CN101687021B - 单克隆抗体8h9的应用 - Google Patents
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Abstract
本发明公开单克隆抗体8H9，其与人B7-同系物3的4Ig结构域同种型，即4Ig-B7H3结合。本发明提供改善具有肿瘤细胞的受试者的预后或延长其存活的方法，所述方法包括向所述受试者施用包括有效量的试剂的组合物，所述试剂能够与由单克隆抗体8H9识别的抗原结合。
Description
本申请要求2007年3月22日提交的美国系列号60/896,416和2007年5月2日提交的美国系列号60/915,672的利益。将前述申请的全部内容和公开内容引入本申请作为参考。
发明领域
本发明涉及单克隆抗体8H9或其衍生物在治疗癌症患者中的应用。
发明背景
肿瘤-限制的表面抗原可以是诊断和基于免疫的疗法的靶标。用于靶向的免疫疗法的理想肿瘤抗原应该不存在于正常组织上，并在肿瘤细胞表面上大量表达。而且，由单克隆抗体识别的在不同谱系肿瘤细胞上表达的“通用”肿瘤-特异性抗原可以在基于抗体的策略中具有更广泛的用途。以前报告了由鼠科单克隆抗体8H9识别的新型58kD表面肿瘤-结合抗原(参见例如美国专利申请公布US 2005/0169932)。由8H9识别的抗原表达在神经外胚层、间充质和上皮来源的广谱肿瘤的细胞膜上，其在正常组织中具有有限的分布。该新型抗体-抗原系统对于肿瘤靶向和免疫疗法是非常有前途的。
单克隆抗体8H9可以用于肿瘤靶向和成像，和清除肿瘤细胞。8H9抗原还是针对广谱人类癌症的基于抗体的免疫疗法的潜在靶标，所述广谱人类癌症包括成神经细胞瘤、脑瘤、促结缔织生成小圆细胞肿瘤、横纹肌肉瘤、骨肉瘤、尤因肉瘤、PNET、黑素瘤、肉瘤、维尔姆斯瘤、肝胚细胞瘤、和多种组织源的癌。还已经记述了8H9单链抗体和抗体-融合构建体的构建(参见例如美国专利申请公布US 2005/0169932)。
本公开内容提供关于利用单克隆抗体8H9改善具有肿瘤细胞的受试者的预后和/或延长其生存的其他数据。
贯穿本申请，引用多份参考文献。将这些出版物的完整公开内容引入本申请作为参考，从而更完整地描述本发明所属领域的现有技术。
发明概述
本发明提供改善具有肿瘤的受试者的预后和/或延长其生存的方法，所述方法包括向该受试者施用包含有效量的试剂的组合物，所述试剂能够结合由单克隆抗体8H9识别的抗原。
本发明还提供改善具有肿瘤的受试者的预后和/或延长其生存的方法，所述肿瘤表达由单克隆抗体8H9识别的抗原。该方法包括向该受试者施用包含有效量的试剂的组合物，所述试剂能够结合由单克隆抗体8H9识别的抗原。
本发明还提供筛选与单克隆抗体8H9具有相同或相似结合特异性的抗体的方法，所述方法包括使候选抗体与包含序列SEQ ID NO.15的多肽、或其片段相接触的步骤，其中与所述多肽结合的抗体是与单克隆抗体8H9具有相同或相似结合特异性的抗体。本发明还提供通过以上筛选方法鉴定的抗体。
本发明还提供由单克隆抗体8H9识别的抗原，其中所述抗原与SEQ ID NO.15具有至少约10％，优选10％-99％的同源性。
附图简述
图1显示8H9 scFv氨基酸序列(SEQ ID NO.7)和基因序列(有义和互补，SEQ ID NOs.8-9)。互补决定区(CDR)以下面的顺序以方框标记：CDR-1(HC，重链)，CDR-2(HC)，CDR-3(HC)，CDR-1(LC，轻链)，CDR-2(LC)，CDR-3(LC)。
图2显示8H9scFv的核苷酸和氨基酸序列(SEQ ID NOs.10-12)。突变的8H9 scFv携带下列定点诱变(VH：K13E和VL：R18Q，R45Q，K103E，K107E)，从而将PI从6.4降至4.8，且净电荷从-1降到-9，这是减少非特异的正常组织粘附的策略。
图3显示8H9蛋白质印迹的非-还原SDS-PAGE。
图4显示8H9亲合纯化(非-还原SDS-PAGE，蛋白质印迹)。
图5显示8H9亲合纯化(非-还原SDS-PAGE，银染色)。
图6显示由FACS分析的HLA-I(MHC I型)和B7H3蛋白在K562细胞表面上的表达。
图7显示NK92细胞针对K562和HTB82细胞的细胞溶解活性(关于细胞-介导的细胞溶解的铬释放测定的结果)。
图8显示NK细胞针对HTB82细胞的细胞溶解活性(关于细胞-介导的细胞溶解的铬释放测定的结果)。NK92MI：亲本NK细胞；NK92MI/NTGLS-8H：用8H9scFv转导的NK92MI。
图9显示NK细胞针对K562细胞的细胞溶解活性(关于细胞-介导的细胞溶解的铬释放测定的结果)。NK92MI：亲本NK细胞；NK92MI/NTGLS-8H：用8H9scFv转导的NK92MI。
发明详述
本发明提供改善具有肿瘤的受试者的预后或延长其生存的方法，所述方法包括向该受试者施用包含有效量的试剂的组合物，所述试剂能够结合由单克隆抗体8H9识别的抗原。用于本文中时，“改善预后”指及早检测癌症和及早起始治疗，这将导致疾病可能复原或治愈的未来疾病过程，而“延长生存”指增长癌症诊断后的预计寿命。在一个实施方案中，所述肿瘤表达由单克隆抗体8H9识别的抗原。
