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CN112426438B - 用于调控酸性环境免疫应答的组合物、其制备方法和用途 - Google Patents

用于调控酸性环境免疫应答的组合物、其制备方法和用途 Download PDF

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CN112426438B
CN112426438B CN202011276641.1A CN202011276641A CN112426438B CN 112426438 B CN112426438 B CN 112426438B CN 202011276641 A CN202011276641 A CN 202011276641A CN 112426438 B CN112426438 B CN 112426438B
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徐建青
张晓燕
廖启彬
丁相卿
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Abstract

本发明提供了一种用于调控酸性环境免疫应答的组合物，所述组合物由酸性环境靶向分子和免疫细胞组成，所述酸性环境靶向分子为低pH插入肽(low pH insertion peptide，pHLIP)或其变体的偶联分子或融合蛋白，所述免疫细胞为内源性或外源性免疫细胞。本发明还提供了本发明的组合物的制备方法和用途。本发明的组合物通过调控酸性环境免疫应答，特别是肿瘤酸性环境的免疫应答，可以用于治疗实体肿瘤。

Description

用于调控酸性环境免疫应答的组合物、其制备方法和用途
技术领域
本发明属于生物医药技术领域，涉及一种用于调控酸性环境免疫应答的组合物，本发明还涉及所述组合物的制备方法和用途。
背景技术
肿瘤的免疫治疗涉及单克隆抗体、疫苗、基因治疗、免疫细胞治疗等，其中靶向肿瘤抗原的单克隆抗体、免疫检查点抑制剂、T细胞受体修饰T细胞(TCR-T)、嵌合抗原受体T细胞(CAR-T)等免疫治疗方法，在临床研究中显示出令人瞩目的抗肿瘤效果。然而，由于肿瘤特异性抗原缺乏而带来的脱靶效应、特别是实体瘤的肿瘤异质性以及肿瘤抑制性微环境对免疫细胞浸润与功能的负向调控等，严重制约了免疫治疗的效果，并引起生物医药技术领域对该种新型肿瘤干预技术的安全性的担忧。
由于肿瘤微环境异常的血管结构以及肿瘤细胞的过度增殖，使得肿瘤组织的代谢更多地依赖于无氧糖酵解，其代谢产物乳酸与呼吸作用产生的CO2共同造成了肿瘤微环境的酸化，肿瘤细胞外pH(6.5-6.8)显著低于外周血或正常组织的pH(7.4)，酸性环境是多种实体肿瘤的一个共有特征。研究表明，肿瘤的酸性环境可以调控多种生物过程，包括细胞增殖、血管新生、免疫抑制、侵袭和耐药等，甚至促使高侵袭表型的肿瘤形成，肿瘤恶性程度更高。因此，靶向肿瘤的酸性环境是一种全新的诊断和治疗方法，其不依赖于特定的肿瘤抗原，不受限于肿瘤异质性，既可以用于预测肿瘤的恶性程度，又可以递送治疗性药物至多种肿瘤组织从而达到治疗的目的。
低pH插入肽(pH low insertion peptide，pHLIP)是来源于细菌的视紫红质跨膜螺旋蛋白C的一种水溶性多肽，能够插入细胞双层脂膜并形成稳定的跨膜α螺旋。pHLIP具有三种主要形态，中性或碱性pH(pH＞7)下形成溶于水的无结构形态I，或粘附到细胞膜表面的无结构形态II，于酸性pH(pH＜7)下插入细胞膜并形成跨膜α螺旋的形态III。传统的细胞穿透肽穿过细胞膜后则直接进入细胞质中。与此截然不同，pHLIP和脂质双分子层之间不存在特异性的相互作用，仅仅受到所处环境下的pH影响即在酸性环境下插入细胞膜，其N端位于细胞膜外而C端则进入细胞质中。因此，pHLIP具有强大的传递能力：通过其N端连接货物分子并系于细胞膜表面，进而通过其C端将不具穿膜能力的货物分子靶向到细胞质中。由于pHLIP所具有的这种特殊的理化性质，它被广泛应用于以下领域：(1)通过荧光染料、成像探针等标记pHLIP并递送至酸性组织，用于疾病诊断或荧光图像导航手术；(2)通过放射性核素标记pHLIP递送至酸性组织，进行核成像(PET/SPECT)；(3)与毒素、药物、治疗性基因偶联以治疗疾病；(4)特异递送纳米材料至酸化组织。
但是，现有技术中并没有pHLIP偶联荧光分子或连接生物活性的细胞因子或趋化因子或各种抗原联合免疫细胞治疗具有酸性特征疾病的相关报道。具体地，关于pHLIP在以下方面的潜在用途尚未见报道：(1)pHLIP连接生物活性的细胞因子或趋化因子或偶联免疫调节药物联合内源性或外源性免疫细胞，特异靶向至酸性环境以调节免疫应答；(2)pHLIP连接肿瘤相关抗原、细菌抗原、病毒抗原等多肽抗原，联合基因工程免疫细胞(T细胞、NK细胞、NKT细胞、巨噬细胞等)的免疫增强型疗法；(3)磁性纳米材料标记pHLIP，递送至酸性组织进行磁共振成像(MRI)。
也就是说，当前对更先进的pHLIP的组合物存在需求。
发明内容
基于此，本发明的目的是提供一种用于调控酸性环境免疫应答的组合物，其包括酸性环境靶向分子和免疫细胞。本发明还提供了所述组合物的制备方法及其用途。
一方面，本发明提供了一种组合物，所述组合物由酸性环境靶向分子和免疫细胞组成，所述酸性环境靶向分子为低pH插入肽(low pH insertion peptide，pHLIP)或其变体的偶联分子或融合蛋白，所述免疫细胞为内源性或外源性免疫细胞；
其中，所述pHLIP或其变体的偶联分子或融合蛋白通过pHLIP或其变体的N端与细胞因子、荧光分子、免疫调节药物、磁共振成像造影剂、磁性纳米材料、肿瘤相关抗原、细菌抗原、病毒抗原、真菌抗原、植物抗原、多肽抗原或抗体Fc片段连接获得。
根据本发明所述的组合物，其中，所述低pH插入肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
优选地，所述低pH插入肽的变体的氨基酸序列如SEQ ID NO:3-SEQ ID NO:18中任一项所示；
优选地，所述pHLIP或其变体的融合蛋白如SEQ ID NO:19-SEQ ID NO:25中任一项所示。
具体地，所述低pHLIP及其变体包括以下多肽：
WT-1(SEQ ID NO:1)；WT-2(SEQ ID NO:2)；Var1(SEQ ID NO:3)；Var2(SEQ ID NO:4)；Var3(SEQ ID NO:5)；Var4(SEQ ID NO:6)；Var5(SEQ ID NO:7)；Var6(SEQ ID NO:8)；Var7(SEQ ID NO:9)；Var8(SEQ ID NO:10)；Var9(SEQ ID NO:11)；Var10(SEQ ID NO:12)；Var11(SEQ ID NO:13)；Var12(SEQ ID NO:14)；Var13(SEQ ID NO:15)；Var14(SEQ ID NO:16)；Var15(SEQ ID NO:17)；Var16(SEQ ID NO:18)。
根据本发明所述的组合物，其中，所述免疫细胞为内源性或外源性免疫细胞，包括但不限于自体免疫细胞、异体免疫细胞、自体基因工程免疫细胞、异体基因工程免疫细胞。
优选地，所述免疫细胞包括但不限于NK细胞、NKT细胞、CD16/64嵌合受体(CD16/64chimeric receptor，CD16/64CR)修饰的NK/NKT/T细胞、嵌合抗原受体(chimericantigen receptor，CAR)修饰的NK/NKT/T细胞；
更优选地，所述免疫细胞为CD16/64CR-T细胞和CAR-T细胞。
根据本发明所述的组合物，其中所述的细胞因子包括但不限于白细胞介素、干扰素、肿瘤坏死因子、集落刺激因子、趋化因子和生长因子。其中，所述白细胞介素包括但不限于IL-2、IL-7、IL-10、IL-12、IL-15和IL-21；所述干扰素包括但不限于IFN-α、IFN-β、IFN-γ、IFN-κ和IFN-λ；其中，所述肿瘤坏死因子包括但不限于TNF-α和TNF-β；其中，所述集落刺激因子包括但不限于SCF、Flt3L、G-CSF、M-CSF和GM-CSF；其中，所述趋化因子包括但不限于CCL2、CCL5、CCL19、CCL21、CXCL8、CXCL9、CXCL10、CXCL11、XCL1和CX3CL1；其中，所述生长因子包括但不限于TGF-β、EGF、FGF、NGF、PDGF和VEGF。
根据本发明所述的组合物，其中，所述免疫调节药物包括免疫增强药物和免疫抑制药物。其中，所述免疫抑制药物包括但不限于环孢素、他克莫司和硫唑嘌呤；所述免疫增强药物包括但不限于胸腺素、转移因子和重组人干扰素。
根据本发明所述的组合物，其中，所述肿瘤相关抗原包括但不限于CD19、CD20、CD22、CD30、CD33、ROR1、ROR2、CD38、CD123、CD133、NKG2D配体、ERBB2、MUC1、CD44v6、CD44v7、CD44v8、CEA、EpCAM、TAG72、KIT、IL-13Rα2、EGFR、EGFRvIII、GD2、GD3、HMW-MAA、MAGE1、MAGEA3、CD171、NCAM、IL-11Rα、FRα、PSCA、PSMA、TARP、CAIX、VEGFR、BCMA、CTLA-4、PD-L1、PD-L12、GPC3、CD47、AXL、FAP、α5β1、αvβ3、TGFβR、ER、PR、P53、IGFR、CD25、CD117、CD34、CD138、BCMA、Mesothelin、S100、CD70、ALK、RANK和HER3。
根据本发明所述的组合物，其中，所述细菌抗原包括但不限于葡萄球菌抗原、链球菌抗原、肺炎链球菌抗原、脑膜炎奈瑟菌抗原和淋病奈瑟菌抗原。
根据本发明所述的组合物，其中，所述病毒抗原包括但不限于巨细胞病毒抗原、巴尔病毒抗原、肝炎病毒抗原、单纯疱疹病毒抗原、HIV抗原、人T-淋巴细胞病毒抗原、风疹病毒抗原、SARS冠状病毒抗原、水痘-带状疱疹病毒、狂犬病毒抗原、流感病毒抗原、轮状病毒抗原和人乳头瘤病毒抗原。
根据本发明所述的组合物，其中，所述真菌抗原包括但不限于皮肤癣真菌抗原、新生隐球菌抗原和白假丝念珠菌抗原。
