CN111848805B - 用于肿瘤免疫治疗的具有双Her2位点的双特异性抗体 - Google Patents
用于肿瘤免疫治疗的具有双Her2位点的双特异性抗体 Download PDFInfo
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Abstract
本发明公开了一种具有双Her2位点的双特异性抗体,其包含:(a)抗CD3的抗原结合片段Fab,其具有轻链可变区VL和轻链恒定区CL,以及重链可变区VH和重链恒定区CH1;(b)抗Her2单域抗原结合片段VHH1,其被连接至所述Fab的CL的C端,并且能够结合至第一Her2表位;以及(c)抗Her2单域抗原结合片段VHH2,其被连接至所述Fab的CH1的C端,并且能够结合至第二Her2表位;其中,所述第一Her2表位和所述第二Her2表位是Her2的非重叠性表位。本发明的具有双Her2位点的双特异性抗体对于IHC评分为+1的Her2肿瘤也具有作用,或能够作用于曲妥珠单抗抗性肿瘤。
Description
技术领域
本发明涉及一种用于肿瘤免疫治疗的双特异性抗体,特别是一种具有双Her2位点的双特异性抗体。本发明还涉及包含该双特异性抗体的药物组合物以及编码所述抗体片段的多核苷酸,包含该多核苷酸的表达载体,包含该表达载体的宿主细胞。
背景技术
人表皮生长因子受体2(Her2,也称为Her2/neu或ErbB2)是跨膜受体酪氨酸激酶的Her家族的一员。Her2包含细胞质酪氨酸激酶区、单一跨膜区以及约630个氨基酸的胞外区,该胞外区含有四种不同结构域(结构域I-IV)。Her2原癌基因在25%–30%的人类原发性乳腺肿瘤和各种其他人类癌症(例如,肺癌、胃癌、口腔癌和结直肠癌)中过表达且具有功能重要性。
Her2在乳腺癌发展中的重要作用促进针对Her2的治疗发展。抗Her2疗法、曲妥珠单抗(Trastuzumab)、拉帕替尼(Lapatinib)、帕妥珠单抗(Pertuzumab)和T-DM1的研发已经为Her2阳性患者带来了临床益处。目前曲妥珠单抗仍然是Her2阳性乳腺癌的主要治疗手段。然而,当前的疗法仍然存在低反应率和耐药性的问题。例如,由于原发耐药性和获得耐药性,仅有15%-30%的Her2阳性患者对曲妥珠单抗治疗有反应。曲妥珠单抗对体内和体外Her2低或中等表达癌细胞的影响极小。差的内化作用也将在T-DM1治疗移性乳腺癌中导致耐药性。为了改善针对Her-2的抗体的效力,日益增多新型的靶向Her2的抗体现在已经被报道,其中包括组合疗法和双特异性抗体,例如曲妥珠单抗、帕妥珠单抗与多烯紫杉醇的联用已经被批准用于Her2阳性转移性乳腺癌的患者的一线治疗;以及靶向Her2和Her3、或靶向Her2的两个不同表位、或通过靶向Her2和CD3将T细胞接合至Her2癌细胞的的双特异性抗体等。但这些抗体和疗法对于Her2低表达细胞(例如,MCF7细胞,IHC评分为1+)仍然没有细胞毒性或仅具有低细胞毒性。
发明内容
本发明要解决的技术问题在于向对Her2靶向治疗耐药或无应答的患者提供治疗方案。本发明要解决的另一技术问题在于提供一种具有更广泛适应性的治疗方案,其对于任何级别的Her2过表达肿瘤(即,IHC评分为3+、2+、1+的Her2肿瘤)均有效。
本发明在一方面提供一种具有双Her2位点的双特异性抗体(在本文中也称为Bp-Bs),其包含:(a)抗CD3的抗原结合片段Fab,其具有轻链可变区VL和轻链恒定区CL,以及重链可变区VH和重链恒定区CH1;(b)抗Her2单域抗原结合片段VHH1,其被连接至所述Fab的CL的C端,并且能够结合至第一Her2表位;以及(c)抗Her2单域抗原结合片段VHH2,其被连接至所述Fab的CH1的C端,并且能够结合至第二Her2表位;其中,所述第一Her2表位和所述第二Her2表位是Her2的非重叠性表位。
在一些实施方式中,所述VHH1和/或VHH2通过接头(GGGGS)3连接至所述Fab。在一些实施方式中,中所述VHH1和VHH2的氨基酸序列独立地选自包含SEQ ID NO.1的序列、包含SEQ ID NO.2的序列、以及与任一所述序列具有70%以上的同一性的序列,优选地与任一所述序列具有至少75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。在一些实施方式中,所述VHH1和VHH2的氨基酸序列独立地选自SEQID NO.1、SEQ ID NO.2、以及与任一所述序列具有70%以上的同一性的序列,优选地与任一所述序列具有至少75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。在一些实施方式中,所述抗CD3的抗原结合片段Fab是来自CD3单克隆抗体UCHT1的抗原结合片段。在一些实施方式中,所述具有双Her2位点的双特异性抗体的分子量为60-100kDa,例如60、65、70、75、80、85、90、95、100kDa或它们之间的任何值。在一些实施方式中,所述具有双Her2位点的双特异性抗体的分子量为79kDa。
本发明另一方面提供一种具有双Her2位点的双特异性抗体,其包含:所述第一多肽链,其包含抗CD3的Fab的轻链恒定区CL、抗CD3 Fab的轻链可变区VL以及抗Her2单域抗原结合片段VHH1,其中所述VL、CL、VHH1按从N端至C端顺次连接,第二多肽链,其包含抗CD3的Fab的重链恒定区CH1、抗CD3 Fab的重链可变区VH以及抗Her2单域抗原结合片段VHH2,其中所述VH、CH1、VHH2按从N端至C端顺次连接;所述第一多肽链与所述第二多肽链通过二硫键连接。
