CN109867725B - PD-1-Fc融合蛋白及其制备方法和用途 - Google Patents
PD-1-Fc融合蛋白及其制备方法和用途 Download PDFInfo
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract
本发明涉及高亲和力PD‑1(HiPD‑1)‑Fc融合蛋白及其制法和用途。具体而言,本发明涉及含高亲和力PD‑1(HiPD‑1)和人免疫球蛋白IgG的Fc片段元件的融合蛋白HiPD‑1‑Fc；编码该融合蛋白的核酸序列；制备该融合蛋白的方法；以及所述融合蛋白在提高T细胞效应功能和杀伤肿瘤活性中的应用。本发明的融合蛋白既保持高亲和力PD‑1蛋白识别PD‑L1的活性，又引入Fc片段以延长HiPD‑1分子半衰期的目标，具有很高的实用价值。
Description
技术领域
本发明涉及肿瘤免疫学领域；更具体地说，本发明涉及高亲和力的可溶性程序性死亡受体(Programmed Death-1,PD-1)分子与Fc片段形成的融合蛋白及其制备方法和在肿瘤治疗中的应用。
背景技术
正常情况下，机体的免疫***能够监视、清除病变的细胞。但是，肿瘤仍然能够继续生长进而影响到生活质量及生命安全，说明肿瘤本身存在一定的机制来逃逸机体的免疫监视。近年来研究发现：肿瘤擅用抑制性协同共刺激分子拮抗T细胞抗肿瘤效应是肿瘤免疫逃逸的重要机制，阻断这类分子成为肿瘤免疫治疗研究新的热点(Hamanishi J et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica,2007,104,3360-3365)。
PD-1(Programmed death 1)及其配体PD-L1(Programmed death-ligand 1)属于CD28/B7超家族蛋白，是一种重要的免疫抑制分子，可截断T细胞活化信号，抑制T细胞增殖及细胞因子的释放，在肿瘤免疫逃逸、自身免疫性疾病及病毒感染过程中发挥重要作用(Keir ME et al.,Annu Rev Immunol,26,677-704)。一系列临床前和临床研究证实针对PD-1/PD-L1信号通路的治疗性单克隆抗体取得了显著疗效，成为肿瘤治疗领域的热点研究方向。这些抗体作用的本质是其针对抗原的高亲和力，及占据了野生型PD-1和PD-L1相互作用的结合位点，从而中断了PD-1与PD-L1结合引起的负性信号的转导，解除了肿瘤微环境中T细胞受抑制的状态(Liu K et al.,Cell Res,27,151-153；Na Z et al.,Cell Res,27,147-150；Tan S et al.,Nat Commun,8,14369)。
研究发现：可溶性PD-1(souble PD-1,sPD-1)能够封闭膜结合性PD-1和PD-L1的相互作用，增强T细胞的抗肿瘤功能。在关节炎患者的血清中发现了高表达的sPD-1，进一步研究表明sPD-1能够封闭膜结合性PD-1对T细胞活化的抑制(Liu et al.,Arthritisresearch&therapy,17,(1),340)，He等也发现sPD-1能够通过结合PD-L1，增强肿瘤特异性的CD8T细胞介导的杀伤(He YF,The Journal of Immunology,173,(8),4919-4928)。通过分子定向进化和噬菌体高通量筛选技术，发现高亲和力PD-1分子(HiPD-1)能够以较野生型PD-1分子更高的亲和力识别肿瘤细胞表面的PD-L1。
尽管HiPD-1分子能够有效的识别PD-L1分子并能封闭PD-1和PD-L1的相互作用，但是由于天然的HiPD-1分子量小，体内应用后半衰期短，限制其体内生物活性的发挥。因此，本领域迫切需要一种改善其体内半衰期，促进其生物学活性发挥的途径。
发明内容
本发明的目的在于提供一种对PD-L1分子具有较高亲和力的PD-1与Fc片段的融合蛋白，所述融合蛋白既能够有效的识别PD-L1分子并封闭PD-1和PD-L1的相互作用，并且能够非常有效地促进效应细胞对肿瘤细胞的杀伤作用；本发明还提供了上述PD-1与Fc片段的融合蛋白的制备方法及用途。
在第一方面，本发明提供一种融合蛋白，所述融合蛋白包含高亲和力PD-1分子和IgG分子的Fc片段，其中：
(i)所述高亲和力PD-1分子与PD-L1分子的亲和力是野生型PD-1分子与PD-L1分子的至少100倍；和
(ii)所述高亲和力PD-1分子的氨基酸序列与野生型PD-1氨基酸序列(SEQ IDNO.1)有至少90％的序列相同性。
在优选的实施方式中，所述高亲和力PD-1分子的氨基酸序列与SEQ ID NO.1所示的氨基酸序列有92％；优选地，至少94％(例如，95％、96％、97％、98％和99％)的序列相同性。
在优选的实施方式中，所述高亲和力PD-1分子与PD-L1分子的亲和力是野生型PD-1分子与PD-L1分子的亲和力的至少200倍；优选地，至少500倍；更优选地，至少1000倍；更优选地，至少2000倍。
在具体的实施方式中，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。
在具体的实施方式中，所述的IgG的Fc片段选自人IgG1、IgG2、IgG3或IgG4的Fc片段；优选地，选自IgG4的Fc片段。
在具体的实施方式中，所述Fc片段包括铰链区、CH2和CH3结构域，优选地，所述Fc片段不包含CH1和CH4结构域，更有选地，所述Fc片段的氨基酸序列为SEQ ID NO:7。
在具体的实施方式中，所述高亲和力PD-1分子的氨基酸序列选自SEQ ID NO:2-6。
在具体的实施方式中，所述融合蛋白中包含两个高亲和力的PD-1分子。
在具体的实施方式中，所述融合蛋白中两个高亲和力的PD-1分子通过二硫键连接形成二聚体结构；优选地，所述二硫键是IgG分子的Fc片段铰链区的二硫键。
在第二方面，本发明提供一种核酸分子，所述核酸分子包含编码第一方面所述融合蛋白的核酸序列或其互补序列。
在第三方面，本发明提供一种载体，所述载体含有第二方面所述的核酸分子。
在第四方面，本发明提供一种宿主细胞，所述宿主细胞中含有第三方面所述载体或染色体中整合有外源的第二方面所述的核酸分子。
在第五方面，本发明提供一种药物组合物，所述组合物中含有第一方面所述的融合蛋白，和任选的药学上可接受的载体或赋形剂。
在第六方面，本发明提供一种治疗疾病的方法，包括给需要治疗的对象施用适量的第一方面所述的融合蛋白或第五方面所述的药物组合物；优选地，所述疾病为肿瘤；更优选地，所述肿瘤表达PD-L1分子。
在第七方面，本发明提供第一方面所述融合蛋白的用途，用于制备***的药物。
在第八方面，本发明提供一种制备第一方面所述融合蛋白的方法，包括步骤：
(a)培养第四方面所述的宿主细胞，从而表达第一方面所述的融合蛋白；
(b)任选分离或纯化所述的融合蛋白。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为野生型PD-1蛋白纯化后的SDS-PAGE胶图。M，蛋白分子量Mark。
图2为野生型PD-1分子与PD-L1分子结合的BIAcore图谱。
图3为PD-1、L5B7识别H1299细胞表面PD-L1的流式检测，显示L5B7识别H1299细胞表面PD-L1的能力高于PD-1。注：A，anti-PD-L1抗体(2.5ul/样品)识别H1299细胞表面的PD-L1；B，不同浓度的PD-1、L5B7(浓度为0.02mg/ml、0.04mg/ml、0.08mg/ml)识别H1299细胞表面的PD-L1的流式检测图，其中SA-PE的用量为0.5ul/样品；C，当浓度为0.08mg/ml时，对照组、PD-1、L5B7识别PD-L1的流式柱状图。
