽劑量一般為〇.001邶至1 000邶，例如0.001邶至1 000 mg，例如0. 1 mg至10 mg，且可於每數天一次至每數個月一次投藥。熟悉此技藝人士可適合地選擇一合適的劑量。 X.使用胜肽、少卜吐小體、抗原呈現細月包與細胞毒殺性 τ淋巴球的方法Expression, A Laboratory Manual, Stockton Press, NY, 1 990. The method of administration may be oral, intradermal, subcutaneous, intravenous, or the like, as well as administration in the vicinity of a systemic administration or topical administration to a target site. A single administration can be performed or by multiple administrations. The dosage of the polynucleotide in a suitable vector or in a cell transformed with a polynucleotide encoding the peptide of the present invention can be suitably adjusted depending on the disease to be treated, the age of the patient, the body weight, the method of administration, and Such a dosage of the peptide of the present invention is generally from 〇.001邶 to 1 000邶, for example from 0.001邶 to 1 000 mg, for example from 0.1 mg to 10 mg, and can be used every few days to every few months. Give it once. Those skilled in the art will be able to suitably select a suitable dosage. X. Method of using a peptide, a small corpuscle, an antigen to present a fine moon pack and a cytotoxic τ lymphocyte

現細胞與細胞毒殺性T淋巴球。甘毁來製備或誘導抗原呈也可使用本發明之外吐小 54 201134480 體與抗原呈現細胞來誘導細胞毒殺性τ淋巴球。胜肽、多核苦酸、外吐小體與抗原呈現細胞可與任何其他化合物: 合使用’只要化合物不抑制其細胞毒殺性τ淋巴球誘發能力。因此，任何上述之本發明藥學物質或組合物可用來誘導細胞毒殺性Τ淋巴球，且除此之外，包括胜肽與多核皆酸的那些也可用來誘導抗原呈現細胞，如於下所討論。 (1)誘導抗原呈現細胞的方法本發明提供使用本發明之胜肽或多核苷酸來誘導具有高細胞毒殺性τ淋巴球誘發能力之抗原呈現細胞的方法。本發明之方法包括h F/FO或7.刀Fi>0將抗原呈現細胞與本發明胜肽接觸的步驟。例如，h 或“ 將抗原呈現細胞與胜肽接觸的方法可包括步驟： a :自一個體收集抗原呈現細胞；以及 b:將步驟a之抗原呈現細胞與胜肽接觸。抗原呈現細胞並不限於特定種類之細胞，且包括樹突細胞、蘭格罕細胞（Langerhans cel 1)、巨嗜細胞、B細胞與活化之T細胞’已知其表現蛋白質（pr〇teinaceous)抗原於其細胞表面以被淋巴球所辨認。較佳為可使用樹突細胞，由於它們於抗原呈現細胞中具有最強的細胞毒殺性丁淋巴球誘發能力。可使用本發明任何胜肽以它們本身或與本發明其他胜肽一起。另一方面，當投予本發明一胜肽至一個體時，抗原呈現細胞//7 F/ 與胜肽接觸，因此於個體之體内誘導具有高細胞毒殺性T淋巴球誘發能力之抗原呈現細胞。因此， 55 201134480 本發明包括投予本發明胜肽至一個體。相似地，當本發明之多核苷酸投予至-個體於一可表達之形式"，本發明 -胜肽被表現且/…。與抗原呈現細胞接觸以因此於個體之體内誘導具有高細胞毒殺性τ淋巴球誘發能力之抗原呈現細胞。因此’本發明也包括投予本發明多核普酸至 -個體。“可表達之形式，，被定義於上述段落“ιχ•藥學物質或組合物、⑺藥學物質或組合物包含多核普酸為活性成分”中。本發明也可包括將本發明一多核苷酸引入一抗原呈現細胞以便誘導具有細胞毒殺性τ淋巴球誘發能力之抗原呈現細胞的步驟。例如，方法可包括步驟： a ·自一個體收集抗原呈現細胞；以及 b :將編碼出本發明胜肽之一多核皆酸引入。可如前述段落“ VI ·抗原呈現細胞，’中所述來執行步驟b。或者本發明提供一製備一專一誘導抗clUAP 1之細胞毒殺性T淋巴球活性的抗原呈現細胞的方法，其中該方法可包括下列步驟之一： (a) 將抗原呈現細胞與本發明一胜肽μ F/iro、α κ/ κσ或/·/? κ/ π接觸；以及 (b) 將編碼出本發明胜肽之一多核苷酸引入抗原呈現細胞。 (2 )誘導細胞毒殺性τ淋巴球的方法本發明也提供使用本發明胜肽、多核苷酸、外吐小體 56 201134480 或抗原呈現細胞來誘導細胞毒殺性τ淋巴球的方法。本發明也提供使用編碼出一多胜肽之多核苦酸來誘導細胞毒殺性τ淋巴球的方法，此多胜肽具形成一 τ細胞受體次單位的能力，而此Τ細胞受體次單位辨認一本發明胜肽與HLA抗原之複合物。較佳為，誘導細胞毒殺性Τ淋巴球的方法可包括至少一步驟擇自由下列之中： a) 將一 CD8陽性Τ細胞與一抗原呈現細胞及/或一外吐小體接觸，該抗原呈現細胞及/或該外吐小體表現一 HLA 抗原與本發明胜肽之複合物於其表面，以及 b) 將一多核苷酸引入一 CD8陽性T細胞，其中該多核苷酸編碼出一多胜肽，該多胜肽具形成一 T細胞受體次單位的能力，而該T細胞受體次單位辨認一本發明胜肽與 HLA抗原之複合物。當本發明之胜肽、多核苷酸、抗原呈現細胞或外吐小體被投予至一個體時’於個體體内誘導細胞毒殺性T淋巴球’且以癌細胞為目標之免疫反應的強度增強。因此，本發明方法包括將本發明之胜肽、多核苷酸、抗原呈現細胞或外吐小體投予至一個體的步驟。或者，藉由ez f/ fo使用它們，也可誘導細胞毒殺性 T淋巴球，且在誘導細胞毒殺性τ淋巴球後，經活化之細胞毒殺性T淋巴球可返回至個體。例如，方法可包括步驟： a :自一個體收集抗原呈現細胞； b :將步驟a之抗原呈現細胞與胜肽接觸；以及 c :將步驟b之抗原呈現細胞與CD8陽性細胞共培養。 57 201134480 於上述步驟C中要與CD8陽性細胞共培養之抗原呈現細胞也可藉由將-包括本發明多料酸之基因轉移進入抗原呈現細胞’如於前述段落“VI•抗原呈現細胞，，中所述來製備；然而，本發明並不限於此，1包括任何有效表現 HLA抗原與本發明胜肽之複合物於其表面的抗原呈現細胞。代替此種抗原呈現細胞’也可使用呈現一 h l a抗原與本發明胜肽之複合物於其表面的外吐小體。換句話說，本發明也包括’與呈現一 HLA抗原與本發明胜肽之複合物於其表面的外吐小體共培養之步驟。此種外吐小體可藉由前述於段落V.外吐小體”中之方法來製備。此外藉由將一包括編碼出與本發明一胜肽結合之τ 細胞受體次單元的多核苦酸的基因引入CD8陽性細胞也可誘導細胞毒殺性τ淋巴球。如於前述段πΠΙ. τ細胞受體（TCR)中所述可執行此轉導。此外，本發明也提供製造-誘導細胞毒殺性了淋巴球之藥學物質或組合物的方法或製程，#中該方法包括將本發明之胜肽與藥學上接受之載體一起混合或配製的步驟。 (3)誘導免疫反應的方法又’本發明提供誘導抗CLUAP1相關疾病之免疫反應的方法。適合的疾病包括癌症，其例子包括，但不限於乳癌、子宮頸癌、大腸直腸癌 '食道癌、[癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰臟癌。本發明方法可包括投予含任何本發明胜肽或編碼出其 58 201134480 之夕核苷酸的物質或組合物们’驟。本發明方法也考慮投予表現任何本發明胜肽之 Α β “ 卜吐J體或抗原呈現細胞。細節參見 Ιχ·藥學物質哎组人私，，之項目，特別是敘述本發明物質或組合物為疫苗之 κ w㈣分。此外，可被使用於本發明誘導免疫反廄之古〜的本發明外吐小體與抗原呈現細胞’被詳細描述在前“ 牡引之於ν.外吐小體，，、“νι抗原呈現細胞”與“ x # m u , al .使用胜肽、外吐小體、抗原呈現細胞與細胞毒殺性T淋巴球的方法，，之⑴與⑺的項目。本發明也提供製造—誘導免疫反應之藥學物質或組合物的方法或製私丨中該方法包括將本發明之胜肽與藥學上接受之載體一起混合或配製的步驟。或者本發明方法可包括投予-疫苗或藥學組合物的步驟，疫苗或藥學組合物包含： (a) 本發明胜肽； (b) 於-可表現之形式，編碼出如此處揭露之此種胜肽的核酸； )表見本毛明⑯肽於其表面上之抗原呈現細胞或外吐小體；或 (d)本發明之細胞毒殺性τ淋巴球。在本^明中’以這些活性成份可治療過度表現i 之癌症。此類癌症的例子包括，但不限於乳癌、子宮頸癌、大腸直腸癌' 食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、騰臟癌。因此，I包括活性成分之疫苗或藥學組合物的投予前，其較佳為確認與相同器官之正常組織相 59 201134480 較’ CLUAP1之表現程度於要被治療之細胞或組織中是否被提高。因此，在一實施例中，本發明提供在—需要的病患中/〇療（過度）表現CLUAP1之癌症的方法，此類方法可包括步驟： 1)測定獲得自具有癌症要治療之個體的細胞或組織中的CLUAP1表現程度； i i )與正常控制組比較CLUAP1表現程度；以及 111)投予擇自由上述（a)至（d)所組成之群組的至少一成份至與正常控制組相較具有過度表現CLUApi之癌症的個體。或者，本發明也可提供包含擇自上述（3)至（(1)中之群組的至少一成份的疫苗或藥學組合物，其要被投予至具有過度表現CLUAP1之癌症的個體中。換句話說，本發明更提供鑑定要被以本發明CLUAP1多胜肽治療之個體的方法，此類方法匕括測疋來自個體之癌細胞或組織中的API表現程度的步驟’其中與基因之正常控制組相較，此程度增加指出個體可具有可以本發明 CLUAP1多胜肽治療之癌症本發明之治療'癌的方法於以下更詳細敛述。可將任何源自個體之細胞或組織用於αυΑρι表現之測定，只要其包括α随之目標轉㈣轉譯產物。適合樣本的例子包括’但不限於身體組織或液體、例如血液、唾與尿液較佳為，生物樣本包含一細胞族群，其包括一上皮細胞’更佳為源自個體之細胞或組織包含一細胞族群，其包括-上皮細胞，更佳為一癌症上皮細胞或一來自 60 201134480 被懷疑癌化之組織的上皮細胞。此外，若需要，細胞可自所獲得之身體組織或液體被純化’且之後使用為源自個體之樣本。本發明之内容中’測定自已知為非癌症之生物樣本的控制組程度被意指為一 “正常控制組程度”。另一方面，控制組程度測定自一癌的生物組織，其意指為一“癌的控制組程度。可將介於樣本表現程度與控制組程度間的不同標準化至控制核酸的表現程度，例如管家基因 (housekeeping gene) ’根據細胞之癌症與非癌程度已知其表現程度並無不同。示範之控制基因包括，但不限於点肌動蛋白（beta actin)、甘油醛j磷酸去氫酶 (glyceraldehyde-3-phosphate dehydr〇genase)與核糖蛋白P1。要藉由本發明治療之個體較佳為一哺乳類動物。示範之哺乳類動物包括，但不限於，例如，人類、非人類靈長類動物、小鼠、大鼠、狗、貓、馬與牛。根據本發明，測定獲得自一個體之細胞或組織中的 CLUAP1表現程度。使用本技術領域已知方法可於轉錄（核酸）產物程度測定表現程度。例如，藉由雜合方法（例^ 北方雜合）使用探針可將CLUAP1的mRNA定量。曰 μ 士 J "、日日片、-陣列如此類或執行❹丨。陣列之使用可較佳為用於偵測CLUAP1表現程度。利用CL_的序列資訊熟技藝人士可製備此種探針。例如，CLUApi #⑽可被用為探針。若需要，可以適合之標誌來標誌探針，例如汰劑、榮光物質與同位素’且基因的表現程度可被侦測二 61 201134480 合標誌的強度。此外，藉由擴大偵測方法 (amplification-base detectin meth〇d)(例如，RT-pcR) 使用引子可將CLUAP1 (例如，序列辨識號：44)的轉錄產物進行定量。根據基因之可獲得序列資訊可製備此種引子。特別是’用於本方法之探針或引子於嚴厲 (stringent)、適度嚴厲、低嚴厲條件下雜合至cluapi的 mRNA。如此處使用，措辭“嚴厲（雜合）條件，，意指在此在條件下探針或引子會雜合至其目標序列，而不是其他序列。嚴厲條件為序列依賴（seqUence_dependent)，且在不同環境下會不同。比起較短之序列，於較高溫度下觀察到較長序列之特定雜合。一般而言，在一定義之離子強度與 pH下所選擇之嚴格條件的溫度為低於一特定序列之熔點 (Tm)約5C °Tm為溫度（在一定義之離子強度與與核酸濃度下），於其下在平衡下50%之互補至目標序列的探針雜合至目標序列。由於目標序列通常存在過量，所以於Tm , 在平衡下50%之探針被佔據。一般而言，嚴苛條件為於其中鹽濃度低於1.0 Μ鈉離子，一般約〇.〇1至1.〇 μ鈉離子（或其他鹽）於pH 7. 0至8. 3，且對於短探針或引子（例如’ 10至50個核苷酸）而言溫度為至少約30〇c，對於較長探針或引子而言溫度為至少約60°C。也可以添加去穩定物質（destabilizing substances)，例如曱醜胺 (for mam ide)來達到嚴苛條件。探針或引子可為特定大小。大小的範圍始於至少1 〇個核苷酸、至少12個核苷酸、至少15個核苷酸、至少20個 62 201134480 =酸、至少25個核Μ、至少3Q個核賴，且探針盘引子延伸大小始於5,個核普酸、1(M5個核芽酸、Μ。個核㈣、2G —25個核普酸與25-30個核苷酸。或者為了本發明之診斷可偵測轉譯產物。例如，可偵測CLUAP1蛋白質（序列辨識號：45)或其免疫片段之量。測定作為轉錄產物之蛋白質的量的方法包括免疫分析方法’其使用-抗體專-辨認此蛋白質。抗體可為單株或多株此外，抗體之任何片段或修飾C例如嵌合型抗體 (chimeric antlbody) 、 scFv 、 Fab ' F(ab’ ）2 、 π 等）可被用來偵測，、要片&或經修飾之抗體維持對蛋白質的結合能力。本發明也提供此類抗本發明胜肽與其片段之此類抗體。這些用於蛋白質读測之這些種類的抗體的製備方法為本技術領域所熟知，且任何方法可被使用於本發明中以製備此種抗體與其等同物（equivalent)。如根據CLUAP1基因轉譯產物偵測CLUApi基因之表現程度的另一方法，使用抗CLUAP1蛋白質之抗體經由免疫組織化學（immunohistochemical)分析可測量到染色強度。即，於此測量中，強的染色指出蛋白質/程度之增加的存在，且同時CLUAP1基因之高表現程度。可確認於癌症細胞中包括CLU API基因之目標基因的表現程度為被提升，若其相較於目標基因之控制組程度（例如，於正常細胞中的程度）增加’例如10%、25%、或50%，或增加大於11倍、大於1.5倍、大於2.0倍、大於5.0倍、大於ι〇·〇倍或更多。 63 201134480 藉由使用先前自一個體/其疾 ,a A 庆届卩1焱（癌的或非癌的）二一的個體收集並儲存的樣本控制組之程度可與癌細胞同時測疋。此外，獲得自具有癌症要被治療之一器官的非 :區=:細胞被使用為正常控制組。或者，根據獲得自刀析先則測定之來自其疾病 ΓτΠΛβ1 ^ m 1卸體之樣本中之 CLUAP1基因的表現程度的結果，稽田統s十方法，可測定控制，.且之程度。此外，控制組程 ^ ± J兩采自自先前測試細胞之表現輪廓的資料庫。並且，根很骒本發明—方面於一生物樣本中之CLUAP1基因的表現鞀许^ > ^ 又，可與多個控制組程度比較，其控制組程度被測定自多個 .Λ 夕1固翏考樣本。較佳為使用一控制組程度測定自一參考樣本，本，、來自—組織形式相似於源自個體生物樣本之組織心式此外，較佳為使用具有已知疾病階段之群組中的CI I丨A Ρ1甘mCell and cell toxic T lymphocytes. It is also possible to use the present invention to induce or induce antigen presentation. It is also possible to use the present invention to induce cells to induce cytotoxic lymphocytes. The peptide, the polynucleotide, the exosome and the antigen-presenting cells can be used in combination with any other compound as long as the compound does not inhibit its cytotoxic trampoline-inducing ability. Thus, any of the above-described pharmaceutical substances or compositions of the present invention can be used to induce cytotoxic sputum lymphocytes, and in addition, those including peptides and polynuclear acids can also be used to induce antigen-presenting cells, as discussed below. . (1) Method for inducing antigen-presenting cells The present invention provides a method of inducing antigen-presenting cells having high cytotoxic tau lymphocyte-inducing ability using the peptide or polynucleotide of the present invention. The method of the present invention comprises the step of contacting the antigen-presenting cells with the peptide of the present invention by h F/FO or 7. Knife Fi>0. For example, h or "the method of contacting the antigen presenting cells with the peptide may comprise the steps of: a: collecting the antigen presenting cells from one body; and b: contacting the antigen presenting cells of step a with the peptide. The antigen presenting cells are not limited Certain types of cells, including dendritic cells, Langerhans cel 1 , macrophages, B cells and activated T cells, are known to express protein (pr〇teinaceous) antigens on their cell surface to be Lymphocytes are recognized. It is preferred to use dendritic cells because they have the strongest cytotoxic lymphocyte-inducing ability in antigen-presenting cells. Any peptide of the present invention can be used as such or in combination with other peptides of the present invention. On the other hand, when a peptide of the present invention is administered to a body, the antigen exhibits a cell//7F/ contact with the peptide, thereby inducing a high cytotoxic T lymphocyte inducing ability in the individual. The antigen exhibits a cell. Thus, the present invention comprises administering a peptide of the invention to a single body. Similarly, when the polynucleotide of the present invention is administered to an individual The form of the present invention--the peptide is expressed and/or. The antigen-presenting cell is contacted to induce an antigen-presenting cell having a high cytotoxic tau lymphocyte-inducing ability in the body. Thus, the present invention also includes The polynucleic acid of the present invention is administered to an individual. The "expressible form" is defined in the above paragraph "a medicinal substance or composition, (7) a pharmaceutical substance or composition comprising a polynucleotide as an active ingredient". The present invention may also include a step of introducing a polynucleotide of the present invention into an antigen-presenting cell to induce an antigen-presenting cell having a cytotoxic trampoline-inducing ability. For example, the method can include the steps of: a: collecting antigen presenting cells from one body; and b: introducing a polynuclear acid encoding one of the peptides of the present invention. Step b may be performed as described in the aforementioned paragraph "VI · Antigen presenting cells," or the present invention provides a method of preparing an antigen presenting cell which specifically induces cytotoxic T lymphocyte activity against clUAP 1 , wherein the method One of the following steps may be included: (a) contacting the antigen presenting cells with a peptide of the present invention, μ F/iro, α κ/κσ or /·/?κ/π; and (b) encoding the peptide of the present invention One of the polynucleotides is introduced into the antigen-presenting cell. (2) Method for inducing cytotoxic tau lymphocytes The present invention also provides for inducing cells using the peptide, polynucleotide, exosome 56 201134480 or antigen-presenting cells of the present invention. Method for intoxicating tau lymphocytes. The present invention also provides a method for inducing cytotoxic tau lymphocytes using a polynucleic acid encoding a multi-peptide, the multi-peptide having the ability to form a tau cell receptor subunit, The sputum cell receptor subunit recognizes a complex of the inventive peptide and the HLA antigen. Preferably, the method of inducing the cytotoxic sputum lymphocyte comprises at least one step of selecting the following: a) placing a CD8 Positive Τ The cell is in contact with an antigen presenting cell and/or an exosome, the antigen presenting the cell and/or the exosome exhibiting a complex of an HLA antigen and a peptide of the present invention on its surface, and b) Nucleotide introduces a CD8-positive T cell, wherein the polynucleotide encodes a multi-peptide that has the ability to form a T cell receptor subunit, and the T cell receptor subunit recognizes an invention a complex of a peptide and an HLA antigen. When the peptide, polynucleotide, antigen-presenting cell or exosome of the present invention is administered to a body, 'inducing a cytotoxic T lymphocyte in an individual' The intensity of the immune response to which the cancer cell is targeted is enhanced. Therefore, the method of the present invention comprises the step of administering the peptide, polynucleotide, antigen presenting cell or exosome of the present invention to a single body. Alternatively, by ez f /fo uses them to also induce cytotoxic T lymphocytes, and after induction of cytotoxic T lymphocytes, the activated cellular toxic T lymphocytes can be returned to the individual. For example, the method can include the steps of: a: One body collecting antigen a cell; b: contacting the antigen-presenting cell of step a with a peptide; and c: co-culturing the antigen-presenting cell of step b with a CD8-positive cell. 57 201134480 An antigen to be co-cultured with CD8-positive cells in the above step C The presenting cells can also be prepared by transferring a gene including the polyacid of the present invention into an antigen presenting cell as described in the aforementioned paragraph "VI•Antigen-presenting cells," however, the present invention is not limited thereto, Any antigen-presenting cell comprising a complex of an HLA antigen and a peptide of the present invention on its surface is included. Instead of such an antigen presenting cell', it is also possible to use an exosome that exhibits a complex of the h1a antigen and the peptide of the present invention on its surface. In other words, the present invention also includes the step of co-cultivating the exosome with a complex of the HLA antigen and the peptide of the present invention on its surface. Such exosome can be prepared by the method described in the above paragraph V. Exosome. In addition, by including a multinuclear bitter cell encoding a tau cell receptor subunit that binds to a peptide of the present invention. The introduction of an acid gene into a CD8 positive cell can also induce a cytotoxic tau lymphocyte. This transduction can be performed as described in the aforementioned paragraph πΠΙ.τ cell receptor (TCR). In addition, the present invention also provides a manufacturing-induced cell poisoning. A method or process for the pharmaceutical substance or composition of a lymphocyte, wherein the method comprises the step of mixing or formulating the peptide of the present invention together with a pharmaceutically acceptable carrier. (3) A method for inducing an immune response The invention provides a method for inducing an immune response against a disease associated with CLUAP1. Suitable diseases include cancer, and examples thereof include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, [cancer, diffuse gastric cancer, lymphoma, nerve Maternal tumor, pancreatic cancer. The method of the invention may comprise administering a substance or composition comprising any of the peptides of the invention or the nucleotides encoding 58 201134480 of the invention. Administration showed any Α β peptide of the present invention wins "Bu J spit or antigen presenting cell. For details, see Ιχ·Pharmaceutical Substances, and, in particular, the substance or composition of the present invention is a κ w (four) of a vaccine. In addition, the exosome and antigen presenting cells of the present invention which can be used in the induction of the immune ruminant of the present invention are described in detail in the front of the sputum, and the "νι antigen" is presented. "Cell" and "x# mu, al. The use of peptides, exosomes, antigen-presenting cells and cytotoxic T-lymphocytes, (1) and (7). The present invention also provides a method of making a pharmaceutical substance or composition for inducing an immune response or a method of making a mixture comprising the step of mixing or formulating the peptide of the present invention together with a pharmaceutically acceptable carrier. Or the method of the invention may comprise the step of administering a vaccine or a pharmaceutical composition comprising: (a) a peptide of the invention; (b) in a form expressible, encoding such a The nucleic acid of the peptide; see the antigen presenting cell or exosome on the surface of the present invention; or (d) the cytotoxic lysate of the present invention. In the present invention, cancers which overexpress i can be treated with these active ingredients. Examples of such cancers include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, and smear cancer. Thus, prior to administration of the vaccine or pharmaceutical composition comprising the active ingredient, it is preferred to confirm whether the normal tissue phase of the same organ is higher than the degree of expression of 'CLUAP1' in the cell or tissue to be treated. Thus, in one embodiment, the invention provides a method of treating (over-expressing) a cancer of CLUAP1 in a patient in need thereof, the method comprising the steps of: 1) determining the individual obtained from the individual having the cancer to be treated Degree of expression of CLUAP1 in cells or tissues; ii) degree of expression of CLUAP1 compared to the normal control group; and 111) administration of at least one component of the group consisting of the above (a) to (d) to the normal control group More individuals with cancers that overexpress CLUApi. Alternatively, the present invention may also provide a vaccine or pharmaceutical composition comprising at least one component selected from the group consisting of (3) to ((1) above, which is to be administered to an individual having a cancer exhibiting excessive expression of CLUAP1. In other words, the present invention further provides a method of identifying an individual to be treated with the CLUAP1 multipeptide of the present invention, such a method comprising the step of measuring the degree of expression of an API in a cancer cell or tissue from an individual, wherein In comparison to the normal control group, this degree of increase indicates that the individual may have a cancer that can be treated with the CLUAP1 multipeptide of the present invention. The method of treating the 'cancer of the present invention is described in more detail below. Any cell or tissue derived from the individual can be used. The measurement of αυΑρι expression is as long as it includes α followed by the target (four) translation product. Examples of suitable samples include, but are not limited to, body tissues or liquids, such as blood, saliva and urine, and biological samples contain a cell population, Including an epithelial cell 'more preferably, the cell or tissue derived from the individual comprises a cell population comprising epithelial cells, more preferably a cancer epithelial cell or one from 60 201134480 Epithelial cells of a tissue suspected of being cancerized. Further, if desired, the cells can be purified from the obtained body tissue or liquid 'and then used as a sample derived from the individual. In the context of the present invention, 'determination is known as non- The degree of control of a biological sample of cancer is referred to as a "normal control group." On the other hand, the degree of control is determined from the biological tissue of a cancer, which means a degree of control of the cancer. The difference between the degree of sample performance and the degree of control group is standardized to control the degree of expression of the nucleic acid, for example, the housekeeping gene 'is not different depending on the degree of cancer and non-cancerous cell expression. The exemplary control genes include, However, it is not limited to beta actin, glyceraldehyde-3-phosphate dehydration genase and riboprotein P 1. The individual to be treated by the present invention is preferably a mammal. Mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses and cattle. According to the present invention, the degree of expression of CLUAP1 in a cell or tissue obtained from a body is determined. The degree of expression can be determined at the degree of transcription (nucleic acid) product using methods known in the art. For example, by hybridization method (example ^ northern miscellaneous The probe can be used to quantify the mRNA of CLUAP1. 曰μ士J ", 日日, Array, or such array. The use of the array can be preferably used to detect the degree of CLUAP1 expression. The sequence information can be prepared by a skilled artisan. For example, CLUApi #(10) can be used as a probe. If necessary, a suitable marker can be used to label the probe, such as a gonad, a glory substance and an isotope, and the expression of the gene. The degree can be detected by the intensity of the flag of 2011 201134480. In addition, transcription products of CLUAP1 (e.g., Sequence ID: 44) can be quantified using primers by amplification-base detectin meth〇d (e.g., RT-pcR). Such primers can be prepared based on the available sequence information of the gene. In particular, the probe or primer used in the method is hybridized to the cluapi mRNA under stringent, moderately severe, and low stringency conditions. As used herein, the phrase "severe (heterozygous) conditions means that the probe or primer will hybridize to its target sequence, but not to other sequences under this condition. Severe conditions are sequence dependent (seqUence_dependent) and in different environments The difference will be different. A specific sequence of longer sequences is observed at higher temperatures than the shorter sequence. In general, the temperature of a stringent condition selected at a defined ionic strength and pH is below a specific The melting point (Tm) of the sequence is about 5 C °Tm is the temperature (under a defined ionic strength and nucleic acid concentration), under which 50% of the probe complementary to the target sequence is hybridized to the target sequence. Usually there is an excess, so at Tm, 50% of the probe is occupied under equilibrium. In general, the harsh conditions are that the salt concentration is less than 1.0 Μ sodium ion, generally about 〇.〇1 to 1. 〇μ sodium Ions (or other salts) at pH 7. 0 to 8.3, and for short probes or primers (eg '10 to 50 nucleotides), the temperature is at least about 30 〇c for longer probes or The temperature of the primer is at least about 60 ° C. Destabilizing conditions can be achieved by the addition of destabilizing substances, such as for mam ide. Probes or primers can be of a specific size. The size ranges from at least 1 nucleotide to at least 12 Nucleotide, at least 15 nucleotides, at least 20 62 201134480 = acid, at least 25 nuclear rafts, at least 3Q nucleosides, and the probe disc primer extension size starts at 5, a nucleoside acid, 1 (M5 Nucleotrate, sputum, nucleus (4), 2G-25 nucleotides and 25-30 nucleotides. Alternatively, the translation product can be detected for the diagnosis of the present invention. For example, the CLUAP1 protein can be detected (SEQ ID NO: :45) or the amount of the immunological fragment thereof. The method for determining the amount of the protein as a transcription product includes an immunoassay method, which uses the antibody-specific antibody to identify the protein. The antibody may be a single plant or multiple strains, and any fragment of the antibody or Modifications C, such as chimeric antlbody, scFv, Fab 'F(ab')2, π, etc., can be used to detect, to support, and to modify the ability of the antibody to maintain binding to the protein. The invention also provides such a peptide resistant to the invention Such antibodies to fragments thereof. These methods of preparing these kinds of antibodies for protein reading are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents. For another method for detecting the degree of expression of the CLUApi gene based on the CLUAP1 gene translation product, the staining intensity can be measured by immunohistochemical analysis using an antibody against the CLUAP1 protein. That is, in this measurement, strong staining indicates the presence of an increase in protein/degree, and at the same time the high degree of expression of the CLUAP1 gene. It can be confirmed that the degree of expression of the target gene including the CLU API gene in the cancer cell is increased, if it is increased by a degree of control group (for example, in a normal cell) of the target gene, for example, 10%, 25%, Or 50%, or an increase greater than 11 times, greater than 1.5 times, greater than 2.0 times, greater than 5.0 times, greater than ι〇·〇 times or more. 63 201134480 The degree of sample control group collected and stored by an individual who has previously been collected from a body (a cancerous or non-cancerous) can be measured simultaneously with cancer cells. In addition, non-region =: cells obtained from an organ having cancer to be treated were used as a normal control group. Alternatively, based on the results obtained from the analysis of the CLUAP1 gene in the sample of the disease ΓτΠΛβ1 ^ m 1 explant, the degree of control can be determined. In addition, the control group ^ ± J was taken from a database of performance profiles of previously tested cells. Moreover, the root is very close to the present invention - the performance of the CLUAP1 gene in a biological sample is ^^^^^, and can be compared with the degree of a plurality of control groups, the degree of which is determined from a plurality of Λ 1 1 solid Refer to the sample. Preferably, the degree of control is determined from a reference sample, and the tissue-derived form is similar to the tissue core derived from the individual biological sample. Further, preferably, CI I is used in a group having a known disease stage.丨A Ρ1 甘m