在一些实施方式中,所述第一多肽链的氨基酸序列包含SEQ ID NO.3所示的序列或与SEQ ID NO.3所示的序列具有90%以上同一性的序列,优选地与该序列具有至少91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。在一些实施方式中,所述第二多肽的氨基酸序列包含SEQ ID NO.5所示的序列或与SEQ ID NO.5所示的序列具有90%以上同一性的序列,优选地与该序列具有至少91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。例如SEQ ID NO.5中第120位之后、第121位之前可以顺次加入赖氨酸(K)和亮氨酸(L)。
本发明在另一方面提供一种用于肿瘤免疫治疗的药物组合物,所述药物组合物包含治疗有效量的上述的具有双Her2位点的双特异性抗体以及药学上可接受的载体。
本发明再一方面提供,本发明的具有双Her2位点的双特异性抗体在制备治疗肿瘤的药物中的应用。
在一些实施方式中,所述肿瘤为通过免疫组化确定的IHC评分为1+、2+或3+Her2肿瘤。在一些实施方式中,其中所述肿瘤选自食管癌、胃癌、结肠癌、直肠癌、胰腺癌、肺癌、乳腺癌、子宫颈癌、子宫体癌、卵巢癌、膀胱癌、头颈癌、子宫内膜癌、骨肉瘤、前列腺癌、神经母细胞瘤。在一些实施方式中,其中所述肿瘤为曲妥珠单抗耐药或无应答肿瘤。
本发明提供了一种编码所述第一多肽链或所述第二多肽链的多核苷酸、包含所述的第一多肽的多核苷酸的质粒、包含所述的第二多肽的多核苷酸的质粒。本发明还提供同时包含两种所述质粒的表达载体以及包含该表达载体的宿主细胞。对于多核苷酸的操作涉及分子生物学、基因工程、蛋白质工程等领域的知识和实验操作,这些都是本领域技术人员所熟知的。
本发明再一方面提供一种治疗肿瘤的方法,其包括将本发明所述具有双Her2位点的双特异性抗体与癌细胞接触。相应地,本发明提供一种治疗对象的癌症的方法,其包括将治疗有效量的本发明具有双Her2位点的双特异性抗体或本发明的药物组合物施用至该对象。
本发明的具有双Her2位点的双特异性抗体具有以下优点中一种或多种:
1)与利用完整IgG进行改造的双特异性抗体相比,基于Fab结构设计的双特异性抗体在表达过程中能降低由于两轻链两重链带来的异源轻链错配的几率,从而减少生产过程中由于错配产物所带来的后续复杂的纯化工艺问题,降低生产成本;
2)基于Fab结构和VHH结构设计的双特异性抗体分子量约79kDa,可增强抗体在肿瘤组织的渗透性,降低抗体与靶点结合的空间限制性,并且降低被肾脏直接排泄的可能性,延长抗体在体内的滞留时间;
3)采用两个结合Her2不同位点的单域抗体设计出Bp-Bs,可提高抗体与Her2阳性肿瘤的结合能力,发挥靶向Her2双位点协同作用效果,对于Her2弱表达肿瘤也具有作用;
4)与曲妥珠单抗作用机制不同,能够作用于曲妥珠单抗抗性肿瘤;或
5)在anti-CD3 Fab的重链恒定区CH1的C末端通过非极性疏水柔性肽(GGGGS)3连接另一个单域抗体,可提高位于Fab C末端两个单域抗体与抗原结合的空间灵活性。
附图说明
图1A-C.Bp-Bs与Bi-Bs结构示意图及Her2结合模式。A.Bi-Bs;B.Bp-Bs;C.结合模式。图1D为还原和非还原条件下Bp-Bs与Bi-Bs的SDS-PAGE电泳图。
图2A流式细胞术检测Bp-Bs和Bi-Bs与CHO、MCF7、LS174T和SKOV3细胞的结合。图2B激光共聚焦显微技术检测Bp-Bs和Bi-Bs在CHO和SKBR3细胞表面的定位效应。图2C.Bp-Bs和Bi-Bs与Her2抗原结合的亲和力常数。
图3Bp-Bs和Bi-Bs促进T细胞介导的细胞毒性杀伤。A.在有或无T细胞情况下,不同浓度抗体对肿瘤细胞的影响;B.剂量依赖的细胞毒性杀伤实验。所有数据为三个重复样本的均值和标准偏差。(***P<0.001vs.肿瘤细胞加T细胞组,Dunnett's multiplecomparisons test)。
图4Bp-Bs对Her2下游信号通路作用较弱。不同肿瘤细胞与抗体共孵30小时,然后提起细胞裂解液总蛋白进行免疫印迹实验。A.SKOV3细胞;B.LS174T细胞;C.MCF7细胞。
图5Bp-Bs和Bi-Bs在小鼠体内的药代动力学特征。上:静脉推注后血清中Bp-Bs和Bi-Bs的浓度。结果为三个重复样本的均值和标准偏差。下:药代动力学参数。Cmax:最高血药浓度;AUC all:药时曲线下面积;CL:总清除率;Vss:表观分布容积;t1/2:消除半衰期。
图6Bp-Bs在LS174T人源结肠癌移植模型中的抗肿瘤活性。A.不同药物治疗对肿瘤的生长抑制效果。B.给药后各个实验组小鼠的体重变化。结果为每组6只小鼠数据的均值和标准误差。(***P<0.001,Dunnett's multiple comparisons test,vehicle vsTrastuzumab和vehicle vs Bp-Bs;***P<0.001,paired t test,Trastuzumab vs Bp-Bs)。
图7Bp-Bs比Bi-Bs具有更强的肿瘤抑制活性。A.不同药物治疗对肿瘤生长的抑制效果;B.给药14天时小鼠皮下肿瘤解剖图;C.给药后各个实验组小鼠的体重变化。结果为每组5只小鼠数据的均值和标准误差。(**P<0.01,vehicle vs Bi-Bs;***P<0.001,vehiclevs Bp-Bs;Dunnett's multiple comparisons test;*P<0.05,paired t test,Bi-Bs vsBp-Bs)。
具体实施方式
定义
“抗体”是指表现所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此,“抗体”在本发明中具有其最广泛的含义,并且明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体)。
如本文所用,术语“组合物”指适于给预期动物对象施用以达到治疗或预防目的的制剂,其含有至少一种药物活性组分,例如化合物。任选地,所述组合物还含在本文中,术语“治疗有效量”和“有效量”表示所述物质和物质的量对于预防、减轻或改善疾病或病症的一种或多种症状,和/或延长接受治疗的对象的生存期是有效的。
