CN109988752A - 一种抗人b7-h4单克隆抗体及其制备和应用 - Google Patents
一种抗人b7-h4单克隆抗体及其制备和应用 Download PDFInfo
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Abstract
本发明涉及一类单克隆抗体,具体涉及一种抗人B7‑H4单克隆抗体及其制备方法,以及所述单克隆抗体在阻断B7‑H4分子抑制T细胞增殖活化中的应用。单抗药物治疗主要是利用其靶向性来干预肿瘤发生发展过程中的各个通路,或是激活宿主对肿瘤的免疫等。目前杂交瘤技术所制备的抗体多用于诊断试剂类产品,而临床应用的抗体类药物则需采用稳定性较好的工程***进行制备,目前亟待解决的问题是如何降低单抗的异源性,单抗的异源性所引起的抗体反应,不但降低了单抗的效价,而且会给患者带来严重的后果,因此,对异源性单抗进行改造以及人源性单抗的研制成为本发明单抗研究的新的技术方向。
Description
技术领域
本发明涉及一类单克隆抗体,具体涉及一种抗人B7-H4单克隆抗体及其制备方法,以及所述单克 隆抗体在阻断B7-H4分子抑制T细胞增殖活化中的应用。
背景技术
T细胞的活化调节受其表面受体与刺激性或抑制性配体的相互作用所控制,细胞活化需要双信号: 即由抗原递呈细胞(APC)表面的MHC-Ag肽复合物与TCR特异性结合传递第一信号和来自于APC表面 的协同刺激分子与T细胞表面的相应受体结合的第二信号。缺乏共刺激信号会导致T细胞无能。B7 家族是免疫球蛋白超家族中一类具有相似结构的协同信号分子的总称,成员包括CD80、CD86与他们 的配体CD28、CTLA-4,在T细胞的活化过程中提供正向或负向的信号。最近,一些B7家族的同源物 被鉴定,包括B7-H1(PD-L1)、B7-DC、B7-H2、B7-H3、B7-H4、B7-H6。B7-H4也称为B7X或B7S1,是 最新发现的新成员之一。
临床研究表明,B7-H4可以作为一种新型的蛋白标记物,同其他标记蛋白结合起来对于一些肿瘤 早期发现具有重要的临床意义,单截止到目前为止,B7-H4的受体尚未被克隆出来,虽然Watanabe 等曽认为B、T淋巴细胞衰减子(B and T lymphocyte attenuator,BTLA)就是B7-H4分子的受体, 但是随后其他研究发现,在分析其结构时发现其与PD-1和CTLA-4相似,分析两受体酪氨酸抑制基序 并比较发现BTLA可能是一个发挥抑制作用的受体,因此BTLA经验证并非是B7-H4的受体,所以B7-H4 在肿瘤抑制的共表达水平难以确定。
肿瘤免疫靶向治疗存在多种机制,理论上,针对互相不重叠的机制进行联合用药可以达到最好的 治疗效果。研究认为B7-H4单抗阻断效应与BTLA及PD-1调节轴均不存在重叠。同时,以往的BTLA 及PD-1抗体的靶向作用机制研究提示,它们分别通过不同的途径来达到抑制肿瘤并杀死肿瘤的目的, 即阻断BTLA与相应配体的结合可减少肿瘤微环境内Tregs数量,和阻断PD-1与PD-L1结合可直接增 强T细胞活化性能,多个研究的数据证明B7-H4单抗阻断效应是独立于前两种途径之外的,多个免疫 检查点的联合用药大大增强了宿主免疫***的抗肿瘤效应。
单抗药物治疗主要是利用其靶向性来干预肿瘤发生发展过程中的各个通路,或是激活宿主对肿瘤 的免疫等。目前杂交瘤技术所制备的抗体多用于诊断试剂类产品,而临床应用的抗体类药物则需采用 稳定性较好的工程***进行制备,目前亟待解决的问题是如何降低单抗的异源性,单抗的异源性所引 起的抗体反应,不但降低了单抗的效价,而且会给患者带来严重的后果,因此,对异源性单抗进行改 造以及人源性单抗的研制成为单抗研究的新的技术方向。
