CN112675202A - 一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞及其制备方法与应用 - Google Patents
一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞制备及其应用,将修饰有生物正交反应基团的非天然糖添加到免疫细胞如NK细胞的培养基中,以获得修饰有生物正交反应基团的免疫细胞;然后将一端为可与上述生物正交反应基团匹配产生连接反应的生物正交反应配对基团的靶向配基如纳米抗体在生理条件下通过生物正交反应修饰到免疫细胞表面,连接方式为转肽酶SrtA介导的化学酶法,靶向配基具有高特异性识别并结合到肿瘤细胞表面高表达受体的特性。本发明公开的靶向配基修饰的免疫细胞可特异性的靶向结合于癌细胞,进而修饰后的免疫细胞产生分泌大量的细胞因子使癌细胞表面发生穿膜孔洞或吞噬裂解癌细胞的效应,具有特异性杀伤癌细胞的作用。
Description
技术领域
本发明属于生物医药工程技术领域,具体涉及一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞及其制备方法与应用。
背景技术
癌症依然是威胁人类健康的头号杀手,是导致人类死亡的第二大因素。据全球医官网报道:预计2020年,全球新发癌症病例1890万例,死亡病例968万例,其中我国新增病例预计将达到385.4万例、死亡病例249.6万例,占据了癌症发病人数、死亡人数全球双榜首。
癌症的免疫治疗方法被誉为癌症治疗的第三次革命,是现今研究最为火热的治疗方式。过继细胞疗法是免疫治疗常见的手段,其中最具代表性的是将免疫细胞进行体外修饰后回输到患者体内进行治疗,这其中包括自体和异体细胞输送。免疫细胞包括自然杀伤性细胞(natural killer cell;NK细胞)、T细胞、巨噬细胞等,尤其是NK细胞是先天免疫系统中最具重要的免疫细胞,它们不依赖于特异性抗原刺激而具有天然的抗肿瘤和抗感染能力。NK细胞在循环中的寿命较短,不会像基于T细胞的免疫疗法中常见的危险细胞因子风暴。但是NK细胞要发挥其功能,需要与肿瘤细胞密切接触。NK细胞与靶细胞结合后,分泌释放穿孔素、颗粒酶等细胞毒性颗粒,并在不受主要组织相容性复合物限制下表达肿瘤坏死因子相关凋亡诱导配体,从而诱导肿瘤细胞表面发生穿孔裂解死亡。
尽管NK细胞、T细胞等免疫细胞有着较好的肿瘤杀伤活性,但是它们缺乏靶向结合于肿瘤细胞的能力,另外尤其是许多肿瘤细胞已进化出了免疫逃逸机制,这给肿瘤的治疗造成了麻烦。
为了解决这一问题,基于嵌合抗原受体(chimeric antigen receptor;CAR)技术,修饰免疫细胞用于肿瘤的治疗受到了广大研究者的关注,其中最具代表性的Kymriah和Yescarta两款CAR-T细胞已被美国FDA在2017年批准用于临床。鉴于此,广大研究工作者考虑将CAR技术用于NK细胞的工程化,尽管目前尚无CAR-NK细胞产品上市,但是通过检索clinicaltrials.gov网站,关于CAR-NK的相关临床研究至少有19项之多,都在不同程度上进入了临床1/2期,治疗的肿瘤涵盖了实体瘤、血液瘤以及淋巴瘤。近期关于CAR修饰的巨噬细胞CAR-M也有了研究报道。CAR技术修饰的靶向配基一般为单链抗体(single-chainvariable Fragment;scFv),但是这样的片段抗体亲和力低、稳定性差,使得scFv-CAR-NK细胞的应用受到了一定限制。另外在前期的报道中也有报道利用适配体、靶向肽对免疫细胞进行修饰,但是适配体作为单链寡核苷酸,在体内环境中易受到核酸酶的攻击,很容易降解而失去其活性;而靶向肽是一类只有几十个氨基酸残基的多肽,其结构简单,在体内多细胞环境中识别和结合癌细胞上受体的能力有限。