本文使用的“治疗”包括给予本申请的化合物、其药学上可接受的盐、或组合物,以减轻疾病或病症的症状或并发症,或消除疾病或病症。本文使用的术语“减轻”用于描述病症的迹象或症状的严重性降低的过程。症状可减轻而没有消除。在一种实施方案中,给予本申请的药物组合物导致迹象或症状的消除。
“对象”或“个体”或“动物”或“患者”或“哺乳动物”是指任何期望诊断、预后或治疗的对象,特别是哺乳动物对象。哺乳动物对象包括人、驯养动物、农畜以及动物园动物、竞技动物或宠物,例如狗、猫、豚鼠、兔、大鼠、小鼠、马、牛、奶牛等。
如试验例中所证实的,双特异性抗体的示意性类型是靶定两个不同抗原的抗体,其中一个抗原存在于肿瘤细胞或微生物上,另一个存在于免疫细胞上。当施用至个体时,这种双特异性抗体特异性地结合至肿瘤细胞或微生物,同时特异性地结合至免疫细胞(如细胞毒细胞)。这种双重结合可导致所结合的肿瘤或微生物被宿主的免疫系统杀死。
“单域抗原结合片段”或“单域抗体片段”或“VHH”是一种能够结合至抗原而不需配备轻链的抗原结合片段。VHH最初以单个抗原结合片段的形式分离自单域抗体(sdAb)。第一个已知的单域抗体分离自骆驼,之后分离自软骨鱼。骆驼产生没有轻链的功能性抗体,它们的单个N端结构域(VHH)结合抗原而无需结构域配对。单域抗体不包括CH1域,在常规抗体中,CH1域与轻链相互作用。VHH包含构成免疫球蛋白结构域的核心结构的四个框架区(FR1-FR4)以及涉及抗原结合的三个互补决定区(CDR1-CDR3)。相比于人VH结构域,VHH框架区显示出与人VH结构域的高序列同源性(>80%)。文献报道称:“VHH的最特征性的特点在于在四个FR2位置(第37、44、45和47位;Kabat编号)处的氨基酸取代,它们在常规VH结构域中是保守的,并且涉及与VL结构域的疏水相互作用”。VHH通常具有在这些以及其他在常规VH中高度保守的位置处的不同氨基酸(如Leu11Ser、Val37Phe或Tyr、Gly44Glu、Leu45Arg或Cys、Trp47Gly)。
Her2的胞外域包括四个结构域,结构域I(ECD1,约1-195的氨基酸残基)、结构域II(ECD2,约196-319的氨基酸残基)、结构域III(ECD3,约320-488的氨基酸残基)和结构域IV(ECD4,约489-630的氨基酸残基)(残基编号,无信号肽)。本发明技术人员可通过本领域已知方法选择Her2的表位,以及根据已知的方法确定与该表位结合的VHH片段。
术语“Her2阳性肿瘤”、“Her2过表达肿瘤”或类似术语是指特征在于Her2蛋白的过表达或Her2基因扩增的肿瘤疾病。术语Her2蛋白的“过表达”是指在来自患者的特定组织或器官内的肿瘤的细胞中,相对于在来自该组织或器官的正常细胞中的表达水平,Her2受体蛋白的表达的异常水平。患有特征在于Her2受体的过量表达的癌症的患者或对象可以通过本领域已知的标准测定确定。Her2阳性癌症特别指如通过免疫组织化学所确定,具有程度1+(Her2 1+)、程度2+(Her2 2+)、或程度3+(Her2 3+)的Her2过表达的癌。在某些实施方案中,Her2阳性癌症是具有如通过免疫组织化学所确定的程度2+或更低、优选地程度1+或更低的Her2表达的癌。如通过实施例显示,患有特征在于在1+,2+,或3+,优选地1+或2+,更优选地1+或更低范围内的Her2蛋白过量表达的癌症的患者将得益于本发明的治疗方法。在这个方面,免疫组织化学指固定的肿瘤样品的免疫组织化学染色和对染色的分析。Her2表达水平0(Her2 0)指在少于10%的肿瘤细胞中无染色或胞膜染色,尤其少于20,000Her2/细胞。Her2 1+指在多于10%的肿瘤细胞中胞膜微弱染色,其中细胞膜仅部分着染,尤其约100,000Her2/细胞。Her2 2+指在多于10%的肿瘤细胞中整个胞膜微弱至中度着染,尤其约500,000Her2/细胞。Her2 3+指在多于10%的肿瘤细胞中完整胞膜强烈着染,尤其约2,000,000Her2/细胞。优选地使用包含癌细胞的组织学样品、尤其福尔马林固定石蜡包埋的癌组织样品,确定Her2表达。用于确定Her2过表达的免疫组织化学测定法优选地包括(i)使包含癌细胞的样品与针对Her2的第一抗体接触,随后(ii)使样品与针对第一抗体并与可视化剂(如催化具有可视终产物的反应的酶,例如辣根过氧化物酶)偶联的第二抗体接触。合适的Her2免疫组织化学试剂盒是HercepTest(Dako Denmark A/S)和Pathway Her2(VentanaMedical Systems,Inc.)。Her2阳性肿瘤疾病还包括如通过荧光原位杂交(FISH)或生色原原位杂交(CISH)确定,对Her2基因扩增呈阳性的癌。根据FISH测定法如果肿瘤细胞中Her2基因的拷贝数是染色体17的拷贝数的至少2倍或如果肿瘤细胞包含至少4个拷贝的Her2基因,则癌症对Her2基因重复呈阳性。根据CISH测定法如果至少50%的肿瘤细胞中存在每个细胞核至少5个拷贝的Her2基因,则癌对Her2基因重复呈阳性。
可使用表达Her2的细胞,例如乳腺癌细胞系可以用于评价本发明的抗体。下面表描述了在几种代表性癌细胞系中Her2的表达水平。
术语“曲妥珠单抗耐药肿瘤”定义为肿瘤细胞对曲妥珠单抗的敏感性降低。将患有这样的肿瘤的患者确定为“曲妥珠单抗耐药肿瘤”患者。由于该抗性可以是原发性的或获得性的,所以将观察到的任一敏感性降低与完全敏感性的“正常”肿瘤细胞(相对于在治疗开始时的最初敏感性,它们对治疗有效剂量的所应用的抗肿瘤药有应答)相比。在后面的情况中,该抗性或耐药性表现为在相同剂量下肿瘤退化量减少或对等量肿瘤退化必需的剂量增加。
本文所用“抑制”或“治疗”包括延缓与疾病有关的症状的发展和/或减轻所述疾病将要或预期发展的这些症状的严重程度。所述术语还包括减缓已有症状、防止另外的症状和减缓或防止这些症状的潜在原因。因此,所述术语表示业已将有益结果赋予患有疾病的脊椎动物对象。