B7-H4在APC(如DC细胞,单核巨噬细胞,小胶质细胞)上表达也抑制了T细胞的增殖。研究表 明,IL-6能诱导肿瘤相关巨噬细胞表达B7-H4。粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白介素 -4(IL-4)等能下调这些细胞上B7-H4的表达。单克隆抗体生产包括腹水制备和细胞转瓶培养两种方法, 例如专利号201810188948.2的单克隆抗体的制备主要采用体内法,将杂交瘤细胞接种于小鼠腹腔内, 杂交瘤细胞在小鼠腹腔内生长,并产生腹水,因而可得到大量的且抗体浓度很高腹水单抗。小鼠体内 杂交瘤细胞培养,腹水中抗体含量浓度高,产量大,可持续抽取,不需要培养基等成本,不需要重复 无菌操作,快速简便,但遗憾的是,腹水中常混有小鼠的各种杂蛋白(包括IgG)和脂类物质等,在很 多情况下必须要提纯后才能使用,还有动物病毒污染的危险,另外由于小鼠个体差异较大,导致产品 批间差较大。
因此在无/低血清条件下的B7-H4的单克隆抗体的体外大量生产成为当前肿瘤领域、抗感染领域, 包括特殊类型感染等领域的主要方向。
发明内容
本发明目的之一在于提供一种B7-H4单克隆抗体。
本发明的目的之二在于提供一种能产生B7-H4单克隆抗体的杂交瘤细胞株。
本发明的目的之三在于提供一种包含B7-H4单克隆抗体的化学免疫偶联物。
本发明的目的之四在于提供一种在无/低血清条件下的B7-H4的单克隆抗体的体外生产方法。
本发明的目的之五在于提供一种治疗或预防疾病的方法。
为达到上述发明目的,本发明采用的技术方案是:
一种杂交瘤细胞株,所述杂交瘤细胞株的制备方法包括以下步骤:
1)从细菌中纯化出不含有信号肽和跨膜序列的B7-H4分子(亦即胞外蛋白序列);
2)用纯化的B7-H4蛋白注射Balb/C小鼠进行初次免疫,在初次免疫2~3周后进行再次免疫,在 再次免疫2周后进行加强免疫。
3)获取融合细胞生长克隆:从免疫后小鼠取其脾细胞作为抗原致敏的B细胞,按常规方法,将B 细胞与骨髓瘤细胞融合,然后利用常规的融合细胞HAT筛选方法进行筛选,进而获取融合细胞生长克 隆;
4)应用流式细胞学技术筛选和鉴定后,挑选出具有高抗体分泌水平的BLJ杂交瘤B7-H4细胞株, 所述杂交瘤细胞株已于2019年2月18日由位于北京市朝阳区北辰西路1号院3号的中国微生物菌种 保藏管理委员会普通微生物中心(CGMCC)保藏,保藏编号为CGMCCNo.17298,参据的生物材料为:BLJ 杂交瘤B7-H4,建议的分类命名为杂交瘤细胞株。
上述技术方案中,步骤1)中纯化的人B7-H4分子具有较强的免疫原性,并且所表达的抗原分子 的空间构型是暴露于细胞膜表面的自然状态,从而可更有效地激发机体的免疫反应,具体构建步骤:
(a)基因合成与亚克隆:
将不含信号肽和跨膜序列的B7-H4克隆到载体pET30a中,并在大肠杆菌中用组氨酸(His)作为 蛋白表达的标签。
(b)表达量评估:
将重组质粒转染至大肠杆菌BL21Star(DE3)中,将单个菌落接种到含有卡那霉素的LB培养基 中;在37℃以200rpm摇菌培养,然后用IPTG诱导培养。用SDS-PAGE和Westernblot检测其表达。
(c)放大表达量:
将存放于甘油中的BL21Star(DE3)接种到含卡那霉素的TB培养基中,15℃培养,当吸光度OD600 达到1.2时,用IPTG在15℃诱导细胞,培养16小时,离心获取细胞。
(d)纯化和分析:
用裂解缓冲液重悬细胞颗粒,然后超声处理。离心后的沉淀物用尿素溶解。离心后的变性上清液 留存以备进一步纯化。将B7-H4蛋白重新折叠后用0.22μm过滤器进行过滤消毒,然后分装保存。标 准BSA法测定其蛋白含量。标准SDS-PAGE法测定蛋白纯度和分子量,同时进行Western blot以验证 结果,所用的凝胶为15%分离胶和5%浓缩胶;先用60V电压持续25min,再用120V电压持续1.5h; 将靶蛋白胶块小心剥离,并剪出同胶块大小一致的滤纸和PVDF膜,将胶块和滤纸浸泡在转膜缓冲液 中,PVDF膜浸泡在甲醇中,以滤纸-胶块-PVDF膜-滤纸的顺序放置于转膜装置,然后以24V的电压运 行120min;转膜结束后,用1×PBST配制的5%牛奶室温下封闭PVDF膜,摇床孵育2h,用PBST洗膜 4次,每次于摇床上10min,用抗His标签抗体为一抗室温下摇床孵育2h,用PBST洗膜4次,每次于 摇床上10min,用HRP标记的羊抗鼠IgG抗体室温下摇床孵育2h,于曝光间用ECL显色法进行曝光。
在杂交瘤培养液中接种上述技术方案所得杂交瘤细胞,培养后培养液中分离纯化所需单克隆抗 体。对于BLJ杂交瘤B7-H4,我们首先通过Western Blotting技术从24个克隆中挑出13个阳性克隆 株(图1);免疫荧光结果显示,所述B7-H4单克隆抗体高度识别B7-H4分子(图2);通过免疫组化 染色发现抗人B7-H4抗体能识别人肠癌、乳腺癌和肝癌多种肿瘤切片上表达的B7-H4分子(图3);竞 争抑制试验结果进一步显示,所述B7-H4单克隆抗体对同一肿瘤切片的B7-H4的亲和力超过现有商品 化抗体(ABCam的ab209242,图4);因此,B7-H4单克隆抗体可制备用于识别人B7-H4的检测抗体, 亦可能成为检测肿瘤细胞的重要标志。
因此,本发明同时要求保护上述B7-H4单克隆抗体作为生物学检测指标,在制备用于肿瘤细胞检 测试剂/药物中的应用。
通过Elispot技术,B7-H4单克隆抗体对人B7-H4蛋白引起的T细胞增殖抑制的阻断作用(图5)。
因此,本发明也同时要求保护上述B7-H4单克隆抗体在制备阻断B7-H4信号对T细胞增殖的抑制 性药物中的应用。
在另一方面,本发明提供了一种治疗或预防疾病的方法,该疾病的特征是会生长且表达B7-H4蛋 白的肿瘤细胞,该方法包括给予患者有效的剂量且包含所述B7-H4单克隆抗体的化学免疫偶联物,以 治疗或预防所述疾病。所述疾病可能为癌症,例如结肠癌、乳腺细胞癌或者卵巢癌等。
因此本发明也同时要求保护一种杂交瘤细胞株在筛选抗肿瘤药物中的应用。
又在另一方面,本发明提供一种治疗自身免疫失调的方法,该方法包括给予患者有效的剂量且包 含所述B7-H4单克隆抗体的化学免疫偶联物,以治疗或预防所述疾病,例如艾滋病,因此本发明同时 要求保护一种杂交瘤细胞株在研究化疗耐药机制中的应用。
由于上述技术方案运用,本发明与现有技术相比具有下列优点:
本发明公开的杂交瘤细胞株的制备方法超越目前的CHO细胞培养方法,该方法可以在无/低血清 条件下的B7-H4的单克隆抗体的体外生产,产生更高的生产力,同时保留与人类免疫***兼容的糖基 化模式。所产生的B7-H4单克隆抗体能特异性地识别B7-H4分子,与现有的单抗相比,具有更高的亲 和力,灵敏性和人源抗原识别力,具有抑制T细胞增殖的作用,可用于临床抗肿瘤和感染炎症领域。
附图说明
图1为24种B7-H4抗体克隆的Western Blot检测;
图2为B7-H4抗原抗体结合荧光检测;
图3为B7-H4单克隆抗体识别人肠癌、乳腺癌和肝癌的B7-H4分子;
图4为B7-H4单克隆抗体(8.2)对同一肿瘤切片的B7-H4的亲和力超过现有商品化抗体;
图5为B7-H4单克隆抗体对B7-H4蛋白引起的T细胞增殖抑制的阻断作用。
具体实施方式
下面结合附图及实施例对本发明作进一步描述:
实施例一:B7-H4单克隆抗体的制备
本实施例描述本发明的抗人B7-H4单克隆抗体的制备。
(1)B7-H4单克隆抗体的制备
