CN110914306B - 检查点抑制物双特异性抗体 - Google Patents
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Abstract
本发明涉及抗体,该抗体为异二聚体且结合人PD‑L1和人PD‑1,并且可单独用于治疗癌症和与化疗和其他癌症治疗剂联合用于治疗癌症。
Description
本发明涉及医药学领域。更具体而言,本发明涉及双特异性抗体,该双特异性抗体结合人程序性死亡1(PD-1)和人PD-1配体1(PD-L1),可单独及与化疗和其他癌症治疗联合用于治疗实体瘤和血液肿瘤。
免疫检查点途径用于维持自身耐受和控制T细胞活化,但癌细胞可用该途径来抑制抗肿瘤反应和防止癌细胞破坏。PD-1/PD-L1途径是一个这种免疫检查点。人PD-1见于T细胞上,人PD-L1由多种肿瘤类型异常表达;PD-L1与PD-1的结合抑制T细胞增殖和细胞因子产生。PD-1/PD-L1抑制轴已为肿瘤所征服,作为在抗肿瘤免疫反应的情景中塑造肿瘤进化的天然选择过程的一部分。
虽然靶向PD-1/PD-L1途径的治疗在临床上得到验证,并已导致癌症治疗的明显的临床进展,但仅部分患者从这种治疗获益(参见例如Sharma,P.和Allison,J.P.,Immunecheckpoint targeting in cancer therapy:toward combination strategies withcurative potential.Cell.2015;161:2015-14)。例如,仅~20%的非小细胞肺癌(NSCLC)患者响应PD-1抗体治疗。
虽然涉及共同施用PD-L1抗体和PD-1抗体的临床试验目前正在进行(参见例如EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY(ESMO)Abstract#2130;Oct.2016),但这些治疗方案涉及按每种抗体相对高的剂量输注两种分开的抗体产品。此外,尚不清楚这类联合治疗与单一治疗相比是否可提供功效的改善而不使不良事件情况恶化。
WO2017/087547特别地公开了抗PD-L1抗体,并一般性地公开了包含其中所述PD-L1结合剂和“第二免疫治疗剂”的异二聚体分子(例如双特异性剂)。在一些实施方案中,该第二免疫治疗剂可以包括“阻断免疫抑制功能的抗体”,如“抗PD-抗体”。但是,这份公开中未公开具体的PD-L1/PD-1双特异性剂。因此,需要这样的双特异性抗体作为某些癌症更有效的药理干预,该双特异性抗体以高亲和力结合PD-L1和PD-1,有效中和所有PDx家族配体的PD-L1和PD-1活化,和/或提供相对已知靶向PD-1/PD-L1途径的治疗剂更优的活性。特别地,希望得到这样的抗PD-L1/PD-1双特异性抗体,其i)可更有效地治疗表征为具有中或高PD-L1或PD-1表达水平的癌症;和ii)显示体内稳定性、物理和化学稳定性,包括但不限于热稳定性、溶解性、低自结合及开发和/或用于治疗癌症可接受的药代动力学特征。
因此,本发明提供新的异二聚双特异性抗体,其通过使两条不同重链和两条不同轻链配对为单个IgG样抗体而可以同时靶向PD-L1和PD-1。此外,本发明提供具有一个或多个以下特征的抗人PD-L1和抗人PD-1异二聚双特异性抗体:阻断PD轴的全部三种相互作用(PD-L1结合PD-1、PD-L2结合PD-1和PD-L1结合CD80),桥连PD-L1和PD-1过量表达的细胞,由于结合的T细胞和肿瘤细胞靠近而增加T细胞活化和肿瘤细胞杀伤,在具有中至高PD配体表达水平的肿瘤细胞中在令人吃惊的低剂量下显示显著的抗肿瘤活性,以及显示出乎意料的物理和化学稳定性,包括但不限于体内稳定性、热稳定性、溶解性、低自结合和药代动力学特征。
因此,本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含:
a)第一重链(HC1),其包含含有氨基酸序列SEQ ID NO:3的重链可变区(HCVR);
b)第一轻链(LC1),其包含含有氨基酸序列SEQ ID NO:4的轻链可变区(LCVR);
c)第二重链(HC2),其包含含有氨基酸序列SEQ ID NO:5的重链可变区;和
d)第二轻链(LC2),其包含含有氨基酸序列SEQ ID NO:8的轻链可变区。
本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含HC1、LC1、HC2和LC2,其中:
a)该HC1在HCVR中包含具有氨基酸序列SEQ ID NO:16的CDR1、具有氨基酸序列SEQID NO:17的CDR2和具有氨基酸序SEQ ID NO:18的CDR3;
b)该LC1在LCVR中包含具有氨基酸序列SEQ ID NO:19的CDR1、具有氨基酸序列SEQID NO:20的CDR2和具有氨基酸序列SEQ ID NO:21的CDR3;
c)该HC2在HCVR中包含具有氨基酸序列SEQ ID NO:22的CDR1、具有氨基酸序列SEQID NO:23或SEQ ID NO:24的CDR2和具有氨基酸序SEQ ID NO:25的CDR3;
d)该LC2在LCVR中包含具有氨基酸序列SEQ ID NO:26的CDR1、具有氨基酸序列SEQID NO:27的CDR2和具有氨基酸序列SEQ ID NO:28的CDR3;
e)HC1的CH1结构域包含氨基酸取代S183K;
f)LC1的恒定区是包含氨基酸取代S176E和Y178E的人λ变体;
g)HC2的CH1结构域包含氨基酸取代L128E、K147T和Q175E;
h)LC2的恒定区是包含氨基酸取代S131R、V133G和S176R的人κ变体;和
i)HC1的CH3结构域包含氨基酸取代T350V、L351Y、F405A和Y407V,HC2的CH3结构域包含氨基酸取代T350V、T366L、K392L和T394W;或HC1的CH3结构域包含氨基酸取代T350V、T366L、K392L和T394W,HC2的CH3结构域包含氨基酸取代T350V、L351Y、F405A和Y407V;和
j)其中HC1和HC2是无免疫效应子功能的人IgG1 Fc变体,其中编号按照EU索引。优选地,HC1和HC2包含氨基酸取代L234A、L235A和D265S。
本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含:
a)HC1,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:3的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:9,CH2结构域中的氨基酸序列SEQ ID NO:10,及CH3结构域中的氨基酸序列SEQ ID NO:11或SEQ ID NO:12;
b)LC1,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:4的LCVR,及恒定区中的氨基酸序列SEQ ID NO:14;
c)HC2,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:5的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:13,CH2结构域中的氨基酸序列SEQ ID NO:10,及CH3结构域中的氨基酸序列SEQ ID NO:12或氨基酸序列SEQ ID NO:11;和
d)LC2,其按从N端起包含:含有氨基酸序列SEQ ID NO:8的LCVR,及恒定区中的氨基酸序列SEQ ID NO:15,条件是氨基酸序列SEQ ID NO:11存在于该HC1的CH3结构域中时,氨基酸序列SEQ ID NO:12存在于该HC2的CH3结构域中;或在氨基酸序列SEQ ID NO:12存在于该HC1的CH3结构域中时,氨基酸序列SEQ ID NO:11存在于该HC2的CH3结构域中。
本发明还提供包含HC1、LC1、HC2和LC2的抗体,其中:
a)该HC1按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:3的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:9,CH2结构域中的氨基酸序列SEQ ID NO:10,CH3结构域中的氨基酸序列SEQ ID NO:11;
b)该LC1按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:4的LCVR,及恒定区中的氨基酸序列SEQ ID NO:14;和
c)该HC2按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:6的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:13,CH2结构域区域中的氨基酸序列SEQ ID NO:10,及CH3结构域中的氨基酸序列SEQ ID NO:12;和
d)该LC2按从N端起包含:含有氨基酸序列SEQ ID NO:8的LCVR,及恒定区中的氨基酸序列SEQ ID NO:15。
本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含:
a)含有氨基酸序列SEQ ID NO:49的HC1;
b)含有氨基酸序列SEQ ID NO:30的LC1;
c)含有氨基酸序列SEQ ID NO:31的HC2;和
d)含有氨基酸序列SEQ ID NO:34的LC2。
本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含:
a)含有氨基酸序列SEQ ID NO:49的HC1;
b)含有氨基酸序列SEQ ID NO:30的LC1;
c)含有氨基酸序列SEQ ID NO:33的HC2;和
d)含有氨基酸序列SEQ ID NO:34的LC2。
本发明提供结合人PD-L1(SEQ ID NO:1)和人PD-1(SEQ ID NO:2)的抗体,其包含:
a)含有氨基酸序列SEQ ID NO:29的HC1;
b)含有氨基酸序列SEQ ID NO:30的LC1;
c)含有氨基酸序列SEQ ID NO:33的HC2;和
d)含有氨基酸序列SEQ ID NO:34的LC2。
本发明提供哺乳动物细胞,该哺乳动物细胞包含这样的DNA分子,该DNA分子包含编码具有氨基酸序列SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:34的多肽的多核苷酸序列,其中该细胞能够表达本发明的抗体。
本发明提供哺乳动物细胞,该哺乳动物细胞包含这样的DNA分子,该DNA分子包含编码具有氨基酸序列SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:33和SEQ ID NO:34的多肽的多核苷酸序列,其中该细胞能够表达本发明的抗体。
