CN116063478B - 干细胞细胞因子的制备方法及其抗体联用的制药用途 - Google Patents
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Abstract
本发明涉及干细胞细胞因子的制备方法及其抗体联用的制药用途。本发明开发了一种特异性针对人BRD4蛋白的单克隆抗体，该抗体能够抑制癌细胞克隆形成以及影响细胞活力，通过与干细胞细胞因子联用后能够达到抑制癌细胞肿瘤生长的效果，具有较好的应用前景。
Description
技术领域
本申请涉及生物领域，还涉及干细胞细胞因子的制备方法及其抗体联用的制药用途。
背景技术
恶性黑色素瘤(malignant melanoma)是起源于胚胎期神经嵴的恶性肿瘤，其恶性度极高，预后较差。可发生于皮肤、眼球、消化道、生殖系统等部位，但其中以皮肤恶性黑色素瘤最常见。由于恶性黑色素瘤恶性度高，易于早期转移，即使早期进行根治手术，患者5年生存率也低于70％。免疫靶向治疗与传统化学疗法相比优势明显，因而日益成为治疗恶性肿瘤的新方向。
嵌合抗原受体(chimericantigenreceptors，CAR)是-种重组抗原受体，具有结合抗原并激活T细胞的功能。利用HERV-K的肿瘤特异性，于T细胞表面设计相关CAR，发现CAR-T可特异性识别HERV-K并与其结合，发挥抗肿瘤作用。恶性黑色素瘤早期即可通过血液转移，传统的手术切除、化学疗法、放射疗法的效果不佳，预临床试验数据显示，嵌合HERV-K受体的T细胞(CAR-T)可准确作用于表达HERV-K的肿瘤细胞表面，并杀伤肿瘤细胞。由于HERV-K在正常细胞表面无表达，其特异性表达于HIV、白血病及多种肿瘤细胞表面，因此，嵌合HERV-K受体的T细胞可用于多种肿瘤的治疗。
外泌体作为-种可以包裹药物的载体以及治疗药物，近年来也被用于肿瘤的靶向治疗以及单独治疗使用。外泌体是经细胞分泌产生的-种自然产物，它具有低免疫原性、无毒副作用；且来源广，具有磷脂双分子层结构，易于与靶细胞的细胞膜融合；分子结构小，分子量为纳米级(10-100nm)，可避免单核细胞的吐噬作用，且易于穿透肿瘤组织毛细血管向深层组织浸润。现有试验发现，由内皮细胞及干细胞分泌的外泌体作为抗肿瘤药物的载体，其优势明显、不良反应小。经未成熟树突状细胞(imDCs)分泌的外泌体因缺乏CD40、CD86、MHC-I、MHC-II等表面标志物，免疫原性低，且imDCs-外泌体表面表达糖蛋白(Lamp2b)，这种表面蛋白融合了iRGD靶向蛋白，可特异性识别肿瘤细胞表面的V整合素。目前，临床上许多抗肿瘤药物因剂量依赖性而应用受限，如阿霉素对多种肿瘤均有抑制作用，但因其对心血管具有剂量依赖性而限制其应用[271。剂量依赖性产生的原因主要是药物缺乏特异性作用于肿瘤细胞的载体，而广泛作用于正常细胞引起。利用外泌体的靶向结合作用，可以将阿霉素等药物直接作用于肿瘤细胞，从而减少对正常细胞造成的损伤。通过基因转染技术过表达iRGD就可以得到大量携带iRGD的外泌体。基于这-原理，将阿霉素整合到imDCs-外泌体中，发现融合iRGD靶向蛋白的外泌体能特异性作用于表达V整合素的乳腺肿瘤细胞，使外泌体通过胞吐释放阿霉素，在体内充分发挥抗肿瘤作用。而且单独使用外泌体来治疗癌症也是目前研究的重要方向。