靶向配基,是能特异性识别并结合肿瘤细胞表面高表达受体的生物大分子,其中包括纳米抗体、适配体、scFv、糖配体、靶向肽等。纳米抗体(Nanobody,Nb)是天然存在于骆驼科及鲨鱼科血清中的一种抗体,分子量约为15kDa左右(分子量是scFv的一半),是现今已知的最小抗体,其基因由人VH家族的III型亚族进化而来,两者具有高度的同源性,但是纳米抗体的CDR3区比人抗体CDR3区要多4~9个氨基酸残基,使得纳米抗体可形成一个特殊的凸环结构,能识别更加隐蔽的抗原决定簇。另外纳米抗体中的FR2区有四个疏水性氨基酸突变为了亲水性氨基酸,大大增加了纳米抗体的水溶性,同时纳米抗体内部的二硫键使其抗热性以及耐酸碱性都大大增强。若将纳米抗体这类靶向配基对NK细胞乃至其他免疫细胞进行修饰,则会显著提高免疫细胞的靶向性,进而产生较佳的肿瘤治疗效果。
2020年年初,已有第一篇关于纳米抗体修饰NK的报道(doi:10.3390/cells9020321),作者借助CAR技术,将纳米抗体修饰到原代NK细胞(分离于人外周血),获得了很好的抗肿瘤活性Nb-CAR-NK细胞。但是CAR技术的实施,需要慢病毒等逆转录病毒的介导,将外源质粒导入NK细胞中,这样的遗传操作会产生病毒基因插入人体正常细胞基因组的风险,具有一定的安全隐患。另外原代NK细胞的分离、扩增耗时耗力。因此,开发一种便捷、安全、行之有效的抗肿瘤NK细胞意义重大。
近些年,发展了一种代谢糖工程与点击化学反应相结合的化学生物学新技术,利用该技术可对许多细胞进行特定的修饰,许多文献报道该技术的一般过程为先将叠氮糖经细胞摄取,使细胞携带叠氮基团,然后将偶连有炔基(如DBCO或端炔基)的适配体、化学药物或荧光基团通过无铜催化或铜催化使细胞连接上特定基团,在上述炔基的修饰过程中均通过纯化学合成,但是由于纯化学合成都需要用到大量有机试剂作为溶剂,使得炔基在修饰纳米抗体、单链抗体以及传统抗体等蛋白类物质时不适用上述方法,因此需要借助更加高效、更加温和的酶方法进行连接。
发明内容
发明目的:为了克服现有技术中存在的不足,本发明提供一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法及应用,尤其是一种基于纳米抗体细胞偶联技术的抗肿瘤NK细胞的制备方法及应用,其中,所述的靶向配基细胞偶联技术在不涉及遗传操作的前提下,通过利用细胞自身的代谢途径、再结合一系列的化学酶法将包括纳米抗体在内的靶向配基共价装载到细胞表面,我们称之为靶向配基细胞偶联技术(Ligand-targetedcell conjugate;LTCC),使用纳米抗体作靶向配基时,该技术则可称作纳米抗体细胞偶联技术(nanobody-cell conjugate;NBCC)。
技术方案:为实现上述目的,本发明采用的技术方案为:
一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,表面分别修饰有生物正交反应配对基团、生物正交反应基团的靶向配基、免疫细胞通过生物正交反应对应连接,形成表面偶联有具有特异性识别并结合癌细胞表面高表达受体的靶向配基的靶向免疫细胞,其中靶向配基为生物大分子。
进一步的,所述靶向配基为包括但不限于纳米抗体、适配体、单链抗体(scFv)、糖配体、靶向肽等在内的生物大分子。
进一步的,所述免疫细胞包括NK细胞、T细胞以及巨噬细胞,所述NK细胞包括但不限于NK92-MI细胞系、NK92细胞系及从人体中分离得到的原代NK细胞等。