图4为表达质粒的凝胶电泳图。其中，M为DNA分子标记；泳道1为野生型PD-1-Fc；泳道2为L2F8-Fc；泳道3为L2F10-Fc；泳道4为L45-Fc；泳道5为L45-123-Fc；泳道6为L5B7-Fc。
图5为融合蛋白纯化后SDS-PAGE电泳图。其中，A为非还原性的；B为还原性的。泳道1为L2F8-Fc；泳道2为L2F10-Fc；泳道3为L45-Fc；泳道4为L45-123-Fc；泳道5为L5B7-Fc。
图6为融合蛋白的Fc片段ELISA检测结果图。
图7显示了本发明融合蛋白促进杀伤的结果。其中A为Mel624；B为H1299；C为A375。
图8显示了本发明融合蛋白促进PBMC释放IFN-γ的Elispot结果。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现将高亲和力的可溶性PD-1与IgGFc片段融合后得到高亲和力PD-1-Fc融合蛋白，所述融合蛋白不仅能够高效识别PD-L1配体，而且能够非常有效地促进效应细胞对肿瘤细胞的杀伤作用。
术语定义
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
本文所用的术语“HiPD-1分子”或“高亲和力PD-1分子”或“高亲和力PD-1”或“本发明的PD-1分子”或“本发明的PD-1”是指对PD-L1的亲和力是野生型PD-1分子对PD-L1的亲和力至少100倍、优选至少200倍、更优选至少500倍、至少1000倍、至少2000倍的可溶性高亲和力PD-1分子，并且所述PD-1分子的氨基酸序列与野生型PD-1氨基酸序列(SEQ ID NO.1)有至少90％、优选至少92％、更优选至少94％(例如，至少95％、96％、97％、98％或99％)的序列相同性；优选地，所述PD-1分子的氨基酸序列选自SEQ ID NO.2-6。
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE(SEQ ID NO.1，野生型PD-1)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSGQVDKLAGFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAE(SEQ ID NO.2，L2F8)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSLNGDKLASFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAE(SEQ ID NO.3，L2F10)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCSAISLAPYIQIKESLRAELRVTERRAE(SEQ ID NO.4，L5B7)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKIQIKESLRAELRVTERRAE(SEQ ID NO.5，L45)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRESPSGQTDKLAAFPEDRSQLGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYMCVAISLAPKIQIKESLRAELRVTERRAE(SEQ ID NO.6，L45-123)。
本文所用的术语“IgG的Fc片段”指免疫球蛋白重链的恒定区，例如IgG的Fc片段包括重链CH1、CH2、CH3、CH4的两个或更多结构域与铰链区的组合。在一个优选的实施例中，所用的免疫球蛋白的Fc片段包括至少一个免疫球蛋白铰链区，一个CH2结构域和一个CH3结构域；优选缺少CH1结构域，更优选缺少CH1、CH4结构域。最优选地，所述Fc片段的氨基酸序列选自SEQ ID NO.7。
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.7，IgG4 Fc片段)
本文所用的术语“HiPD-1-Fc融合蛋白”或“高亲和力PD-1-Fc融合蛋白”或“高亲和力PD-1-Fc”或“本发明的PD-1-Fc融合蛋白”或“本发明的PD-1-Fc”是指由高亲和力PD-1分子与人的IgG Fc片段融合形成的蛋白。优选地，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。优选地，所述融合蛋白中包含两个高亲和力PD-1分子。更优选地，所述两个高亲和力PD-1分子是通过免疫球蛋白Fc片段二硫键形成的稳定的二聚体结构。最优选地，所述HiPD-1-Fc融合蛋白分子的氨基酸序列选自SEQ ID NO.10、12、14、16或18；其编码核苷酸序列分别对应于SEQ ID NO.11、13、15、17或19。
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.8，野生型PD-1-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTAACCAGACCGATAAGCTGGCTGCTTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCGGTGCTATTTCTCTGGCTCCTAAAGCTCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.9，野生型PD-1-Fc)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSGQVDKLAGFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.10，L2F8-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTGGTCAGGTTGATAAGCTGGCTGGTTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCGTTGCTATTTCTCTGGCTCCTAAACCGCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.11，L2F8-Fc)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSLNGDKLASFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.12，L2F10-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTCTGAATGGTGATAAGCTGGCTAGCTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCGTTGCTATTTCTCTGGCTCCTAAACCGCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