CN117677701A - 抗原结合分子与链霉亲和素突变体的融合蛋白 - Google Patents
抗原结合分子与链霉亲和素突变体的融合蛋白 Download PDFInfo
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Abstract
本发明的课题在于提供用于在癌症的治疗或诊断中使用的、识别癌细胞等的分子与链霉亲和素突变体的融合蛋白。根据本发明，提供一种融合蛋白，其是分子量为20,000以下的抗原结合分子经由接头序列与链霉亲和素突变体的N末端侧和/或C末端侧结合而成，该链霉亲和素突变体在SEQ ID NO:17所记载的核心链霉亲和素的氨基酸序列(其中，C末端的由Pro‑Ser‑Ala‑Ala‑Ser构成的氨基酸序列可缺失一部分或全部)中包含具有下述(1)～(6)的突变的氨基酸序列、且与野生型链霉亲和素相比免疫原性降低。(1)第10位的氨基酸残基的酪氨酸取代成了丝氨酸或苏氨酸的突变；(2)第71位的氨基酸残基的酪氨酸取代成了丙氨酸或丝氨酸的突变；(3)第72位的氨基酸残基的精氨酸取代成了赖氨酸的突变；(4)第89位的氨基酸残基的谷氨酸取代成了天冬氨酸的突变；(5)第91位的氨基酸残基的精氨酸取代成了赖氨酸的突变；(6)第104位的氨基酸残基的谷氨酸取代成了谷氨酰胺或天冬酰胺的突变。
Description
技术领域
本发明涉及抗原结合分子与链霉亲和素突变体的融合蛋白、以及其利用。
背景技术
亲和素与生物素、或者链霉亲和素与生物素之间的亲和性非常高(Kd＝10-15～10-14M)，作为生物双分子间的相互作用，这是最强的相互作用之一。目前，亲和素/链霉亲和素-生物素相互作用在生物化学、分子生物学或医学领域被广泛应用。已经设计了将亲和素/链霉亲和素与生物素的高结合能力和抗体分子组合的药物递送方法和预靶向法。
专利文献1中记载了通过向氨基酸中导入突变使免疫原性降低的链霉亲和素突变体。专利文献1中还记载了治疗或诊断试剂盒，其中包含上述链霉亲和素突变体和用生物素或其衍生物标记的诊断用或治疗用物质。
现有技术文献
专利文献
专利文献1：国际公开WO2010/95455。
发明内容
发明所要解决的课题
本发明以提供用于在癌症的治疗或诊断中使用的识别癌细胞等的分子与链霉亲和素突变体的融合蛋白作为所应解决的课题。本发明还以使用了上述的融合蛋白的癌症的治疗方法或癌症的诊断方法作为所应解决的课题。
用于解决课题的手段
为了解决上述课题，本发明人进行了深入研究，结果选择分子量较抗体小的分子作为识别癌细胞的分子，调制了上述分子与链霉亲和素突变体的融合蛋白。而且，还发现了：通过使用了上述的融合蛋白和生物素与酞菁染料的缀合物的光免疫疗法可抑制癌细胞的生长，从而完成了本发明。
即，根据本发明，提供以下的发明。
<1>融合蛋白，其是分子量为20,000以下的抗原结合分子经由接头序列与链霉亲和素突变体的N末端侧和/或C末端侧结合而成，该链霉亲和素突变体在SEQ ID NO:17所记载的核心链霉亲和素的氨基酸序列(其中，C末端的由Pro-Ser-Ala-Ala-Ser构成的氨基酸序列可缺失一部分或全部)中包含具有下述(1)～(6)的突变的氨基酸序列、且与野生型链霉亲和素相比免疫原性降低。
(1)第10位的氨基酸残基的酪氨酸取代成了丝氨酸或苏氨酸的突变；
(2)第71位的氨基酸残基的酪氨酸取代成了丙氨酸或丝氨酸的突变；
(3)第72位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(4)第89位的氨基酸残基的谷氨酸取代成了天冬氨酸的突变；
(5)第91位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(6)第104位的氨基酸残基的谷氨酸取代成了谷氨酰胺或天冬酰胺的突变。
<2><1>所述的融合蛋白，其从N末端侧到C末端侧依次具有分子量为20,000以下的抗原结合分子、接头序列和上述链霉亲和素突变体的氨基酸序列。
<3><1>或<2>所述的融合蛋白，其中，抗原结合分子是与在癌细胞中表达的抗原结合的分子。
<4><1>～<3>中任一项所述的融合蛋白，其中，抗原结合分子是与Her2结合的分子。
<5><1>～<4>中任一项所述的融合蛋白，其中，抗原结合分子具有SEQ ID NO:2所记载的氨基酸序列。
<6><1>～<5>中任一项所述的融合蛋白，其中，接头序列由甘氨酸残基和丝氨酸残基构成，氨基酸残基的数量为5～25个。
<7><1>～<6>中任一项所述的融合蛋白，其中，接头序列是[(Gly)m-Ser]n(式中，m表示1～10的整数，n表示1～5的整数)所示的氨基酸序列。
<8><1>～<7>中任一项所述的融合蛋白，其具有SEQ ID NO:3或9所记载的氨基酸序列。
<9>核酸，其编码具有SEQ ID NO:3或9所记载的氨基酸序列的融合蛋白。
<10>癌症治疗药或癌症诊断药，其包含<1>～<8>中任一项所述的融合蛋白。
<11>癌症的治疗或诊断试剂盒，其包含：(1)<1>～<8>中任一项所述的融合蛋白；以及(2)生物素与诊断用物质或治疗用物质的缀合物。
<12><11>所述的试剂盒，其中，诊断用物质或治疗用物质为酞菁染料。
<13><1>～<8>中任一项所述的融合蛋白的制造方法，其包括：使编码<1>～<8>中任一项所述的融合蛋白的核酸在宿主中表达的步骤。
<14><13>所述的方法，其中，使上述融合蛋白在细菌的封入体内表达并回收。
发明效果
通过使用本发明的抗原结合分子与链霉亲和素突变体的融合蛋白，例如可抑制癌细胞的生长。
附图说明
[图1]图1显示MFL2-G5S×3-His、MFL2-G5S×3-del5的开卷(巻戻し)研究的结果。
[图2]图2显示Biacore测定(MFL2-His vs.HER2的结果。
[图3]图3显示Biacore测定(MFL2-His vs.生物素-SiPc)的结果。
[图4]图4显示MFL2-G5S×3-His-SiPc细胞毒性测定的结果。
[图5]图5显示MFL2-G5Sx1-PSAAS、MFL2-G5Sx2-PSAAS、MFL2-G5S×3-PSAAS的表达/纯化和性能评价的结果。
具体实施方式
以下，进一步对本发明进行详细说明。
<抗原结合分子与链霉亲和素突变体的融合蛋白>
本发明的融合蛋白是分子量为20,000以下的抗原结合分子经由接头序列与链霉亲和素突变体的N末端侧和/或C末端侧结合而成，该链霉亲和素突变体在SEQ ID NO:17所记载的核心链霉亲和素的氨基酸序列(其中，C末端的由Pro-Ser-Ala-Ala-Ser构成的氨基酸序列可缺失一部分或全部)中包含具有下述(1)～(6)的突变的氨基酸序列、且与野生型链霉亲和素相比免疫原性降低。
(1)第10位的氨基酸残基的酪氨酸取代成了丝氨酸或苏氨酸的突变；
(2)第71位的氨基酸残基的酪氨酸取代成了丙氨酸或丝氨酸的突变；
(3)第72位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(4)第89位的氨基酸残基的谷氨酸取代成了天冬氨酸的突变；