在一个实施方案中，由单克隆抗体8H9识别的抗原是包含序列SEQ ID NO.15的多肽。在另一个实施方案中，所述抗原是SEQ ID NO.15的多肽同系物。通常，与SEQ ID NO.15存在至少约10％同源性，或至少约15％同源性，或至少约25％同源性，或至少约35％同源性，或至少约45％同源性，或至少约55％同源性，或至多100％同源性。本领域中的普通技术人员应该容易SEQ ID NO.15的同系物或直向同源物(参见例如表1)。表1CD276的直向同源物
显示核苷酸水平(n)或氨基酸水平(a)的％同源性的人相似性。
| 生物体 | 基因 | 描述 | 人相似性 | NCBI登记号 |
| 狗(Canisfamiliaris) | CD2761 | CD276分子 | 90.65(n)93.97(a) | 487638 XM_849111.1 XP_854204.1 |
| 黑猩猩(Pantroglodytes) | LOC4678181 | CD276分子 | 99(n)98.88(a) | 467818 XM_523213.2 XP_523213.2 |
| 大鼠(褐家鼠(Rattusnorvegicus)) | Cd2761 | CD276抗原 | 89.35(n)93.47(a) | 315716 NM_182824.2 NP_877976.1 |
| 小鼠(小家鼠(Musmusculus)) | Cd2764Cd2761 | CD276抗原1，4 | 88.89(n)192.78(a)1 | 1026571 NM_133983.31 NP_598744.11AI4156254 AI5936404(均参见16) |
| 鸡(原鸡(Gallusgallus)) | CD2761 | CD276分子 | 73.36(n)68.51(a) | 415315 XM_413702.2 XP_413702.2 |
| 斑马鱼(Danio rerio) | LOC5721931 | 与CD276抗原类似 | 62.37(n)55.68(a) | 572193 XM_695881.2 XP_700973.2 |
| 非洲爪蛙(Xenopus laevis) | X1.153871～ | 非洲爪蟾 的具有较 弱相似性 的转录序 列 | 68.63(n) | CB207657.1 |
在一个实施方案中，以上组合物中的试剂是包含源自单克隆抗体8H9的互补决定区(CDR)的多肽。所述多肽的实例包括，但不仅限于，单链抗体或抗体-融合构建体。用于本文中时，“单链抗体”指将免疫球蛋白分子(4个肽链)缩减为单肽，该单肽保持针对抗原或针对肿瘤的免疫反应性和特异性，其通常采用合并免疫球蛋白重链和轻链的单肽形式，而“抗体-融合构建体”指将所述单链抗体化学地或遗传地连接于另一种蛋白或肽，形成新型抗体-融合构建体。
在一个实施方案中，所述多肽包括SEQ ID NOs.1-3，4-6，或1-6的CDR。优选地，以上多肽上除CDR外的序列是人来源的。在另一个实施方案中，所述多肽具有氨基酸序列SEQ ID NO.7或12。而且，以上组合物中的试剂可以直接或间接偶联于标记试剂或细胞毒性试剂。所述标记试剂或细胞毒性试剂的代表性实例包括，但不仅限于，放射性同位素和毒素诸如假单孢菌外毒素。
通常，以上组合物可以腹膜内，静脉内，鞘内，通过奥马耶贮器或通过腰椎穿刺(spinal tap)，实质内(intraparenchymally)施用于肿瘤(原发的或转移的)，或施用于肿瘤周围的组织中。
以上组合物的试剂，在用放射性同位素标记时，可以用于治疗目的和用于成像目的。在一个实施方案中，以上组合物中的这种试剂以0.01mg-20mg/注射施用，其携带1mCi-100mCi的131-碘，且在优选的实施方案中，治疗使用。
在另一个实施方案中，以上组合物中的试剂以0.01mg-20mg/注射施用，其携带1mCi-100mCi的124-碘，且在优选的实施方案中，用于成像和剂量测定的目的。
在另一个实施方案中，以上组合物中的试剂以0.01mg-20mg/注射施用，其携带与1mCi-100mCi的131-碘生物学等价放射性剂量的β-放射物或α放射物，其中所述β-放射物或α放射物可以是213-铋，212-铋，111-铟，118-铼，90-钇，225-锕，and 177-镥，或85-砹。
在另一个实施方案中，以上组合物中的试剂以0.01mg-20mg/注射施用，其携带与1mCi-100mCi的124-碘生物学等价放射性剂量的正电子-放射物，其中所述正电子放射物可以是94m-锝，64-铜，89-锆，68-镓，66-镓，76-溴，86-钇，82-铷，110m-铟，13-氮，11-碳或18-氟。
在优选的实施方案中，以上组合物在受试者接受一种或多种其他癌症治疗进行治疗后施用。在另一个实施方案中，以上组合物在受试者接受一种或多种其他癌症治疗进行治疗时同时或顺序地施用。所述其他癌症治疗的实例包括，但不仅限于，手术、化学疗法、和辐射。
本发明还提供具有上述特征(例如，能够结合由单克隆抗体8H9识别的抗原)的试剂作为药物改善具有肿瘤的受试者的预后或延长其生存的用途。在一个实施方案中，所述肿瘤表达由单克隆抗体8H9识别的抗原。施用包含所述试剂的组合物的途径和剂量可由本领域中的普通技术人员容易地确定。例如，所述组合物可以按照上述施用的剂量和途径施用。
本发明还提供筛选与单克隆抗体8H9具有相同或相似的结合特异性的抗体的方法，所述方法包括使候选抗体与包含序列SEQ ID NO.15的多肽、或其片段相接触的步骤，其中与所述多肽结合的抗体是与单克隆抗体8H9具有相同或相似的结合特异性的抗体。本发明还提供通过本文中所述的方法鉴定的抗体。
