CN111662383A - 抗pd-l1抗体及其应用 - Google Patents
抗pd-l1抗体及其应用 Download PDFInfo
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- CN111662383A CN111662383A CN201910173886.2A CN201910173886A CN111662383A CN 111662383 A CN111662383 A CN 111662383A CN 201910173886 A CN201910173886 A CN 201910173886A CN 111662383 A CN111662383 A CN 111662383A
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Abstract
本发明提供一种针对PD‑L1的抗体分子或其结合片段，包括抗体分子或其结合片段的编码核酸和包含抗体分子或其结合片段的组合物，以及它们增强T细胞免疫应答、治疗疾病的用途。本发明提供的抗体分子或其结合片段能够特异性结合人PD‑L1，阻断PD‑L1和PD‑1的结合，增强T细胞活化，并且显著抑制肿瘤生长。
Description
技术领域
本发明属于生物医药领域，涉及一种新的抗PD-L1抗体或其功能性片段。本发明还涉及所述抗体或其功能性片段的应用。
背景技术
T细胞在对外源抗原做出应答时需要抗原递呈细胞APC向静止的T淋巴细胞提供两个信号：第一个信号是，T细胞借助TCR识别与MHC分子结合的抗原肽、通过TCR/CD3复合体传递的抗原识别信号；第二信号是由一系列的协同刺激分子提供的信号。根据第二信号产生效应不同，可将协同刺激分子分为正性共刺激分子和负性共刺激分子，而正性和负性协同刺激信号的调节及两者之间的平衡在机体免疫应答的整个过程中起着重要的调节作用。
PD-1是CD28受体家族的一员，该家族还包括CTLA4、CD28、ICOS和BTLA。PD-1的配体包括PD-L1和PD-L2，已有的研究结果表明，受体和配体结合后会下调T细胞的活化和相关细胞因子的分泌(Freeman等，(2000)J Exp Med 192:1027-34；Latchman等，(2001)NatImmunol 2:261-8；Carter等，(2002)Eur J Immunol 32:634-43；Ohigashi等，(2005)Clincancer Res11:2947-53)。
PD-L1(B7-H1)是细胞表面糖蛋白，属于B7家族，具有IgV和IgC样区、跨膜区及胞浆区尾部。该基因于1999年首次被发现并克隆(Dong H等，(1999)Nat Med 5:1365-1369)，它与T细胞上的受体PD1相互作用，在免疫应答的负性调控方面发挥着重要作用。PD-L1除了在巨噬细胞谱系的细胞上表达外，在人类正常的组织中表达量较低，但是在一些肿瘤细胞系上却有着较高的表达，例如肺癌、卵巢癌、结肠癌和黑色素瘤(Iwai等，(2002)PNAS99:12293-7；Ohigashi等，(2005)Clin Cancer Res 11:2947-53)。已有的结果显示，肿瘤细胞高表达的PD-L1通过增加T细胞的凋亡从而在肿瘤的免疫逃逸中起着重要的作用。研究者发现，转染PD-L1基因的P815肿瘤细胞系在体外可抵制特异性CTL的裂解，将其接种小鼠体内后具有更强的致瘤性和侵袭性。这些生物学特性均可通过阻断PD-L1而逆转。敲除PD1基因的小鼠，阻断PD-L1/PD-1通路，则接种肿瘤细胞不能形成肿瘤(Dong H等，(2002)Nat Med8:793-800)。
发明内容
本发明的目的是提供针对PD-L1的抗体分子或其结合片段，包括抗体分子或其结合片段的编码核酸和包含抗体分子或其结合片段的组合物，以及它们增强T细胞免疫应答、治疗疾病的用途。本发明提供的抗体分子或其结合片段能够特异性结合人PD-L1，阻断PD-L1和PD-1的结合，增强T细胞活化，并且显著抑制肿瘤生长。
本发明所述“抗体分子或其结合片段”涵盖全长抗体以及其各种功能性片段，例如其抗原结合部分，如Fab、F(ab’)2或scFv片段，以及经过修饰的抗体，例如人源化、糖基化等。
具体而言，本发明提供以下技术方案。
一方面，本发明提供一种能够特异性结合PD-L1的抗体分子或其结合片段，所述抗体分子或其结合片段包含轻链可变区(VL)和/或重链可变区(VH)，其中所述轻链可变区包含以下CDR组合之一：
a.SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；
b.SEQ ID NO:7所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；
c.SEQ ID NO:9所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和
d.SEQ ID NO:29所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；
和/或
所述重链可变区包含以下CDR组合之一：
A.SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:6所示的VH-CDR3；
B.SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:8所示的VH-CDR3；和
C.SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3。
优选地，本发明提供的能够特异性结合PD-L1的抗体分子或其结合片段包含轻链可变区和重链可变区，所述轻链可变区和重链可变区包含以下CDR组合之一：
1.SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；和，SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:6所示的VH-CDR3；
2.SEQ ID NO:7所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；和，SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:8所示的VH-CDR3；
3.SEQ ID NO:9所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和，SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3；
4.SEQ ID NO:29所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和，SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3。
优选地，在所述抗体分子或其结合片段中，所述轻链可变区包含选自SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:24和SEQ IDNO:28中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和/或
所述重链可变区包含选自SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ IDNO:22、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列。
更优选地，所述抗体分子或其结合片段包含以下轻链可变区和重链可变区组合之一：
I.SEQ ID NO:15所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:16所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
II.SEQ ID NO:17所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:18所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
III.SEQ ID NO:19所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:20所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
IV.SEQ ID NO:21所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:22所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
V.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:22所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VI.SEQ ID NO:24所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:25所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VII.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:26所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VIII.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:27所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
IX.SEQ ID NO:28所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:25所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列。
本发明所述的“至少75％同一性”包括至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少100％以及前述范围内的任何数值。
本发明所述抗体分子或其结合片段可以是具有相应活性的任何抗体形式或片段。例如，所述抗体分子或其结合片段为半抗体或半抗体的抗原结合片段，优选地，为Fab、Fab’、F(ab’)2、Fv或单链Fv(scFv)片段；
优选地，本发明所述抗体分子或其结合片段还包含人或鼠恒定区；
优选地，所述抗体分子或其结合片段为抗体，优选为鼠源抗体、人源化抗体或者经脱氨突变或糖基化位点突变的优化抗体；
其中，所述优化抗体为将人源化抗体的VH中具有高脱氨风险的位点进行突变得到；根据本发明的具体实施方式，所述优化抗体相对于未经优化的抗体具有VH中的N43Q或G44D突变；
或者，所述优化抗体为将人源化抗体的VL中糖基化位点进行突变得到；根据本发明的具体实施方式，所述优化抗体相对于未经优化的抗体具有VL中的N28G突变。
优选地，所述抗体分子或其结合片段还包含轻链恒定区(CL)和/或重链恒定区(CH)。
优选地，所述抗体分子或其结合片段包含选自(例如人的)IgG(包括IgG1、IgG2、IgG3和IgG4)、IgA、IgM、IgD或IgE的重链恒定区和/或(例如人的)κ或λ型轻链恒定区。
根据本发明的具体实施方式，所述抗体分子或其结合片段选自抗体5A5、7A3、8G1、7A3 1-3、7A3 3-3、8G1 3-3、7A3 3-3ND、7A3 3-3QG和8G1 3-3NG。
另一方面，本发明还提供一种核酸分子，其编码本发明提供的抗体分子或其结合片段或者编码所述抗体分子或其结合片段中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链。
还一方面，本发明提供一种载体，其包含本发明提供的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或以任何方式进入宿主细胞内，用于保存或表达抗体等目的。因此，另一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
又一方面，本发明还提供一种药物组合物，其包含本发明提供的抗体分子或其结合片段、核酸分子或载体，以及任选地药学上可接受的载体、赋形剂和/或稳定剂。
再一方面，本发明提供所述的抗体分子或其结合片段、核酸分子、载体或药物组合物在制备药物中的用途，所述药物用于治疗与PD-L1高表达相关的疾病；