(5)第91位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(6)第104位的氨基酸残基的谷氨酸取代成了谷氨酰胺或天冬酰胺的突变。
在上述链霉亲和素突变体的N末端侧和C末端侧经由接头序列均结合有分子量为20,000以下的抗原结合分子的情况下，该抗原结合分子可相同也可不同。
优选地，本发明的融合蛋白是从N末端侧到C末端侧依次具有分子量为20,000以下的抗原结合分子、接头序列和上述链霉亲和素突变体的氨基酸序列的融合蛋白。
本发明中的链霉亲和素突变体是国际公开WO2010/95455中记载的通过向氨基酸中导入突变而使免疫原性降低的链霉亲和素突变体。即，本发明中的链霉亲和素突变体是在SEQ ID NO:17所记载的核心链霉亲和素的氨基酸序列(其中，C末端的由Pro-Ser-Ala-Ala-Ser构成的氨基酸序列可缺失一部分或全部)中包含具有以下突变的氨基酸序列的链霉亲和素突变体。
(1)第10位的氨基酸残基的酪氨酸取代成了丝氨酸或苏氨酸的突变；
(2)第71位的氨基酸残基的酪氨酸取代成了丙氨酸或丝氨酸的突变；
(3)第72位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(4)第89位的氨基酸残基的谷氨酸取代成了天冬氨酸的突变；
(5)第91位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(6)第104位的氨基酸残基的谷氨酸取代成了谷氨酰胺或天冬酰胺的突变。
本发明中所说的与野生型链霉亲和素相比免疫原性降低是指在对人等哺乳动物给予链霉亲和素突变体的情况下免疫原性降低。免疫原性降低例如可通过以下的方法确认。即，关于本发明的突变体链霉亲和素，分析对用野生型链霉亲和素免疫食蟹猴而获取的抗链霉亲和素抗血清的反应性，如果对上述抗链霉亲和素抗血清的反应性与野生型链霉亲和素相比降低，则可判断为与野生型链霉亲和素相比免疫原性降低。在通过上述方法判断免疫原性降低的情况下，与野生型链霉亲和素相比，本发明的链霉亲和素突变体的免疫原性降低至优选80％以下、进一步优选60％以下、进一步优选20％以下、进一步优选15％以下、进一步优选10％以下、特别优选5％以下。
如上所述，本发明的融合蛋白是分子量为20,000以下的抗原结合分子与链霉亲和素突变体的融合蛋白。本发明的融合蛋白通过链霉亲和素突变体的氨基酸序列之间的亲和性形成四聚体。例如在使用具有实施例2中记载的氨基酸序列的蛋白作为抗原结合分子的情况下，本发明的融合蛋白的四聚体的分子量为约96kDa。在对生物体给予本发明这样的融合蛋白以进行癌症治疗的情况下，重要的是可同时实现向肿瘤中的摄取(tumor uptake)良好、清除速度(clearance rate)良好、以及良好的向肿瘤中的渗透(tumor penetration)良好。设想本发明中的上述四聚体的分子量(约96kDa)是可同时实现上述3个参数的分子量。
抗原结合分子的分子量只要是20,000以下即可，但抗原结合分子的分子量通常为4,000以上且20,000以下、优选为4,000以上且10,000以下、更优选为4,000以上且8,000以下。
本发明中的抗原结合分子是不同于抗体的概念的分子。IgG抗体的分子量通常为150,000左右，相对于此，本发明中的抗原结合分子是具有远小于IgG抗体的分子量的分子。作为本发明中的抗原结合分子，可以是模拟抗体而制作的分子。例如可从改变蛋白A的IgG结合结构域(VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL AEAKKLNDAQAPK)(SEQID NO:18)的一部分而制作的分子量为约6,000的蛋白中通过筛选适当选择与所期望的抗原结合的分子。
对抗原结合分子中的抗原没有特别限定，优选在癌细胞中表达的抗原。作为特异性地在癌中表达的抗原，可列举以下的抗原。
表皮调节素(EREG)、ROBO1,2,3,4、1-40-β-淀粉样蛋白、4-1BB、5AC、5T4、ACVR2B、腺癌抗原、甲胎蛋白(α-fetoprotein)、血管生成素2、炭疽毒素、AOC3(VAP-1)、B-淋巴瘤细胞、B7-H3、BAFF、β淀粉样蛋白、C242抗原、C5、CA-125、碳酸酐酶9(CA-IX)、心脏肌球蛋白(心肌肌球蛋白)、CCL11(eotaxin-1，嗜酸性粒细胞趋化因子-1)、CCR4、CCR5、CD11、CD18、CD125、CD140a、CD147(basigin，基础免疫球蛋白)、CD147(basigin，基础免疫球蛋白)、CD15、CD152、CD154(CD40L)、CD154、CD19、CD2、CD20、CD200、CD22、CD221、CD23(IgE受体)、CD25(IL-2受体的α链)、CD28、CD3、CD30(TNFRSF8)、CD33、CD37、CD38(环状ADP核糖水解酶)、CD4、CD40、CD41(整联蛋白α-IIb)、CD44 v6、CD5、CD51、CD52、CD56、CD6、CD70、CD74、CD79B、CD80、CEA、CFD、ch4D5、CLDN18.2、艰难梭菌(Clostridium difficile)、凝聚因子(Clumpingfactor)A、CSF2、CTLA-4、巨细胞病毒、巨细胞病毒糖蛋白B、DLL4、DR5、大肠杆菌志贺毒素1型、大肠杆菌志贺毒素2型、EGFL7、EGFR、内毒素、EpCAM、上皮唾液蛋白(Episialin)、ERBB3、大肠杆菌(Escherichia coli)、呼吸道合胞病毒(respiratory syncytial virus)的F蛋白、FAP、纤维蛋白IIβ链、纤连蛋白额外结构域-B(Extra-domain B of fibronectin)、叶酸受体1、卷曲(Frizzled)受体、GD2、GD3神经节苷脂、GMCSF受体α链、GPNMB、乙型肝炎表面抗原、乙型肝炎病毒、HER1、HER2/neu、HER3、HGF、HIV-1、HLA-DRβ、HNGF、Hsp90、人β淀粉样蛋白、人分散因子(Scatter factor)受体激酶、人TNF、ICAM-1(CD54)、IFN-α、IFN-γ、IgE、IgEFc区、IGF-1受体、IGF-I、IgG4、IGHE、IL-1β、IL-12、IL-13、IL-17、IL-17A、IL-22、IL-23、IL-4、IL-5、IL-6、IL-6受体、IL-9、ILGF2、甲型流感血凝素、胰岛素样生长因子I受体、整联蛋白α4、整联蛋白α4β7、整联蛋白α5β1、整联蛋白α7β7、整联蛋白αIIbβ3、整联蛋白αvβ3、整联蛋白γ诱导蛋白、干扰素受体、干扰素α/β受体、ITGA2、ITGB2(CD18)、KIR2D、L-选择素(CD62L)、Lewis-Y抗原、LFA-1(CD11a)、脂磷壁酸(Lipoteichoic acid)、LOXL2、LTA、MCP-1、间皮素、MS4A1、MUC1、粘蛋白CanAg、肌生长抑制素、N-羟乙酰神经氨酸(N-glycolylneuraminic acid)、NARP-1、NCA-90(粒细胞抗原)、NGF、NOGO-A、NRP1、穴兔(Oryctolagus cuniculus)、OX-40、oxLDL、PCSK9、PD-1、PDCD1、PDGF-Rα、磷脂酰丝氨酸、前列腺癌细胞、绿脓杆菌(Pseudomonas aeruginosa)、狂犬病病毒糖蛋白、RANKL、呼吸道合胞病毒、RHD、Rh(Rhesus，猕猴)因子、RON、RTN4、骨硬化蛋白(Sclerostin)、SDC1、选择素P、SLAMF7、SOST、鞘氨醇-1-磷酸(Sphingosine-1-phosphate)、TAG-72、TEM1、肌腱蛋白(Tenascin)C、TFPI、TGFβ1、TGFβ2、TGF-β、TNF-α、TRAIL-R1、TRAIL-R2、肿瘤抗原CTAA16.88、MUC1的肿瘤特异性糖基化、TWEAK受体、TYRP1(糖蛋白75)、VEGF-A、VEGFR-1、VEGFR2、波形蛋白、VWF。