本发明还提供由单克隆抗体8H9识别的抗原，其中所述抗原与SEQ ID NO.15具有至少约10％，优选10％-99％的同源性。
本发明还提供上调NK/T细胞中抗-转移免疫应答的方法，所述方法包括用适当的试剂阻断NK/T细胞上存在的B7H3受体的步骤。
本发明还提供用于筛选竞争抑制单克隆抗体8H9与其靶标结合的试剂的方法，所述方法包括使候选者与靶标在容许所述候选者与靶标结合的条件下相接触的步骤。在优选的实施方案中，以上方法还包括检测复合物的形成和所述候选者和所述靶标。在该实施方案中，所述靶标是B7H3，也称为CD276，且所述试剂可以抗体、肽、细胞表面蛋白、或配体。
本发明将通过参考其后的实验详情更好地理解，但本领域中的技术人员应该容易理解具体的实验详情仅是例证性的，且不旨在限制如本文中所述的发明，所述发明由其后所附权利要求定义。实施例1通过包括经由脑脊髓液递送的131-碘-8H9放射性免疫疗法的组合药征的改进结果
背景：原发脑瘤和转移到CNS(脑实质或柔脑膜[LM])的癌症是难以控制的。经由脑脊髓液(CSF)区室施用的基于抗体的靶向疗法具有治疗潜力。单克隆抗体8H9是与广谱人实体瘤反应的鼠科IgG1抗体。经由奥马耶施用的131-碘-8H9在非人灵长类动物中具有最小毒性的有利的药物动力学。
方法：在I阶段研究中，15名患者(pts，年龄2-34岁)(1名黑素瘤，3名复发性室管膜瘤，8名复发的CNS成神经细胞瘤[NB]，3名复发性成神经管细胞瘤)接受2mCi奥马耶-内(intra-Ommaya)131I-8H9以用于剂量测定，其后1周后接受奥马耶-内治疗剂量10(n＝3pts)，20(n＝3)，30(n＝6)，或40(n＝3)mCi。采样连续的脑脊髓液(CSF)和血液，以用于剂量测定计算。在24小时后进行核扫描以研究131I-8H9定位。如果pt无PD，则在1个月后重复131-碘-8H9剂量测定和治疗剂量。
结果：副作用包括1或2级发烧，头痛或呕吐；人在第一次注射(30mCi)时具有短暂的3级ALT升高。计算的对CSF的平均放射剂量是35.7(范围15-79)cGy/mCi；平均血液剂量是2.4cGy/mCi。在15名pt中，8名(组#1)具有成神经细胞瘤的初步诊断。当他们形成CNS转移时(中值年限3.8年)，用包括131-碘-8H9的抢救方案治疗他们。全部8名pts保持无存活级数(自131-碘-8H9以后3+，10+，16+，16+，18+，18+，20+，30+个月，和自CNS/LM复发以后5-43+个月)；在1名pt中，131-碘-8H9实现LM疾病的CR。相反，关于27名有病史的对照，从CNS/LM NB发作到死亡的中值时间是5.4个月。急性副作用是自身限制；在40mCi剂量时，没有观察到DLT。
结论：类似于大多数其他实体瘤中的CNS转移，常规疗法对NB-CNS无效。奥马耶-内131-碘-8H9(1)是安全的，(2)对CSF和骨髓具有有利的剂量测定，和(3)当加入到利用常规药征的抢救疗法中时可以在8H9-阳性LM/CNS癌症治疗中具有临床效用。实施例2在PET/CT扫描中使用124-碘-8H9改进CNS肿瘤的分辨率和反差成像
背景：如实施例1中所述，经由脑脊髓液(CSF)区室施用的基于抗体的靶向疗法具有治疗潜力，且放射性标记的131-碘-8H9可以用于治疗转移疾病。患者的预后应该不仅通过改进的治疗而且还通过改进的转移疾病检测和改进的剂量测定而得到改善。下面的实施例描述检测成神经细胞瘤的改进方法。
方法：对5名患者鞘内注射124-碘-8H9，并进行连续PET/CT成像和CSF取样。患者具有CNS肿瘤(脉络丛癌，转移性横纹肌肉瘤，和转移性成神经细胞瘤)。将1.7-2mCi 124-碘-8H9通过奥马耶贮器施用。获得注射后约4，24，和48小时的PET/CT。获得经过48小时内的连续脑脊髓液(CSF)样品。通过在全部3个时间点时将目的区域置于脊柱上，分析图像。PET图像提供CSF区室内的直接活性测量。
结果：124-碘-8H9 PET扫描通过靶向2名在MRI上患有结构性病灶的患者，提供抗体分布的高分辨率图像。24小时的时候，大部分抗体从脑室中清除，并遍及鞘囊和脑凸面周围分布。该分布与预-处理111In-DTPA脑池造影术充分对应。在24和48小时时观察到肝、脾和膀胱中的系统活性。生物学T1/2清除在8.9小时-64.6小时的范围内，其具有对CSF的相应剂量14.1-92.9cGy/mCi。
结论：对于分布、靶向和剂量测定，124-碘-8H9PCT/CT比利用131-碘-8H9的SPECT提供更高的分辨率和反差图像。实施例38H9抗体识别人B7-同系物3的4Ig结构域同种型，即4Ig-B7H3
下面的实施例描述由8H9抗体识别的抗原的生物化学特征。该抗原的身份是人B7-同系物3的4Ig结构域同种型，即4Ig-B7H3。
细胞培养。人成神经细胞瘤细胞系LAN-1由Robert Seeger博士提供(洛杉矶儿童医院(Children’s Hospital of Los Angeles)，洛杉矶，CA)。人横纹肌肉瘤细胞系HTB82，骨肉瘤细胞系U2OS，和伯基特淋巴瘤细胞系Daudi购自美国典型培养物保藏中心(American Type CultureCollection)(Bethesda，MD)。全部细胞系在增补了10％胎牛血清、2mM谷氨酰胺、100U/ml青霉素、和100μg/ml链霉素的RPMI 1640培养基中，在37℃，5％CO2培养箱中生长。