.13，L2F10-Fc)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCSAISLAPYIQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.14，L5B7-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTAACCAGACCGATAAGCTGGCTGCTTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCAGCGCTATTTCTCTGGCTCCTTATATACAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.15，L5B7-Fc)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCVAISLAPKIQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.16，L45-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTAACCAGACCGATAAGCTGGCTGCTTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCGTTGCTATTTCTCTGGCTCCTAAAATTCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.17，L45-Fc)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRESPSGQTDKLAAFPEDRSQLGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYMCVAISLAPKIQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.18，L45-123-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGGAATCTCCTTCTGGTCAGACCGATAAGCTGGCTGCTTTCCCTGAAGATAGATCTCAGCTGGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACATGTGCGTTGCTATTTCTCTGGCTCCTAAAATTCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.19，L45-123-Fc)。
本发明还涉及编码本发明HiPD-1-Fc融合蛋白的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明融合蛋白的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。例如，本文所用的“简并的变异体”表示编码具有野生型PD-1-Fc(SEQ ID NO:8)的蛋白序列，但与SEQ ID NO:9的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明Hi-PD-1-Fc融合蛋白的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明中所述的表达载体可以是原核表达载体及真核表达载体。载体的选择及构建是本领域技术人员通常所掌握的。由于原核表达体系所表达的产物常以不溶性的包涵体形式存在，表达的目的蛋白需经过复杂的变性及复性过程后，才能获得具有天然生物学活性的目的蛋白。而Fc融合蛋白分子较大，分子中含有较大二硫键，结果及功能也较复杂，一般均采用真核表达***表达，以便通过糖基化修饰和自然折叠，产生具有天然蛋白或多肽生物学活性功能的糖基化重组融合蛋白。因此本发明优选了哺乳动物真核表达***。在优选的一个方案中，本发明人使用了真核分泌型表达载体pFUSE-hIgG1e1-Fc2(InvivoGen)。在此载体基础上，构建了pFUSE-HiPD-1-Fc真核表达载体。
在本发明所述的宿主细胞包括原核细胞和真核细胞，常用的原核细胞包括大肠杆菌等，常用的真核宿主细胞包括酵母细胞，昆虫细胞和哺乳动物细胞。在一个优选的实施例中，本发明人使用了293T贴壁哺乳动物细胞，更优选的使用expi293FTM表达宿主细胞(life)，一种改良型的293细胞系，具有更高的转染效率和蛋白产量。
在本发明所述的HiPD-1-Fc融合蛋白制备方法包括将含有编码HiPD-1-Fc融合蛋白的核酸序列，***到合适的载体中，得到相应的合适载体，转染适宜的宿主细胞；并在在适宜的培养条件下，培养转染细胞，并从中分离纯化出表达的HiPD-1-Fc蛋白。
表达载体的转化可采用常规的方法，如氯化钙法、电穿孔法、脂质体转染法等。优选地，本发明人使用了Lipofectamine 2000转染试剂，更优选地使用了ExpiFectamineTM293Reagent。
纯化工艺可采用本领域中常规使用的纯化工艺，包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。也可采用本发明人专门设计的纯化工艺。优选地，包括以下步骤：(a)收集培养上清；(b)镍柱亲和层析分离；(c)Superdex 200分子筛分离。用本发明人选择的工艺进行纯化，最终可获得纯度大于85％的HiPD-1-Fc双价融合蛋白。
本发明的主要优点在于：
(1)本发明的HiPD-1-Fc融合蛋白不仅对PD-L1具有高亲和力，还具备优异的稳定性；和
(2)本发明的HiPD-1-Fc融合蛋白能够有效提高效应细胞的功能和杀伤肿瘤活性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例中的实验方法，如无特别说明，均采用本领域常规技术，实验试剂均为市售产品。虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
实施例1.野生型PD-1的表达、复性和纯化
野生型PD-1的胞外氨基酸序列及核苷酸序列分别为SEQ ID NO.1和20(CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATACGAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAGATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTGGTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGAATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA)，将携带野生型PD-1的胞外序列的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体(Novagen)该载体经过优化后带有biotin tag标签)连接。连接产物转化至E.coli DH5α(Vazyme)，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Rosetta菌株(TIANGEN)中用于表达。