抗体治疗黑色素瘤目前是研究的-个热点，而且随着人们对免疫系统的研究。越来越多的黑色素瘤免疫治疗相关靶点被发现。其中.T细胞通路的研究最为广泛，研制了细胞毒性T淋巴细胞相关抗原4和程序性死亡分子-1(PD-1)等抗体。Ipilimumab是抗CTLA-4的人IgG1单克隆抗体，通过结合CTLA-4从而阻断由CTLA-4产生的抑制信号早期的临床试验确定了Ipilimumab的治疗剂量.并被FDA批准用于黑色素瘤的Ⅲ期临床试验.也是第-个因总生存期获益而被FDA批准用于中晚期黑色素瘤治疗的药物。Tremelimumab是抗CTLA-4的人IgG2单克隆抗体.它的半衰期(22d)比Ipilimumab更长(15.4d)。但是，Tremelimumab目前尚未被FAD认证用于肿瘤治疗Ⅲ期临床试验表明，相比较达卡巴嗪、替莫唑胺等化疗药物，Tremelimumab并未显示出明显的优势.总体生存周期为12.4个月(化疗组为10.7个月)，但是其肿瘤缓解期较化疗组更长。抗PD-1抗体是Pembrolizumab.也是人源性IgG4.其靶点作用于PD-l、PD-Ll和PD-L2之间的相互作用。173例经过至少2个疗程Ipilimumab治疗的不能切除或已发生转移的黑色素瘤患者被纳入研究，治疗剂量分为2mg/Kg(n＝89)和10mg/Kg(n＝84)，两组的有效率均为26％。而其免疫毒性虽然被发现.但发生率和严重程度较抗CTLA-4抗体低。疲劳、瘙痒和皮疹是最常见的副作用，在两组间比较并未发现副作用发生率的差别。FDA于2014年9月批准了该药物用于经过Ipilimumab或者BARF抑制剂治疗后的带有BARF变异的黑色素瘤的后续治疗。
Bromodomain(BRDS)是能够特异性识别蛋白中乙酰化赖氨酸残基的保守蛋白结构域，根据结构和序列的相似性，61个人类溴结构域被分为8个家族，其中的BET家族蛋白最具代表性并包括BRD2,BRD3,BRD4,和BRDT，BET家族溴结构域的蛋白质BRD4蛋白含有能够结合组蛋白和其他蛋白的乙酰化赖氨酸残基，在调控基因和控制细胞生长方面起着重要作用，BRD4蛋白与调控基因转录方面的大蛋白质复合物相关，包括介体、PAFc和超级伸长复合物等。BRD4的激酶活性可直接磷酸化并激活RNA聚合酶II,从而调节基因的转录表达。人类许多疾病都与BRD4蛋白有着密切的联系，如肿瘤、自体免疫性或炎症性疾病、病毒感染等。BRD4抑制剂靶向BRD4，对其进行抑制，在抗癌和抗炎以及多个领域有着极大的价值，-直在吸引各大制药公司和科研机构的关注。BRD4蛋白含有两个亚型BD1和BD2，鉴于它们的高序列相似性，在BET家族中获得选择性抑制是具有挑战性的，因此，泛BET抑制剂(如(+)-JQ1和iBET-151等)通过同时抑制多个溴域而被广泛应用于BET蛋白的功能研究。
但是目前，针对黑色素瘤的治疗靶点的研究还不够多，特别是针对BRD4的抑制剂开发还不够多，治疗黑色素瘤的可选方案也不多。
发明内容
本发明一方面，提供了一种特异性针对人BRD4蛋白的单克隆抗体。
一方面，所述人BRD4蛋白的单克隆抗体是BRD4-3F14，其轻链可变区的氨基酸序列如SEQ ID NO.1所示：
DLVMTQTAPSVPVTPGESVSISCRSTAWWYVWWMLWQLYWFLQRPGQSPQLLIYVYHNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCWHFPRHKCSFGSGTKLEIK
重链可变区的氨基酸序列如SEQ ID NO.2所示：