单克隆抗体。8H9和对照MoAb 5F9是鼠科IgG1，且是针对人成神经细胞瘤产生的。它们在使用前通过蛋白质A(GE卫生保健(GEHealthcare)，Piscataway，NJ)亲合层析法纯化。
全细胞裂解物和蛋白质印迹。8H9-阳性细胞系(LAN-1，HTB82和U2OS)和8H9-阴性细胞系(Daudi)生长至～80％汇合。细胞利用2mMEDTA收获并用冰冷的PBS清洗。
非变性PAGE利用非变性PAGE Novex Bis-Tris凝胶系统(NativePAGE Novex Bis-Tris Gel System)(Invitrogen，Carlsbad，CA)，按照制造商的说明进行。在冰上在非变性PAGE 1X样品缓冲液加1％洗涤剂(Triton-X100或正十二烷基-β-D-maltoside(DDM))和蛋白酶抑制剂混合片(罗氏应用科学(Roche Applied Science)，德国)中，对细胞进行裂解(20分钟)。裂解产物通过在14,000rpm，4℃离心20分钟净化。通过非变性PAGE Novex 4-16％Bis-Tris凝胶分析50μg全细胞裂解产物。
在非还原或还原条件下的SDS-PAGE利用Tris-甘氨酸即用凝胶系统(Tris-甘氨酸Ready Gel System)(Bio-Rad，Hercules，CA)进行。简要地，在冰上在Triton裂解缓冲液(50mM Tris-HCl，pH 7.2，50mM NaCl，10％甘油，1％Triton X-100，和蛋白酶抑制剂混合片)中，对细胞进行裂解(20分钟)。如上述净化裂解产物。通过4-15％Tris-HCl凝胶分析25～50μg全细胞裂解产物。
在任一PAGE中电泳后，将样品转移到免疫-印迹PVDF膜(Bio-Rad)上，室温(RT)下用处于TBST中的10％乳粉封闭1小时，并与一抗(处于10-20μg/ml的8H9，处于20μg/ml的5F9)一起在RT下温育3小时。然后用TBST清洗该膜，并与二级过氧化物酶-缀合的亲合纯山羊抗-小鼠IgG(H+L)(杰克逊免疫研究(Jackson ImmunoResearch)，West Grove，PA)一起温育。用SuperSignal West Pico化学发光物质(PIERCE，Rockford，IL)检测条带。
亚细胞分级。为了粗膜的制备，将LAN-1细胞吸移出组织培养皿，用冰冷的PBS洗涤，并在蔗糖缓冲液(0.25M蔗糖，5mM Tris-HCl，pH7.2，和蛋白酶抑制剂混合片)中用杜恩斯匀浆器(Kontes，Vineland，NJ)在冰上裂解。在1000g离心10分钟沉淀所有的细胞核，如通过显微镜法鉴定的。将1000g的上清液在Beckman L-70K(25,000rpm，SW41Ti转子)中以100,000g进行超离心30分钟，以提供膜颗粒(P100)和胞质(S100)馏分。将胞质级分调整到1％Triton，而将粗细胞核和膜馏分重悬在Triton裂解缓冲液中，并在使用前净化。
8H9抗原亲合纯化。通过利用MoAb 8H9的免疫-亲合层析法从LAN-1细胞提取物中纯化8H9抗原。利用Pierce蛋白G IgG加定向试剂盒(Orientation Kit)(PIERCE，Rockford，IL)，按照制造商的说明，制备8H9亲合柱。
4mg如上制备的LAN-1全细胞裂解产物或等量的膜级分与20μl 8H9-蛋白G琼脂糖(与二琥珀酰亚胺基辛二酸酯(DSS)共价交联，3mg结合的8H9/ml珠)一起在4℃温育过夜。用Triton裂解缓冲液广泛洗涤后，用50mM包含1M NaCl的Tris-HCl，pH 7.2，0.1M甘氨酸-HCl，pH 2.8和pH 2.0，SDS样品缓冲液(62.5mM Tris-HCl，pH 6.8，2％SDS，10％甘油，0.005％溴苯酚蓝)，和在水中沸腾5分钟的SDS样品缓冲液加顺序地洗脱该柱。通过利用8H9抗体在非还原条件下进行的蛋白质印迹分析，监测洗出液小等分试样的8H9存在性。还通过银染色(SilverQuest银染色试剂盒，Invitrogen)分析1/4的洗出液。最后，通过胶状考马斯蓝染色(凝胶编码蓝染色剂(GelCode Blue Stain Reagent)，PIERCE)分析一半的8H9抗原-阳性洗出液(0.1M甘氨酸-HCl，pH 2.0洗脱的馏分)，并将8H9抗原-阳性条带送去由MSKCC微量化学和蛋白质组学核心研究所(MSKCC Microchemistryand Proteomics Core Facility)进行质谱鉴定。
结果
8H9抗原的蛋白质印迹检测。8H9抗原首先利用非变性PAGENovex Bis-Tris凝胶系统在非变性条件下由8H9MoAb检测。在所有8H9-阳性细胞系(LAN-1，HTB82和U2OS)，而非8H9-阴性细胞系(Daudi)中检测到单一条带，如通过利用1％非离子型洗涤剂(Triton-X100或DDM)的流式细胞计数法分析定义的(数据未显示)。该检测是特异性的，因为对5F9，即针对Ku70蛋白的对照MoAb，检测到具有不同尺寸的条带(数据未显示)。