本文所用术语“治疗有效量”或“有效量”是指当将本发明具有双Her2位点的双特异性抗体或其片段单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时,其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量,所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态,或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时,治疗有效量是指该单独的成分。当施用组合时,治疗有效量是指产生治疗效果的活性成分的联合的量,而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10%;通常至少20%;优选至少约30%;更优选至少40%和最优选至少50%。
药物制剂或药物组合物
本发明包括本发明的具有双Her2位点的双特异性抗体或抗体片段的药物制剂。为了制备药物组合物或无菌组合物,让抗体或其片段与可药用载体或赋形剂混合。可通过与生理学上可接受的载体、赋形剂或稳定剂混合,来制备呈例如冻干粉、浆液、水溶液剂或混悬剂形式的治疗及诊断药物的制剂。
可通过标准药物方法,在细胞培养物或实验动物中测定单独给予或与免疫抑制剂联合给予的抗体组合物的毒性和治疗功效,所述方法例如为用于测定LD50(使群体的50%致死的剂量)和ED50(有效治疗群体的50%的剂量)的方法。毒性和治疗效果之间的剂量比是治疗指数,可表示为LD50与ED50之比。从这些细胞培养物测定及动物研究中获得的数据可用于调配用于人的剂量范围。所述化合物的剂量优选在包括毒性极少或无毒性的ED50的循环浓度范围内。可根据采用的剂型及所用的给药途径,使剂量在该范围内变化。
合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)及口服给药。可按多种常规方式给予用于药物组合物或用于实践本发明方法的抗体,这些方法例如有经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。在一个实施方案中,静脉内给予本发明的结合化合物。在另一个实施方案中,皮下给予本发明的结合化合物。或者,人们可以以局部而非全身方式(通常为长效制剂或缓释制剂)给予抗体,例如经由将抗体直接注射到作用位点。此外,人们可在靶向药物递送系统中给予抗体。
由临床医生例如用本领域已知或怀疑影响治疗或预期影响治疗的参数或因子来测定合适的剂量。通常,开始剂量比最佳剂量稍低,此后少量增加直到达到相对于任何不良副作用所要的或最佳的作用效果。重要的诊断测量包括测量例如炎性症状或所产生的炎性细胞因子的水平。
可通过连续输注或通过以一定间隔(例如一天、一周或每周1-7次)给药来提供抗体、抗体片段和细胞因子。可通过静脉内、皮下、腹膜内、经皮肤、局部、经口、经鼻、经直肠、肌内、大脑内、脊柱内或通过吸入来提供剂量。优选剂量方案是包括避免显著的不合乎需要的副作用的最大剂量或给药频率的方案。周总剂量通常为至少0.05μg/kg体重,更通常为至少0.2μg/kg,最通常为至少0.5μg/kg,典型地为至少1μg/kg,更典型地为至少10μg/kg,最典型地为至少109μg/kg,优选为至少0.2mg/kg,更优选为至少1.0mg/kg,最优选为至少2.0mg/kg,理想地为至少10mg/kg,更理想地为至少25mg/kg,而最理想地为至少50mg/kg。基于摩尔/kg计算,小分子治疗剂例如肽模拟物、天然产物或有机化学药剂的所需剂量与抗体或多肽的剂量接近相同。
本发明药物组合物还可含有其它药剂,包括但不限于细胞毒剂、细胞生长抑制剂、抗血管形成药物或抗代谢药物、靶向肿瘤药物、免疫刺激剂或免疫调节剂或与细胞毒剂、细胞生长抑制剂或其它毒性药物缀合的抗体。也可与其它治疗形式(例如手术、化疗及放射)一起施用所述药物组合物。典型的兽医、实验或研究对象包括猴、狗、猫、大鼠、小鼠、兔、豚鼠、马和人。
肿瘤
本发明抗体可用于治疗肿瘤(即抑制肿瘤细胞的生长或存活)。可用本发明抗体抑制其生长的优选的肿瘤包括通常对免疫疗法有反应的肿瘤。用于治疗的优选癌症的非限制性实例包括Her2过表达癌症。该Her2过表达癌症可包括Her2高过表达癌症、Her2中等过表达癌症或Her2低过表达癌症。Her2过表达癌症的例子包括但不限于食管癌、胃癌、结肠癌、直肠癌、胰腺癌、肺癌、乳腺癌、子宫颈癌、子宫体癌、卵巢癌、膀胱癌、头颈癌、子宫内膜癌、骨肉瘤、前列腺癌、神经母细胞瘤。如前所述,Her2过表达癌症可以根据IHC分类为Her2 1+、Her2 2+、Her2 3+过表达癌症。本发明的抗体适用于Her2 1+、Her2 2+、Her2 3+过表达癌症。实验证实,本发明的抗体对于Her2 1+肿瘤仍具有显著杀伤效果。
本发明抗体可单独使用或与以下其它物质联合使用:抗肿瘤药或免疫原剂(例如减弱的癌细胞、肿瘤抗原(包括重组蛋白质、肽和糖类分子)、抗原呈递细胞例如用来源于肿瘤的抗原或核酸刺激的树突细胞、免疫刺激细胞因子(例如IL-2、IFNa2、GM-CSF)和用编码免疫刺激细胞因子(例如但不限于GM-CSF)的基因转染的细胞;标准癌症治疗(例如化疗、放疗或手术);或其它抗体,包括但不限于针对以下物质的抗体:VEGF、EGFR、VEGF受体、其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS,例如曲妥珠单抗或帕托珠单抗。
免疫佐剂
其它组合疗法如上文所述,可共同给予本发明具有双Her2位点的双特异性抗体及一种或多种其它治疗剂(例如细胞毒剂、放射性毒性剂或免疫抑制剂)。所述抗体可与所述药剂连接(作为免疫复合体),或可与治疗剂分开给予。在后一种情况下(分开给予)下,可在给予治疗剂之前、之后或并行给予抗体,或可与其它已知的疗法共同给予。
抗体还可用于体内诊断测定。通常用放射性核素(例如111In、99Tc、4C、31I、125I、3H、32P、35S或18F)标记抗体,以使可用免疫显像或正电子成像术定位抗原或表达抗体的细胞。