VKPGGSLKLSCAASRGWVDPYRMSWVRQTPDKRLEWVAIQITGHDFDYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCLGPKAVKHLELWGQGTTVTVS
干细胞的培养上清液中富含外泌体以及各种细胞因子，此前的研究已经表明干细胞上清液具有一定的增加癌症治疗效果的功效，本发明就特异性的制备了人表皮干细胞上清液，并通过实验证实了所述上清液冻干粉具有治疗黑色素瘤的效果，并能够基于预期，也用于其他癌症的治疗。
在一些实施方案中，本文所提供的BRD4抗体包含与SEQ ID NO:1中提供的说明性VL序列具有至少约50％、60％、70％、80％、90％、95％或99％同一性的VL序列。在一些实施方案中，本文所提供的BRD4抗体包含SEQ ID NO:1中提供的VL序列，其具有多达1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个氨基酸取代。在一些方面，氨基酸取代是保守氨基酸取代。在一些实施方案中，在本段中所述的BRD4抗体在本文中被称为“变体”。在一些实施方案中，这种变体例如通过亲和力成熟、定点诱变、随机诱变或本领域中已知或本文所述的任何其它方法衍生自本文所提供的序列。在一些实施方案中，这种变体不是衍生自本文所提供的序列，并且可例如根据本文所提供的用于获得BRD4抗体的方法从头分离。
在一些实施方案中，本文所提供的BRD4抗体包含与SEQ ID NO:2中提供的说明性VH序列具有至少约50％、60％、70％、80％、90％、95％或99％同一性的VH序列。在一些实施方案中，本文所提供的BRD4抗体包含SEQ ID NO:2中提供的VH序列，其具有多达1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个氨基酸取代。在一些方面，氨基酸取代是保守氨基酸取代。在一些实施方案中，在本段中所述的BRD4抗体在本文中被称为“变体”。在一些实施方案中，这种变体例如通过亲和力成熟、定点诱变、随机诱变或本领域中已知或本文所述的任何其它方法衍生自本文所提供的序列。在一些实施方案中，这种变体不是衍生自本文所提供的序列，并且可例如根据本文所提供的用于获得BRD4抗体的方法从头分离。
在一些实施方案中，衍生自本文所提供的抗体的抗体片段对BRD4的亲和力(如通过KD所测量)保留在所述抗体亲和力的约1.5倍、约2倍、约3倍、约4倍、约5倍、约6倍、约7倍、约8倍、约9倍或约10倍内。在一些实施方案中，衍生自本文所提供的抗体的抗体片段对重组的BRD4的亲和力(如通过KD所测量)保留在所述抗体亲和力的约1.5倍、约2倍、约3倍、约4倍、约5倍、约6倍、约7倍、约8倍、约9倍或约10倍内。
本发明还提供一种核酸分子，其包含能够编码靶向人BRD4蛋白的单克隆抗体的重链互补决定区或轻链互补决定区的核酸序列。
本发明还提供一种载体，其含有上述核酸分子。
本发明还提供一种宿主细胞，该宿主细胞含有上述靶向人BRD4蛋白的兔重组单克隆抗体、上述核酸分子或上述载体。
本发明还提供一种偶联物，含有上述抗体。
本发明还提供一种药物组合物，含有主成分和辅成分，其中：主成分上述靶向人BRD4蛋白的单克隆抗体、上述核酸分子、上述载体、上述宿主细胞、上述偶联物中的一种或多种，辅成分选自药学上可接受的载体或赋形剂，以及任选的其它生物活性物质。
进一步的，本发明还提供了一种能够用于美容的化妆品，所述化妆品中含有本发明所述的单克隆抗体，所述化妆品能够抑制黑色素细胞的生长。