根据本发明所述的组合物，其中，所述多肽抗原包括但不限于AviTag(SEQ ID NO:26)、Calmodulin tag(SEQ ID NO:27)、polyglutamate tag(SEQ ID NO:28)、E-tag(SEQ IDNO:29)、FLAG tag(SEQ ID NO:30)、HA-tag(SEQ ID NO:31)、His-tag(SEQ ID NO:32)、Myc-tag(SEQ ID NO:33)、S-tag(SEQ ID NO:34)、SBP-tag(SEQ ID NO:35)、Sof-tag 1(SEQ IDNO:36)、Softag3(SEQ ID NO:37)、Strep-tag(SEQ ID NO:38)、TC tag(SEQ ID NO:39)、V5tag(SEQ ID NO:40)、T7 tag(SEQ ID NO:41)、VSV tag(SEQ ID NO:42)、Xpress tag(SEQID NO:43)、3X FLAG tag(SEQ ID NO:44)、Isopeptag(SEQ ID NO:45)、Spytag(SEQ ID NO:46)、Snooptag(SEQ ID NO:47)和PNE tag(SEQ ID NO:48)。
根据本发明所述的组合物，其中，所述荧光分子包括但不限于FITC、FAM、PE、APC、PB、Cy3、Cy5、Texas Red、TRITC、GFP、RFP、CFP和BFP。
根据本发明所述的组合物，其中，所述抗体Fc片段包括但不限于IgG1的Fc片段、IgG2的Fc片段、IgG3的Fc片段和IgG4的Fc片段。
更优选地，所述磁性纳米材料包括但不限于钆喷酸葡胺(Gd-DTPA)、镝喷酸葡胺(Dy-DTPA)、锰福地吡三钠(Mn-DPDP)为代表的顺磁性金属离子螯合物和Fe2O3、Fe3O4为核心的超顺磁性金属氧化物粒子。
根据本发明所述的组合物，其中，所述酸性环境靶向分子选自FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP、Fc-pHLIP或CCL19-pHLIP；所述免疫细胞为FITC CAR-T细胞、CAR-T细胞、CD16/64CR-T细胞、T细胞。
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白CCL19-pHLIP和T细胞，CCL19-pHLIP的氨基酸序列如SEQ ID NO:49所示。具体地，趋化因子CCL19通过GS柔性连接子氨基酸序列(GSSGGSGGSGGSG)连接至野生型pHLIP的N端，中间以蛋白酶切割序列(LSGRSDNH)间隔，以便在肿瘤微环境内切割并释放。另外，组氨酸标签序列(HHHHHH)则用于体外检测和纯化CCL19-pHLIP。
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP偶联分子FITC-pHLIP和FITC CAR-T细胞。具体地，所述pHLIP为野生型pHLIP，氨基酸序列如SEQ ID NO:1所示。所述偶联分子为荧光分子异硫氰酸荧光素FITC。其中，FITC通过氨基己酸(ACP)与pHLIP偶联获得FITC-pHLIP，结构式为：FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT。
其中，所述组合物的免疫细胞为靶向FITC的FITC CAR-T细胞，FITC CAR序列如SEQID NO:50所示。
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白FLAG-pHLIP和CAR-T细胞。
具体地，pHLIP的氨基酸序列如SEQ ID NO:1所示，所述融合蛋白为多肽抗原FLAGtag，FLAG tag连接至pHLIP的N端获得，序列如SEQ ID NO:51所示。或者，多肽抗原为PNEtag，PNE tag连接至pHLIP的N端获得PNE-pHLIP，序列如SEQ ID NO:52所示。所述免疫细胞分别为靶向FLAG tag或PNE tag的FLAG CAR-T细胞或PNE CAR-T细胞，其中，所述PNE CAR的氨基酸序列如SEQ ID NO:53。
根据本发明的一个实施方案，所述调控酸性环境免疫应答的组合物为酸性环境靶向分子pHLIP融合蛋白和CD16/64CR-T细胞。所述融合蛋白为pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc，其序列如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示。所述CD16/64CR-T细胞为靶向IgG1的Fc片段的CD16 CR-T细胞，CD16 CR的序列如SEQ ID NO:54所示。
另一方面，本发明还提供了本发明的组合物制备用于诊断和/或治疗肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病的药物的用途。
再一方面，本发明还提供了用于诊断和/或治疗各种疾病的方法，所述方法包括给予有需要的患者治疗有效量的组合物。
其中，所述疾病选自肿瘤、自身免疫性疾病、伴有缺血缺氧的疾病和/或炎性疾病等。
虽然本发明仅仅以SEQ ID NO:1所示的pHLIP进行了说明，但由于pHLIP及其变体理化性质的相似性，所以本领域技术人员可以毫无疑义地地确认，所有的pHLIP及其变体均可以应用于本发明的技术方案并获得相同的效果。
具体地，发明人发现，本发明的组合物具有以下的用途：(1)所述组合物中的融合蛋白为具有生物活性的细胞因子时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，加强或抑制免疫应答；(2)所述组合物中的融合蛋白为具有生物活性的趋化因子时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，招募免疫细胞，加强或抑制免疫应答；(3)所述组合物中的偶联分子为免疫调节药物时，联合内源性或外源性免疫细胞，将其特异靶向至酸性环境，发挥局部免疫增强或免疫抑制的作用；(4)所述组合物中的融合蛋白为肿瘤相关抗原、细菌抗原、病毒抗原、荧光分子等抗原，联合基因工程免疫细胞(T细胞、NK细胞、NKT细胞、巨噬细胞等)，将其特异靶向至肿瘤酸性环境，达到广谱抗肿瘤的目的；(5)所述组合物中的偶联分子为磁性纳米材料时，递送至酸性组织进行磁共振成像(MRI)。
再另一方面，本发明还提供了所述的调控酸性环境免疫应答的组合物的制备方法，所述方法包括以下步骤：
利用多肽自动合成仪，通过固相法合成上述组合物；或
用生物合成法，通过工程菌和细胞表达并纯化上述组合物。
与现有技术相比，本发明具有以下优点和有益效果:
本发明的组合物可以偶联或连接肿瘤相关抗原、病原体来源的抗原、荧光分子、多肽抗原等抗原，通过联合内源性或外源性免疫细胞调控酸性环境的免疫应答，开发出通用型的创新细胞免疫疗法，有望治疗几乎所有的实体肿瘤。此外，除了实体肿瘤，细胞外酸化还与许多病理状态相关，包括缺血性中风、神经损伤、感染、组织撕裂受损等多种病理状态。细胞在损伤或病变组织中的异常代谢活动常常造成细胞外环境的酸化，所以胞外酸化有望成为多种疾病的诊断和治疗通用标志物，利用pHLIP可靶向酸性环境的特征，亦可用于上述病理状态的诊断和治疗。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示利用流式细胞仪检测本发明的酸性环境靶向分子在不同pH条件的微环境下，在肿瘤细胞(A549)上的表达情况，其中，所述偶联分子或融合蛋白分别为FITC(异硫氰酸荧光素)、FLAG标签多肽和PNE多肽(来源于酵母转录因子GCN4的14个氨基酸多肽)；
图2显示利用流式细胞仪检测本发明的酸性环境靶向分子在不同pH条件的微环境下，在肿瘤细胞(A549)上的表达情况，其中，所述融合蛋白为免疫细胞募集分子，分别为抗体可结晶片段(Fc)、携带三聚化foldon序列的抗体可结晶片段(foldon-Fc)和携带三聚化p24序列的可结晶片段(p24-Fc)；
图3显示本发明的酸性环境靶向分子pHLIP融合蛋白CCL19-pHLIP趋化T细胞的效果；
图4显示利用免疫组化/荧光分析系统TissueFAXS观察酸性环境靶向分子FITC-pHLIP在肿瘤组织和其它脏器上的定位，白色三角指示处为锚定在肿瘤细胞上的FITC-pHLIP；
图5显示酸性环境下本发明的组合物FITC-pHLIP联合FITC CAR-T细胞特异杀伤肿瘤细胞，其中A549为肺腺癌细胞(肺泡基底上皮细胞)，NCI-H292为肺癌细胞(淋巴结转移)；
图6显示本发明的组合物FITC-pHLIP联合FITC CAR-T细胞控制荷瘤小鼠的肿瘤生长曲线。
具体实施方式
实施例1 本发明组合物的酸性环境靶向分子在不同pH条件的微环境下，在肿瘤细 胞(A549)上的表达
细胞培养：A549细胞(美国模式培养物集存库)使用RPMI1640完全培养基培养(简写为R10)培养，R10为RPMI1640培养基(Corning)含有10％(体积比)胎牛血清(BiologicalIndustries)、1X链霉素/青霉素(100X链霉素/青霉素，Gibco)的完全培养基，在37℃，5％CO2的细胞培养箱中培养。细胞长满后按1:5传代。
pHLIP表达分析：A549细胞经过胰酶细胞消化液消化1～2分钟，采用10倍体积的R10培养基中和，500xg，离心5分钟。弃上清，采用R10培养基重悬制备A549单细胞悬液，各取1x105细胞置于3个1.5mL EP管中，800xg，离心3分钟，弃去培养基分别加入pH6.0、pH6.5和pH7.4的1xPBS，加入本发明的酸性环境靶向分子5μg/mL，分别为FITC-pHLIP(结构式为：
FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT)，FLAG-pHLIP(SEQ ID NO:51)和PNE-pHLIp(SEQ ID NO:52)(上海昕浩生物科技有限公司化学合成多肽)。室温孵育1小时，弃上清用相应pH的1xPBS洗涤3次，加入pH7.4的1xPBS重悬细胞，采用流式细胞仪检测FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP在A549细胞吸附水平。所检测细胞样本共分为6组：(1)pH6.0未处理组；(2)pH6.0+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP处理组；(3)pH6.5未处理组；(4)pH6.5+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP处理组；(5)pH7.4未处理组：(6)pH7.4+FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP。