将上述含有重组质粒pET28a-PD-1的Rosetta菌落接种于含有卡那霉素的LB培养基中，37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.7mM，37℃继续培养4h。6000g离心15min收获细胞沉淀物，用BugbusterMaster Mix(Merck)裂解细胞沉淀物，6000g离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000g离心15min,收集包涵体。将包涵体溶解在缓冲液(50mM Tris-HCl，200mM NaCl,2mM EDTA，6Mguanidine HCl，pH 8.1)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向7mg溶解的PD-1包涵体蛋白中，加入2mL缓冲液(50mM Tris-HCl，200mM NaCl，2mM EDTA，6M guanidine HCl，pH 8.1)，再加入DTT至终浓度为20mM，37℃处理1h。向100mL复性缓冲液(50mM HEPES，pH 7.5，500mM L-arginine，9mM glutathione，1mM glutathionedisulfide，24mM NaCl，1mM KCl)中滴加上述处理后的PD-1混合液，4℃搅拌30min，然后将复性液装入截留量为3.5KD的纤维素膜透析袋,透析袋置于2L预冷的水中，4℃缓慢搅拌过夜。24小时后，将透析液换成2L预冷的缓冲液(10mMTris-HCl，pH 8.5)，4℃继续透析24h，然后将透析液换成相同的新鲜缓冲液继续透析24小时，样品经0.45μm滤膜过滤，真空脱气后进样至阴离子交换柱(HiTrap Q HP，GE Healthcare)。用10mMTris-HCl pH 8.5配制的0-1MNaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析。根据分析结果，收集目标PD-1组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300，GE Healthcare)纯化，目标组分也进行SDS-PAGE分析，结果如图1所示。
实施例2.结合表征
BIAcore分析
使用BIAcore T200实时分析***检测野生型PD-1分子与PD-L1的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将生物素化的PD-1流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将PD-1用HEPES-EP缓冲液(10mM HEPES，150mMNaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mMGly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
本实施例中所用的PD-L1的氨基酸序列和核苷酸序列分别如SEQ ID NO.21(FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPEL)、SEQID NO.22(TTTACGGTTACGGTTCCGAAAGACCTGTATGTGGTTGAATACGGCTCTAATATGACGATTGAATGCAAATTCCCGGTTGAAAAACAACTGGATCTGGCGGCCCTGATTGTGTATTGGGAAATGGAAGACAAAAACATCATCCAATTCGTGCATGGCGAAGAAGATCTGAAAGTTCAGCACAGCTCTTACCGTCAACGCGCACGTCTGCTGAAAGACCAGCTGAGCCTGGGCAATGCAGCTCTGCAGATCACGGATGTTAAACTGCAAGACGCCGGTGTCTATCGCTGCATGATTTCTTATGGCGGTGCAGACTACAAACGTATCACCGTCAAAGTGAACGCTCCGTACAACAAAATTAATCAGCGCATCCTGGTGGTTGATCCGGTTACGTCCGAACATGAACTGACCTGTCAAGCGGAAGGCTATCCGAAAGCCGAAGTCATTTGGACCAGTTCCGATCACCAGGTGCTGTCAGGTAAAACCACGACCACGAACTCGAAACGCGAAGAAAAACTGTTTAATGTCACGAGCACCCTGCGTATTAACACCACGACCAATGAAATCTTCTACTGCACCTTTCGTCGTCTGGACCCGGAAGAAAATCATACGGCGGAACTGGTTATCCCGGAACTG)所示，其表达、复性和纯化过程与实施例1中野生型PD-L1的表达、复性和纯化过程相同。其生物素化的过程如下：
a.生物素化
用Millipore超滤管将纯化的PD-L1分子浓缩，同时将缓冲液置换为10mMTris pH8.0，然后加入生物素化试剂0.05MBicine pH 8.3、10mM ATP、10mMMgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
b.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的PD-L1分子浓缩至500μl，采用凝胶过滤层析纯化生物素化的PD-L1，先用过滤过的PBS预平衡Superdex 75 10/300凝胶过滤柱(GE通用电气公司)，再加载500μl浓缩过的生物素化PD-L1分子，然后用PBS以1ml/min流速洗脱,将收集到的组分进行SDS-PAGE分析，根据结果合并含有目标蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，将生物素化的PD-L1分子分装保存在-80℃。
通过本实施例上述过程检测到野生型PD-1分子与PD-L1分子的结合亲和力的KD值为2.815E-06M，其BIAcore结合图谱如图2所示。
实施例3.高亲和力PD-1分子的产生
将实施例1中所述的野生型PD-1的胞外序列作为模板链，根据Li等，(2005)NatureBiotech 23(3):349-354)描述的噬菌体展示和筛选方法，进行高亲和力PD-1的筛选。经过几轮筛选后的噬菌体文库均和PD-1有较强的结合信号，从中挑取单克隆，并进行序列分析。
按照实施例1中所述方法表达、复性和纯化本发明高亲和性PD-1分子，并按实施例2中所述方法测定其与PD-L1分子的亲和力。本发明中获得的高亲和力PD-1分子与PD-L1分子的亲和力是野生型PD-1分子与PD-L1分子的亲和力至少100倍，其氨基酸序列及其与PD-L1分子的亲和力数值如下表1所示。
表1高亲和力克隆对PD-L1分子的BIAcore结果
实施例4.L5B7识别H1299细胞表面PD-L1的能力高于PD-1
Biacore结果显示，经过筛选后确实得到了亲和力提高的PD-1突变体，但这种亲和力的改变是否会影响其与生理条件下细胞表面PD-L1的结合仍需实验确认。因此，我们选用PD-L1表达阳性的H1299细胞，加入不同浓度的生物素化PD-1、L5B7蛋白，流式细胞术分析PD-1、L5B7识别细胞表面PD-L1的能力。