Pi基因的表現程度的標準值 cStandard value)。標準值可獾 ρ ώ u 知早值了獲侍自本技術領域任何已知的方法。例如，平均值+/_?妒 • Z ‘準差或平均值+/-3標準差，了被使用為標準值。當與正常控制組程度相較CLUAP1基因科表現程度被增加或相似/等同於癌控制組程度，可診斷個體為具有癌症要被治療。本發明也提供（i)啥齡β τ U) 0斷疋否一個體被懷疑具有要被治療之癌症，及/或（ϋ )准選擇要癌症治療之個體的方法，其方法包括步驟：、 a)測定在癌症細胞或組織中，cl随的表現程度，癌症細胞或組織獲得自破敗疑具有要被治療之癌症的個體； 64 201134480 b)與正常控制組比較CLUAP1之表現程度；加 :丨)若CLUAP1之表現程度與正常控制組程度相較被增則診斷個體為具有要被治療之癌症；以及 d)若個體於步驟）中祜峰症鄉C;〒破5乡斷為具有要被治療之癌 JJ選擇要癌症治療之個體。或者，此種方法可包括步驟： a) 測定在癌症細胞或組織中，⑽Αρι的表現程度，癌症細胞或組織獲得自體；敗疑“要被治療之癌症的個 b) 與癌症控制組比較CLUAP1之表現程度；度 )右CLUAP1之表現程度相似或等於癌症控制組程則診斷個體為具有要被治療之癌症；以及症 d)若個體於步驟c)中被診斷為具有要被治療之癌則選擇要癌症治療之個體。本發明也提供-診斷套組以診斷或測定一個體其為或被懷疑遭受可被以本發明_多胜肽治療之癌症，盆也在㈣癌症之預後（㈣卿叫及/或監控癌症治療的功效與適用性中為提供用途，特行钔馮癌症免疫治療。適合癌症之說明例子包括，但不限於乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、騰臟癌。更特別的是，套組較佳包括王v 用以偵測來自個體癌細胞中之謝U因的表現程度的試劑，此被擇自： (〇 —試劑用以偵測CLUApi基因的mRNA; 65 201134480 (b) —試劑用以偵測CLUAP1蛋白質或其免疫活性片段；以及 (c) 一試劑用以偵測CLUAP1蛋白質的生物活性。適合用以偵測CLUAP1基因之mRNA之試劑的例子可包括核酸其專一結合或辨認CLUAP1 mRNA，例如，具有對於 CLUAP1 mRNA之一部分互補的序列的寡核苷酸。這些種類之寡核苷酸以專一於CLUAP1 mRNA之引子與探針為例子。根據本技術領域所熟知的方法可製備這些種類之寡核苷酸。若需要，用以偵測CLUAPl mRNA之試劑可被固定於固體基質（matrix)上。此外，大於一個之用以偵測 mRNA的試劑可被包含於套組中。另一方面，適合用以偵測CLUAP1蛋白質或其免疫活性片段之試劑的例子可包括對於CLUAP1蛋白質或其免疫活性片段的抗體。抗體可為單株或多株。此外，抗體之任何片段或修飾（例如嵌合型抗體（chimeric antib〇dy)、 scFv、Fab、F(ab’ ）2、Fv等）可被用來作為試劑，只要片段或經修飾之抗體維持對CLUAP1蛋白質或其免疫活性片段的結合能力。這些用於蛋白質偵測之這些種類的抗體的製備方法為本技術領域所熟知，且任何方法可被使用於本發明中以製備此種抗體與其等同物（equivalent)。另外，可以訊號產生分子經由直接連接或一間接標誌技術來將抗體進行標誌。標誌與標誌抗體之方法與偵測抗體對其目標的結合為本技術領域所熟知，且任何標誌、與方法可被使用於本發明。另外’大於一個之用於偵測CLUAP1蛋白質的試 66 201134480 劑可被包括於套組中。且可包含多於一個之前述試劑。套組可更包括用以、=口對於CLUAP1基因之探針或對於CLUApi胜肽之抗體的體土質與试劑、用以培養細胞之培養基與容器、正與負控制組試劑與用以偵測對於CUApi 肽之抗體的二次抗體例如，獲得自沒有癌症或遭受癌症之個體的組織樣本可作為有用的控制組試劑。本發明之套組可更包括商業或者角度所4之其他材料，包括緩衝溶液、稀釋液、濾裔/主射針、主射器與具有使用之操作指南的包裝插入物 (例如’書面、磁帶或。卜_等）。這些試劑或此類可保持於-具有標誌之容器。適合之容器包括瓶子、小玻璃瓶 (V1al)與試驗試管。容器可形成自多樣化之材料，例如玻璃或塑膠。在本發明一實施例中，當試劑為抗CLUApi 之探針時’試劑可被固定於—固體基質上，例如一多孔條 (P㈣US strip)以形成至少—㈣位。多孔條之測量或債測區可包括複數個位置，各含有—㈣（探針）。一測試條也可含有負及/或正控制組的位置。或者’控制組之位置可位於與測試條分離之一條。視需要而定不同之偵測位可包含不同量之經固定之核冑’即一較高量於第一偵測位中且-較低含量於隨後之位置中。藉由測試樣本的加入，顯示可㈣訊號之-些位置提供—於樣本巾⑽ρι雜存在之量較量指示。偵測位可被設置於任何適合之可偵測形狀且-般為在橫跨-測試條之寬度的條狀物或點的形 67 201134480 狀中。本發明之套組可更包括一正控制組樣本或CLUAP1標準樣本。藉由收集CLUAP1正之樣本可製備本發明之正控制組樣本且之後分析它們的CLUAP1程度。或者，可將經純化之CLUAP1蛋白質或多核苷酸加至不表現αυΑρι之細胞以形成正樣本（positive sample)或CUApi標準樣本。於本發明中，經純化之CLUAP1可為重組蛋白質。正控制組樣本之CLUAP1程度為，例如，大於臨界值（cut vaiue)。在-實施例中’本發明更提供一診斷套組，包括一蛋白質或其-部份蛋白質’專__辨認本發明抗體或其片段之能力。此處所考慮之本發明之蛋白質之部分胜肽與免疫活性片段的例子包括多胜肽，其係由在本發明蛋白f之胺基酉夂序列中之至）8個，較佳15個、更佳2Q個連續胺基酸所組成。冑用本發明之一蛋白質或—胜肽（多胜肽），藉由偵測於-樣本（例如，血液、組織）中之一抗體可診斷癌症。製備本發明胜肽或蛋白質的方法如上所述。如上所述，藉由測定介於抗CLUAP1抗體與其在對應控制、、且中之的量的^異可執行本發明診斷癌症之方法。若個體之細胞或組織含有抗基因之表現產物（CLUApi)抗體且抗 CLUAP1抗體的量被測定大於在相較於其在正常控制組之程度中的截斷值時，被體被懷疑遭受癌症。在另一實施例中，本發明之診斷套組可包括本發明之胜肽與結合至其之HLA分子。使用抗原胜肽與HLA分子摘測抗原專一細胞毒殺性τ淋巴球的適合方法已被建立（例 68 201134480 如，Altman JD et al.’ Science. 1 996, 274(5284)： 94-6 )。因此，本發明之胜肽與HLA分子的複合物可應用至偵測腫瘤抗原專一細胞毒殺性τ淋巴球的偵測方法，藉此使早期偵測癌症之復發及/或轉移為可能。此外，其可被用來個體之篩選，個體適合包含本發明胜肽為一活性成分的藥物，或藥物治療功效的評估。特別是，根據已知方法 (參見，例如 Altman JD et al·，Science. 1996，274(5284): 94 6)可製備放射標諸之HLA分子與本發明胜肽之寡聚複合物，例如四聚體。可使用複合物來對來自被懷疑遭受癌症之個體的周邊血液淋巴球（peripheral bl〇〇d lymphocytes)中之抗原-胜肽專一細胞毒殺性τ淋巴球進行定量。本發明更k供藉由使用此處敘述之胜肽抗原決定位，用以評估免疫反應之診斷試劑。在本發明一實施例中，如上述之HLA限制胜肽被使用為評估或預測一個體之免疫反應的試劑。藉由將免疫抗原（immun〇gen)與免疫活性細胞 (immUnocompetent)/；7 或 “汴^接觸可誘導要被評估之免疫反應。在較佳實施例中，用以評估一免疫反應的免疫活性細胞可選擇自周邊血液、周邊血液淋巴球 (PBL)、與周邊血液單核細胞（PBMCh收集或分離此類免疫活性細胞的方法為本技術領域所熟知。在特定實施例中，使用為試劑之物質或組合物可為任何物質或組合物其可導致抗原專-細胞毒殺性T淋巴球的產±，細胞毒殺性了淋巴球辨認與結合至胜肽抗原決定位。胜肽試劑必須不被使 69 201134480 用為免疫抗原。用於此類分析之分析系統包括相當新近之技術發展，例如四聚體，對細胞内淋巴激素（lymph〇kines) 之染色與干擾素釋放分析或EL I SPOT分析。在較佳實施例中’要與胜肽試劑接觸之免疫活性細胞可為包括樹突細胞之抗原呈現細胞。例如’本發明胜肽可使用於四聚體染色分析以評估為了抗原專一細胞毒殺性τ淋巴球存在之周邊血液單核細胞’在暴露至腫瘤抗原或一免疫抗原後。Hla四聚體複合物可被使用來直接顯現抗原專一細胞毒殺性T淋巴球（參見’例如〇gg et a 1.，Science 279: 2103-2106，1998; and Altman et al，Science 174 : 94-96，1996 )，並測定於周邊血液單核細胞之樣本中的抗原專一細胞毒殺性T淋巴球族群的頻率。使用本發明胜肽之四聚體試劑可如下被產生。在對應之HL Α重鏈與/S2-微球蛋白存在下重新折疊結合至HLA之胜肽，以產生三分子複合物。在複合物中，重鏈之縮端為經生物素化於一預先設計進入蛋白質之位置。將卵白素加至複合物以形成由三分子複合物與印白素 (streptavidin)所組成之四聚體。藉由以螢光標誌卵白素’可使用四聚體來對抗原呈現細胞染色。之後可鑑定細胞，例如藉由流式細胞技術。此類分析可被用於診斷與預後（prognostic)目的。藉由此程序之細胞鑑定也可備用於治療目的。The standard value of the degree of expression of the Pi gene is cStandard value). The standard value 獾 ρ ώ u is known to be any method known in the art. For example, the mean +/_?妒 • Z ‘quasi-difference or mean +/-3 standard deviation is used as the standard value. When the degree of CLUAP1 gene expression is increased or similar/equal to the degree of cancer control group compared with the normal control group, the individual can be diagnosed as having cancer to be treated. The present invention also provides a method for (i) age-inducing β τ U) 0 to break a body suspected of having a cancer to be treated, and/or (ϋ) to select an individual to be treated for cancer, the method comprising the steps of: a) determining the extent to which cl is expressed in a cancer cell or tissue, and the cancer cell or tissue is obtained from an individual with a cancer that is to be treated; 64 201134480 b) Comparison of the degree of expression of CLUAP1 compared to the normal control group; If the degree of performance of CLUAP1 is increased compared with the degree of the normal control group, then the individual is diagnosed as having cancer to be treated; and d) if the individual is in the step), the peak of the township C; Treated cancer JJ chooses individuals who want cancer treatment. Alternatively, such a method may comprise the steps of: a) determining the degree of expression of (10) Αρι in cancer cells or tissues, obtaining autologous cancer cells or tissues; ignoring "a b of cancer to be treated" compared with cancer control group CLUAP1 Degree of performance; degree) the degree of performance of the right CLUAP1 is similar or equal to the cancer control group, the individual is diagnosed as having the cancer to be treated; and the disease d) if the individual is diagnosed as having the cancer to be treated in step c) Selecting an individual to be treated for cancer. The invention also provides a diagnostic kit for diagnosing or determining a body that is or is suspected of suffering from a cancer that can be treated with the invention, the pot is also (4) the prognosis of the cancer ((4) For the purpose of providing and/or monitoring the efficacy and applicability of cancer treatment, special treatments include cancer immunotherapy. Examples of suitable cancer include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, Diffuse gastric cancer, lymphoma, neuroblastoma, visceral cancer. More specifically, the kit preferably includes a table for detecting the cause of X-ray from individual cancer cells. The degree of reagent, which is selected from: (〇 - reagent for detecting mRNA of CLUApi gene; 65 201134480 (b) - reagent for detecting CLUAP1 protein or immunologically active fragment thereof; and (c) a reagent for detection The biological activity of the CLUAP1 protein is measured. Examples of a reagent suitable for detecting mRNA of the CLUAP1 gene may include a nucleic acid which specifically binds or recognizes CLUAP1 mRNA, for example, an oligonucleotide having a sequence partially complementary to one of the CLUAP1 mRNAs. Oligonucleotides are exemplified by primers and probes specific to CLUAP1 mRNA. These kinds of oligonucleotides can be prepared according to methods well known in the art. If necessary, reagents for detecting CLUAP1 mRNA can be immobilized. In addition to a reagent for detecting mRNA, more than one reagent for detecting mRNA may be included in the kit. On the other hand, examples of reagents suitable for detecting the CLUAP1 protein or an immunologically active fragment thereof may include An antibody against a CLUAP1 protein or an immunologically active fragment thereof. The antibody may be single or multiple. In addition, any fragment or modification of the antibody (eg, a chimeric antibody (c) Himeric antib〇dy), scFv, Fab, F(ab')2, Fv, etc.) can be used as reagents as long as the fragment or modified antibody maintains binding ability to the CLUAP1 protein or an immunologically active fragment thereof. Methods for the preparation of these types of antibodies for protein detection are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents. Alternatively, the signal generating molecules can be directly linked or An indirect labeling technique to label antibodies. Methods of labeling and labeling antibodies and detecting the binding of antibodies to their targets are well known in the art, and any markers, methods and methods can be used in the present invention. In addition, more than one test for detecting CLUAP1 protein 66 201134480 can be included in the kit. More than one of the foregoing reagents may be included. The kit may further include a body material and a reagent for the CLUAP1 gene probe or an antibody against the CLUApi peptide, a culture medium and a container for culturing the cells, a positive and negative control group reagent, and a reagent for detecting Secondary antibodies to antibodies to CUApi peptides, for example, tissue samples obtained from individuals without cancer or suffering from cancer can serve as useful control panel reagents. The kit of the present invention may further include other materials of commercial or angle 4, including buffer solutions, diluents, filter/primary needles, primary emitters, and package inserts having operational guidelines for use (eg, 'written, tape Or. Bu_etc.). These agents or the like can be held in a container with a logo. Suitable containers include bottles, vials (V1al) and test tubes. The container can be formed from a variety of materials such as glass or plastic. In one embodiment of the invention, when the reagent is an anti-CLUApi probe, the reagent can be immobilized on a solid substrate, such as a porous strip (P(IV) US strip) to form at least - (four) positions. The measurement or defect zone of the porous strip can include a plurality of locations, each containing - (iv) (probe). A test strip can also contain negative and/or positive control group positions. Or the location of the control group can be located in one of the separate strips from the test strip. Depending on the need, different detection bits may contain different amounts of fixed kernels', ie a higher amount in the first detection position and a lower content in the subsequent position. By the addition of the test sample, the position indications of the (4) signal are provided - the quantity comparison indication of the sample towel (10) ρι is present. The detection bit can be placed in any suitable detectable shape and is generally in the shape of a strip or point across the width of the test strip. The kit of the present invention may further comprise a positive control group sample or a CLUAP1 standard sample. The positive control group samples of the present invention can be prepared by collecting positive samples of CLUAP1 and then analyzing their degree of CLUAP1. Alternatively, purified CLUAP1 protein or polynucleotide can be added to cells that do not exhibit αυΑρι to form a positive sample or a CUApi standard sample. In the present invention, the purified CLUAP1 may be a recombinant protein. The degree of CLUAP1 of the positive control group sample is, for example, greater than the cut vaiue. In an embodiment, the invention further provides a diagnostic kit comprising a protein or a portion thereof protein capable of recognizing an antibody of the invention or a fragment thereof. Examples of partial peptides and immunologically active fragments of the protein of the present invention contemplated herein include polypeptides, which are up to 8, preferably 15, more in the aminopurine sequence of the protein f of the present invention. It is composed of 2Q continuous amino acids. Using one of the proteins or peptides of the present invention (polypeptide), cancer can be diagnosed by detecting one of the antibodies in the sample (e.g., blood, tissue). The method of preparing the peptide or protein of the present invention is as described above. As described above, the method of diagnosing cancer can be carried out by measuring the amount of the anti-CLUAP1 antibody and its corresponding control, and the amount thereof. If the individual's cells or tissues contain an anti-gene expression product (CLUApi) antibody and the amount of the anti-CLUAP1 antibody is determined to be greater than the cut-off value in the extent of its normal control group, the subject is suspected of suffering from cancer. In another embodiment, a diagnostic kit of the invention can comprise a peptide of the invention and an HLA molecule bound thereto. A suitable method for extracting antigen-specific cytotoxic lytic lymphocytes using antigenic peptides and HLA molecules has been established (Example 68 201134480, for example, Altman JD et al.' Science. 1 996, 274(5284): 94-6). Therefore, the complex of the peptide of the present invention and the HLA molecule can be applied to a method for detecting a tumor cell-specific cytotoxic lymphocyte, thereby enabling early detection of cancer recurrence and/or metastasis. Furthermore, it can be used for screening of individuals, and the individual is suitable for the drug comprising the peptide of the present invention as an active ingredient, or an evaluation of the efficacy of the drug treatment. In particular, an oligomeric complex of a radiolabeled HLA molecule and a peptide of the present invention, such as four, can be prepared according to known methods (see, for example, Altman JD et al., Science. 1996, 274(5284): 94 6). Polymer. Complexes can be used to quantify antigen-peptide specific cytotoxic tau lymphocytes from peripheral bl〇〇d lymphocytes of individuals suspected of having cancer. The invention further provides diagnostic reagents for assessing immune responses by using the peptide epitopes described herein. In one embodiment of the invention, the HLA-restricted peptide as described above is used as an agent for assessing or predicting the immune response of a subject. The immune response to be assessed can be induced by contacting an immunizing antigen with an immunocompetent cell (immUnocompetent)/; or "汴^. In a preferred embodiment, the immunological activity for assessing an immune response is evaluated. The cells may be selected from peripheral blood, peripheral blood lymphocytes (PBL), and peripheral blood mononuclear cells (PBMCh methods for collecting or isolating such immunocompetent cells are well known in the art. In a particular embodiment, the reagents are used. The substance or composition may be any substance or composition which results in the production of antigen-specific cytotoxic T lymphocytes, cytotoxic lymphocyte recognition and binding to the peptide epitope. The peptide reagent must not be rendered 69 201134480 Used as an immunizing antigen. Analytical systems for such analysis include fairly recent technological developments such as tetramers, staining of lymphocytes in lymphocytes and interferon release assays or EL I SPOT assays. In a preferred embodiment, the immunologically active cell to be contacted with the peptide reagent may be an antigen presenting cell comprising dendritic cells. For example, the peptide of the present invention may The tetramer staining assay is used to evaluate peripheral blood mononuclear cells in the presence of antigen-specific cytotoxic lytic lymphocytes. After exposure to tumor antigens or an immune antigen, Hla tetrameric complexes can be used to directly visualize antigen specificity. Cytotoxic T lymphocytes (see 'eg 〇gg et al 1., Science 279: 2103-2106, 1998; and Altman et al, Science 174: 94-96, 1996) and assayed in peripheral blood mononuclear cells The frequency of antigen-specific cytotoxic T lymphocyte populations in the sample. The tetrameric reagent using the peptide of the present invention can be produced as follows. Refolding binding in the presence of the corresponding HL Α heavy chain and /S2-microglobulin HLA peptide to produce a three-molecule complex. In the complex, the end of the heavy chain is biotinylated at a position pre-designed into the protein. Adding avidin to the complex to form a complex of three molecules And a tetramer composed of streptavidin. The antigen can be stained with a tetramer by fluorescently labeling avidin. The cells can then be identified, for example by flow cytometry. Such analysis can be used to diagnose pre (prognostic) object. By this procedure of identification of cells may also be prepared for therapeutic purposes.