稍后，还利用Tris-甘氨酸即用凝胶SDS-PAGE系统，在非还原条件下由8H9MoAb检测8H9抗原。正如在非变性条件下，利用1％Triton裂解缓冲液，在所有8H9-阳性细胞系(LAN-1，HTB82和U2OS)，而非8H9-阴性细胞系(Daudi)中检测到单一条带(～85KD，利用Invitrogen SeeBluePlus2预-染色的标准物作为蛋白质分子量标记)(图3，且数据未显示)。该检测是特异性的，因为对5F9(对Ku70特异性的IgG1)没有检测到具有相同尺寸的条带(数据未显示)。检测到的8H9抗原的尺寸与以前利用8H9放射性-免疫沉淀法的数据一致。我们没能在还原条件下通过蛋白质印迹检测到8H9抗原(数据未显示)，这提示8H9识别构象敏感的表位。
亚细胞分级后，检测到8H9抗原主要在膜级分中(图3)，这与以前8H9抗原是细胞表面抗原的数据一致。再利用亲合纯化进行抗原在膜级分中的8H9的富集。
8H9抗原的亲合纯化。选择LAN-1细胞系用于抗原纯化，因为其相对高水平表达8H9抗原并能够在组织培养基中迅速生长。8H9亲合柱通过以确定的方向将8H9的Fc部分共价缀合于凝胶基质的蛋白G而制备，这容许暴露更多数量的自由抗体结合位点，以进行抗原结合。使用NHS-酯DSS代替传统的亚氨酸酯DMP进行交联也显著防止抗体从支持物中沥滤。
用8H9-蛋白G琼脂糖过夜温育LAN-1(和作为阴性对照的Daudi)全细胞裂解产物或LAN-1膜级分后，显著部分(＞50％)的8H9抗原与该琼脂糖结合(图4，且数据未显示)。如通过蛋白质印迹分析所述监测地，将8H9抗原特异地且主要洗脱在0.1M甘氨酸-HCl，pH 2.0中(图4，且数据未显示)，这提示在8H9抗体和其抗原之间非常强的相互作用。对相同的洗出液银染色后，相应地仅在LAN-1细胞提取物而非Daudi细胞提取物中检测到清晰的条带(图5)。该洗出液也在85KD附近足够干净以进行质谱分析。最后，收集该条带中足量的8H9抗原(～10ng，利用胶状考马斯蓝染色可显现，数据未显示)，并送去进行质谱鉴定。
质谱鉴定利用包装在Eppendorf凝胶加样头中的2μL床-体积的Poros 50R2(PerSeptive)反相珠，对胰蛋白酶消化物进行微量-清除程序。利用具有延时提取的Bruker Ultraflex TOF/TOF仪器，对由RP-微量头柱回收的肽集合(16&30％MeCN)进行质谱法(MALDI-ReTOF)。为了质量指纹识别，使用由两次MALDI-ReTOF试验组合的试验质量(m/z)，利用肽研究(PeptideSearch)(生物化学马赛厄斯·曼恩，马克斯-普朗克学院(Matthias Mann，Max-Planck Institute for Biochemistry)，Martinsried，德国)算法，来搜索非-冗余的人蛋白质数据库(NR；～192,489个条目；NCBI；Bethesda，MD)。涵盖是预期分子量的2倍的分子量范围，其质量精确性限制优于50ppm，且容许最多一个遗漏的裂解位点/肽。选自部分分级集合的肽的质谱测序(MALDI-TOF-MS/MS)在Bruker Ultraflex TOF/TOF仪器中以“LIFT”模式进行，并采用片段离子光谱，利用MASCOT MS/MS离子搜索程序(矩阵科学(Matrix Science))搜索人数据库。鉴定了两种来自肽消化物的肽序列：NPVLQQDAHSSVTITPQR(SEQ ID NO.13)，和SPTGAVEVQVPEDPVVALVGTDATLR(SEQ ID NO.14)。
这些产生这样的明确鉴定：将该抗原明确鉴定为4Ig-B7H3，即人B7-同系物3的4Ig结构域同种型，也称为CD276，登记号NM_001024736.1，其编码534个氨基酸、分子量57235kD的肽。基因位于染色体15q24.1上。成年人蛋白的氨基酸序列如下(在潜在的N-糖基化位点下划线表示)：
MLRRRGSPGMGVRVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA(SEQ IDNO.15)表2
B7成员 对免疫性的作用 受体B7H1(CD274) 抑制 PD-1(CD279)B7DC(CD273) 刺激/抑制 PD-1(CD279)B7H2(LICOS) TH2时滞 ICOSB7H3(CD276) 刺激/抑制 ？？B7H4(B7X) 抑制 ？？
| L1-26 | IgV127-140 | IgC1141-244 | IgV2245-358 | IgC2359-461 | TM462-492 | CT493-534 |
迄今为止，新的B7包括B7H1，B7DC，B7H2，B7H3，和B7H4(参见表2)。4尽管它们的mRNA相当普遍存在，但是这些蛋白分子可能在转录后水平受到不同的调节。B7H3首先由Chapoval等5克隆为B7共刺激性蛋白家族的成员。其后，确定其作为具有四个而非两个Ig-样结构域的I型膜蛋白存在，并且由此指定新名称为4Ig-B7H3(参见表2)。6对于T细胞活化，体外4Ig-B7H3更具有抑制性而不是共刺激性。6已经在胃、NSCLC、成神经细胞瘤和许多人肿瘤细胞系中检测到B7H3蛋白的表达。5，7，8表达4Ig-B7H3的人成神经细胞瘤肿瘤和细胞系可以抑制NK-介导的免疫应答。7发现B7H3在59％胃癌和100％胃腺瘤样品上表达，9且似乎与更好的存活相关。在鼠科模型10，11和人黑素瘤中，12B7H3似乎显示抗-肿瘤应答。鼠科B7H3促进急性和慢性同种异体移植排斥。13B7H3可能在加强肿瘤免疫监视中起作用，而4Ig-B7H3发挥抑制作用。4感兴趣的是，4Ig-B7H3是除脑和胎盘以外大部分问题中的主要同种型。14在胎盘中，通过蛋白质印迹，B7H3是110kd的双条带和60kd的单条带。15其贯穿妊娠始终，在绒毛外滋养层上最显著。还认为B7H3在骨形成中起作用。16