图3结果显示，随着加入的PD-1、L5B7蛋白浓度的升高，其对PD-L1的识别能力逐渐增强；在同一浓度条件下，L5B7蛋白识别PD-L1的能力高于PD-1，这种识别能力的改变可能是由于L5B7的亲和力较高所引起的，与生化结果一致。
实施例5.HiPD-1-Fc融合蛋白的设计及表达载体构建
HiPD-1-Fc融合蛋白是由两条单链肽段经链间二硫键形成的二聚体可溶性蛋白，其主要结构包含两个高亲和力PD-1分子(HiPD-1)的头部和一个IgG4的Fc尾部。组成该融合蛋白的单链肽段包含315个氨基酸，从N端到C端依次为：HiPD-1分子、IgG4 Fc片段、Histag，其中，1-116为HiPD-1分子；117-345为IgG4的Fc片段；118-128，Fc片段的铰链区；129-236，Fc片段的CH2的结构域；237-345，Fc片段的CH3结构域；346-351，6个组氨酸组成Histag用于蛋白纯化。两条肽段在124位和127位(铰链区)各自形成两个链间二硫键，保证HiPD-1-Fc二聚体化。
基于此设计，野生型PD-1分子和IgG4 Fc片段的融合氨基酸序列(SEQ ID NO:8)经真核表达***核苷酸序列优化后，由南京金斯瑞公司体外合成核苷酸片段(SEQ ID NO:9)，采用同源重组技术(参照ClonExpress II One Step Cloning Kit标准技术流程，南京诺唯赞生物科技有限公司)由EcoRⅠ和NheⅠ酶切位点整合到pFUSE-hIgG1e1-Fc2(InvivoGen)表达载体(该载体采用IL-2信号肽，融合蛋白分泌表达在上清)，并化转到Top10大肠杆菌(广州真知生物科技有限公司)中，涂布含博莱霉素的LB平板，37℃倒置培养过夜，对阳性重组子进行测序，鉴定正确的野生型PD-1-Fc融合蛋白质粒。在此基础上，通过PCR突变技术构建HiPD-1-Fc重组克隆子及His tag，并转化到Top10感受态，测序鉴定构建成功的重组子，菌液冻存-20℃备用。
将鉴定正确的含有HiPD-1-Fc质粒的菌液按照1:100接种于200ml LB培养基(含100μg/ml氨苄青霉素)中，37℃过夜培养后，4500g，30min离心收集沉淀。按照大提质粒标准流程(参照PureLinkTMHiPure Plasmid Filter Maxiprep Kit标准技术流程，life)抽提质粒，如图4所示，OD260/280测定质粒浓度，调整质粒浓度为1mg/ml，分装后于-20℃保存。
实施例6.HiPD-1-Fc融合蛋白的表达和纯化
复苏expi293FTM细胞株(购自Life)，并于37℃,125rpm,5％CO2稳定培养2-3代。于转染前一天，调整细胞密度为2*106个/ml，过夜培养。转染当天(以30ml转染体系为例)，首先调整过夜培养细胞的密度为2.5*106个/ml于25.5ml(要求细胞活率大于95％)中；其次，将30μg质粒和81μl ExpiFectamineTM293Reagent(购自Life)分别稀释到opti-MEM培养基(各共1.5ml)中，静置5min后，将转染试剂混合液缓慢滴加到质粒混合液中，静置放置20min。最后，将质粒和转染试剂的混合液滴加到细胞培养物中，于37℃,125rpm,5％CO2条件下培养16-18h，加入150μl enhancer 1(试剂盒，购自life)和1.5ml enhancer 2(试剂盒，购自life)，继续培养3-4天(摸索条件发现，在培养4-5天后，细胞活率在40-50％)，4℃，12000g离心收集上清，0.45μm过滤。
以2ml/min的进样流速经过镍柱(GE Healthcare)，用500mM咪唑磷酸盐缓冲液(pH7.2)线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析。根据分析结果，收集目标HiPD-1-Fc组分浓缩后进一步用分子筛Superdex 200(GE Healthcare)纯化，收集目标组分并进行SDS-PAGE分析，鉴定蛋白纯度。结果如图5所示。
实施例7.HiPD-1-Fc融合蛋白的结合活性
为了评估实施例2中制备和纯化的HiPD-1-Fc融合蛋白的活性，确定该融合蛋白是否既可以识别HiPD-1分子特异性配体PD-L1，又具有Fc片段活性。首先，通过基于生物膜干涉技术的Octet system测定HiPD-1-Fc融合蛋白对PD-L1的识别能力。将HiPD-1-Fc融合蛋白稀释成0.5μM，加载到NTA探(PALL)针上。将PD-L1抗原按照2倍梯度稀释成不同浓度，HBS稀释缓冲液(70mM NaCl，750mM Na2HPO4·2H2O，25mM HEPS)作为基线，结合到HiPD-1-Fc融合蛋白上。利用Data Acquisition软件收集数据，Data Analysis软件分析数据，并计算动力学参数。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。HiPD-1-Fc融合蛋白与PD-L1的抗原结合的亲和力如表1。结果显示，融合蛋白的高亲和力PD-1段仍然能够以相当高的亲和力结合PD-L1，与Fc片段融合后并没有改变高亲和力PD-1结合PD-L1的能力。其次，HiPD-1-Fc分子是以可溶性、二聚体形式存在，这要求Fc需要具有活性。本发明人通过酶联免疫吸附测定(ELISA)证明融合蛋白的Fc片段是完整有活性的。前一天，将HiPD-1-Fc融合蛋白用PBS稀释到20μg/ml，每孔100μl加入到ELISA板中，4℃过夜。第二天，PBS洗3次后，用5％milk-PBS室温封闭ELISA板中非特异性结合位点2h。0.05％PBST洗板三次，加入1:750稀释的HRP偶联的抗人IgG抗体并室温孵育1h。0.05％PBST洗三次，加入100μl/孔(TMBBioPanda Diagnostics)显色10min，1M H2SO4 50μl/孔终止显色，OD450读数。结果如图6，HiPD-1-Fc融合蛋白结合anti-IgG抗体的信号明显高于阴性对照组，说明HiPD-1-Fc融合蛋白的Fc片段是有活性的。
表1.HiPD-1-Fc融合对PD-L1分子的亲和力测定(Octet system)
实施例8.HiPD-1-Fc融合蛋白促进了PBMC对PD-L1+肿瘤细胞的特异性杀伤
为了进一步检测HiPD-1-Fc融合蛋白对PBMC特异性杀伤的效应，我们使用乳酸酶脱氢法(LDH)来检测PBMC对PD-L1+的Mel624、A375、H1299肿瘤细胞系的特异性杀伤。ImmTACs分子能够重定向T细胞特异性杀伤肿瘤细胞已在多篇研究中报道(Jakobsen,2013；Oates et al.,2015)。其基本原理是ImmTACs可以模拟T细胞活化发挥效应功能的关键信号，一方面通过其高亲和的特异性TCR识别肿瘤细胞表面的MHC-肽复合物，另一方面通过其anti-CD3抗体端激活T细胞活化的下游信号通路，从而定向T细胞特异性杀伤肿瘤细胞。本发明人首先将10μg/ml的HiPD-1-Fc融合蛋白与肿瘤细胞(2*104个/孔)提前孵育30-60min，再按照PBMC:肿瘤细胞为5:1的细胞比例加入PBMC细胞，同时调整ImmTAC-IG4的浓度为2*10-10M，共150μl体系，放置培养箱48h后，取上清检测LDH(Promega)的释放。结果如图7所示，HiPD-1-Fc融合蛋白能显著促进PBMC对肿瘤细胞的特异性杀伤。并且，对于不同的肿瘤细胞系都有明显的促进作用。相对于PBMC+肿瘤细胞系+ImmTAC组，对肿瘤的杀伤至少提高了30％，在Mel624肿瘤细胞中，加入L45-123-Fc后对肿瘤的杀伤提高了245.2％。