本發明也k供5乎估免疫收回反應（immune recaH 70 201134480 responses)之試劑（參見’例如 Bert〇ni et al，J. Clin Invest. 100: 5〇3-5i3，1 997 與 penna ai，j EXp. 174: 1565-1 570，1991 )，其包括本發明之胜肽。例如，為了抗原-專一細胞毒殺性T淋巴球，使用特定專一胜肽，可分析來自具有要被治療之癌症之個體的病患PBMC樣本。藉由培養PBMC與以本發明胜肽刺激細胞可評估含單核細胞之金液樣本。在適合之培養期間後，例如為了細胞毒殺性T淋巴球活性，分析經擴張之細胞族群。胜肽也可使用為評估一疫苗功效之試劑。使用例如上述方法可分析獲自一以一免疫抗原接種之病患的PBMC。病患為經HLA分型’且選擇辨認表現於病患中之對偶基因 (allelespecific)分子的胜肽抗原試劑以分析。藉由於 PBMC樣本中之抗原決定位-專一細胞毒殺性τ淋巴球的存在’可h出疫苗之免疫抗原性（immun〇geni City)。本發明之胜狀也可用來製造抗體，使用本技術領域已熟知的技術 (參見’例如，CURRENT PROTOCOLS IN IMMUNOLOGY， Wiley/Greene， NY;與 Antibodies A Laboratory Manual,The invention also provides reagents for assessing immune retake reactions (immune reca H 70 201134480 responses) (see 'eg Bert〇ni et al, J. Clin Invest. 100: 5〇 3-5i3, 1 997 and penna ai, j EXp. 174: 1565-1 570, 1991), which comprises the peptide of the present invention. For example, for antigen-specific cytotoxic T lymphocytes, a patient-specific PBMC sample from an individual having a cancer to be treated can be analyzed using a specific specific peptide. A gold sample containing monocytes can be evaluated by culturing PBMC and stimulating cells with the peptide of the present invention. The expanded cell population is analyzed, for example, for cytotoxic T lymphocyte activity during a suitable culture period. The peptide can also be used as an agent to evaluate the efficacy of a vaccine. PBMC obtained from a patient vaccinated with one immunogen can be analyzed using, for example, the above method. The disease is an HLA typing' and the peptide antigen reagents that identify the allele specific molecules in the patient are selected for analysis. By the presence of the epitope in the PBMC sample - the presence of a specific cytotoxic lytic lymphocyte, the immunogenic antigenicity of the vaccine can be obtained. The success of the invention can also be used to make antibodies using techniques well known in the art (see, for example, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual,

Harlow and Lane, Cold Spring Harbor Laboratory Press, 1 989 ) ’其可有效作為診斷、偵測或監測癌症之試劑。此類抗體可包括辨認於HLA分子内容中之胜肽的那些，即，結合至胜肽-MHC複合物的抗體。本發明胜肽或組合物具有一些額外之用途，其之一些為此處所述。例如，本發明提供診斷或偵測以CLUAPi免疫活性胜狀之表現為特徵的一疾病。此類方法包含測定於一 71 201134480 生物樣本中之CLUAPl HLA結合胜肽的表現或CLUAPl HLA 結合胜肽之一複合物與HLA class I分子。藉由以對於胜肽或複合物之結合伙伴（binding partner)分析可測定或偵測一胜肽之表現或胜肽與HLA class I分子之複合物。在一較佳實施例中’對於胜肽或複合物之結合伙伴可為一抗體其辨且專一結合至胜狀。藉由使用CLUAPl引子之標準PCR放大步驟也可測試於一生物樣本，例如一腫瘤切片中之CLUAP1的表現。腫瘤表現之例子於此被呈現且示範之用於CLUAPl放大的條件與引子的更進一步揭露可被發現於 W02003/27322 中。較佳診斷方法包含將分離自一個體的生物樣本與專一於CLUAPl HLA結合胜肽之一試劑接觸以偵測於生物樣本中之CLUAPl HLA結合胜肽的存在。如此處所使用“接觸”意指以有效接近試劑方式放置生物樣本且在適合之例如，濃度、溫度 '時間、離子強度條件下，以允許介於試劑與存在生物樣本中之CLUAPl HLA結合胜肽的專一互相作用。一般而言’將試劑接觸生物樣本之條件為熟悉此技藝人士所知之條件以促進介於分子及於生物樣本中之其同類物 (cognate)(例如，一蛋白質與其受體同類物、一抗體與其蛋白質抗原同類物、一核酸與其互補序列同類物）之間的專一互相作用。促進介於分子與其同類物之間的專一互相作用的不範條件敘述於Low et al所提出之u s. p^entHarlow and Lane, Cold Spring Harbor Laboratory Press, 1 989 ) 'is effective as a reagent for diagnosing, detecting or monitoring cancer. Such antibodies may include those recognized as peptides in the HLA molecular content, i.e., antibodies that bind to the peptide-MHC complex. The peptide or composition of the invention has some additional uses, some of which are described herein. For example, the invention provides a disease that is characterized by the diagnosis or detection of a manifestation of a viral activity of CLUAPi. Such methods comprise assaying the performance of a CLUAP1 HLA-binding peptide or a complex of a CLUAP1 HLA-binding peptide with an HLA class I molecule in a 71 201134480 biological sample. The performance of a peptide or the complex of a peptide and an HLA class I molecule can be determined or detected by analysis of a binding partner for a peptide or complex. In a preferred embodiment, the binding partner for the peptide or complex can be an antibody that is uniquely bound to a winning form. The performance of CLUAP1 in a biological sample, such as a tumor slice, can also be tested by using the standard PCR amplification step of the CLUAP1 primer. Examples of tumor manifestations The further disclosure of conditions and primers for CLUAP1 amplification presented and exemplified herein can be found in W02003/27322. A preferred diagnostic method involves contacting a biological sample isolated from a body with one of the CLUAP1 HLA-binding peptides to detect the presence of a CLUAP1 HLA-binding peptide in the biological sample. As used herein, "contacting" means placing a biological sample in an effective proximity reagent and, for example, concentration, temperature 'time, ionic strength conditions, to allow binding of the peptide to the CLUAP1 HLA in the biological sample. Specific interaction. In general, the conditions under which a reagent is contacted with a biological sample are those known to those skilled in the art to promote cognate between the molecule and the biological sample (eg, a protein and its receptor analog, an antibody) A specific interaction with its protein antigen congener, a nucleic acid and its complementary sequence congener. The unconditional conditions that promote the specific interaction between the molecule and its congeners are described in the u s. p^ent proposed by Low et al.

No. 5, 1 08, 921。可在切或以F/ 之一或兩者執行本發明之診 72 201134480 斷方法。因此，在太發明φ + 隹I贫明中生物樣本可位於/77以⑺或中。例如，生物樣本可為一如組織且專—於 CLUAP1免疫活性多胜肽的試劑可被用來偵測於組織中此類分子的存在d或者’可//7 _收集或分離生物樣本（例如血液樣本、腫瘤切片、組織萃取物）。在一特別較佳實施例中，生物樣本可為一含細胞樣本，更佳為一樣本含有收集自要被診斷或測試之個體的腫瘤細胞。No. 5, 1 08, 921. The method of the present invention can be performed in either or both of F/ or both. Therefore, in the invention of φ + 隹I poorly, the biological sample can be located at /77 in (7) or in. For example, a biological sample can be a tissue-specific and specialized CLUAP1 immunologically active peptide that can be used to detect the presence of such molecules in a tissue d or 'may//7 _ collect or isolate biological samples (eg, Blood samples, tumor sections, tissue extracts). In a particularly preferred embodiment, the biological sample can be a cell-containing sample, more preferably the same as the tumor cells collected from the individual to be diagnosed or tested.

或者，藉由使用一方法可執行診斷，此方法藉由以螢光素（fluoresce in)標誌HLA多聚複合物染色允許抗原一專一 T細胞的直接定量（例如’ Ai tman, J. D. et al.，1996， Science 274 : 94; Altman, J. D. et al., 1993, Proc. Natl. Acad· Sci. USA 90 : 1 0330)。也已提供細胞内淋巴激素（lymphokines)之染色與干擾素釋放分析或ELISPOT 分析。多聚體染色、細胞内淋巴激素染色與EL I SPOT分析皆顯露比一般分析靈敏至少多10倍（Murali-Krishna，K. et al. , 1 998, Immunity 8 : 177; Lalvani, A. etal., 1997, J. Exp. Med. 186: 859; Dunbar, P. R. etal., 1998, Curr. Biol. 8: 413)。也可使用五聚體（例如，US 2004-209295A )、右聚體（dextramer)(例如，WO 02/072631)、鏈聚體（st rep tamer)(例如，Nature medicine 6· 631-637 (2002))。例如，在一些實施例中，本發明提供診斷或評估被投予本發明至少一 CLUAP1胜肽之個體的免疫反應的方法’方法包括步驟： 73 201134480 (a) 在適合誘導專一於免疫原之細胞毒殺性T淋巴球的條件下，將免疫原與免疫活性細胞接觸； (b) 偵測或測定於步驟（a )中所誘導之細胞毒殺性τ 淋巴球的誘導程度；以及 (c) 使個體之免疫反應與細胞毒殺性Τ淋巴球的誘導程度互相關連。在本發明中，免疫原為擇自序列辨識號：2至23與25 至43之胺基酸序列中的CLUAP1胜肽、具有此種胺基酸序列的胜肽與具有此種胺基酸序列於其中已被以卜2或更多胺基酸取代所修飾的胜肽的至少一個。於期間，適合誘導免疫原專一之細胞毒殺性Τ淋巴球的條件為本技術領域所熟知。例如，可i η ν i t ro培養免疫活性細胞在免疫存在下以誘導免疫原專一之細胞毒殺性T淋巴球。為了誘導免疫原專一之細胞毒殺性T淋巴球’可加入任何刺激因子於細胞培養物中。例如’ IL-2為細胞毒殺性τ淋巴球誘導之較佳刺激因子。在一些實施例中’可在治療前、期間及/或後執行監測或s平估要被以胜肽癌症治療之個體的免疫反應的步驟。一般而言，在癌症治療的程序中，一再地將免疫原性胜肽投予至要被治療的個體。例如，可每週投予免疫原性胜肽達 3 1 〇週。因此，在癌症治療程序期間可評估或監測個體之免疫反應。或者，對於癌症治療之評估或監測的步驟可在~療程序元成時。根據本發明，當與控制組相較時，經增強之免疫原專一之細胞毒殺性τ淋巴球的誘導指出要被 74 201134480 評估或診斷之個體免疫性地對已被投予之免疫原反應。用以評估s疫反應之適合的控制組包括，例如當免疫活性細胞未與胜肽接觸或與具有除了任冑cLUApi胜肽之胺基酸序列的控制組胜肽（例如，隨機胺基酸序列）#觸時的細胞毒殺性τ淋巴球的誘導程度。在一較佳實施例中，藉由將介於投予至個體之各個免疫原間的免疫反應進行比較，以序列專—方式來評估個體之免疫反應。特別是，甚至當將CLUAP1胜肽之一些種類的此合物投予至個體時，依據胜肽免疫反應可成多樣化。在此例子巾冑由將介於各個胜肽間的免疫反應進行比較，可確認出對於其個體顯示較高反應之胜肽。 XI.抗體本發明提供抗體其結合至本發明胜肽。較佳抗體專一結合至本發明胜肽且不會結合（或微弱結合）至非本發明胜肽。或者抗體結合本發明胜肽與其同源物（h〇m〇1〇gs)。抗本發明胜肽之抗體可提供用途於癌症診斷與預後分析及成像方法（丨1^§丨叱1^讣〇(1〇1(^45)。相似地，對於在癌症病患中也表現或過度表現CLUAP1程度而言，此類抗體可提供使用於其他癌症之治療、診斷，及/或預後。此外，細胞内表現之抗體（例如，單鏈抗體）可在治療與CLUApi之表現相關的癌症中提供治療用途，與CLUApi之表現相關的癌症例子包括，但不限於，乳癌、子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型胃癌、淋巴癌、神經母細胞瘤、胰 75 201134480 臟癌。本發明也提供CLUAP1蛋白質（序列辨識號：45)或其片段之偵測或定量的各種免疫活性分析，CLUAP1蛋白質（序列辨識號：45)或其片段包括具有擇自序列辨識號：3'4、 5、8、9、10、14、15、16、18、23、30、33、34、35、36 與38中之胺基酸序列之多胜肽。此類分析可包括一或多個具辨認及結合CLUAP1蛋白質或其片段之能力之抗CLUAP1 抗體為適當的。在本發明内容中，與CLUAP1多胜肽結合之抗CLUAP1抗體，較佳為辨認一多胜肽，此多胜肽為具有擇自序列辨識號：3、4、5、8、9、10、14、15、16、18、23、 30、33、34、35、36與38中的胺基酸序列。以抑制測試 (inhibition test)可確認抗體之結合專一性。其為，在具有擇自序列辨識號：3、4、5、8、9、10、14、15、16、18、 23 ' 30、33、34、35、36與38中之胺基酸序列之任何片段多胜肽存在下’當介於要被分析之抗體與全長之CLUAP1 胜肽之間的結合被抑制時，其顯示此抗體專一結合至片段。在本發明内容中，在包括，但不限於放射免疫分析 (radioimmunoassays)、免疫色層分析技術 (immuno-chromatgraph technique)、酵素連結免疫吸附分析（enzyme-linked immunosorbent assays，ELISA)、酵素連結免疫螢光分析（enzyme- linked immunofluorescent assays，ELIFA)專之多種本技術領域熟知之免疫分析形式中執行此類免疫分析。本發明之關於免疫，但非抗體分析也可包括T細胞致 76 201134480 免疫性分析（immunogenicity assay)(抑制或刺激）與主要組織相容性複合物（ma jor hi stocompat ibi 1 i ty CQmpiex 結合分析。此外’本發明考慮具偵測表現CLUAP1之癌症之能力的免疫成像分析，其例子包括，但不限於使用本發明之經標誌的抗體的放射顯像成像（radio sCintigFaphie 11^§丨叫）方法。此類可分析在（^1^?1表現之癌症谓測、監控與預後中提供臨床用途，CLUAP1表現之癌症，其例子包括’但不限於’乳癌、子宮頸癌、大腸直腸癌、食道癌、月癌、/彌漫型月癌、淋巴癌、神經母細胞瘤 '胰臟癌。本發明也提供結合至本發明胜肽的抗體。本發明抗體可使用於任何形式中’例如單株或多株抗體，且可更包括獲得自將動物，例如兔子，以本發明胜肽進行免疫之抗血清、所有類型之多株或單株抗體、人類抗體與由基因重組產生之人源化抗體。使用為一抗原以獲得一抗體之本發明胜肽可來自任何動物種類，但較佳為來自哺乳動物例如，類老鼠或大鼠’更佳為一人類。人類來源胜狀可獲得自此處揭露之核苷酸或胺基酸序列。根據本發明，使用為免疫抗原之胜肽可為一完整之蛋白質或部分胜肽之蛋白冑。部分胜肽可包括，例如，本發明之一胜肽之胺基（N)端或羧基（C)端片段。此處，一抗體被定義為-蛋白質其與CLUApi胜肽之全長或片段反應。在車又佳實鈿例中，本發明之抗體辨認CLUApi片段胜肽其具有擇自序列辨識號：3、4、5、8、9、10、14、15、16: M 、35、36與38中的胺基酸序列。合 77 201134480 成券胜狀的方法為本技術領域所熟知。在合成後’在使用為免疫抗原（immunogen)前，胜肽可視需要被純化。在本發明内容中，寡胜肽（例如9或員）可與載體結合或連結以增強致免疫性。鑰孔血藍蛋白（Keyhol卜limpet hemocyanin，KLH)被熟知為載體。結合鑰孔血藍蛋白與胜狀的方法為本技術領域所熟知。或者’可將編碼出本發明一胜肽或其片段的一基因插入一已知的表現載體’其之後被轉形至一宿主細胞，如於此所敘述。藉由任何標準方法，所需之胜肽或其片段可自宿主細胞之外部或内部被重新獲得，且之後可被使用為一抗原。或者，可將表現胜肽之整個細胞或其細胞萃出物或一化學合成胜肽使用為抗原。可以抗原將任何哺乳動物進行免疫，但較佳為考慮與用來細胞融合之親代細胞的相容性。一般而言使用嚅齒目（Rodentia)、兔形目（Lagomorpha)或靈長目 (Primates)。嗡齒目的動物包括，例如小鼠、大鼠與倉鼠。兔形目的動物包括，例如兔子。靈長目的動物包括，例如狹鼻類（舊世界猴），子，例如馬來猴（Macaca fascicularis)、獼猴（rhesus monkey)、聖狒狒（sacr d baboon)與黑猩猩（chimpanzees)。以抗原免疫動物之方法為本技術領域所熟知。抗原之腹腔内注射（intraperit〇neai injecti〇n)或皮下注射 (subcutaneous in jection)為免疫哺乳動物之一標準方法。更特別地，可將抗原稀釋或懸浮於一適合量的：酸鹽 78 201134480 緩衝/合液、生理食鹽水等。若需要可將抗原懸浮液與適合量之標準佐劑，例如佛氏完全佐劑（Freund's complete d juvant)/tti合，製成乳狀液（emuisi〇n)並且之後投予至哺乳動物。較佳為其之後投予與適合量之佛氏不完全佐劑 (Freund’s incomplete adjuvant)混合的抗原每 4 至 21 天也可使用一適合之載體來免疫。於上述免疫後，藉由為了增加所需之抗體量標準方法可檢驗血清。藉由自被檢驗以增加於血清中之抗體量的經免疫動物收集血液解藉由以任何一般方法自血液分離血清，可製備抗本發明胜肽之多株抗體。多株抗體包括含多株抗體的血 β，與含可自血清分離多株抗體的部分（fracti〇n)。例如使用與本發明胜肽結合之親合管柱且更進一步使用蛋白質或蛋白質G管柱純化此部分，可自僅辨認本發明胜肽之部分純化免疫球蛋白G或Μ。為了製備單株抗體，自如上所述經以抗原免疫並確認於血清中所需抗體之增加程度的哺乳動物收集免疫細胞且使免疫細胞遭遇細胞融合。用來細胞融合之免疫細胞，較佳為獲自脾臟。其他要被與上述免疫細胞融合之親代細胞匕括例如’哺乳動物之骨髓瘤（mye 1 oma)，且較佳為具有乂藥物筛選之融合細胞之獲得特性的骨髓瘤細胞。根據已法例如 Milstein et al. (Galfre and Milstein，Alternatively, the diagnosis can be performed by using a method that allows direct quantification of antigen-specific T cells by staining with a fluorescein-labeled HLA multimeric complex (eg, 'Ai tman, JD et al., 1996, Science 274: 94; Altman, JD et al., 1993, Proc. Natl. Acad. Sci. USA 90: 1 0330). Intracellular lymphokines staining and interferon release assays or ELISPOT assays have also been provided. Multimer staining, intracellular lymphokine staining, and EL I SPOT analysis were all shown to be at least 10 times more sensitive than general analysis (Murali-Krishna, K. et al., 1 998, Immunity 8: 177; Lalvani, A. et al. , 1997, J. Exp. Med. 186: 859; Dunbar, PR et al., 1998, Curr. Biol. 8: 413). Pentamers (for example, US 2004-209295A), dextramers (for example, WO 02/072631), and st rep tamers (for example, Nature medicine 6· 631-637 (2002) can also be used. )). For example, in some embodiments, the invention provides a method of diagnosing or assessing an immune response in an individual administered to at least one CLUAP1 peptide of the invention. The method comprises the steps of: 73 201134480 (a) in a cell suitable for inducing specificity to the immunogen The immunogen is contacted with the immunologically active cells under conditions of a toxic T lymphocyte; (b) detecting or determining the degree of induction of the cytotoxic tau lymphocytes induced in step (a); and (c) enabling the individual The immune response is correlated with the degree of induction of cytotoxic lymphocytes. In the present invention, the immunogen is a CLUAP1 peptide selected from the amino acid sequence of sequence identification numbers 2 to 23 and 25 to 43, a peptide having such an amino acid sequence, and having such an amino acid sequence At least one of the peptides modified therein has been substituted with 2 or more amino acids. Conditions suitable for inducing immunogenic specific cytotoxic lymphocytes during the period are well known in the art. For example, immunocompetent cells can be cultured in the presence of immunization to induce immunogen-specific cytotoxic T lymphocytes. To induce immunogenic cytotoxic T lymphocytes, any stimulating factor can be added to the cell culture. For example, 'IL-2 is a better stimulating factor for cytotoxic tau lymphocyte induction. In some embodiments, the step of monitoring or sifting the immune response of an individual to be treated with a peptide cancer can be performed before, during, and/or after treatment. In general, in the course of cancer treatment, the immunogenic peptide is repeatedly administered to the individual to be treated. For example, an immunogenic peptide can be administered weekly for 3 1 week. Thus, an individual's immune response can be assessed or monitored during the cancer treatment procedure. Alternatively, the steps for assessing or monitoring cancer treatment may be at the time of the treatment procedure. According to the present invention, the induction of the enhanced immunogen-specific cytotoxic τ lymphocytes indicates that the individual to be assessed or diagnosed by 74 201134480 immunologically responds to the administered immunogen when compared to the control group. Suitable control sets for assessing s-epide reactions include, for example, when the immunocompetent cells are not in contact with the peptide or with a control group peptide having an amino acid sequence other than the CLUApi peptide (eg, a random amino acid sequence) The degree of induction of cytotoxic tibia lymphocytes at the time of exposure. In a preferred embodiment, the individual's immune response is assessed in a sequence-specific manner by comparing the immune responses administered between the individual immunogens administered to the individual. In particular, even when some of the compounds of the CLUAP1 peptide are administered to an individual, the immune response according to the peptide can be diversified. In this example, the immunological reaction between the individual peptides was compared, and the peptide which showed a higher response to the individual was confirmed. XI. Antibodies The invention provides antibodies which bind to the peptides of the invention. Preferably, the antibody specifically binds to the peptide of the present invention and does not bind (or weakly bind) to the non-inventive peptide. Alternatively, the antibody binds to the peptide of the invention and its homolog (h〇m〇1〇gs). The antibody against the peptide of the present invention can be used for cancer diagnosis and prognosis analysis and imaging methods (丨1^§丨叱1^讣〇(1〇1(^45). Similarly, it is also expressed in cancer patients) Such antibodies may provide for the treatment, diagnosis, and/or prognosis of other cancers, in addition to the extent of overexpression of CLUAP1. In addition, antibodies that are expressed intracellularly (eg, single chain antibodies) may be associated with treatment of CLUApi expression. For cancer therapy, examples of cancers associated with the performance of CLUApi include, but are not limited to, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, lymphoma, neuroblastoma, pancreas 75 201134480 Dirty cancer. The present invention also provides various immunological activity assays for the detection or quantification of the CLUAP1 protein (SEQ ID NO: 45) or a fragment thereof, the CLUAP1 protein (SEQ ID NO: 45) or a fragment thereof comprising the sequence identification number: Polypeptides of amino acid sequences in 3'4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38. Such analysis may include Or multiple identified and combined CLUA An anti-CLUAP1 antibody capable of a P1 protein or a fragment thereof is suitable. In the context of the present invention, an anti-CLUAP1 antibody that binds to a CLUAP1 polypeptide, preferably recognizes a multi-peptide, which has an alternative sequence Identification numbers: amino acid sequences in 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23, 30, 33, 34, 35, 36 and 38. Inhibition test The binding specificity of the antibody can be confirmed, which is obtained by having the sequence identification numbers: 3, 4, 5, 8, 9, 10, 14, 15, 16, 18, 23 ' 30, 33, 34, 35, 36 In the presence of any fragment of the amino acid sequence of 38, when the binding between the antibody to be analyzed and the full-length CLUAP1 peptide is inhibited, it shows that the antibody specifically binds to the fragment. The invention includes, but is not limited to, radioimmunoassays, immuno-chromatgraph techniques, enzyme-linked immunosorbent assays (ELISA), and enzyme-linked immunofluorescence assays. (enzyme-linked immunofluorescent assays, ELIFA Such immunoassays are performed in a variety of immunoassay formats well known in the art. The present invention relates to immunization, but non-antibody analysis can also include T cell-induced 76 201134480 immunogenicity assay (inhibition or stimulation) and Major histocompatibility complex (ma jor hi stocompat ibi 1 i ty CQmpiex binding assay. Furthermore, the present invention contemplates immunological imaging assays having the ability to detect cancers that exhibit CLUAP1, examples of which include, but are not limited to, radiographic imaging (radio sCintigFaphie 11^§ barking) methods using the labeled antibodies of the invention. Such can be analyzed for clinical use in cancer presupposition, monitoring and prognosis, and CLUAP1 manifests in cancer, examples of which include 'but not limited to' breast cancer, cervical cancer, colorectal cancer, esophageal cancer , lunar cancer, diffuse lunar cancer, lymphoma, neuroblastoma 'pancreatic cancer. The present invention also provides an antibody that binds to the peptide of the present invention. The antibody of the present invention can be used in any form, such as single or multiple An antibody, and may further comprise an antiserum obtained by immunizing an animal, such as a rabbit, with the peptide of the present invention, a multi-strain or monoclonal antibody of all types, a human antibody, and a humanized antibody produced by genetic recombination. The peptide of the present invention which is an antigen to obtain an antibody may be derived from any animal species, but is preferably from a mammal such as a mouse or a rat, preferably a human. The human source is obtained from the disclosure herein. Nucleotide or amino acid sequence. According to the present invention, the peptide used as the immunizing antigen may be a protein of a complete protein or a partial peptide. The partial peptide may include, for example, one of the present inventions. An amino (N) terminal or a carboxyl (C) terminal fragment of a peptide. Here, an antibody is defined as a protein which reacts with a full length or fragment of a CLUApi peptide. In the case of the car, the present invention The antibody recognizes the CLUApi fragment peptide having the sequence identification number: 3, 4, 5, 8, 9, 10, 14, 15, 16: amino acid sequences in M, 35, 36 and 38. 77 77. The method of winning a ticket is well known in the art. After synthesis, the peptide can be purified as needed before being used as an immunogen. In the context of the present invention, an oligopeptide (e.g., 9 or a member) can be used. The vector is conjugated or linked to enhance immunogenicity. Keyhole limpet hemocyanin (KLH) is known as a vector. Methods for binding keyhole limpet hemocyanin and victoria are well known in the art. A gene encoding a peptide of the invention or a fragment thereof is inserted into a known expression vector, which is then transformed into a host cell, as described herein. The desired peptide or its desired method by any standard method Fragments can be retrieved from outside or inside the host cell, And can be used as an antigen later. Alternatively, the entire cell expressing the peptide or its cell extract or a chemically synthesized peptide can be used as an antigen. The antigen can be immunized to any mammal, but is preferably considered The compatibility of the parental cells used for cell fusion. Generally speaking, Rodentia, Lagomorpha or Primates are used. Animals of the caries include, for example, mice, rats and hamsters. Rabbit-shaped animals include, for example, rabbits. Primate animals include, for example, the narrow-nose (old world monkey), sons, such as Macaca fascicularis, rhesus monkey, sacr d baboon, and Chimpanzees (chimpanzees). Methods for immunizing animals with antigens are well known in the art. Intraperitoneal injection (intraperit〇neai injecti〇n) or subcutaneous in jection is a standard method for immunizing mammals. More particularly, the antigen can be diluted or suspended in an appropriate amount: acid salt 78 201134480 buffer/liquid, physiological saline, and the like. If necessary, the antigen suspension can be combined with a suitable amount of a standard adjuvant such as Freund's complete d juvant/tti to prepare an emulsion (emuisi〇n) and then administered to a mammal. Preferably, the subsequent administration of an antigen mixed with a suitable amount of Freund's incomplete adjuvant may also be immunized every 4 to 21 days using a suitable carrier. After the above immunization, the serum can be tested by a standard method for increasing the amount of antibody required. By collecting blood from an immunized animal that has been tested to increase the amount of antibody in serum, a plurality of antibodies against the peptide of the present invention can be prepared by separating serum from blood by any general method. A plurality of antibodies include blood beta containing a plurality of antibodies, and a fraction containing a plurality of antibodies which can be isolated from serum (fracti〇n). For example, using an affinity column coupled to a peptide of the present invention and further purifying the portion using a protein or protein G column, the immunoglobulin G or hydrazine can be purified from only a portion of the peptide of the present invention. To prepare a monoclonal antibody, the immune cells are harvested from the mammals immunized with the antigen and confirmed to the extent of the desired antibody in the serum as described above and the immune cells are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other parental cells to be fused with the above-mentioned immune cells include, for example, 'mammalian myeloma, and preferably myeloma cells having acquired characteristics of conjugated cells for drug screening. According to the law, for example, Milstein et al. (Galfre and Milstein,