结果：图1A、B、C显示，在中性溶液环境中(pH7.4)，FITC-pHLIP、FLAG-pHLIP、PNE-pHLIP插入A549的细胞膜的效率较低，分别为30％、30％和15％。在酸性溶液环境中，FITC-pHLIP高效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为85％和99％，显著高于pH7.4；本发明的FLAG-pHLIP多肽亦可高效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为73％和91％，显著高于pH7.4；本发明的PNE-pHLIP多肽亦能有效插入A549细胞膜，pH6.5和pH6.0的插入效率分别为57％和53％，显著高于pH7.4。
实施例2 本发明的酸性环境靶向分子在不同pH条件的微环境下，在肿瘤细胞 (A549)上的表达
2.1pHLIP-Fc、pHLIP-foldon-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的体外表达和纯化
2.1.1真核表达载体构建：将根据本发明的融合蛋白氨基酸序列(SEQID NO:19、SEQ ID NO:20和SEQ ID NO:21)设计DNA序列交付上海捷瑞生物工程有限公司进行全基因合成制备真核表达载体。
2.1.2pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的体外表达：隔天铺HEK293T细胞于15cm培养皿，每个培养皿铺1.5x107细胞，在37℃，5％CO2的细胞培养箱中培养过夜。第二天，采用转染试剂PEIpro以30μg/皿的DNA总量转染携带融合蛋白DNA序列的真核表达载体，6～8小时更换为无血清培养基，在37℃，5％CO2的细胞培养箱中继续培养5～7天。
2.1.3pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白的纯化：收集细胞培养上清，2000rpm，4℃，离心10分钟，弃去细胞沉淀。离心收集的培养上清采用Protein G琼脂糖柱子(GE公司，货号28-9031-34)进行纯化，纯化后的抗体用PBS缓冲液溶解。
2.2pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc的pHLIP融合蛋白在A549细胞上表达
细胞培养：A549细胞使用R10培养基，在37℃，5％CO2的细胞培养箱中培养。细胞长满后按1:5传代。
流式细胞仪观察表达：A549细胞经过胰酶细胞消化液消化1～2分钟，10倍体积的R10培养基中和，500xg，离心5分钟，R10培养基重悬，最终获得A549单细胞悬液。取1x105细胞置于3个1.5mL EP管中，800xg，离心3分钟，弃去培养基分别加入pH6.0和pH7.4的1xPBS，加入上述制备的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白(20μg/mL)，室温孵育1小时，弃上清用相应pH的1xPBS洗涤3次，加入相应pH的1xPBS重悬细胞，采用流式细胞仪检测pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白在A549细胞上的表达水平。分组：(1)pH6.0未处理组；(2)pH6.0+pHLIP-Fc组；(3)pH6.0+pHLIP-folden-Fc组；(4)pH6.0+pHLIP-p24-Fc组；(5)pH7.4未处理组：(6)pH7.4+pHLIP-Fc组；
(7)pH7.4+pHLIP-folden-Fc组；(8)pH7.4+pHLIP-p24-Fc组。
结果：图2显示，在中性溶液环境中(pH7.4)，本发明的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白插入A549的细胞膜的效率极低，低于4％。而在酸性溶液环境中，本发明的pHLIP-Fc、pHLIP-folden-Fc和pHLIP-p24-Fc融合蛋白均能插入A549细胞膜，pH6.0的插入效率分别为21％、19.1％和21.4％，均高于pH7.4。
实施例3酸性环境靶向分子CCL19-pHLIP高效趋化T细胞
3.1 CCL19-pHLIP融合蛋白的体外表达和鉴定
3.1.1真核表达载体构建
将根据本发明的融合蛋白氨基酸序列(SEQ ID NO:49)设计DNA序列交付上海捷瑞生物工程有限公司进行全基因合成制备真核表达载体。
3.1.2 CCL19-pHLIP融合蛋白的体外表达
HEK293T细胞(美国模式培养物集存库)使用DMEM完全培养基培养(简写为D10)，D10为DMEM培养基(Corning)含有10％(体积比)胎牛血清(Biological Industries)、1X链霉素/青霉素(100X链霉素/青霉素，Gibco)的完全培养基，在37℃，5％CO2的细胞培养箱中培养。细胞长满后按1:5传代。
HEK293T细胞经过胰酶细胞消化液消化1-2分钟，10倍体积的D10培养基中和，500xg，离心5分钟，DMEM完全培养基重悬，最终获得HEK293T单细胞悬液。将HEK293T细胞于六孔板中，每孔铺1x106细胞，在37℃，5％CO2的细胞培养箱中培养过夜。第二天，采用转染试剂Turbofect以4μg/孔的DNA总量转染携带融合蛋白DNA序列的真核表达载体，在37℃，5％CO2的细胞培养箱中继续培养2天，收集培养上清，2000rpm，4℃，离心10分钟，收集细胞沉淀用于WB检测，收集离心后的培养上清，冻存备用。
3.2T细胞趋化试验
取一块5μm孔径的Transwell培养板，下室加入上述离心收集的培养上清600μL，上室加入100μL的T细胞悬液，细胞密度为1x106/mL，在37℃，5％CO2的细胞培养箱中分别培养2、4或6小时取出计数下室细胞数目。
结果：图3A显示，携带CCL19-pHLIP融合蛋白DNA序列的真核表达载体在HEK293T细胞中高效表达。图3B显示，本发明的酸性环境靶向分子CCL19-pHLIP融合蛋白可以有效地趋化T细胞，趋化效率与阳性对照CCL19相当。
实施例4酸性环境靶向分子的FITC-pHLIP在体内肿瘤组织和脏器的分布
5x105个黑色素B16细胞接种于C57BL/6小鼠(购自上海史莱克实验动物有限责任公司)右侧腹部皮下，待肿瘤直径长至0.5-1cm时，静脉注射1mg/kg的FITC-pHLIP，分别于注射后4、12、24、48、72和96小时处死小鼠，取肿瘤组织、心脏、肝脏、脾脏、肺脏和肾脏制作冰冻切片，4％多聚甲醛固定后采用PE-anti-FITC(Abcam，ab25539)和DAPI染色，采用免疫组化/荧光分析系统TissueFAXS进行分析。
结果：图4显示，FITC-pHLIP于回输后至少6小时富集在肿瘤组织，一直持续到回输后24小时均可见明显的FITC-pHLIP定位在肿瘤细胞上。然而，FITC-pHLIP回输后4、12、24、48小时均不在心脏、肝脏、脾脏、肺脏和肾脏富集。
实施例5酸性环境靶向分子FITC-pHLIP联合抗FITC CAR-T细胞的体外抗肿瘤作用
5.1细胞培养和铺板
稳定表达荧光素酶的肺癌细胞A549-luciferase/NCI-H292-luciferase(上海鑫湾生物科技有限公司)使用含有10％胎牛血清、1x链霉素/青霉素的R10培养基，在37℃，5％CO2的细胞培养箱中培养。细胞长满后按1:5传代。
A549-luciferase/NCI-H292-luciferase细胞经过胰酶细胞消化液消化1～2分钟，10倍体积的R10培养基中和，500xg，离心5分钟，R10培养基重悬，最终获得A549-luciferase/NCI-H292-luciferase单细胞悬液。将A549-luciferase/NCI-H292-luciferase细胞铺在96孔黑底平板中，每孔1x104个细胞，总体积为100μL，在37℃，5％CO2的细胞培养箱中培养过夜。
5.2 FITC-pHLIP处理
A549-luciferase/NCI-H292-luciferase细胞在培养板上培养过夜，弃去培养上清分别加入pH6.5和pH6.0的R10培养基，加入实施例1的FITC-pHLIP(5μg/mL)，37℃孵育1小时，弃去上清用相应pH的1xPBS洗涤3次，加入pH6.5和pH6.0的R10培养基100μL。分组：pH6.5未处理组；(2)pH6.5+FITC-pHLIP处理组；(3)pH6.0未处理组；(4)pH6.0+FITC-pHLIP处理组。
5.3抗FITC CAR-T细胞处理
收集静息期的抗FITC CAR-T细胞，用R10培养基重悬至相应细胞浓度。以效应细胞:靶细胞为5:1的比例加入抗FITC CAR-T细胞至上述4个处理组，每孔加入100μL的抗FITCCAR-T细胞悬液。
结果：图5A显示，在酸性溶液环境中，FITC-pHLIP联合抗FITC CAR-T细胞可以特异杀伤肺腺癌细胞A549-luciferase。图5B显示，在酸性溶液环境中，FITC-pHLIP联合抗FITCCAR-T细胞可以特异杀伤高转移肺癌细胞NCI-H292-luciferase。
实施例6 酸性环境靶向分子FITC-pHLIP联合抗FITC CAR-T细胞的体内抗肿瘤效 果评价
将NCI-H292肺癌细胞(上海鑫湾生物科技有限公司)接种于B-NDG免疫缺陷小鼠(购自北京百奥赛图基因生物技术有限公司)的右侧腹部皮下，2x106个细胞/只，待肿瘤直径长至0.5-1cm，剔除过大和过小的肿瘤，将肿瘤大小基本一致的小鼠随机分组。共分为3组：PBS单独注射组，5只；抗FITC CAR-T细胞单独注射组，5只；FITC-pHLIP联合抗FITC CAR-T细胞注射组，5只。每隔3天测量肿瘤大小。
给药方法：FITC-pHLIP给药：每次每只小鼠静脉注射20μg(125μL)，从分组完成后当天开始注射，一天注射一次，每隔一天注射一次，连续给药10次。抗FITC CAR-T细胞给药：每次每只小鼠静脉回输抗FITC CAR-T细胞107个/125μL，总共2次。从分组完成后当天开始回输第一次，注射时间为第一次给FITC-pHLIP后12小时，九天后进行第二次细胞回输。
结果：图6显示，FITC-pHLIP联合抗FITC CAR-T细胞显著抑制肺癌荷瘤小鼠的肿瘤生长，PBS和单独抗FITC CAR-T细胞均不能控制荷瘤小鼠的肿瘤生长。