上述之中，特别优选HER2。
作为抗原结合分子的一例，可与HER2结合。可列举：具有SEQ IDNO:2所记载的氨基酸序列的蛋白。
对接头序列没有特别限定，只要可实现本发明的效果即可，氨基酸数优选5～25个氨基酸、更优选10～25个氨基酸、进一步优选15～20个氨基酸。
作为接头序列的具体例子，可列举：由甘氨酸残基和丝氨酸残基构成的序列。作为接头序列，例如可使用[(Gly)m-Ser]n(式中，m表示1～10的整数，n表示1～5的整数)所示的氨基酸序列。作为接头序列的具体例子，可列举：Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser，没有特别限定。
作为本发明的融合蛋白的具体例子，可列举：具有SEQ ID NO:3或9所记载的氨基酸序列的融合蛋白。
根据本发明，还提供编码上述本发明的融合蛋白的核酸(例如，DNA)。作为本发明的核酸的具体例子，可列举：编码具有SEQ ID NO:3或9所记载的氨基酸序列的融合蛋白的核酸。作为本发明的核酸的一例，可列举：具有SEQ ID NO:4或10所记载的核苷酸序列的核酸。
编码本发明的融合蛋白的核酸(例如，DNA)可插入到载体中使用。为了制造本发明的融合蛋白，可将编码本发明的融合蛋白的核酸插入到表达载体中，并将该表达载体转化到宿主中，从而使本发明的融合蛋白表达。即，根据本发明，提供本发明的融合蛋白的制造方法，其包括：使编码本发明的融合蛋白的核酸在宿主中表达的步骤。融合蛋白可优选地在细菌的封入体内表达并回收。
在以大肠杆菌为宿主的情况下，作为载体，优选具有复制起点(ori)、还具有用于选择所转化的宿主的基因(例如，耐氨苄青霉素、四环素、卡那霉素或氯霉素等药剂的耐药性基因等)。另外，在表达载体的情况下，优选具有可使本发明的链霉亲和素突变体在宿主中高效率地表达的这样的启动子、例如lacZ启动子或T7启动子等。作为这样的载体，载体的例子可列举：M13系载体、pUC系载体、pBR322、pBluescript、pCR-Script、pGEX-5X-1(Pharmacia)、“QIAexpress system”(Qiagen)、pEGFP或pET(这种情况下，宿主优选使用表达T7 RNA聚合酶的BL21)等。
向宿主细胞中引入载体例如可采用氯化钙法、电穿孔法进行。另外，可添加用于提高可溶性的标签、例如编码谷胱甘肽S-转移酶或硫氧还蛋白、麦芽糖结合蛋白的序列。另外，还可添加以容易纯化为目的而设计的标签、例如聚组氨酸标签、Myc表位、血凝素(HA)表位、T7表位、Xpress标签或FLAG肽标签、编码其他已知的标签序列的序列。
除大肠杆菌以外，还可列举：来自哺乳动物的表达载体(例如，pcDNA3(Invitrogen公司制造)或pEGF-BOS(Nucleic Acids.Res.1990,18(17),第5322页)、pEF、pCDM8)、来自昆虫细胞的表达载体(例如“Bac-to-BAC杆状病毒表达系统”(Gibco-BRL公司制造)、pBacPAK8)、来自植物的表达载体(例如，pMH1、pMH2)、来自动物病毒的表达载体(例如，pHSV、pMV、pAdexLcw)、来自逆转录病毒的表达载体(例如，pZIPneo)、来自酵母的表达载体(例如，“毕赤酵母表达试剂盒”(Invitrogen公司制造)、pNV11、SP-Q01)、来自枯草杆菌的表达载体(例如，pPL608、pKTH50)。
在以CHO细胞、COS细胞、NIH3T3细胞等动物细胞中的表达为目的的情况下，具有在细胞内表达所必需的启动子、例如SV40启动子(Mulligan等人,Nature(1979)277,108)、MMLV-LTR启动子、EF1α启动子(Mizushima等人,Nucleic Acids Res.(1990)18,5322)、CMV启动子等是必不可少的，进一步优选具有用于选拔向细胞内转化的基因(例如，可通过药剂(新霉素、G418等)判别的这样的耐药性基因)。作为具有这种特性的载体，例如可列举：pMAM、pDR2、pBK-RSV、pBK-CMV、pOPRSV、pOP13等。
对引入载体的宿主细胞没有特别限定，可以是原核生物和真核生物的任一种。例如，可使用大肠杆菌或各种动物细胞等。
在使用真核细胞的情况下，例如在宿主中可使用动物细胞、植物细胞、真菌细胞。作为动物细胞，可使用哺乳类细胞、例如CHO细胞、COS细胞、3T3细胞、HeLa细胞、Vero细胞、或昆虫细胞、例如Sf9、Sf21、Tn5等。在动物细胞中，以大量表达为目的的情况下，特别优选CHO细胞。向宿主细胞中引入载体例如可通过磷酸钙法、DEAE葡聚糖法、使用了阳离子脂质体DOTAP(Boehringer Mannheim公司制造)的方法、电穿孔法、脂质转染等方法来进行。
作为植物细胞，例如来自烟草(Nicotiana tabacum)的细胞作为蛋白生产系统而已知，只要对其进行愈伤组织培养即可。作为真菌细胞，已知有酵母、例如酵母(Saccharomyces)属、例如酿酒酵母(Saccharomyces cerevisiae)；丝状菌(丝状真菌)、例如曲霉(Aspergillus)属、例如黑曲霉(Aspergillus niger)。
在使用原核细胞的情况下，可列举：大肠杆菌(E.coli)、例如JM109、DH5α、HB101等，除此以外，还已知枯草杆菌。
利用本发明的核酸转化这些细胞，将所转化的细胞在体外进行培养，从而得到本发明的融合蛋白。培养可按照已知的方法来进行。例如，作为动物细胞的培养液，例如可使用DMEM、MEM、RPMI1640、IMDM。此时，还可并用胎牛血清(FCS)等血清补液，也可进行无血清培养。培养时的pH优选为约6～8。培养通常是在约30～40℃下进行约15～200小时，根据需要进行培养基的交换、通气、搅拌。另外，还可添加用于促进细胞生长的生长因子。
<癌症治疗药或癌症诊断药>
本发明的融合蛋白可用作癌症治疗药或癌症诊断药。
根据本发明，提供癌症的治疗或诊断试剂盒，该试剂盒包含：(1)本发明的融合蛋白；以及(2)生物素与诊断用物质或治疗用物质的缀合物。
在使用与存在于癌细胞内的抗原结合的分子作为抗原结合分子的情况下，通过对患者给予本发明的融合蛋白，可使链霉亲和素突变体特异性地聚集于癌细胞。接下来，通过对患者给予生物素与诊断用物质或治疗用物质的缀合物，可使诊断用物质或治疗用物质准确地向癌细胞聚集。