实施例9.HiPD-1-Fc融合蛋白促进了PBMC细胞IFN-γ的释放
为了进一步检测HiPD-1-Fc融合蛋白对PBMC活化时细胞因子释放的影响，我们使用酶联免疫斑点测定法(Elispot，BD)来检测PBMC特异性的IFN-γ释放。首先将15μg/ml的Hi-PD-1-Fc融合蛋白与肿瘤细胞(1*104个/孔150μl体系)提前孵育30-60min，再按照PBMC:肿瘤细胞为2:1的细胞比例加入PBMC细胞，同时调整ImmTAC-IG4的浓度为4*10-9M，放置培养箱36h后，按照标准流程检测IFN-γ释放。结果如图5所示HiPD-1-Fc融合蛋白能促进PBMC特异性释放IFN-γ。结果如图8所示，HiPD-1-Fc融合蛋白能促进PBMC特异性释放IFN-γ,其中L2F8-Fc促进ImmTAC的IFN-γ释放为77.42％,L2F10-Fc促进ImmTAC的IFN-γ释放为96.1％，L45-Fc促进ImmTAC的IFN-γ释放为82.9％，L45-123-Fc促进ImmTAC的IFN-γ释放为100.3％，L5B7-Fc促进ImmTAC的IFN-γ释放为67.4％。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 广东香雪精准医疗技术有限公司
<120> PD-1-Fc融合蛋白及其制备方法和用途
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Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 11
<211> 1035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cctcctacat tttctcctgc tctgctggtc gtgacagagg gcgacaacgc cacctttacc 60
tgctctttct ccaacacctc tgagtctttc gtgctgaact ggtacaggat gtctccttct 120
ggtcaggttg ataagctggc tggtttccct gaagatagat ctcagcctgg acaggattgc 180
aggtttagag tcacacagct gcctaacggt cgcgattttc atatgtccgt cgtcagagct 240
aggagaaacg attctggaac ctacctgtgc gttgctattt ctctggctcc taaaccgcag 300
atcaaagagt ccctgagagc tgaactgaga gtcacagaaa gaagggctga atcaaagtat 360
ggaccacctt gcccatcctg tccagcacca gagtttctgg gcggaccctc cgtgttcctg 420
tttccaccca agcctaaaga tacactgatg atttcccgca cacctgaagt cacttgcgtg 480
gtcgtggacg tgagccagga ggatccagaa gtccagttca actggtacgt ggacggagtc 540
gaggtgcaca atgccaagac caaaccccgg gaggaacagt ttaacagtac atacagagtc 600
gtgtcagtcc tgactgtgct gcatcaggac tggctgaacg gcaaggagta taagtgcaaa 660
gtgtctaata agggactgcc ttcatccatc gagaaaacaa ttagtaaggc aaaaggccag 720
cctagagaac cacaggtgta cactctgcct ccaagtcagg aggaaatgac taagaaccag 780
gtctcactga cctgtctggt gaaagggttc tatccaagcg atatcgctgt ggagtgggaa 840
tctaatggtc agcccgagaa caattacaag acaactcccc ctgtgctgga cagcgatggc 900
tctttctttc tgtattcccg tctgactgtg gacaagagca ggtggcagga gggaaacgtc 960
tttagctgtt ctgtgatgca cgaagctctg cacaatcatt acacccagaa gagtctgtca 1020
ctgtccctgg gcaaa 1035
<210> 12
<211> 345
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Leu Asn Gly Asp Lys Leu Ala Ser
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 13
<211> 1035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cctcctacat tttctcctgc tctgctggtc gtgacagagg gcgacaacgc cacctttacc 60
tgctctttct ccaacacctc tgagtctttc gtgctgaact ggtacaggat gtctccttct 120
ctgaatggtg ataagctggc tagcttccct gaagatagat ctcagcctgg acaggattgc 180
aggtttagag tcacacagct gcctaacggt cgcgattttc atatgtccgt cgtcagagct 240
aggagaaacg attctggaac ctacctgtgc gttgctattt ctctggctcc taaaccgcag 300
atcaaagagt ccctgagagc tgaactgaga gtcacagaaa gaagggctga atcaaagtat 360
ggaccacctt gcccatcctg tccagcacca gagtttctgg gcggaccctc cgtgttcctg 420
tttccaccca agcctaaaga tacactgatg atttcccgca cacctgaagt cacttgcgtg 480
gtcgtggacg tgagccagga ggatccagaa gtccagttca actggtacgt ggacggagtc 540
gaggtgcaca atgccaagac caaaccccgg gaggaacagt ttaacagtac atacagagtc 600
gtgtcagtcc tgactgtgct gcatcaggac tggctgaacg gcaaggagta taagtgcaaa 660
gtgtctaata agggactgcc ttcatccatc gagaaaacaa ttagtaaggc aaaaggccag 720
cctagagaac cacaggtgta cactctgcct ccaagtcagg aggaaatgac taagaaccag 780