虽然以上仅描述了本发明的具体实施方式的范例，但本领域的技术人员应当理解，以上这些仅为举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均应落入本发明的保护范围。
序列表
<110> 上海鑫湾生物科技有限公司
<120> 用于调控酸性环境免疫应答的组合物、其制备方法和用途
<130> DIC19110078R
<150> 2019111120438
<151> 2019-11-14
<160> 54
<170> SIPOSequenceListing 1.0
<210> 1
<211> 35
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe
1 5 10 15
Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp
20 25 30
Glu Gly Thr
35
<210> 2
<211> 36
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
1 5 10 15
Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala
20 25 30
Asp Glu Gly Thr
35
<210> 3
<211> 32
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu
1 5 10 15
Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly
20 25 30
<210> 4
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe
1 5 10 15
Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly
20 25 30
<210> 5
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe
1 5 10 15
Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp
20 25
<210> 6
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe
1 5 10 15
Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp
20 25
<210> 7
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe
1 5 10 15
Pro Thr Asp Thr Leu Leu Leu Asp Leu
20 25
<210> 8
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro
1 5 10 15
Thr Asp Thr Leu Leu Leu Asp Trp
20
<210> 9
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe
1 5 10 15
Pro Thr Glu Thr Leu Leu Leu Glu Leu
20 25
<210> 10
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Cys Glu Glu Gln Gln Pro Trp Ala Gln Tyr Leu Glu Leu Leu Phe Pro
1 5 10 15
Thr Glu Thr Leu Leu Leu Glu Trp
20
<210> 11
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Cys Glu Glu Gln Gln Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe Pro
1 5 10 15
Thr Glu Thr Leu Leu Leu Glu Trp
20
<210> 12
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe
1 5 10 15
Pro Thr Thr Leu Leu Leu Leu Asp
20
<210> 13
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe
1 5 10 15
Pro Thr Thr Leu Leu Leu Leu Glu
20
<210> 14
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe
1 5 10 15
Pro Thr Thr Leu Ala Trp
20
<210> 15
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe
1 5 10 15
Pro Thr Thr Leu Ala Trp
20
<210> 16
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro
1 5 10 15
Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu
20 25 30
Cys Ala
<210> 17
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Asn
1 5 10 15
Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asn Gly Ala Leu Leu Val
20 25 30
Glu Ala Glu Glu Thr
35
<210> 18
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Pro
1 5 10 15
Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Pro Gly Ala Leu Leu Val
20 25 30
Glu Ala Glu Glu Thr
35
<210> 19
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
50 55 60
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
65 70 75 80
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
85 90 95
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
100 105 110
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
115 120 125
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
130 135 140
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
145 150 155 160
Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly
165 170 175
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
180 185 190
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
195 200 205
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
210 215 220
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
225 230 235 240
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
245 250 255
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
260 265 270
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
275 280
<210> 20
<211> 324
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val
50 55 60
Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Gly
65 70 75 80
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser
85 90 95
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
100 105 110
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
115 120 125
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
130 135 140
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
145 150 155 160
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
165 170 175
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro
195 200 205
Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
210 215 220
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
225 230 235 240
