CN112194723A - 一种免疫细胞在治疗癌症中应用 - Google Patents
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Abstract
本发明涉及一种免疫细胞在治疗癌症中的应用。本发明制备了特异性针对PD‑L1的单克隆抗体，其具有较好的抑制肝癌细胞增殖的效果，此外，还通过分离以及优化获得了能够抑制肝癌细胞增殖的免疫细胞，通过将免疫细胞与PD‑L1细胞组合使用，可以有效的治疗肝癌，具有协同的作用，具有极好的应用前景。
Description
技术领域
本发明涉及生物制药领域，具体的涉及用于肝癌治疗的领域，更近一步的涉及一种免疫细胞在治疗癌症中应用。
背景技术
肝细胞癌(HCC)占原发性肝癌的70％～80％，世界范围内是第6位常见的癌症，术后的复发以及转移引起的死亡排名第3。在亚洲国家尤其是中国和日本，在明确诊断后的数周至数月内死亡率高，5年生存率小于14％，术后5年的复发率大于50％，术后5年生存率只有30％～40％。
随着医学技术的发展，免疫细胞治疗直接识别并杀伤患者血液和淋巴中的肿瘤细胞，在不损伤和破坏机体免疫系统和功能的前提下杀伤患者血液、淋巴中的肿瘤细胞，恢复并增强机体自然抗癌免疫系统。免疫细胞治疗可减轻肝癌患者的放化疗毒副作用，降低患者复发率，延长无进展生存时间和总生存期。
NK(natural killer cell，自然杀伤细胞)细胞和NKT细胞是固有免疫细胞的重要组成部分，均从骨髓衍生。在肝脏中定居的NK细胞发挥宿主免疫作用来有效阻止细胞的肿瘤化。NK细胞通过分泌细胞因子来调节其他免疫细胞的活性以及发挥细胞毒性。
自然杀伤细胞(NK)占人肝脏内淋巴细胞的25％～40％，根据CD56分子的表达量可分为CD56bright和CD56dim两个亚群。其中CD56bright亚群可被IL-2刺激后扩增，约10％表达杀伤细胞免疫球蛋白样受体(KIR)，可分泌合成TNF相关的凋亡诱导配体(TRAIL)；而CD56dim亚群对IL-2刺激不敏感，85％的CD56dim为KIR+，分泌合成穿孔素和颗粒酶B。当HCC发生时，一方面肝癌细胞表面表达Rae1，该因子作为NK细胞活化受体NKG2D的配体可以活化NK细胞，促使其发挥抗肿瘤免疫的作用。另一方面，NK细胞的免疫功能受到限制，HCC患者外周血中CD56dimNK细胞亚群明显少于健康对照组。同时HCC患者肿瘤区域中的CD56dimNK细胞表达的IFN-γ少于非肿瘤区域，有体外实验表明这与CD4+CD25+调节性T细胞(Treg)相关。肝癌发生时，细胞外基质微环境的改变、HSC分泌的TGF-β等可以抑制NK细胞活性及功能，进而使其对肝细胞的监视功能减弱。在正常的生理状态下，NK细胞通过产生“细胞溶解颗粒”来发挥生理作用，这种颗粒中包含有穿孔素、颗粒酶、肿瘤坏死因子相关凋亡诱导配体和INF-γ。NKT细胞是T淋巴细胞的一个亚群，细胞功能与T细胞和NK细胞相重合，也表达αβT细胞受体和多种NK细胞受体，是一种IL-4，IFN-γ和TNF-α等细胞因子的重要分泌源。NK细胞的抗肿瘤能力与NKG2D和MICA的表达水平密切相关。Chen等证实了蝎毒多肽(PESV)可通过调节MICA-NKG2D通路增强NK细胞的抗肿瘤能力。Jiang等也发现人IL-15基因修饰的NK细胞NKL_IL15)在HCC荷瘤小鼠中表现出较强的抑瘤活性。研究显示，RFA可有效激活HCC患者外周血中NK细胞的活性。
目前已有研究表明CIK细胞联合贝伐单抗在体内外对HepG2细胞增殖、侵袭、迁移均有抑制作用，且优于单独治疗组。但是抗体应用仅限于贝伐单抗，成本较高。随着分子生物学、免疫学、基因工程等研究的不断深入，肿瘤的免疫细胞治疗得到了长足的发展和广泛的应用。然而，如何进一步提高疗效，仍然是肿瘤免疫细胞治疗所亟待解决的问题。将单抗与免疫细胞进行组合使用已经显示了治疗法可行性，为肝癌的治疗提供了新手段。但是目前研究较少。
发明内容
本发明克服了现有技术的缺陷，提供了一种免疫细胞联合单抗治疗肝癌的方法。
一方面，本发明提供了一种特异性的PD-L1单克隆抗体，其名称为5-D4。其重链可变区序列为：
QSLEESGGRLVVPDETLTITCTVSGIDLSGNFLDWVRQAPGEGLEWIGEDNKDAYTYYASWAAERLTISKPSSTKVDLKITSPTTETTATYFCGRSAFKGGTDLWGPGTLVTVSS(SEQ ID NO：1)。