从细菌中纯化出不含有信号肽和跨膜序列的B7-H4蛋白,用纯化的B7-H4蛋白注射Balb/C小鼠 进行初次免疫:称取上述制备好的抗原,用PBS稀释,选取4-6只周龄的雌性Balb/C小鼠,弗氏完 全佐剂乳化以后,加入玻璃注射器中,按照200μL/只的剂量,于小鼠腹部、腹股沟***、背部等处 皮下多点注射,在首次免疫后第21天,用不完全弗氏佐剂按照首次免疫的免疫方法进行免疫,15天 后,再用不完全弗氏佐剂按照首次免疫的免疫方法进行免疫,并在免疫结束一周后,采血检测,融合 前2-3天进行最后一次免疫(直接注射,不加佐剂,不乳化)。
具体构建B7-H4蛋白步骤:(a)基因合成与亚克隆:
将不含信号肽和跨膜序列的B7-H4克隆到载体pET30a中,并在大肠杆菌中用组氨酸(His)作为 蛋白表达的标签。
(b)表达量评估:
将重组质粒转染至大肠杆菌BL21Star(DE3)中。将单个菌落接种到含有卡那霉素的LB培养基 中;在37℃以200rpm摇菌培养,然后用IPTG诱导培养。用SDS-PAGE和Westernblot检测其表达。
(c)放大表达量:
将存放于甘油中的BL21Star(DE3)接种到含卡那霉素的TB培养基中,15℃培养。当吸光度OD600 达到1.2时,用IPTG在15℃诱导细胞,培养16小时,离心获取细胞。
(d)纯化和分析:
用裂解缓冲液重悬细胞颗粒,然后超声处理。离心后的沉淀物用尿素溶解。离心后的变性上清液 留存以备进一步纯化。将B7-H4蛋白重新折叠后用0.22μm过滤器进行过滤消毒,然后分装保存。标 准BSA法测定其蛋白含量。标准SDS-PAGE法测定蛋白纯度和分子量,同时进行Western blot以验证 结果,所用的凝胶为15%分离胶和5%浓缩胶;先用60V电压持续25min,再用120V电压持续1.5h; 将靶蛋白胶块小心剥离,并剪出同胶块大小一致的滤纸和PVDF膜,将胶块和滤纸浸泡在转膜缓冲液 中,PVDF膜浸泡在甲醇中,以滤纸-胶块-PVDF膜-滤纸的顺序放置于转膜装置,然后以24V的电压运 行120min;转膜结束后,用1×PBST配制的5%牛奶室温下封闭PVDF膜,摇床孵育2h,用PBST洗膜 4次,每次于摇床上10min,用抗His标签抗体为一抗室温下摇床孵育2h,用PBST洗膜4次,每次于 摇床上10min,用HRP标记的羊抗鼠IgG抗体室温下摇床孵育2h,于曝光间用ECL显色法进行曝光。
(2)B7-H4单克隆抗体的生产与同种性鉴定
为了确定纯化的抗体的同种性,可以使用对特定同种型抗体具有特异性的试剂进行同种型ELISA。
用1μg/ml的人免疫球蛋白抗体于4℃下过夜涂覆微量滴定孔盘的孔。用1%的BSA遮蔽后,该孔 盘于1μg/ml或更低的测试单克隆抗体或纯化的同种型对照抗体在温室下反应1-2小时。该孔盘然后 于人IgG1或人IgM-特异的碱性磷酸酶连接探针进行反应。
用化学发光的免疫印迹Westernblot分析的方法鉴定BLJ杂交瘤B7-H4识别特异性。预备B7-H4 蛋白并进行SDS聚丙烯酰胺凝胶电泳。在电泳后将分离的抗原转移至硝酸纤维素膜上,用10%的胎牛 血清遮蔽后,用待检测的B7-H4单克隆抗体进行探测。人IgG的结合作用可用碱性磷酸酶标记的抗人 IgG的抗体来检测并且用BCIP/NBT片剂来显影。
也可由细胞免疫荧光的方法鉴定B7-H4单克隆抗体识别特异性:收集生长良好的293T/mock、 293T/B7-H4-GFP细胞进行离心甩片,1×104细胞/片,丙酮固定后按常规免疫组化步骤进行,阴性对 照组的一抗为鼠IgG,实验组一抗为1:100稀释后纯化的mAb,二抗为罗丹红标记羊抗鼠IgG,封片 观察。细胞免疫组化的结果也显示B7-H4单克隆抗体仅能特异性识别转基因细胞表面B7-H4分子的表 达。
以商品化的抗体为阳性对照组(参照图1),为24种B7-H4抗体克隆的Western Blot检测,1,2, 3,4,5,6,9,11,13,19,21为Anti-B7-H4阴性克隆,7,8,10,12,14,15,16,17,18,20,22,23,24(图中用倒角矩形框标识)为13种Anti-B7-H4阳性克隆。1-24代表24种不同克隆号的 抗体,A代表商业化抗体。C代表阴性样本,B代表过表达B7-IgG样本。图中直角矩形框内条带为B7H4 抗原抗体结合位置,图中用倒角矩形框标识的为Anti-B7-H4阳性克隆。