Methods Enzym〇i 73: 3-46 (1981 ))的方法，可將上述免疫細胞與骨髓瘤細胞融合。獲自細胞融合之產生的融合瘤，藉由將它們培養於標 79 201134480 準篩選培養基，例如hat培養基（含亞黃嘌呤 (hypoxanthine)、氨蝶呤（amin〇pterin)和胸腺嘧啶 (thymidine)之培養基），可被篩選。通常持續細胞培養於 HAT培養基數天至數週，時間為允許除了所需融合瘤外之其他細胞（非融合細胞）死亡。之後，可執行標準限制稀釋以篩選並複製產生所需抗體之融合瘤。除了於其中為了製備融合瘤、以一抗原免疫一非人類動物的上述方法，可以胜肽、表現胜肽之細胞或其細胞萃取物//7 hiro免疫人類淋巴細胞，例如被eb病毒感染的那些。之後，將經免疫之淋巴細胞與具不明確分裂能力之來自人類之骨髓瘤’例如U266融合，以產生一產生所需人類抗體之融合瘤，所需之人類抗體可與可被獲得之胜肽結合（Unexamined Published japanese Patent AppUcati〇n No. Sho 63-17688)。將所獲得之融合瘤之後轉植進入小鼠之腹腔且萃取腹水。可純化所獲得之單株抗體，藉由例如硫酸銨沉殿、一蛋白質A或蛋白質G管柱、DEAE離子交換色層分析或一與本發明胜肽結合之親和管柱。本發明抗體不只可被使用來純化偵測本發明胜肽，也可作為本發明胜肽之促進劑與拮抗劑的候選物。或者，一產生抗體之免疫細胞，例如一經免疫之淋巴細胞可藉由一致癌基因以永生或被使用來製備單株抗體。Methods Enzym〇i 73: 3-46 (1981)), which combines the above-mentioned immune cells with myeloma cells. Fusion tumors obtained from cell fusion by culturing them in standard 79 201134480 quasi-screening media, such as hat medium (containing hypoxanthine, amin〇pterin, and thymidine) Medium) can be screened. The cells are usually maintained in HAT medium for several days to several weeks for the death of other cells (non-fused cells) other than the desired fusion tumor. Thereafter, standard restriction dilutions can be performed to screen and replicate fusion tumors that produce the desired antibodies. In addition to the above methods in which a non-human animal is immunized with an antigen in order to prepare a fusion tumor, a peptide, a peptide-expressing cell or a cell extract thereof, and/or a 7 hiro-immunized human lymphocyte, such as those infected with an eb virus, can be used. . Thereafter, the immunized lymphocytes are fused with a human-derived myeloma such as U266 with ambiguous ability to produce a fusion tumor producing the desired human antibody, and the desired human antibody can be obtained with the peptide obtained. Binding (Unexamined Published japanese Patent App Ucati〇n No. Sho 63-17688). The obtained fusion tumor was then transferred into the abdominal cavity of the mouse and the ascites was extracted. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate sink, a protein A or protein G column, DEAE ion exchange chromatography or an affinity column coupled to the peptide of the present invention. The antibody of the present invention can be used not only to purify and detect the peptide of the present invention, but also as a candidate for the promoter and antagonist of the peptide of the present invention. Alternatively, an antibody-producing immune cell, such as an immunized lymphocyte, can be prepared by immortalization or use of a consensus oncogene to prepare a monoclonal antibody.

也可使用基因工程技術來重組製備因此獲得之單株抗體 (參見，例如 B〇rrebaeck and UrHck，TherapeutU 80 201134480Genetic engineering techniques can also be used to recombinantly prepare the thus obtained monoclonal antibodies (see, for example, B〇rrebaeck and UrHck, TherapeutU 80 201134480)

Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD ( 1 990))。例如，一編碼出抗體的DNA可自一免疫細胞，例如產生抗體之— 融合瘤或一經免疫的淋巴細胞被複製，插入一適合之載體’且引入一宿主細胞以製備重組抗體。本發明也提供如上述製備之重組抗體。此外，本發明抗體可為一抗體之片段或經修飾之抗體’只要其結合一或多個本發明之胜肽。例如，抗體片段可為Fab、F(ab )2、Fv或單鍵Fv (scFv)，於其中來自重與輕鏈的Fv片段藉由合適的連結器來連接（Hustcm etMonoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1 990)). For example, a DNA encoding an antibody can be replicated from an immune cell, e.g., an antibody-producing tumor or an immunized lymphocyte, inserted into a suitable vector and introduced into a host cell to produce a recombinant antibody. The present invention also provides a recombinant antibody prepared as described above. Furthermore, the antibody of the present invention may be a fragment of an antibody or a modified antibody as long as it binds one or more of the peptides of the present invention. For example, the antibody fragment can be Fab, F(ab)2, Fv or a single bond Fv (scFv) in which the Fv fragments from the heavy and light chains are joined by a suitable linker (Hustcm et

Proc Natl Acad Sci USA 85: 5879-83 ( 1 988))。更特別是’藉由以酵素例如木瓜酵素或胃蛋白酶處理抗體可產生抗體片段。或者’編碼出抗體之基因可被構築、插入一表現載體且表現於一適合的宿主細胞中（參見，例如C〇 e t a J . J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991))。藉由與各種分子，例如聚乙二醇（polyethylene glyc〇i，PEG)結合可修飾抗體。本發明提供此類經修飾之抗體。藉由化學修飾一抗體可獲知·經修飾之抗體。這些修飾方法為本技術領域中所常見。 81 201134480 或者’本發明之抗體可被獲得為嵌合抗體，介於來自非人抗體之可變區與來自人類抗體之固定區之間，或為人源化抗體’包括來自非人抗體之互補決定區、架構作用區 (frame work region，FR)與來自人類抗體之固定區。根據已知方法可製備此類抗體。藉由齧齒類互補決定區之序列取代人類抗體對應之互補序列可執行人源化（參見，例如Proc Natl Acad Sci USA 85: 5879-83 (1 988)). More specifically, antibody fragments can be produced by treating antibodies with enzymes such as papain or pepsin. Alternatively, the gene encoding the antibody can be constructed, inserted into a expression vector and expressed in a suitable host cell (see, for example, C〇eta J. J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 ( 1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)). The antibody can be modified by binding to various molecules such as polyethylene glycol (PEG). The invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. These modifications are common in the art. 81 201134480 or 'The antibody of the invention can be obtained as a chimeric antibody, between a variable region derived from a non-human antibody and a fixed region derived from a human antibody, or a humanized antibody comprising complementary to a non-human antibody The decision region, the framework work region (FR) and the fixed region from human antibodies. Such antibodies can be prepared according to known methods. Humanization can be performed by substituting the sequence of the rodent complementarity determining region for the complementary sequence corresponding to the human antibody (see, for example,

Verhoeyen et al·， Science 239:1534-1536 (1988))。因此，此類人源化抗體為嵌合抗體，其中實質上少於完整之人類可變區已被以來自非人種類之對應序列取代。也可使用包括人類可變區、架構作用區與固定區的全人類抗體。使用各種本技術領域所知的技術可產生此類抗體。例如，in vitro方法包括呈現於噬菌體上之人類抗體片段的重組資料庫的使用（例如，H〇〇genb〇〇m & Winter， Μ〇1· Biol· 227:381 ( 1 991 ))。相似地，藉由將人類免疫球蛋白基因座（l〇ci)引入轉殖動物，例如於其中内生免疫球蛋白基因已被部分或完全去活化的小鼠，可製造人類抗體。此方法被敘述，例如於U. s. Patent Nos. 6, 1 50, 584, 5’545,807; 5, 545, 806; 5, 569, 825; 5, 625, 126； 5, 633, 425; 5, 661，016。獲自上述之抗體可被純化至同質（homogenei ty)。例如，根據用於一般蛋白質的分離與純化方法可執行抗體之分離與純化。例如，藉由合適地選擇與結合管柱色層分析、過濾超過濾、鹽析、透析、對鈉十二烷基的硫酸鹽聚丙烯酰胺凝膠電泳（SDS polyacrylamide gel 82 201134480 electrophoresis)、等電焦集法（isoelectric focusing) 的使用可分開與分離抗體（Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988))，但不限於此。蛋白質A管柱與蛋白質G管柱A可被使用為親合管柱。要被使用之示範的蛋白質 A 管柱包括，例如 Hyper D、P0R0S 與 Sepharose F. F. (Pharmacia) ° 除了親合，示範之色層分析包括，例如離子交換色層分析、疏水色層分析、膠體過濾、逆向色層分析、吸附色層分析等（Strategies for Protein Purification andVerhoeyen et al., Science 239: 1534-1536 (1988)). Thus, such humanized antibodies are chimeric antibodies in which substantially less than intact human variable regions have been substituted with corresponding sequences from non-human species. Whole human antibodies including human variable regions, framework active regions, and immobilization regions can also be used. Such antibodies can be produced using a variety of techniques known in the art. For example, the in vitro method involves the use of a recombinant database of human antibody fragments presented on phage (e.g., H〇〇genb〇〇m & Winter, Μ〇1· Biol. 227:381 (1 991)). Similarly, human antibodies can be made by introducing a human immunoglobulin locus (l〇ci) into a transgenic animal, such as a mouse in which the endogenous immunoglobulin gene has been partially or completely deactivated. This method is described, for example, in U.s. Patent Nos. 6, 1 50, 584, 5'545, 807; 5, 545, 806; 5, 569, 825; 5, 625, 126; 5, 633, 425; , 661,016. The antibodies obtained from the above can be purified to homogeneity. For example, isolation and purification of antibodies can be performed according to methods for isolation and purification of general proteins. For example, by suitable selection and combined column chromatography, filtration ultrafiltration, salting out, dialysis, sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS polyacrylamide gel 82 201134480 electrophoresis), isoelectric The use of isoelectric focusing can be used to separate and isolate antibodies (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but is not limited thereto. Protein A column and protein G column A can be used as affinity columns. Exemplary protein A columns to be used include, for example, Hyper D, P0R0S and Sepharose FF (Pharmacia) °. In addition to affinity, exemplary chromatographic analysis includes, for example, ion exchange chromatography, hydrophobic chromatography, colloid filtration, Reverse Chromatography, Adsorption Chromatography, etc. (Strategies for Protein Purification and

Characterization： A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press ( 1 996))。藉由液相色層分析’例如Ηριχ 與FPLC可執行色層分析步驟。例如，可使用吸收之測量、酵素連結免疫吸附分析 (ELISA)、酵素免疫分析（EIA)、放射免疫分析及/或免疫螢光以測量本發明抗體之抗原結合活性。在酵素連結免疫吸附分析中，本發明抗體為固定於一培養盤上，提供本發明胜肽至一培養盤，且之後提供含所需抗體之樣本，如，產生抗體之細胞的培養懸浮液或經純化的抗體。之後，提供辨認第一抗體且被標誌酵素，例如鹼性磷酸酶之第二抗體，且之後培養培養盤。接著在清洗後，將酵素受質，例如對硝基苯磷酸（P-nitr〇phenyl ph〇sphate)，加至培養盤，並測量吸收以評估樣本之抗原結合活性。胜肽之片段， 83 201134480 例如c端或N端之片段可被使用為抗原以評估抗體結合活隹根據本發明，可使用BIAcore (Pharmacia)來評估抗體的活性。上述方法允許本發明胜肽之偵測或測量，藉由露出本發明抗體至假定含本發明胜肽之樣本並偵測或測量由抗體與胜肽所形成之免疫複合物。由於根據本發明之偵測或測量方法可專一偵測或測量胜肽，方法在使用胜肽之各種實驗中為有用的。 X11 ·載體與宿主細胞本發明也提供載體與宿主細胞，於其中將編碼出本發明胜肽之核苷酸引入。本發明之載體可用於維持本發明核苷酸，特別是DNA於宿主細胞中以表現本發明胜肽，或為基因治療以投予本發明本發明核苷酸。Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996). The color layer analysis step can be performed by liquid chromatography analysis, such as Ηριχ and FPLC. For example, the measurement of absorption, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay, and/or immunofluorescence can be used to measure the antigen-binding activity of the antibody of the present invention. In an enzyme-linked immunosorbent assay, the antibody of the present invention is immobilized on a culture plate, providing the peptide of the present invention to a culture plate, and then providing a sample containing the desired antibody, such as a culture suspension of the antibody-producing cells or Purified antibody. Thereafter, a second antibody that recognizes the first antibody and is labeled with an enzyme such as alkaline phosphatase is provided, and then the culture plate is cultured. Next, after washing, an enzyme substrate such as p-nitr〇phenyl ph〇sphate was added to the culture plate, and absorption was measured to evaluate the antigen binding activity of the sample. Fragment of a peptide, 83 201134480 For example, a c-terminal or N-terminal fragment can be used as an antigen to evaluate antibody binding activity. According to the present invention, BIAcore (Pharmacia) can be used to assess the activity of an antibody. The above method allows the detection or measurement of the peptide of the present invention by exposing the antibody of the present invention to a sample presumed to contain the peptide of the present invention and detecting or measuring the immune complex formed by the antibody and the peptide. Since the detection or measurement method according to the present invention can specifically detect or measure the peptide, the method is useful in various experiments using the peptide. X11 - Vector and Host Cell The present invention also provides a vector and a host cell into which a nucleotide encoding the peptide of the present invention is introduced. The vector of the present invention can be used to maintain the nucleotide of the present invention, particularly DNA in a host cell to express the peptide of the present invention, or to be genetically treated to administer the nucleotide of the present invention.

當E. coli為宿主細胞且載體被放大且大量製造於E coll (例如，JM109、DH5 alpha、HB101 或 XLlBlue)中時，載體具有要被放大於E_ c〇li中之“ori”且篩選轉形E. c〇li之標誌基因（例如，藉由安比西林（ampiciUin)、四環黴素（tetracycline)、卡那黴素（kanamycin)、氯黴素 (&1〇1^111汕611泌〇1)或類似物篩選之一抗藥基因）。例如，可使用M13-系列載體、PUC-系列載體、pBR322、 pBlueScript、pCR-Script 等。此外，pGEM_T、pDIRECT 與 pT7也可被用來次複製與萃取cDNA與上述載體。當載體被用來產生本發明蛋白質時，一表現載體可提供使用。 84 201134480 例如，要被表現於E. col i中之一表現載體應具有上述特徵以被放大於E. col i中。當使用E. c〇i i，例如 JM109、DH5 alpha、HB101 或 XLlBlue 為宿主細胞時，載體應具有啟動子（promoter)，例如iacz啟動子（Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)) 、araB 啟動子（Better et al.， Science 240: 1041-3 (1988))、T7啟動子或類似物，其可有效表現所需基因於E. col i.中。基於那方面，可使用，例如pGEpsxi (Pharmacia), "QIAexpress system" (Qiagen)、pEGFP 與 PET(於此例子，宿主較佳為BL21，其表現T7RNA聚合酶），取代上述載體。另外’載體也可含用於胜肽分泌之訊號序列。一引導要被分泌之胜肽至E. col i的胞膜間區 (periplasm)的示範之訊號序列為pelB訊號序列（Lei et al·，J Bacteriol 1 69: 4379 ( 1 987))。將載體引入目標宿主細胞的方式包括，例如，氯化辦方法，與電穿孔 (electroporation)方法。除了 E. col i，例如來自哺乳動物之表現載體（例如 pcDNA3 (Invitrogen)與 pEGF-BOS (Nucleic Acids Res 18( 1 7): 5322 ( 1 990))，PEF，PCDM8)、來自昆蟲細胞之表現載體（例如、"Bac-to-BAC桿狀病毒表現系統 (baculovirus expression system)" (GIBC0 BRL)、 pBacPAK8)、來自植物之表現載體（例如，pMH1、pMH2)、來自動物病毒之表現載體（例如、PHSV、pMV、pAdexLcw )、來自反轉錄病毒之表現載體（例如，pZipne0)、來自酵母 85 201134480 菌之表現載體（例如，"Pichia Expression Kit” (Invitrogen)、pNVU、SP-Q〇i )與來自枯草桿菌（Baci i lus subtilis)之表現載體（例如，pPL6〇8，pKTH5〇)可被用來產生本發明之多胜肽。為了在於動物細胞’例如CH0、C0S或NIH3T3細胞中表現載體，載體應具有表現於此類細胞中所必須的啟動子，例如 SV40 啟動子（Mul 1 igan et al.，Nature 277: 108 ( 1 979))、MMLV-LTR 啟動子、EF1 alpha 啟動子（Mizushima et al.’ Nucleic Acids Res 18: 5322 (1990)) 、 CMV 啟動子等，與篩選轉形物的標誌基因（例如藉由藥物篩選（例如，新黴素（neomycin)、G418之抗藥基因）較佳。具又這些載體特徵的已知載體的例子包括，例如pMAM、pDR2、 pBK-RSV 、 pBK-CMV 、 pOPRSV 與 p0P13 。呈現下列實施例以說明本發明與以協助熟悉此技藝人士製造與使用本發明。實施例並不傾向於在其他方面限制本發明範圍任何方式中。【實施例】材料與方法細胞株When E. coli is a host cell and the vector is amplified and mass produced in E coll (for example, JM109, DH5 alpha, HB101 or XLlBlue), the vector has an "ori" to be amplified in E_c〇li and screened for a marker gene of E. c〇li (for example, by ampicillin, tetracycline, kanamycin, chloramphenicol (&1〇1^111汕611 secretion) 〇1) or an analog screen for one of the drug resistance genes). For example, an M13-series carrier, a PUC-series carrier, pBR322, pBlueScript, pCR-Script, or the like can be used. In addition, pGEM_T, pDIRECT and pT7 can also be used for secondary replication and extraction of cDNA with the above vectors. A performance vector can be provided for use when the vector is used to produce a protein of the invention. 84 201134480 For example, one of the performance vectors to be represented in E. col i should have the above characteristics to be magnified in E. col i. When using E. c〇ii, such as JM109, DH5 alpha, HB101 or XLlBlue as the host cell, the vector should have a promoter, such as the iacz promoter (Ward et al., Nature 341: 544-6 (1989) FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or analog, which can effectively express the desired gene in E. Col i. Based on that aspect, for example, pGEpsxi (Pharmacia), "QIAexpress system" (Qiagen), pEGFP and PET (in this case, the host preferably BL21, which expresses T7 RNA polymerase) can be used instead of the above vector. Alternatively, the vector may contain a signal sequence for peptide secretion. An exemplary signal sequence that directs the peptide to be secreted to the periplasm of E. col i is the pelB signal sequence (Lei et al., J Bacteriol 1 69: 4379 (1 987)). The manner in which the vector is introduced into the host cell of interest includes, for example, a method of chlorination, and an electroporation method. In addition to E. col i, for example, mammalian expression vectors (eg, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (1 7): 5322 (1 990)), PEF, PCDM8), performance from insect cells Vector (for example, "Bac-to-BAC baculovirus expression system" (GIBC0 BRL), pBacPAK8), plant-derived expression vector (e.g., pMH1, pMH2), expression vector derived from animal virus (eg, PHSV, pMV, pAdexLcw), expression vectors from retroviruses (eg, pZipne0), expression vectors from yeast 85 201134480 (eg, "Pichia Expression Kit" (Invitrogen), pNVU, SP-Q〇 i) and a expression vector derived from Bacillus subtilis (for example, pPL6〇8, pKTH5〇) can be used to produce the multi-peptide of the present invention. In order to be in animal cells such as CH0, COS or NIH3T3 cells A expression vector that has a promoter necessary for expression in such cells, such as the SV40 promoter (Mul 1 igan et al., Nature 277: 108 (1 979)), the MMLV-LTR promoter, EF1 alpha (Mizushima et al. ' Nucleic Acids Res 18: 5322 (1990)), CMV promoter, etc., and marker genes for screening transformants (eg, by drug screening (eg, neomycin, G418) The drug gene) is preferred. Examples of known vectors having these carrier characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13. The following examples are presented to illustrate the invention and to assist in the familiarization of the art. The invention is made and used, and the examples are not intended to limit the scope of the invention in any way. [Examples] Materials and Methods Cell Lines

HLA-A 外匕母細胞株 (lymphoblastoidcell line)為購自職細胞與基因銀行 (SeaUie，WA)q2、HLA_A*〇2〇1陽性人類b淋巴母細胞株與C0S7、非洲綠猴腎細胞株為自ATCC所購得。 86 201134480 來自CLUAPI之胜肽的候選物選擇使用結合預測軟體 "BIMAS" (www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al.(J Immunol 1994， 152(1): 163-75)， Kuzushima et al.(Blood 2001， 98(6): 1872-81))與"NetMHC 3.0" (www.cbs.dtu.dk/services/NetMHC/) (Buus et a 1. (Tissue Antigens., 62:378-84， 2003)， Nielsen et al. (Protein Sc i., 1 2:1 007-1 7, 2003, Bio i nformat i cs, 20(9):1388-97, 2004))預測來自 CLUAP1 之 9 員與 10 員胜狀’其結合至HLA-A 2402或HLA_A*0201分子。這些胜狀係根據一標準固相合成方法由Biosynthesis (Lewisville, TX)來合成且藉由逆相高效能液體層析（reverse(j phase high performance liquid chromatography, HPLC)來純化。分別藉由分析型HLPC與質譜分析確認這些胜肽之純度 (>90%)與身份（identity)。將胜肽溶解於二曱基亞砜 (dimethylsulfoxide, DMS0)中於 20 mg/ml 且儲存於 -80〇C。 I n v i t r 〇細胞毒殺性τ淋巴球誘導使用來自單核白血球之樹突細胞做為抗原呈現細胞以誘導抗表現於人類白血球組織抗原（HLA)上之胜肽的細胞毒殺性T淋巴球反應。//?".⑽產生樹突細胞如別處所述 (Nakahara S et al., Cancer Res 2003 Jul 15, 63(14)： 87 201134480 4112 8)特別地，由 Ficol 1-Plaque (Pharmacia)溶液分離自一正常自願者（Hla-A* 2402或HLA-A* 020 1陽性）之周邊血液單核細胞，藉由貼附至一塑膠組織培養盤（Becton Dickinson)來分離以豐富其如一單核白血球部分。將經豐 s單核白血球之族群培養在1〇〇〇 u/ml之人類顆粒-巨噬細胞群落刺激因子（granulocyte-macrophage colony-stimulating factor， GM-CSF) (R&D System)與 1 000 U/ml 之白細胞介素（inter ieukin，IL)_4 (R&1) System) 存在下於含2%之熱去活性自身取得血清（aut〇1〇g〇us serum, AS)之 AIM-V 培養基（invi trogen)中。培養 7 天後，於AIM-V培養基中，於3 μ g /ml之召-2微球蛋白（beta 2-microglobulin)存在下以20 vg/ml之各合成胜肽脈衝 (pulsed)細胞激素誘導之樹突細胞3小時於37°c。所產生之細胞顯示表現樹突細胞相關分子，例如 CD80、CD83、CD86與HLA Class 11於其細胞表面（資料未顯示）。之後以X-射線（20 Gy)將這些胜肽脈衝之樹突細胞去活性且將其以1 : 2 0之比例與自身取得c D 8陽性T 細胞混合’ CD8陽性T細胞藉由以CD8 P〇sitive Isolatiori Ki t (Dynal)正選擇獲得。這些培養物設置於48孔盤 (Corning);各孔含1. 5 X 104胜肽脈衝之樹突細胞、3 χ ι〇5 CD8 陽性 Τ 細胞與 1 0 ng/ml 之 IL-7 (R&D System)於 0· 5 ml之AIM-V/2%自身取得血清培養基中。三天之後，以 IL_2(CHIR0N)添加至培養物至終濃度為20 IU/mh第7天與第14天更以自身取得胜狀脈衝之樹突細胞進一步刺激τ 88 201134480 細胞。以與上述相同之方法每次製備樹突細胞。於第21天，第三輪之胜肽刺激後，將細胞毒殺性T淋巴球進行抗胜肽脈衝之TISI細胞（A24)或T2細胞（A2)測試（Tanaka H et al., Br J Cancer 2001 Jan 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al.，Cl in Cancer Res 2004 Dec 15，10(24): 8577-86;The HLA-A lymphoblastoid cell line is purchased from the self-owned cell and gene bank (SeaUie, WA) q2, HLA_A*〇2〇1 positive human b lymphoblastoid cell line and COS7, African green monkey kidney cell line. Purchased from ATCC. 86 201134480 Candidates for peptides from CLUAPI were selected using the combination prediction software "BIMAS" (www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al. (J Immunol 1994, 152(1): 163) -75), Kuzushima et al. (Blood 2001, 98(6): 1872-81)) and "NetMHC 3.0" (www.cbs.dtu.dk/services/NetMHC/) (Buus et a 1. ( Tissue Antigens., 62:378-84, 2003), Nielsen et al. (Protein Sc i., 1 2:1 007-1 7, 2003, Bio i nformat i cs, 20(9):1388-97, 2004 )) It is predicted that 9 members and 10 members from CLUAP1 will be conjugated to HLA-A 2402 or HLA_A*0201 molecules. These traits were synthesized by Biosynthesis (Lewisville, TX) according to a standard solid phase synthesis method and purified by reverse phase high performance liquid chromatography (HPLC). HLPC and mass spectrometry confirmed the purity (>90%) and identity of these peptides. The peptide was dissolved in dimethylsulfoxide (DMS0) at 20 mg/ml and stored at -80 °C. I nvitr 〇 cytotoxic tau lymphocytes induce the use of dendritic cells derived from mononuclear leukocytes as antigen presenting cells to induce cytotoxic T lymphocyte responses against peptides expressed on human leukocyte antigen (HLA). //?" (10) produces dendritic cells as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul 15, 63(14): 87 201134480 4112 8), in particular, by Ficol 1-Plaque (Pharmacia) solution Peripheral blood mononuclear cells isolated from a normal volunteer (Hla-A* 2402 or HLA-A* 020 1 positive) are separated by a plastic tissue culture plate (Becton Dickinson) to enrich it as a single core White blood cell part. The human granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D System) and 1 000 U were cultured in a population of 1 〇〇〇u/ml. /ml interleukin (IL)_4 (R&1) System) AIM-V medium in the presence of 2% heat deactivated serum (aut〇1〇g〇us serum, AS) (invi trogen). After 7 days of culture, induced by pulsed cytokines in 20 μg/ml of each peptide in AIM-V medium in the presence of 3 μg / ml of beta 2-microglobulin The dendritic cells were at 37 ° C for 3 hours. The resulting cells were shown to exhibit dendritic cell-associated molecules such as CD80, CD83, CD86 and HLA Class 11 on their cell surface (data not shown). These peptide-pulsed dendritic cells were then deactivated by X-ray (20 Gy) and mixed with c D 8 positive T cells in a ratio of 1:20. CD8 positive T cells by CD8 P 〇sitive Isolatiori Ki t (Dynal) is choosing to get. These cultures were placed in 48-well plates (Corning); each well contained 1.5 X 104 peptide-pulsed dendritic cells, 3 χ ι〇5 CD8-positive sputum cells and 10 ng/ml of IL-7 (R& D System) was obtained in serum medium at 0. 5 ml of AIM-V/2% itself. Three days later, dendritic cells, which were added to the culture with IL_2 (CHIR0N) to a final concentration of 20 IU/mh on day 7 and day 14 to obtain a victory pulse, further stimulated tau 88 201134480 cells. Dendritic cells were prepared each time in the same manner as described above. On day 21, after the third round of peptide stimulation, the cytotoxic T lymphocytes were tested for anti-peptide pulsed TISI cells (A24) or T2 cells (A2) (Tanaka H et al., Br J Cancer 2001). Jan 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr 20, 84(8): 1052-7; Uchida N et al., Cl in Cancer Res 2004 Dec 15,10( 24): 8577-86;