进一步的,所述生物正交反应配对基团为与非天然糖上的生物正交反应基团成对应用;所述生物正交反应基团包括叠氮基团、酮/醛基、烯烃基、环炔基等,对应的所述生物正交反应配对基团即为含炔基化合物、羟胺、含四氮唑化合物、含四嗪化合物等,所述含炔基化合物包括适合无铜催化的二苯并环辛炔DBCO、二氟化环辛炔DIFO、双芳基环辛炔酮BERAC和适合铜催化的末端为炔基的化合物。本发明涉及的功能化纳米抗体在结构组成上表现为:纳米抗体-PEGn-生物正交反应配对基团,其中生物正交反应配对基团为能与非天然糖上修饰的生物正交反应基团发生生物正交反应(也称活细胞化学修饰)的功能基团:当生物正交反应基团为叠氮基团时,生物正交反应配对基团则对应为包括二苯并环辛炔(DBCO)、二氟化环辛炔(DIFO)、双芳基环辛炔酮(BERAC)、端基为炔基等在内的炔基化合物;当标记基团为酮/醛基时,生物正交反应配对基团则对应为包括肼、羟胺等等;当生物正交反应基团为烯烃基时,生物正交反应配对基团则应为四氮唑基;当生物正交反应基团为环炔基时,生物正交反应配对基团则应为四嗪基,以此类推。
进一步的,所述非天然糖包括但不限于N-叠氮乙酰基乙酰化甘露糖胺Ac4ManNAz、N-叠氮乙酰基乙酰化半乳糖胺Ac4GalNAz、N-叠氮乙酰基乙酰化葡萄糖胺Ac4GlcNAz、N-叠氮乙酰基乙酰化甘露糖胺ManNAz、N-叠氮乙酰基乙酰化唾液酸SiaNAz、N-丙酰基乙酰化甘露糖胺ManLev、N-丙酰基乙酰化甘露糖胺Ac4ManLev在内的修饰有生物正交反应基团的单糖。
本发明的另一目的,是提供一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法,将修饰有生物正交反应基团的非天然糖添加到免疫细胞培养基中,培养获得修饰有生物正交反应基团的免疫细胞;再将经过化学酶法合成的功能化靶向配基在生理条件下通过生物正交反应修饰到免疫细胞表面,形成靶向免疫细胞;其中,所述功能化靶向配基为一端携带有生物正交反应配对基团的靶向配基。
进一步的,所述功能化靶向配基的合成方法为:先通过化学有机合成将生物正交反应配对基团的一端连接上聚乙二醇(PEG)链接子以及具有游离氨基的三甘氨酸肽,使其满足转肽酶Sortase A连接的条件,其中PEG包括但不限于四个聚合度的PEG4;原始的靶向配基可通过生物表达或化学合成,使其一端含有LPXTG转肽酶Sortase A识别位点,X为任意氨基酸,在转肽酶Sortase A作用下,将连接有具有游离氨基的三甘氨酸肽、PEG4的生物正交反应配对基团与一端携带有氨基酸序列为LPXTG的原始的靶向配基进行连接,16℃反应5~9h,然后借助镍离子磁珠快速进行纯化,合成所述功能化靶向配基。
更进一步的,所述生物正交反应配对基团的一端与PEG链接子、具有游离氨基的三甘氨酸肽通过酰胺缩合反应相连。
进一步的,所述生物正交反应条件为:修饰有生物正交反应基团的免疫细胞与功能化靶向配基混合,在包括但不限于PBS或生理盐水体系中进行生物正交反应。
本发明的第三个目的,是提供基于靶向配基细胞偶联技术的抗肿瘤免疫细胞应用于制备癌症治疗药物,所述癌症治疗药物所治疗的癌症需含有所述靶向配基特异性识别的靶点,包括实体瘤、血液瘤以及淋巴瘤。
有益效果:相比现有技术,本发明具备以下优点:
1、本发明提供的靶向配基细胞偶联技术尤其是纳米抗体细胞偶联技术为一种以发酵工程、代谢糖工程、化学酶法合成等技术为基础的新型技术,该技术不涉及到任何遗传操作手段和病毒转染技术的使用,具有很好的安全性;同时该技术可将纳米抗体这类可作为靶向配基的蛋白类生物活性分子快速便捷的与免疫细胞进行连接,可综合靶向配基和免疫细胞各自的优势,起到“1+1>2”的效果,显著提高免疫细胞靶向结合杀伤肿瘤的能力。