进一步的，本发明还提供了一种能够用于美容的化妆品，所述化妆品中含有本发明所述的单克隆抗体以及表皮干细胞细胞因子，所述化妆品能够抑制黑色素细胞的生长。
进一步的，本发明还提供了本发明所述的单克隆抗体以及干细胞细胞因子在制备通过抑制黑色素细胞生长从而达到美白的化妆品中的用途。
本发明还提供上述靶向人BRD4蛋白的单克隆抗体、上述酸分子上述载体、上述宿主细胞、上述偶联物在制备治疗疾病的药物或检测试剂中的应用。
进一步的，本发明还提供了靶向人BRD4蛋白的单克隆抗体和干细胞细胞因子在制备用于治疗黑色素瘤的药物组合物或者药盒中的用途。
所述干细胞细胞因子是将表皮干细胞进行培养后收集的培养液冷冻干燥制备得到的。
进一步的，所述药物组合物可包含一种或多种药物赋形剂。可使用任何合适的药物赋形剂，并且本领域普通技术人员能够选择合适的药物赋形剂。因此，下文提供的药物赋形剂意为说明性，而非限制性的。另外的药物赋形剂包括在例如Handbook ofPharmaceutical Excipients,Rowe等人(编者)第6版(2009)中所述的那些，该文献以引用的方式整体并入。
在一些实施方案中，药物组合物包含消泡剂。可使用任何合适的消泡剂。在一些方面，消泡剂选自醇、醚、油、蜡、硅氧烷、表面活性剂及其组合。在一些方面，消泡剂选自矿物油、植物油、乙烯双硬脂酰胺、石蜡、酯蜡、脂肪醇蜡、长链脂肪醇、脂肪酸皂、脂肪酸酯、硅乙二醇、氟硅酮、聚乙二醇-聚丙二醇共聚物、聚二甲硅氧烷-二氧化硅、乙醚、辛醇、辛醇、去水山梨糖醇三油酸酯、乙醇、2-乙基己醇、二甲基硅油、油醇、二甲基硅油及其组合。
在一些实施方案中，药物组合物包含助溶剂。助溶剂的说明性实例包括乙醇、聚(乙二醇)、丁二醇、二甲基乙酰胺、甘油、丙二醇及其组合。
在一些实施方案中，本文提供了一种通过向受试者施用有效量的本文所提供的抗体来治疗有此需要的受试者的疾病或病状的方法。在一些方面，所述疾病或病状是癌症。在一些方面，所述疾病或病状是病毒感染。
任何合适的癌症都可用本文所提供的抗体治疗。说明性合适的癌症包括例如：急性淋巴母细胞性白血病(ALL)、急性骨髓性白血病(AML)、肾上腺皮质癌、肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、脑肿瘤、胆管癌、膀胱癌、骨癌、乳腺癌、支气管肿瘤、未知原发起源的癌瘤、心脏肿瘤、子宫颈癌、脊索瘤、结肠癌、结肠直肠癌、颅咽管瘤、导管癌瘤、胚胎性瘤、子宫内膜癌、室管膜瘤、食道癌、鼻腔神经胶质瘤、纤维组织细胞瘤、尤因肉瘤、眼癌、生殖细胞肿瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠基质肿瘤、妊娠期滋养层疾病、神经胶质瘤、头颈癌、肝细胞癌、组织细胞增多症、何杰金氏淋巴瘤、下咽癌、眼内黑色素瘤、胰岛细胞瘤、卡波氏肉瘤、肾癌、郎格罕氏细胞组织细胞增多症、喉癌、唇和口腔癌、肝癌、小叶原位癌、肺癌、巨球蛋白血症、恶性纤维组织细胞瘤、黑色素瘤、梅克尔细胞癌、间皮瘤、具有涉及NUT基因的隐匿性原发性中线呼吸道癌瘤的转移性鳞状颈癌、口腔癌、多发性内分泌瘤综合征、多发性骨髓瘤、阿利贝尔氏病、脊髓发育不良综合征、骨髓发育不良/骨髓增生性肿瘤、鼻腔和副鼻窦癌、鼻咽癌、神经母细胞瘤、非小细胞肺癌、口咽癌、骨肉瘤、卵巢癌、胰腺癌、乳头状瘤病、副神经节瘤、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、垂体瘤、胸膜肺胚细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管癌、成视网膜细胞瘤、横纹肌样瘤、唾液腺癌、赛谢综合征、皮肤癌、小细胞肺癌、小肠癌、软组织肉瘤、脊髓肿瘤、胃癌、T细胞淋巴瘤、畸胎样瘤、睾丸癌、咽喉癌、胸腺瘤和胸腺癌瘤、甲状腺癌、尿道癌、子宫癌、阴道癌、外阴癌及维尔姆斯氏肿瘤。