5-D4的轻链可变区序列为：
AIVMTQTPSPVSAAVGGTVTINCTESGAIYSQAYLGWFQQKPGQPPKLLIYDSSTLNCGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQFGKSNNTDGCGFGGGTEVVVR(SEQ ID NO：2)。
本发明另外一方面，提供一种免疫细胞的制备方法，具体包括以下步骤：
(a)将刺激因子包被培养瓶；其中刺激因子由IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL、IL-15浓度2000IU/mL、GM-CSF浓度2000IU/mL、IL-4浓度750IU/mL、TNF-α浓度1000IU/mL以生理盐水配置组成。
(b)将血液采用密度梯度离心法分离单个核细胞；
(c)在刺激因子包被的培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基，再将单个核细胞接种于刺激因子包被的培养瓶中在37℃、二氧化碳浓度5％条件下进行培养，其中单个核细胞的接种浓度为5×106个/ml；
(d)待培养3天后，将单个核细胞移入空培养瓶中，再向空培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基继续在37℃、二氧化碳浓度5％条件下进行培养；
(e)待培养7天后，将单个核细胞移入培养袋中，再向培养袋中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基在37℃、二氧化碳浓度5％条件下进行培养，待培养15天后，收集单个核细胞。采用流式细胞检测方法对步骤(e)中收集的单个核细胞中NK免疫细胞(CD3-CD56+)的含量进行检测。
本发明另外一方面，提供一种药物组合物，其含有分离的免疫细胞以及所述的PD-L1单克隆抗体。
跟进一步的，抗PD-L1抗体或其PD-L1-结合功能片段可单独配制成药物组合物，该药物组合物适于通过预期的给药途径给予靶受试者，如下文更详细地讨论的。
适用于本文所述方法的药物组合物可包括治疗活性剂(例如抗PD-L1抗体或其功能片段)和药学上可接受的载体或稀释剂。
该组合物可制成静脉内，皮下，腹膜内，肌肉内，口服，鼻，肺，眼，阴道或直肠给药。在一些实施例中，配制抗PD-L1抗体或其功能性片段用于静脉内，皮下，腹膜内或肌肉内给药，例如溶液，悬浮液，乳剂，脂质体制剂等。药物组合物可使用本领域已知的技术配制成速释组合物，缓释组合物，延迟释放组合物等。
用于各种剂型的药理学上可接受的载体是本领域已知的。例如，用于固体制剂的赋形剂，润滑剂，粘合剂和崩解剂是已知的；用于液体制剂的溶剂，增溶剂，悬浮剂，等渗剂，缓冲剂和安抚剂是已知的。在一些实施方案中，药物组合物包括一种或多种附加组分，例如一种或多种防腐剂，抗氧化剂，稳定剂等。
另外，所公开的药物组合物可以配制成溶液，微乳液，脂质体或其它适合于高药物浓度的有序结构。载体可以是包含例如水，乙醇，多元醇(例如甘油，丙二醇和液体聚乙二醇等)及其适当混合物的溶剂或分散介质。例如，可以通过使用诸如卵磷脂的涂层，通过在分散的情况下保持所需的颗粒尺寸以及通过使用表面活性剂来保持适当的流动性。在一些实施方案中，优选在组合物中包括等渗剂，例如糖，多元醇如甘露醇，山梨醇或氯化钠。可注射组合物的延长吸收可通过在组合物中包括延迟吸收的试剂例如单硬脂酸盐和明胶来实现。
无菌可注射溶液可通过将所需量的活性化合物与上述一种或多种组分的组合按需混合在适当的溶剂中，然后进行灭菌微滤来制备。通常，分散体是通过将活性化合物掺入无菌载体来制备的，所述无菌载体包含碱性分散介质和来自以上列举的那些的所需其它组分。在用于制备无菌可注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥(冷冻干燥)，其从先前无菌过滤的活性成分溶液中产生活性成分粉末和任何附加的所需成分。