优选地，所述疾病为癌症；更优选地，所述疾病选自肺癌、卵巢癌、结肠癌、黑色素瘤、膀胱癌、前列腺癌、肝癌、胃癌、肾癌、乳腺癌、头颈癌、淋巴瘤和Merkel细胞癌等。
或者，本发明提供所述的抗体分子或其结合片段、核酸分子或载体在制备药物中的用途，所述药物用于增强T细胞免疫应答或增强T细胞活化；
优选地，所述药物用于增加T细胞的细胞因子产生，所述细胞因子优选为IFN-γ。
又一方面，本发明提供了一种用于在有此需要的受试者中治疗或预防疾病的方法，其包括将本发明提供的抗体分子或其结合片段、核酸分子、载体或药物组合物施用给所述受试者。优选地，所述疾病为癌症；更优选地，所述疾病选自肺癌、卵巢癌、结肠癌、黑色素瘤、膀胱癌、前列腺癌、肝癌、胃癌、肾癌、乳腺癌、头颈癌、淋巴瘤和Merkel细胞癌等。
另一方面，本发明提供了用于在有此需要的受试者中增强T细胞免疫应答或增强T细胞活化的方法，其包括将本发明提供的抗体分子或其结合片段、核酸分子、载体或药物组合物施用给所述受试者。优选地，所述方法用于增加T细胞的细胞因子产生，所述细胞因子优选为IFN-γ。
在本发明提供的方法中，受试者可以是哺乳动物，尤其是人；可选地。本发明提供的方法为体外方法。
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示出了抗体5A5、7A3、8G1特异性结合PD-L1能力检测(ELISA法)结果。
图2示出了抗体5A5、7A3、8G1特异性结合细胞表面PD-L1能力检测(FACS法)结果。
图3示出了抗体5A5、7A3、8G1阻断能力检测(FACS法)结果。
图4示出了抗体5A5、7A3、8G1阻断能力检测(ELISA法)结果。
图5示出了抗体5A5、7A3、8G1特异性结合PD-L1能力检测(FACS法)结果。
图6示出了抗体5A5、7A3、8G1体外增加T细胞活化能力检测(MLR)结果。
图7示出了人源化抗体7A3 1-3、7A3 3-3和8G1 3-3特异性结合PD-L1能力检测(ELISA法)结果。
图8示出了人源化抗体7A3 1-3、7A3 3-3和8G1 3-3阻断能力检测(ELISA法)结果。
图9示出了人源化抗体7A3 1-3、7A3 3-3和8G1 3-3特异性结合PD-L1能力检测(FACS法)结果。
图10示出了人源化抗体7A3 1-3、7A3 3-3和8G1 3-3体外增加T细胞活化能力检测(MLR)结果。
图11示出了人源化抗体7A3 1-3(11-A)、7A3 3-3(11-B)和8G1 3-3(11-C)热稳定性检测(DSC)结果。
图12示出了人源化抗体7A3 3-3经脱氨突变后特异性结合PD-L1能力检测(ELISA法)结果。
图13示出了人源化抗体7A3 3-3经脱氨突变后体外增加T细胞活化能力检测(MLR)结果。
图14示出了人源化抗体7A3 3-3(14-A)以及经脱氨突变后(14-B、14-C)的脱氨分析结果。
图15示出了人源化抗体8G1 3-3NG和7A3 1-3抗肿瘤活性检测结果。
图16示出了人源化抗体7A3 3-3ND和7A3 3-3QG抗肿瘤活性检测结果。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中，
实施例1重组人PD-L1、PD-1的表达和相关EGFP细胞制备
根据蛋白数据库Uniprot上人PD-L1的氨基酸序列(Q9NZQ7)，得到人PD-L1胞外结构域的氨基酸序列(即Q9NZQ7中第1位残基至238位残基)；根据蛋白数据库Uniprot上人免疫球蛋白gamma1(IgG1)的恒定区氨基酸序列(P01857)，得到人IgG1-Fc的结构域氨基酸序列(即P01857中第104位残基至330位残基)；根据蛋白数据库Uniprot上鼠免疫球蛋白gamma1(IgG1)的恒定区氨基酸序列(P01868)，得到鼠IgG1-Fc的结构域氨基酸序列(即P01868中第98位残基至324位残基)。利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到hPD-L1-Fc、hPD-L1-muFc融合蛋白的基因，按照同样的方法得到hPD-1-Fc的基因。根据蛋白数据库Uniprot上信息得到增强型绿色荧光蛋白EGFP氨基酸序列(C5MKY7)、人PD-L1的氨基酸序列(Q9NZQ7)、鼠PD-L1的氨基酸序列(Q9EP73)、人PD-1的氨基酸序列(Q15116)。利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到hPD-L1-EGFP融合蛋白的基因，按照同样的方法得到hPD-1-EGFP和mPD-L1-EGFP的基因。通过人工合成的方式得到其DNA片段。合成好的基因序列分别经Fermentas公司的HindIII与EcoRI双酶切亚克隆到商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-hPD-L1-Fc、pcDNA4-hPD-L1-muFc、pcDNA4-hPD-1-Fc、pcDNA4-hPD-L1-EGFP、pcDNA4-hPD1-EGFP、pcDNA4-mPD-L1-EGFP。
利用反转录-聚合酶链式反应RT-PCR技术从实验室培养的树突状细胞(DC细胞)(该DC细胞由PBMC中分离的单核细胞经过TNF-α成熟而来)中扩增人的PD-L2、B7H3和B7H4基因，扩增引物如下：
PD-L2-F HindIII：
GCGCAAGCTTGCCACCATGATCTTCCTCCTGCTAATG(SEQ ID NO:44)，
PD-L2-R EcoI：GCCGAATTCGATAGCACTGTTCACTTCCCTC(SEQ ID NO:45)；
hB7H3-F HindIII：
GCGCAAGCTTGCCACCATGCTGCGTCGGCGGGGCAGC(SEQ ID NO:46)，
hB7H3-R EcoRI：
GCGCGAATTCGGCTATTTCTTGTCCATCATCTTC(SEQ ID NO:47)；
hB7H4-F HindIII：
GCGCaagcttGCCACCATGGCTTCCCTGGGGCAGATCC(SEQ ID NO:48)，
hB7H4-R EcoRI：
GCCgaattcTTTTAGCATCAGGTAAGGGCT(SEQ ID NO:49)
得到的PCR产物经Fermentas公司的HindIII与EcoRI双酶切亚克隆到已构建好的pcDNA4-hPD-L1-EGFP中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-hPD-L2-EGFP、pcDNA4-hB7H3-EGFP、pcDNA4-hB7H4-EGFP。
将相关的EGFP重组质粒转染到HEK293(ATCC，CRL-1573TM)细胞中，转染48h后通过荧光激活信号分选(FACS)确认hPD-1、hPD-L1、mPD-L1、hPD-L2、hB7H3和hB7H4的表达。
将pcDNA4-hPD-L1-Fc、pcDNA4-hPD-L1-muFc、pcDNA4-hPD1-Fc瞬时转染至HEK293细胞中用于蛋白生产。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L类胰岛素生长因子-1(insulin-like growth factor-1，IGF-1)。4小时后再补加EX293培养基，2mM谷氨酰胺和50μg/L IGF-1，135rpm培养。24小时后加3.8mM丙戊酸钠(VPA)。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，初步纯化得到hPD-L1-Fc、hPD-L1-muFc和hPD-1-Fc蛋白样品，用于以下各实施例。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰的看到目的条带。
实施例2抗PD-L1抗体的产生
用rhPD-L1蛋白(Acro Biosystems,Cat#PD1-H5229)免疫8周龄Balb/c小鼠(中山大学实验动物中心)，两次免疫后用ELISA方法进行抗体效价检测。
ELISA检测方法：在ELISA板上包被1μg/ml的rhPD-L1，100μl/孔，37℃孵育2h。用10mM，pH7.4PBS/Tween(0.5％)洗1次，用含有1％BSA的PBST封闭，37℃孵育2h；洗板3次后加入梯度稀释的小鼠血清，37℃孵育1h，洗板3次，加入100μl羊抗鼠IgG-HRP酶标二抗(CellSignaling,
Cat#7076S,Lot#31)，37℃孵育45min，清洗3次后，用TMB底物(TIANGEN,Cat#PA107-02,Lot#P5230)37℃孵育10min，加入50μl 2M H2SO4终止反应，在OD 450nm/620nm处进行分光光度分析(TECAN,INFINITE F50)。
将产生抗PD-L1抗体滴度最高的4号小鼠强化免疫，1周后用于融合，按照下文所述进行融合，并通过ELISA和FACS检测杂交瘤上清液。
按照标准方案，使用PEG融合方法将脾脏细胞和小鼠骨髓瘤细胞SP2/0进行融合。取脾细胞和SP2/0细胞按照脾细胞:SP2/0细胞＝10:1的比例进行融合，400g离心5min后，弃上清。加入1ml预热的PEG，混匀后静置90s，缓慢加入30ml预热的不加血清的IMDM培养基，400g离心5min，弃上清。分多次加入30ml HAT培养液，轻轻吹吸沉淀，使细胞分散，将分散后的细胞悬液加入96孔培养板中，然后将培养板放置于37℃，5％CO2培养箱中进行培养。1周后利用ELISA方法对杂交瘤上清进行结合抗原能力检测。对具有结合能力的杂交瘤上清进行阻断能力检测，各检测方法如下：
ELISA结合检测方法：在ELISA板上包被1μg/ml的rhPD-L1，100μl/孔，37℃孵育2h。用10mM，pH7.4PBS/Tween(0.5％)洗1次，用含有1％BSA的PBST封闭，37℃孵育2h；洗板3次后加入杂交瘤上清，37℃孵育1h，洗板5次，加入抗小鼠IgG-HRP酶标二抗，37℃孵育40min，清洗后，用TMB底物对板进行显色，并在OD 450nm/620nm处进行分光光度分析。挑选OD>1.2的克隆进行FACS结合检测。
阻断PD-L1和PD-1结合能力检测：以包被缓冲液(50mM Na2CO3，NaHCO3，pH9.6)稀释hPD-L1-Fc至5μg/ml，100μl/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将含抗hPD-L1抗体的杂交瘤上清梯度稀释至含10μg/ml的PD-1-Biotin的缓冲液中，抗体起始浓度是100μg/ml，6倍稀释，共11个浓度，37℃孵育2h。加入SA-HRP(eBioscience，Cat.No:18-4100)，100μl/孔，37℃孵育1h。R&D显色液显色7min，加入2N H2SO4 50ul/孔，终止显色反应。置MDSpectraMax Plus384酶标仪上读OD 450nm-570nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析。
经过以上三种活性检测方法测定，经三轮亚克隆后最后挑选来自三株杂交瘤克隆细胞的抗体5A5、7A3、8G1进行鼠源抗体的表征。
实施例3抗PD-L1鼠源抗体的表征
复苏冻存杂交瘤细胞，在含10％FBS(Gibco，Cat:10437-028)的IMDM(Gibco，Cat:SH30228.01)中培养，待细胞状态恢复，以1×105～1.5×105个细胞/ml的密度接种于CDHybridoma Medium(Gibco，Cat:11279-023)中，37℃8％CO2条件培养6～7天，待细胞活率低于80％，收培养上清、protein A珠子(Tribipscience,Cat#TBS9210-25)纯化。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰的看到目的条带。
抗PD-L1抗体特异性结合PD-L1能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3，pH9.6)稀释hPD-L1-Fc至5μg/ml，100μL/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将抗hPD-L1抗体分别从10μg/ml开始，进行3倍梯度稀释，共11个浓度，稀释液(1％BSA-PBS)作对照，37℃孵育2h。加入羊抗鼠IgG-HRP(Goatanti-mouse IgG-HRP conjugated)，37℃孵育1h。加可溶性单组分TMB底物显色液，室温避光显色5-10min。2N H2SO4 50μl/孔，终止显色反应。置MD SpectraMaxPlus384酶标仪上读OD450nm-650nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图1所示。从结果中可以看出来自三株杂交瘤的抗体均能结合抗原。