另外的治疗剂可通过任何合适的方法施用。在一些实施方案中，本文所提供的抗体和所述另外的治疗剂是包括在同一药物组合物中。在一些实施方案中，本文所提供的抗体和所述另外的治疗剂是包括在不同的药物组合物中。
有益效果
本发明开发了一种特异性针对人BRD4蛋白的单克隆抗体，该抗体能够抑制癌细胞克隆形成以及影响细胞活力，通过与干细胞细胞因子联用后能够达到抑制癌细胞肿瘤生长的效果，具有较好的应用前景。
附图说明
图1不同小鼠的血清效价结果图
图2单抗的亚型鉴定结果图
图3单抗对黑色素瘤细胞克隆形成的影响结果图
图4单抗对黑色素瘤细胞活力影响结果图
图5单抗对黑色素瘤细胞的基因表达影响结果图
图6单抗治疗小鼠黑色素瘤实验结果图
具体实施方式
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
实施例1 BRD4单克隆抗体的制备
1、杂交瘤细胞的制备
选择免疫原性较强的区段(BRD4)重组人Bromodomain结构域结合蛋白4(BRD4,区域49-170aa)(Biovision，货号：7643-100)免疫4只发育6-7周龄的雌性Balb/C小鼠,4次免疫均采用腹部皮下分点注射，剂量为100μg免疫时采用尾静脉注射；首免采用完全弗氏佐剂乳化，二免至三免采用不完全弗氏佐剂乳化按照抗原与佐剂1:1的比例)；操作时间:首免3周后进行第二次免疫，二免至三免，间隔均为2周，在三免之后第20天进行冲击免疫，免疫原用生理盐水混匀，剂量为200μg/只。在冲击免疫3d后进行采血，血清离心，1500r/min，离心10min，取上清。用间接ELISA检测抗体效价。包被BRD4，用包被缓冲液稀释至1μg/ml，用1×PBS按照以下梯度稀释免疫后小鼠血清:1:1000,1:2000,1：4000，1：8000，1：16000，1：32000，1：64000，1：128000,100μl/孔，按照稀释倍数从小到大依次加入到酶标板中，同时设置阳性血清、未免疫血清阴性对照和PBS空白对照，进行ELISA检测，计算阳性血清与阴性血清之比(positive/negative,P/N>，当P/N≥2.1时出现相近比值的最大稀释倍数，为抗体效价。结果如图1所示。
由图1可知，1号和4号小鼠血清效价均可达到1：128000，第2和第3号小鼠血清效价较低，因此，选择效价最好的4号小鼠用于细胞融合。
细胞融合、阳性细胞筛选等方法均按照常规方法进行。将SP2/0细胞与脾细胞在PEG1500作用下融合，融合后使用HAT及HT培养基进行换液。待杂交瘤细胞生长至底面积1/3左右，吸取上清液，用间接ELISA方法测定其抗体效价。选择测定为强阳性的孔的细胞株共计6株，进行亚克隆筛选，获得能够分泌抗BRD4蛋白抗体杂交瘤细胞株2株，分别命名为BRD4-2E23和BRD4-3F14。