本发明提供哺乳动物细胞,该哺乳动物细胞包含这样的DNA分子,该DNA分子包含编码具有氨基酸序列SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:33和SEQ ID NO:34的多肽的多核苷酸序列,其中该细胞能够表达本发明的抗体。
本发明提供用于产生本发明的抗体的方法,其包括在使得表达抗体的条件下培养本发明的哺乳动物细胞,并回收所表达的抗体。
本发明提供通过本发明的方法产生的抗体。
本发明提供药物组合物,其包含本发明的抗体和可接受的载体、稀释剂或赋形剂。
本发明提供治疗癌症的方法,其包括对有需要的患者施用有效量的本发明的抗体。本发明还提供治疗癌症的方法,其中该方法包括对有需要的患者施用有效量的本发明的抗体,其中癌症是霍奇金或非霍奇金淋巴瘤、黑素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃和食管癌、结直肠癌、肝癌、肝细胞癌、胆管癌、胰腺癌、乳腺癌、三阴乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈癌的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性成胶质细胞瘤。
本发明提供治疗癌症的方法,其中癌症是黑素瘤。本发明还提供治疗癌症的方法,其中癌症是肺癌。本发明还提供治疗癌症的方法,其中肺癌是NSCLC(鳞状和非鳞状)、小细胞肺癌或间皮瘤。本发明还提供治疗癌症的方法,其中癌症是头颈癌。本发明还提供治疗癌症的方法,其中癌症是肝癌。本发明还提供治疗癌症的方法,其中癌症是结直肠癌。本发明还提供治疗癌症的方法,其中癌症是胰腺癌。本发明还提供治疗癌症的方法,其中癌症是胃癌。本发明还提供治疗癌症的方法,其中癌症是肾癌。本发明还提供治疗癌症的方法,其中癌症是膀胱癌。本发明还提供治疗癌症的方法,其中癌症是前列腺癌。本发明还提供治疗癌症的方法,其中癌症是乳腺癌。本发明还提供治疗癌症的方法,其中癌症是卵巢癌。本发明还提供治疗癌症的方法,其中癌症是子宫内膜癌。本发明还提供治疗癌症的方法,其中癌症是食管癌。本发明还提供治疗癌症的方法,其中癌症是软组织肉瘤。本发明还提供治疗癌症的方法,其中癌症是胆管癌。本发明还提供治疗癌症的方法,其中癌症是肝细胞癌。
本发明还提供这样的方法,其包括与选自以下的一种或多种抗肿瘤剂同时、分开或顺次联合施用有效量的本发明的抗体:顺铂、卡铂、达卡巴嗪(dacarbazine)、多柔比星脂质体(liposomal doxorubicin)、多西紫杉醇(docetaxel)、环磷酰胺(cyclophosphamide)和多柔比星(doxorubicin)、长春瑞滨(navelbine)、艾力布林(eribulin)、紫杉醇(paclitaxel)、紫杉醇蛋白结合颗粒注射悬液、伊沙匹隆(ixabepilone)、卡培他滨(capecitabine)、FOLFOX(亚叶酸(leucovorin)、氟尿嘧啶(fluorouracil)和奥沙利铂(oxaliplatin))、FOLFIRI(亚叶酸、氟尿嘧啶和伊立替康(irinotecan))、吉西他滨(gemcitabine)、拓扑替康(topotecan)、伊立替康脂质体(liposomal irinotecan)、培美曲塞(pemetrexed)、西妥昔单抗(cetuximab)、纳武单抗(nivolumab)、伊匹单抗(ipilimumab)、pidilizumab、pembrolizumab、tremelimumab、urelumab、lirilumab、atezolizumab、epacadostat和durvalumab。
本发明还提供这样的方法,其包括与电离辐射同时、分开或顺次联合施用有效量的本发明的抗体。
本发明提供本发明的抗体用于疗法中。本发明提供本发明的抗体用于治疗癌症。本发明提供本发明的抗体用于治疗癌症,其中癌症是霍奇金或非霍奇金淋巴瘤、黑素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃和食管癌、结直肠癌、肝癌、肝细胞癌、胆管癌、胰腺癌、乳腺癌、三阴乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈癌的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性成胶质细胞瘤。
本发明提供本发明的抗体用于治疗黑素瘤。本发明提供本发明的抗体用于治疗肺癌。本发明还提供本发明的抗体,其中肺癌是NSCLC(鳞状和非鳞状)、小细胞肺癌或间皮瘤。本发明提供本发明的抗体用于治疗头颈癌。本发明提供本发明的抗体用于治疗肝癌。本发明提供本发明的抗体用于治疗结直肠癌。本发明提供本发明的抗体用于治疗胰腺癌。本发明提供本发明的抗体用于治疗胃癌。本发明提供本发明的抗体用于治疗肾癌。本发明提供本发明的抗体用于治疗膀胱癌。本发明提供本发明的抗体用于治疗前列腺癌。本发明提供本发明的抗体用于治疗乳腺癌。本发明提供本发明的抗体用于治疗卵巢癌。本发明提供本发明的抗体用于治疗子宫内膜癌。本发明提供本发明的抗体用于治疗食管癌。本发明提供本发明的抗体用于治疗软组织肉瘤。本发明提供本发明的抗体用于治疗胆管癌。本发明提供本发明的抗体用于治疗肝细胞癌。
本发明提供本发明的抗体用于与选自以下的一种或多种抗肿瘤剂同时、分开或顺次联合用于治疗癌症:顺铂、卡铂、达卡巴嗪、多柔比星脂质体、多西紫杉醇、环磷酰胺和多柔比星、长春瑞滨、艾力布林、紫杉醇、紫杉醇蛋白结合颗粒注射悬液、伊沙匹隆、卡培他滨、FOLFOX(亚叶酸、氟尿嘧啶和奥沙利铂)、FOLFIRI(亚叶酸、氟尿嘧啶和伊立替康)、吉西他滨、拓扑替康、伊立替康脂质体、培美曲塞、西妥昔单抗、纳武单抗、伊匹单抗、pidilizumab、pembrolizumab、tremelimumab、urelumab、lirilumab、atezolizumab、epacadostat和durvalumab。
本发明提供本发明的抗体与电离辐射同时、分开或顺次联合用于治疗癌症。
本发明提供药物组合物用于治疗癌症,其包含有效量的本发明的抗体。本发明还提供药物组合物用于治疗癌症,其包含有效量的本发明的抗体,其中癌症是霍奇金或非霍奇金淋巴瘤、黑素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃和食管癌、结直肠癌、肝癌、肝细胞癌、胆管癌、胰腺癌、乳腺癌、三阴乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈癌的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性成胶质细胞瘤。本发明还提供药物组合物用于治疗癌症,其包含有效量的本发明的抗体,其中肺癌是NSCLC(鳞状和非鳞状)、小细胞肺癌或间皮瘤。
本发明提供药物组合物用于治疗黑素瘤,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗包括但不限于NSCLC(鳞状和非鳞状)、小细胞肺癌或间皮瘤的肺癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗头颈癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗肝癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗结直肠癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗胰腺癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗胃癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗肾癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗膀胱癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗前列腺癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗乳腺癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗卵巢癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗子宫内膜癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗食管癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗软组织肉瘤,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗胆管癌,其包含有效量的本发明的抗体。本发明提供药物组合物用于治疗肝细胞癌,其包含有效量的本发明的抗体。
本发明还提供药物组合物用于治疗癌症,其中该药物组合物与选自以下的一种或多种抗肿瘤剂同时、分开或顺次联合施用:顺铂、卡铂、达卡巴嗪、多柔比星脂质体、多西紫杉醇、环磷酰胺和多柔比星、长春瑞滨、艾力布林、紫杉醇、紫杉醇蛋白结合颗粒注射悬液、伊沙匹隆、卡培他滨、FOLFOX(亚叶酸、氟尿嘧啶和奥沙利铂)、FOLFIRI(亚叶酸、氟尿嘧啶和伊立替康)、吉西他滨、拓扑替康、伊立替康脂质体、培美曲塞、西妥昔单抗、纳武单抗、伊匹单抗、pidilizumab、pembrolizumab、tremelimumab、urelumab、lirilumab、atezolizumab、epacadostat和durvalumab。
本发明还提供药物组合物用于治疗癌症,其中该药物组合物与电离辐射同时、分开或顺次联合施用。
本发明提供本发明的抗体在制备用于治疗癌症的药物中的用途。本发明还提供本发明的抗体在制备用于治疗癌症的药物中的用途,其中癌症是霍奇金或非霍奇金淋巴瘤、黑素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃和食管癌、结直肠癌、肝癌、肝细胞癌、胆管癌、胰腺癌、乳腺癌、三阴乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈癌的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性成胶质细胞瘤。本发明还提供本发明的抗体在制备用于治疗癌症的药物中的用途,其中肺癌是NSCLC(鳞状和非鳞状)、小细胞肺癌或间皮瘤。
本发明还提供本发明的抗体在制备用于治疗癌症的药物中的用途,其中该药物将与选自以下的一种或多种抗肿瘤剂同时、分开或顺次施用:顺铂、卡铂、达卡巴嗪、多柔比星脂质体、多西紫杉醇、环磷酰胺和多柔比星、长春瑞滨、艾力布林、紫杉醇、紫杉醇蛋白结合颗粒注射悬液、伊沙匹隆、卡培他滨、FOLFOX(亚叶酸、氟尿嘧啶和奥沙利铂)、FOLFIRI(亚叶酸、氟尿嘧啶和伊立替康)、吉西他滨、拓扑替康、伊立替康脂质体、培美曲塞、西妥昔单抗、纳武单抗、伊匹单抗、pidilizumab、pembrolizumab、tremelimumab、urelumab、lirilumab、atezolizumab、epacadostat和durvalumab。