图2为B7-H4抗原抗体结合荧光检测,高表达B7-H4-IgG样本组与对照组相比,表现出较强的GFP 和Anti-B7-H4,绿色荧光GFP代表转染B7H4-IgG的阳性细胞,红色荧光为Anti-B7H4。
图3为B7-H4单克隆抗体识别人肠癌、乳腺癌和肝癌的B7-H4分子。
图4为B7-H4单克隆抗体(8.2)对同一肿瘤切片的B7-H4的亲和力超过现有商品化抗体(ABCam 的ab209242)。
实施例二:在无/低血清条件下的B7-H4的单克隆抗体的体外生产方法
(a)采用逐步降低血清的方法驯化中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)的保 藏编号为CGMCCNo.17298的BLJ杂交瘤B7-H4细胞株以适应低浓度血清培养。将含10%新生牛血清的 MEM培养液中稳定生长的BLJ杂交瘤B7-H4细胞株长至对数生长中期时,更换为含5%新生牛血清的 MEM培养液,待细胞长至80%~90%的汇合度时,胰蛋白酶消化细胞,以细胞密度为2.0×105 cells/ml传代于含5%新生牛血清的MEM培养液中。经过数代后,BLJ杂交瘤B7-H4细胞株在含5% 新生牛血清的MEM培养液中的存活率维持在90%以上,并保持较快生长速率,作进一步降低血清驯 化培养。以同样的方法使BLJ杂交瘤B7-H4细胞株逐步适应含1%新生牛血清的MEM培养条件。
(b)将适应了1%新生牛血清培养条件的BLJ杂交瘤B7-H4细胞株在细胞三角瓶中进行悬浮培 养驯化适应。细胞培养液为自行研制的BLJ杂交瘤B7-H4细胞株无血清培养基,细胞初始接种密度为 1.0×106cells/ml,转速设置为160r/min,置于5%CO2培养箱中进行悬浮培养。
(c)将血清含量分别为无血清、1%、2%和3%新生牛血清的BLJ杂交瘤B7-H4细胞株无血清培 养基与密度为0.5×106cells/ml细胞悬液在细胞培养瓶进行BLJ杂交瘤B7-H4细胞株的悬浮培养, 培养过程中每隔24小时取样计细胞数。
(d)将BLJ杂交瘤B7-H4细胞株分别以0.3×106cells/ml、0.5×106cells/ml和0.75×106cells/ml三个初始密度接种于细胞培养瓶中进行悬浮培养,培养基为含1%新生牛血清的 BLJ杂交瘤B7-H4细胞株无血清培养基,培养过程中每隔24小时取样计细胞数。
随着血清使用浓度的降低,BLJ杂交瘤B7-H4细胞株贴壁生长的形态逐步发生改变,最终经无血 清驯化适应后细胞呈现出单细胞悬浮生长状态。当血清浓度降低至1%时,BLJ杂交瘤B7-H4细胞株生 长速度减慢,经过传代后,细胞适应了血清浓度为1%的营养条件,生长速度恢复。在悬浮培养驯化后 期,悬浮BLJ杂交瘤B7-H4细胞株呈现较好的单细胞悬浮生长状态,细胞呈圆球形,结团现象较少, 胞体大小基本一致,生长速度正常,说明BLJ杂交瘤B7-H4细胞株已经适应了1%血清悬浮状态生长。
将不同血清浓度的BLJ杂交瘤B7-H4细胞株无血清培养基于细胞培养瓶中悬浮培养BLJ杂交瘤B7-H4细胞株,每隔24小时取样计数,结果表明血清浓度分别为1%、2%和3%的BLJ杂交瘤B7-H4 细胞株无血清培养基在整个生长周期细胞密度相似,细胞培养密度优于无血清培养基组,因此选择 BLJ杂交瘤B7-H4细胞株无血清培养基中添加1%血清浓度为最适培养基。