本公开的药物组合物可与其它治疗剂联合给药。例如，所述联合治疗可包括药物组合物，所述药物组合物包含至少一种所公开的抗PD-L1抗体或其功能片段与至少一种或多种另外的治疗剂，包括但不限于，CAR-T细胞(例如，表达抗CD19的修饰T细胞，抗HER2，抗BCMA，抗CS-1，抗PSCA，抗CaIX，抗IL13R，或抗PD-L1 CAR)，其它肿瘤靶向抗体(例如，抗CAIX抗体)，免疫应答增强模式(例如抗-GITR抗体，抗-OX40抗体，抗-CD137抗体或TLR激动剂)和小分子药物(例如BTK抑制剂，EGFR抑制剂，BET抑制剂，PI3K抑制剂，BRAF抑制剂或PARP抑制剂)。本公开的药物组合物也可以与放射治疗联合给药。
本文提供了用所公开的抗PD-L1抗体治疗癌症，恶性疾病或癌细胞增殖的方法。更具体地，本公开抗肿瘤免疫的方法，包括给予治疗有效量的任何上述抗PD-L1抗体或组合物。
增强T细胞功能和抗肿瘤免疫提供了治疗癌症，恶性疾病或癌细胞增殖的广谱途径。因此，通过施用所公开的抗PD-L1抗体及其功能片段，可以治疗肝癌。
在一些实施方案中，根据所公开的方法治疗的癌症是高度免疫原性的癌症。并且在一些实施方案中，根据所公开的方法治疗的癌症是表达PD-L1的癌症。
调节给药方案以提供最佳所需反应(例如，治疗反应如肿瘤消退或缓解)。例如，在一些实施方案中，可以施用单个丸，而在一些实施方案中，可以随时间施用几个分开的剂量，或者可以根据情况按比例减少或增加剂量。例如，在一些实施方案中，所公开的抗体或功能片段可以通过皮下注射或静脉内注射每周施用一次或两次。在一些实施方案中，所公开的抗体或功能片段可以通过皮下注射每月施用一次或两次。在一些实施方案中，所公开的抗体或功能片段可以每周施用一次，每隔一周施用一次，每三周施用一次，每四周施用一次，每隔一个月施用一次，每三个月施用一次，每四个月施用一次，每五个月施用一次，或每六个月施用一次。
示例性的剂量可以根据被治疗个体的大小和健康状况以及被治疗的病症而变化。例如，在一些实施方案中，所公开的抗体或功能片段可以1-100mg/kg的剂量给药。在一些实施例中，所公开的抗体和功能片段可以以1的剂量给药，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，25，30，35，40，45，50，55，60，65，70，75，80，85，90，95或100mg/kg。
此外，所公开的治疗方法还可包括给予除抗PD-L1抗体或其功能片段外的第二治疗化合物。例如，在一些实施方案中，附加的治疗化合物是CAR-T细胞，肿瘤靶向抗体，免疫应答增强程式或小分子药物。
因此，为了本公开的目的，如果获得一个或多个有益的或期望的结果，包括期望的临床结果，则治疗对象。例如，有益的或所需的临床结果包括，但不限于，以下的一种或多种：减少由所述疾病引起的一种或多种症状，提高患有所述疾病的人的生活质量，减少治疗所述疾病所需的其它药物的剂量，延迟所述疾病的发展，和/或延长个体的生存期。
此外，虽然所述方法的主题通常是癌症患者，但是患者的年龄不受限制。所公开的方法可用于治疗癌症，恶性疾病或癌细胞增生，其具有各种不同的复发和预后结果，所述复发和预后结果跨越所有年龄段和群体。因此，在一些实施方案中，受试者可以是儿科受试者，而在其它实施方案中，受试者可以是成人受试者。
有益效果
本发明制备了特异性针对PD-L1的单克隆抗体具有较好的抑制肝癌细胞增殖的效果，此外，还通过分离以及优化获得了能够抑制肝癌细胞增殖的免疫细胞，通过将免疫细胞与PD-L1细胞组合使用，可以有效的治疗肝癌，具有协同的作用，具有极好的应用前景。
附图说明
图1各抗体ELISA检测结果图
图2不同剂量的单抗均对肝癌细胞增殖抑制图
图3不同药物对肿瘤体积影响图
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1PD-L1单克隆抗体的制备
人工合成针对PD-L1的KLH偶联的羧基端肽RLRKGRMMDVKKCGIQDTNSKKQSDTHLEET。以该合成肽作为免疫原进行小鼠的免疫。
选用6～8周龄雌性BALB/c小鼠，按照首次在皮下注射混合有Freund完全佐剂的合成肽100μg，以后每隔3周腹腔注射混有Freund不完全佐剂的抗原30μg(加强免疫)，第3次加强免疫后3周，腹腔注射抗原20μg(冲击免疫)。冲击免疫完成后，在96h内处死小鼠，无菌操作取出脾脏，分离B细胞。用于完成细胞融合。