本发明提供本发明的抗体在制备用于治疗癌症的药物中的用途,其中该药物与电离辐射同时、分开或顺次施用。
本发明的抗体是经改造的(engineered)非天然存在的多肽复合物。本发明的DNA分子是非天然存在的DNA分子,其包含编码多肽的多核苷酸序列,所述多肽具有本发明抗体中的多肽之一的氨基酸序列。
本发明的抗体是IgG型抗体,具有通过链内和链间二硫键交联的“重”链和“轻”链。每条重链包含N端HCVR和重链恒定区(“HCCR”)。每条轻链包含N端LCVR和轻链恒定区(“LCCR”)。轻链各与重链形成二硫键,两条重链相互之间形成两个二硫键。
重链的恒定区包含CH1、CH2和CH3结构域。CH1在HCVR之后;CH1和HCVR形成Fab的重链部分。CH2在铰链区之后,CH3之前。CH3在CH2之后,是重链的羧基端。
轻链的恒定区包含一个结构域CL。CL在LCVR之后;CL和LCVR形成Fab的轻链部分。
本发明的抗体是异二聚体,这是因为,由于两条不同的重链和两条不同的轻链形成抗体,抗体的每条臂显示与其关联抗原的选择性单价结合。在本发明中,抗体的一条臂结合人PD-L1(SEQ ID NO:1),另一条臂结合PD-1(SEQ ID NO:2)。这类多肽链的CH2和/或CH3结构域无需在序列上一致,且有利地修饰以促进两条多肽链之间的复合。例如,可以在CH2或CH3结构域中引入氨基酸取代(优选地用形成“结(knob)”的包含大侧基团的氨基酸(例如色氨酸)取代),使得空间位阻将阻止与相似突变的结构域相互作用,迫使该突变结构域与已在其中改造了互补或容纳突变(即“扣(hole)”)(例如用甘氨酸取代)的结构域配对。这类突变组改造入任意一对包含形成Fc区的CH2-CH3结构域的多肽。使得偏向于异二聚化而不是同二聚化的蛋白质改造方法为本领域公知,尤其是对于免疫球蛋白样分子的改造(参见例如WO 96/27011,WO 98/050431,EP 1870459,WO 2007/110205,WO 2007/147901,WO2009/089004,WO 2010/129304,WO 2011/90754,WO 2011/143545,WO 2012/058768,WO2013/157954,WO 2013/096291,EP 1 870 459A1,以及Ridgway等(1996)"'Knobs-Into-Holes'Engineering Of Antibody CH3 Domains For Heavy ChainHeterodimerization,"Protein Engr.9:617-621,Atwell等(1997)"Stable HeterodimersFrom Remodeling The Domain Interface Of A Homodimer Using A Phage DisplayLibrary,"J.Mol.Biol.270:26-35和Xie等(2005)"A New Format Of BispecificAntibody:Highly Efficient Heterodimerization,Expression And Tumor CellLysis,"J.Immunol.Methods 296:95-101)。通常,在本领域已知的方法中,以互补方式改造第一重链的CH3结构域和第二重链的CH3结构域这两者,使得包含一个改造的CH3结构域的重链不再与另一具有相同结构的重链同二聚化(例如,CH3改造的第一重链可以不再与另一CH3改造的第一重链同二聚化;CH3改造的第二重链可以不再与另一CH3改造的第二重链同二聚化)。由此迫使包含一个改造的CH3结构域的重链与另一包含以互补方式改造的CH3结构域的重链异二聚化。对于本发明的此实施方案,通过氨基酸取代以互补方式改造第一重链的CH3结构域和第二重链的CH3结构域,使得迫使第一重链和第二重链异二聚化,而第一重链和第二重链不再可以同二聚化(例如由于位阻原因)。
考虑本领域已知的支持重链异二聚化的不同方法作为用于本发明的双特异性抗体的不同替代方法。在本发明的一些实施方案中,在重链序列中在CH1和CH3区内及在轻链序列中在轻链恒定区内掺入突变。进行CH1和LC突变以利于所需轻链和重链对的天然配对,不利于轻链错配。进行CH3突变以利于两条不同重链的异二聚配对,不利于同二聚体形成。
优选地,在抗体的抗PD-L1部分的CH3区中的突变包括EU编号的350、351、405和407位时,抗体的抗PD-1部分的CH3区中的突变包括EU编号的350、366、392和394位;在抗体的抗PD-L1部分的CH3区中的突变包括EU编号的350、366、392和394位时,抗体的抗PD-1部分的CH3区中的突变包括EU编号的350、351、356、405和407位(如下文序列比对中所示)。
野生型人IgG1及抗体v3.2和v13884重链恒定区的氨基酸序列比对(对优选的修饰下划线):
IgG1-wt ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
3.2PD-L1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
844PD-1 ASTKGPSVFPEAPSSKSTSGGTAALGCLVTDYFPEPVTVSWNSGALTSGV
3.2PD-1 ASTKGPSVFPEAPSSKSTSGGTAALGCLVTDYFPEPVTVSWNSGALTSGV
844PD-L1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
IgG1-wt HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
3.2PD-L1 HTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
844PD-1 HTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
3.2PD-1 HTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
844PD-L1 HTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
IgG1-wt KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
3.2PD-L1 KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVS
844PD-1 KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVS
3.2PD-1 KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVS
844PD-L1 KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVS
IgG1-wt HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
3.2PD-L1 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
844PD-1 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
3.2PD-1 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
844PD-L1 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
IgG1-wt EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
3.2PD-L1 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTC
844PD-1 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTC
3.2PD-1 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLC
844PD-L1 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLC
IgG1-wt LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
3.2PD-L1 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRW
844PD-1 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRW
3.2PD-1 LVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRW
844PD-L1 LVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRW
IgG1-wt QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40)
3.2PD-L1 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:41)
844PD-1 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:42)
3.2PD-1 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43)
844PD-L1 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:44)
优选地,如上文比对中下划线所示,抗体的抗PD-L1部分的CH1区中的突变优选包括EU编号的183位,而抗体的抗PD-1部分的CH1区中的突变优选包括EU编号的128、147和175位。
抗体的抗PD-L1部分的CL区优选是人λ亚型。更优选地,抗体的抗PD-L1部分的CL区是在EU编号的176和178位包含氨基酸取代的人λ变体(参见下文比对)。
野生型人λ氨基酸序列与v3.2、v3.0和v13844抗体的PD-L1轻链的恒定区的比对(对优选的修饰下划线)
λ-wt QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVK
PD-L1 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK
λ-wt AGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV
PD-L1 AGVETTTPSKQSNNKYAAESELSLTPEQWKSHRSYSCQVTHEGSTVEKTV
λ APTECS(SEQ ID NO:45)
PD-L1 APAECS(SEQ ID NO:46)
抗体的抗PD-1部分的CL区优选是人κ亚型。本发明的抗体的抗PD-1部分的CL区优选是在EU编号的131、133和176位包含氨基酸取代的人κ变体。
野生型人κ和针对PD-1的抗体的轻链恒定区的氨基酸序列比对(对优选的修饰下划线)
κ-wt RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
PD-1 RTVAAPSVFIFPPSDEQLKSGTARVGCLLNNFYPREAKVQWKVDNALQSG
κ-wt NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
PD-1 NSQESVTEQDSKDSTYSLRSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
κ-wt SFNRGEC(SEQ ID NO:47)
PD-1 SFNRGEC(SEQ ID NO:48)
在本发明的某些抗体中,重链异二聚体配对突变产生高于78℃的CH3热稳定性。
在某些生物系统中表达时,具有Fc序列的抗体在Fc区中糖基化。通常,糖基化发生在抗体Fc区中高度保守的N糖基化位点处。N聚糖通常连接至天冬酰胺。抗体也可以在其他位置糖基化。
优选地,本发明的抗体包含源自人IgG1的Fc区变体。众所周知,IgG1结合Fc-γ受体家族(FcγR)以及C1q。与这些受体的相互作用可诱导依赖抗体的细胞毒性(ADCC)和依赖补体的细胞毒性(CDC)。因此,在本发明的抗体的人IgG1 Fc区中引入某些氨基酸取代以去除免疫效应子功能。抗体重链CH2区中的突变可以包括EU编号的234、235和265位,以减少或消除免疫效应子功能。
通过使HCVR编码DNA与另一编码重链恒定区或其变体的DNA分子有效连接,可将分离的编码HCVR区的DNA转化为全长重链基因。人以及其他哺乳动物重链恒定区基因的序列为本领域已知。涵盖这些区域的DNA片段可以例如通过标准PCR扩增获得。优选地,对于本发明的抗体,重链的重链恒定区是人IgG1的变体。
通过使LCVR编码DNA与另一编码轻链恒定区或其变体的DNA分子有效连接,可将分离的编码LCVR区的DNA转化为全长轻链基因。人以及其他哺乳动物轻链恒定区基因的序列为本领域已知。涵盖这些区域的DNA片段可以通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区。优选地,对于本发明的抗体,抗体的抗PD-L1部分的轻链恒定区是λ轻链的变体,抗体的抗PD-1部分的轻链恒定区是κ轻链的变体。
在本发明的多核苷酸序列与表达控制序列有效连接后,本发明的多核苷酸可以在宿主细胞中表达。表达载体通常可作为附加体或作为宿主染色体DNA的整合部分在宿主生物中复制。通常,表达载体将包含选择标记,例如四环素、新霉素、谷氨酰胺合成酶和二氢叶酸还原酶,以允许检测用目的DNA序列转化的那些细胞。
本发明的抗体可以容易地在哺乳动物细胞如CHO、NS0、HEK293或COS细胞中产生。用本领域公知的技术培养宿主细胞。
包含目的多核苷酸序列(例如编码抗体的多肽的多核苷酸和表达控制序列)的载体可以通过公知的方法转移入宿主细胞,该方法取决于细胞宿主的类型而变。
可以利用多种蛋白质纯化方法,这类方法为本领域已知,并描述于例如Deutscher,Methods in Enzymology 182:83-89(1990)and Scopes,ProteinPurification:Principles and Practice,第3版,Springer,NY(1994)中。
在本发明的另一实施方案中,以分离的形式提供抗体或编码抗体的核酸。本文所用的术语“分离的”指不含或基本不含见于细胞环境中的任何其他大分子种类的蛋白质、肽或核酸。本文所使用的“基本不含”指目的蛋白质、肽或核酸包含超过80%(以摩尔为基础)的大分子种类存在,优选超过90%,更优选超过95%。
本发明的抗体或包含本发明的抗体的药物组合物可以通过胃肠外途径(例如皮下和静脉内)施用。本发明的抗体可以以单个或多个剂量仅与可药用载体、稀释剂或赋形剂一起对患者施用。本发明的药物组合物可以通过本领域公知的方法制备(例如Remington:TheScience and Practice of Pharmacy,第22版(2012),A.Loyd等,Pharmaceutical Press),包含本文中公开的抗体和一种或多种可药用载体、稀释剂或赋形剂。
术语“治疗”指减慢、中断、中止、缓解、阻止、减少或逆转现有症状、障碍、病症或疾病的进展或严重度。
除非另有说明,本文中提到抗体对人PD-L1(SEQ ID NO:1)、人PD-1(SEQ ID NO:2)或二者的亲和力时所用的“结合”旨在指通过本领域已知的常用方法(包括通过基本按本文所述在37℃使用表面等离振子共振(SPR)生物传感器)测定的小于约1x10-6 M、优选小于约1x 10-9M的KD。
“有效量”指本发明的抗体或包含本发明的抗体的药物组合物的量,其引出研究人员、医学博士或其他临床医生所寻求的生物学或医学反应或希望得到的对组织、系统、动物、哺乳动物或人的治疗作用。抗体的有效量可根据诸如个体的疾病状态、年龄、性别和体重及抗体在个体中引出所希望得到的反应的能力的因素而变。有效量还是这样的量,其中治疗有益作用超出抗体的毒性或有害作用。
通过以下非限制性实施例进一步说明本发明。
实施例1:抗体表达和纯化
抗体v3.2、v3.0和v13844的重链和轻链的可变区多肽、完整重链和轻链氨基酸序列及编码它们的核苷酸序列在下文标题为“氨基酸和核苷酸序列”的章节中列出。此外,抗体v3.2、v3.0和v13844的轻链、重链、轻链可变区和重链可变区的氨基酸序列的SEQ ID NO在下文表1(a)中显示。此外,抗体v3.2、v3.0和v13844的PD-L1和PD-1结合可变区的CDR的氨基酸序列的SEQ ID NO分别在表1(b)和表1(c)中显示。
本发明的抗体(包括但不限于抗体v3.2、v3.0和v13844)可以基本按以下制备和纯化。可以使用四元载体(即编码两条轻链和两条重链的表达的单个载体)、双载体(即两个载体,其一起编码两条不同的轻链和两条不同的重链)或四个单载体(其中两个编码不同的轻链,其中两个编码不同的重链),使用用于分泌抗体的表达系统瞬时或稳定转染适当的宿主细胞,如CHO。可以用任意许多常用的技术纯化抗体已分泌进入其中的培养基。例如,可将培养基加至已用相容的缓冲液如磷酸缓冲盐溶液(pH 7.4)平衡的用于Fab片段的MabSelect柱(GE Healthcare)或KappaSelect柱(GE Healthcare)。可以洗涤柱以去除非特异性结合成分。结合的抗体可以例如通过pH梯度(如20mM Tris缓冲液pH 7.0至10mM柠檬酸钠缓冲液pH 3.0,或磷酸缓冲盐溶液pH 7.4至100mM甘氨酸缓冲液pH 3.0)洗脱。抗体级分可以如通过SDS-PAGE检测,然后合并。可溶性聚集体和多聚体可以通过常用技术有效去除,包括大小排阻、疏水相互作用、离子交换、多模式(multimodal)或羟基磷灰石层析。抗体可以用常用技术浓缩和/或除菌过滤。产物可立即冷冻于-70℃或可以冷冻干燥。
表1(a)-(c):检查点抑制物双特异性抗体(BsAb)序列的SEQ ID No
1(a)
1(b)
1(c)
实施例2:通过ELISA测量的与人PD-L1和PD-1这二者的结合
本发明的抗体结合人PD-L1和人PD-1二者的能力可以在夹心法ELISA测定中测量。对于PD-1结合测定,可以用人PD-1-Fc(R&D Systems,cat.#1086-PD)在4℃过夜包被96孔板(Nunc)。可以用封闭缓冲液(含5%脱脂奶粉的PBS)封闭孔2小时。可以用含0.1%Tween-20的PBS洗涤孔三次。然后可以加入抗PD-1抗体或对照IgG(100μl),然后可以在室温孵育平板1小时。洗涤后,可以用100μl山羊抗人IgG F(ab’)2-HRP缀合物(Jackson ImmunoResaerch,cat.#109-035-097)在室温下孵育平板1小时。可以洗涤平板,然后可以用100μl3,3’,5,5’-四甲基联苯胺孵育。可以在微板读数仪上读取450nm吸光度。可以用GraphPadprism 6软件计算半数最大有效浓度(EC50)。
对于PD-L1结合测定,除可以用人PD-L1-Fc(R&D Systems,cat#156-B7)在4℃过夜包被96孔板(Nunc)外,可以应用相似的流程。
在本实施例2中基本按上文所述进行的实验中,抗体v3.2以0.0802nM的EC50结合人PD-1。相比较而言,亲本PD-1抗体(作为IgG4-PAA同二聚体)的结合亲和力为0.1318nM。抗体v3.2以0.4554nM的EC50结合人PD-L1。相比较而言,亲本PD-L1抗体(作为IgG1-EN同二聚体)的结合亲和力为0.4702nM。
实施例3:桥连PD-1表达细胞与PD-L1表达细胞
使用表达PD-1或PD-L1的转染CHO细胞,通过流式细胞术分析测定了本发明的抗体桥连PD-1和PD-L1表达细胞的能力。简言之,可以用CFSE(羧基荧光素二乙酸琥珀酰亚胺酯)(BD Horizon)或Cell Tracker Deep Red(CTDR/Thermo)差异标记CHO-PD1和CHOK1-PDL1过量表达细胞。用测试抗体如抗体v3.2分开孵育CHO-PD1细胞和CHOK1-PDL1细胞2小时(在PBS+1%BSA+0.09%叠氮化钠中于冰上)。未结合的抗体可以通过洗涤(2X,用200μl PBS+1%BSA+0.09%叠氮化钠)去除。用含45μg/ml亲本PD-L1抗体或hIgG1对照的PBS+1%BSA+0.09%叠氮化钠冰上孵育CHOK1-PDL1细胞2小时。用含45μg/ml亲本PD-1抗体或huIgG4-PAA的PBS+1%BSA+0.09%叠氮化钠冰上孵育CHO-PD1细胞2小时。将CHO-PD1/抗体v3.2细胞与CHOK1-PDL1+终浓度22.5ug/ml的亲本PD-L1抗体或hIgG1混合。可以将CHOK1-PDL1/抗体v3.2细胞与CHO-PD1+终浓度22.5μg/ml的亲本PD-1抗体或huIgG4-PAA混合。孵育(4℃)约72小时后,可以在Fortessa X20(具有HTS采样器)上在适合用于CFSE和CTDR的通道测量细胞。使用软件(FlowJo,LLC,Ashland,OR),可以设门(gated)双阳性事件(CFSE+/CTDR+),并可以计算和报告总事件的%(对于2个复孔)。可以用Graphpad Prism用非线性回归(可变斜率,4参数)产生拟合和统计。