或者，还可调制使“本发明的融合蛋白”和“生物素与诊断用物质或治疗用物质的缀合物”结合而成的复合物，对患者给予该复合物。
<生物素与诊断用物质或治疗用物质的缀合物>
通过使诊断用物质或治疗用物质与生物素结合，可调制生物素与诊断用物质或治疗用物质的缀合物。作为诊断用物质或治疗用物质，可列举：荧光色素、化学发光剤、放射性同位素、由金属化合物等构成的敏化剂、由金属化合物等构成的中子捕捉剤、酞菁染料、低分子化合物、微气泡或纳米气泡、蛋白等。可优选使用酞菁染料。
<酞菁染料>
作为酞菁染料，具体而言，可使用下述式(1)所示的化合物或其盐。
[化学式1]
X表示末端具有亲水性基团的取代基。作为亲水性基团，优选磺酸基、磷酸基、铵基等。
X优选为末端具有磺酸基的取代基。
作为X的一例，可列举：以下所示的基团。
[化学式2]
在本说明书中，Me表示甲基。
L3、L4、L5和L6分别独立地表示二价连接基。
L3优选为：
[化学式3]
(式中，m表示1～5的整数)。
L4优选为-[(CH2)p-O)]q-
(式中，p和q分别独立地表示1～5的整数)。
L5优选为-CONH-、-NHCO-、-COO-或-OCO-，更优选为-CONH-。
L6优选为-(CH2)r-、-(CH2)r-O-或-(CH2)r-Si(R1)(R2)-O-(式中，r表示1～5的整数。R1和R2分别独立地表示碳数为1～4的烷基)。R1和R2特别优选为甲基。
Y表示可与抗原结合分子结合的基团。Y优选为羧基的活性酯体，更优选为羧基的琥珀酰亚胺酯。
R3表示碳数为1～6的烷基、碳数为1～6的烷氧基、卤原子、-SR11、-SOR12、碳数为6～10的芳基、-N(R13)(R14)或-NO2，或者相邻的2个R3可一起形成碳数为6～10的芳基。R11、R12、R13和R14分别独立地表示碳数为1～6的烷基或碳数为1～6的烷氧基。
这里，芳基可被碳数为1～6的烷基或碳数为1～6的烷氧基取代。
这里，烷基和烷氧基可被卤原子取代。
在存在多个R3的情况下，各自可相同也可不同。
s表示0～4的整数。优选s为0。
作为卤原子，可以是氟、氯、溴、碘的任一者，优选为氟、氯或溴。
酞菁染料的化合物可按照实施例的制造例1～5中记载的方法合成。
<光免疫疗法>
光免疫疗法是指为了在体内破坏特定的细胞而使用光敏剂和照射光的治疗方法。光敏剂在暴露于特异性波长的光时产生可诱发附近的细胞的凋亡、坏死和/或自噬作用的细胞毒性的活性氧物种(Reactive Oxygen Species)。例如，在日本专利6127045号公报中记载了使细胞死亡的方法，该方法包括：使包含细胞表面蛋白的细胞与治疗有效量的1种或多种抗体-IR700分子接触的步骤，且该抗体与该细胞表面蛋白特异性地结合的步骤；以660～740nm的波长、且以至少1Jcm-2的放射剂量(辐射剂量)对该细胞进行照射的步骤；以及在对该细胞照射的约0～8小时后，使该细胞与1种或多种治疗剂接触，由此使该细胞死亡的步骤。在日本特表2017-524659号公报中记载了在患有疾病或病态的受试者中诱发细胞毒性的方法，该方法包括：(a)对该受试者给予对治疗有效的药剂，该药剂包含在与受试者的细胞特异性地结合的探针上缀合的IRDye(注册商标)700DX等酞菁染料；以及(b)以有效诱发细胞死亡的量对上述细胞照射适当的激发光。
对受试者给予本发明的融合蛋白、和生物素修饰二聚体与酞菁染料的缀合物，以有效地抑制细胞生长或诱发细胞死亡的量对细胞照射激发光，从而可抑制细胞生长或诱发细胞死亡，治疗受试者。
优选对受试者给予本发明的融合蛋白、和生物素与酞菁染料的缀合物，以有效地抑制细胞生长或诱发细胞死亡的量对细胞照射激发光，从而可抑制细胞生长或诱发细胞死亡，治疗受试者。
作为受试者，包括人和非人哺乳动物，可列举：人或小鼠等实验动物。作为受试者，优选患有希望抑制细胞生长或诱发细胞死亡的疾病的受试者，例如可列举：具有癌症或实体瘤的受试者。
“癌症”可列举：癌瘤、淋巴瘤、母细胞瘤、肉瘤和白血病或恶性淋巴瘤。作为癌症的具体例子，可列举：鳞状上皮癌(例如，上皮性鳞状细胞癌)、包括小细胞肺癌在内的肺癌、非小细胞肺癌(“NSCLC”)、肺腺癌和肺鳞状细胞癌、腹膜癌、肝细胞性癌、包括消化系统癌症在内的胃体癌或胃癌、胰腺癌、成胶质细胞瘤(Glioblastoma，神经胶质母细胞瘤)、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞癌、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌或肾部癌、前列腺癌、外阴部癌、甲状腺癌、肝细胞癌瘤、肛门癌、阴茎癌、以及头颈部癌。
实体瘤无论是良性还是恶性通常是指不含包囊的异常细胞团块。作为实体瘤，可列举：神经胶质瘤、星形细胞瘤、成神经管细胞瘤(Medulloblastoma，髓母细胞瘤)、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、黑色素瘤、成神经细胞瘤(Neuroblastoma，神经母细胞瘤)和视网膜母细胞瘤。
作为对受试者的给予(给药)方法，可列举：局部途径、注射(皮下注射、肌肉内注射、皮内注射、腹腔内注射、肿瘤内注射和静脉内注射等)、口服途径、眼途径、舌下途径、直肠途径、经皮途径、鼻腔内途径、阴道途径和吸入途径等，但并不限于这些。
生物素修饰二聚体与酞菁染料的缀合物、和本发明的融合蛋白分别优选以治疗有效量进行给药。
关于上述的缀合物和融合蛋白的各自的治疗有效量，为每60千克至少0.5毫克(mg/60kg)、至少5mg/60kg、至少10mg/60kg、至少20mg/60kg、至少30mg/60kg、至少50mg/60kg。例如，在静脉内给药的情况下，为1mg/60kg、2mg/60kg、5mg/60kg、20mg/60kg或50mg/60kg的用量(剂量)等、例如0.5～50mg/60kg。在其他例子中，治疗有效量为至少100μg/kg、至少500μg/kg或至少500μg/kg等、至少10μg/kg，例如，在肿瘤内给药或腹腔内给药的情况下，为100μg/kg、250μg/kg、约500μg/kg、750μg/kg或1000μg/kg的用量等、例如10μg/kg～1000μg/kg。在一个例子中，治疗有效量为以局部用溶液进行给药的10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml、60μg/ml、70μg/ml、80μg/ml、90μg/ml或100μg/ml等、20μg/ml～100μg/ml之间等、至少500μg/ml等、至少1μg/ml。
可将上述的给药量通过1次或多次的分割用量(2、3或4次的用量等)或单一制剂进行给药。
生物素与酞菁染料的缀合物、和本发明的融合蛋白可分别单独给药，也可在药学上可接受的载体的存在下给药，还可在其他治疗剂(其他抗癌药等)的存在下给药。
生物素与酞菁染料的缀合物、和本发明的融合蛋白可与循环的肿瘤细胞或实体瘤细胞等靶细胞或靶组织结合。之后，若照射光，则上述缀合物或复合物可吸收光，损伤或破坏靶细胞或组织。