Suda T et al., Cancer Sci 2006 May, 97(5): 411-9； Watanabe T et al., Cancer Sci 2005 Aug, 96(8)： 498-506)。細胞毒殺性T淋巴球擴張步驟使用與由 Riddel letal. (Walter EAetal.，N Engl J Med 1 995 Oct 19, 333(16): 1038-44; Riddel 1 SR et al., Nat Med 1996 Feb, 2(2): 216-23)敘述之相似方法於培養中擴張細胞毒殺性T淋巴球。全部5 x 1 〇4細胞毒殺性τ 淋巴球懸浮於25 ml之含有由MMC去活化之兩種人類β類淋巴母細胞株之AIM-V/5%自身取得血清培養基，在40 ng/ml之抗-CD3單株抗體（Pharmingen)存在下。在開始培養1天後，120IU/ml之IL-2加入培養中。於第5、8、11 天以新鮮之含30 IU/ml之IL-2的AIM-V/5%自身取得血清培養基提供給培養物（Tanaka H et al.，Br J Cancer 2001 Jan 5, 84(1)： 94-9; Umano Y et al., Br J Cancer 200 1 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer 89 201134480Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506). The cytotoxic T lymphocyte expansion step is used with Riddel Letal. (Walter EA et al., N Engl J Med 1 995 Oct 19, 333(16): 1038-44; Riddel 1 SR et al., Nat Med 1996 Feb, 2 (2): 216-23) A similar method described to expand cytotoxic T lymphocytes in culture. All 5 x 1 〇4 cytotoxic τ lymphocytes were suspended in 25 ml of AIM-V/5% self-derived serum medium containing two human β-lymphocyte strains deactivated by MMC at 40 ng/ml In the presence of anti-CD3 monoclonal antibody (Pharmingen). One day after the start of cultivation, 120 IU/ml of IL-2 was added to the culture. Serum medium was obtained from fresh AIM-V/5% self containing 30 IU/ml of IL-2 on days 5, 8, and 11 (Tanaka H et al., Br J Cancer 2001 Jan 5, 84). (1): 94-9; Umano Y et al., Br J Cancer 200 1 Apr 20, 84(8): 1052-7; Uchida N et al., Clin Cancer 89 201134480

Res 2004 Dec 1 5, 1 0(24): 8577-86; Suda T et al. , CancerRes 2004 Dec 1 5, 1 0(24): 8577-86; Suda T et al. , Cancer