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
245 250 255
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
260 265 270
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
275 280 285
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
290 295 300
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
305 310 315 320
Ser Pro Gly Lys
<210> 21
<211> 528
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Pro
50 55 60
Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys
65 70 75 80
Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly
85 90 95
Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His
100 105 110
Gln Ala Ala Met Gln Ile Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala
115 120 125
Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
130 135 140
Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Asn
145 150 155 160
Leu Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Val Pro Val
165 170 175
Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
180 185 190
Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys
195 200 205
Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala
210 215 220
Glu Gln Ala Thr Gln Gly Val Lys Asn Trp Met Thr Asp Thr Leu Leu
225 230 235 240
Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly
245 250 255
Pro Gly Ala Ser Ile Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly
260 265 270
Gly Pro Ser His Lys Ala Lys Val Leu Gly Gly Gly Gly Ser Gly Gly
275 280 285
Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr
290 295 300
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
305 310 315 320
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
325 330 335
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
340 345 350
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
355 360 365
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
370 375 380
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
385 390 395 400
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr
405 410 415
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
420 425 430
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
435 440 445
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
450 455 460
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
465 470 475 480
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
485 490 495
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
500 505 510
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
515 520 525
<210> 22
<211> 382
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu
50 55 60
Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile
65 70 75 80
Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
145 150 155 160
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
180 185 190
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
195 200 205
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
210 215 220
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
225 230 235 240
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser
260 265 270
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
275 280 285
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
305 310 315 320
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
325 330 335
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
340 345 350
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
355 360 365
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 23
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu
50 55 60
Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile
65 70 75 80
Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala
145 150 155 160
Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg
165 170 175
Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly
180 185 190
Gly Gly Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu
195 200 205
Leu Ala Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala
210 215 220
Tyr Arg Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp
245 250 255
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
275 280 285
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
290 295 300
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
325 330 335
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
340 345 350
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu
355 360 365
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
370 375 380
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
385 390 395 400
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
420 425 430