融合前2周开始施行，在含100mL/L胎牛血清RPMI1640培养基的培养皿中加入骨髓瘤细胞，放入37℃孵箱。融合前24h换液1次，确保在融合时争少1x108个细胞。
将小鼠脾细胞与骨髓瘤细胞按5：1比例混合，加入40mL RPMI1640液，1000r/min离心6min弃上清。吸取1mL PEG缓慢滴入离心管内，37℃静5min。沿管壁滴加RPMI1640培养液20mL，然后加至50mL使PEG作用终止。800r/min离心5rain，弃上清。将沉淀胞轻轻悬浮于HAT培养液中，接种至96孔板，将培养板放入37℃50mL/L CO2孵箱。5d后用HAT培养换出1/2培养基，10d后换为HT培养基，14d后用ELISA筛选阳性克隆。
将抗原以NaCO3-NaHCO3(pH9.6)包被，每块酶标板20μg合成肽、cTnI和H-FABP，每孔包被体积为100μL，4℃过夜。弃抗原，拍干酶标板孔内液体，以l0mg/mL BSA于37℃封闭2h。弃去孔内液体，拍干，加入融合后的细胞培养上清液100μL。37℃孵育1h，弃去液体，拍干；PBST洗涤5min*3次，每孔加入100μLHRP标记山羊抗小鼠IgG，37℃孵育1h，弃去液体，加入TMB显色，适时用50μL 2mol/L H2SO4终止反应，用酶标仪读出各孔A450值。结果如图1所示。筛选得到二株效果较好的单克隆抗体分别是5-D4和4-G3。通过效价鉴定，得到5-D4的效价为3.2×106和4-G3的效价为3.9×106。
实施例2PD-L1 mAb抗体特性鉴定
非竞争酶免疫实验测定其亲和常数(Affinity constant)，Ka值。参考Raghava的方法，按照公式Ka＝(n－1)/2(n[Ab']t－[Ab]t)计算亲和常数Ka值。结果其亲和力常数分别是5-D4为4.3×109和4-G3为1.32×109。
用SBA小鼠mAb分型试剂盒测定2种单克隆抗体Ig类及其亚类。结果显示5-D4为IgG2b，4-G3抗体均为IgG1。
通过轻重链序列分析，鉴定得到5-D4的重链可变区序列为：
QSLEESGGRLVVPDETLTITCTVSGIDLSGNFLDWVRQAPGEGLEWIGEDNKDAYTYYASWAAERLTISKPSSTKVDLKITSPTTETTATYFCGRSAFKGGTDLWGPGTLVTVSS。
5-D4的轻链可变区序列为：
AIVMTQTPSPVSAAVGGTVTINCTESGAIYSQAYLGWFQQKPGQPPKLLIYDSSTLNCGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQFGKSNNTDGCGFGGGTEVVVR。
实施例3 5-D4单抗对HepG2细胞增殖影响的测定
采用MTT法，每孔加100μL HepG2细胞悬液(2*104/孔)孔平底细胞培养板中，培养12h后，共分二组：实验组，加入不同浓度的5-D4单抗溶液；对照组，仅加入RPMI 1640培养液。每孔终体积200μL。37℃，50ml/L CO2继续培养12h，离心更换不含抗体的培养液，终体积200μL。加入MTT工作也20μl/孔，继续培养4h。离心弃上清，每孔加入150μl DMSO震荡，用全自动酶标仪在波长570nm下测D吸收值。每一个浓度涉及4个复孔，实验重复3次。计算细胞抑制率＝(1-实验组D值/对照组D值)*100％。结果如图2所示。
不同剂量的单抗均对肝癌细胞增殖有抑制作用，并呈明显的剂量相关性。MTT法检测单抗浓度为0.01mg/L时对HepG2细胞增殖抑制率不到30％，当浓度为10mg/L时，增殖抑制率可接近75％，具有较好的抑制效率。
实施例4治疗肝癌的免疫细胞的制备
(a)将刺激因子包被培养瓶；其中刺激因子由IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL、IL-15浓度2000IU/mL、GM-CSF浓度2000IU/mL、IL-4浓度750IU/mL、TNF-α浓度1000IU/mL以生理盐水配置组成。
(b)将血液采用密度梯度离心法分离单个核细胞；
(c)在刺激因子包被的培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基，再将单个核细胞接种于刺激因子包被的培养瓶中在37℃、二氧化碳浓度5％条件下进行培养，其中单个核细胞的接种浓度为5×106个/ml；
