CN115947848B - Dc细胞和单克隆抗体制备的抗癌组合物 - Google Patents
Dc细胞和单克隆抗体制备的抗癌组合物 Download PDFInfo
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及DC细胞和单克隆抗体制备的抗癌组合物。本发明通过杂交瘤技术制备了小鼠抗PD‑L1单克隆抗体，筛选出能稳定分泌高特异性的抗PD‑L1的杂交瘤细胞株，制备腹水获得高特异性、高亲和力的抗PD‑L1单克隆抗体，所述抗体能够有效的抑制肿瘤细胞的增殖以及降低PD‑L1蛋白的表达；同时，将PD‑L1单克隆抗体与负载了肿瘤抗原的DC细胞一起使用，能够有效的抑制肿瘤组织的生长以及提高机体的肿瘤杀伤活性。
Description
技术领域
本申请涉及生物领域，更具体的涉及DC细胞和单克隆抗体制备的抗癌组合物。
背景技术
树突状细胞(也称DC细胞)是功能最强的抗原提呈细胞，因其成熟时伸出许多树突样或伪足样突起而得名。树突状细胞(Dendritic cells,DC)是机体功能最强的专职抗原递呈细胞(Antigen presenting cells,APC)，它能高效地摄取、加工处理和递呈抗原，未成熟DC具有较强的迁移能力，成熟DC能有效激活初始T细胞，处于启动、调控、并维持免疫应答的中心环节。
人树突状细胞起源于造血干细胞(hemopoieticstemcell)。DC的来源有两条途径：①髓样干细胞在GM-CSF的刺激下分化为DC，称为髓样DC(myeloid dendritic cells，MDC)，也称DCl，与单核细胞和粒细胞有共同的前体细胞；包括朗格汉斯细胞(Langerhans cells，LCs)，间皮(或真皮)DCs以及单核细胞衍生的DCs等②来源于淋巴样干细胞，称为淋巴样DC(Lymophoid dendritic cells，LDC)或浆细胞样DC(plasmacytoid dendritic cells，piX；)，即DC2，与T细胞和NK细胞有共同的前体细胞。树突状细胞(DC)尽管数量不足外周血单核细胞的1％，但表面具有丰富的抗原递呈分子(MHC-Ⅰ和MHC-Ⅱ)、共刺激因子(CD80/B7-1、CD86/B7-2、CD40、CD40L等)和粘附因子(ICAM-1、ICAM-2、ICAM-3、LFA-1、LFA-3等)，是功能强大的专职抗原递呈细胞(APC)。DC自身具有免疫刺激能力，是惟一能激活未致敏的初始型T细胞的APC。
DC细胞可以用于肿瘤的治疗。DC抗肿瘤的机制如下：①DC可以高表达MHC-Ⅰ类和MHC-Ⅱ类分子，MHC分子与其捕获加工的肿瘤抗原结合，形成肽-MHC分子复合物，并递呈给T细胞，从而启动MHC-I类限制性CTL反应和MHC-Ⅱ类限制性的CD4+Thl反应。同时，DC还通过其高表达的共刺激分子(CD80/B7-1、CD86/B7-2、CD40等)提供T细胞活化所必须的第二信号，启动了免疫应答。②DC与T细胞结合可大量分泌IL-12、IL-18激活T细胞增殖，诱导CTL生成，主导Th1型免疫应答，利于肿瘤清除；激活穿孔素P颗粒酶B和FasL/Fas介导的途径增强NK细胞毒作用；③DC分泌趋化因子(Chemotactic Cytokines，CCK)专一趋化初始型T细胞促进T细胞聚集，增强了T细胞的激发。保持效应T细胞在肿瘤部位长期存在，可能通过释放某些抗血管生成物质(如IL-12、IFN-γ)及前血管生成因子而影响肿瘤血管的形成。上述CCK进一步以正反馈旁分泌的方式活化DC，上调IL-12及CD80、CD86的表达；同时DC也直接向CD8+T细胞呈递抗原肽，在活化的CD4+T细胞辅助下使CD8+T细胞活化，CD4+和CD8+T细胞还可以进一步通过分泌细胞因子或直接杀伤，增强机体抗肿瘤免疫应答。