抗PD-L1抗体特异性结合细胞表面PD-L1能力检测(FACS法)：
取新鲜制备的PD-L1-EGFP细胞，PBS洗2次，加入纯化后的抗体，起始浓度是20μg/ml，8倍稀释，5个梯度；冰上孵育30min，PBST洗2次，加入抗mIg-PE二抗，0.3μl/测试，冰上孵育30min，PBS洗2次，FACS检测。结果如下表1和图2所示。从结果可以看出来自三株杂交瘤的抗体均能结合细胞表面的PD-L1。
表1抗PD-L1抗体结合细胞表面PD-L1能力检测
| 5A5 | 7A3 | 8G1 | |
| EC50(μg/ml) | 1.329 | 2.758 | 2.247 |
抗PD-L1抗体阻断能力检测(FACS法)：
取新鲜制备的hPD-L1-EGFP细胞，PBS洗2次，将20μg/ml的抗PD-L1抗体(5A5、7A3、8G1)稀释至5μg/ml的PD-1-Biotin中，冰上孵育30min，PBS洗2次，加入二抗SA-APC，0.3μl/测试，冰上孵育30min，PBS洗2次，FACS检测，计算各组的MFI。结果如图3所示。
抗PD-L1抗体阻断能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3，pH9.6)稀释hPD-L1-Fc至5μg/ml，100μL/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将抗hPD-L1抗体梯度稀释至含10μg/ml的PD-1-Biotin的缓冲液中，抗体起始浓度是100μg/ml，6倍稀释，共11个浓度，37℃孵育2h。加入SA-HRP(eBioscience，Cat.No:18-4100)，100μl/孔，37℃孵育1h。R&D显色液显色7min，加入2NH2SO4 50μl/孔，终止显色反应。置MD SpectraMax Plus384酶标仪上读OD 450nm-570nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图4所示。
抗PD-L1抗体特异性结合PD-L1能力检测(FACS法)：
取新鲜制备的hPD-L1-EGFP细胞、hPD-1-EGFP细胞、hB7H3-EGFP细胞、hB7H4细胞、mPD-L1细胞，PBS洗2次，加入纯化后的抗体，浓度是10μg/ml，冰上孵育30min，PBS洗2次，加入抗mIg-PE二抗，0.3μl/测试，冰上孵育30min，PBS洗2次，FACS检测。结果如图5所示。从结果可以看出，来自三株杂交瘤的抗体均可以特异性结合hPD-L1，不与同家族的其他抗原结合，同时不能结合mPD-L1。
抗PD-L1抗体结合PD-L1动力学检测(BLI)：
抗PD-L1抗体针对重组的人PD-L1-His的结合动力学通过生物膜干涉(BLI)方法，使用Octet K2仪器测量。将待测抗体用SD缓冲液稀释至20μg/ml，并偶联至AMC传感器上。hPD-L1-His抗原用SD缓冲液梯度稀释，从100nM开始，2倍稀释，6个梯度。设置Octet程序进行动力学检测，用分析软件打开数据，分析结果，拟合动力学曲线，结果如下表2所示。
表2抗PD-L1抗体结合hPD-L1动力学检测
| 名称 | Ka(1/Ms) | Kd(1/s) | KD(M) |
| 8G1 | 5.04E+05 | 1.17E-03 | 2.32E-09 |
| 7A3 | 8.40E+05 | 3.60E-03 | 4.29E-09 |
| 5A5 | 7.88E+05 | 4.53E-03 | 5.75E-09 |
抗PD-L1抗体体外活性检测(MLR)：
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC。然后将其重悬于无血清的RPMI 1640中，在10cm培养皿中培养1-2小时，除去未贴壁的细胞，并将细胞培养于含10％FBS的RPMI中。以250ng/ml GM-CSF(上海普欣，货号：102-03)和100ng/ml IL-4(上海普欣，货号：101-04)的终浓度添加细胞因子，每2-3天添加含细胞因子的新鲜培养基。在培养的第6天，用50ng/ml的TNF-alpha(上海普欣，货号：103-01)使细胞成熟并使其孵育24小时。收获成熟的树突细胞，用HLA-DR抗体染色确定其成熟。将其重悬于RPMI完全培养基中，20万个细胞/ml，然后在96孔U形底板(Costar，Cat.No:3799)中每孔加入50μl，放培养箱中培养。
利用磁珠分离试剂盒(Miltenyi Biotec，Cat.No：130-096-533)按照说明书方法从另一个供体PBMC中分离CD4+T细胞。计数重悬到RPMI完全培养基中，浓度为200万个细胞/ml，然后加入到含有树突细胞的96孔U形底板，每孔加入50μl。每孔加入梯度稀释于RPMI完全培养基中的PD-L1抗体100μl，抗体终浓度分别为100、10、1、0.1、0.01、0.001、0μg/ml。培养5天后取上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平结果，如下表3和图6所示。从结果可以看出，抗PD-L1抗体可以增加混合淋巴细胞反应中IFN-γ的分泌，也就是说抗PD-L1抗体增加了T细胞的活化。
表3抗PD-L1抗体体外活性检测
| 5A5 | 7A3 | 8G1 | |
| EC50(μg/ml) | 0.1039 | 0.05876 | ～0.03001 |
综合以上结果可以得出，抗PD-L1抗体5A5、7A3、8G1可以阻断hPD-L1和hPD-1的结合，并且能在体外刺激T细胞的增殖，使IFN-γ的分泌增加。对抗体5A5、7A3、8G1进行测序，序列如下所示，下划线区域为CDR。
>5A5VH
重链可变区编码序列：
GAGGTGCAACTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAATACATGGGGTACATAAGCTACACTGGTAGCACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTGACTACTGAAGACACAGCCACATATTACTGTGCAAAATACGGGCTATGGCACCTACCGGCGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:31)
重链可变区序列：
EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCAKYGLWHLPAAMDYWGQGTSVTVSS(SEQ ID NO:16)
>5A5VL
轻链可变区编码序列：
GATATCGTTCTCACTCAATCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTATAAGTTACATGCACTGGTATCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGCACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGTTACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ ID NO:30)轻链可变区序列：
DIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPFTFGSGTKLEIK(SEQ ID NO:15)
>7A3VH
重链可变区编码序列：
GAGGTGCAGCTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATGATCTTGAATACATGGGGTACATAAGCTACACTGGTAGCACTTACTACAATCCGTCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTCGGCCTATGGCACCTACCGGCGGCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:33)
重链可变区序列：
EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNDLEYMGYISYTGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARFGLWHLPAALDYWGQGTSVTVSS(SEQ ID NO:18)
>7A3VL
轻链可变区编码序列：
GATATCGTGCTCACTCAATCTCCAGCAATCATGTCTGCTTCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAAGTCAAGTATAAGTTACATGCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGCTACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ ID NO:32)轻链可变区序列：
DIVLTQSPAIMSASPGEKVTMTCSAKSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPFTFGSGTKLEIK(SEQ ID NO:17)
>8G1VH
重链可变区编码序列：
AAGGTCCAGCTGCGGCAGTCTGGAGCTGAGCTGGTGAAACCCGGGACATCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACTGAATATATTATACACTGGGTAAAGCAGAGGTCTGGACAGGGTCTTGAGTGGATTGGGTGGTTTTACCCTGGAAGTGGTAATATAAGGTACAATGAGAAATTCAAGGACAAGGCCACATTGACTGCGGACAAATCCTCCAGCACAGTCTATATGGAACTTAGTAGATTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACACGAAGATAAAGGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:35)
重链可变区序列：
KVQLRQSGAELVKPGTSVKLSCKASGYTFTEYIIHWVKQRSGQGLEWIGWFYPGSGNIRYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHEDKGAWFAYWGQGTLVTVSA(SEQ ID NO:20)
>8G1VL
轻链可变区编码序列：
GATATCGTGCTCACCCAATCTCCAGCACTCTTGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAATGTAAGTTACATGTACTGGTACCAGCAGAAGCCAAGATCCTCCCCCAAACCCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCACTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGGTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGGTGAAA(SEQ ID NO:34)
轻链可变区序列：
DIVLTQSPALLSASPGEKVTMTCSASSNVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFTGSGSGTSYSLTISSMEAEDGATYYCQQWSSNPLTFGAGTKLEVK(SEQ ID NO:19)
将抗体5A5、7A3、8G1的轻链可变区与重链可变区以及其中的CDR总结于下表4。
表4抗体5A5、7A3、8G1的相关序列
实施例4鼠源抗体的人源化及表征
抗PD-L1抗体的人源化及相关质粒构建
将两株杂交瘤克隆的抗体7A3、8G1按照传统的杂交瘤测序的方法进行VH和VL序列鉴定，得到序列后，将7A3和8G1的轻链可变区VL和重链可变区VH各人源化两个版本，即7A3VHV1，7A3VHV3，7A3VLV1，7A3VLV3，8G1VHV1，8G1VHV3，8G1VLV1，8G1VLV3。相关基因合成后，与之前的人IgG1-Fc基因融合，然后经Fermentas公司的HindIII和EcoRI双酶切克隆至商业化载体pcDNA3.1中，按照分子克隆的标准进行克隆和质粒提取，提取后的质粒在293细胞中瞬时表达，并通过protein A柱纯化。人源化后载体命名及轻重链搭配情况见下表5。