在本实施例3中基本按上文所述进行的实验中,PD-1/PD-L1双特异性抗体介导可通过流式细胞术检测为双阳性事件的细胞桥连。抗体v3.2与CHO-PD1或CHOK1-PDL1细胞结合(随后去除未结合的抗体),然后分别与CHOK1-PDL1或CHO-PD1细胞混合,导致相对于背景的双阳性事件的剂量依赖性增加(与仅缓冲液相比高达增加4倍)。通过按高浓度加入过量的竞争PD-L1和/或PD-1mAb阻断此双阳性事件的增加,但匹配的非特异性IgG对照则不然,证明特异性和对靶抗原表达的依赖性。
实施例4:阻断PD-L1与PD-1、PD-L1与CD80和PD-L2与PD-1的相互作用
通过将不同量的抗PD-1抗体或对照IgG与固定量的生物素化PD-1-Fc融合蛋白(100ng/mL)混合并在室温孵育约1小时,可以进行PD-L1/PD-1阻断测定。然后,可以将混合物转移至用PD-L1-Fc(100ng/孔)或PD-L2-Fc(100ng/孔)预包被的96孔板,然后再在室温孵育约1小时。洗涤后,可以加入链霉抗生物素蛋白HRP缀合物,并可以读取450nm吸光度。IC50代表50%抑制PD-1与PD-L1结合或与PD-L2结合所需的抗体浓度。
类似地,通过使用1μg/ml B7-1-Fc(R&D Systems,cat#140-B1-100)包被的平板,可以进行PD-L1/B7-1阻断测定。用GraphPad prism 6软件计算50%抑制PD-L1与B7-1结合所需的抗体浓度(IC50)。
在本实施例4中基本按上文所述进行的实验中,抗体v3.2在中和更高浓度下似乎阻断PD-1受体和PD-L1配体之间的相互作用,在较低和中浓度下看起来以强于天然配体-受体相互作用的亲和力以双结合桥连受体和配体。此外,抗体v3.2似乎以0.75nM的IC50阻断PD-L1与B7-1的相互作用,以2.27nM的IC50阻断PD-L2与PD-1的相互作用。
实施例5:混合白细胞反应(MLR)中的抗体体外功能分析
CD14+单核细胞可以用MACS珠(Miltenyi,cat.#130-091-153)从获自健康供体的冷冻人PBMC(AllCells cat.#PB005F)分离。未成熟树突细胞(DC)可以通过在1000IU/mlhGM-CSF和500IU/ml hIL-4存在下在12ml完全RPMI-1640培养基中培养这些单核细胞4天来产生。CD4+T细胞可以通过负选择(Miltenyi 130-096-533)从不同健康供体的新鲜人PBMC(AllCells cat.#PB002)纯化。然后,可以在含100μl完全AIM-V培养基的96孔板的单个孔中混合两种类型的细胞,每孔含5x 104个CD4+T细胞和4x 103个未成熟DC(E:T=12.5:1)。可以一式八份加入100μl含人IgG1-EN、亲本抗PD-1抗体、亲本抗PD-L1抗体、亲本抗体的组合、或抗体v3.2(分别从32nM开始3:1系列稀释)的完全AIM-V培养基。37℃、5%CO2孵育72小时后,可以收集上清,然后用ELISA试剂盒(R&D cat#SIF50和R&D cat#S2050)测量人IFN-γ和IL-2。
在本实施例5中基本按上文所述进行的实验中,与亲本PD-L1 Ab、亲本PD-1Ab和二者的组合分别为0.026nM、0.029nM和0.115nM相比,向同种异体CD4+T细胞和DC的共培养中加入抗体v3.2以0.005nM的EC50导致响应CD4 T细胞淋巴细胞的IFN-γ产生的增加。类似地,与PD-L1 Ab、PD-1Ab和PD-L1 Ab+PD-1Ab组合(分别为0.034nM、0.023nM和0.046nM)相比,抗体v3.2还以0.011nM的EC50增加共培养物中的IL-2产生。结果表明,抗体v3.2在其增强体外T细胞活化的能力上优于单独的亲本PD-L1 Ab、单独的亲本PD-1Ab或其组合。
实施例6:重振T细胞
PD-L1阳性人T活化CHOK1细胞(Promega part#CS187108)和PD-L1阴性人T活化CHOK1细胞(Promega part#CS187110)可获自Promega。PD-L1/PD-L2双阳性人T活化CHOK1细胞可以通过用编码全长PD-L2的载体转染PD-L1阳性人T活化CHOK1细胞来建立。可以将这些细胞于100μL培养基(10%FBS F-12,0.2mg/ml潮霉素B和0.2mg/ml G418)中按每孔40,000个细胞接种在96孔白色不透明组织培养板中,并在37℃、5%CO2下过夜孵育。次日从测定平板去除培养基,按每孔40μl加入在测定缓冲液(2%FBS RPMI)中系列稀释的测试和对照抗体。可以将GloResponse NFAT-luc2/PD1 Jurkat细胞(Promega part#CS187102)按1.25x106个/mL的浓度重悬在测定缓冲液中,并按每孔40μl加至平板。孵育6小时后,从培养箱中取出测定平板,室温平衡5至10分钟。可以按照厂家说明书配制Bio-GloTM Reagent(PromegaG7941),并按每孔80μl加入。然后可以室温孵育平板5至10分钟。可以在微板读数仪中测量发光,可以用GraphPad Prism 7分析数据。
在本实施例6中基本按上文所述进行的PD-1/NFAT报告Jurkat T细胞测定中,观察到PD-L1阳性人T活化物CHO细胞抑制T细胞活化。PD-1Ab或PD-L1 Ab通过逆转PD-L1-PD-1介导的抑制重新活化T细胞。但是,抗体v3.2(EC50 0.12nM)在其重振T细胞的能力上似乎优于单独的亲本PD-L1 Ab(1.92nM)、单独的亲本PD-1Ab(1.01nM)、或亲本PD-1Ab和亲本PD-L1Ab的组合(0.796nM)。在将PD-L1/PD-L2双阳性人T活化物CHO细胞用于此系统时,PD-1阳性报告T细胞的活化也受抑制。亲本PD-1Ab而不是亲本PD-L1 Ab能够重新活化T细胞。但是,抗体v3.2(EC50 0.181nM)在其重振T细胞的能力上优于单独的亲本PD-1Ab(0.946nM)、或亲本PD-1Ab和亲本PD-L1 Ab的组合(1.251nM)。
实施例7:体内功效测定
可以在用人免疫细胞重建的免疫缺陷小鼠异种移植模型中测试本发明的抗体的功效,以在模型中评估通过增强对异型抗原的T细胞反应来延迟或破坏已建立的肿瘤的能力。研究中的所有动物由机构动物管理及使用委员会(Institutional Animal Care andUse Committee)批准,并按照美国农业部(United States Department of Agriculture)和美国国立卫生研究院(National Institute of Health)的当前法规和标准进行。
A部分:NCI-H292人NSCLC异种移植模型
用HBSS中的2x 106个NCI-H292细胞或2x 106个NCI-H292细胞和1x 106个新鲜分离的人PBMC的混合物(总体积0.2ml)皮下移植入来自Jackson Laboratories的NOD scidγ(NSG)小鼠(7周龄,雌性,每组7-8只)的胁腹。从第1天开始,每周一次腹膜内注射人IgG1-EN(对照)、亲本抗PD-L1抗体(0.25mg/kg)、或亲本抗PD-1抗体(0.25mg/kg)、或亲本抗体的组合(各0.25mg/kg)、或抗体v3.0、v3.2或v13844(0.5mg/kg)治疗小鼠,共三个剂量。每周至少两次监测动物健康和行为,包括理毛(grooming)和走动(ambulation)。每周两次测量体重和肿瘤体积。
在实施例7的A部分中基本按上文所述进行的实验中,如通过体重和临床观察监测,按10mg/kg给药的抗体v3.0、v3.2和v13844耐受良好且安全。在移植入NCI-H292肿瘤细胞和PBMC的混合物的小鼠中,与用对照分子人IgG1-EN治疗相比,按各10mg/kg用抗PD-L1或抗PD-1抗体、或抗PD-L1和抗PD-1抗体的组合治疗全都延迟肿瘤生长。在移植入NCI-H292肿瘤细胞和PBMC的混合物的小鼠中,按10mg/kg qw用PD-1/PD-L1双特异性Ab(v13844、v3.0或v3.2)治疗的完全消退(complete regression;CR)分别为7/8、5/8和5/8,全都比联合治疗(亲本PD-L1抗体+亲本PD-1抗体)(2/8CR)更有效。
B部分:HCC827人NSCLC异种移植模型
用HBSS中的10x 106个HCC827细胞(总体积0.2ml)皮下移植入来自JacksonLaboratories的NSG小鼠(7周龄,雌性,每组8只)的胁腹。用人T-Activator CD3/CD28扩增来自全血的大量人T细胞(New York Blood Center)10天并冻存。在移植后第44天,融化、洗涤、计数T细胞,静脉内输注(每只小鼠0.2ml PBS中的3.5x 106个T细胞)入带有HCC827肿瘤的小鼠。从次日起,每周一次腹膜内注射人IgG1-EN(对照)、亲本抗PD-L1抗体、亲本抗PD-1抗体、或亲本抗PD-L1抗体和亲本抗PD-1抗体的组合(各2mg/kg)、或三个剂量水平(0.02、0.2或2mg/kg)的抗体v3.2治疗小鼠,持续4周。每周至少两次监测动物健康和行为,包括理毛和走动。每周两次测量体重和肿瘤体积。每周一次按标题“IM肿瘤生长测量”的SOP中所述用电子卡尺测量肿瘤体积。用以下公式计算肿瘤体积:肿瘤体积(mm3)=π/6*长度*宽度2。
在本实施例7的B部分中基本按上文所述进行的实验中,全部三个剂量水平(0.02、0.2或2mg/kg)的抗体v3.2在扩增T细胞的存在下在已建立的HCC827肿瘤模型中具有相似的抗肿瘤反应。此外,在肿瘤细胞移植后第69天,0.02mg/kg的抗体v3.2比抗PD-L1抗体(2mg/kg,p=0.05)、抗PD-1抗体(2mg/kg,p<0.001)和两种抗体的组合(各2mg/kg,p<0.001)更显著地延迟肿瘤生长(参见表2)。
表2:HCC827人NSCLC建立的肿瘤模型中第69天的肿瘤抑制
SEM:平均值的标准估计
氨基酸和核苷酸序列
SEQ ID NO:1(人PD-L1)
MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET
SEQ ID NO:2(人PD-1)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
SEQ ID NO:3(PD-L1 Ab的HCVR)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPDYSPYYYYGMDVWGQGTTVTVSS
SEQ ID NO:4(PD-L1 Ab的LCVR)
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCQSYDSSLSGSVFGGGIKLTVLG
SEQ ID NO:5(PD-1Ab-Xaa的HCVR)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPXaaFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS
Xaa:M或S.