肿瘤抗原肽负载的DC肿瘤疫苗是常用的DC疫苗类型。该类型的DC肿瘤疫苗，肿瘤抗原肽(包括合成肽)或肿瘤细胞相关抗原与自身或同种异体的DC共育，DC与肿瘤表位肽结合之后，可诱导CD8+CTL及CD4+T辅助细胞的应答及Th1和Th2细胞因子的产生，从而激发机体的抗肿瘤细胞免疫反应。目前实验室和临床应用较多的抗原肽来源于众多肿瘤相关抗原(tumor-associatedanti-gen，TAA)，包括人端粒酶逆转录酶(hTERT)，黑色素瘤抗原，癌胚抗原(CEA)，p53，Survivin，Her-2/Neu等。用肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)多肽致敏DC具有很好的靶向性，不易产生自身免疫性疾病，在体外和动物模型中得到广泛应用，而且已经用于人类临床试验。研究发现慢病毒介导hTERT修饰制备的DC疫苗，其刺激T细胞增殖的能力增强，诱导的CTL对端粒酶阳性的HepG2肿瘤细胞具有特异而且显著增强的杀伤效应。使用DC负载肝癌相关抗原HCA661多肽，其表面成熟标志高度表达，能有效激活自体CD8+淋巴细胞产生对HCA661+肝癌细胞的特异性CTL反应。用T细胞识别的黑色素瘤抗原(MAR-T)致敏DC，然后回输病人体内发现DC负载的MAR-T比一般的细胞因子有更高和持久的抗肿瘤免疫反应。研究发现，用含有人癌胚抗原(CEA)基因的酵母菌处理过的DC，能有效刺激T淋巴细胞增殖并增强其活性，杀伤CEA(+)的人肿瘤细胞。但是，肿瘤抗原肽负载的DC肿瘤疫苗仍然存在很多不足，主要因为已确定的肿瘤抗原较少，且单一抗原的免疫攻击尚无法完全杀伤肿瘤细胞；受人MHC限制，肿瘤细胞易通过抗原变异逃避针对单一抗原表位的免疫作用。由于存在此缺陷，人们选择了不确定的肿瘤抗原肽致敏DC诱导抗肿瘤免疫。肿瘤全细胞抗原负载的DC肿瘤疫苗目前只有一部分肿瘤的肿瘤相关抗原或肿瘤特异性抗原已知，大部分肿瘤尚未明确。肿瘤全细胞抗原含有全部的肿瘤抗原，因而无需明确肿瘤抗原，致敏DC后能表达MHC-Ⅰ、Ⅱ类分子及共刺激分子，会使疫苗的抗瘤作用明显提高。此类抗原包括凋亡坏死肿瘤细胞裂解物或提取物、完整肿瘤细胞等。用热休克凋亡食管癌细胞负载自体树突状细胞，实验发现其能有效刺激T细胞的增殖，明显提高对自体肿瘤细胞及细胞株的杀伤作用。Burdorf等[14]应用患者自体外周血单个核细胞培养DC负载肿瘤裂解物治疗进展期结直肠癌6例，具体方法为：皮内注射DC疫苗，每2周1次，共5次，其中有5例患者完成5次疫苗注射。患者有良好耐受性，所有患者未发现与疫苗有关的不良反应，表明DC疫苗安全、无毒。首先开展了DC肿瘤细胞融合技术，到目前为止，已经开展了黑色素瘤、肝癌、乳腺癌、白血病、肾癌、前列腺癌等肿瘤细胞与DC的融合研究。将绿色荧光标记的DC与用红色荧光标记的自身肿瘤细胞融合，然后用即时发光细胞分选技术纯化该融合细胞，将该细胞作为疫苗，治疗转移黑色素瘤患者取得了较好效果。研究发现，对35例晚期肝癌患者进行Ⅱ期临床实验，使用肝癌细胞系HepG2致敏肝癌患者DC，结果显示疾病控制率(部分临床反应和病情稳定3个月)为28％，17例AFP＞1000g/L的患者中有4例AFP水平下降70％以上。