通过参考以下实施例将更充分地理解本发明。然而,这些实施例不应该理解为限制本发明范围。本文提及的所有文献和专利引用都明确地通过引用并入本文。
实施例
实施例1.双特异性抗体Bp-Bs及其对照Bi-Bs设计和纯化
二价抗Her2双特异抗体(Bi-Bs)和与Her2双位点结合的双特异性抗体Bp-Bs的结构分别图1A和1B所示。将DNA改组和连接技术用于克隆各自的基因。其中,Bi-Bs:单链结构域抗-Her2 VHH1(SEQ ID NO.1,GenBank:JX047590.1;Even-Desrumeaux,K.,P.Fourquet,V.Secq,D.Baty and P.Chames(2012)."Single-domain antibodies:a versatile andrich source of binders for breast cancer diagnostic approaches."Mol Biosyst 8(9):2385-2394.)连接至抗CD3UCHT1克隆的VH-CH1(具有接头:(GGGGS)3))以及VL-CL的C端;而对于Bp-Bs:则用另外的抗-Her2 VHH2来替换在Bi-Bs的VH-CH1处的抗-Her2 VHH1(SEQ ID NO.2;Wu,X.,S.Chen,L.Lin,J.Liu,Y.Wang,Y.Li,et al.(2018)."A SingleDomain-Based Anti-Her2Antibody Has Potent Antitumor Activities."Transl Oncol11(2):366-373.)。将生成的重链和轻链基因分别克隆到pET26b载体(重链HC)pET21a载体(轻链LC)。通过VH-CH1-VHH2(SEQ ID NO.5)和VL-CL-VHH1(SEQ ID NO.3)的异源二聚作用来形成Bp-Bs抗体。而Bi-Bs抗体则是通过VH-CH1-VHH1(SEQ ID NO.3)和VL-CL-VHH1(SEQIDNO.4)的异源二聚作用形成。因此,将分子克隆技术获得的对应重组质粒以1:1的比例共转入BL21大肠杆菌感受态细胞,在卡那霉素和氨苄霉素双重抗性的琼脂糖平板中生长获得单克隆双转子菌落;挑取单菌落接种于LB培养基培养后扩大至M9培养基培养,用IPTG诱导大肠杆菌表达Bi-Bs和Bp-Bs蛋白;收集培养基上清液通过Ni Sepharose亲和纯化得到提纯的Bi-Bs和Bp-Bs蛋白。然后将纯化的抗体Bi-Bs和Bp-Bs蛋白在还原条件下和非还原条件下进行SDS-PAGE电泳,随后通过考马斯亮蓝染色。如图1D所示,纯化蛋白在SDS-PAGE上的相对迁移率与在还原条件下预期的39kDa的单链Bi-Bs或Bp-Bs抗体的分子量,而在非还原条件下79kDa的Bi-Bs或Bp-Bs抗体的分子量是一致的。
实施例2.Bp-Bs抗体的结合特性
实验方法:
细胞系:CHO,MCF7,LS174T,SKOV3,SKBR3细胞均来源于中科院典型培养物保藏委员会细胞库;细胞培养所用培养基、胎牛血清、胰酶、青霉素-链霉素抗生素混合液等添加剂均购自于Gibco公司;细胞培养所用耗材均购自于Corning Costar公司。所有细胞系在37℃,5%CO2条件下在含有10%HI胎牛血清(Thermo,USA)和1%青霉素/链霉素(Hyclone)的DMEM(用于MCF7、SKBR-3和SKOV3)或RPMI-1640(Thermo,China)(用于LS174T和CHO)中培养。
亲和力测定:使用OctetQKe仪器(Pall Life Sciences)测定抗Her2抗体对Her2蛋白胞外区的亲和力。简而言之,将PBST中具有Fc标签的人Her2(AcroBiosystem,货号HE2-H5253)上样到ProteinA Capture Biosensors(ProA)的表面上。达到0.8nM至1.2nM的固化水平。然后应用60秒生物传感器基线步骤,之后分析生物传感器上的抗原/抗体association 180秒,以测试抗体/抗原。然后以两倍浓度梯度施加被测试分子。使用数据分析软件8.2版(PALL/ForteBio)评估Octet数据,并使用全拟合1:1模态来确定Kd值。
流式细胞术分析:将流式细胞术用于评估双特异性抗体在Her2阳性或阴性细胞上的结合。培养不同的细胞系并在胰蛋白酶消化后重悬浮。然后洗涤细胞并重悬于0.1%BSA的PBS溶液中。在不存在或存在抗体的情况下,将每个样品总共100μL的5×10 5个细胞在冰上孵育1小时。用冰冷的PBS洗涤两次后,将细胞在冰上与山羊抗人IgG(H+L)-AF488(Invitrogen,货号A11013)一起孵育1小时。用Cytomics FC500流式细胞仪(BeckmanCoulter)分析细胞相关荧光,并使用FlowJo(http://www.flowjo.com)绘图。
免疫荧光试验:为了进一步分析抗体与Her2在细胞表面上的结合,如前所述进行免疫荧光测定(Xing,J.,L.Lin,J.Li,J.Liu,C.Zhou,H.Pan,et al.(2017)."BiHC,a T-Cell-Engaging Bispecific Recombinant Antibody,Has Potent Cytotoxic ActivityAgainst Her2 Tumor Cells."Transl Oncol 10(5):780-785.)简言之,将CHO和SKBR3细胞在玻璃底培养皿(Cellvis)上培养过夜。用PBS洗涤三次之后,用4%多聚甲醛固定细胞。在室温下用PBS加1%BSA封闭1小时后,将细胞与抗体在室温下孵育1小时。用PBS洗涤三次后,将样品与山羊抗人IgG(H+L)-AF488在4℃温育1小时。用PBS洗涤后,使用共聚焦激光扫描显微镜(Zeiss EC Plan-Neofluar 40x/1.30Oil DIC M27物镜)检查样品并通过ZEN软件进行分析。
实验结果:
为了检测抗体与Her2抗原的结合能力,我们采用生物膜干涉技术(BLI)分析抗体与Her2抗原的相互作用。如图2C所示,作为对照的曲妥珠单抗、抗Her2-VHH1-Fc或抗Her2-VHH2-Fc分别具有0.213nM、8.85nM或3.02nM的亲和力。亲和力数据(KD)(图2C)示出了,基于anti-Her2 VHH1改造的单位点双价双特异性抗体Bi-Bs亲和力(3.06nM)与单位点双价抗体anti-Her2 VHH1-Fc(8.85nM)类似,说明双特异性抗体的改造不影响抗体与Her2的结合能力。然而,具有双Her2位点的双特异性抗体Bp-Bs的亲和力(0.109nM)比单位点双价抗体anti-Her2 VHH1-Fc、anti-Her2 VHH2-Fc(3.02nM)或Bi-Bs强30倍,并且与Trastuzumab亲和力相当,说明基于Her2双位点改造的抗体Bp-Bs对Her2抗原具有更高的亲和力。
为了检测抗体与细胞表面抗原的结合能力,我们采用流式细胞术(FACS)对Bp-Bs和Bi-Bs进行分析。采用已经被鉴定为Her2阴性细胞株CHO,Her2高表达细胞株SKOV3,Her2中等表达细胞株LS174T和Her2弱表达表达细胞株MCF7进行实验,实验结果表明(图2A):Bp-Bs和Bi-Bs均不结合Her2及CD3阴性细胞CHO;在Her2阳性细胞株中,Bp-Bs和Bi-Bs阳性荧光信号位移与细胞Her2表达量呈正相关,提示Bp-Bs和Bi-Bs对SKOV3,LS174T和MCF7细胞均有不同程度结合;与Bi-Bs相比,Bp-Bs在Her2阳性细胞中表现出更强的结合能力。
接着我们采用激光共聚焦显微技术(Confocal microscopy)对Bp-Bs和Bi-Bs与Her2阳性SKBR3细胞表面Her2蛋白的结合情况进行定位分析,设Her2阴性CHO细胞作为对照组。SKBR3和CHO细胞分别与Bp-Bs或Bi-Bs孵育后,在SKBR3细胞膜表面呈现明显的荧光定位,但CHO细胞膜表面不出现荧光定位,提示Bp-Bs或Bi-Bs可结合于SKBR3细胞表面Her2蛋白,且Bp-Bs比Bi-Bs具有更强的细胞结合能力(图2B)。流式细胞实验和激光共聚焦实验结果共同说明Bp-Bs能与Her2阳性肿瘤细胞特异性结合,且结合能力更强。
实施例3.Bp-Bs抗体诱导T细胞介导的细胞毒性
实验方法:
为了测量体外的双特异性抗体的细胞毒性,使用Ficoll-Plaque Plus(GEhealth)梯度离心法从新鲜捐献的血液中新鲜制备人外周血单核细胞(PBMC)。人外周血采集于健康的志愿者并获得书面许可。然后使用EasySepTM人类CD3阳性选择试剂盒(StemcellTechnologies,Inc.,Vancouver,BC,Canada)根据制造商的说明书从PBMC分离人CD3+T细胞。如前所述地进行细胞毒性测定(Li,L.,P.He,C.Zhou,L.Jing,B.Dong,S.Chen,et al.(2015)."A novel bispecific antibody,S-Fab,induces potent cancer cellkilling."J Immunother 38(9):350-356.)。简言之,将SKOV3,MCF7,LS174T或CHO癌细胞用胰蛋白酶消化并以5000个细胞/孔的密度接种在96孔组织培养板中作为靶细胞,并在37℃,5%CO2中孵育过夜。然后加入50,000个未经预先刺激的人CD3+T细胞作为效应细胞。向不同的孔中加入不同浓度的抗Her2抗体。孵育72小时后,使用细胞计数试剂盒-8试剂(Dojindo,CK04)根据制造商的实验方案定量细胞活力。使用下式计算靶细胞的存活率(%):[(活靶细胞(样品)-培养基)/(活靶细胞(对照)-培养基)]×100%。
实验结果:
为了确定Bp-Bs和Bi-Bs能否募集T细胞杀伤Her2阳性肿瘤细胞,我们进行了细胞毒性杀伤实验。实验结果显示(图3A),Bp-Bs和Bi-Bs均不能募集T细胞杀伤Her2阴性细胞CHO。在不加T细胞的实验组,高低浓度的Bp-Bs和Bi-Bs均不能抑制Her2阳性细胞SKOV3和LS174T生长;在加入T细胞的实验组,15.6nM和156nM给药浓度的Bp-Bs或Bi-Bs都对Her2阳性细胞显示出显著的肿瘤杀伤作用。并且,在同等条件下,低浓度的Bp-Bs募集T细胞对Her2中等表达细胞LS174T的杀伤作用略强于Bi-Bs。
为了进一步探讨Bp-Bs和Bi-Bs对肿瘤细胞毒性作用的剂量-效应关系,我们检测了梯度浓度Bp-Bs和Bi-Bs对肿瘤细胞的杀伤作用。根据细胞毒性杀伤实验的结果(图3A),我们确定抗体给药浓度范围为1.56×102nM~1.56×10-3nM。选取SKOV3和MCF7细胞作为剂量依赖的细胞毒性杀伤实验的靶细胞,CHO细胞为对照。实验结果显示(图3B),在一定浓度范围内,Bp-Bs或Bi-Bs的细胞毒性作用与肿瘤细胞表面Her2的表达水平呈正相关;Bp-Bs或Bi-Bs对SKOV3细胞的杀伤作用与剂量呈正相关。然而,对于MCF7细胞,最高浓度的Bi-Bs仍没有明显杀伤效果,Bp-Bs剂量大于1.56nM时开始出现杀伤效果,且与剂量呈正相关。这些结果表明,与Bi-Bs相比,Bp-Bs募集T细胞特异性杀伤Her2阳性特别是Her2弱表达肿瘤细胞效果更强。
实施例4.Bp-Bs利用Her2双位点设计募集T细胞的双特异性抗体用于治疗Her2阳性肿瘤实验方法:将SKOV3、LS174T或MCF7细胞接种(300,000细胞/孔)于6孔板中,并在37℃下孵育过夜。然后用或不用100nM抗Her2抗体在37℃下处理细胞30小时。温育后,用冷PBS洗涤细胞两次,并使用RIPA裂解缓冲液(Beyotime,货号P0013B)根据制造商的说明书进行裂解。通过BCA方法(Thermo Fisher Scientific)测定蛋白质浓度,并通过8%SDS-PAGE分析各20μg的蛋白质样品,并用针对ErbB2、磷酸-ErbB2-Tyr1221/1222、AKT、磷酸-AKT-Ser473、p44/42MAPK、磷酸-p44/42MAPK-Thr202/Tyr204和Tubulin(Cell Signaling Technology,货号4290、2243、4691、4060、4695、9101和2144)的抗体进行免疫印迹。