表5抗PD-L1人源化抗体的信息
| 人源化抗体名称 | 轻链名称 | 重链名称 |
| 7A3 1-1 | 7A3VLV1 | 7A3VHV1 |
| 7A3 1-3 | 7A3VLV1 | 7A3VHV3 |
| 7A3 3-1 | 7A3VLV3 | 7A3VHV1 |
| 7A3 3-3 | 7A3VLV3 | 7A3VHV3 |
| 8G1 1-1 | 8G1VLV1 | 8G1VHV1 |
| 8G1 1-3 | 8G1VLV1 | 8G1VHV3 |
| 8G1 3-1 | 8G1VLV3 | 8G1VHV1 |
| 8G1 3-3 | 8G1VLV3 | 8G1VHV3 |
人源化抗体特异性结合PD-L1能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3，pH9.6)稀释hPD-L1-Fc至5μg/ml，100μL/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将人源化后的8株抗体分别从10μg/ml开始，进行3倍梯度稀释，共11个浓度，稀释液(1％BSA-PBS)作对照，37℃孵育2h。加入羊抗人IgG-HRP(Goat anti-human IgG-HRP conjugated)，37℃孵育1h。加可溶性单组分TMB底物显色液，室温避光显色5-10min。2N H2SO4 50μl/孔，终止显色反应。置MD SpectraMax Plus384酶标仪上读OD 450nm-650nm值，应用软件SoftMax Prov5.4进行数据处理和作图分析。ELISA结果显示只有7A3 1-3、7A3 3-3和8G1 3-3保留了对PD-L1抗原高亲和力的结合，结果如图7所示。
人源化抗体阻断能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3，pH9.6)稀释hPD-L1-Fc至5μg/ml，100μL/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将8G1 3-3、7A3 3-3和7A3 1-3三株抗体分别梯度稀释至含10μg/ml的PD-1-Biotin的缓冲液中，抗体起始浓度是100μg/ml，6倍稀释，共11个浓度，37℃孵育2h。加入SA-HRP(eBioscience，Cat.No:18-4100)，100μl/孔，37℃孵育1h。R&D显色液显色7min，加入2N H2SO4 50μl/孔，终止显色反应。置MDSpectraMax Plus384酶标仪上读OD 450nm-570nm值，应用软件SoftMax Prov5.4进行数据处理和作图分析，结果如下表6和图8所示。
表6抗PD-L1人源化抗体阻断能力的IC50
| 抗体名称 | IC50(μg/ml) |
| 7A3 1-3 | 1.817 |
| 7A3 3-3 | 1.442 |
| 8G1 3-3 | 1.244 |
人源化抗体特异性结合PD-L1能力检测(FACS法)：
按照实施例3中的实验方法对人源化抗体特异性结合进行检测，结果如图9所示。
人源化后抗体结合PD-L1动力学检测(BLI)：
人源化后抗体针对重组的人PD-L1-His的结合动力学通过生物膜干涉(BLI)方法，使用Octet K2仪器测量。将待测抗体用SD缓冲液稀释至20μg/ml，并偶联至AHC传感器上。hPD-L1-His抗原用SD缓冲液梯度稀释，从100nM开始，2倍稀释，6个梯度。设置Octet程序进行动力学检测，用分析软件打开数据，分析结果，拟合动力学曲线，结果如下表7所示。
表7人源化抗体结合hPD-L1动力学检测
| 名称 | KD(M) | Kon(1/Ms) | Kd(1/s) |
| 7A3 3-3 | 2.85E-09 | 4.49E+05 | 1.28E-03 |
| 8G1 3-3 | 1.63E-11 | 3.55E+05 | 5.79E-06 |
| 7A3 1-3 | 2.36E-08 | 5.04E+05 | 1.19E-02 |
人源化抗体体外活性检测(MLR)：
按照实施例3中检测活性的方法对人源化抗体的活性进行检测，结果如下表8和图10所示。从结果可以看出，7A3 1-3、7A3 3-3和8G1 3-3三株人源化抗体在体外可以促进T细胞的活化，使IFN-γ的分泌增加。
表8人源化抗体结合体外活性检测
| 7A3 1-3 | 7A3 3-3 | 8G1 3-3 | |
| EC50(μg/ml) | ～0.1758 | 0.4490 | ～1.115e+011 |
人源化抗体稳定性检测(DSC)：
对7A3 1-3、7A3 3-3、8G1 3-3三株抗体进行热稳定性检测，结果如图11所示。
由以上结果可以看出，抗体7A3 1-3，7A3 3-3和8G1 3-3保留了高亲和力结合抗原的能力，并能阻断PD-1和PD-L1的结合。三株抗体序列如下：
>7A3 1-3VH
重链可变区编码序列：
CAGGTGCAGCTGCAGGAGAGCGGACCTGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGACAGCATCACCAGCGGCTACTGGAACTGGATCAGGAAGCCCCCCGGCAATGGCCTGGAGTACATGGGCTACATCAGCTACACCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGATCACCATCACCAGGGACACCAGCAAGAACCAGTACAGCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACCGCCGTGTACTACTGCGCCAGATTCGGCCTGTGGCACCTGCCTGCCGCCCTGGATTACTGGGGACAGGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNO:37)
重链可变区序列：
QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRKPPGNGLEYMGYISYTGSTYYNPSLKSRITITRDTSKNQYSLKLSSVTAADTAVYYCARFGLWHLPAALDYWGQGTLVTVSS(SEQ ID NO:22)
>7A3 1-3VL
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCCACACTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGCGCCAAGAGCAGCATCAGCTACATGCACTGGTATCAACAGAAGCCTGGACAGGCCCCCAGGCTGCTGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCTGCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTTACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGAGGAGCAGCTACCCCTTCACCTTCGGCCAGGGCACAAAGGTGGAGATCAAG(SEQ ID NO:36)
轻链可变区：
DIVLTQSPATLSLSPGERATLSCSAKSSISYMHWYQQKPGQAPRLLIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPFTFGQGTKVEIK(SEQ ID NO:21)
>7A3 3-3VH
重链可变区编码序列：
CAGGTGCAGCTGCAGGAGAGCGGACCTGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGACAGCATCACCAGCGGCTACTGGAACTGGATCAGGAAGCCCCCCGGCAATGGCCTGGAGTACATGGGCTACATCAGCTACACCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGATCACCATCACCAGGGACACCAGCAAGAACCAGTACAGCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACCGCCGTGTACTACTGCGCCAGATTCGGCCTGTGGCACCTGCCTGCCGCCCTGGATTACTGGGGACAGGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNO:37)
重链可变区序列：
QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRKPPGNGLEYMGYISYTGSTYYNPSLKSRITITRDTSKNQYSLKLSSVTAADTAVYYCARFGLWHLPAALDYWGQGTLVTVSS(SEQ ID NO:22)
>7A3 3-3VL
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCTACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGCGCCAAGAGCAGCATCAGCTACATGCACTGGTATCAACAGAAGCCCGGCACCAGCCCTAAGAGGTGGATCTACGACACAAGCAAGCTGGCCAGCGGCGTGCCTGCCAGATTTAGCGGCAGCGGCAGCGGCACCAGCTACACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGAGGAGCAGCTACCCCTTCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG
(SEQ ID NO:38)
轻链可变区序列：
DIVLTQSPATLSLSPGERATLSCSAKSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCHQRSSYPFTFGQGTKVEIK(SEQ ID NO:23)
>8G1 3-3VH
重链可变区编码序列：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCTGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGAGTACATCATCCACTGGGTGAAGCAGGCCCCTGGCCAGGGCCTGGAATGGATCGGCTGGTTCTACCCCGGCAGCGGCAACATCAGGTACAACGAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTTCTGCGCCAGACACGAGGACAAGGGCGCCTGGTTTGCCTACTGGGGCCAGGGCACACTGGTGACCGTGAGCAGC(SEQ ID NO:40)
重链可变区序列：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYIIHWVKQAPGQGLEWIGWFYPGSGNIRYNEKFKDKATLTADKSSSTVYMELSSLRSEDTAVYFCARHEDKGAWFAYWGQGTLVTVSS(SEQ ID NO:25)
>8G1 3-3VL
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCTACCCTGAGCCTGAGCCCCGGAGAGAGAGCCACCCTGAGCTGCAGCGCCAGCAGCAACGTGAGCTACATGTACTGGTATCAACAGAAGCCCGGCCAGAGCCCCAAACCCTGGATCTACCTGACCAGCAATCTGGCCAGCGGCGTGCCTGCCAGATTTACCGGCAGCGGCAGCGGCACCAGCTACACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:39)
轻链可变区序列：
DIVLTQSPATLSLSPGERATLSCSASSNVSYMYWYQQKPGQSPKPWIYLTSNLASGVPARFTGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK(SEQ ID NO:24)
将人源化抗体7A3 1-3、7A3 3-3和8G1 3-3的轻链可变区与重链可变区以及其中的CDR总结于下表9。
表9人源化抗体7A3 1-3、7A3 3-3和8G1 3-3的相关序列