近几年来，应用DC肿瘤疫苗治疗肿瘤，被广泛研究并部分应用于临床治疗，取得了一定的成绩。研究表明，以DC为基础的免疫疗法在当今肿瘤治疗中是最有价值也是最有前途的方法之一。此前的研究表明，DC细胞疫苗联合CD27抗体或者PD-L1抗体或者PD-L1抗体或者PD137抗体一起联用后，对于肿瘤细胞的杀伤率都显著的高于单一治疗组，表明联合治疗具有提高T细胞活性。但是目前，联合治疗可提供的选择形式还不够多。
发明内容
本发明的目的在于提供抗PD-L1单克隆抗体及其应用。
为了达到上述目的，本发明采用以下技术方案予以实现：
本发明公开了一株抗PD-L1单克隆抗体，包括轻链和重链。
优选地，该单克隆抗体轻链可变区的氨基酸序列如SEQ ID NO.1，重链可变区的氨基酸序列如SEQ ID NO.2所示。
进一步的，本发明提供了PD-L1单克隆抗体在制备治疗癌症的药物中的用途。
具体的，所述癌症是乳腺癌。
更具体的，所述的癌症是乳腺癌细胞MDA-MB-231导致的。
更进一步的，本发明还提供一种成熟化的负载肿瘤抗原的DC细胞。
具体的，所述DC细胞是采用如下方法制备并获得：在无菌条件下，小心游离并提取BALB/c雌性小鼠的双侧后肢股骨和胫骨，冲洗骨髓腔后，将骨髓组织冲碎并溶于NaCl中，离心，弃上清，加入无菌水，低渗迫使红细胞裂解，随后加入高渗生理盐水恢复其渗透压，离心，过滤，记数。NaCl洗涤，培养基重悬，后再加入rmGM-CSF和rmIL-4，铺在24孔板上，后置于CO2培养箱，并在第3天及时更换培养基，补充细胞因子，然后按培养细胞与肿瘤细胞比例为1:10向RPMI-1640培养液中加入肿瘤抗原裂解物，继续培养，然后轻轻吹吸下豁附于板底的聚集体，收集得到了负载肿瘤抗原的DC细胞。
更一步的，所述的肿瘤抗原裂解物是采用如下方法制备：取对数生长期MDA-MB-231癌细胞株离心后42℃温浴1h，反复冻融，离心后取上清，-80℃冰箱保存备用。
更进一步的，本发明还提供了负载肿瘤抗原的DC细胞联合PD-L1单克隆抗体在制备治疗癌症的药物组合物中的用途。
具体的，所述癌症是乳腺癌。
更具体的，所述的癌症是乳腺癌细胞MDA-MB-231导致的。
本发明也提供了组合物(例如药物组合物)，其包含有效量的在任何合适载体中的任何本发明的抗体，包括其片段和衍生物。
可用于这些组合物中的药学上可接受的载体包括，但不限于，离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白如人类血清白蛋白、缓冲物质如磷酸盐、甘氨酸、山梨酸、山梨酸钾、饱和的植物脂肪酸的偏甘油酯混合物、水、盐或电解质类，如硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、胶体硅、三硅酸镁、聚乙烯吡咯烷酮、纤维素基物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧化丙烯-嵌段聚合物、聚乙二醇和羊毛脂。
本发明的组合物可以口服、肠胃外给药，通过吸入喷雾、局部地、经直肠地、经鼻地、向颊地、经阴道地或通过植入池给药。如本文中所使用的术语“肠道外的”包括皮下的、静脉内的、肌肉内的、关节内的、胸骨内的、胸内的、肝内的、病灶内和颅内注射或输注技术。
本发明的组合物的无菌可注射形式可以是水性或油质悬液。这些悬液可以根据本领域已知的技术，利用合适的分散剂或润湿剂和悬浮剂配制。无菌的可注射制剂也可以是在非毒性肠道外可接受的稀释液或溶剂中(例如在1，3-丁二醇中)的无菌可注射溶液或悬液。这些可采用的可接受载体或溶剂是水、林格溶液和等渗氯化钠溶液。另外，传统地使用无菌不挥发性油作为溶剂或悬浮介质。为此，可以使用任何温和的不挥发性油，包括合成的单甘油酯或二甘油酯。脂肪酸如油酸和其甘油衍生物在制备可注射制剂是有用的，因为是天然的药学上可接受的油，如橄榄油或蓖麻油，尤其是它们的聚氧乙基化形式。这些油溶液或悬浮液也可以包含长链醇稀释剂或分散剂，如羟甲基纤维素或类似的分散剂(其通常用于药学上可接受的剂量形式，包括乳液和悬浮液)。其他经常使用的表面活性剂，如吐温、司盘和其他的乳化剂或生物利用度增强剂(其通常用于药学上可接受的固体、液体或其他剂型)也可用于配制制剂的目的。