2、本发明提供的靶向配基细胞偶联技术尤其是纳米抗体细胞偶联技术具有很好的普适性,只需更换靶向配基的种类如纳米抗体,就可以使得免疫细胞完成对几乎所有肿瘤细胞的杀伤。另外,由于所有细胞的糖代谢通路高度保守,因此,该技术可应用于几乎所有细胞的靶向配基如纳米抗体修饰。
3、本发明所用的免疫细胞包括各种NK细胞、T细胞以及巨噬细胞等,其中,NK细胞一般为NK92-MI等易于培养的细胞系,相比于现今大多数研究用的从人体外周血中的NK细胞,NK92-MI等细胞系容易大规模培养,更易于产业化,具有良好的应用前景。
附图说明
图1为本发明的原理过程示意图。
图2为最佳Ac4ManNAz浓度的筛选;A为流式细胞术位移图,B为荧光定量分析图。
图3为Ac4ManNAz对NK92-MI细胞活力的影响。
图4为化学酶法合成功能化的纳米抗体靶向基团;A为化学合成DBCO-PEG4-GGG全过程,B为sortaseA酶法将DBCO-PEG4-GGG与纳米抗体7D12连接合成DBCO-PEG4-7D12,C为连接产物DBCO-PEG4-7D12的质谱鉴定(理论分子量为16391.5;质谱观测值为16390.4)。
图5为纳米抗体7D12装载细胞后的检测;A为激光共聚焦观察,B为最适装载DBCO-PEG4-7D12浓度的筛选。
图6为7D12-NK92MI体外靶向性验证;A为EGFR高表达靶细胞与NK92-MI的结合,B为EGFR高表达靶细胞与7D12-NK92MI的结合,C为上述两组结合率的统计。
图7为纳米抗体修饰NK92-MI细胞后的体外抗肿瘤活性;A为不同效靶比时的抗肿瘤活性比较,B为纳米抗体在杀伤活性中重要性的验证。
图8为纳米抗体修饰NK92-MI细胞广泛的抗瘤谱分析。
图9为纳米抗体修饰NK92-MI细胞后的体内抗肿瘤活性;A为动物实验过程示意图,B为治疗前后肿瘤实物图,C为治疗过程中肿瘤体积的变化,D为治疗前后瘤重比较,E为治疗前后的肿瘤抑制率,F为治疗过程中小鼠体重变化。
具体实施方式
为了使本发明技术手段、达到的目的与效果易于了解,通过下面的实例进一步阐释本发明,但本发明不受实例的限制。
实施例
根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
本发明意在公开一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法及应用,实施例主要选取7D12纳米抗体作为靶向配基、NK细胞作为免疫细胞作为示例,充分解释说明利用NBCC进行抗肿瘤NK细胞的制备及其应用。所利用的NK细胞一般为被称为“现成试剂”(off-the-shelf reagent)的NK92-MI或NK92细胞系,此方式细胞获取便捷、易大规模扩大培养,便于后续的转化生产。以下实施例不仅限于这两种NK细胞,还包括其他NK细胞、T细胞以及巨噬细胞等免疫细胞;同理,靶向配基也不仅局限于7D12纳米抗体,也适用于其他纳米抗体,也适用于其他诸如适配体、scFv、糖配体、靶向肽等具有靶向及特异性识别功能性质的生物大分子。
实施例1:功能化纳米抗体的制备
功能化纳米抗体的制备需要三个过程,即纳米抗体发酵与纯化、化学合成带三甘氨酸肽的生物正交功能基团、Sortase A酶法进行连接。
纳米抗体发酵纯化:
取100μL前期构建好的能生产纳米抗体7D12(靶向于EGFR)的大肠杆菌工程菌的甘油冻存菌接种于25mL含100μg/mL卡那霉素的LB培养基于37℃、200rpm进行过夜培养活化,第二天按2%接种量转接到含100μg/mL卡那霉素的TB培养基中于37℃、200rpm进行培养,TB的配方为:蛋白胨12g/L,酵母粉24g/L,甘油5g/L,K2HPO4·3H2O 16.4g/L,KH2PO4 2.31g/L,待OD600为0.6~0.8时,加入终浓度为0.2mM的IPTG进行诱导表达,诱导温度为16℃,诱导时间为24h。发酵结束后,8000rpm离心收集菌体,加入超声破碎裂解缓冲液进行超声破碎(该缓冲液选择根据后续所用镍柱说明书进行配置),之后在12000rpm、4℃离心15min,收集上清,反复离心3次,随后将上清用镍柱对纳米抗体7D12进行纯化,最后再用G25凝胶柱进行脱盐处理除去咪唑盐。
化学合成:
1)叠氮糖合成:取1.22g甘露糖胺盐酸盐(Mannosamine hydrochloride)溶于50mLMeOH,随后加入1.06mL 30%NaOMe/MeOH溶液室温搅拌1h,之后加入0.815mL TEA和2.9g氯乙酸酐,室温搅拌反应6h,接下来通过硅胶柱用CH2Cl2:MeOH从6:1到5:1进行梯度洗脱获得N-氯乙酰基甘露糖胺,将溶剂挥发后,加入16mL DMF和3.68g NaN3,80℃反应4h,冷却到室温后,将溶剂挥发,再加入30mL吡啶和15mL醋酸酐,室温搅拌过夜进行反应,随后在反应液中加入200mL乙酸乙酯混匀,有机层中加入100mL、1M的盐酸溶液进行清洗,共洗三次进行分液,然后加入100mL饱和NaHCO3进行清洗,共洗三次进行分液,最后加入100mL饱和NaCl进行清洗,共洗两次进行分液,之后加入无水Na2SO4进行干燥,再通过硅胶柱用正己烷:乙酸乙酯从1:1到1:2进行梯度系统获得产品Ac4ManNAz。
除了上述制备得到的N-叠氮乙酰基甘露糖胺-四酰基化(Ac4ManNAz),本发明可适用的非天然糖还包括:N-叠氮乙酰基乙酰化半乳糖胺(Ac4GalNAz)、N-叠氮乙酰基乙酰化葡萄糖胺(Ac4GlcNAz)、N-叠氮乙酰基乙酰化甘露糖胺(ManNAz)、N-叠氮乙酰基乙酰化唾液酸(SiaNAz)、N-丙酰基乙酰化甘露糖胺(ManLev)、N-丙酰基乙酰化甘露糖胺(Ac4ManLev)等,分别以对应糖的母体作为原料,同理可通过上述化学合成反应制得。
本发明的实施例中,如图2所示,结合流式细胞术位移效果和荧光定量分析结果,可以看出,添加非天然糖的最适浓度为50~100μM。
2)DBCO-PEG4-GGG合成:整个过程如图4A所示,先用一端为Cbz保护的乙二胺与一端为Boc保护的三甘氨酸肽按4:5~4:7的当量比进行酰胺缩合反应,在脱去Cbz后与原料DBCO-PEG4-NHS按2:1~1:1的当量比进行反应,最后再脱去Boc基团后获得产物DBCO-PEG4-GGG。
具体方法如下:
取310mg Cbz-EDA-NH2与578mg Boc-GGG-COOH(Boc:t-Butyloxy carbonyl,叔丁氧羰基)、411mg HOBt、0.6mL TEA、573mg EDC·HCl于10mL DMF在0℃混匀,室温搅拌反应4h,之后用DCM和饱和NaCl进行抽提分液,再用无水Na2SO4进行干燥,随后用DCM:MeOH=50:1作为层析液通过硅胶层析进行纯化;然后加入20mL MeOH、120mg Pd/C在氢气的氛围下室温搅拌12h进行Cbz的脱去,纯化旋干以后获得Boc-GGG-EDA-NH2,取70mg Boc-GGG-EDA-NH2溶于10mL DMF,之后加入140mg DBCO-PEG4-NHS,室温搅拌反应30min,反应完成后用DCM:MeOH=10:1作为层析液通过硅胶层析进行纯化;之后再用20%TFA/DCM在0℃搅拌反应1.5h以脱去Boc基团,再加入适量甲苯进行减压蒸馏获得DBCO-PEG4-GGG-NH2。