Sci 2006 May, 97(5): 41 卜9; Watanabe T et al.，Cancer Sci 2005 Aug, 96(8): 498-506)。細胞毒殺性T淋巴球複製（ci〇ne)的建立稀釋以使細胞毒殺性T淋巴球以0. 3、1與3細胞毒殺性Τ淋巴球/孔的含量於96圓底（round-bottomed)微效價盤（Nalge Nunc International)中。細胞毒殺性τ淋巴球與1 X 104細胞/孔之2種兩種人類B類淋巴母細胞株、 30 ng/ml之抗-CD抗體與125 U/ml之IL-2於全部150 μ 1 /孔之含5%自身取得之血清的AIM-V培養基中一起培養。 10天後將50 // 1/孔之IL-2加入培養基中以達到i25U/ml IL-2之終濃度。於第14天測試細胞毒殺性τ淋巴球之活性，且使用上述相同方法擴張細胞毒殺性T淋巴球複製 (Uchida N et al. , Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506)。專一之細胞毒殺性T淋巴球活性為了測試專一之細胞毒殺性T淋巴球活性，執行 IFN-τ酵素結合免疫斑點（ELISp〇T)分析與酵素結合免疫吸附（ELISA)分析。特別地’製備胜肽脈衝之 TISKA24)或T2(A2)(1 X i〇4/weii)為刺激細胞。培養之細 201134480 胞於48孔中做為應答細胞。在製造商步驟下執行ifn— γ 酵素結合免疫斑點分析與IFN_ r酵素結合免疫吸附分析。強有力地表現目標基因與HLA_24或Hu_A2任一或兩者的細胞的建立藉由PCR將編碼出目標基因之開放讀框或hla_a24或 HLA-A2之cDNA放大。將PCR放大產物複製進一載體。使用 lipofectamine 2000 (invitrogen)，根據製造商建議步驟將質體轉染進C0S7，其為一目標基因與HU_A24與A2 無效細胞株。於自轉染後2天，以versene (Invitr〇gen) 收集經轉染的細胞且使用為細胞毒殺性τ淋巴球活性分析之目標細胞（5 X l〇4/well)。結果1 於癌症中增強之CLUAP1表現使用c-DNA微陣列獲得自各種癌症之廣泛基因表現概况（profile)顯示 CLUAPl ( GenBank Accession No. 匪一01 5041或NM—024793;例如，序列辨識號：44)被提升。與對應之正常組織比較，乳癌中7 7個有17個、子宮頸癌中1 9個有1 5個、大腸直腸癌中1 9個有12個、食道癌中 19個有12個、胃癌中25個有7個、瀰漫型胃癌中5個有 5個、淋巴癌中18個有5個、神經母細胞瘤中16個有2 個、騰臟癌中9個有4個，有根據地提升CLUAP1表現（表 1) ° 91 201134480 表1、與對應之正常組織比較，於癌症組織中觀察到 CLUAP1之向上調控之例子的比例癌症/腫瘤比乳癌 17/77 子宮頸癌 15/19 大腸直腸癌 12/19 食道癌 12/19 _胃癌 7/25 觸漫型胃癌 5/5 淋巴癌 5/18 神經母細胞瘤 2/16 騰臟癌 4/9 結果2 來自CLUAP1之HLA-A24結合胜肽的預測表2a與2b以最高之結合親和力之順序顯示CLUAP1之 HLA-A24結合9員與1〇員胜肽。藉由BIMAS來預測序列辨識號：1至11與序列辨識號：15至22的胜肽而藉由 NetMHC3. 0來預測序列辨識號：12至14的胜肽。總共22 個具有潛在HLA-A24結合能力之胜狀被選擇且將其試驗以確定抗原決定位胜肽。 92 201134480 表2a、來自CLUAP1之HLA-A24結合9員胜肽起始位置胺基酸序列分數序列辨識號 258 TYLEKFQNL 518. 4 1 244 EYEKTEEEL 330 2 255 QYDTYLEKF 110 3 283 RFEEAKNTL 86. 4 4 31 NFGLVSEVL 28 5 8 NFTEMMRAL 24 6 26 NFRTPNFGL 20 7 168 RTEAIARPL 16. 8 8 64 FFIKAIAQF 15 9 216 KIEKRKLEL 13. 2 10 335 KPQTAMEML 12 11 起始位置胺基酸序列胜肽 Kd (nM) 序列辨識號 86 LYQADGYAV 473 12 71 QFMATKAHI 1477 13 154 LYDLLGMEV 1564 14 表2b、來自CLUAP1之HLA-A24結合10員胜肽起始位置胺基酸序列分數序列辨識號 91 GYAVKELLKI 55 15 104 LYNAMKTKGM 37. 5 16 31 NFGLVSEVLL 20 17 125 KFKFDLGSKI 13. 2 18 17 GYPRHISMEN 11. 6 19 289 NTLCLIQNKL 11. 1 20 158 TYLEKFQNLT 10. 8 21 63 VFFIKAIAQF 10 22 起始位置指自CLUAP1之N端的胺基酸殘基數目。結合分數與分離常數（dissociation constant)[Kd (nM)]來自 “BIMAS” 與 “NetMHC3. 0” 。 93 201134480 以HLA- A* 2402限制之來自CLUAP1之預測胜肽誘導細胞毒殺性T淋巴球根據敘述於“材料與方法”之步驟產生對於那些來自 CLUAP1之胜肽的細胞毒殺性τ淋巴球。藉由IFN_ γ酵素結合免疫斑點分析測定胜肽專一細胞毒殺性τ淋巴球活性 (第1 a- j圖）。與控制組孔洞相較，下列孔洞編號顯示強而有力的IFN-r產生：以CLUAP卜A24-9-255 (序列辨識號：3)刺激之孔洞編號#6 (a)、以CLUAP1-A24-9-283 (序列辨識號：4)刺激之#2 (b)、以CLUAP1-A24-9-31 (序列辨識號：5 )刺激之#2 (c)、以CLUAP1-A24-9-168 (序列辨識號：8)刺激之#4 (d)、以CLUAP卜A24-9-64 (序列辨識號：9)刺激之#4 (e)、以CLUAP卜A24-9-216 (序列辨識號：10)刺激之#1 (〇、以CLUAP1-A24-9-1 54 (序列辨識號：14)刺激之#6 (g)、以CLUAP1-A24-10-91 (序列辨識號：15)刺激之#2 (h);以CLUAP卜A24-10-104 (序列辨識號：16)刺激之#4 (i)與以CLUAP1-A24-1 0-125 (序列辨識號.18)刺激之#5 (j)。另一方面，藉由以顯示於表2a與表2b中之其他胜肽刺激沒有測定出專一之細胞毒殺性T淋巴球活性’儘管那些胜肽具有與HLA-A* 2402之可能結合活性。如負資料的典型例子，從以CLUAP1-A24-9-258 (序列辨識號：1)刺激之細胞毒殺性T淋巴球，並無觀察到專一的IFN- r產生（第lk圖）。因此，其指出筛選出來自CLUAP1之10個胜肽為可誘導強而有力之細胞毒殺性 T淋巴球的胜肽。 94 201134480 抗C L U A P1衍生胜肽之細胞毒殺性τ淋巴球細胞株與複製的建立藉由IFN- 7"酵素結合免疫斑點分析偵測，於以 CLUAP1-A24-9-255 (序列辨識號：3 )刺激之孔洞編號#6 (a)、以CLUAP卜Α24-9-1 68(序列辨識號：8 )刺激之(b)、以CLUAP1-A24-9-64 (序列辨識號：9)刺激之#4 (c)、以 CLUAP1-A24-9-1 54 (序列辨識號：14)刺激之#6 (d)與以 CLUAP1-A24-10-91 (序列辨識號：15)刺激之#2 (e)中顯示胜肽專一細胞毒殺性T淋巴球活性的細胞被擴張，並藉由限制稀釋來建立細胞毒殺性τ淋巴球細胞株，於上方段落“材料與方法”中所述。藉由IFN-T酵素結合免疫吸附分析來測定那些細胞毒殺性T淋巴球細胞株的細胞毒殺性 T淋巴球活性（第2a-e圖）。與無胜肽脈衝之目標細胞相較’所有細胞毒殺性T淋巴球細胞株顯示強的抗以對應胜肽脈衝之目標細胞的IFN- r產生。此外，如於“材料與方法所述藉由來自細胞毒殺性τ淋巴球細胞株的限制稀釋建立細胞毒殺性T淋巴球複製，且藉由IFN_ r酵素結合免疫吸附分析測定來自抗經胜肽脈衝目標細胞之細胞毒殺性 T淋巴球複製的IFN- r產生。在第3圖中，從以 CLUAP1-A24-9-255 (序列辨識號：3) 、CLUAP1-A24-9-64 (序列辨識號：9)與CLUAP1-A24-10-91 (序列辨識號： 1 5 )刺激之細胞毒殺性T淋巴球複製測定出強的丨FN_ γ產生0 95 201134480 抗外生表現CLUAP1與HLA-A* 2402之目標細胞的專一細胞毒殺性T淋巴球活性檢驗經提升抗這些胜肽之所建立的細胞毒殺性T淋巴球細胞株其對於辨認内生表現CLUAP1與HLA-A5" 2402分子之目標細胞的能力。使用由對應之胜肽提升的細胞毒殺性 T淋巴球細胞株做來測試抗C0S7細胞的專一細胞毒殺性T 淋巴球活性，而C0S7細胞經全長之CLUAP1與HLA-A* 2402 分子基因轉染（對於外生表現CLUAP1與HLA-A# 2402基因之目標細胞的特定模式）。COS7細胞以全長CLUAP1基因或HLA-A* 2402轉染製備為控制組。於第4圖中，以 CLUAP1-A24-9-255 (序列辨識號：3)刺激之細胞毒殺性T 淋巴球顯示強的抗表現CLUAP1與HLA-A# 2402兩者之COS7 細胞的細胞毒殺性T淋巴球活性。另一方面，沒有偵測到抗控制組之顯著專一之細胞毒殺性T淋巴球活性。因此，這些資料清楚證明CLUAP1-A24-9-255 (序列辨識號：3 ) 被自然表現於具有HLA-A* 2402分子之目標細胞上且由細胞毒殺性T淋巴球所辨認。這些結果顯示此來自CLUAP1之胜肽為可適合為具CLU API表現之腫瘤之病患的癌症疫苗。抗原胜肽之同源分析以 CLUAP1-A24-9-255 (序列辨識號：3 )、 CLUAP1-A24-9-283C 序列辨識號：4)、CLUAP卜A24-9-31 (序列辨識號：5)、 CLUAP1-A24-9-1 68 (序列辨識號：8)、 96 201134480 CLUAP1-A24-9-64 (序列辨識號：9)、 CLUAP1-A24-9-216 (序列辨識號：10)、 CLUAP卜A24-9-1 54 (序列辨識號： 14 ) 、 CLUAP1-A24-10-91 (序列辨識號：15 )、 CLUAP1-A24-10-104 (序列辨識號：16 )與 CLUAP1-A24-1 0-1 25 (序列辨識號：1 8 )刺激之細胞毒殺性 T淋巴球顯示顯著且專一之細胞毒殺性T淋巴球活性。此結果可能起因於CLUAP1-A24-9-255 (序列辨識號：3)、 CLUAP 卜 A24-9-283 (序列辨識號：4)、 CLUAP1-A24-9-31 (序列辨識號：5)、 CLUAP1-A24-9-1 68 (序列辨識號： 8)、CLUAPl-A24-9-64(序列辨識號：9)、CLUAP1-A24-9-216 (序列辨識號：10 )、 CLUAP1-A24-9-1 54 (序列辨識號： 14 ) 、 CLUAP卜A24-10-91 (序列辨識號：15 )、 CLUAP1-A24-10-104 (序列辨識號：16)與 CLUAP卜A24-1 0-1 25 (序列辨識號：18)之序列為與源自已知使人類免疫系統敏感之其他分子的胜肽同源的事實。為了排除此可能性，對於使用為關鍵字向BLAST演算法 (http://www.ncbi.nlm.nih.gov/blast/blast.cgi)查詢之這些胜肽序列執行同源性分析，而BLAST演算法顯示沒有序列顯示顯著之同源性。同源性分析之結果指出 CLUAP卜A24-9-2 55 (序列辨識號：3)、CLUAP卜A24-9-283 (序列辨識號：4)、CLUAP卜A24-9-31 (序列辨識號：5)、 CLUAP卜A24-9-168 C 序列辨識號：8)、 CLUAP卜A24-9-64 (序列辨識號：9)、CLUAPl-A24-9-216(序列辨識號：10)、 CLUAP卜A24-9-1 54C 序列辨識號：14)、CLUAP卜A24-10-91 97 201134480 (序列辨識號：15)、 CLUAP卜A24-1 0-1 04 (序列辨識號： 16)與CLUAP1-A24-1 0- 1 25 (序列辨識號：18)之序列為獨特的，且因此只有很小可能性分子會對於一些非相關分子提高非傾向之免疫反應。因此，確認新穎之來自CLUAP1之HLA-A24抗原決定位胜肽。此外此處結果證明CLUAP1之抗原決定位胜肽對使用於癌症免疫治療中而言可為適合的。結果3 來自CLUAP1之HLA-A02結合胜肽的預測表3a與3b以最高之結合親和力之順序顯示CLUAPI之 HLA-A02結合9員與10員胜肽。總共21個具有潛在HLA-A02 結合能力之胜肽被選擇且將其試驗以確定抗原決定位胜肽0 表3a、來自CLUAP1之HLA-A02結合9員胜肽起始位置胺基酸序列分數序列辨識號 34 LVSEVLLWL 164. 809 23 58 TEQDRVFFI 61.024 24 95 KELLKITSV 40.281 25 85 KLYQADGYA 39.784 26 290 TLCLIQNKL 21.362 27 265 NLTYLEQQL 21.362 28 297 KLKEEEKRL 10. 729 29 99 KITSVLYNA 5. 499 30 188 AIKEILTQV 3. 156 31 96 ELLKITSVL 2. 431 32 98 201134480 表3b、來自CLUAP1之HLA-A02結合10員胜肽起始位置胺基酸序列分數序列辨識號 153 SLYDLLGMEV 912.522 33 85 KLYQADGYAV 778. 982 34 34 LVSEVLLWLV 731. 711 35 33 GLVSEVLLWL 270.234 36 72 FMATKAHIKL 70.971 37 80 KLNTKKLYQA 39. 992 38 41 WLVKRYEPOT 34.279 39 233 TLQSVRPCFM 27. 324 40 183 KVMRIAIKEI 27. 19 41 343 LMQGRPGKRI 12. 809 42 222 LELERNRKRL 10. 712 43 起始位置指自CLUAP1之N端的胺基酸殘基數目。結合分數來自“BIMAS” 。以HLA- A* 020 1限制之來自CLUAP1之預測胜肽誘導細胞毒殺性T淋巴球根據敘述於“材料與方法”之步驟產生對於那些來自 C L U A P1之胜肽的細胞毒殺性T淋巴球。藉由IF N - γ酵素結合免疫斑點分析測定胜肽專一細胞毒殺性Τ淋巴球活性 (第5a-g圖）。與控制組孔洞相較下列孔洞編號顯示強而有力的IFN- r產生：以CLUAP1-A02-9-34 (序列辨識號： 23 )刺激之孔洞編號#3 (a)、以CLUAP1-A02-9-99 (序列辨識號：30)刺激之#6(13)、以（^1^?卜六02-1 0-1 53 (序列辨識號：33)刺激之#2 (c)、以CLUAP卜A02- 1 0-85 (序列辨識號：34)刺激之#3 (d)、以CLUAP1-A0 2- 1 0-34 (序 99 201134480 列辨識號：35)刺激之#2 (e)、以CLUAP卜A02-1 0-33 (序列辨識號：36)刺激之#2 (f)與以CLUAP卜A02-1 0-80 (序列辨識號.38)刺激之#5 (g)。另一方面’藉由以顯示於表3a與表3b中之其他胜狀刺激沒有測定出專一之細胞毒殺性T淋巴球活性，儘管那些胜肽具有與HLA-A* 0201之可能結合活性。如負資料的典型例子，從以CLUAP1-A02-9-58 (序列辨識號：24 ) (h)刺激之細胞毒殺性T淋巴球，並無觀察到專一的IFN- 7產生。因此，其指出篩選來自CLUAP1 之7個胜肽為可誘導強而有力之細胞毒殺性T淋巴球的胜肽。抗CLUAP1衍生胜肽之細胞毒殺性T淋巴球細胞株與複製的建立藉由IFN- r 酵素結合免疫斑點分析偵測，於以 CLUAP1-A02-9-34 (序列辨識號：23 )刺激之孔洞編號#3 (a) 、以 CLUAP卜A02-9-99 (序列辨識號：30 )刺激之#6 (b) 、以 CLUAP1-A02-10-1 53 (序列辨識號：33)刺激之#2 (c) 、以CLUAP卜A02-1 0-85 (序列辨識號：34)刺激之#3 (d) 、以 CLUAP1-A02-10-34C 序列辨識號：35)刺激之#2 (e) 與以CLUAP 1-A02-1 0-33 (序列辨識號：36)刺激之#2 (f) 中顯示胜肽專一細胞毒殺性T淋巴球活性的細胞被擴張，並藉由限制稀釋來建立細胞毒殺性T淋巴球細胞株，於上方段落“材料與方法，’中所述。藉由IFN-τ酵素結合免疫吸附分析來測定這些細胞毒殺性T淋巴球細胞株的細胞毒 100 201134480 殺性T淋巴球活性（第6a-f圖）。與無胜肽脈衝之目標細胞相較，細胞毒殺性T淋巴球細胞株顯示強的抗以對應胜狀脈衝之目標細胞的IF N - τ產生。此外，如於“材料與方法”所述藉由來自細胞毒殺性T淋巴球細胞株的限制稀釋建立細胞毒殺性T淋巴球複製，且藉由IFN- 7酵素結合免疫吸附分析測定來自抗經胜肽脈衝目標細胞之細胞毒殺性 T淋巴球複製的IFN-r產生。從以CLUAP1-A02-9-34 (序列辨識號：23) (a)、CLUAP1-A02-9-99 (序列辨識號： 30 ) (b)、CLUAP1-A02-1 0-1 53 (序列辨識號：33 ) (c)與 CLUAP卜A02-1 0-85 (序列辨識號：34) (d)刺激之細胞毒殺性T淋巴球複製測定出強的ifn- 7產生（第7a-d圖）。抗外生表現CLUAP1與HLA-A* 020 1之目標細胞的專一細胞毒殺性T淋巴球活性檢驗經提升抗各胜肽之所建立的細胞毒殺性T淋巴球細胞株與複製其對於辨認内生表現CLUAP1與HLA-A* 0201 分子之目標細胞的能力。使用由對應之胜肽提升的細胞毒殺性τ淋巴球細胞株與複製做為影響細胞來測試抗c〇S7細胞的專一細胞毒殺性T淋巴球活性，而c〇S7細胞經全長之 CLUAP1與HLA-A* 0201基因轉染（對於外生表現CLUApi與 HLA-A* 020 1基因之目標細胞的特定模式）。c〇S7細胞以全長CLUAP1或HLA-A* 0201轉染製備為控制組。於第8圖中，以CLUAP1-A24-9-255 (序列辨識號：3 ) 刺激之細胞毒殺性τ淋巴球細胞株顯示強的抗表現CLUAP1 101 201134480 與HLA-A* 0201兩者之C0S7細胞的細胞毒殺性τ淋巴球活性。另一方面，沒有偵測到抗控制組之顯著專一之細胞毒殺性 Τ淋巴球活性。因此’這些資料清楚證明 CLUAPl-A24-9-255 (序列辨識號：3)被内生處理並表現於具有H L A _ A * 0 2 0 1分子之目標細胞上且由細胞毒殺性τ淋巴球所辨認。這些結果顯示這些來自CLUAP1之胜肽為可得以提供具CLUAP1表現之腫瘤之病患的癌症疫苗。抗原胜肽之同源分析以 CLUAP1-A02-9-34 (序列辨識號：23 )、 CLUAP卜A02-9-99C 序列辨識號：30)、CLUAP卜A02-10-153 (序列辨識號：33)、 CLUAP卜A02- 1 0-85 C序列辨識號： 34 ) 、 CLUAP1-A02-10-34 (序列辨識號：35 )、 CLUAP1-A02-1 0-33C 序列辨識號：36)與 CLUAP1-A02-10-80 (序列辨識號：38 )刺激之細胞毒殺性T淋巴球顯示顯著且專一之細胞毒殺性T淋巴球活性。此結果可能起因於 CLUAP1-A02-9-34 C 序列辨識號：23)、 CLUAP卜A02-9-99 (序列辨識號：30 )、 CLUAP1-A02-1 0-1 53 (序列辨識號： 33 ) 、 CLUAP1-A02-1 0-85 (序列辨識號：34 )、 CLUAP1-A02-1 0-34(序列辨識號：35)、CLUAP卜A02-10-33 (序列辨識號：36)與CLUAP1-A02-1 0-80 (序列辨識號： 3 8 )之序列為與源自已知使人類免疫系統敏感之其他分子的胜肽同源的事實。為了排除此可能性，對於使用為關鍵字向BLAST演算 102 201134480 法(http://www.ncbi.nlm.nih.gov/blast/blast.cgi)查詢之這些胜肽序列執行同源性分析，而BLAST演算法顯示沒有序列顯示顯著之同源性。同源性分析之結果指出 CLUAP1-A02-9-34C 序列辨識號：23)、CLUAPl-A02-9-99 (序列辨識號：30)、CLUAP1-A0 2-1 0-1 53 C 序列辨識號：33)、 CLUAP卜A0 2-1 0-85(序列辨識號：34)、CLUAP卜A0 2-10-34 (序列辨識號：35)、 CLUAP1-A02- 1 0-33 C序列辨識號： 36 )與CLUAP1-A02-1 0-80 (序列辨識號：38 )之序列為獨特的，且因此只有很小可能性分子會對於一些非相關分子提高非傾向之免疫反應。因此’我們確認新穎之來自CLUAP1之HLA- A* 020 1 抗原決定位胜肽。此外，其證明CLUAP1之抗原決定位胜肽對癌症免疫治療而言可為適用。產業利用性本發明提供新的腫瘤相關抗原，特別是來自CLMpi的那些，其可誘導強且專一的抗腫瘤免疫.反應，且對於癌症形弋之廣泛種類而&具有應用性。此腫瘤相關抗原可提供效用為抗與CLUAPI相關之疾病的胜肽疫苗與AH相關之疾病，例如癌症，更拉&丨a a十文特別疋礼癌 '子宮頸癌、大腸直腸癌、食道癌、胃癌、瀰漫型f ^又i月届、淋巴癌、砷經母細胞瘤、騰臟癌。當於此詳述本#明與提及其特定實施例時需瞭解的 103 201134480 是’前述敘述本質為示範與解釋且意為說明本發明與其較佳實施例。於例行實驗之中，熟悉此技藝人士可立即瞭解在不脫離本發明之精神下其可實行各種改變與修飾。因此本發明不被前述所定義，而被下列申請專利範圍與其等同物所定義。【圖式簡單說明】 [第la-f圖] 第la-f圖由一系列照片，（a)至（f)所組成，其顯示在以來自CLUAP1之胜肽誘導之細胞毒殺性τ淋巴球上之 IFN-τ 酵素結合免疫斑點分析（ELISP0T)的結果。與控制組孔洞相較，於下列孔洞編號中之細胞毒殺性T淋巴球顯示強的IFN- r產生：以CLUAP1-A24-9-255 (序列辨識號： 3)刺激之孔洞編號#6 (a)、以CLUAP卜A24-9-283 (序列辨識號：4)刺激之#2 (b)、以CLUAP卜A24-9-31 (序列辨識號：5)刺激之#2 (c)、以CLUAP1-A24-9-168 (序列辨識號：8)刺激之#·4 (d)、以CLUAP1-A24-9-64 (序列辨識號：9)刺激之#4 (e)與以CLUAP1-A24-9-216 (序列辨識號：10 )刺激之#1 (f)。以矩形盒狀所指出於孔洞中之細胞被擴張來建立細胞毒殺性T淋巴球細胞株。相對的，如負資料的典型例子，其未顯示來自以CLUAP1-A24-9-258C序列辨識號：1)刺激之細胞毒殺性T淋巴球之專一的IFN- γ 產生（k)。於圖中，“ 指出抗以適合之胜肽脈衝的目標細胞的IF N - 7產生，而“’指出抗未以任何胜肽脈衝的 104 201134480 目標細胞的IFN- T產生。 [第lg-k圖] 第1 g _ k圖由·一糸列照片’（g)至（k)所組成，其顯示在以來自CLUAP1之胜肽誘導之細胞毒殺性τ淋巴球上之 IFN-τ酵素結合免疫斑點分析（ELISP0T)的結果。與控制組孔洞相較，於下列孔洞編號中之細胞毒殺性T淋巴球顯示強的IFN-τ產生：以CLUAPl-A24-9-154(序列辨識號： 14)刺激之孔洞#6 (g)、以CLUAP1-A24-10-91 (序列辨識號：15)刺激之#2 (h)、以CLUAP卜A24-1 0-104 (序列辨識號：16)刺激之#4 (i)與以CLUAP1-A24-1 0-125 (序列辨識號：1 8 )刺激之#5 ( j)。以矩形盒狀所指出於孔洞中之細胞被擴張來建立細胞毒殺性T淋巴球細胞株。相對的，如負資料的典型例子’其未顯示來自以 CLUAP1-A24-9-258 (序列辨識號：1)刺激之細胞毒殺性T 淋巴球之專一的IFN-γ產生（k)。於圖中，“+”指出抗以適合之胜肽脈衝的目標細胞的IFN- τ產生’而“指出抗未以任何胜肽脈衝的目標細胞的IFN- τ產生。 [第2圖] 第2圖由一系列線圖，（a)至（e)所組成’顯示IF N - 7 酵素結合免疫吸附分析偵測的結果，其顯示以 CLUAP1-A24-9-255 (序列辨識號：3 ) (a)、以 CLUAP1-A24-9-168 (序列辨識號：8 ) (b)、以 105 201134480 CLUAP卜A24-9-64 (序列辨識號：9 ) (c)、以 CLUAP1-A24-9-154 (序列辨識號：14 ) (d)與以 CLUAP卜A24-10-91 (序列辨識號：15) (e)刺激之細胞毒殺性T淋巴球細胞株的IFN- τ產生。結果顯示與控制組相較’藉由以各胜肽刺激建立之細胞毒殺性T淋巴球細胞株顯示強而有力之IFN- r產生。於圖中，“ +，，指出抗以適合之胜肽脈衝的目標細胞的IFN- 7產生，而“指出抗未以任何胜肽脈衝的目標細胞的IFN- 7產生。 [第3圖] 第3圖由一系列線圖，（a)至（d)所組成，顯示IFN- 7· 酵素結合免疫吸附分析偵測的結果’其顯示藉由來自以 CLUAPl-A24-9-255(序列辨識號：3 ) (a)、CLUAP1-A24-9-64 (序列辨識號：9) (b)與CLUAP卜A24-10-91 (序列辨識號：15 ) (c)刺激之細胞毒殺性τ淋巴球細胞株的限制稀釋所建立之細胞毒殺性T淋巴球複製的IFN- 7產生。結果顯示與控制組相較，藉由以各胜肽刺激所建立之細胞毒殺性T淋巴球複製顯示強而有力之iFN_ 7產生。於圖中，指出抗以適合之胜肽脈衝的目標細胞的丨FN_ 7產生’而指出抗未以任何胜肽脈衝的目標細胞的IFN_ γ 產生。 [第4圖]Sci 2006 May, 97(5): 41 Bu 9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506). The cytotoxic T lymphocyte replication (ci〇ne) was established to dilute the cytotoxic T lymphocytes with 0.3, 1 and 3 cell toxic sputum lymphocytes/holes at a round-bottomed level. In the micro-valence plate (Nalge Nunc International). Two types of human B lymphoblastoid cells with 1 x 104 cells/well, 30 ng/ml anti-CD antibody and 125 U/ml IL-2 at all 150 μl / The wells were cultured together in AIM-V medium containing 5% of the serum obtained by themselves. After 10 days, 50 // 1/well of IL-2 was added to the medium to reach a final concentration of i25 U/ml IL-2. The activity of the cytotoxic T lymphocytes was tested on day 14 and the cytotoxic T lymphocyte replication was expanded using the same method described above (Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506). Specific Cytotoxic T Lymphocyte Activity To test the specific cytotoxic T lymphocyte activity, an IFN-τ enzyme-binding immunospot (ELISp〇T) assay and an enzyme-binding immunosorbent assay (ELISA) were performed. Specifically, 'preparation of peptide peptide TISKA24) or T2 (A2) (1 X i〇4/weii) is to stimulate cells. Cultured fine 201134480 Cells were used as responder cells in 48 wells. The ifn-gamma enzyme binding immunospot assay was combined with the IFN-r enzyme-binding immunosorbent assay at the manufacturer's step. Establishment of cells that strongly express the target gene and either or both of HLA_24 or Hu_A2 The cDNA encoding the open reading frame of the target gene or hla_a24 or HLA-A2 is amplified by PCR. The PCR amplification product was copied into a vector. The plastids were transfected into COS7 using lipofectamine 2000 (invitrogen) according to the manufacturer's recommended procedure, which is a target gene and HU_A24 and A2 null cell lines. Two days after transfection, transfected cells were harvested as versene (Invitr〇gen) and target cells (5 X l〇4/well) analyzed for cytotoxic t lymphocyte activity were used. Results 1 Enhanced CLUAP1 expression in cancer The broad gene expression profile obtained from various cancers using c-DNA microarrays shows CLUAP1 (GenBank Accession No. 匪01 5041 or NM-024793; for example, sequence ID: 44 ) was promoted. Compared with the corresponding normal tissues, there are 17 of 7 in breast cancer, 15 in 19 of cervical cancer, 12 in colorectal cancer, 12 in 19 of esophageal cancer, and 12 in gastric cancer. There are 7 in 25, 5 in diffuse gastric cancer, 5 in lymphoma, 5 in neuroblastoma, 2 in neuroblastoma, and 4 in 9 in sputum cancer, and CLUAP1 is promoted. Performance (Table 1) ° 91 201134480 Table 1. Proportion of examples of upward regulation of CLUAP1 observed in cancer tissues compared with the corresponding normal tissues Cancer/tumor than breast cancer 17/77 Cervical cancer 15/19 Colorectal cancer 12 /19 Esophageal cancer 12/19 _ gastric cancer 7/25 Diffuse gastric cancer 5/5 Lymphoma 5/18 Neuroblastoma 2/16 Stem cancer 4/9 Result 2 Prediction of HLA-A24 binding peptide from CLUAP1 Tables 2a and 2b show that HLA-A24 binding of 9 and 1 member peptide of CLUAP1 is in the order of highest binding affinity. The sequence identification number: 1 to 11 and the sequence identification number: 15 to 22 peptides were predicted by BIMAS and the sequence identification number: 12 to 14 peptides were predicted by NetMHC3.0. A total of 22 traits with potential HLA-A24 binding capacity were selected and tested to determine epitopes. 92 201134480 Table 2a, HLA-A24 from CLUAP1 binding 9-member peptide start position amino acid sequence fractional sequence identification number 258 TYLEKFQNL 518. 4 1 244 EYEKTEEEL 330 2 255 QYDTYLEKF 110 3 283 RFEEAKNTL 86. 4 4 31 NFGLVSEVL 28 5 8 NFTEMMRAL 24 6 26 NFRTPNFGL 20 7 168 RTEAIARPL 16. 8 8 64 FFIKAIAQF 15 9 216 KIEKRKLEL 13. 2 10 335 KPQTAMEML 12 11 Starting position amino acid sequence peptide Kd (nM) Sequence identification number 86 LYQADGYAV 473 12 71 QFMATKAHI 1477 13 154 LYDLLGMEV 1564 14 Table 2b, HLA-A24 from CLUAP1 binding 10 member peptide start position amino acid sequence fraction sequence identification number 91 GYAVKELLKI 55 15 104 LYNAMKTKGM 37. 5 16 31 NFGLVSEVLL 20 17 125 KFKFDLGSKI 13. 2 18 17 GYPRHISMEN 11. 6 19 289 NTLCLIQNKL 11. 1 20 158 TYLEKFQNLT 10. 8 21 63 VFFIKAIAQF 10 22 The starting position is the number of amino acid residues from the N-terminus of CLUAP1. The binding fraction and the dissociation constant [Kd (nM)] are from "BIMAS" and "NetMHC3.0". 93 201134480 Predicted peptides from CLUAP1 restricted by HLA-A* 2402 induced cytotoxic T lymphocytes Cytotoxic tau lymphocytes were generated for those peptides derived from CLUAP1 according to the procedure described in Materials and Methods. The peptide-specific cytotoxic lymphosphere activity was determined by IFN_γ enzyme binding immunospot assay (Fig. 1 a- j). Compared with the control group, the following hole numbers show strong IFN-r production: hole number #6 (a) stimulated by CLUAP A24-9-255 (SEQ ID NO: 3), with CLUAP1-A24- 9-283 (Sequence ID: 4) Stimulus #2 (b), CLUAP1-A24-9-31 (Serial Identification Number: 5) Stimulated #2 (c), with CLUAP1-A24-9-168 ( Sequence identification number: 8) Stimulus #4 (d), CLUAP A24-9-64 (sequence identification number: 9) Stimulation #4 (e), CLUAP A24-9-216 (sequence identification number: 10) Stimulus #1 (〇, stimulated by CLUAP1-A24-9-1 54 (SEQ ID NO: 14) #6 (g), stimulated by CLUAP1-A24-10-91 (SEQ ID NO: 15) #2 (h); #4 (i) stimulated by CLUAP A24-10-104 (sequence identification number: 16) and #5 (stimulated by CLUAP1-A24-1 0-125 (sequence identification number. 18) j). On the other hand, specific cytotoxic T lymphocyte activity was not determined by stimulation with other peptides shown in Tables 2a and 2b 'although those peptides have the potential to bind to HLA-A* 2402 Activity. A typical example of negative data from a cytotoxic T lymphocyte stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1) The ball, no specific IFN- r production was observed (line lk). Therefore, it was pointed out that 10 peptides from CLUAP1 were screened to be peptides that induce strong and potent cytotoxic T lymphocytes. 94 201134480 The cytotoxic T lymphocyte cell line and replication of anti-CLUA P1-derived peptides were detected by IFN-7" enzyme-binding immunospot assay and stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) Hole number #6 (a), stimulated by CLUAP dip 24-9-1 68 (sequence identification number: 8), (b), stimulated by CLUAP1-A24-9-64 (sequence identification number: 9) #4 (c) #6 (d) stimulated with CLUAP1-A24-9-1 54 (sequence identification number: 14) and #2 (e) stimulated with CLUAP1-A24-10-91 (sequence identification number: 15) The cells showing the activity of the peptide-specific cytotoxic T lymphocyte expansion are expanded, and the cytotoxic TNF lymphocyte cell line is established by limiting dilution, as described in the above section "Materials and Methods". By IFN-T Enzymes combined with immunosorbent assays were used to determine the cytotoxic T lymphocyte activity of those cytotoxic T lymphocyte cell lines (Fig. 2a-e). All cytotoxic T lymphocyte cell lines showed strong resistance to IFN- r production by target cells corresponding to peptide pulses compared to target cells with peptide-free pulses. In addition, cytotoxic T lymphocyte replication was established by limiting dilution from cytotoxic tau lymphocyte cell lines as described in Materials and Methods, and anti-synaptic peptide pulses were determined by IFN-R enzyme binding immunosorbent assay. IFN- r production by cytotoxic T lymphocyte replication in target cells. In Figure 3, from CLUAP1-A24-9-255 (SEQ ID NO: 3), CLUAP1-A24-9-64 (SEQ ID NO: :9) and CLUAP1-A24-10-91 (SEQ ID NO: 15) stimulated cytotoxic T lymphocyte replication to determine strong 丨FN_ γ production 0 95 201134480 Anti-exogenous performance CLUAP1 and HLA-A* 2402 The specific cytotoxic T lymphocyte activity assay of the target cells was enhanced by the ability of the cytotoxic T lymphocyte cell line established against these peptides to recognize endogenously expressed CLUAP1 and HLA-A5" 2402 molecules The specific cytotoxic T lymphocyte activity of anti-CORS7 cells was tested using a cytotoxic T lymphocyte cell line boosted by the corresponding peptide, while the C0S7 cells were transfected with the full-length CLUAP1 and HLA-A* 2402 genes. (For exogenous performance CL Specific patterns of target cells of UAP1 and HLA-A# 2402 genes. COS7 cells were prepared as control group by transfection with full-length CLUAP1 gene or HLA-A* 2402. In Figure 4, CLUAP1-A24-9-255 (sequence Identification number: 3) Stimulated cytotoxic T lymphocytes showed strong anti-lymphocyte activity against COS7 cells expressing both CLUAP1 and HLA-A# 2402. On the other hand, no anti-control group was detected. Significantly specific cytotoxic T lymphocyte activity. Therefore, these data clearly demonstrate that CLUAP1-A24-9-255 (SEQ ID NO: 3) is naturally expressed on target cells with HLA-A* 2402 molecules and is sterilized by cells. The T lymphocytes were identified. These results indicate that the peptide derived from CLUAP1 is a cancer vaccine suitable for patients with tumors with CLU API expression. The homologous analysis of the antigen peptide is CLUAP1-A24-9-255 (sequence Identification number: 3), CLUAP1-A24-9-283C Sequence identification number: 4), CLUAP A24-9-31 (sequence identification number: 5), CLUAP1-A24-9-1 68 (sequence identification number: 8) , 96 201134480 CLUAP1-A24-9-64 (sequence identification number: 9), CLUAP1-A24-9-216 (sequence identification number: 10), CLUAP A24-9-1 54 (sequence identification number: 14), CLUAP1-A24-10-91 (sequence identification number: 15), CLUAP1-A24-10-104 (sequence identification number: 16) and CLUAP1 -A24-1 0-1 25 (SEQ ID NO: 1 8 ) Stimulated cytotoxic T lymphocytes showed significant and specific cytotoxic T lymphocyte activity. This result may be due to CLUAP1-A24-9-255 (sequence identification number: 3), CLUAP A24-9-283 (sequence identification number: 4), CLUAP1-A24-9-31 (sequence identification number: 5), CLUAP1-A24-9-1 68 (sequence identification number: 8), CLUAP1-A24-9-64 (sequence identification number: 9), CLUAP1-A24-9-216 (sequence identification number: 10), CLUAP1-A24- 9-1 54 (sequence identification number: 14), CLUAP A24-10-91 (sequence identification number: 15), CLUAP1-A24-10-104 (sequence identification number: 16) and CLUAP A24-1 0-1 The sequence of 25 (SEQ ID NO: 18) is a fact homologous to a peptide derived from other molecules known to be sensitive to the human immune system. To rule out this possibility, homology analysis was performed for these peptide sequences that were searched for the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) for the keyword, while BLAST The algorithm shows that no sequences show significant homology. The results of the homology analysis indicated that CLUAP A24-9-2 55 (sequence identification number: 3), CLUAP A24-9-283 (sequence identification number: 4), CLUAP A24-9-31 (sequence identification number: 5), CLUAP A24-9-168 C Sequence identification number: 8), CLUAP A24-9-64 (sequence identification number: 9), CLUAPl-A24-9-216 (sequence identification number: 10), CLUAP A24-9-1 54C Sequence identification number: 14), CLUAP A24-10-91 97 201134480 (sequence identification number: 15), CLUAP A24-1 0-1 04 (sequence identification number: 16) and CLUAP1-A24 The sequence of -1 0- 1 25 (SEQ ID NO: 18) is unique, and therefore there is only a small likelihood that the molecule will increase the non-prone immune response to some non-related molecules. Therefore, the novel HLA-A24 epitope peptide derived from CLUAP1 was confirmed. Furthermore, the results herein demonstrate that the epitope of CLUAP1 is suitable for use in cancer immunotherapy. Results 3 Prediction of HLA-A02 binding peptide from CLUAP1 Tables 3a and 3b show the HLA-A02 binding of 9 and 10 member peptides of CLUAPI in the order of highest binding affinity. A total of 21 peptides with potential HLA-A02 binding ability were selected and tested to determine epitopes. peptide 3 Table 3a, HLA-A02 from CLUAP1 binding 9-member peptide starting position amino acid sequence fraction sequence Identification number 34 LVSEVLLWL 164. 809 23 58 TEQDRVFFI 61.024 24 95 KELLKITSV 40.281 25 85 KLYQADGYA 39.784 26 290 TLCLIQNKL 21.362 27 265 NLTYLEQQL 21.362 28 297 KLKEEEKRL 10. 729 29 99 KITSVLYNA 5. 499 30 188 AIKEILTQV 3. 156 31 96 ELLKITSVL 2. 431 32 98 201134480 Table 3b, HLA-A02 from CLUAP1 binding 10 member peptide start position amino acid sequence fraction sequence identification number 153 SLYDLLGMEV 912.522 33 85 KLYQADGYAV 778. 982 34 34 LVSEVLLWLV 731. 711 35 33 GLVSEVLLWL 270.234 36 72 FMATKAHIKL 70.971 37 80 KLNTKKLYQA 39. 992 38 41 WLVKRYEPOT 34.279 39 233 TLQSVRPCFM 27. 324 40 183 KVMRIAIKEI 27. 19 41 343 LMQGRPGKRI 12. 809 42 222 LELERNRKRL 10. 712 43 Starting position means the amino acid residue from the N-terminus of CLUAP1 The number of bases. The combined score is from "BIMAS". Predicted peptides from CLUAP1 restricted by HLA-A* 020 1 induced cytotoxic T lymphocytes Cytotoxic T lymphocytes were generated for those peptides derived from C L U A P1 according to the procedure described in Materials and Methods. The peptide-specific cytotoxic lymphocyte activity was determined by IF N - γ-enzyme binding immunospot assay (Fig. 5a-g). Compared with the control group, the following hole numbers show strong IFN- r production: hole number #3 (a) stimulated by CLUAP1-A02-9-34 (sequence identification number: 23), with CLUAP1-A02-9 -99 (sequence identification number: 30) #6(13) of the stimulus, #2 (c) with (^1^? Bu 6 02-1 0-1 53 (sequence identification number: 33), with CLUAP A02- 1 0-85 (Sequence ID: 34) Stimulus #3 (d), with CLUAP1-A0 2- 1 0-34 (Sequence 99 201134480 column ID: 35) Stimulus #2 (e), CLUAP A02-1 0-33 (sequence identification number: 36) Stimulus #2 (f) and #5 (g) stimulated by CLUAP A02-1 0-80 (sequence identification number. 38). 'The specific cytotoxic T lymphocyte activity was not determined by the other stimuli shown in Tables 3a and 3b, although those peptides have a possible binding activity to HLA-A* 0201. A typical example, from the cytotoxic T lymphocytes stimulated with CLUAP1-A02-9-58 (SEQ ID NO: 24) (h), no specific IFN-7 production was observed. Therefore, it was indicated that the screening was from CLUAP1. 7 peptides are potent and potent cytotoxic T lymphocytes Peptide. The cytotoxic T lymphocyte cell line and replication of anti-CLUAP1-derived peptide were detected by IFN-r enzyme binding immunospot assay, with CLUAP1-A02-9-34 (SEQ ID NO: 23) Stimulus hole number #3 (a), stimulated by CLUAP A02-9-99 (sequence identification number: 30) #6 (b), stimulated by CLUAP1-A02-10-1 53 (sequence identification number: 33) #2 (c), #3 (d) stimulated by CLUAP A02-1 0-85 (sequence identification number: 34), serial number of CLUAP1-A02-10-34C: 35) Stimulus #2 ( e) cells in the #2 (f) stimulated with CLUAP 1-A02-1 0-33 (SEQ ID NO: 36) showing cell-specific cytotoxic T lymphocyte activity are expanded and restricted by limiting dilution Establishment of a cytotoxic T lymphocyte cell line, as described in the paragraph "Materials and Methods," above. The cytotoxicity of these cytotoxic T lymphocyte cell lines was determined by IFN-τ enzyme binding immunosorbent assay 100 201134480 T lymphocyte activity (Fig. 6a-f). The cytotoxic T lymphocyte cell line showed a strong resistance to the corresponding victory pulse compared to the target cell without the peptide pulse. The IF N - τ of the target cells is produced. In addition, cytotoxic T lymphocyte replication was established by limiting dilution from cytotoxic T lymphocyte cell lines as described in Materials and Methods, and was determined by IFN-7 enzyme-binding immunosorbent assay. Peptide-pulsed target cells cytotoxic T lymphocyte replication of IFN-r production. From CLUAP1-A02-9-34 (sequence identification number: 23) (a), CLUAP1-A02-9-99 (sequence identification number: 30) (b), CLUAP1-A02-1 0-1 53 (sequence identification No.: 33) (c) with CLUAP A02-1 0-85 (SEQ ID NO: 34) (d) Stimulated cytotoxic T lymphocyte replication to determine strong ifn-7 production (Fig. 7a-d) . Anti-exogenous performance of CLUAP1 and HLA-A* 020 1 target cells with specific cytotoxic T lymphocyte activity assays by increasing the resistance of each peptide to the established cytotoxic T lymphocyte cell line and replicating it for identification of endogenous The ability to express target cells of CLUAP1 and HLA-A* 0201 molecules. The cytotoxic T lymphocyte cell line promoted by the corresponding peptide was used as a cell to test the specific cytotoxic T lymphocyte activity of anti-c〇S7 cells, while the c〇S7 cells were subjected to full-length CLUAP1 and HLA. -A* 0201 gene transfection (specific pattern for target cells exogenously expressing CLUApi and HLA-A* 020 1 genes). c〇S7 cells were prepared as control group by transfection with full length CLUAP1 or HLA-A* 0201. In Figure 8, the cytotoxic tau lymphocyte cell line stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) showed a strong anti-COS7 cell with both CLUAP1 101 201134480 and HLA-A* 0201. Cytotoxic T. Lymphocyte Activity On the other hand, no significant specific cytotoxic sputum lymphocyte activity was detected in the anti-control group. Therefore, 'these data clearly demonstrate that CLUAP1-A24-9-255 (SEQ ID NO: 3) is endogenously processed and expressed on target cells with HLA_A*0 2 0 1 molecule and is cytotoxic by the cytotoxic lymphocytes identify. These results show that these peptides derived from CLUAP1 are cancer vaccines that can provide patients with tumors with CLUAP1 expression. Homologous analysis of the antigen peptide is CLUAP1-A02-9-34 (SEQ ID NO: 23), CLUAP A02-9-99C sequence identification number: 30), CLUAP A02-10-153 (sequence identification number: 33) ), CLUAP A02- 1 0-85 C sequence identification number: 34), CLUAP1-A02-10-34 (sequence identification number: 35), CLUAP1-A02-1 0-33C sequence identification number: 36) and CLUAP1- A02-10-80 (SEQ ID NO: 38) Stimulated cytotoxic T lymphocytes showed significant and specific cytotoxic T lymphocyte activity. This result may be due to CLUAP1-A02-9-34 C sequence identification number: 23), CLUAP Bu A02-9-99 (sequence identification number: 30), CLUAP1-A02-1 0-1 53 (sequence identification number: 33 ), CLUAP1-A02-1 0-85 (sequence identification number: 34), CLUAP1-A02-1 0-34 (sequence identification number: 35), CLUAP A02-10-33 (sequence identification number: 36) and CLUAP1 The sequence of -A02-1 0-80 (SEQ ID NO: 3 8 ) is a fact homologous to a peptide derived from other molecules known to be sensitive to the human immune system. To rule out this possibility, homology analysis was performed on these peptide sequences that were searched for by the BLAST calculus 102 201134480 method (http://www.ncbi.nlm.nih.gov/blast/blast.cgi), The BLAST algorithm showed no sequence showing significant homology. The results of homology analysis indicated that CLUAP1-A02-9-34C sequence identification number: 23), CLUAP1-A02-9-99 (sequence identification number: 30), CLUAP1-A0 2-1 0-1 53 C sequence identification number :33), CLUAP Bu A0 2-1 0-85 (sequence identification number: 34), CLUAP Bu A0 2-10-34 (sequence identification number: 35), CLUAP1-A02- 1 0-33 C sequence identification number: 36) The sequence with CLUAP1-A02-1 0-80 (SEQ ID NO: 38) is unique, and therefore there is only a small probability that the molecule will increase the non-prone immune response to some non-related molecules. Therefore, we confirmed the novel HLA-A* 020 1 epitope peptide from CLUAP1. Furthermore, it is demonstrated that the epitope of CLUAP1 is applicable to cancer immunotherapy. Industrial Applicability The present invention provides novel tumor-associated antigens, particularly those derived from CLMpi, which induce strong and specific anti-tumor immunity, response, and have applicability to a wide variety of cancer forms. This tumor-associated antigen can provide a therapeutic effect against CLUAPI-related diseases of peptide vaccines and AH-related diseases, such as cancer, more pull & 丨aa ten special 疋癌 cancer 'cervical cancer, colorectal cancer, esophageal cancer , gastric cancer, diffuse f ^ and i month, lymphoma, arsenic blastoma, sputum cancer. 103 201134480, the disclosure of which is hereby incorporated by reference in its entirety in its entirety herein in its entirety herein in In the course of routine experimentation, it will be readily apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. Therefore, the invention is not to be defined as described above, but is defined by the scope of the following claims and their equivalents. [Simple diagram] [La-f diagram] The first la-f diagram consists of a series of photographs, (a) to (f), showing cytotoxic thymocytes induced by peptides derived from CLUAP1. The results of IFN-τ enzyme combined with immunospot assay (ELISP0T). The cytotoxic T lymphocytes in the following well numbers showed strong IFN-r production compared to the control group wells: Hole number #6 (a) stimulated with CLUAP1-A24-9-255 (SEQ ID NO: 3) ) #2 (b) stimulated by CLUAP A24-9-283 (sequence identification number: 4), #2 (c) stimulated by CLUAP A24-9-31 (sequence identification number: 5), with CLUAP1 -A24-9-168 (Sequence ID: 8) Stimulus #·4 (d), CLUAP1-A24-9-64 (Serial Identification Number: 9) Stimulation #4 (e) and CLUAP1-A24- 9-216 (sequence identification number: 10) Stimulus #1 (f). The cells in the wells were expanded in a rectangular box to establish a cytotoxic T lymphocyte cell line. In contrast, as a typical example of negative data, it does not show IFN-γ production (k) derived from the CLUAP1-A24-9-258C sequence identification number: 1) stimulated cytotoxic T lymphocytes. In the figure, "Indicative IF N-7 production against target cells pulsed with a suitable peptide is indicated, while "' indicates IFN-T production against 104 201134480 target cells not pulsed with any peptide. [Fig. lg-k] The 1 g _ k map consists of a 糸-column picture '(g) to (k) showing IFN- on the cytotoxic lytic lymphocytes induced by the peptide derived from CLUAP1 The results of tau enzyme combined with immunospot analysis (ELISP0T). Compared to the control group, the cytotoxic T lymphocytes in the following well numbers showed strong IFN-τ production: holes #6 (g) stimulated by CLUAP1-A24-9-154 (SEQ ID NO: 14) #2 (h) stimulated by CLUAP1-A24-10-91 (sequence identification number: 15), #4 (i) and CLUAP1 stimulated by CLUAP A24-1 0-104 (sequence identification number: 16) -A24-1 0-125 (Sequence ID: 1 8) Stimulus #5 ( j). The cells in the pores indicated by the rectangular box were expanded to establish a cytotoxic T lymphocyte cell line. In contrast, a typical example of negative data ' does not show specific IFN-γ production (k) from cytotoxic T lymphocytes stimulated with CLUAP1-A24-9-258 (SEQ ID NO: 1). In the figure, "+" indicates that IFN-[tau] production against a target cell pulsed with a suitable peptide is 'and' indicates that IFN-[tau] production against a target cell not pulsed with any peptide is produced. [Fig. 2] The figure consists of a series of line graphs, consisting of (a) to (e), showing the results of IF N-7 enzyme binding immunosorbent assay, which is shown as CLUAP1-A24-9-255 (SEQ ID NO: 3) ( a), with CLUAP1-A24-9-168 (sequence identification number: 8) (b), 105 201134480 CLUAP A24-9-64 (sequence identification number: 9) (c), with CLUAP1-A24-9- 154 (SEQ ID NO: 14) (d) IFN-τ produced by cytotoxic T lymphocyte cell line stimulated with CLUAP A24-10-91 (SEQ ID NO: 15) (e). Results display and control The group showed stronger IFN-r production by the cytotoxic T lymphocyte cell line established by stimulation with each peptide. In the figure, "+,, indicates the target cell against the pulse of the appropriate peptide. IFN-7 is produced, and "indicates that IFN-7 is produced against target cells that are not pulsed with any peptide. [Fig. 3] Figure 3 consists of a series of line graphs, (a) to (d), showing IFN- 7 · The results of enzyme-linked immunosorbent assay detection are shown by CLUAP1-A24-9-255 (SEQ ID NO: 3) (a), CLUAP1-A24-9-64 (SEQ ID NO: 9) b) IFN-7 production by cytotoxic T lymphocyte replication established by CLUAP A24-10-91 (SEQ ID NO: 15) (c) restricted dilution of stimulated cytotoxic T lymphocyte cell lines. It was shown that the cytotoxic T lymphocyte replication established by stimulation with each peptide showed strong iFN-7 production compared to the control group. In the figure, the target cells against the target peptide pulsed with the peptide were indicated. FN_7 produces 'and indicates that IFN_γ is produced against target cells that are not pulsed with any peptide. [Fig. 4]