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
435 440 445
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
450 455 460
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
465 470 475 480
Gly Lys
<210> 24
<211> 382
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp
50 55 60
Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp
65 70 75 80
Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
145 150 155 160
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
180 185 190
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
195 200 205
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
210 215 220
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
225 230 235 240
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser
260 265 270
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
275 280 285
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
305 310 315 320
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
325 330 335
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
340 345 350
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
355 360 365
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 25
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu
1 5 10 15
Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Ile Pro Asn
20 25 30
Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp
50 55 60
Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp
65 70 75 80
Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu Leu Ala Leu Asp
100 105 110
Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp
115 120 125
Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg
145 150 155 160
Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala
165 170 175
Leu Leu Val Asp Ala Asp Glu Gly Thr Gly Gly Gly Gly Ser Gly Gly
180 185 190
Gly Gly Ser Gly Gly Gly Gly Ser Thr Gly Glu Asp Ala Asp Val Leu
195 200 205
Leu Ala Leu Asp Leu Leu Leu Leu Pro Thr Thr Phe Leu Trp Asp Ala
210 215 220
Tyr Arg Ala Trp Tyr Ile Pro Asn Gln Glu Ala Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp
245 250 255
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
275 280 285
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
290 295 300
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
325 330 335
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
340 345 350
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu
355 360 365
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
370 375 380
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
385 390 395 400
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
420 425 430
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
435 440 445
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
450 455 460
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
465 470 475 480
Gly Lys
<210> 26
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
<210> 27
<211> 26
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg
1 5 10 15
Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu
20 25
<210> 28
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Glu Glu Glu Glu Glu Glu
1 5
<210> 29
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg
1 5 10
<210> 30
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 31
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 32
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
His His His His His His
1 5
<210> 33
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 34
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
1 5 10 15
<210> 35
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly
1 5 10 15
Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro
20 25 30
Gln Gly Gln Arg Glu Pro
35
<210> 36
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser
1 5 10
<210> 37
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Thr Gln Asp Pro Ser Arg Val Gly
1 5
<210> 38
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 39
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Cys Cys Pro Gly Cys Cys
1 5
<210> 40
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr
1 5 10
<210> 41
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly
1 5 10
<210> 42
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys
1 5 10
<210> 43
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Asp Leu Tyr Asp Asp Asp Asp Lys
1 5
<210> 44
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
1 5 10 15
Tyr Lys Asp Asp Asp Asp Lys
20
<210> 45
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Thr Asp Lys Asp Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu
1 5 10 15