实施例5人源化后的抗体7A3 3-3的优化
对抗体7A3 3-3分子重链可变区序列分析发现，该序列FR2中存在着高脱氨风险的位点即N43位，进一步对该位点进行突变，突变成Q得到7A3 3-3QG，或者对G44位进行点突变，突变成D得到7A3 3-3ND，序列如下所示。并且对优化后的蛋白进行活性，稳定性及脱氨情况的分析。结果如下所示。>7A3 3-3ND VH
重链可变区序列：
QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRKPPGNDLEYMGYISYTGSTYYNPSLKSRITITRDTSKNQYSLKLSSVTAADTAVYYCARFGLWHLPAALDYWGQGTLVTVSS(SEQ ID NO:26)
重链可变区编码序列：
CAGGTGCAGCTGCAGGAGAGCGGACCTGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGACAGCATCACCAGCGGCTACTGGAACTGGATCAGGAAGCCCCCCGGCAATGACCTGGAGTACATGGGCTACATCAGCTACACCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGATCACCATCACCAGGGACACCAGCAAGAACCAGTACAGCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACCGCCGTGTACTACTGCGCCAGATTCGGCCTGTGGCACCTGCCTGCCGCCCTGGATTACTGGGGACAGGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:41)
>7A3 3-3ND VL
轻链可变区序列：
DIVLTQSPATLSLSPGERATLSCSAKSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCHQRSSYPFTFGQGTKVEIK(SEQ ID NO:23)
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCTACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGCGCCAAGAGCAGCATCAGCTACATGCACTGGTATCAACAGAAGCCCGGCACCAGCCCTAAGAGGTGGATCTACGACACAAGCAAGCTGGCCAGCGGCGTGCCTGCCAGATTTAGCGGCAGCGGCAGCGGCACCAGCTACACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGAGGAGCAGCTACCCCTTCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:38)>7A3 3-3QG VH
重链可变区序列：
QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRKPPGQGLEYMGYISYTGSTYYNPSLKSRITITRDTSKNQYSLKLSSVTAADTAVYYCARFGLWHLPAALDYWGQGTLVTVSS(SEQ ID NO:27)
重链可变区编码序列：
CAGGTGCAGCTGCAGGAGAGCGGACCTGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGACAGCATCACCAGCGGCTACTGGAACTGGATCAGGAAGCCCCCCGGCCAAGGCCTGGAGTACATGGGCTACATCAGCTACACCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGATCACCATCACCAGGGACACCAGCAAGAACCAGTACAGCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACCGCCGTGTACTACTGCGCCAGATTCGGCCTGTGGCACCTGCCTGCCGCCCTGGATTACTGGGGACAGGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:42)
>7A3 3-3QG VL
轻链可变区序列：
DIVLTQSPATLSLSPGERATLSCSAKSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCHQRSSYPFTFGQGTKVEIK(SEQ ID NO:23)
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCTACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGCGCCAAGAGCAGCATCAGCTACATGCACTGGTATCAACAGAAGCCCGGCACCAGCCCTAAGAGGTGGATCTACGACACAAGCAAGCTGGCCAGCGGCGTGCCTGCCAGATTTAGCGGCAGCGGCAGCGGCACCAGCTACACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGAGGAGCAGCTACCCCTTCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:38)
将人源化抗体7A3 3-3ND和7A3 3-3QG的轻链可变区与重链可变区总结于下表10。
表10人源化抗体7A3 3-3优化后的抗体7A3 3-3ND和7A3 3-3QG的相关序列
7A3 3-3优化后生物学活性检测：
按照实施例4中的操作方法对7A3 3-3ND和7A3 3-3QG两株抗体结合抗原能力、亲和力、体外活性进行了检测。结合抗原能力检测结果见图12，可以看出脱氨突变并没有影响7A3 3-3结合抗原的能力。亲和力的结果如下表11所示，可以看出脱氨突变对抗体的亲和力并没有什么影响。体外阻断PD-1和PD-L1结合的活性结果如下表12和图13所示，可以看出脱氨突变后的两株抗体在高浓度的情况下可以上调IFN-γ的分泌。
表11抗体7A3 3-3突变后亲和力检测
| 名称 | KD(M) | kon(1/Ms) | kd(1/s) |
| 7A3 3-3 | 1.32E-08 | 4.15E+05 | 5.47E-03 |
| 7A3 3-3ND | 1.44E-08 | 4.38E+05 | 6.31E-03 |
| 7A3 3-3QG | 1.39E-08 | 4.67E+05 | 6.51E-05 |
表12抗体7A3 3-3突变后体外活性检测
| 7A3 3-3 | 7A3 3-3ND | 7A3 3-3QG | |
| EC50(μg/ml) | 0.02848 | 0.5757 | 1.048 |
7A3 3-3优化后脱氨分析：
对7A3 3-3及突变后的蛋白进行脱氨分析，样品的处理方法是：样品换液至20mMTris，pH 8.0缓冲液中，并浓缩样品至浓度为10mg/ml；取换液后样品250μg，加入400μl500mM NH4HCO3，不足部分用超纯水补齐，使终体积为500μl；平行处理2份；在37℃下反应，分别于5h和15.5h取样；取样后立即换液至20mM Tris，pH 8.0缓冲液中；换液后冻存至-80℃，至所有样品备齐后一起进行iCIEF检测。结果如图14所示。
实施例6人源化后的抗体8G1 3-3的优化
8G1 3-3分子轻链可变区CDR1中存在着糖基化位点，N28，进一步对该位点进行点突变，N突变成Q，得到8G1 3-3NG，序列如下所示。并且对优化后的蛋白进行活性分析。
>8G1 3-3NG VL
轻链可变区序列：
DIVLTQSPATLSLSPGERATLSCSASSQVSYMYWYQQKPGQSPKPWIYLTSNLASGVPARFTGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK(SEQ ID NO:28)
轻链可变区编码序列：
GACATCGTGCTGACCCAGAGCCCTGCTACCCTGAGCCTGAGCCCCGGAGAGAGAGCCACCCTGAGCTGCAGCGCCAGCAGCCAGGTGAGCTACATGTACTGGTATCAACAGAAGCCCGGCCAGAGCCCCAAACCCTGGATCTACCTGACCAGCAATCTGGCCAGCGGCGTGCCTGCCAGATTTACCGGCAGCGGCAGCGGCACCAGCTACACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:43)
>8G1 3-3NG VH
重链可变区序列：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYIIHWVKQAPGQGLEWIGWFYPGSGNIRYNEKFKDKATLTADKSSSTVYMELSSLRSEDTAVYFCARHEDKGAWFAYWGQGTLVTVSS(SEQ ID NO:25)
重链可变区编码序列：
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCTGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGAGTACATCATCCACTGGGTGAAGCAGGCCCCTGGCCAGGGCCTGGAATGGATCGGCTGGTTCTACCCCGGCAGCGGCAACATCAGGTACAACGAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTTCTGCGCCAGACACGAGGACAAGGGCGCCTGGTTTGCCTACTGGGGCCAGGGCACACTGGTGACCGTGAGCAGC(SEQ ID NO:40)
将人源化抗体8G1 3-3NG的轻链可变区与重链可变区总结于下表13。
表13人源化抗体8G1 3-3优化后的抗体8G1 3-3NG的相关序列
8G1 3-3优化后亲和力检测：
按照实施例4中亲和力检测方法对8G1 3-3NG的亲和力进行检测，结果如下表14所示。
表14抗体8G1 3-3突变后亲和力检测
| 名称 | KD(M) | Kon(1/Ms) | Kd(1/s) |
| 8G1 3-3NG | 6.89E-09 | 2.52E+05 | 1.74E-03 |
从结果中可以看出，去糖基化的突变对亲和力的影响较大。
实施例6抗hPD-L1抗体对免疫缺陷小鼠A375黑色素瘤皮下移植瘤肿瘤生长的抑制活性
由于筛选到的抗体均不能识别小鼠PD-L1，采用免疫缺陷NOD/SCID(非肥胖型糖尿病/重症联合免疫缺陷)小鼠研究其体内活性。通过对NOD/SCID小鼠皮下移植表达人PD-L1的黑色素瘤细胞系A375(ATCC，CRL-1619TM)和人的外周血单个核细胞PBMC的实验来实现此研究目的。A375和PBMC按5：1的比例在注射前混合，总体积100μl皮下注射(含500万个A375，100万个PBMC)，抗体在肿瘤接种第0、7、10、14、21、24、28天腹腔注射给药，剂量为3mg/kg，PBS作为阴性对照。每个实验组4-6只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图，结果如图15所示。可以看出抗体8G13-3NG和7A31-3可以显著抑制肿瘤生长。
实施例7抗hPD-L1抗体对B-hPD-L1人源化小鼠MC38-hPD-L1结肠癌皮下移植瘤肿瘤生长的抑制活性
由于筛选到的抗体均不能识别小鼠PD-L1，因此利用B-hPD-L1人源化小鼠测定抗hPD-L1抗体的抗肿瘤活性。将MC38-hPD-L1结肠癌细胞2×105个/0.1mL接种于雌性B-hPD-1人源化小鼠右侧前胁肋部皮下，待肿瘤生长到约105mm3时按瘤体积分层、随机分组，每组8只，共4组，分别为：溶剂(NaCl)阴性对照组、Atezolizumab(Tecentriq)阳性对照组、7A3 3-3QG组、7A3 3-3ND组。所有组给药途径均为腹腔注射，10mg/kg，每两天给药1次，连续给药8次，末次给药结束后7天结束实验。每周测量肿瘤体积及体重3次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤体积(RTV)、相对肿瘤抑制率(TGI％)、瘤重抑制率(IRTW％)。以TGI％≥60％，且治疗组RTV显著低于溶剂对照组RTV(P<0.05)作为参考标准，即对肿瘤生长具有显著抑制作用。