本发明的组合物可以以任何口服可接受的剂型(包括，但不限于胶囊剂、片剂、水性悬液或溶液)口服给药。如果是口服使用的片剂，通常使用的载体包括乳糖和玉米淀粉。通常还加入润滑剂，例如硬脂酸镁。对于以胶囊形式的口服给药，有用的稀释剂包括乳糖和干燥的玉米淀粉。当要求口服用水性悬液时，将活性成分与乳化剂和悬浮剂联合。如果需要，也可以添加某些甜味剂、调味剂或着色剂。
可替换地，本发明的组合物可以以注射给药的形式给药。这些可以通过混合该药物与合适的无刺激性赋形剂进行制备。这样的物质包括可可油、蜂蜡、聚乙二醇。这样的组合物是根据药剂领域熟知的技术制备的。
根据本发明另一个重要的具体实施方式，抗PD-L1抗体和DC细胞以及/或其他化合物可以与一种或多种另外的治疗剂一起配制，其中所述另外的治疗剂包括对于特定治疗目的(该抗体或化合物被给予)而正常使用的药物。另外的治疗剂通常按照在单一治疗中用于治疗特定疾病或症状所常用的剂量给药。这样的治疗剂包括，但不限于：治疗癌症中所使用的治疗剂(“抗癌化合物”，包括化学治疗化合物、激素、血管形成抑制剂、凋亡剂等)；用于治疗传染病的治疗剂(包括抗病毒化合物)；用于其他免疫治疗的治疗剂，例如自身免疫性疾病、炎性障碍和移植排斥的治疗剂；细胞因子；免疫调节剂；辅助化合物；或针对激活和抑制NK细胞受体的其他抗体和其他抗体的片段。除非另有指明，下面列出的联合组合物可以包括本发明的活化抗体、抑制抗体或细胞毒素-抗体结合物。
用于治疗癌症的治疗剂包括化学治疗剂(包括干扰DNA复制、有丝分裂和染色体分离的药物，以及破坏多核苷酸前体合成和精确度的试剂)、激素治疗剂、抗血管形成剂以及诱导凋亡的试剂。
作为示例性考虑的化学治疗药包括但不限于：烷化剂，抗代谢药，细胞毒性抗生素，长春花生物碱，例如阿霉素、更生霉素、自力霉素、洋红霉素、道诺霉素、亚德里亚霉素、三苯氧胺、红豆杉醇、紫杉特尔(taxotere)、长春新碱、长春碱、长春瑞滨、依托扑沙(VP-16)、5-氟尿嘧啶(5FU)、阿糖胞苷、环磷酰胺、硫替派、氨甲叶酸、喜树碱、放线菌素-D、丝裂霉素C、顺铂(CDDP)、氨喋呤、考布他汀(combretastatin)及其衍生物和前物。
有益效果
本发明本发明通过杂交瘤技术制备了小鼠抗PD-L1单克隆抗体，筛选出能稳定分泌高特异性的抗PD-L1的杂交瘤细胞株，制备腹水获得高特异性、高亲和力的抗PD-L1单克隆抗体，所述抗体能够有效的抑制肿瘤细胞的增殖以及降低PD-L1蛋白的表达；同时，将PD-L1单克隆抗体与负载了肿瘤抗原的DC细胞一起使用，能够有效的抑制肿瘤组织的生长以及提高机体的肿瘤杀伤活性。
附图说明
图1 PD-L1单克隆抗体特异性鉴定结果
图2 PD-L1单克隆抗体对癌细胞细胞活力的影响
图3 PD-L1单克隆抗体对PD-L1蛋白表达的影响
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，本发明的说明书和权利要求书中术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
实施例1 PD-L1单克隆抗体的制备