将4Ig-B7H3鉴定为8H9的抗原提示该糖蛋白在人实体瘤中高度表达。相对于正常组织，8H9识别的表位似乎受限于肿瘤。基于至今为止公开的mRNA研究，人们应该合情合理地推断该抗原是普遍存在的，且不适合于作为肿瘤靶标。然而，我们发现并非如此。我们假定针对4Ig-B7H3的抗体可以在无主要副作用的条件下安全施用，如近期对靶向T-细胞的抗-CD28抗体或抗-CTLA4所观察到的。我们认为4Ig-B7H3是免疫共抑制性分子，且抗体如8H9可以在一系列人类癌症中调节其功能并加强宿主抗-肿瘤免疫应答。实施例4利用8H9单克隆抗体分离和鉴定活化的NK/T细胞上的B7H3受体
单克隆抗体8H9识别人B7-同系物3的4Ig结构域同种型，即4Ig-B7H3。人B7-同系物3(B7H3)，也称为CD276，是被认为为免疫系统提供负信号，特别是为NK/T细胞提供负信号，容许肿瘤细胞逃脱免疫应答的分子。鉴定的受单克隆抗体8H9靶向的抗原4Ig-B7H3是B7H3(CD276)的主要变体形式。4Ig-B7H3是包含剪接变化的人B7H3的主要表达形式，其复制V-样和C-样Ig结构域。14，6
作为免疫调节剂，报告了B7H3的阳性和阴性免疫功能。描述2Ig-B7H3变体的报告证明B7H3的作用是促进T细胞活化和通过与活化的T细胞上的推定受体结合而促进IFN-γ生成。5抗肿瘤应答在鼠科肿瘤模型中通过B7H3表达增强。11在患者中，胃癌中的B7H3正性与增加的存活相关。9相反地，B7H3的共抑制作用受到这样的报告的支持，所述报告是2Ig-B7H3和4Ig-B7H3均抑制T细胞增殖和细胞因子产生6，B7H3在B7H3-缺陷型小鼠中优选下调TH1-介导的免疫应答17，且4Ig-B7H3通过与NK细胞表面中推定的抑制受体相互作用而抑制NK-介导的成神经细胞瘤细胞的裂解7。该矛盾性发现可能通过拮抗性B7H3受体解释。
以下实施例描述可以如何识别和分离活化的NK/T细胞上的B7H3受体。该实验尚未进行。
NK/T细胞上的B7H3受体通过利用2Ig-B7H3-Fc和4Ig-B7H3-Fc作为诱饵的亲合层析法纯化。B7H3-Fc融合蛋白以下述方式构建：2Ig-B7H3-Fc购自R&D系统(R&D Systems)，而利用pFUSE-mlg-G2a-Fc2表达载体，使编码人4Ig-B7H3的细胞外结构域的cDNA序列融合于小鼠IgG2a的Fc区。使该融合蛋白在CG44-CHO细胞系中表达，并通过利用蛋白A琼脂糖的亲合层析法纯化。融合蛋白的纯度和功能性通过考马斯蓝染色和抗-B7H3蛋白质印迹评估。
选择对B7H3受体阳性的NK/T细胞。确定的NK/T细胞系NK92，NKL，NK3.3，YT，TALL-104，以及由新鲜外周血单核细胞(PBMC)富集的活化的NK/T细胞与B7H3-Fc一起温育，随后用荧光-缀合的二级抗体染色，并通过荧光活化的细胞分类(FACS)分析。阳性细胞进一步通过B7H3-Fc蛋白质印迹验证。
作为对B7H3受体阳性选择的NK/T细胞用于亲合纯化。B7H3-Fc亲合柱通过利用蛋白G IgG加定位试剂盒(Pierce生物技术(Pierce Biotechnology))使B7H3-Fc的Fc部分与凝胶基质上的蛋白G共价缀合而制备。来自B7H3受体-阳性细胞的细胞提取物与柱上的琼脂糖珠一起温育。将柱彻底洗涤并洗脱。B7H3受体的存在和纯度通过B7H3-Fc蛋白质印迹和银染色监测。将超过20ng的B7H3受体-阳性条带送去进行质谱鉴定。实施例58H9单克隆抗体用于阻断抑制性B7H3(CD276)和随后增强NK/T细胞-介导的肿瘤细胞细胞溶解的用途
发现对肿瘤的免疫应答通过使用单克隆抗体阻断T细胞上的抑制性受体得到增强，所述单克隆抗体特异于所述抑制性受体。该现象的已知实例是通过利用抗-CTLA-4单克隆抗体阻断T细胞上的CTLA-4抑制性受体而增强免疫应答。以下实施例描述用8H9抗体阻断NK/T细胞上的B7H3受体如何使肿瘤细胞针对NK/T细胞-介导的毒性敏感。该实验尚未进行。