整个实验过程中，除1只动物因未知原因死亡以外，其余动物精神状态良好。实验结束时(首次给药21天后)，各组动物体重均出现增长，与溶剂对照组相比，不同组别动物体重变化无显著性差异(P>0.05)。
实验结束时，溶剂对照组平均肿瘤体积为1410mm3。阳性对照Atezolizumab组平均肿瘤体积为762mm3，TGI％为54.91％，IRTW％为48.53％，出现肿瘤消退的小鼠比例为16.7％，且该组的RTV显著低于溶剂对照组RTV(P<0.05)，表明Atezolizumab具有明显的抑瘤作用。受试品7A3 3-3QG和7A3 3-3ND各组平均肿瘤体积分别为642mm3、505mm3，TGI％分别为50.12％、61.26％，IRTW％分别为57.77％、56.31％，出现肿瘤消退的小鼠比例分别为0％、0％，且7A3 3-3ND组的RTV显著低于溶剂对照组RTV(P<0.05)，表明7A3 3-3ND具有显著的抑瘤作用，7A3 3-3QG有一定的抑瘤作用，但抑瘤效果不明显。在相同给药剂量(10mg/kg)下，7A3 3-3ND和Atezolizumab组间的RTV比较无显著性差异(P>0.05)，表明7A3 3-3ND和Atezolizumab抑瘤作用相似。
在本实验条件下，受试品7A3 3-3ND可显著抑制肿瘤生长。受试品7A33-3QG具有一定的抑瘤作用，与7A3 3-3ND相比抑瘤作用相对较弱。在相同剂量10mg/kg下7A3 3-3ND与阳性对照Atezolizumab的抑瘤作用相似。两个受试品均未对动物产生毒性作用。抗hPD-L1抗体对B-hPD-L1人源化小鼠MC38-hPD-L1结肠癌皮下移植瘤生长的抑制效果如图16所示。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
序列表
<110> 珠海市丽珠单抗生物技术有限公司
苏州丁孚靶点生物技术有限公司
<120> 抗PD-L1抗体及其应用
<130> LC18110024
<160> 49
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR1
<400> 1
Ser Ala Ser Ser Ser Ile Ser Tyr Met His
1 5 10
<210> 2
<211> 7
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR2
<400> 2
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 3
<211> 9
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR3
<400> 3
His Gln Arg Ser Ser Tyr Pro Phe Thr
1 5
<210> 4
<211> 5
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR1
<400> 4
Ser Gly Tyr Trp Asn
1 5
<210> 5
<211> 16
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR2
<400> 5
Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 6
<211> 12
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR3
<400> 6
Tyr Gly Leu Trp His Leu Pro Ala Ala Met Asp Tyr
1 5 10
<210> 7
<211> 10
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR1
<400> 7
Ser Ala Lys Ser Ser Ile Ser Tyr Met His
1 5 10
<210> 8
<211> 12
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR3
<400> 8
Phe Gly Leu Trp His Leu Pro Ala Ala Leu Asp Tyr
1 5 10
<210> 9
<211> 10
<212> PRT
<213> VL CDR1
<400> 9
Ser Ala Ser Ser Asn Val Ser Tyr Met Tyr
1 5 10
<210> 10
<211> 7
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR2
<400> 10
Leu Thr Ser Asn Leu Ala Ser
1 5
<210> 11
<211> 9
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR3
<400> 11
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210> 12
<211> 5
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR1
<400> 12
Glu Tyr Ile Ile His
1 5
<210> 13
<211> 17
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR2
<400> 13
Trp Phe Tyr Pro Gly Ser Gly Asn Ile Arg Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 14
<211> 10
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH CDR3
<400> 14
His Glu Asp Lys Gly Ala Trp Phe Ala Tyr
1 5 10
<210> 15
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 15
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 16
<211> 120
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 16
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys Tyr Gly Leu Trp His Leu Pro Ala Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 17
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 17
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Lys Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 18
<211> 120
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 18
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Asp Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Phe Gly Leu Trp His Leu Pro Ala Ala Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 19
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 19
Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Asn Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Gly Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Val Lys
100 105
<210> 20
<211> 119
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 20
Lys Val Gln Leu Arg Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Phe Tyr Pro Gly Ser Gly Asn Ile Arg Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg His Glu Asp Lys Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 21
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 21
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Lys Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 22
<211> 120
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Pro Pro Gly Asn Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Phe Gly Leu Trp His Leu Pro Ala Ala Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 23
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 23
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Lys Ser Ser Ile Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 24
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 24
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Asn Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 25
<211> 119
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Ile Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Phe Tyr Pro Gly Ser Gly Asn Ile Arg Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg His Glu Asp Lys Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 26
<211> 120
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 26
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Pro Pro Gly Asn Asp Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Phe Gly Leu Trp His Leu Pro Ala Ala Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 27
<211> 120
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VH
<400> 27
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Pro Pro Gly Gln Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Phe Gly Leu Trp His Leu Pro Ala Ala Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 28
<211> 106
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL
<400> 28
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Gln Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 29
<211> 10
<212> PRT
<213> 人工(artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> VL CDR1
<400> 29
Ser Ala Ser Ser Gln Val Ser Tyr Met Tyr
1 5 10
<210> 30
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 30
gatatcgttc tcactcaatc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aagtataagt tacatgcact ggtatcagca gaagccaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg cacctcttat tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccatcagcgg agtagttacc cattcacgtt cggctcgggg 300
acaaagttgg aaataaaa 318
<210> 31
<211> 360
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 31
gaggtgcaac ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc 60
acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc 120
ccagggaata aacttgaata catggggtac ataagctaca ctggtagcac ttactacaat 180
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gtactacctg 240
cagttgaatt ctgtgactac tgaagacaca gccacatatt actgtgcaaa atacgggcta 300
tggcacctac cggcggctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
<210> 32
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 32
gatatcgtgc tcactcaatc tccagcaatc atgtctgctt ctccagggga gaaggtcacc 60
atgacctgca gtgccaagtc aagtataagt tacatgcact ggtaccagca gaagccaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccatcagcgg agtagctacc cattcacgtt cggctcgggg 300
acaaagttgg aaataaaa 318
<210> 33
<211> 360
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 33
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc 60
acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc 120
ccagggaatg atcttgaata catggggtac ataagctaca ctggtagcac ttactacaat 180
ccgtctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gtactacctg 240
cagttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag attcggccta 300
tggcacctac cggcggctct ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
<210> 34
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 34
gatatcgtgc tcacccaatc tccagcactc ttgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aaatgtaagt tacatgtact ggtaccagca gaagccaaga 120
tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180
ttcactggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatggtgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggg 300
accaagctgg aggtgaaa 318
<210> 35
<211> 357
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 35
aaggtccagc tgcggcagtc tggagctgag ctggtgaaac ccgggacatc agtgaagctg 60
tcctgcaagg cttctggcta caccttcact gaatatatta tacactgggt aaagcagagg 120
tctggacagg gtcttgagtg gattgggtgg ttttaccctg gaagtggtaa tataaggtac 180
aatgagaaat tcaaggacaa ggccacattg actgcggaca aatcctccag cacagtctat 240
atggaactta gtagattgac atctgaagac tctgcggtct atttctgtgc aagacacgaa 300
gataaagggg cctggtttgc ttactggggc caagggactc tggtcactgt ctctgca 357
<210> 36
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 36
gacatcgtgc tgacccagag ccctgccaca ctgagcctga gccctggcga gagagccacc 60
ctgagctgca gcgccaagag cagcatcagc tacatgcact ggtatcaaca gaagcctgga 120
caggccccca ggctgctgat ctacgacacc agcaagctgg ccagcggcgt gcctgctagg 180
tttagcggca gcggcagcgg caccgacttt accctgacca tcagcagcct ggagcccgag 240
gacttcgccg tgtactactg ccaccagagg agcagctacc ccttcacctt cggccagggc 300
acaaaggtgg agatcaag 318
<210> 37
<211> 360
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 37
caggtgcagc tgcaggagag cggacctggc ctggtgaagc ccagcgagac cctgagcctg 60
acctgcaccg tgagcggcga cagcatcacc agcggctact ggaactggat caggaagccc 120
cccggcaatg gcctggagta catgggctac atcagctaca ccggcagcac ctactacaac 180
cccagcctga agagcaggat caccatcacc agggacacca gcaagaacca gtacagcctg 240
aagctgagca gcgtgacagc cgccgatacc gccgtgtact actgcgccag attcggcctg 300
tggcacctgc ctgccgccct ggattactgg ggacagggca ccctggtgac cgtgagcagc 360
<210> 38
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 38
gacatcgtgc tgacccagag ccctgctacc ctgagcctga gccctggcga gagagccacc 60
ctgagctgca gcgccaagag cagcatcagc tacatgcact ggtatcaaca gaagcccggc 120
accagcccta agaggtggat ctacgacaca agcaagctgg ccagcggcgt gcctgccaga 180
tttagcggca gcggcagcgg caccagctac accctgacca tcagcagcct ggagcccgag 240
gacttcgccg tgtactactg ccaccagagg agcagctacc ccttcacctt cggccagggc 300
accaaggtgg agatcaag 318
<210> 39
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 39
gacatcgtgc tgacccagag ccctgctacc ctgagcctga gccccggaga gagagccacc 60
ctgagctgca gcgccagcag caacgtgagc tacatgtact ggtatcaaca gaagcccggc 120
cagagcccca aaccctggat ctacctgacc agcaatctgg ccagcggcgt gcctgccaga 180
tttaccggca gcggcagcgg caccagctac accctgacca tcagcagcct ggagcccgag 240
gacttcgccg tgtactactg ccagcagtgg agcagcaacc ccctgacctt cggccagggc 300
accaaggtgg agatcaag 318
<210> 40
<211> 357
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 40
caggtgcagc tggtgcagag cggcgccgaa gtgaagaagc ctggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc gagtacatca tccactgggt gaagcaggcc 120
cctggccagg gcctggaatg gatcggctgg ttctaccccg gcagcggcaa catcaggtac 180
aacgagaagt tcaaggacaa ggccaccctg accgccgaca agagcagcag caccgtgtac 240
atggagctga gcagcctgag gagcgaggac accgccgtgt acttctgcgc cagacacgag 300
gacaagggcg cctggtttgc ctactggggc cagggcacac tggtgaccgt gagcagc 357
<210> 41
<211> 360
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 41
caggtgcagc tgcaggagag cggacctggc ctggtgaagc ccagcgagac cctgagcctg 60
acctgcaccg tgagcggcga cagcatcacc agcggctact ggaactggat caggaagccc 120
cccggcaatg acctggagta catgggctac atcagctaca ccggcagcac ctactacaac 180
cccagcctga agagcaggat caccatcacc agggacacca gcaagaacca gtacagcctg 240