Balb/C小鼠3只，首次免疫，弗氏完全佐剂与重组PD-L1蛋白(ProSci，PSI-92-487)按1：1混合乳化，背部皮下多点免疫小鼠(剂量：融合蛋白100μg/只)；2周后第一次加强免疫(70μg/只，不完全佐剂)；3天后第二次加强免疫(60μg/只，不完全佐剂)；3天后尾静脉采血，间接ELISA法检测抗体效价，取抗体效价最高的小鼠，追加免疫一次，(80μg/只，不完全佐剂)，2周后细胞融合前3天，脾内加强免疫(80μg/只，1×PBS溶解)。
免疫小鼠脾细胞与骨髓瘤细胞Sp2/0以10：1混合，用50％PEG4000为融合剂，融合细胞用含20％小牛血清的HAT选择性培养基悬浮后，接种于以小鼠腹腔巨噬细胞作饲养层的96孔培养板中，培养于5％CO2、37℃培养箱，在融合后的第3、第6天更换HAT培养液；于融合后第7-14天后，通过问接ELISA法筛选有杂交瘤克隆生长的培养孔的上清液，阳性孔需经融合蛋白中标签蛋白复筛，选择与标签蛋白呈阴性反应的阳性孔，通过限稀释法进行单克隆化，直至所有克隆孔阳性率为100％时，定杂交瘤细胞株获得了具有较好特异性的PD-L1单抗4株，分别是P2A7，P3D5，P5H2，P6F9。
将8周龄雌性BALB/c小鼠腹腔注射0.5mL液体石蜡致敏。7d后分别腹腔注射5×106的P2A7，P3D5，P5H2，P6F9杂交瘤细胞，待小鼠腹部明显膨大，抽取腹水。采用间接ELISA法检测杂交瘤腹水效价，四个杂交瘤细胞对应的腹水的效价均大于1：2.048×105。IgG类单克隆抗体纯化试剂盒纯化单抗，并将抗体浓度调整为1mg/mL备用。
实施例2 PD-L1单克隆抗体P2A7特异性鉴定
取5Oμg重组PD-L1蛋白及His标签蛋白经1O％SDS—PAGE电泳完后半干转移至PVDF膜上。5％(W/V)脱脂奶粉37℃封闭lh，加入1：200倍稀释的PD-L1单克隆抗体P2A7，4℃过夜，TBS-Tween20洗膜3次，每次15min，加入1：5000倍稀释的羊抗鼠HRP-IgG，37℃下放置1h，TBST洗膜3次，每次15min，化学发光，用化学发光检测系统采集图像。结果如图1所示。
Western blot显示，单抗P2A7只能与PD-L1蛋白泳道检测到与抗原带大小一致的特异条带，而His标签蛋白的泳道则无条带，见图1，这说明所述单抗P2A7具有较好的特异性。
实施例3 PD-L1单克隆抗体P2A7亲和力鉴定
使用SPR测量抗体的结合动力学：BiacoreT200S系列传感器芯片CM5，胺偶联试剂盒和10×HBS-EP购自GEHealthcare。在固定步骤中，在注射前立即混合400mM EDC和100mMNHS来制备活化缓冲液，CM5传感器芯片通过激活缓冲液激活440秒，然后以5μL/分钟的流速将在10mMNaAc(pH4.5)中的30μg/mL山羊抗人IgGFcγ抗体注射到Fc1-Fc4通道中200秒，该芯片由1M乙醇胺-HCl(GE)去活化，然后将抗体捕获在芯片上。将运行缓冲液(HBS-EP+)中的4μg/mL抗体以10μL/分钟的流速分别注射到Fc3通道30秒。八种不同浓度(20nM、10nM、5nM、2.5nM、1.25nM、0.625nM、0.3125nM和0.15625nM)的分析物PD-L1重组蛋白和空白运行缓冲液以35μL/分钟的流速对Fc1-Fc4通道有序地进行120秒的结合阶段，随后是2400秒的解离阶段。在每个解离阶段之后，以10μL/分钟的速度注入再生缓冲液(10mM甘氨酸pH1.5)35秒。结果显示，抗体P2A7以1.85nM的亲和力结合人PD-L1重组蛋白，显示出较好的亲和特性。
实施例4 PD-L1单克隆抗体P2A7生物学特性实验