将BLJ杂交瘤B7-H4细胞株分别以初始密度为0.3×106cells/ml、0.5×106cells/ml和0.75× 106cells/ml接种于细胞培养瓶中进行悬浮培养,结果表明细胞初始接种密度为0.75×106cells/ml, 培养48小时细胞密度分别为1.1×106、1.8×106和3.1×106,因此选择0.75×106cells/ml为 细胞初始接种密度。
自行研制的BLJ杂交瘤B7-H4细胞株无血清培养基使用浓缩的XerumFreeTM XF205基础上,添加 多种血清和蛋白替代组分,同时补充氨基酸、微量元素和维生素等营养成分(配制在电阻率为18.2M Ωcm,25℃,无热原的超纯水中,并经过0.22μm微孔滤膜过滤除菌)。
复苏冻存的肿瘤浸润淋巴细胞(TIL),用TIL初始培养基培养过夜,第二天收集TIL,离心弃上 清,PBS洗后用不完全TIL培养基重悬,以2×104/200ul铺板于已包被IFN-γ抗体的Elispot板, 以CD3(OKT3)为TIL细胞激活阳性对照组,以B7H4蛋白10ug/ml,B7H4抗体浓度为10ug/ml分别加入 实验组,培养24h后,PBS洗涤5次,7-B6-ALP二抗37℃孵育1h,PBS洗涤5次,BCIP/NBT-plus显 色,出现适量大小斑点后流水终止,晾干后Elispot仪读板。每个斑点代表一个IFN-γ+的TIL细胞, 通过记录斑点数目反应出激活的T细胞数目,以此评估B7-H4蛋白对TIL的抑制作用以及B7-H4抗体 对此抑制作用的阻断作用,参见图5。
实施例三:B7-H4单克隆抗体制备的抗肿瘤药物
B7-H4单克隆抗体是药物良好的靶向性载体,通过药物分子上特殊的功能基团如:羟基、巯基、 氨基等,将治疗药物与单抗相连接而组成化学免疫偶联物,避免了药物对其他正常组织的毒害作用, 选择性地发挥治疗作用。
作为一种选择,与本发明所得B7-H4单克隆抗体进行偶联的可以是阿霉素、柔红霉素、平阳霉素、 博安霉素、丝裂霉素、新制癌菌素、氨甲喋呤等。
作为一种选择,与本发明所得B7-H4单克隆抗体进行偶联的可以是标记物,例如放射性标记物、 荧光标记物。
作为一种选择,与本发明所得B7-H4单克隆抗体进行偶联的可以是细胞毒素,例如紫衫醇、细胞 松弛素B、短杆菌肽D、溴化乙锭、吐根碱、丝裂霉素、依托泊苷、替尼泊苷、长春新碱、长春碱、 秋水仙素、多柔比星、柔红霉素、二羟基炭疽菌素二酮、放线菌素D、白喉毒素、利多卡因及其类似 物或同系物。
利用本发明所得B7-H4单克隆抗体制备的抗肿瘤药物,结合PD-1抗肿瘤药物(或其他免疫性治 疗药物)进行组合治疗。治疗的组合方式包括:抗B7-H4和抗PD-L1/PD-1顺序给药,如先给患者服 用抗B7-H4抗体,隔一段时间服用抗PD-L1抗体;或者先给患者服用抗PD-L1/PD-1抗体,隔一段时 间服用抗B7-H4抗体;或者第一次同时服用抗B7-H4抗体和抗PD-L1/PD-1抗体,第二次再分开服用, 分开服用的方式多种,可以按照先抗B7-H4抗体后抗PD-L1/PD-1抗体的顺序服用,也可以按照不同 剂量的配比服用,例如80%的高剂量抗B7-H4抗体和20%的低剂量抗PD-L1/PD-1抗体,服用一周后, 再变为80%的高剂量抗PD-L1/PD-1抗体和20%的低剂量抗B7-H4抗体,因为我们发现在胶质瘤样本中 PD-L1和B7-H4之间存在互斥的表达模式,这有助于评估显著和不重叠的免疫逃逸途径。PD-L1和B7-H4 模块之间的差异性免疫微环境表明,有助于指导选择将PD-L1和B7-H4作为肿瘤生物标志物的免疫治 疗策略。未来的研究有必要揭示PD-L1低表达肿瘤(包括B7-H4)替代免疫检查点的生物学机制,这 显示了免疫治疗中临床B7-H4单克隆抗体生物标记物的潜在优势。
本领域的技术人员在不脱离权利要求书确定的本发明的精神和范围的条件下,还可以对以上内容 进行各种各样的修改。因此本发明的范围并不仅限于以上的说明,而是由权利要求书的范围来确定的。
Claims (9)