(d)待培养3天后，将单个核细胞移入空培养瓶中，再向空培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基继续在37℃、二氧化碳浓度5％条件下进行培养；
(e)待培养7天后，将单个核细胞移入培养袋中，再向培养袋中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基在37℃、二氧化碳浓度5％条件下进行培养，待培养15天后，收集单个核细胞。培养15天后的单个核细胞数量相比接种时扩增了162倍。采用流式细胞检测方法对步骤(e)中收集的单个核细胞中NK免疫细胞(CD3-CD56+)的含量进行检测，检测结果为97.43％。
实施例5免疫细胞与单抗联合给药实验
收集对数生长期HepG2细胞及培养的实施例4扩增的免疫细胞；
利用HepG2细胞构建小鼠皮下移植瘤模型，观察比较抗体治疗组、免疫细胞单药治疗组、抗体联合免疫细胞治疗组小鼠皮下肿瘤的体积，体内检测小鼠对抗体和免疫细胞治疗的敏感性。
本实施例的具体步骤为：消化、洗涤HepG2细胞株，调整其浓度为1*107个/ml备用，4-6周龄C57BL/6J免疫完全小鼠皮下接种100ul含有1*106个HepG2细胞的悬液。待皮下瘤平均体积达到约50-60mm3时随机分为四组，分别给予5-D4抗体治疗组(n＝10)、免疫细胞单药治疗组、5-D4抗体联合免疫细胞治疗组(n＝10)治疗；模型组空白对照组(n＝10)。其中5-D4抗体按照10mg/kg体重的剂量皮下注射方式给药,免疫细胞单药按照5*109个/kg体重的剂量皮下注射给药；5-D4抗体和免疫细胞联合治疗给药方式为按照5mg/kg体重的剂量每日皮下注射给药,免疫细胞单药按照1*109个/kg体重的剂量皮下注射给药；模型组空白对照不给药物。给药2周后，测量肿瘤体积。
结果如图3所示，5-D4抗体单药治疗以及免疫细胞单独治疗均能够抑制小鼠体内的肿瘤生长，而5-D4抗体联合免疫细胞治疗可显著的协同抑制肿瘤的生长增殖，2周后肿瘤体积只有40mm3左右，比没有治疗的模型组的450mm3要远远缩小。该结果显示本发明的5-D4单抗联合免疫细胞治疗显示出明显的优于两单药治疗组的抑制肿瘤效应，进而说明两药联合可有效的用于肝癌的治疗。
序列表
<110> 北京广未生物科技有限公司
<120> 一种免疫细胞在治疗癌症中应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 115
<212> PRT
<213> 人工序列(Artificial Sequence)
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Glu Asp Asn Lys Asp Ala Tyr Thr Tyr Tyr Ala Ser Trp Ala Ala Glu
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Arg Leu Thr Ile Ser Lys Pro Ser Ser Thr Lys Val Asp Leu Lys Ile
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Thr Ser Pro Thr Thr Glu Thr Thr Ala Thr Tyr Phe Cys Gly Arg Ser
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Ala Phe Lys Gly Gly Thr Asp Leu Trp Gly Pro Gly Thr Leu Val Thr
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Val Ser Ser
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ile Val Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
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Gly Thr Val Thr Ile Asn Cys Thr Glu Ser Gly Ala Ile Tyr Ser Gln