乳腺癌细胞MDA-MB-231进行培养后，在96孔板每孔接种8000个细胞，待细胞贴壁后，分别加入不同浓度梯度的PD-L1单克隆抗体P2A7，浓度分别设置为0、1μg/mL、10μg/mL、50μg/mL、100μg/mL、200μg/mL的终浓度。每个浓度梯度设置3个复孔。PD-L1单克隆抗体P2A7分别处理72h后，每孔加入5mg/mLMTT溶液20μL，37℃细胞培养箱继续培养4h，弃孔板内液体，每孔加入200μLDMSO，水平振荡器上振荡10min，酶标仪测定490nm处吸光度。
从图2的结果可有看出，通过MTT法检测单抗对MDA-MB-231细胞活力的影响随着单抗的浓度增加而活力逐渐降低。当单抗的浓度为200μg/mLP2A7时，细胞活力降低到(41.84±0.92)％，与空白对照相比，差异极其显著(P<0.05)。
MDA-MB-231细胞经100μg/mL单抗处理72h后收集细胞，制备蛋白样品，免疫印迹实验(Westernblot)检测PD-L1蛋白水平变化。空白对照采用同样操作。结果如图3显示，乳腺癌细胞系MDA-MB-231能够高表达PD-L1，在不用抗体处理时，PD-L1高表达，而采用单抗处理后，单抗能够抑制细胞PD-L1蛋白表达。
实施例5 DC细胞的制备
MDA-MB-231癌抗原裂解物的制备：取对数生长期MDA-MB-231癌细胞株离心后42℃温浴1h，反复冻融，离心后取上清，-80℃冰箱保存备用。
在无菌条件下，小心游离并提取BALB/c雌性小鼠的双侧后肢股骨和胫骨，冲洗骨髓腔后，将骨髓组织冲碎并溶于0.9％NaCl中，离心，弃上清，加入10ml无菌水，低渗迫使红细胞裂解，随后加入10％高渗生理盐水1ml恢复其渗透压，离心，过滤，记数。0.9％NaCl洗涤2遍，培养基重悬，使细胞密度达到2×106/ml，后再加入rmGM-CSF(5ng/mL)和rmIL-4(5ng/mL)，1mL/孔，铺在24孔板上，后置于CO2培养箱，并在第3天及时更换培养基，补充细胞因子，然后按培养细胞与肿瘤细胞比例为1:10向RPMI-1640培养液中加入肿瘤抗原裂解物，继续培养24h，然后轻轻吹吸下豁附于板底的聚集体，收集备用。取部分收集细胞送鉴定。采用流式细胞仪检测PE标记的鼠抗人CD80、CD86抗体，检测显示，在收获的成熟DC中，CD80+为82.3％，CD86+为88.3％，说明制备得到了负载肿瘤抗原的DC细胞。
实施例6 DC细胞联合P2A7单抗的治疗实验
取C57BL/76J小鼠，将5×105的MDA-MB-231癌细胞接种于每只小鼠左侧腋下3d后，小鼠随机分成4组，每组10只，分组如下：
模型组：注射生理盐水作为对照；
DC疫苗治疗组：在肿瘤接种后第3天与第10天，将1×106经过肿瘤全抗原刺激后的成熟DC细胞注射进小鼠右侧腹皮下；
抗PD-L1抗体治疗组：在肿瘤接种厉第3天与第10天，将100μg/mL PD-L1抗体注入小鼠腹腔内。
DC疫苗联合抗PD-L1抗体治疗组：在肿瘤接种后第3天与第10天，将1×106经过肿瘤全抗原刺激后的成熟DC细胞注射进小鼠右侧腹皮下，在肿瘤接种厉第3天与第10天，将100μg/mL PD-L1抗体注入小鼠腹腔内。
肿瘤接种后28d，处死小鼠，用游标卡尺测量肿瘤长、短径，按照公式计算肿瘤大小。结果如表1所示。
表1不同治疗组肿瘤大小
| 实验组别 | 肿瘤大小(mm2) |
| 模型对照组 | 195.6±12.5 |
| DC疫苗治疗组 | 98.6±10.3* |
| 抗PD-L1抗体治疗组 | 117.6±11.7* |
| DC疫苗联合抗PD-L1抗体治疗组 | 68.3±9.3* |
*与对照组比较，P<0.01.