SEQ ID NO:6(PD-1Xaa-S的HCVR)
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGL IIPSFDTAGYAQKFQGRVAI TVDESTSTAY MELSSLRSED TAVYYCARAE HSSTGTFDYW GQGTLVTVSS
SEQ ID NO:7(PD-1Xaa-M的HCVR)
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGL
IIPMFDTAGY AQKFQGRVAI TVDESTSTAY MELSSLRSED TAVYYCARAE HSSTGTFDYWGQGTLVTVSS
SEQ ID NO:8(PD-1Ab的LCVR)
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIK
SEQ ID NO:9(来自PD-L1 Ab v3.2和v13844 HC的CH1结构域的区域)
SLKSV
SEQ ID NO:10(来自PD-L1 Ab HC的CH2结构域的区域)
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVS
SEQ ID NO:11(来自PD-1v13844和PD-L1 v3.2的HC的CH3结构域的区域)
VYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALV
SEQ ID NO:12(来自PD-1v3.2和PD-L1 v13844的HC的CH3结构域的区域)
VLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTW
SEQ ID NO:13(来自PD-1Ab v3.2和PD-1v13844 HC的CH1结构域的区域)
EAPSSKSTSGGTAALGCLVTDYFPEPVTVSWNSGALTSGV HTFPAVLE
SEQ ID NO:14(来自PD-L1 v3.2和v13844 Abs的LC恒定区的区域)
AAESELS
SEQ ID NO:15(来自PD-1v3.2和v13844 Ab的LC恒定区的区域)
RVGCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLR
SEQ ID NO:16(PD-L1 Ab的HCDR1)
KASGGTFSSYAIS
SEQ ID NO:17(PD-L1 Ab的HCDR2)
GIIPIFGTANYAQKFQG
SEQ ID NO:18(PD-L1 Ab的HCDR3)
ARSPDYSPYYYYGMDV
SEQ ID NO:19(PD-L1 Ab的LCDR1)
SGSSSNIGSNTVN
SEQ ID NO:20(PD-L1 Ab的LCDR2)
YGNSNRPS
SEQ ID NO:21(PD-L1 Ab的LCDR3)
QSYDSSLSGSV
SEQ ID NO:22(PD-1Ab的HCDR1)
KASGGTFSSYAIS
SEQ ID NO:23(PD-1Ab;v3.2的HCDR2)
LIIPSFDTAGYAQKFQG
SEQ ID NO:24(PD-1Ab;v3.0的HCDR2)
LIIPMFDTAGYAQKFQG
SEQ ID NO:25(PD-1Ab的HCDR3)
ARAEHSSTGTFDY
SEQ ID NO:26(PD-1Ab的LCDR1)
RASQGISSWLA
SEQ ID NO:27(PD-1Ab的LCDR2)
SAASSLQS
SEQ ID NO:28(PD-1Ab的LCDR3)
QQANHLPFT
SEQ ID NO:29(PD-L1 Ab v13844的HC)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPDYSPYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:30(PD-L1 Ab v3.2,v3.0和v13844的LC)
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCQSYDSSLSGSVFGGGIKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAESELSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS
SEQ ID NO:31(PD-1Ab v3.2的HC)
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGL IIPSFDTAGYAQKFQGRVAI TVDESTSTAY MELSSLRSED TAVYYCARAE HSSTGTFDYW GQGTLVTVSS ASTKGPSVFPEAPSSKSTSG GTAALGCLVT DYFPEPVTVS WNSGALTSGV HTFPAVLESS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVSVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYVLPPSRDE LTKNQVSLLC LVKGFYPSDI AVEWESNGQP ENNYLTWPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
SEQ ID NO:32(PD-1v3.0的HC)
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGL IIPMFDTAGYAQKFQGRVAI TVDESTSTAY MELSSLRSED TAVYYCARAE HSSTGTFDYW GQGTLVTVSS ASTKGPSVFPEAPSSKSTSG GTAALGCLVT DYFPEPVTVS WNSGALTSGV HTFPAVLESS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVSVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYVLPPSRDE LTKNQVSLLC LVKGFYPSDI AVEWESNGQP ENNYLTWPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
SEQ ID NO:33(PD-1Ab v13844的HC)
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGL IIPMFDTAGYAQKFQGRVAI TVDESTSTAY MELSSLRSED TAVYYCARAE HSSTGTFDYW GQGTLVTVSS ASTKGPSVFPEAPSSKSTSG GTAALGCLVT DYFPEPVTVS WNSGALTSGV HTFPAVLESS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVSVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYVYPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFALVSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
SEQ ID NO:34(PD-1Ab v3.2,v3.0和v13844的LC)
DIQMTQSPSS VSASVGDRVT ITCRASQGIS SWLAWYQQKP GKAPKLLISA ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ ANHLPFTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTARVGCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLRSTLT LSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC
SEQ ID NO:35 PD-L1 LC的DNA序列
CAGTCCGTCC TGACTCAGCC ACCTTCCGCT AGCGGTACCC CCGGCCAGAG AGTGACAATCTCATGCTCCG GTTCCAGCTC TAACATTGGC TCTAACACTG TCAATTGGTA CCAGCAGCTG CCAGGAACCGCACCAAAGCT GCTGATCTAT GGAAACTCAA ATAGGCCTAG CGGGGTGCCA GACCGGTTTA GCGGATCTAAAAGTGGGACT TCAGCTTCCC TGGCAATTTC TGGACTGCAG AGTGAGGACG AAGCTGATTA CTATTGCCAGTCCTACGATA GTTCACTGAG CGGTTCCGTG TTCGGCGGAG GGATCAAGCT GACAGTCCTG GGCCAGCCCAAGGTGAGTTC TAGAGGATCC ATCTGGGATA AGCATGCTGT TTTCTGTCTG TCCCTAACAT GCCCTGTGATTATCCGCAAA CAACACACCC AAGGGCAGAA CTTTGTTACT TAAACACCAT CCTGTTTGCT TCTTTCCTCAGGCCGCTCCT TCCGTGACTC TGTTTCCCCC TTCCAGCGAG GAACTGCAGG CCAATAAGGC CACCCTGGTGTGCCTGATTA GCGACTTCTA TCCTGGAGCT GTGACAGTCG CATGGAAGGC CGATTCTAGT CCAGTGAAAGCAGGGGTCGA GACCACAACT CCCTCCAAGC AGAGCAACAA CAAGTACGCA GCCGAGTCTG AACTGAGTCTGACCCCAGAA CAGTGGAAGT CCCACAGGAG TTATTCATGC CAGGTGACCC ATGAGGGCTC CACAGTGGAGAAGACCGTGG CCCCTGCTGA GTGTAGC
SEQ ID NO:36 PD-1LC的DNA序列
GACATTCAGATGACCCAGAGCCCAAGCAGCGTGAGCGCCAGCGTCGGGGACCGAGTGACCATCACATGCAGGGCCAGCCAGGGTATTTCTAGTTGGCTGGCTTGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGCTGCTGATCTCCGCCGCTTCAAGCCTGCAGTCCGGAGTGCCCTCTCGATTCTCTGGTAGTGGCTCAGGAACAGACTTTACTCTGACCATTTCCTCTCTGCAGCCTGAGGATTTCGCTACTTACTATTGCCAGCAGGCAAACCACCTGCCATTCACCTTTGGCGGAGGGACAAAAGTGGAGATCAAGAGAACCGTCGCGGCGCCCAGTGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAGTCTGGGACAGCCAGAGTGGGCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCTAAAGTGCAGTGGAAGGTCGATAACGCACTGCAGTCCGGAAATTCTCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGAGAAGCACACTGACTCTGAGCAAGGCCGACTACGAGAAGCATAAAGTGTATGCTTGTGAAGTCACCCACCAGGGGCTGAGTTCACCAGTCACAAAATCATTCAACAGAGGGGAGTGC