gtctcactga cctgtctggt gaaagggttc tatccaagcg atatcgctgt ggagtgggaa 840
tctaatggtc agcccgagaa caattacaag acaactcccc ctgtgctgga cagcgatggc 900
tctttctttc tgtattcccg tctgactgtg gacaagagca ggtggcagga gggaaacgtc 960
tttagctgtt ctgtgatgca cgaagctctg cacaatcatt acacccagaa gagtctgtca 1020
ctgtccctgg gcaaa 1035
<210> 14
<211> 345
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Tyr Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 15
<211> 1035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cctcctacat tttctcctgc tctgctggtc gtgacagagg gcgacaacgc cacctttacc 60
tgctctttct ccaacacctc tgagtctttc gtgctgaact ggtacaggat gtctccttct 120
aaccagaccg ataagctggc tgctttccct gaagatagat ctcagcctgg acaggattgc 180
aggtttagag tcacacagct gcctaacggt cgcgattttc atatgtccgt cgtcagagct 240
aggagaaacg attctggaac ctacctgtgc agcgctattt ctctggctcc ttatatacag 300
atcaaagagt ccctgagagc tgaactgaga gtcacagaaa gaagggctga atcaaagtat 360
ggaccacctt gcccatcctg tccagcacca gagtttctgg gcggaccctc cgtgttcctg 420
tttccaccca agcctaaaga tacactgatg atttcccgca cacctgaagt cacttgcgtg 480
gtcgtggacg tgagccagga ggatccagaa gtccagttca actggtacgt ggacggagtc 540
gaggtgcaca atgccaagac caaaccccgg gaggaacagt ttaacagtac atacagagtc 600
gtgtcagtcc tgactgtgct gcatcaggac tggctgaacg gcaaggagta taagtgcaaa 660
gtgtctaata agggactgcc ttcatccatc gagaaaacaa ttagtaaggc aaaaggccag 720
cctagagaac cacaggtgta cactctgcct ccaagtcagg aggaaatgac taagaaccag 780
gtctcactga cctgtctggt gaaagggttc tatccaagcg atatcgctgt ggagtgggaa 840
tctaatggtc agcccgagaa caattacaag acaactcccc ctgtgctgga cagcgatggc 900
tctttctttc tgtattcccg tctgactgtg gacaagagca ggtggcagga gggaaacgtc 960
tttagctgtt ctgtgatgca cgaagctctg cacaatcatt acacccagaa gagtctgtca 1020
ctgtccctgg gcaaa 1035
<210> 16
<211> 345
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 17
<211> 1035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cctcctacat tttctcctgc tctgctggtc gtgacagagg gcgacaacgc cacctttacc 60
tgctctttct ccaacacctc tgagtctttc gtgctgaact ggtacaggat gtctccttct 120
aaccagaccg ataagctggc tgctttccct gaagatagat ctcagcctgg acaggattgc 180
aggtttagag tcacacagct gcctaacggt cgcgattttc atatgtccgt cgtcagagct 240
aggagaaacg attctggaac ctacctgtgc gttgctattt ctctggctcc taaaattcag 300
atcaaagagt ccctgagagc tgaactgaga gtcacagaaa gaagggctga atcaaagtat 360
ggaccacctt gcccatcctg tccagcacca gagtttctgg gcggaccctc cgtgttcctg 420
tttccaccca agcctaaaga tacactgatg atttcccgca cacctgaagt cacttgcgtg 480
gtcgtggacg tgagccagga ggatccagaa gtccagttca actggtacgt ggacggagtc 540
gaggtgcaca atgccaagac caaaccccgg gaggaacagt ttaacagtac atacagagtc 600
gtgtcagtcc tgactgtgct gcatcaggac tggctgaacg gcaaggagta taagtgcaaa 660
gtgtctaata agggactgcc ttcatccatc gagaaaacaa ttagtaaggc aaaaggccag 720
cctagagaac cacaggtgta cactctgcct ccaagtcagg aggaaatgac taagaaccag 780
gtctcactga cctgtctggt gaaagggttc tatccaagcg atatcgctgt ggagtgggaa 840
tctaatggtc agcccgagaa caattacaag acaactcccc ctgtgctgga cagcgatggc 900
tctttctttc tgtattcccg tctgactgtg gacaagagca ggtggcagga gggaaacgtc 960
tttagctgtt ctgtgatgca cgaagctctg cacaatcatt acacccagaa gagtctgtca 1020
ctgtccctgg gcaaa 1035
<210> 18
<211> 345
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Glu Ser Pro Ser Gly Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Met Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 19