从表1的结果可以看出，DC细胞疫苗和抗PD-L1抗体联合治疗组肿瘤大小明显小于模型对照组，以及单独使用疫苗组和单独使用抗体治疗组。
肿瘤接种后28d处死小鼠后，在无菌环境下，取对照组和不同治疗组小鼠的脾脏在无菌200筛网上研磨，获得单细胞悬液，裂解红细胞后，加入到淋巴细胞分离液，离心后按照说明书取淋巴细胞层，获得小鼠脾淋巴细胞，一部分作为效应细胞，进行肿瘤杀伤实验；另一部分用来检测干扰素γ的分泌，MDA-MB-231癌细胞作为靶细胞，根据LDH说明书确定最佳靶细胞数量接种到U型底96孔板中，严格按照说明书操作。结果如表2所示。
表2不同治疗组对淋巴细胞杀伤率和干扰素γ的分泌水平的影响
*和**表示与对照组比较，P<0.01，差异显著
从表2的结果可以看出，DC细胞疫苗和抗PD-L1联合治疗组的脾淋巴细胞对癌细胞杀伤率明显高于对照组，以及单独疫苗组和抗体单独治疗组；DC细胞和抗PD-L1抗体单独治疗组分别与対照组相比，杀伤率也显著提高。此外，DC细胞和抗PD-L1抗体联合治疗组的脾淋巴细胞与癌细胞共孵育分泌的扰素γ显著高于对照组，以及疫苗和抗体单独治疗组，说明本发明制备的DC疫苗与抗体组联合使用后具有较好的肿瘤治疗效果。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (7)
1.抗PD-L1性单克隆抗体P2A7，其特征在于，该单克隆抗体的轻链可变的氨基酸序列如SEQ ID NO.1所示，重链可变区的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的抗PD-L1性单克隆抗体P2A7在制备抑制乳腺癌的药物中的应用。
3.一种用于癌症治疗的药物组合物，其包含权利要求1所述的抗PD-L1性单克隆抗体P2A7和一种以上药学可接受的载体。
4.一种用于癌症治疗的药物组合物，其特征在于含有权利要求1所述的抗PD-L1性单克隆抗体P2A7和负载有肿瘤抗原的DC细胞，所述负载肿瘤抗原的DC细胞是将癌细胞42℃温浴1h，反复冻融，离心后取上清后与DC细胞混合孵育24h后，轻轻吹吸下豁附于板底的聚集体，收集得到了负载肿瘤抗原的DC细胞。
5.权利要求1所述的抗PD-L1性单克隆抗体P2A7和负载有肿瘤抗原的DC细胞在制备治疗乳腺癌的药物组合物中的用途；其中，所述负载肿瘤抗原的DC细胞是将癌细胞42℃温浴1h，反复冻融，离心后取上清后与DC细胞混合孵育24h后，轻轻吹吸下豁附于板底的聚集体，收集得到了负载肿瘤抗原的DC细胞。
6.如权利要求5所述的用途，其特征在于所述药物组合物还含有药学上可接受的载体。
7.如权利要求6所述的用途，其特征在于所述药物组合物可选的还添加有癌症治疗其他治疗剂。
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| CN109776678A (zh) * | 2019-03-08 | 2019-05-21 | 安徽安科生物工程（集团）股份有限公司 | 一种人源化pd-l1单克隆抗体、其制备方法和应用 |
| CN110575537A (zh) * | 2019-09-06 | 2019-12-17 | 刘慧宁 | Dc疫苗和nkg2a拮抗剂的组合物及在抗乳腺癌或肝癌中的应用 |
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