将BALB/c小鼠进行腹腔石蜡致敏，7d后分别注射1×106个BRD4-2E23和BRD4-3F14杂交瘤细胞后，观察到小鼠腹部明显隆起，收集腹水离心后的上清，用1μg免疫原作为包被抗原，封闭后将腹水单克隆抗体从1:8000开始连续稀释测定抗体效价。进行间接ELISA效价测定，结果显示，BRD4-2E23和BRD4-3F14效价均达到了1:512000，效果较好。
利用硫酸铵盐析法对制备的腹水进行粗提，再使用ProteinG亲和层析柱进一步纯化得到纯度较高的IgG抗体，并将纯化的单克隆抗体进行SDS-PAGE鉴定纯度均较纯，并调整抗体浓度为5mg/mL备用。
2、抗体亚型检测
使用SouthernBiotech公司的SBA亚型检测试剂盒，按照说明书进行检测，具体检测方法如下：
(1)包被：用CBS缓冲液将试剂盒中的包被抗原稀释成5μg/ml，100μl/孔包被到酶标板中，4℃包被过夜。用PBST缓冲液洗板5次，拍干酶标板。
(2)封闭：用含2％甘氨酸的PBST缓冲液封闭酶标板，200μl/孔，37℃孵育2h。用PBST缓冲液洗板5次，拍干酶标板。
(3)检测：将待检测的抗体加入到酶标板中(每个抗体做8个检测)，100μl/孔，37℃孵育1h。用PBST缓冲液洗板5次，拍干酶标板。
(4)加酶标二抗：用PBST缓冲液将HRP标记的羊抗鼠抗体(包含IgA-HRP、IgM-HRP、IgG1-HRP、IgG2a-HRP、IgG2b-HRP、IgG3-HRP、κ-HRP、λ-HRP)500倍稀释，加入到酶标板中，100μl/孔，37℃孵育1h。用PBST缓冲液洗板5次，拍干酶标板。
(5)显色：用TMB显色液避光显色，100μl/孔，37℃孵育15min；用2M硫酸终止液进行终止，50μl/孔，于450nm波长下检测。结果如下表1所示：
表1亚型检测结果
| 亚型 | BRD4-2E23单抗 | BRD4-3F14单抗 |
| IgG1 | 0.31 | 0.32 |
| IgG2a | 1.69 | 0.22 |
| IgG2b | 0.32 | 1.56 |
| IgG3 | 0.24 | 0.24 |
| IgM | 0.24 | 0.21 |
| IgA | 0.22 | 0.25 |
| Kappa | 1.56 | 1.48 |
| Lambda | 0.32 | 0.20 |
从表1的结果可以看出，BRD4-2E23单抗是IgG2a亚型，而BRD4-3F14单抗是IgG2b亚型。
实施例2BRD4-3F14单抗特异性及特性鉴定
分别选取BRD4蛋白、PD-1蛋白、PD-L1蛋白、BSA、正常小鼠血清用包被缓冲液稀释至1μg/ml，用1×PBS按照1:2000稀释BRD4-3F14单抗,100μl/孔加入到酶标板中，同时设置PBS空白对照，进行ELISA检测，计算各实验组与阴性空白对照之比(positive/negative,P/N>，当P/N≥2.1时表示特异性结合。结果如图2所示。
从图2的结果可以看出，BRD4-3F14单抗只能特异性结合BRD4蛋白，而与其他几种物质：PD-1蛋白、PD-L1蛋白、BSA、正常小鼠血清均无明显的结合特性，显示了本发明制备的BRD4-3F14单抗具有较好的特异性。
利用Fortibio Octet生物大分子相互作用分析仪分析测定BRD4-3F14抗体与BRD4抗原的结合动力学。分析结果发现BRD4-3F14单抗与BRD4蛋白具有较好的亲和力，其KD值达到了1.07nM。
通过扩增BRD4-3F14杂交瘤细胞的轻重链序列，鉴定获得了该抗体的轻重链可变区分别如SEQ ID NO：1和2所示。