第4圖為一線圖，其顯示抗外生表現CLUAP1與HLA-A 106 201134480 * 2402*之目標細胞的專一細胞毒殺性τ淋巴球活性。將以 HLA-A* 2402或全長之CLUAP1基因轉染之c〇控制組。…―255 (序列辨識號細胞毒殺性τ淋巴球細胞株顯示抗以cluapi肖心—c 2402兩者轉染之⑽7細胞的專—細胞毒殺性了淋巴球活性（黑色菱形）。3 -方面’沒有相到顯著專—之細胞毒殺性τ淋巴球活性，其抗表現HLA_A*24〇2 (三角形）或 CLUAP1 (圓形）任一的目標細胞。 [第5a-f圖] 第5a-f圖由一系列照片，（3)至（〇所组成，其顯示在以來自CLUAP1之胜肽誘導之細胞毒殺性τ淋巴球上之 IFN-r酵素結合免疫斑點分析（ELISPOT)的結果。與控制組孔洞相較，於下列孔洞編號中之細胞毒殺性T淋巴球顯示強的IFN-T"產生：以CLUAP1-A02-9-34 (序列辨識號： 23 )刺激之孔洞編號#3 (a)、以CLUAP卜A02-9-99 (序列辨識號：30)刺激之 #6 (b)、以 CLUAP1-A02-10-153 (序列辨識號：33)刺激之#2 (c)、以CLUAP1-A02-1 0-85 (序列辨識號：34)刺激之#3 (d)、以CLUAP1-A02-1 0-34 (序列辨識號：35 )刺激之#2 (e)與以CLUAP1-A02-1 0-33 (序列辨識號：36 )刺激之#2 (f)。以矩形盒狀所指出於孔洞中之細胞被擴張來建立細胞毒殺性T淋巴球細胞株。相對的，如負資料的典型例子，其未顯示來自以 CLUAP1-A02-9-58 (序列辨識號：24 ) (h)刺激之細胞毒殺 107 201134480 性τ淋巴球之專一的IFN_ r產生。於圖中，“ +”指出抗以適合之胜肽脈衝的目標細胞的IFN- 7產生，而“指出抗未以任何胜肽脈衝的目標細胞的IFN- 7產生。 [第5g-h圖] 第5g-h圖由一系列照片所組成，（g)其顯示在以來自 CLUAP1之胜肽誘導之細胞毒殺性τ淋巴球上之IFN-7*酵素結合免疫斑點分析（EL I SPOT)的結果。與控制組孔洞相較’於下列孔洞編號中之細胞毒殺性T淋巴球顯示強的 IFN- γ產生：以CLUAP1-A02-1 0-80 (序列辨識號：38)刺激之#5 (g)。以矩形盒狀所指出於孔洞中之細胞被擴張來建立細胞毒殺性T淋巴球細胞株。相對的，如負資料的典型例子’其未顯示來自以CLUAPh〇2 —9-58(序列辨識號： 2 4 ) (h )刺激之細胞毒殺性τ m丄 * , ’双汪i淋巴球之專一的IFN_ r產生。於圖中，“ +”指出抗以说人、5之胜肽脈衝的目標細胞的 IF N - 7產生，而“”指由屯^未以任何胜肽脈衝的目標細胞的IFN-γ產生。 [第6圖] 酵素結合免疫吸附分析 CLUAP卜A02-9-34 (序歹,】 CLUAP卜A02-9-99 (序歹,】 CLUAP卜A02-1 0-153 (序歹顯示IF N - 7" 其顯示以 (a) 、以 (b) 、以 (c) 、以第6圖由一系列線圖 (a)至（f)所組成，偵測的結果，辨識號：23 ) 辨識號：30 ) 辨識號：33 ) 108 201134480 CLUAP1-A02-10_85 (序列辨識號：34 ) (d)、以 CLUAP1-A02-1 0-34 (序列辨識號：35 ) (e)與以 CLUAPl-A02-10-33 (序列辨識號：36) (f)刺激之細胞毒殺性T淋巴球細胞株之IF N_ 7"產生。結果顯示與控制組相較，藉由以各胜肽刺激建立之細胞毒殺性T淋巴球細胞株顯示強而有力之IF N - τ產生。於圖中，“ +，，指出抗以適合之胜肽脈衝的目標細胞的IFN- 7產生，而“ 指出抗未以任何胜肽脈衝的目標細胞的IFN- γ產生。 [第7圖] 第7圖由一系列線圖，（a)至（d)所組成’其顯示藉由來自以CLUAP1-A02-9-34 (序列辨識號：23) (a)、 CLUAP1-A02-9-99 (序列辨識號：3〇 ) (b)、 CLUAP1-A02-1 0-1 53 (序列辨識號：33 ) 與 CLUAPH02-1 0-85 (序列辨識號：34)⑷刺激之細胞毒殺性τ淋巴球細胞株的限制稀釋所建立之細胞毒殺性τ淋巴球複製的IFN- r產生。結果顯示與控制組相較藉由以各胜肽刺激所建立之細胞毒殺性T淋巴球複製顯示強而有力之IFN- r產生。於圖中，“ +”指屮扞丨、丨、*人出抗以適合之胜肽脈衝指出抗未以任何胜肽的目標細胞的IF N - 7產生，而‘、脈衝的目標細胞的IFN- τ產生。 [第8圖] (a)至（d)所組成，其顯示抗外第8圖由一系列線圖 109 201134480 生表現CLUAP1與HLA-A* 020 1之目標細胞的專一細胞毒殺性T淋巴球活性。將以HLA-A* 0201或全長之CLUAP1基因轉染之COS7細胞製備為控制組。以CLUAP1-A02-9-99 (序列辨識號.30) (a)、CLUAP1-A02-10-153 (序列辨識號： 33) (b)、CLUAP1-A02-1 0-85 (序列辨識號：34 ) (c) 與CLUAP1-A02-10-33 (序列辨識號·· 36) (d)建立之細胞毒殺性τ淋巴球細胞株顯示抗以CLUApi與HLA_A* 兩者轉染之COS7細胞的專一方形）。一細胞毒殺性T淋巴球活性（正Figure 4 is a line graph showing specific cytotoxic tau lymphosphere activity against target cells of exogenous expression CLUAP1 and HLA-A 106 201134480 * 2402*. The c〇 control group transfected with HLA-A* 2402 or the full-length CLUAP1 gene. ...-255 (Sequence identification number cytotoxic TNF lymphocyte cell line showed anti-cytotoxicity of lymphocyte activity (black diamond) against (10)7 cells transfected with both cluapi xiaoxin-c 2402. 3 - aspect' There is no significant specific cytotoxic tau lymphocyte activity, which is resistant to target cells of either HLA_A*24〇2 (triangle) or CLUAP1 (circle) [5a-f] 5a-f From a series of photographs, consisting of (3) to (〇, which is shown in the results of IFN-r enzyme binding immunospot assay (ELISPOT) on cytotoxic tau lymphocytes induced by peptides derived from CLUAP1. Compared with the pores, the cytotoxic T lymphocytes in the following well numbers showed strong IFN-T"Generation: Hole number #3 (a) stimulated by CLUAP1-A02-9-34 (SEQ ID NO: 23), #6 (b) stimulated by CLUAP A02-9-99 (sequence identification number: 30), #2 (c) stimulated by CLUAP1-A02-10-153 (sequence identification number: 33), with CLUAP1-A02 -1 0-85 (Sequence ID: 34) Stimulus #3 (d), CLUAP1-A02-1 0-34 (Serial Identification Number: 35) Stimulated #2 (e) and with CLUAP1-A02- 1 0-33 (SEQ ID NO: 36) Stimulus #2 (f). The cells in the pores are expanded in a rectangular box to establish a cytotoxic T lymphocyte cell line. In contrast, negative data A typical example, which does not show the specificity of IFN_r production from cells catalyzed by CLUAP1-A02-9-58 (SEQ ID NO: 24) (h), catalyzed by 107 201134480, is shown in the figure, "+" indicates IFN-7 production against target cells pulsed with a suitable peptide, and "indicates production of IFN-7 against target cells not pulsed with any peptide. [5g-h] The 5g-h diagram consists of a series of Photographs, (g) results showing IFN-7* enzyme binding immunospot assay (EL I SPOT) on cytotoxic tau lymphocytes induced by peptides derived from CLUAP1. The cytotoxic T lymphocytes in the following well numbers showed strong IFN-γ production: #5 (g) stimulated with CLUAP1-A02-1 0-80 (SEQ ID NO: 38). The cells in the pores are expanded to establish a cytotoxic T lymphocyte cell line. Relatively, as typical of negative data The example ' does not show cytotoxicity τ m丄* stimulated by CLUAPh〇2-9-58 (SEQ ID NO: 2 4 ) (h), and the specific IFN_r production of the double lymphocytes. In the figure, "+" indicates that IF N-7 is produced against a target cell pulsed with a human, 5 peptide, and "" refers to IFN-γ produced by a target cell which is not pulsed with any peptide. [Picture 6] Enzyme-binding immunosorbent assay CLUAP A02-9-34 (Preface, CLUAP A02-9-99 (Serial, CLUAP) A02-1 0-153 (Preface shows IF N - 7&quot The display consists of (a), (b), (c), and Figure 6 from a series of line graphs (a) to (f), the result of the detection, identification number: 23) Identification number: 30) Identification number: 33) 108 201134480 CLUAP1-A02-10_85 (sequence identification number: 34) (d), with CLUAP1-A02-1 0-34 (sequence identification number: 35) (e) and with CLUAPl-A02- 10-33 (SEQ ID NO: 36) (f) IF N_ 7" produced by stimulated cytotoxic T lymphocyte cell lines. The results showed that the cytotoxic T lymphocyte cell line established by stimulation with each peptide showed strong IF N - τ production as compared with the control group. In the figure, "+," indicates production of IFN-7 against target cells pulsed with a suitable peptide, and "indicates IFN-γ production against target cells not pulsed with any peptide. [Fig. 7] Figure 7 consists of a series of line graphs, consisting of (a) to (d), which are shown by CLUAP1-A02-9-34 (Serial Identification Number: 23) (a), CLUAP1- A02-9-99 (sequence identification number: 3〇) (b), CLUAP1-A02-1 0-1 53 (sequence identification number: 33) and CLUAPH02-1 0-85 (sequence identification number: 34) (4) Stimulation IFN- r production by cytotoxic tau lymphocyte replication established by limiting dilution of cytotoxic tau lymphocyte cell lines. The results showed that the cytotoxic T lymphocyte replication established by stimulation with each peptide showed strong IFN-r production compared to the control group. In the figure, "+" refers to 屮捍丨, 丨, * human EGFR, which is indicated by a suitable peptide pulse indicating that IF N-7 is not produced by any target cell of the peptide, and ', pulsed target cell IFN - τ is generated. [Fig. 8] Compositions consisting of (a) to (d), showing an anti-external figure 8 consisting of a series of line diagrams 109 201134480 showing specific cell toxic T lymphocytes of target cells of CLUAP1 and HLA-A* 020 1 active. COS7 cells transfected with HLA-A* 0201 or the full-length CLUAP1 gene were prepared as control groups. CLUAP1-A02-9-99 (sequence identification number.30) (a), CLUAP1-A02-10-153 (sequence identification number: 33) (b), CLUAP1-A02-1 0-85 (sequence identification number: 34) (c) CLUAP1-A02-10-33 (SEQ ID NO: 36) (d) The cytotoxic tau lymphocyte cell line was shown to be specific to COS7 cells transfected with both CLUApi and HLA_A*. Square). Cytotoxic T lymphocyte activity

任一的目標細胞。淋巴球活* (圓形）任一【主要元件符號說明】益 ««»> 110 201134480 序列表 <110> 腫瘤療法·科學股份有限公司 <120> CLUAP1胜肽及其疫苗〈130〉 0NC-A1011-TW 〈150〉 US 61/322,099 〈151〉 2010-04-08 <160〉 49 〈170〉 Patent In version 3.5 〈210〉 1 <211〉 9 〈212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列〈400〉 1Any target cell. Lymphocyte activity* (circular) either [main component symbol description] 益««»> 110 201134480 Sequence Listing <110> Oncology Therapy Science Co., Ltd. <120> CLUAP1 peptide and its vaccine <130> 0NC-A1011-TW <150> US 61/322,099 <151> 2010-04-08 <160> 49 <170> Patent In version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220〉 <223> Synthetic peptide sequence <400> 1

Thr Tyr Leu Glu Lys Phe Gin Asn Leu 1 5 <210> 2 <211〉 9 <212〉 PRT <213> 人工序列 <220> <223> 人工合成胜肽序列 <400> 2Thr Tyr Leu Glu Lys Phe Gin Asn Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide sequence <400> 2

Glu Tyr Glu Lys Thr Glu Glu Glu Leu 1 5 <210〉 3 <211> 9 <212〉 PRT 〈213>人工序列 <220> 201134480 <223〉人工合成胜肽序列 <400> 3Glu Tyr Glu Lys Thr Glu Glu Glu Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> 201134480 <223> Synthetic peptide sequence <400> 3

Gin Tyr Asp Thr Tyr Leu Glu Lys Phe 1 5 <210〉 4 <211> 9 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 4Gin Tyr Asp Thr Tyr Leu Glu Lys Phe 1 5 <210> 4 <211> 9 <212> PRT <213>Artificial sequence <220> <223>Synthetic peptide sequence <400> 4

Arg Phe Glu Glu Ala Lys Asn Thr Leu 1 5 <210〉 5 <211〉 9 〈212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 5Arg Phe Glu Glu Ala Lys Asn Thr Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 5

Asn Phe Gly Leu Val Ser Glu Val Leu <210〉 6 <211〉 9 〈212〉 PRT <213〉人工序列 <220> <223〉人工合成胜肽序列 <400> 6Asn Phe Gly Leu Val Ser Glu Val Leu <210> 6 <211> 9 <212> PRT <213>Artificial sequence <220><223>Synthetic peptide sequence <400> 6

Asn Phe Thr Glu Met Met Arg Ala Leu 1 5 〈210〉 7 <211〉 9 2 201134480 <212> PRT 〈213>人工序列 <220> <223〉人工合成胜肽序列 <400〉 7Asn Phe Thr Glu Met Met Arg Ala Leu 1 5 <210> 7 <211> 9 2 201134480 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide sequence <400〉 7

Asn Phe Arg Thr Pro Asn Phe Gly Leu 1 5 <210〉 8 <211> 9 <212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 8Asn Phe Arg Thr Pro Asn Phe Gly Leu 1 5 <210〉 8 <211> 9 <212> PRT <213>Artificial sequence <220> <223>Synthetic peptide sequence <400〉 8

Arg Thr Glu Ala lie Ala Arg Pro Leu 1 5 〈210〉 9 <211〉 9 〈212〉 PRT <213〉人工序列〈220> <223〉人工合成胜肽序列 <400〉 9Arg Thr Glu Ala lie Ala Arg Pro Leu 1 5 <210> 9 <211> 9 <212> PRT <213>Artificial sequence <220> <223> Synthetic peptide sequence <400〉 9

Phe Phe lie Lys Ala lie Ala 6ln Phe 〈210〉 10 〈211〉 9 <212〉 PRT <213〉人工序列〈220> <223〉人工合成胜肽序列 <400> 10Phe Phe lie Lys Ala lie Ala 6ln Phe <210> 10 <211> 9 <212> PRT <213>Artificial sequence <220> <223> Synthetic peptide sequence <400>

Lys lie Glu Lys Arg Lys Leu Glu Leu 3 201134480 <210> 11 <211〉 9 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 11Lys lie Glu Lys Arg Lys Leu Glu Leu 3 201134480 <210> 11 <211> 9 <212> PRT <213>Artificial sequence <220> <223>Synthetic peptide sequence <400> 11

Lys Pro Gin Thr Ala Met Glu Met Leu <210〉 12 <211> 9 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 12Lys Pro Gin Thr Ala Met Glu Met Leu <210> 12 <211> 9 <212> PRT <213>Artificial Sequence <220> <223>Synthetic Peptide Sequence <400> 12

Leu Tyr Gin Ala Asp Gly Tyr Ala Val 1 5 〈210〉 13 <211〉 9 〈212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 13Leu Tyr Gin Ala Asp Gly Tyr Ala Val 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 13

Gin Phe Met Ala Thr Lys Ala His lie 1 5 <210> 14 〈211〉 9 <212〉 PRT 〈213〉人工序列〈220〉〈223〉人工合成胜肽序列 4 201134480 <400〉 14Gin Phe Met Ala Thr Lys Ala His lie 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence 4 201134480 <400〉 14

Leu Tyr Asp Leu Leu Gly Met Glu Val 1 5 <210> 15 <211> 10 〈212〉 PRT <213〉人工序列 <220> <223〉人工合成胜肽序列 <400> 15 6ly Tyr Ala Val Lys Glu Leu Leu Lys lie 1 5 10 <210> 16 <211> 10 〈212〉 PRT <213〉人工序列 <220〉〈223>人工合成胜肽序列 <400> 16Leu Tyr Asp Leu Leu Gly Met Glu Val 1 5 <210> 15 <211> 10 <212> PRT <213>Artificial Sequence <220><223> Artificial Synthetic Peptide Sequence <400> 15 6ly Tyr Ala Val Lys Glu Leu Leu Lys lie 1 5 10 <210> 16 <211> 10 <212> PRT <213>Artificial sequence <220> <223> Synthetic peptide sequence <400> 16

Leu Tyr Asn Ala Met Lys Thr Lys Gly Met 1 5 10 〈210〉 17 <211〉 10 <212〉 PRT 〈213>人工序列 <220> 〈223>人工合成胜肽序列 <400> 17Leu Tyr Asn Ala Met Lys Thr Lys Gly Met 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide sequence <400>

Asn Phe Gly Leu Val Ser Glu Val Leu Leu 1 5 10 〈210〉 18 〈211〉 10 <212〉 PRT 〈213〉人工序列 201134480 <220〉 <223〉人工合成胜肽序列 <400〉 18Asn Phe Gly Leu Val Ser Glu Val Leu Leu 1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial sequence 201134480 <220> <223> Synthetic peptide sequence <400> 18

Lys Phe Lys Phe Asp Leu Gly Ser Lys lie 1 5 10 <210> 19 <211〉 10 <212〉 PRT 〈213〉人工序列〈220> 〈223>人工合成胜肽序列 <400> 19Lys Phe Lys Phe Asp Leu Gly Ser Lys lie 1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400>

Gly Tyr Pro Arg His lie Ser Met Glu Asn 1 5 10 〈210〉 20 <211〉 10 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 20Gly Tyr Pro Arg His lie Ser Met Glu Asn 1 5 10 <210> 20 <211> 10 <212> PRT < 213 > 213 > artificial sequence < 220 < 223 > 223 > artificially synthesized peptide sequence <400> 20

Asn Thr Leu Cys Leu Ile Gin Asn Lys Leu 1 5 10 <210〉 21 <211〉 10 <212> PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 21Asn Thr Leu Cys Leu Ile Gin Asn Lys Leu 1 5 10 <210> 21 <211> 10 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > 223 > synthetic peptide sequence <400>; twenty one

Thr Tyr Leu Glu Lys Phe Gin Asn Leu Thr 1 5 10 6 201134480 * <210〉 <211> <212> <213〉 22 10 PRT 人工序列 <220> <223> 人工合成胜肽序列〈400〉 22Thr Tyr Leu Glu Lys Phe Gin Asn Leu Thr 1 5 10 6 201134480 * <210〉 <211><212><213> 22 10 PRT Artificial Sequence <220><223> Synthetic peptide sequence <400> 22

Val Phe Phe lie Lys Ala lie Ala Gin Phe 1 5 10 <210> <211〉 <212> <213> 23 9 PRT 人工序列 <220〉 <223〉人工合成胜肽序列 <400> 23Val Phe Phe lie Lys Ala lie Ala Gin Phe 1 5 10 <210><211><212><213> 23 9 PRT artificial sequence <220〉 <223> Synthetic peptide sequence <400>; twenty three