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Leu Ile Tyr Asp Ser Ser Thr Leu Asn Cys Gly Val Pro Ser Arg Phe
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Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Phe Gly Lys Ser Asn
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Claims (6)
1.一种特异性结合PD-L1的单克隆抗体，其名称为5-D4，其特征在于：重链可变区序列如SEQ ID NO：1所示，轻链可变区序列如SEQ ID NO：2所示。
2.一种药物组合物，其含有权利要求1所述的单克隆抗体以及提取的免疫细胞，所述的免疫细胞为采用下述方法制备得到：
(a)将刺激因子包被培养瓶；其中刺激因子由IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL、IL-15浓度2000IU/mL、GM-CSF浓度2000IU/mL、IL-4浓度750IU/mL、TNF-α浓度1000IU/mL以生理盐水配置组成；
(b)将血液采用密度梯度离心法分离单个核细胞；
(c)在刺激因子包被的培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基，再将单个核细胞接种于刺激因子包被的培养瓶中在37℃、二氧化碳浓度5％条件下进行培养，其中单个核细胞的接种浓度为5×106个/ml；
(d)待培养3天后，将单个核细胞移入空培养瓶中，再向空培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基继续在37℃、二氧化碳浓度5％条件下进行培养；
(e)待培养7天后，将单个核细胞移入培养袋中，再向培养袋中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基在37℃、二氧化碳浓度5％条件下进行培养，待培养15天后，收集单个核细胞。
3.权利要求1所述的单克隆抗体以及免疫细胞在制备用于治疗肝癌的药物组合物中的用途；其中所述免疫细胞为采用下述方法制备得到：
(a)将刺激因子包被培养瓶；其中刺激因子由IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL、IL-15浓度2000IU/mL、GM-CSF浓度2000IU/mL、IL-4浓度750IU/mL、TNF-α浓度1000IU/mL以生理盐水配置组成；
(b)将血液采用密度梯度离心法分离单个核细胞；
(c)在刺激因子包被的培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基，再将单个核细胞接种于刺激因子包被的培养瓶中在37℃、二氧化碳浓度5％条件下进行培养，其中单个核细胞的接种浓度为5×106个/ml；
(d)待培养3天后，将单个核细胞移入空培养瓶中，再向空培养瓶中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI1640培养基继续在37℃、二氧化碳浓度5％条件下进行培养；
(e)待培养7天后，将单个核细胞移入培养袋中，再向培养袋中添加含有亚油酸2mg/L，氨基酸20mg/L，维生素50mg/L、IL-2浓度5000IU/mL、IFN-γ浓度5000IU/mL的RPMI 1640培养基在37℃、二氧化碳浓度5％条件下进行培养，待培养15天后，收集单个核细胞。
4.如权利要求2所述的组合物，其特征在于该组合物可制成静脉内，皮下，腹膜内，肌肉内，口服，鼻，肺，眼，阴道或直肠给药。
5.如权利要求4所述的药物组合物，其中组合物的剂型为溶液，悬浮液，乳剂，脂质体制剂。
6.如权利要求4所述的组合物，其中在组合物中包括等渗剂，所述等渗剂选择糖，甘露醇，山梨醇或氯化钠。
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