在光免疫疗法中，照射光的波长优选为660～740nm，例如具有660nm、670nm、680nm、690nm、700nm、710nm、720nm、730nm或740nm的波长。光的照射可通过使用具备近红外(NIR)发光二极管的装置来进行。
光的照射量为至少1J/cm2、例如至少4J/cm2、至少10J/cm2、至少15J/cm2、至少20J/cm2、至少50J/cm2、或至少100J/cm2、例如1～500J/cm2。光照射可实施多次(例如，2、3、4、5、6、7、8、9或10次)。
通过以下的实施例来进一步具体地说明本发明，但本发明并不受实施例限定。
实施例
<制造例1>
[化学式4]
在0.5mg(1.2μmol)生物素-PEG3-NH2中加入1.8mg(1.0μmol)NHS-SiPc、200μL磷酸氢二钠缓冲液(pH8.4)和200μL二甲基亚砜，用铝箔遮光，在室温下搅拌12小时。将反应液用水稀释至1mL，所得稀释液通过反相HPLC(梯度：30％5分钟、30-80％30分钟，乙腈的50mM三乙基乙酸铵水溶液(pH7.0)，保留时间＝23.8分钟，YMC-Triart C18，流速＝3.5mL/分钟)进行纯化，从而得到了0.7mg(0.34μmol)目标化合物生物素-SiPc(Biotin-SiPc)(34％、深蓝色)。
LRMS(ESI):m/z 941.45[M-2H]2-
<HER2识别蛋白的调制方法>
将LISA314分子(SEQ ID NO:1)和识别HER2抗原的分子(Lofblom,J.FEBSLett.584(12):2670-80(2010))(SEQ ID NO:2)融合，在大肠杆菌中合成对生物素分子和Her2/ErbB2两者具有结合性的蛋白(以下，显示为MFL2-G5S×3-His)(SEQ ID NO:3)。由Eurofins Genomics公司进行MFL2-G5S×3-His的基因序列(SEQ ID NO:4)的人工基因合成。使用pET45b(+)作为表达载体，按照常规方法进行表达载体的制作。具体而言，使用引物组(Rv:catggtatatctccttcttaaagttaaac(SEQ ID NO:5),Fw:cgcagcttaattaacctaggctgctgccac(SEQ ID NO:6))，通过PCR反应进行载体的直链化。对于通过人工基因合成调制的序列，使用引物组(Fw:aggagatataccatgGTGGACAACAAATTCAACAAAGAG(SEQ ID NO:7),Rv:gttaattaagctgcgTTAATGATGGTGGTGATGATG CGATG(SEQ ID NO:8))，通过PCR反应进行扩增。PCR反应物均通过琼脂糖凝胶电泳并切取谱带进行纯化。使用In-Fusion HD克隆试剂盒(TaKaRa Bio)，按照说明书的用法容量，对纯化的载体与插入物的连接反应实施克隆。
另外，以缺损SEQ ID NO:3所记载的氨基酸序列的C末端氨基酸序列PSAASHHHHHH、且表达具有SEQ ID NO:9所示的氨基酸序列的MFL2-G5S×3-del5的方式，进行MFL2-G5S×3-His表达载体的缺失突变体的制作(SEQ ID NO:10)。具体而言，使用引物组(Fw：AAGTCAAATAACGCAGCTTAATTAACCTAG(SEQ ID NO:11)、Rw：TGCGTTATTTGACTTTGGTAAAGGTGTCATG(SEQ ID NO:12))，以表达载体为模板进行PCR反应。之后，在37℃下进行1小时的反应液的DpnI处理，在该反应液中进行大肠杆菌的转化，进行克隆。克隆后，进行序列分析，确认插入了SEQ ID NO:10所记载的基因序列，将载体命名为pET45-MFL2-G5S×3-del5。
将确认插入了SEQ ID NO:4和10所记载的基因序列的质粒载体(pET45-MFL2-G5S×3-His、pET45-MFL2-G5S×3-del5)引入到感受态细胞BL21(DE3)(ECOS感受态大肠杆菌BL21(DE3)、Nippon Gene公司)中进行转化。将在100mL的2×YT培养基中培养了一夜的培养液接种在1升的培养液中，在37℃下进行培养，在600nm的OD值达到0.5-0.8的时间点添加IPTG使最终浓度为0.5mM，在37℃下进行4小时的培养后，通过离心(7500×g、20分钟、4℃)回收菌体。
关于由菌体回收IB的方法如下所示。在B-PER(Thermo SCIENTIFIC公司)中添加Benzonase(Merck)，悬浮后，在室温下培育10分钟，通过离心来分离不溶性组分(以下，将内含体(inclusion body)记作IB)和可溶性组分，回收IB。接下来，将已回收的IB在10倍稀释的B-PER缓冲液(未添加Benzonase)中重新悬浮，离心后，废弃上清，重复进行3次IB的洗涤，洗涤3次后，通过离心而回收的IB用超纯水(MilliQ)重新悬浮，向1.5mL管中分别注入1mL，在-80℃下冷冻保存。
关于IB的改性和开卷如下所示。向IB中添加改性缓冲液(denature buffer，变性缓冲液)(0.1M的Tris-HCl、pH8.5、6M的盐酸胍、10mM的EDTA)，通过移液使其溶解，边用转子搅拌边在4℃下培育一夜，进行离心(15,000×g、20分钟、4℃)，回收上清。用改性缓冲液进行调制使回收的上清的蛋白浓度(OD280nm)为30-50mg/mL，边搅拌再折叠缓冲液(0.05M的磷酸钠、0.4M的精氨酸盐酸盐、pH6.8)边滴加浓度调制后的改性蛋白溶液，其用量为约50倍稀释。之后，在4℃下培育24-48小时，进行离心(15,000×g、20分钟、4℃)，回收上清。对于回收的上清，通过SDS-PAGE电泳，根据CBB染色确认4聚体的形成。其结果，如图1A所示，确认到：与MFL2-G5S×3-His相比，MFL2-G5S×3-del5的大部分以1聚体的形式存在。关于MFL2-G5S×3-His的浓缩结果，如图1B所示，确认获取了MFL2-G5S×3-His。
<MFL2-G5S×3-His的性能评价>
关于MFL2-G5S×3-His，使用SPR(BiacoreT200,Cytiva)，对与HER2(ErbB2)的结合活性进行分析。通过胺偶联法(胺偶联试剂盒,Cytiva)在传感器芯片CM5(Cytiva)上进行Her2抗原(重组人ErbB2/Her2Fc嵌合蛋白,R&D SYSTEMS)的固定。接下来，使用FL的稀释系列(1E-08M～6.25E-10M的2倍稀释系列)实施结合活性的测定。实施结果见图2。由结果确认到：显示稳定的结合。分析的结果，ka＝8.509E+6、kd＝0.006598、KD＝7.753E-10。
关于MFL2-G5S×3-His，使用SPR(BiacoreT200,Cytiva)，对与Psyche的结合活性进行分析。利用胺偶联法将MFL2-G5S×3-His固定在传感器芯片CM5(Cytiva)上，利用单循环动力学法实施与以生物素-SiPc(上述制造例X中制造的化合物Y)稀释系列(1E-08M～6.25E-10M的2倍稀释系列、5个系列)为分析物的结合活性的分析。实施结果见图3。由结果确认到：显示稳定的结合。分析的结果，ka＝1.160E+6、kd＝0.002770、KD＝2.387E-9。