Leu Val Ser Glu Val Leu Leu Trp Leu 1 5 〈210> 24 <211> <212> <213> 9 PRT 人工序列〈220> <223〉人工合成胜肽序列 <400> 24Leu Val Ser Glu Val Leu Leu Trp Leu 1 5 <210> 24 <211><212><213> 9 PRT artificial sequence <220> <223> Synthetic peptide sequence <400> 24

Thr Glu Gin Asp Arg Val Phe Phe lie <210〉 25 <211> <212〉 <213> 9 PRT 人工序列 <220〉 <223> 人工合成胜肽序列〈400〉 25 201134480Thr Glu Gin Asp Arg Val Phe Phe lie <210> 25 <211><212><213> 9 PRT artificial sequence <220> <223> Synthetic peptide sequence <400> 25 201134480

Lys Glu Leu Leu Lys lie Thr Ser Val 1 5 〈210〉 26 <211〉 9 <212〉 PRT 〈213>人工序列 <220〉 <223〉人工合成胜肽序列 <400> 26Lys Glu Leu Leu Lys lie Thr Ser Val 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 26

Lys Leu Tyr Gin Ala Asp Gly Tyr Ala 1 5 <210〉 27 <211> 9 <212〉 PRT 〈213〉人工序列 <220> 〈223>人工合成胜肽序列 <400〉 27Lys Leu Tyr Gin Ala Asp Gly Tyr Ala 1 5 <210> 27 <211> 9 <212> PRT <213>Artificial sequence <220><223> Synthetic peptide sequence <400> 27

Thr Leu Cys Leu lie Gin Asn Lys Leu <210〉 28 <211〉 9 <212> PRT <213〉人工序列 <220〉〈223>人工合成胜肽序列 <400〉 28Thr Leu Cys Leu lie Gin Asn Lys Leu <210> 28 <211> 9 <212> PRT <213>Artificial Sequence <220> <223> Synthetic peptide sequence <400> 28

Asn Leu Thr Tyr Leu Glu Gin Gin Leu 〈210〉 29 <211〉 9 〈212〉 PRT <213〉人工序列 8 〈220〉 201134480 • <223〉人工合成胜肽序列 <400> 29Asn Leu Thr Tyr Leu Glu Gin Gin Leu <210> 29 <211> 9 <212> PRT <213> Artificial sequence 8 <220> 201134480 • <223> Synthetic peptide sequence <400> 29

Lys Leu Lys Glu Glu Glu Lys Arg Leu 〈210〉 30 <211〉 9 〈212〉 PRT 〈213〉人工序列 <220〉〈223>人工合成胜肽序列 <400〉 30Lys Leu Lys Glu Glu Glu Lys Arg Leu <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 30

Lys lie Thr Ser Val Leu Tyr Asn Ala 1 5 <210> 31 <211> 9 〈212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列〈400〉 31Lys lie Thr Ser Val Leu Tyr Asn Ala 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 31

Ala lie Lys Glu lie Leu Thr 6ln Val 1 5 <210〉 32 <211〉 9 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 32Ala lie Lys Glu lie Leu Thr 6ln Val 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 32

Glu Leu Leu Lys lie Thr Ser Val Leu 1 5 <210〉 33 〈211〉 10 201134480 <212〉 PRT 〈213>人工序列 <220> <223〉人工合成胜肽序列 <400> 33Glu Leu Leu Lys lie Thr Ser Val Leu 1 5 <210> 33 <211> 10 201134480 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide sequence <400> 33

Ser Leu Tyr Asp Leu Leu Gly Met Glu Val 1 5 10 <210〉 34 <211> 10 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 34Ser Leu Tyr Asp Leu Leu Gly Met Glu Val 1 5 10 <210> 34 <211> 10 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > 223 > synthetic peptide sequence <400 〉 34

Lys Leu Tyr Gin Ala Asp Gly Tyr Ala Val 1 5 10 <210〉 35 <211〉 10 〈212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 35Lys Leu Tyr Gin Ala Asp Gly Tyr Ala Val 1 5 10 <210> 35 <211> 10 <212> PRT <213>Artificial Sequence <220> <223>Synthetic Peptide Sequence <400〉 35

Leu Val Ser Glu Val Leu Leu Trp Leu Val 1 5 10 <210〉 36 <211> 10 〈212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 36Leu Val Ser Glu Val Leu Leu Trp Leu Val 1 5 10 <210> 36 <211> 10 <212> PRT <213>Artificial sequence <220> <223> Synthetic peptide sequence <400> 36

Gly Leu Val Ser Glu Val Leu Leu Trp Leu 1 5 10 10 201134480 <210> 37 <211> 10 <212> PRT 〈213〉人工序列 <220> 〈223>人工合成胜肽序列 <400〉 37Gly Leu Val Ser Glu Val Leu Leu Trp Leu 1 5 10 10 201134480 <210> 37 <211> 10 <212> PRT <213>Artificial sequence <220><223> Synthetic peptide sequence <400 〉 37

Phe Met Ala Thr Lys Ala His lie Lys Leu 1 5 10 〈210〉 38 〈211〉 10 <212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 38Phe Met Ala Thr Lys Ala His lie Lys Leu 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 38

Lys Leu Asn Thr Lys Lys Leu Tyr Gin Ala 1 5 10 〈210〉 39 〈211〉 10 〈212〉 PRT 〈213〉人工序列 <220> 〈223>人工合成胜肽序列 <400> 39Lys Leu Asn Thr Lys Lys Leu Tyr Gin Ala 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence <400> 39

Trp Leu Val Lys Arg Tyr Glu Pro Gin Thr 1 5 10 <210〉 40 <211> 10 〈212〉 PRT 〈213〉人工序列 <220〉 <223〉人工合成胜肽序列 201134480 <400〉 40Trp Leu Val Lys Arg Tyr Glu Pro Gin Thr 1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide sequence 201134480 <400〉 40

Thr Leu Gin Ser Val Arg Pro Cys Phe Met 1 5 10 <210> 41 <211〉 10 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400〉 41Thr Leu Gin Ser Val Arg Pro Cys Phe Met 1 5 10 <210> 41 <211> 10 <212> PRT < 213 > 213 > artificial sequence < 220 < 223 > 223 > synthetic peptide sequence <400 〉 41

Lys Val Met Arg lie Ala lie Lys Glu lie 1 5 10 <210〉 42 〈211〉 10 〈212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 42Lys Val Met Arg lie Ala lie Lys Glu lie 1 5 10 <210> 42 <211> 10 <212> PRT <213>Artificial sequence <220> <223> Synthetic peptide sequence <400>

Leu Met Gin Gly Arg Pro Gly Lys Arg lie 1 5 10 <210〉 43 <211〉 10 <212〉 PRT <213〉人工序列 <220〉 <223〉人工合成胜肽序列 <400> 43Leu Met Gin Gly Arg Pro Gly Lys Arg lie 1 5 10 <210> 43 <211> 10 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > 223 > artificially synthesized peptide sequence <400>; 43

Leu Glu Leu Glu Arg Asn Arg Lys Arg Leu 1 5 10 <210〉 44 <211〉 1681 <212> DNA <213〉人類 12 201134480 參 <400> 44 gccgtccaag ggtccattgg ttgccataga aggggcgagc agttgcgacc ctgggctcct ctccgcaatt tcacagagat gatgagagcc aatttccgta cacccaattt tggacttgta tatgagcccc agactgacat cccgcctgac attaaggcaa ttgcccagtt catggccacc ctttatcaag cagatgggta tgcggtaaaa aatgctatga agaccaaggg gatggagggc ttcaagtttg atcttggctc aaagattgca gaaatcacct ccaaaggagc atctctgtat gaaatgagaa cagaagccat tgccagacct agaattgcaa taaaagagat tttgacacag gtggcctctg atgaagctaa tttagaagcc agaaatcgga agcgactaga gactctgcag gagaagactg aggaagaatt acaaaagcag ctgacttatc tggaacaaca gcttgaagac gaagctaaaa acactctctg cctgatacag ctcaagagtg gaagtaacga tgactcggac agtgagttgg aagaaaggcg gctgcccaag ggaagacctg gcaaacgcat tgtgggcacg gactcggagg agagtgaaat tgacatggaa gacgagagca tttctctctc accaaccaag ctggatgaga gtgacaatga cttctgaccc cctcagactt gtaggtaaat gggaacttag actaagctgg ctggactcat gatcactgaa gatcgtcgag cgctgggcct gtgatcgctg 60 ggggacctga gcgttatgtc tttccgcgac 120 ctgggatacc ctcgacatat ttctatggaa 180 tctgaagtgc ttctctggct tgtgaaaaga 240 gtggatactg aacaggaccg agttttcttc 300 aaggcacata taaaactcaa cactaagaag 360 gagctgctga agatcacatc tgtcctttat 420 tctgaaatag tagaggaaga tgtcaacaag 480 gatttgaagg cagccaggca gcttgcgtct 540 gacttgctcg gcatggaagt agagttgagg 600 ctggaaataa acgagactga aaaagtgatg 660 gttcagaaga ctaaagacct gctcaataat 720 aaaatcgaaa agagaaaatt agaactggaa 780 agtgtcaggc catgttttat ggatgagtat 840 tatgacactt atctggagaa atttcaaaat 900 catcatagga tggagcaaga aaggtttgag 960 aacaagctca aggaggaaga gaagcgcctg 1020 atagacatcc aggaggacga tgaatccgac 1080 ccacagacag ccatggagat gctcatgcaa 1140 atgcaaggtg gagactccga tgacaatgag 1200 gatgatgatg acgaggatga cgatttggaa 1260 cccaatcgaa gggtccggaa atctgaaccc 1320 ttttgccaag ggaccctggc agattaaaac 1380 aaggttagga aggtaacccc tgttttgttt 1440 gcaatactta tttctgcttt agcctcctat 1500 13 201134480 gtttgcattc catgaagctt aaataagaat tgaagcaaat ccctaagatt tatttttttc 1560 caccttattt atcttctaaa acttgaggaa tgcatgtgtt cttagtgatt cacatccacg 1620 ggacaaaaac tcaagaagaa ataagagctg acgccacaca aaaaaaaaaa aaaaaaaaaa 1680 a 1681 3 T類 5 1 R 4 4 p > > > > 0 12 3 1— 1— 2 2 2 2 <400> 45Leu Glu Leu Glu Arg Asn Arg Lys Arg Leu 1 5 10 <210> 44 <211> 1681 <212> DNA <213>Human 12 201134480 参<400> 44 gccgtccaag ggtccattgg ttgccataga aggggcgagc agttgcgacc ctgggctcct ctccgcaatt tcacagagat gatgagagcc aatttccgta cacccaattt tggacttgta tatgagcccc agactgacat cccgcctgac attaaggcaa ttgcccagtt catggccacc ctttatcaag cagatgggta tgcggtaaaa aatgctatga agaccaaggg gatggagggc ttcaagtttg atcttggctc aaagattgca gaaatcacct ccaaaggagc atctctgtat gaaatgagaa cagaagccat tgccagacct agaattgcaa taaaagagat tttgacacag gtggcctctg atgaagctaa tttagaagcc agaaatcgga agcgactaga gactctgcag gagaagactg aggaagaatt acaaaagcag ctgacttatc tggaacaaca gcttgaagac gaagctaaaa acactctctg cctgatacag ctcaagagtg gaagtaacga tgactcggac agtgagttgg aagaaaggcg gctgcccaag ggaagacctg gcaaacgcat Tgtgggcacg gactcggagg agagtgaaat tgacatggaa gacgagagca tttctctctc accaaccaag ctggatgaga gtgacaatga cttctgaccc cctcagactt gtaggtaaat gggaacttag actaagctgg ctggactcat gatcactgaa gatcgtcgag cgctgggcct gtgatcgctg 60 ggggacctga gcgttatgtc tttccgcgac 120 ctgggatacc ctcgacatat ttctatggaa 180 tctgaagtgc ttctctggct tgtgaaaaga 240 gtggatactg aacaggaccg agttttcttc 300 aaggcacata taaaactcaa cactaagaag 360 gagctgctga agatcacatc tgtcctttat 420 tctgaaatag tagaggaaga tgtcaacaag 480 gatttgaagg cagccaggca gcttgcgtct 540 gacttgctcg gcatggaagt agagttgagg 600 ctggaaataa acgagactga aaaagtgatg 660 gttcagaaga ctaaagacct gctcaataat 720 aaaatcgaaa agagaaaatt agaactggaa 780 agtgtcaggc catgttttat ggatgagtat 840 tatgacactt atctggagaa atttcaaaat 900 catcatagga tggagcaaga aaggtttgag 960 aacaagctca aggaggaaga gaagcgcctg 1020 atagacatcc aggaggacga tgaatccgac 1080 ccacagacag ccatggagat gctcatgcaa 1140 atgcaaggtg gagactccga tgacaatgag 1200 gatgatgatg acgaggatga cgatttggaa 1260 cccaatcgaa gggtccggaa atctgaaccc 1320 ttttgccaag ggaccctggc agattaaaac 1380 aaggttagga aggtaacccc tgttttgttt 1440 gcaatactta tttctgcttt agcctcctat 1500 13 201134480 gtttgcattc Catgaagctt aaataagaat tgaagcaaat ccctaagatt tatttttttc 1560 caccttattt atctt Ctaaa acttgaggaa tgcatgtgtt cttagtgatt cacatccacg 1620 ggacaaaaac tcaagaagaa ataagagctg acgccacaca aaaaaaaaaa aaaaaaaaaa 1680 a 1681 3 T class 5 1 R 4 4 p >>>> 0 12 3 1 - 1 2 2 2 2 <400> 45

Met Ser Phe Arg Asp Leu Arg Asn Phe Thr Glu Met Met Arg Ala Leu 1 5 10 15Met Ser Phe Arg Asp Leu Arg Asn Phe Thr Glu Met Met Arg Ala Leu 1 5 10 15

Gly Tyr Pro Arg His lie Ser Met Glu Asn Phe Arg Thr Pro Asn Phe 20 25 30Gly Tyr Pro Arg His lie Ser Met Glu Asn Phe Arg Thr Pro Asn Phe 20 25 30

Gly Leu Val Ser Glu Val Leu Leu Trp Leu Val Lys Arg Tyr Glu Pro 35 40 45Gly Leu Val Ser Glu Val Leu Leu Trp Leu Val Lys Arg Tyr Glu Pro 35 40 45

Gin Thr Asp lie Pro Pro Asp Val Asp Thr Glu Gin Asp Arg Val Phe 50 55 60Gin Thr Asp lie Pro Pro Asp Val Asp Thr Glu Gin Asp Arg Val Phe 50 55 60

Phe lie Lys Ala lie Ala Gin Phe Met Ala Thr Lys Ala His lie Lys 65 70 75 80Phe lie Lys Ala lie Ala Gin Phe Met Ala Thr Lys Ala His lie Lys 65 70 75 80

Leu Asn Thr Lys Lys Leu Tyr Gin Ala Asp Gly Tyr Ala Val Lys Glu 85 90 95Leu Asn Thr Lys Lys Leu Tyr Gin Ala Asp Gly Tyr Ala Val Lys Glu 85 90 95

Leu Leu Lys lie Thr Ser Val Leu Tyr Asn Ala Met Lys Thr Lys Gly 100 105 110Leu Leu Lys lie Thr Ser Val Leu Tyr Asn Ala Met Lys Thr Lys Gly 100 105 110

Met Glu Gly Ser Glu Me Val Glu Glu Asp Val Asn Lys Phe Lys Phe 115 120 125Met Glu Gly Ser Glu Me Val Glu Glu Asp Val Asn Lys Phe Lys Phe 115 120 125

Asp Leu Gly Ser Lys lie Ala Asp Leu Lys Ala Ala Arg Gin Leu Ala 130 135 140 14 201134480Asp Leu Gly Ser Lys lie Ala Asp Leu Lys Ala Ala Arg Gin Leu Ala 130 135 140 14 201134480

Ser 6lu lie Thr Ser Lys Gly Ala Ser Leu Tyr Asp Leu Leu 6ly Met 145 150 155 160Ser 6lu lie Thr Ser Lys Gly Ala Ser Leu Tyr Asp Leu Leu 6ly Met 145 150 155 160

Glu Val Glu Leu Arg Glu Met Arg Thr Glu Ala lie Ala Arg Pro Leu 165 170 175Glu Val Glu Leu Arg Glu Met Arg Thr Glu Ala lie Ala Arg Pro Leu 165 170 175

Glu lie Asn Glu Thr Glu Lys Val Met Arg lie Ala lie Lys Glu lie 180 185 190Glu lie Asn Glu Thr Glu Lys Val Met Arg lie Ala lie Lys Glu lie 180 185 190

Leu Thr Gin Val Gin Lys Thr Lys Asp Leu Leu Asn Asn Val Ala Ser 195 200 205Leu Thr Gin Val Gin Lys Thr Lys Asp Leu Leu Asn Asn Val Ala Ser 195 200 205

Asp Glu Ala Asn Leu Glu Ala Lys lie Glu Lys Arg Lys Leu Glu Leu 210 215 220Asp Glu Ala Asn Leu Glu Ala Lys lie Glu Lys Arg Lys Leu Glu Leu 210 215 220

Glu Arg Asn Arg Lys Arg Leu Glu Thr Leu Gin Ser Val Arg Pro Cys 225 230 235 240Glu Arg Asn Arg Lys Arg Leu Glu Thr Leu Gin Ser Val Arg Pro Cys 225 230 235 240

Phe Met Asp Glu Tyr Glu Lys Thr Glu Glu Glu Leu Gin Lys Gin Tyr 245 250 255Phe Met Asp Glu Tyr Glu Lys Thr Glu Glu Glu Leu Gin Lys Gin Tyr 245 250 255

Asp Thr Tyr Leu Glu Lys Phe Gin Asn Leu Thr Tyr Leu Glu Gin Gin 260 265 270Asp Thr Tyr Leu Glu Lys Phe Gin Asn Leu Thr Tyr Leu Glu Gin Gin 260 265 270

Leu Glu Asp His His Arg Met Glu Gin Glu Arg Phe Glu Glu Ala Lys 275 280 285Leu Glu Asp His His Arg Met Glu Gin Glu Arg Phe Glu Glu Ala Lys 275 280 285

Asn Thr Leu Cys Leu lie Gin Asn Lys Leu Lys Glu Glu Glu Lys Arg 290 295 300Asn Thr Leu Cys Leu lie Gin Asn Lys Leu Lys Glu Glu Glu Lys Arg 290 295 300

Leu Leu Lys Ser Gly Ser Asn Asp Asp Ser Asp lie Asp Me Gin Glu 305 310 315 320Leu Leu Lys Ser Gly Ser Asn Asp Asp Ser Asp lie Asp Me Gin Glu 305 310 315 320

Asp Asp Glu Ser Asp Ser Glu Leu Glu Glu Arg Arg Leu Pro Lys Pro 325 330 335Asp Asp Glu Ser Asp Ser Glu Leu Glu Glu Arg Arg Leu Pro Lys Pro 325 330 335

Gin Thr Ala Met Glu Met Leu Met Gin Gly Arg Pro Gly Lys Arg lie 340 345 350 15 201134480Gin Thr Ala Met Glu Met Leu Met Gin Gly Arg Pro Gly Lys Arg lie 340 345 350 15 201134480

Val Gly Thr Met Gin Gly Gly Asp Ser Asp Asp Asn Glu Asp Ser Glu 355 360 365Val Gly Thr Met Gin Gly Gly Asp Ser Asp Asp Asn Glu Asp Ser Glu 355 360 365

Glu Ser Glu lie Asp Met Glu Asp Asp Asp Asp Glu Asp Asp Asp Leu 370 375 380Glu Ser Glu lie Asp Met Glu Asp Asp Asp Asp Glu Asp Asp Asp Leu 370 375 380

Glu Asp Glu Ser lie Ser Leu Ser Pro Thr Lys Pro Asn Arg Arg Val 385 390 395 400Glu Asp Glu Ser lie Ser Leu Ser Pro Thr Lys Pro Asn Arg Arg Val 385 390 395 400

Arg Lys Ser Glu Pro Leu Asp Glu Ser Asp Asn Asp Phe 405 410 <210〉 46 <211〉 22 <212〉 DNA <213〉人工 <220〉〈223>人工序列 <400〉 46 gtctaccagg cattcgcttc at 22 <210> 47 <211〉 24 <212〉 DNA 〈213〉人工 <220〉〈223>人工序列 <400〉 47 tcagctggac cacagccgca gcgt 24 <210〉 48 <211〉 21 <212〉 DNA <213〉人工〈220〉〈223>人工序列 <400〉 48 tcagaaatcc tttctcttga c 21 16 201134480 ^ <210〉 <211> 〈212〉 <213〉 49 24 DNA 人工 <220〉 <223> 人工序列 <400> 49 ctagcctctg gaatcctttc tctt 24 17Arg Lys Ser Glu Pro Leu Asp Glu Ser Asp Asn Asp Phe 405 410 <210> 46 <211> 22 <212> DNA <213>Manual<220> <223>>Artificial Sequence<400> 46 gtctaccagg Cattcgcttc at 22 <210> 47 <211> 24 <212> DNA <213>manual<220> <223>> artificial sequence<400> 47 tcagctggac cacagccgca gcgt 24 <210> 48 <211> 21 <212> DNA <213>Artificial <220> <223>Artificial sequence<400> 48 tcagaaatcc tttctcttga c 21 16 201134480 ^ <210> <211> <212> <213> 49 24 DNA Artificial <;220><223> Manual Sequence <400> 49 ctagcctctg gaatcctttc tctt 24 17

Claims

201134480 VII. Patent application scope: 1. An isolated peptide with cytotoxic TNF-inducing ability, wherein the peptide consists of the amino acid sequence of CLUAP1 or an immunologically active fragment thereof. 2. The isolated peptide according to claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of 2 to 2 3 and 2 5 to 4 3 3. The separated peptides as described in any one of claims 1 and 2, wherein 1, 2 or several amino acids are substituted, deleted or added. 4. The isolated peptide as described in claim 3, wherein in the context of human leukocyte antigen-A24, the peptide has one or both of the following characteristics: (a) a second from the N-terminus The amino acid is modified to be an amino acid, which is selected from the group consisting of amphetamine, tyrosine, thioglycolic acid or tryptophan; and (b) the C-terminal amino acid is Or modified to an amino acid, which is selected from the group consisting of amphetamine, leucine, isoleucine, tryptophan or methionine. 5. If the patent application scope is in one or both of human leukocyte antigen-A2: the isolated peptide described in item 3, wherein the peptide has the following characteristics: a π-mail with Hita leucoside a group consisting of thiobutyric acid; and (b) a C-terminal amino acid is a group of free hydrazines associated with decanoic acid and leucine at the group 201134480 > group. 6. The isolated peptide according to any one of claims 1 to 5, wherein the peptide is a nine peptide or a ten peptide. An isolated polynucleotide which encodes the peptide as described in any one of claims 1 to 6. A composition for inducing a cytotoxic T lymphocyte, wherein the composition comprises one of the peptides according to any one of claims 6 to 6 or the peptide, or as claimed in claim 7 One or more of the polynucleotides described in the item. A pharmaceutical composition for use in the treatment and/or prevention of cancer, and/or its prevention of recurrence after surgery, wherein the composition comprises one of any one of claims 1 to 6 or A plurality of the peptides, or as described in claim 7 of the patent application, or a plurality of the polynucleic acid. The pharmaceutical composition according to claim 9 of the present invention, which is formulated for administration of one (four), wherein the human leukocyte antigen is human leukocyte antigen-A24 or human leukocyte antigen _8. U. The pharmaceutical composition of claim 9 or claim 10, wherein the composition is formulated for the treatment of cancer. 12. A method of inducing an antigen-presenting cell having a cytotoxic lymphoblast-inducing ability, wherein the method comprises a step of selecting a group consisting of: (a) in vitro, cell and patent application a range of touches; and α ~ 叩 or / /? The antigen is represented by any one of the above-mentioned items 1 to 6 to 201134480 (b) will be coded as claimed in claims 1 to 6 - A polynucleotide in which the peptide is introduced into an antigen-presenting cell.斤 13. A method for inducing cytotoxic lymphocytes by a method comprising the step of selecting a group consisting of: (a) co-cultivating CD8-positive sputum cells with antigen-presenting cells, antigen-seeking, The cell exhibits a complex of the human leukocyte tissue antigen and the peptide as described in any one of claims 1 to 6 on the surface thereof; (8) co-culturing the CD8-positive tau cell with the exosome The exosome exhibits a complex of the human leukocyte tissue antigen and the peptide as described in any one of claims 6 to 6; and (c) encodes a tau cell The gene of the polynucleotide of the multi-peptide of the subunit is introduced into a tau cell, and the tau cell receptor subunit multi-peptide is combined with the peptide as described in any one of claims 1 to 6. It is an isolated antigen-presenting cell which exhibits a complex of a human leukocyte tissue antigen and the peptide described in the above-mentioned item of the scope of the patent application No. 6 to 6 on the surface thereof. 15. The antigen-presenting cell of claim 14, which is induced by the method of claim 12 of the patent application. 16. An isolated cytotoxic tau lymphocyte, which is characterized by the peptide as described in any one of claims 1 to 6. 17. The cytotoxic T lymphocyte as described in claim 6 of the Shenqing patent scope, which is induced by the method as described in claim 13 of the patent application. 18. A method of inducing an immune response against a cancer in a body, wherein the method comprises administering to the individual a composition comprising 3 201134480: a wide range of claims 1 to 6 of the patent application - The peptide, the immunologically active fragment thereof, or the polynucleotide encoding the peptide or the fragment. An antibody or a fragment thereof, which is any one of the peptides described in any one of the above-mentioned items. A vector comprising a nucleotide sequence encoding the peptide as described in any one of claims 6 to 6. A host cell which is transformed or transfected with an expression vector as described in claim 20 of the patent application. 22. A diagnostic kit comprising the peptide according to any one of the claims of the patent scope, the nucleic acid of the seventh aspect of the patent scope, or the antibody of claim 19 of the patent application. 23. The isolated peptide according to any one of claims 4-6, wherein the free sequence identification number is: 3, 4, 5, 8, 9, 丨Q, 14, 15, 16, Group of 18, 23, 30, 33, 34, 35, 36, and 38. 4