1.一种杂交瘤细胞株,其特征在于:所述杂交瘤细胞株为分泌抗人B7-H4单克隆抗体的BLJ杂交瘤细胞株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,其简称为CGMCC,保藏地址是北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNo.17298,保藏日期为2019年2月18日。
2.一种抗人B7-H4单克隆抗体,其特征在于,所述抗人B7-H4单克隆抗体为保藏编号CGMCCNo.17298的杂交瘤细胞株产生的单克隆抗体。
3.根据权利要求2所述的一种抗人B7-H4单克隆抗体,其特征在于,所述抗人B7-H4单克隆抗体的制备是从细菌中纯化出不含有信号肽和跨膜序列的B7-H4蛋白,用纯化的B7-H4蛋白注射Balb/C小鼠进行初次免疫:称取上述制备好的抗原,用PBS稀释,选取4-6只周龄的雌性Balb/C小鼠,弗氏完全佐剂乳化以后,加入玻璃注射器中,按照200μL/只的剂量,于小鼠腹部、腹股沟***、背部等处皮下多点注射,在首次免疫后第21天,用不完全弗氏佐剂按照首次免疫的免疫方法进行免疫,15天后,再用不完全弗氏佐剂按照首次免疫的免疫方法进行免疫,并在免疫结束一周后,采血检测,融合前2-3天进行最后一次免疫。
4.根据权利要求3所述的一种抗人B7-H4单克隆抗体,其特征在于,所述B7-H4蛋白的构件步骤:
(a)基因合成与亚克隆:
将不含信号肽和跨膜序列的B7-H4克隆到载体pET30a中,并在大肠杆菌中用组氨酸(His)作为蛋白表达的标签;
(b)表达量评估:
将重组质粒转染至大肠杆菌BL21 Star(DE3)中,将单个菌落接种到含有卡那霉素的LB培养基中;在37℃以200rpm摇菌培养,然后用IPTG诱导培养,用SDS-PAGE和Western blot检测其表达;
(c)放大表达量:
将存放于甘油中的BL21 Star(DE3)接种到含卡那霉素的TB培养基中,15℃培养;当吸光度OD600达到1.2时,用IPTG在15℃诱导细胞,培养16小时,离心获取细胞;
(d)纯化和分析:
用裂解缓冲液重悬细胞颗粒,然后超声处理,离心后的沉淀物用尿素溶解,离心后的变性上清液留存以备进一步纯化,将B7-H4蛋白重新折叠后用0.22μm过滤器进行过滤消毒,然后分装保存,标准BSA法测定其蛋白含量,标准SDS-PAGE法测定蛋白纯度和分子量,同时进行Western blot以验证结果,所用的凝胶为15%分离胶和5%浓缩胶;先用60V电压持续25min,再用120V电压持续1.5h;将靶蛋白胶块小心剥离,并剪出同胶块大小一致的滤纸和PVDF膜,将胶块和滤纸浸泡在转膜缓冲液中,PVDF膜浸泡在甲醇中,以滤纸-胶块-PVDF膜-滤纸的顺序放置于转膜装置,然后以24V的电压运行120min;转膜结束后,用1×PBST配制的5%牛奶室温下封闭PVDF膜,摇床孵育2h,用PBST洗膜4次,每次于摇床上10min,用抗His标签抗体为一抗室温下摇床孵育2h,用PBST洗膜4次,每次于摇床上10min,用HRP标记的羊抗鼠IgG抗体室温下摇床孵育2h,于曝光间用ECL显色法进行曝光。
5.权利要求2所述抗人B7-H4单克隆抗体在制备用于肿瘤细胞检测的药物中应用。
6.一种权利要求2所述抗人B7-H4单克隆抗体的生产方法,包括悬浮培养法、微载体培养法、中空纤维细胞培养***、微囊化细胞培养***中的任意一种。
7.权利要求1所述的一种杂交瘤细胞株在研究化疗耐药机制中的应用。
8.权利要求1所述的一种杂交瘤细胞株在筛选抗肿瘤药物中的应用。
9.权利要求2所述抗人B7-H4单克隆抗体联合PD-L1抗体在抑制肿瘤细胞免疫逃逸中应用。
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