实验结果:临床使用的Her2单克隆抗体Trastuzumab抑制肿瘤生长的机制之一是抑制Her2蛋白的表达,并下调Her2下游PI3K信号通路。结果表明(图4),Trastuzumab可以抑制LS174T和MCF7细胞Her2蛋白表达及Her2蛋白磷酸化,并下调Her2下游信号通路MAPK及AKT蛋白的磷酸化水平(图3)。相比Trastuzumab,Bp-Bs或Bi-Bs仅能轻微的下调SKOV3、LS174T和MCF7细胞Her2和MAPK蛋白的磷酸化,提示Bp-Bs和Bi-Bs对Her2下游信号通路作用较弱,其抗肿瘤机制主要依赖于其anti-CD3 Fab片段募集T细胞对肿瘤进行杀伤。因此,Bp-Bs能够用于Trastuzumab抗性的肿瘤的治疗。
实施例5.Bp-Bs的体内药代动力学实验方法:药代动力学(PK)研究:在雌性CB-17SCID小鼠中进行Bi-Bs和Bp-Bs的单剂量PK研究。将动物随机分成不同的治疗组(每组n=9,每个时间点3只动物),静脉内注射1mg/kg Bi-B或Bp-Bs。在注射后0.25、0.5、1、2、4、8、12、24和48小时收集血清样品用于生物分析测量。如前所述,通过ELISA方法测定血清中的抗体浓度(Pan,H.,J.Liu,W.Deng,J.Xing,Q.Li and Z.Wang(2018)."Site-specificPEGylation of an anti-CEA/CD3bispecific antibody improves its antitumorefficacy."Int J Nanomedicine 13:3189-3201.)。使用Kinetica(v.5.1SP1,ThermoFisher Scientific),以非房室模型将检测得到的各时间点血药浓度数据进行分析。
实验结果:如图5所示的药代动力学参数,Bp-Bs在注射后10小时示出了稍高的剩余浓度,Bi-Bs和Bp-Bss在SCID小鼠体内的消除半衰期是相似的,提示针对Fab结构C末端的VHH改造不会显著影响Bp-Bs和Bi-Bs体内代谢情况。
实施例6.Bp-Bs体内抗肿瘤活性研究
实验方法:
对于体内异种移植研究,从细胞培养物中收获LS174T人结肠癌细胞,用PBS洗涤两次,然后重悬于PBS中。以每只小鼠总体积200μl,含有1×106个LS174T细胞,皮下注射到NOD/SCID小鼠右后肢处。当肿瘤大小达到50至100m3时,将小鼠随机分组,每组5或6只,腹膜内施用5×106个新鲜分离的人PBMC(根据实施例3方法制备)。后用不同剂量的抗体或对照载体处理动物。称重小鼠,并在两个垂直维度上测定肿瘤体积,并使用下式计算:(长度×宽度2)/2。当肿瘤体积达到1500mm3时处死小鼠。所有结果均以每组的算术平均值表示。
实验结果:
对小鼠进行分组(n=6)后给予1mg/kg剂量的Bp-Bs进行治疗,以剂量2mg/kg的Trastuzumab治疗组作为阳性对照,PBS溶媒组为阴性对照,每两天腹腔注射给药一次。通过五次治疗,给药后第14天,溶媒组的肿瘤体积均值为1568mm3,2mg/kg的Trastuzumab治疗组为886mm3,1mg/kg的Bp-Bs治疗组为551mm3;即,1mg/kg的Bp-Bs能抑制65%的肿瘤生长,相比临床使用的Her2单抗Trastuzumab,Bp-Bs抗肿瘤效果明显更强(图6A)。同时,各实验组小鼠体重变化无明显差异,提示Bp-Bs在此模型中无明显毒副作用(图6B)。
体外实验表明Bp-Bs募集T细胞特异性杀伤Her2阳性特别是Her2弱表达肿瘤细胞的效果比Bi-Bs更强,我们想进一步探讨Bp-Bs在小鼠荷瘤模型是否能更有效的抑制肿瘤生长。同样在NOD/SCID小鼠上构建人源结肠癌皮下荷瘤模型并建立其人源免疫系统,我们将与Her2单位点双价结合的双特异性抗体Bi-Bs以及与Her2单位点单价结合的双特异性抗体CD3-S-Fab(参见,Lin,L.,L.Li,C.Zhou,J.Li,J.Liu,R.Shu,et al.(2018)."AHer2bispecific antibody can be efficiently expressed in Escherichia coli withpotent cytotoxicity."Oncol Lett 16(1):1259-1266.)加入实验中进行比较。采用1.5mg/kg剂量的Bp-Bs、Bi-Bs或CD3-S-Fab对模型小鼠进行治疗,设PBS溶媒组为阴性对照,每三天腹腔注射给药一次,给药五次。如图7A所示,给药后第14天,溶媒组肿瘤体积均值为1424mm3,CD3-S-Fab组为1073mm3,Bi-Bs组为857mm3,Bp-Bs组为413mm3,提示Bp-Bs比Bi-Bs或CD3-S-Fab更能抑制肿瘤生长。统计学结果差异显示,给药后第14天,Bp-Bs组和Bi-Bs组肿瘤体积大小与溶媒组相比存在显著性差异,且配对T检验结果显示Bp-Bs组肿瘤大小和Bi-Bs组存在显著性差异(P<0.05)。实验终点对小鼠皮下肿瘤进行解剖后可见(图7B),在Bp-Bs给药组中,有2只小鼠肿瘤组织极小,提示Bp-Bs给药有40%的肿瘤完全抑制的可能性。Bp-Bs、Bi-Bs或CD3-S-Fab给药均未对小鼠体重造成显著性变化(图7C)。这些结果表明在此动物荷瘤模型中,相同剂量下,Bp-Bs比Bi-Bs表现出更强的肿瘤抑制效果。Bp-Bs能更有效的聚集于Her2阳性肿瘤组织,进而募集更多T细胞对肿瘤细胞进行杀伤。