实施例3BRD4-3F14单抗对黑色素瘤细胞克隆形成的影响
A875人黑色素瘤细胞培养在添加10％FBS和1％青霉素和硫酸链霉素的混合抗生素的DMEM培养基中。细胞在37℃、5％CO2的培养箱中培养。将细胞接种于6孔细胞培养板(接种密度为100个分散的单细胞/孔)，设置阴性对照组、50nM BRD4-3F14单抗、100nM BRD4-3F14单抗、200nM BRD4-3F14单抗组和200nM的JQ1阳性对照组(BRD4的抑制剂，翌圣生物，货号：51503ES08)，培养7天，每2天换一次培养液。结束培养弃培养液后用PBS洗两次，加入4％的多聚甲醛约1ml，于室温中进行固定30min，之后用0.1％的结晶紫溶液染色10分钟，弃染液后用双蒸H2O清洗3次，观察克隆计数克隆数量，拍照后进行统计。结果如图3所示。
从图3的平板克隆实验结果显示，无论是BRD4-3F14单抗还是JQ1处理后7天后克隆形成的数量明显减少(P<0.01)，以200nM剂量的单抗和JQ1抑制效果更为明显，并且相同浓度下，本发明制备的BRD4-3F14单抗具有更好的抑制克隆形成效果，200nM剂量的情况下，单抗对应的克隆数只有(9±1)个，而JQ1克隆数有(24±3)个。
实施例4BRD4-3F14单抗对黑色素瘤细胞活力及基因表达的影响
取对数生长期的A875人黑色素瘤细胞接种于96孔板，通过细胞计数确保5×103个cells/100μL的细胞密度。向培养液中加入不同浓度梯度的JQ1，每组5个复孔。在给药48小时，向每个培养孔加入20μL的MTT(终浓度为5mg/ml)，继续在培养箱孵育4h，移出培养液，每孔加150μL的DMSO。在570nm处测定吸光度，计算相对于空白对照组的细胞活力(％)。结果如图4所示。
MTT结果如图4显示，JQ1以及BRD4-3F14单抗对A875人黑色素瘤细胞活力具有剂量依赖性抑制作用，且药物浓度越大，细胞活力越低，在200nM剂量的BRD4-3F14单抗处理情形下，细胞活力只有(28.4±1.3)％，而JQ1对应的细胞活力为(54.2±3.1)％。
用冷PBS洗涤药物处理结束后的各组细胞，用细胞刮刮下细胞，裂解细胞后提取细胞总蛋白，通过western blot检测JQ1作用48小时后黑色素瘤细胞中c-myc的蛋白水平。结果如图5所示。
从图5的结果可以看出，BRD4-3F14单抗以及JQ1相对于阴性对照组可有效抑制c-myc的表达(P<0.01)。c-myc是BRD4下游公认的作用靶点，通过检测c-myc的水平可以明确BRD4单抗的作用效果。结果显示单抗确实有效的抑制了BRD4的功能。
实施例5表皮间充质干细胞细胞因子的制备
人表皮干细胞(钦诚生物，货号：QCB1171)冻存管在37℃水浴中迅速摇晃解冻，加入4mL DMEM+10％FBS培养基混合均匀。在1000RPM条件下离心4分钟，弃去上清液，补加1-2mL DMEM+10％FBS培养基后吹匀。然后将所有细胞悬液加入培养瓶中培养过夜。第二天换液继续培养，经14天培养长至70％融合度进行传代培养。选用生长状况良好的干细胞，按10000个/cm2密度接种于康宁T175培养瓶中，加入30mL LONZA12-725F间充质干细胞无血清培养基正常培养。培养3-4天后细胞生长至90％融合度时吸出上清液用于收集细胞因子，收集的细胞上清液3000转/分钟离心20分钟去除细胞碎片，收集上清液。细胞上清液加入15mL超滤管中浓缩，去除多余水分使体积浓缩为原体积的一半，浓缩液过0.22μm滤膜除菌，得到干细胞细胞因子上清液，制备好的细胞因子上清液加入2％的海藻糖冷冻干燥，于-80℃条件下保存待用。