<使用MFL2-G5S×3-His和光敏剂Psyche的细胞毒活性>
在96孔板上接种Her2阳性细胞(KPL-4)使细胞数为1×104个细胞/孔、培养液为50μL/孔，培养一夜。混合已纯化的MFL2-G5S×3-His和生物素-SiPc(制造例1中制造的物质)使摩尔比为1:4(以下，表示为MFL2-G5S×3-His复合物)，制作了10μg/mL～10倍稀释系列的8个系列(包括零浓度)。调整稀释系列后，在细胞中添加复合物，24小时后使用发射690nm的光的LED从培养板底面进行光照射使达到100J/cm2。之后，培养24小时，去除含有MFL2-G5S×3-His复合物的培养基后，用PBS洗涤细胞1次，添加新的培养基，使用细胞计数试剂盒-8(同仁化学公司)进行活细胞数的比较。用法用量按照操作说明书添加试剂，在37℃、CO2培养箱中培养1.5小时，之后测定450nm的吸光度，计算平均值，在背景校正后，以对照为100％，计算各条件下相对于对照的细胞生长的比例。实施结果见图4。确认到：FL与光敏剂生物素-SiPc化合物的复合物具有光照射依赖性和浓度依赖性的细胞毒性。
<MFL2-G4Sx1-PSAAS、MFL2-G4Sx2-PSAAS、MFL2-G4Sx3-PSAAS的表达/纯化和性能评价>
接下来，进行蛋白表达载体的构建，其中，MFL2-G5S×3-del5中的连接识别HER2抗原的分子与Cupid-del5的氨基酸接头的长度不同、且C末端添加有5个氨基酸PSAAS。具体而言，如图5A所示，以4个甘氨酸和1个丝氨酸组合而成的肽为基本单位，制作了具有1个单位、2个单位、3个单位的长度的接头、且C末端为PSAAS的表达载体。制作方法如下。
通过人工基因合成制作了编码具有SEQ ID NO:11～13所记载的各接头的长度和C末端的氨基酸序列的基因序列(SEQ ID NO:14-16)。接下来，使用引物组(Rv:catggtatatctccttcttaaagttaaac(SEQ ID NO:5)、Fw:cgcagcttaattaacctaggctgctgccac(SEQ ID NO:6))，通过PCR反应进行pET45b载体的直链化。通过人工基因合成而调制的序列，利用引物组(Fw:aggagatataccatgGTGGACAACAAATTCAACAAAGAG(SEQ ID NO:7)、Rv:gttaattaagctgcgTTAATGATGGTGGTGATGATGCGATG(SEQ ID NO:8))，经PCR反应进行扩增。PCR反应物均通过琼脂糖凝胶电泳并切取谱带进行纯化，使用In-Fusion HD克隆试剂盒(TaKaRaBio)，按照说明书的用法容量对载体与插入物的连接反应实施克隆。进行序列分析，将确认插入了SEQ ID NO:14-16所记载的基因序列的表达载体分别命名为pET45-MFL2-G4Sx1-PSAAS、pET45-MFL2-G4Sx2-PSAAS、pET45-MFL2-G4Sx3-PSAAS。
<IB的回收、改性和开卷纯化>
将pET45-MFL2-G4Sx1-PSAAS、pET45-MFL2-G4Sx2-PSAAS、pET45-MFL2-G4Sx3-PSAAS引入到感受态细胞BL21(DE3)(ECOS感受态大肠杆菌BL21(DE3),Nippon Gene公司)中，进行转化。将用100mL的2×YT培养基培养了一夜的培养液接种在1升的培养液中，在37℃下进行培养，在600nm的OD值达到0.5-0.8的时间点添加IPTG使最终浓度为0.5mM，在37℃下进行4小时的培养后，通过离心(7500×g、20分钟、4℃)回收菌体。
关于由菌体回收IB的方法如下所示。向B-PER(Thermo SCIENTIFIC公司)中添加Benzonase(Merck)，悬浮后，在室温下培育10分钟，通过离心来分离不溶性组分(以下，将内含体(inclusion body)记作IB)和可溶性组分，回收IB。接下来，将已回收的IB在10倍稀释的B-PER缓冲液(未添加Benzonase)中重新悬浮，离心后，废弃上清，重复进行3次IB的洗涤，洗涤3次后，通过离心而回收的IB用超纯水(MilliQ)重新悬浮，向1.5mL管中分别注入1mL，在-80℃下冷冻保存。
关于IB的改性如下所示。在IB中添加改性缓冲液(0.1M的Tris-HCl、pH8.5、6M的盐酸胍、10mM的EDTA)，通过移液使其溶解，边用转子搅拌边在4℃下培育一夜，进行离心(15,000×g、20分钟、4℃)，回收上清。用改性缓冲液调制使回收的上清的蛋白浓度(OD280nm)为30-50mg/mL。
关于开卷如下所示。再折叠缓冲液是在McIlvaine缓冲液中添加0.4M的精氨酸盐酸盐，用NaOH调制成pH4.0、pH4.5、pH5.0、pH6.5、pH7.5、pH8.0等各pH值。相对于1mL各pH的再折叠缓冲液，添加25μL的改性溶液上清，通过稀释进行开卷。样品在4℃下培育，通过SDS-PAGE电泳来确认4聚体的形成。
关于MFL2-G4Sx2-PSAAS、MFL2-G4Sx3-PSAAS，在各pH下开卷1小时后、24小时后的4聚体形成确认结果见图5B。由该结果确认到：在pH5.0-pH6.5下促进由单体形成4聚体。然而，72小时后确认到在所有pH下4聚体的谱带均消失。
同样，如图5C所示，对于MFL2-G4Sx1-PSAAS、MFL2-G4Sx2-PSAAS、MFL2-G4Sx3-PSAAS，即使是pH6.0下的开卷多物，72小时后也无法确认到形成稳定的四聚体。
SEQ ID NO:1LISA314的氨基酸序列
AEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLT
GRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGG
ADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*；
SEQ ID NO:2识别HER2抗原的分子
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLL AEAKKLNDAQAPK；
SEQ ID NO:3MFL2-G5S×3-His的氨基酸序列
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANL
LAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSN
QLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDG
SGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLT
SGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*；
SEQ ID NO:4MFL2-G4Sx3-His的基因序列