SEQ ID NO:37 PD-L1 HC的DNA序列
CAGGTCCAGC TGGTGCAGAG CGGAGCCGAA GTGAAGAAAC CCGGTAGCAG CGTCAAAGTGTCATGTAAAG CCTCAGGGGG AACATTCTCC AGCTACGCCA TCTCCTGGGT GAGACAGGCT CCAGGACAGGGACTGGAGTG GATGGGAGGA ATCATCCCTA TCTTCGGCAC CGCCAACTAC GCTCAGAAGT TTCAGGGCCGCGTGACCATC ACAGCCGACA AGAGCACCTC TACAGCTTAT ATGGAGCTGT CTTCCCTGAG AAGCGAGGATACAGCCGTGT ACTATTGCGC TCGCTCCCCC GACTACAGCC CTTACTATTA CTATGGCATG GACGTGTGGGGCCAGGGCAC CACAGTGACC GTGAGCTCTG CTAGCACAAA GGGCCCATCC GTGTTCCCAC TGGCTCCATCCAGCAAGTCC ACCAGCGGAG GAACAGCCGC TCTGGGCTGT CTGGTGAAGG ACTATTTCCC AGAGCCAGTGACCGTGTCCT GGAACAGCGG CGCCCTGACC TCTGGAGTGC ACACATTTCC CGCTGTGCTG CAGTCTTCCGGCCTGTACTC TCTGAAGTCC GTGGTGACCG TGCCTAGCTC TTCCCTGGGC ACCCAGACAT ATATCTGCAACGTGAATCAC AAGCCTTCCA ATACAAAGGT GGACAAGAGG GTGGAGCCAA AGAGCTGTGA TAAGACCCATACATGCCCCC CTTGTCCTGC TCCAGAGGCT GCTGGAGGAC CAAGCGTGTT CCTGTTTCCA CCCAAGCCCAAGGACACCCT GATGATCTCT AGGACCCCTG AGGTGACATG CGTGGTGGTG TCCGTGTCCC ACGAGGACCCAGAGGTGAAG TTTAACTGGT ACGTGGATGG CGTGGAGGTG CATAATGCTA AGACCAAGCC TAGGGAGGAGCAGTACAACA GCACCTATCG GGTGGTGTCT GTGCTGACAG TGCTGCATCA GGATTGGCTG AACGGCAAGGAGTATAAGTG CAAGGTGTCT AATAAGGCCC TGCCCGCTCC TATCGAGAAG ACCATCTCCA AGGCCAAGGGCCAGCCTAGG GAGCCACAGG TGTACGTGCT GCCTCCAAGC CGGGACGAGC TGACAAAGAA CCAGGTGTCTCTGCTGTGCC TGGTGAAGGG CTTCTATCCA TCTGATATCG CTGTGGAGTG GGAGTCCAAT GGCCAGCCCGAGAACAATTA CCTGACCTGG CCCCCTGTGC TGGACAGCGA TGGCTCTTTC TTTCTGTATT CCAAGCTGACAGTGGATAAG AGCCGGTGGC AGCAGGGCAA CGTGTTCTCC TGTTCTGTGA TGCACGAGGC ACTGCACAATCATTACACCC AGAAATCCCT GTCACTGAGC CCCGGCAAG
SEQ ID NO:38 PD-1HC(v3.2)的DNA序列
CAGGTCCAGCTGGTGCAGAGCGGGGCAGAGGTCAAGAAACCCGGTAGCTCCGTGAAGGTCAGCTGCAAGGCTTCCGGCGGAACCTTCTCTAGTTACGCCATCAGCTGGGTGAGACAGGCTCCTGGCCAGGGACTGGAATGGATGGGCCTGATCATTCCATCTTTTGATACCGCTGGCTACGCACAGAAGTTTCAGGGACGGGTGGCAATTACAGTCGATGAGTCAACCAGCACAGCCTATATGGAGCTGTCAAGCCTGCGGTCCGAAGACACTGCCGTGTACTATTGCGCAAGGGCCGAACACTCCTCTACTGGAACCTTCGATTACTGGGGGCAGGGTACCCTGGTGACAGTCAGTTCAGCCAGCACTAAGGGACCCAGCGTGTTTCCAGAGGCCCCCTCTAGTAAATCCACTTCTGGAGGCACCGCTGCACTGGGCTGTCTGGTGACCGATTACTTCCCAGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCCCTGACCAGCGGAGTCCATACATTTCCTGCTGTGCTGGAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCTTCAAATACAAAAGTTGACAAACGTGTGGAACCCAAGAGTTGTGATAAAACCCATACATGCCCCCCTTGTCCGGCGCCAGAGGCTGCAGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACACTGATGATTTCCCGAACCCCCGAAGTCACATGCGTGGTCGTGTCTGTGAGTCACGAGGACCCTGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACTAAACCTAGGGAGGAACAGTACAACTCAACCTATCGCGTCGTGAGCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATATAAGTGCAAAGTGAGCAATAAGGCCCTGCCCGCTCCTATCGAGAAAACCATTTCCAAGGCTAAAGGGCAGCCTCGCGAACCACAGGTCTACGTGCTGCCTCCATCCCGGGACGAGCTGACAAAGAACCAGGTCTCTCTGCTGTGCCTGGTGAAAGGCTTCTATCCATCAGATATTGCTGTGGAGTGGGAAAGCAATGGGCAGCCCGAGAACAATTACCTGACTTGGCCCCCTGTGCTGGACTCTGATGGGAGTTTCTTTCTGTATTCTAAGCTGACCGTGGATAAAAGTAGGTGGCAGCAGGGAAATGTCTTTAGTTGTTCAGTGATGCATGAAGCCCTGCATAACCACTACACCCAGAAAAGCCTGTCCCTGTCCCCCGGAAAA
SEQ ID NO:39 PD-1HC(v13844)的DNA序列
CAGGTCCAGCTGGTGCAGAGCGGGGCAGAGGTCAAGAAACCCGGTAGCTCCGTGAAGGTCAGCTGCAAGGCTTCCGGCGGAACCTTCTCTAGTTACGCCATCAGCTGGGTGAGACAGGCTCCTGGCCAGGGACTGGAATGGATGGGCCTGATCATTCCAATGTTCGACACCGCTGGCTACGCACAGAAGTTTCAGGGACGGGTGGCAATTACAGTCGATGAGTCAACCAGCACAGCCTATATGGAGCTGTCAAGCCTGCGGTCCGAAGACACTGCCGTGTACTATTGCGCAAGGGCCGAACACTCCTCTACTGGAACCTTCGATTACTGGGGGCAGGGTACCCTGGTGACAGTCAGTTCAGCCAGCACTAAGGGACCCAGCGTGTTTCCAGAGGCCCCCTCTAGTAAATCCACTTCTGGAGGCACCGCTGCACTGGGCTGTCTGGTGACCGATTACTTCCCAGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCCCTGACCAGCGGAGTCCATACATTTCCTGCTGTGCTGGAGTCAAGCGGGCTGTACTCCCTGTCCTCTGTGGTCACCGTGCCAAGTTCAAGCCTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCTTCAAATACAAAAGTTGACAAACGTGTGGAACCCAAGAGTTGTGATAAAACCCATACATGCCCCCCTTGTCCGGCGCCAGAGGCTGCAGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACACTGATGATTTCCCGAACCCCCGAAGTCACATGCGTGGTCGTGTCTGTGAGTCACGAGGACCCTGAAGTCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAGACTAAACCTAGGGAGGAACAGTACAACTCAACCTATCGCGTCGTGAGCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATATAAGTGCAAAGTGAGCAATAAGGCCCTGCCCGCTCCTATCGAGAAAACCATTTCCAAGGCTAAAGGGCAGCCTCGCGAACCACAGGTCTACGTGTATCCTCCAAGCCGGGACGAGCTGACAAAGAACCAGGTCTCCCTGACTTGTCTGGTGAAAGGGTTTTACCCTAGTGATATCGCTGTGGAGTGGGAATCAAATGGACAGCCAGAGAACAATTATAAGACTACCCCCCCTGTGCTGGACAGTGATGGGTCATTCGCACTGGTCTCCAAGCTGACAGTGGACAAATCTCGGTGGCAGCAGGGAAATGTCTTTTCATGTAGCGTGATGCATGAAGCACTGCACAACCATTACACCCAGAAGTCACTGTCACTGTCACCAGGAAAA
SEQ ID NO:40 IgG1 CH wt
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:41 v3.2 PD-L1 CH
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:42 v13844 PD-1CH
ASTKGPSVFPEAPSSKSTSGGTAALGCLVTDYFPEPVTVSWNSGALTSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:43 v3.2 PD-1CH
ASTKGPSVFPEAPSSKSTSGGTAALGCLVTDYFPEPVTVSWNSGALTSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:44 v13844 PD-L1 CH
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:45 CLλ-野生型
QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO:46 CL PD-L1
QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAESELSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS
SEQ ID NO:47 CLκ-野生型
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:48 CLκ-PD-1