说明书中引用的所有专利和其他参考文献都是本发明所属领域普通技术人员的水平的表示,通过引用将它们以整体形式合并至本文中,包括其中的任何表格和附图,就如同每个参考文献都单独通过引用以其整体形式合并至本文中一样。本领域技术人员会容易意识到,本发明可容易改造而获得本文所述的那些目的和优点以及隐含在本文中的那些目的和优点。在本文中以当前优选实施方式的代表的形式描述的方法、变体和组合物是示例性的,并不意在限制本发明的范围。对于本领域技术人员来说,可对它们做出改变或将其用于其他用途,但这都包括在如所附权利要求定义的本发明的范围内。
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Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
245 250 255
Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly
260 265 270
Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Pro Pro Gly
275 280 285
Lys Glu Leu Glu Trp Val Ser Ala Ile Trp Gly Gly Gly Asp Ser Gln
290 295 300
His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
305 310 315 320
Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp
325 330 335
Thr Gly Met Tyr Tyr Cys Val Lys Asp Arg Gly Pro Phe Phe Ser Gly
340 345 350
Ser Lys Tyr Val Leu Gly Ala Ala Asp Arg Gly Gln Gly Thr Gln Val
355 360 365
Thr Val Ser Ser
370
Claims (18)
1.一种具有双Her2位点的双特异性抗体,其包含:
(a)抗CD3的抗原结合片段Fab,其具有轻链可变区VL和轻链恒定区CL,以及重链可变区VH和重链恒定区CH1;
(b)抗Her2单域抗原结合片段VHH1,其被连接至所述Fab的CL的C端,并且能够结合至第一Her2表位;以及
(c)抗Her2单域抗原结合片段VHH2,其被连接至所述Fab的CH1的C端,并且能够结合至第二Her2表位;
其中,所述第一Her2表位和所述第二Her2表位是Her2的非重叠性表位,并且所述VHH1和VHH2中的一个的氨基酸序列包含SEQ ID NO.2的序列,
所述VHH1和VHH2中的另一个的氨基酸序列包含SEQ ID NO.1的序列。
2.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述VHH1和/或VHH2通过接头(GGGGS)3连接至所述Fab。
3.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述VHH1和VHH2中的一个的氨基酸序列为SEQ ID NO.2的序列。
4.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述VHH1和VHH2中的一个的氨基酸序列为SEQ ID NO.2的序列,所述VHH1和VHH2中的另一个的氨基酸序列为SEQID NO.1的序列。
5.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述抗CD3的抗原结合片段Fab是来自CD3单克隆抗体UCHT1的抗原结合片段。
6.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述具有双Her2位点的双特异性抗体的分子量为60-100kDa。
7.根据权利要求1所述的具有双Her2位点的双特异性抗体,其中所述具有双Her2位点的双特异性抗体的分子量为79kDa。
8.一种具有双Her2位点的双特异性抗体,包含:
第一多肽链,其包含抗CD3的Fab的轻链恒定区CL、抗CD3的Fab的轻链可变区VL以及抗Her2单域抗原结合片段VHH1,其中所述VL、CL、VHH1按从N端至C端顺次连接,以及
第二多肽链,其包含抗CD3的Fab的重链恒定区CH1、抗CD3的Fab的重链可变区VH以及抗Her2单域抗原结合片段VHH2,其中所述VH、CH1、VHH2按从N端至C端顺次连接;
所述第一多肽链与所述第二多肽链通过二硫键连接;
其中,所述第二多肽链的氨基酸序列包含SEQ ID NO.5所示的序列,所述第一多肽链的氨基酸序列包含SEQ ID NO.3所示的序列。
9.一种用于肿瘤免疫治疗的药物组合物,所述药物组合物包含治疗有效量的权利要求1-8的任一项所述的具有双Her2位点的双特异性抗体以及药学上可接受的载体。
10.权利要求1或8所述的具有双Her2位点的双特异性抗体在制备治疗Her2阳性肿瘤的药物中的应用。
11.根据权利要求10所述的应用,其中所述Her2阳性肿瘤为通过免疫组化确定的IHC评分为1+、2+或3+Her2阳性肿瘤。
12.根据权利要求11所述的应用,其中所述Her2阳性肿瘤选自食管癌、胃癌、结肠癌、直肠癌、胰腺癌、肺癌、乳腺癌、子宫颈癌、子宫体癌、卵巢癌、膀胱癌、头颈癌、子宫内膜癌、骨肉瘤、前列腺癌、神经母细胞瘤。
13.根据权利要求12所述的应用,其中所述肿瘤为曲妥珠单抗耐药或无应答肿瘤。
14.一种编码权利要求8中所述的第一多肽链或第二多肽链的多核苷酸。
15.一种包含权利要求14所述的编码第一多肽链的多核苷酸的质粒。
16.一种包含权利要求14所述的编码第二多肽链的多核苷酸的质粒。
17.一种包含权利要求15所述的质粒和权利要求16所述的质粒的表达载体。
18.一种包含权利要求17所述的表达载体的宿主细胞。
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