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACT
GGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACG
TGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGA
ATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCC
GAAAAGCGGAGGCGGCGGCGGGTCTGGTGGTGGCGGTGGCAG
TGGCGGAGGTGGCGGTTCAGCGGAAGCCGGTATTACCGGGACC
TGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGC
GGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAAT
GCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCC
GGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCG
TGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGT
CAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCA
GTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAA
TCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGA
GTGCAGCATCGCATCATCACCACCATCATTAA；
SEQ ID NO:9MFL2-G5S×3-del5的氨基酸序列
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANL
LAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSN
QLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDG
SGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLT
SGTTNANAWKSTLVGHDTFTKVK；
SEQ ID NO:10MFL2-G5S×3-del5的基因序列
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACT
GGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACG
TGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGA
ATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCC
GAAAAGCGGAGGCGGCGGCGGGTCTGGTGGTGGCGGTGGCAG
TGGCGGAGGTGGCGGTTCAGCGGAAGCCGGTATTACCGGGACC
TGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGC
GGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAAT
GCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCC
GGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCG
TGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGT
CAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCA
GTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAA
TCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAA；
SEQ ID NO:11MFL2-G4Sx1-PSAAS的氨基酸序列
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANL
LAEAKKLNDAQAPKGGGGSAEAGITGTWSNQLGSTFIVTAGADG
ALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWK
NNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKST
LVGHDTFTKVKPSAAS*；
SEQ ID NO:12MFL2-G4Sx2-PSAAS的氨基酸序列
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANL
LAEAKKLNDAQAPKGGGGSGGGGSAEAGITGTWSNQLGSTFIVTA
GADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTV
AWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANA
WKSTLVGHDTFTKVKPSAAS*；
SEQ ID NO:13MFL2-G4Sx3-PSAAS的氨基酸序列
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANL
LAEAKKLNDAQAPKGGGGSGGGGSGGGGSAEAGITGTWSNQLGS
TFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTA
LGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTT
NANAWKSTLVGHDTFTKVKPSAAS*；
SEQ ID NO:14FL2-G4Sx1-PSAAS的基因序列
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACT
GGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACG
TGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGA
ATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCC
GAAAGGAGGCGGAGGGTCTGCGGAAGCCGGTATTACCGGGAC
CTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCG
CGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAA
TGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTC
CGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGC
GTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGG
TCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTC
AGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAA
ATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCG
AGTGCAGCATCGTAA；
SEQ ID NO:15MFL2-G4Sx2-PSAAS的基因序列
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACT
GGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACG
TGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGA
ATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCC
GAAAGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGCGGAAGC
CGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTA
TCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGA
GTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGT
CGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTT
AGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCAC
AGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATG
CGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAA
TGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGTAA；
SEQ ID NO:16MFL2-G4Sx3-PSAAS的基因序列ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGGTGGCGGTGGCAGTGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGTAA；
SEQ ID NO:17
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr
Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu
Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr
Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr
Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr TrpSerGly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp
Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu
Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser。
Claims (14)
1.融合蛋白，其是分子量为20,000以下的抗原结合分子经由接头序列与链霉亲和素突变体的N末端侧和/或C末端侧结合而成，该链霉亲和素突变体在SEQ ID NO:17所记载的核心链霉亲和素的氨基酸序列(其中，C末端的由Pro-Ser-Ala-Ala-Ser构成的氨基酸序列可缺失一部分或全部)中包含具有下述(1)～(6)的突变的氨基酸序列、且与野生型链霉亲和素相比免疫原性降低，
(1)第10位的氨基酸残基的酪氨酸取代成了丝氨酸或苏氨酸的突变；
(2)第71位的氨基酸残基的酪氨酸取代成了丙氨酸或丝氨酸的突变；
(3)第72位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(4)第89位的氨基酸残基的谷氨酸取代成了天冬氨酸的突变；
(5)第91位的氨基酸残基的精氨酸取代成了赖氨酸的突变；
(6)第104位的氨基酸残基的谷氨酸取代成了谷氨酰胺或天冬酰胺的突变。
2.权利要求1所述的融合蛋白，其从N末端侧到C末端侧依次具有分子量为20,000以下的抗原结合分子、接头序列和上述链霉亲和素突变体的氨基酸序列。
3.权利要求1或2所述的融合蛋白，其中，抗原结合分子是与在癌细胞中表达的抗原结合的分子。
4.权利要求1～3中任一项所述的融合蛋白，其中，抗原结合分子是与Her2结合的分子。
5.权利要求1～4中任一项所述的融合蛋白，其中，抗原结合分子具有SEQ ID NO:2所记载的氨基酸序列。
6.权利要求1～5中任一项所述的融合蛋白，其中，接头序列由甘氨酸残基和丝氨酸残基构成，氨基酸残基的数量为5～25个。
7.权利要求1～6中任一项所述的融合蛋白，其中，接头序列是[(Gly)m-Ser]n所示的氨基酸序列，式中，m表示1～10的整数，n表示1～5的整数。
8.权利要求1～7中任一项所述的融合蛋白，其具有SEQ ID NO:3或9所记载的氨基酸序列。
9.核酸，其编码具有SEQ ID NO:3或9所记载的氨基酸序列的融合蛋白。
10.癌症治疗药或癌症诊断药，其包含权利要求1～8中任一项所述的融合蛋白。
11.癌症的治疗或诊断试剂盒，其包含：(1)权利要求1～8中任一项所述的融合蛋白；以及(2)生物素与诊断用物质或治疗用物质的缀合物。
12.权利要求11所述的试剂盒，其中，诊断用物质或治疗用物质为酞菁染料。
13.权利要求1～8中任一项所述的融合蛋白的制造方法，该制造方法包括：使编码权利要求1～8中任一项所述的融合蛋白的核酸在宿主中表达的步骤。
14.权利要求13所述的方法，其中，使上述融合蛋白在细菌的封入体内表达并回收。
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