细胞-介导的细胞溶解(铬释放)测定：为了NK细胞-介导的细胞溶解测定，将人CML细胞系K562选为靶细胞。如通过FACS分析证明地，K562在HLA-1和B7H3蛋白上具有低表达。横纹肌肉瘤HTB82细胞用作对照。在标准4小时51Cr-释放测定中，尽管只有少于10％的横纹肌肉瘤HTB82细胞被NK 92细胞裂解，但是至多60％的K562细胞被NK92效应子细胞有效地杀死。用编码剪接形式4Ig-B7H3的核酸转染一组K562靶细胞群体，以使B7H3在该细胞群体中过表达。用100μCi51Cr/106细胞，在37℃放射性标记K562靶细胞1小时。单克隆抗体8H9与转染的靶细胞一起温育，而对照与HLA-1mAb HB95一起温育，且利用针对共抑制性B7H3受体阳性的效应子细胞进行细胞裂解测定。NK92效应子细胞与250μl靶细胞一起在96-孔板中在37℃温育4小时。与非-转染的K562相比，NK92效应子细胞针对B7H3转染的K562的细胞溶解活性应该减小。阻断共抑制性B7H3后，应该观察到复原的细胞溶解活性。参考文献1.Modak S，Kramer K，Gultekin SH，等：Monoclonal antibody 8H9 targets anovel cell surface antigen expressed by a wide spectrum of human solid tumors(单克隆抗体8H9靶向由广谱人实体瘤表达的新型细胞表面抗原).CancerRes.(癌症研究)61：4048-54，2001.2.Modak S，Gerald W，Cheung NK：Disialoganglioside GD2 and a noveltumor antigen：potential targets for immunotherapy of desmoplastic smallround cell tumor(双唾液神经节苷脂GD2和新型肿瘤抗原：成结缔组织小圆细胞肿瘤免疫疗法的潜在靶标).Med.Pediatr.Oncol.(医学儿科肿瘤学)39：547-51，2002.3.Modak S，Guo HF，Humm JL，等：Radioimmunotargeting of humanrhabdomyosarcoma using monoclonal antibody 8H9(利用单克隆抗体8H9放射性免疫靶向人横纹肌肉瘤).Cancer Biother.Radiopharm.(癌症生物疗法和放射性药物学)20：534-46，2005.4.Flies DB，Chen L：The new B7s：playing a pivotal role in tumor immunity(新B7：在肿瘤免疫中起关键作用).J.Immunother.(免疫疗法杂志)30：251-60，2007.5.Chapoval AI，Ni J，Lau JS，等：B7-H3：a costimulatory molecule for T cellactivation and IFN-gamma production(B7-H3：用于T细胞活化和IFN-γ生成的共刺激性分子).Nat.Immunol.(自然免疫学)2：269-74，2001.6.Steinberger P，Majdic O，Derdak SV，等：Molecular characterization ofhuman 4Ig-B7-H3，a member of the B7 family with four Ig-like domains(人4Ig-B7-H3，即具有四个Ig-样结构域的B7家族成员的分子特征).J.Immunol.(免疫学杂志)172：2352-9，2004.7.Castriconi R，Dondero A，Augugliaro R，等：Identification of 4Ig-B7-H3 asa neuroblastoma-associated molecule that exerts a protective role from an NKcell-mediated lysis(鉴定4Ig-B7-H3为发挥防止NK细胞-介导的裂解的保护作用的成神经细胞瘤-相关分子).Proc.Natl.Acad.Sci.USA(美国国家科学院学报)101：12640-5，2004.8.Sun Y，Wang Y，Zhao J，等：B7-H3 and B7-H4 expression in non-small-celllung cancer(非-小细胞肺癌中B7-H3和B7-H4的表达).Lung Cancer(肺癌)53：143-51，2006.9.Wu CP，Jiang JT，Tan M，等：Relationship between co-stimulatory moleculeB7-H3 expression and gastric carcinoma histology and prognosis(共刺激性分子B7-H3表达和胃癌组织学和预后之间的关系).World J.Gastroenterol.(世界肠胃病学杂志)12：457-9，2006.10.Luo L，Chapoval AI，Flies DB，等：B7-H3 enhances tumor immunity invivo by costimulating rapid clonal expansion of antigen-specific CD8+cytolytic T cells(B7-H3通过共刺激抗原-特异性CD8+细胞溶解性T细胞的快速无性繁殖而增强体内肿瘤免疫性).J.Immunol.(免疫学杂志)173：5445-50，2004.11.Sun X，Vale M，Leung E，等：Mouse B7-H3 induces antitumor immunity(小鼠B7-H3诱导抗肿瘤免疫性).Gene Ther.(基因疗法)10：1728-34，2003.12.Lupu CM，Eisenbach C，Kuefner MA，等：An orthotopic colon cancermodel for studying the B7-H3 antitumor effect in vivo(用于研究B7-H3体内抗肿瘤作用的同位结肠癌模型).J.Gastrointest.Surg.