<210> 46
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210> 47
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys
1 5 10
<210> 48
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu
1 5 10
<210> 49
<211> 139
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
His His His His His His Glu Asp Cys Cys Leu Ser Val Thr Gln Lys
1 5 10 15
Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr Leu Leu Ile Lys
20 25 30
Asp Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr Leu Arg Gly Arg
35 40 45
Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln
50 55 60
Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser Gly Ser
65 70 75 80
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Arg Ser
85 90 95
Asp Asn His Gly Ser Ser Gly Thr Ala Glu Gln Asn Pro Ile Tyr Trp
100 105 110
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
115 120 125
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
130 135
<210> 50
<211> 806
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Asn Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Gly Ser Phe Ser Met Ser Trp Val Arg Gln Ala Pro Gly Gly
50 55 60
Gly Leu Glu Trp Val Ala Gly Leu Ser Ala Arg Ser Ser Leu Thr His
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Arg Ser Tyr Asp Ser Ser Gly Tyr Trp
115 120 125
Gly His Phe Tyr Ser Tyr Met Asp Val Trp Gly Gln Gly Thr Leu Val
130 135 140
Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Ser Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Gly
165 170 175
Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asn
180 185 190
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
195 200 205
Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Gly Leu
225 230 235 240
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser
245 250 255
Leu Ser Glu Phe Leu Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly
260 265 270
Ala Ser Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys
275 280 285
Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser
290 295 300
Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val
305 310 315 320
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
325 330 335
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr
340 345 350
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
355 360 365
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser
370 375 380
Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
385 390 395 400
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
405 410 415
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
420 425 430
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
435 440 445
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
450 455 460
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg
465 470 475 480
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
485 490 495
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
500 505 510
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
515 520 525
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly
530 535 540
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
545 550 555 560
Glu Asn Pro Gly Pro His Met Met Val Ser Lys Gly Glu Glu Leu Phe
565 570 575
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
580 585 590
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
595 600 605
Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
610 615 620
Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser
625 630 635 640
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
645 650 655
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
660 665 670
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
675 680 685
Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile
690 695 700
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
705 710 715 720
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg
725 730 735
His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
740 745 750
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
755 760 765
Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
770 775 780
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
785 790 795 800
Met Asp Glu Leu Tyr Lys
805
<210> 51
<211> 43
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Glu Gln Asn Pro Ile Tyr Trp
1 5 10 15
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
20 25 30
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
35 40
<210> 52
<211> 59
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
Gly Gly Gly Gly Ser Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu
1 5 10 15