aagctgagca gcgtgacagc cgccgatacc gccgtgtact actgcgccag attcggcctg 300
tggcacctgc ctgccgccct ggattactgg ggacagggca ccctggtgac cgtgagcagc 360
<210> 42
<211> 360
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VH
<400> 42
caggtgcagc tgcaggagag cggacctggc ctggtgaagc ccagcgagac cctgagcctg 60
acctgcaccg tgagcggcga cagcatcacc agcggctact ggaactggat caggaagccc 120
cccggccaag gcctggagta catgggctac atcagctaca ccggcagcac ctactacaac 180
cccagcctga agagcaggat caccatcacc agggacacca gcaagaacca gtacagcctg 240
aagctgagca gcgtgacagc cgccgatacc gccgtgtact actgcgccag attcggcctg 300
tggcacctgc ctgccgccct ggattactgg ggacagggca ccctggtgac cgtgagcagc 360
<210> 43
<211> 318
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> VL
<400> 43
gacatcgtgc tgacccagag ccctgctacc ctgagcctga gccccggaga gagagccacc 60
ctgagctgca gcgccagcag ccaggtgagc tacatgtact ggtatcaaca gaagcccggc 120
cagagcccca aaccctggat ctacctgacc agcaatctgg ccagcggcgt gcctgccaga 180
tttaccggca gcggcagcgg caccagctac accctgacca tcagcagcct ggagcccgag 240
gacttcgccg tgtactactg ccagcagtgg agcagcaacc ccctgacctt cggccagggc 300
accaaggtgg agatcaag 318
<210> 44
<211> 37
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 44
gcgcaagctt gccaccatga tcttcctcct gctaatg 37
<210> 45
<211> 31
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 45
gccgaattcg atagcactgt tcacttccct c 31
<210> 46
<211> 37
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 46
gcgcaagctt gccaccatgc tgcgtcggcg gggcagc 37
<210> 47
<211> 34
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 47
gcgcgaattc ggctatttct tgtccatcat cttc 34
<210> 48
<211> 38
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 48
gcgcaagctt gccaccatgg cttccctggg gcagatcc 38
<210> 49
<211> 30
<212> DNA
<213> 人工(artificial)
<220>
<221> gene
<222> ()..()
<223> 引物
<400> 49
gccgaattct tttagcatca ggtaagggct 30
Claims (10)
1.一种能够特异性结合PD-L1的抗体分子或其结合片段，所述抗体分子或其结合片段包含轻链可变区(VL)和/或重链可变区(VH)，其中所述轻链可变区包含以下CDR组合之一：
a.SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；
b.SEQ ID NO:7所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；
c.SEQ ID NO:9所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和
d.SEQ ID NO:29所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；
和/或
所述重链可变区包含以下CDR组合之一：
A.SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:6所示的VH-CDR3；
B.SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:8所示的VH-CDR3；和
C.SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3。
2.根据权利要求1所述的抗体分子或其结合片段，其特征在于，所述轻链可变区和重链可变区包含以下CDR组合之一：
1)SEQ ID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；和，SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:6所示的VH-CDR3；
2)SEQ ID NO:7所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2、SEQ ID NO:3所示的VL-CDR3；和，SEQ ID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2、SEQ ID NO:8所示的VH-CDR3；
3)SEQ ID NO:9所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和，SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3；
4)SEQ ID NO:29所示的VL-CDR1、SEQ ID NO:10所示的VL-CDR2、SEQ ID NO:11所示的VL-CDR3；和，SEQ ID NO:12所示的VH-CDR1、SEQ ID NO:13所示的VH-CDR2、SEQ ID NO:14所示的VH-CDR3；
优选地，所述轻链可变区包含选自SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQID NO:21、SEQ ID NO:23、SEQ ID NO:24和SEQ ID NO:28中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和/或
所述重链可变区包含选自SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列。
3.根据权利要求1或2所述的抗体分子或其结合片段，其特征在于，所述抗体分子或其结合片段包含以下轻链可变区和重链可变区组合之一：
I.SEQ ID NO:15所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:16所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
II.SEQ ID NO:17所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:18所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
III.SEQ ID NO:19所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:20所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
IV.SEQ ID NO:21所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:22所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
V.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:22所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VI.SEQ ID NO:24所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:25所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VII.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:26所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
VIII.SEQ ID NO:23所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:27所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；
IX.SEQ ID NO:28所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列；和SEQ ID NO:25所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的序列。
4.根据权利要求1至3中任一项所述的抗体分子或其结合片段，其特征在于，所述抗体分子或其结合片段为半抗体或半抗体的抗原结合片段，优选地，为Fab、Fab’、F(ab’)2、Fv或单链Fv片段(scFv)；
优选地，所述抗体分子或其结合片段还包含人或鼠恒定区；
优选地，所述抗体分子或其结合片段为抗体，优选为鼠源抗体、人源化抗体或者经脱氨突变或糖基化位点突变的优化抗体；
优选地，所述抗体分子或其结合片段还包含轻链恒定区(CL)和/或重链恒定区(CH)；
优选地，所述抗体分子或其结合片段包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
5.根据权利要求1至4中任一项所述的抗体分子或其结合片段，所述抗体分子或其结合片段选自抗体5A5、7A3、8G1、7A3 1-3、7A3 3-3、8G1 3-3、7A3 3-3ND、7A3 3-3QG和8G1 3-3NG。
6.一种核酸分子，其编码权利要求1至5中任一项所述的抗体分子或其结合片段或者编码所述抗体分子或其结合片段中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链。
7.一种载体，其包含权利要求6所述的核酸分子。
8.一种药物组合物，其包含权利要求1至5中任一项所述的抗体分子或其结合片段、权利要求6所述的核酸分子或权利要求7所述的载体，以及任选地药学上可接受的载体、赋形剂和/或稳定剂。
9.权利要求1至5中任一项所述的抗体分子或其结合片段、权利要求6所述的核酸分子或权利要求7所述的载体在制备药物中的用途，所述药物用于治疗与PD-L1高表达相关的疾病；
优选地，所述疾病为癌症；更优选地，所述疾病选自肺癌、卵巢癌、结肠癌、黑色素瘤、膀胱癌、前列腺癌、肝癌、胃癌、肾癌、乳腺癌、头颈癌、淋巴瘤和Merkel细胞癌。
10.权利要求1至5中任一项所述的抗体分子或其结合片段、权利要求6所述的核酸分子或权利要求7所述的载体在制备药物中的用途，所述药物用于增强T细胞免疫应答或增强T细胞活化；
优选地，所述药物用于增加T细胞的细胞因子产生，所述细胞因子优选为IFN-γ。
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| CN108250296A (zh) * | 2018-01-17 | 2018-07-06 | 长春金赛药业股份有限公司 | 全人源抗人pd-l1单克隆抗体及其应用 |
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