<211> 1035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cctcctacat tttctcctgc tctgctggtc gtgacagagg gcgacaacgc cacctttacc 60
tgctctttct ccaacacctc tgagtctttc gtgctgaact ggtacaggga atctccttct 120
ggtcagaccg ataagctggc tgctttccct gaagatagat ctcagctggg acaggattgc 180
aggtttagag tcacacagct gcctaacggt cgcgattttc atatgtccgt cgtcagagct 240
aggagaaacg attctggaac ctacatgtgc gttgctattt ctctggctcc taaaattcag 300
atcaaagagt ccctgagagc tgaactgaga gtcacagaaa gaagggctga atcaaagtat 360
ggaccacctt gcccatcctg tccagcacca gagtttctgg gcggaccctc cgtgttcctg 420
tttccaccca agcctaaaga tacactgatg atttcccgca cacctgaagt cacttgcgtg 480
gtcgtggacg tgagccagga ggatccagaa gtccagttca actggtacgt ggacggagtc 540
gaggtgcaca atgccaagac caaaccccgg gaggaacagt ttaacagtac atacagagtc 600
gtgtcagtcc tgactgtgct gcatcaggac tggctgaacg gcaaggagta taagtgcaaa 660
gtgtctaata agggactgcc ttcatccatc gagaaaacaa ttagtaaggc aaaaggccag 720
cctagagaac cacaggtgta cactctgcct ccaagtcagg aggaaatgac taagaaccag 780
gtctcactga cctgtctggt gaaagggttc tatccaagcg atatcgctgt ggagtgggaa 840
tctaatggtc agcccgagaa caattacaag acaactcccc ctgtgctgga cagcgatggc 900
tctttctttc tgtattcccg tctgactgtg gacaagagca ggtggcagga gggaaacgtc 960
tttagctgtt ctgtgatgca cgaagctctg cacaatcatt acacccagaa gagtctgtca 1020
ctgtccctgg gcaaa 1035
<210> 20
<211> 351
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 21
<211> 211
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu
210
<210> 22
<211> 633
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tttacggtta cggttccgaa agacctgtat gtggttgaat acggctctaa tatgacgatt 60
gaatgcaaat tcccggttga aaaacaactg gatctggcgg ccctgattgt gtattgggaa 120
atggaagaca aaaacatcat ccaattcgtg catggcgaag aagatctgaa agttcagcac 180
agctcttacc gtcaacgcgc acgtctgctg aaagaccagc tgagcctggg caatgcagct 240
ctgcagatca cggatgttaa actgcaagac gccggtgtct atcgctgcat gatttcttat 300
ggcggtgcag actacaaacg tatcaccgtc aaagtgaacg ctccgtacaa caaaattaat 360
cagcgcatcc tggtggttga tccggttacg tccgaacatg aactgacctg tcaagcggaa 420
ggctatccga aagccgaagt catttggacc agttccgatc accaggtgct gtcaggtaaa 480
accacgacca cgaactcgaa acgcgaagaa aaactgttta atgtcacgag caccctgcgt 540
attaacacca cgaccaatga aatcttctac tgcacctttc gtcgtctgga cccggaagaa 600
aatcatacgg cggaactggt tatcccggaa ctg 633
Claims (13)
1.一种融合蛋白，其特征在于，所述融合蛋白包含两个高亲和力PD-1分子和IgG分子的Fc片段，其中：
所述高亲和力PD-1分子的氨基酸序列选自SEQ ID NO: 6并且所述融合蛋白中两个高亲和力的PD-1分子通过IgG分子的Fc片段铰链区的二硫键连接形成二聚体结构。
2.如权利要求1所述的融合蛋白，其特征在于，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。
3.如权利要求1所述的融合蛋白，其特征在于，所述的IgG的Fc片段选自人IgG1、IgG2、IgG3或IgG4的Fc片段。
4.如权利要求3所述的融合蛋白，其特征在于，所述的IgG的Fc片段选自IgG4的Fc片段。
5.如权利要求1所述的融合蛋白，其特征在于，所述Fc片段包括铰链区、CH2和CH3结构域。
6.如权利要求5所述的融合蛋白，其特征在于，所述Fc片段不包含CH1和CH4结构域。
7.如权利要求6所述的融合蛋白，其特征在于，所述Fc片段的氨基酸序列为SEQ ID NO:7。
8.一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1-7中任一项所述融合蛋白的核酸序列或其互补序列。
9.一种载体，其特征在于，所述载体含有权利要求8所述的核酸分子。
10.一种宿主细胞，其特征在于，所述宿主细胞中含有权利要求9中所述载体或染色体中整合有外源的权利要求8中所述的核酸分子。
11.一种药物组合物，其特征在于，所述组合物中含有权利要求1-7中任一项所述的融合蛋白，和任选的药学上可接受的载体或赋形剂。
12.权利要求1-7中任一项所述的融合蛋白或权利要求11中所述的药物组合物在制备治疗表达PD-L1分子的肿瘤的药物中的用途。
13.一种制备权利要求1-7中任一所述融合蛋白的方法，包括步骤：
(a) 培养权利要求10中所述宿主细胞，从而表达权利要求1-7中任一所述融合蛋白；
(b) 分离或纯化所述的融合蛋白。
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