实施例6细胞因子联合BRD4-3F14单抗治疗黑色素瘤实验
将6-8周龄的WT小鼠随机分组。于小鼠腹部右侧皮下分别接种小鼠黑色素瘤细胞A875人黑色素瘤细胞，接种细胞量为2×105cell/50μl/只，从接种部位出现肉眼可见肿瘤开始，也就是肿瘤细胞接种第6天，将荷瘤小鼠分别按照下组进行治疗：
单抗治疗组：BRD4-3F14单抗200μg/只，4d/次，共2次；
细胞因子组：实施例4细胞因子200μg/只，4d/次，共2次；
细胞因子联合单抗组：实施例4细胞因子200μg/只，4d/次，共1次；
BRD4-3F14单抗200μg/只，4d/次，共3次；
阳性对照组：JQ1200μg/只，4d/次，共3次；
模型组：等量生理盐水，剂量同上。最后一次注射4d后处死小鼠，测量肿瘤的体积结果如图6所示。
从图6的结果可以看出，本发明的BRD4-3F14单抗具有较好的肿瘤抑制生长效果，特别是与细胞因子联用后，相对于单抗、细胞因子以及阳性对照三组单独治疗效果，差异较为显著(P<0.05),其肿瘤体积只有(124.1±11.2)mm3，而阳性对照组为(435±22.3)mm3，从这个结果也可以看出，细胞因子能够较好的协同BRD4-3F14抗体发挥作用。
应当理解的是，本发明在其应用上并不一定局限于在以下说明中所描述和/或在附图中所说明的组件的构造和布置的细节。本发明能够具有除所述和以不同方式实践或进行的那些实施方案之外的实施方案。而且，应理解本文所采用的短语和术语以及摘要出于描述目的并且不应视为限制性的。
Claims (5)
1.一种特异性针对人BRD4蛋白的单克隆抗体，该抗体是BRD4-3F14，其
轻链可变区的氨基酸序列为：
DLVMTQTAPSVPVTPGESVSISCRSTAWWYVWWMLWQLYWFLQRPGQSPQLLIYVYHNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCWHFPRHKCSFGSGTKLEIK
重链可变区的氨基酸序列为：
VKPGGSLKLSCAASRGWVDPYRMSWVRQTPDKRLEWVAIQITGHDFDYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCLGPKAVKHLELWGQGTTVTVS。
2.如权利要求1所述的BRD4蛋白的单克隆抗体BRD4-3F14在制备用于治疗黑色素瘤的药物组合物中的应用。
3.如权利要求1所述的BRD4蛋白的单克隆抗体BRD4-3F14和表皮干细胞细胞因子的组合在制备用于治疗黑色素瘤的药物组合物中的应用，其中表皮干细胞细胞因子是通过培养表皮干细胞，然后收集的细胞上清液3000转/分钟离心20分钟去除细胞碎片，收集上清液，细胞上清液加入超滤管中浓缩，去除多余水分使体积浓缩为原体积的一半，浓缩液过0.22μm滤膜除菌，得到浓缩的表皮干细胞细胞因子上清液，制备好的浓缩的细胞因子上清液加入2%的海藻糖冷冻干燥即得表皮干细胞细胞因子。
4.如权利要求2或3所述的应用，其特征在于所述药物组合物中含有药学上可接受的载体。
5.如权利要求2或3所述的应用，其特征在于所述药物组合物包含助溶剂。
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