(胃肠病学外科杂志)10：635-45，2006.13.Wang L，Fraser CC，Kikly K，等：B 7-H3 promotes acute and chronicallograft rejection(B7-H3促进急性和慢性同种异体移植排斥).Eur.J.Immunol.(欧洲免疫学杂志)35：428-38，2005.14.Sun M，Richards S，Prasad DV，等：Characterization of mouse and humanB7-H3 genes(小鼠和人B7-H3基因的特征).J.Immunol.(免疫学杂志)168：6294-7，2002.15.Petroff MG，Kharatyan E，Torry DS，等：The immunomodulatory proteinsB7-DC，B7-H2，and B7-H3 are differentially expressed across gestation in thehuman placenta(免疫调节蛋白B7-DC，B7-H2，和B7-H3在妊娠过程中在人胚胎中区别性表达).Am.J.Pathol.(美国病理学杂志)167：465-73，2005.16.Suh WK，Wang SX，Jheon AH，等：The immune regulatory protein B7-H3promotes osteoblast differentiation and bone mineralization(免疫调节蛋白B7-H3促进造骨细胞分化和骨矿化).Proc.Natl.Acad.Sci.USA(美国国家科学院学报)101：12969-73，2004.17.Suh WK，Gajewska BU，Okada H，Gronski MA，Bertram EM，DawickiW，Duncan GS，Bukczynski J，Plyte S，Elia A，Wakeham A，Itie A，Chung S，Da Costa J，Arya S，Horan T，Campbell P，Gaida K，Ohashi PS，Watts TH，Yoshinaga SK，Bray MR，Jordana M，Mak TW：The B7 family members B7H3preferentially down-regulates T helper type 1-mediated immune responses(B7家族成员B7H3优选下调1型T辅助细胞-介导的免疫应答).Nat.Immunol.(自然免疫学)4：899-906，2003.
Claims (9)
1.组合物用于制备改善具有肿瘤的受试者的预后或延长其存活的药物的应用，其中所述组合物包含含有单克隆抗体8H9的互补决定区(CDRs)的抗体构建体，其中所述CDRs的序列是SEQ ID NOs.1-6。
2.权利要求1的应用，其中所述肿瘤选自由转移性成神经细胞瘤、和成神经细胞瘤组成的组。
3.权利要求1的应用，其中所述抗体构建体是单链抗体或抗体-融合构建体。
4.权利要求1的应用，其中所述抗体构建体直接或间接地与标记试剂或细胞毒性试剂偶联。
5.权利要求4的应用，其中所述细胞毒性试剂是放射性同位素。
6.权利要求1的应用，其中所述组合物在受试者已经用一种或多种其他癌症治疗进行治疗后施用。
7.权利要求6的应用，其中所述其他癌症治疗选自由手术、化学疗法、和放射组成的组。
8.权利要求1的应用，其中所述组合物通过选自由下列各项组成的组的方法施用：静脉内注射，鞘内注射，通过奥马耶贮器或通过腰椎穿剌注射，实质内注射入肿瘤或肿瘤周围的组织中，和腹膜内注射。
9.权利要求1的应用，其中所述抗体构建体以0.01mg-20mg/注射进行施用，其携带1mCi-100mCi的131-碘、124-碘、或生物学等价放射性剂量的β-放射物、α放射物或正电子放射物。
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| JP2016020346A (ja) | 2016-02-04 |
| JP2014088411A (ja) | 2014-05-15 |
| CN101687021A (zh) | 2010-03-31 |
| KR20100014527A (ko) | 2010-02-10 |
| CA2680111A1 (en) | 2008-09-25 |
| US20100143245A1 (en) | 2010-06-10 |
| WO2008116219A2 (en) | 2008-09-25 |
| WO2008116219A3 (en) | 2008-12-11 |
| EP2121008A4 (en) | 2010-03-31 |
| JP2010523478A (ja) | 2010-07-15 |
| EP2121008A2 (en) | 2009-11-25 |
| CA2680111C (en) | 2018-05-08 |
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