Lys Lys Leu Gly Gly Gly Gly Ser Ala Glu Gln Asn Pro Ile Tyr Trp
20 25 30
Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp
35 40 45
Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr
50 55
<210> 53
<211> 497
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 53
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ala Val
20 25 30
Val Thr Gln Glu Ser Ala Leu Thr Ser Ser Pro Gly Glu Thr Val Thr
35 40 45
Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala
50 55 60
Ser Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly
65 70 75 80
Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser
85 90 95
Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu
100 105 110
Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp Tyr Ser Asp His Trp Val
115 120 125
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
145 150 155 160
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
165 170 175
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Leu Leu Thr Asp Tyr Gly
180 185 190
Val Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly
195 200 205
Val Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys Ser
210 215 220
Arg Leu Ser Val Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
225 230 235 240
Met Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg Tyr Tyr Cys Val Thr
245 250 255
Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
260 265 270
Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
275 280 285
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
290 295 300
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
305 310 315 320
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
325 330 335
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
340 345 350
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
355 360 365
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
370 375 380
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
385 390 395 400
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
405 410 415
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
420 425 430
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
435 440 445
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
450 455 460
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
465 470 475 480
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
485 490 495
Arg
<210> 54
<211> 433
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
35 40 45
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
85 90 95
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
115 120 125
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
145 150 155 160
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val
165 170 175
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190
Gly Leu Ala Val Pro Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
195 200 205
Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
210 215 220
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
225 230 235 240
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
245 250 255
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
260 265 270
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
275 280 285
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
290 295 300
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
305 310 315 320
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
325 330 335
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
340 345 350
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
355 360 365
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
370 375 380
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
385 390 395 400
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
405 410 415
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
420 425 430
Arg

Claims (3)

1.一种用于调控酸性环境免疫应答的组合物，所述组合物由酸性环境靶向分子和免疫细胞组成；
其中，所述酸性环境靶向分子是结构式为：FITC-ACP-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT的FITC-pHLIP；所述免疫细胞是靶向FITC的FITC CAR-T细胞。
2.如权利要求1所述的组合物在制备用于治疗肿瘤的药物中的用途。
3. 如权利要求1所述的组合物的制备方法，所述方法包括以下步骤：
利用多肽自动合成仪，通过固相法合成；或
用生物合成法，通过工程菌和细胞表达并纯化。
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CN114149510B (zh) * 2021-10-29 2024-01-30 上海鑫湾生物科技有限公司 一种条件控制的可剪接嵌合抗原受体分子及其应用
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CN114181319A (zh) * 2021-11-26 2022-03-15 北京双赢科创生物科技有限公司 用于靶向肿瘤细胞的多肽偶联物及其制备方法与应用
CN114716566A (zh) * 2022-02-22 2022-07-08 广东粤港澳大湾区国家纳米科技创新研究院 一种融合蛋白及其在制备肿瘤药物中的应用
CN116640229B (zh) * 2023-04-10 2024-01-30 中国人民解放军总医院第五医学中心 一种低pH靶向性CAR-T细胞的构建及应用
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