RTVAAPSVFIFPPSDEQLKSGTARVGCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLRSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:49(PD-L1 Ab v3.2的HC)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPDYSPYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:50 PD-L1 LC的DNA序列(密码子变体1)
CAGTCTGTGC TGACTCAGCC ACCTTCCGCC TCTGGAACCC CAGGACAGAG GGTCACAATCAGTTGCTCAG GGAGCTCCTC TAACATTGGA AGCAACACTG TGAATTGGTA CCAGCAGCTG CCTGGGACCGCTCCAAAGCT GCTGATCTAT GGCAACTCCA ATCGACCATC TGGAGTGCCT GACCGGTTCA GCGGCTCCAAATCTGGCACC AGTGCTTCAC TGGCAATTAG TGGCCTGCAG TCCGAGGACG AAGCCGATTA CTATTGCCAGAGCTACGATA GTTCACTGAG CGGCTCCGTG TTCGGCGGGG GAATCAAGCT GACAGTCCTG GGACAGCCAAAAGCGGCGCC CAGCGTGACT CTGTTTCCAC CCAGCTCCGA GGAACTGCAG GCCAATAAGG CTACCCTGGTCTGTCTGATT TCCGACTTCT ACCCCGGGGC TGTGACAGTC GCATGGAAGG CCGATTCTAG TCCTGTGAAAGCAGGAGTCG AGACCACAAC TCCATCAAAG CAGAGCAACA ACAAGTACGC AGCCGAGAGC GAGCTGTCTCTGACACCTGA ACAGTGGAAA AGCCACCGGT CTTATAGTTG TCAGGTGACT CACGAGGGCT CAACAGTGGAAAAGACAGTC GCACCCGCAG AATGCTCA
SEQ ID NO:51 PD-L1 HC的DNA序列(密码子变体1)
CAGGTCCAGC TGGTGCAGAG CGGAGCCGAA GTGAAGAAAC CAGGCAGCTC CGTCAAGGTGTCATGCAAAG CCAGCGGCGG GACTTTCTCT AGTTACGCTA TCTCCTGGGT GAGACAGGCA CCAGGACAGGGACTGGAGTG GATGGGAGGA ATCATTCCTA TCTTCGGGAC AGCTAACTAC GCACAGAAGT TTCAGGGAAGGGTGACTATT ACCGCCGACA AATCTACAAG TACTGCTTAT ATGGAGCTGT CAAGCCTGAG GAGCGAAGATACCGCAGTGT ACTATTGCGC CCGCTCCCCC GACTACTCTC CTTACTATTA CTATGGCATG GACGTGTGGGGGCAGGGAAC CACAGTCACA GTGTCCTCTG CCAGCACTAA GGGGCCTTCA GTGTTTCCAC TGGCACCCAGTTCAAAATCA ACAAGCGGAG GAACTGCCGC TCTGGGATGT CTGGTGAAGG ACTATTTCCC AGAGCCAGTCACCGTGAGCT GGAACTCCGG CGCACTGACT TCCGGAGTCC ACACCTTTCC AGCCGTGCTG CAGAGCTCCGGACTGTACTC TCTGAAGAGT GTGGTCACAG TGCCTTCAAG CTCCCTGGGC ACCCAGACAT ATATCTGCAACGTGAATCAC AAGCCTAGTA ATACTAAGGT TGACAAACGT GTGGAACCAA AGAGCTGTGA TAAAACTCATACCTGCCCCC CTTGTCCGGC GCCAGAGGCA GCAGGAGGAC CAAGCGTGTT CCTGTTTCCA CCCAAGCCCAAAGACACCCT GATGATTAGC CGAACCCCTG AAGTCACATG CGTGGTCGTG TCCGTGTCTC ACGAGGACCCAGAAGTCAAG TTCAACTGGT ACGTGGATGG CGTCGAGGTG CATAATGCCA AGACAAAACC CCGGGAGGAACAGTACAACA GCACCTATAG AGTCGTGTCC GTCCTGACAG TGCTGCACCA GGATTGGCTG AACGGCAAGGAATATAAGTG CAAAGTGTCC AATAAGGCCC TGCCCGCTCC TATCGAGAAA ACCATTTCTA AGGCAAAAGGCCAGCCTCGC GAACCACAGG TCTACGTGTA TCCTCCAAGC CGGGACGAGC TGACAAAGAA CCAGGTCTCCCTGACTTGTC TGGTGAAAGG GTTTTACCCT AGTGATATCG CTGTGGAGTG GGAATCAAAT GGACAGCCAGAGAACAATTA TAAGACTACC CCCCCTGTGC TGGACAGTGA TGGGTCATTC GCACTGGTCT CCAAGCTGACAGTGGACAAA TCTCGGTGGC AGCAGGGAAA TGTCTTTTCA TGTAGCGTGA TGCATGAAGC ACTGCACAACCATTACACCC AGAAGTCACT GTCACTGTCA CCAGGAAAA
Claims (16)
1.结合人PD-L1和人PD-1的抗体,其包含:
e)第一重链(HC1),其包含含有氨基酸序列SEQ ID NO:3的重链可变区(HCVR);
f)第一轻链(LC1),其包含含有氨基酸序列SEQ ID NO:4的轻链可变区(LCVR);
g)第二重链(HC2),其包含含有氨基酸序列SEQ ID NO:5的重链可变区;和
h)第二轻链(LC2),其包含含有氨基酸序列SEQ ID NO:8的轻链可变区。
2.权利要求1的抗体,其包含:
a)HC1,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:3的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:9,CH2结构域中的氨基酸序列SEQ ID NO:10,及CH3结构域中的氨基酸序列SEQ ID NO:11或SEQ ID NO:12;
b)LC1,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:4的LCVR,及恒定区中的氨基酸序列SEQ ID NO:14;
c)HC2,其按从N端至C端的顺序包含:含有氨基酸序列SEQ ID NO:5的HCVR,CH1结构域中的氨基酸序列SEQ ID NO:13,CH2结构域中的氨基酸序列SEQ ID NO:10,及CH3结构域中的氨基酸序列SEQ ID NO:12或氨基酸序列SEQ ID NO:11;和
d)LC2,其按从N端起包含:含有氨基酸序列SEQ ID NO:8的LCVR,及恒定区中的氨基酸序列SEQ ID NO:15,条件是氨基酸序列SEQ ID NO:11存在于所述HC1的CH3结构域中时,氨基酸序列SEQ ID NO:12存在于所述HC2的CH3结构域中;或在氨基酸序列SEQ ID NO:12存在于所述HC1的CH3结构域中时,氨基酸序列SEQ ID NO:11存在于所述HC2的CH3结构域中。
3.权利要求2的抗体,其中所述HC1在CH3结构域中包含氨基酸序列SEQ ID NO:11,且其中所述HC2包含含有氨基酸序列SEQ ID NO:6的HCVR,和所述HC2在CH3结构域中包含氨基酸序列SEQ ID NO:12。
4.权利要求3的抗体,其中所述HC1、LC1、HC2和LC2分别包含氨基酸序列SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO 31和SEQ IDNO:34。
5.权利要求3的抗体,其中所述HC1、LC1、HC2和LC2分别包含氨基酸序列SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:33和SEQ IDNO:34。
6.哺乳动物细胞,其包含含有编码具有氨基酸序列SEQ ID NO:49、SEQ ID NO:30、SEQID NO:31和SEQ ID NO:34的多肽的多核苷酸序列的DNA分子,其中所述细胞能够表达权利要求4的抗体。
7.哺乳动物细胞,其包含含有编码具有氨基酸序列SEQ ID NO:29、SEQ ID NO:30、SEQID NO:33和SEQ ID NO:34的多肽的多核苷酸序列的DNA分子,其中所述细胞能够表达权利要求5的抗体。
8.用于产生抗体的方法,其包括在使得表达抗体的条件下培养权利要求6或7的哺乳动物细胞,并回收所表达的抗体。
9.药物组合物,其包含权利要求1-5中任一项所述的抗体和可接受的载体、稀释剂或赋形剂。
10.权利要求9所述的药物组合物,其还包含选自以下的一种或多种抗肿瘤剂:顺铂、卡铂、达卡巴嗪、多柔比星脂质体、多西紫杉醇、环磷酰胺和多柔比星、长春瑞滨、艾力布林、紫杉醇、紫杉醇蛋白结合颗粒注射悬液、伊沙匹隆、卡培他滨、FOLFOX(亚叶酸、氟尿嘧啶和奥沙利铂)、FOLFIRI(亚叶酸、氟尿嘧啶和伊立替康)、吉西他滨、拓扑替康、伊立替康脂质体、培美曲塞、西妥昔单抗、纳武单抗、伊匹单抗、pidilizumab、pembrolizumab、tremelimumab、urelumab、lirilumab、atezolizumab、epacadostat和durvalumab。
11.权利要求1-5中任一项所述的抗体的用途,用于制备治疗癌症的药物组合物。
12.权利要求11的用途,其中所述癌症是霍奇金或非霍奇金淋巴瘤、黑素瘤、肾癌、肺癌、膀胱癌、胃和食管癌、结直肠癌、肝癌、胆管癌、胰腺癌、乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、间皮瘤、头颈癌的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性成胶质细胞瘤。
13.权利要求12的用途,其中肺癌是非小细胞肺癌(NSCLC)或小细胞肺癌。
14.权利要求12的用途,其中乳腺癌是三阴乳腺癌。
15.权利要求11的用途,其中所述癌症是肾细胞癌。
16.权利要求11的用途,其中所述癌症是肝细胞癌。
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PCT/US2018/041205 WO2019014091A1 (en) | 2017-07-10 | 2018-07-09 | BISPECIFIC ANTIBODIES AS INHIBITORS OF CONTROL POINTS |
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CN110520445B (zh) | 2017-04-01 | 2023-03-14 | 北京韩美药品有限公司 | 抗pd-l1/抗pd-1天然抗体结构样异源二聚体形式双特异抗体及其制备 |
WO2019153200A1 (zh) | 2018-02-08 | 2019-08-15 | 北京韩美药品有限公司 | 抗pd-1/抗her2天然抗体结构样异源二聚体形式双特异抗体及其制备 |
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