Sortase A酶法连接:整个过程如图4B所示。纳米抗体是经大肠杆菌表达的,羧基端携带有LPXTG(X为任意氨基酸,如E/A/V等)转肽酶Sortase A识别位点和组氨酸(Histag)标签。在进行Sortase A介导的酶连反应时,靶向配基即纳米抗体投料为20~25μM、含生物正交基团小分子底物为250~500μM、Sortase A酶为3~5μM,在pH为7.4~7.5且含有CaCl2的Tris-HCl/NaCl缓冲液中反应,反应条件为16℃、5~9h。之后再用镍离子螯合磁珠或镍离子螯合琼脂糖柱进行纯化,最后用G25凝胶柱按照除盐的步骤进行生物正交基团等小分子的去除。
反应体系为:20μM 7D12、500μM DBCO-PEG4-GGG-NH2、5μM Srt A酶、1×Srt A反应缓冲液(配方:50mM Tris、150mM NaCl、5mM CaCl2、pH 7.4~7.5),按照上述体系进行加样,混匀后于16℃反应5h,之后用镍离子螯合磁珠抓取未反应完的7D12、Srt A以及切割下的His标签,取出上清用G25凝胶柱出去过量的DBCO-PEG4-GGG-NH2,即可获得DBCO-PEG4-7D12。
实施例2:叠氮糖对NK细胞的活性的影响
本实施例验证了添加的非天然糖对细胞活力的影响,具体方法为:非天然糖经细胞摄取后,培养一定的时间,然后用CCK8试剂盒(碧云天)进行测定OD450,分析加糖组和未加糖组细胞活性的差异。
具体方法如下:将1×105个NK细胞铺于96孔板中,对照组添加0.1%DMSO,实验组添加终浓度为50μM Ac4ManNAz(DMSO的量相当于0.1%),之后37℃细胞培养箱进行培养,然后分别在24h、48h加入CCK8试剂,孵育2h后通过酶标仪检测OD450,结果如图3显示,非天然糖添加后对细胞并无毒性,反而有微弱的促生长作用。
实施例3:NK细胞等免疫细胞的纳米抗体修饰
本实施例为叠氮标记NK细胞的获得及7D12的修饰,原理过程如图1所示。非天然糖经细胞摄取后,利用糖酵解、唾液酸代谢途径等代谢糖工程过程,将糖上修饰的生物正交反应基团装载到细胞表面,之后收集细胞,在类似生理条件下,向细胞中加入功能化纳米抗体(1×104个NK细胞用8~10μg功能化纳米抗体处理),37℃反应1~1.5h。通过免疫荧光验证细胞是否成功修饰上了纳米抗体。
具体方法如下:
将上述化学合成的Ac4ManNAz溶于DMSO中配置成50mM的母液,过滤除菌后按照1‰的比例加入NK细胞专用培养基中,使培养基含非天然糖量为50μM,用该培养基培养NK92-MI细胞48h,之后600rpm离心5min收集细胞,用PBS清洗细胞3次得到修饰了生物正交反应基团N3的NK细胞(N3-NK92MI)。
然后加入8.2μg DBCO-PEG4-7D12/10000个细胞,在PBS作为缓冲体系,37℃反应1.5h,之后用PBS再清洗3次即为7D12功能化的NK细胞(7D12-NK92MI)。
所得到的功能化的NK细胞7D12-NK92MI通过图5免疫荧光显示,7D12修饰成功。
实施例4:功能化纳米抗体的免疫细胞的体外靶向性检测
本实施例是对实施例2得到的7D12-NK92MI功能化纳米抗体的免疫细胞进行体外靶向性及抗肿瘤活性分析,具体方法为:将NK细胞与肿瘤靶细胞分别用两种颜色的脂溶性染料进行标记,在肿瘤靶细胞贴壁后,按照效靶比为1:1将未修饰纳米抗体的NK细胞和修饰纳米抗体的NK细胞分别与肿瘤靶细胞共培养,通过荧光成像,观测纳米抗体修饰的免疫细胞的肿瘤细胞黏附能力。
具体过程如下:
先将NK92MI/7D12-NK92MI用绿色荧光的DiO进行标记,过夜贴壁的高表达EGFR的LOVO细胞用红色荧光的DiR进行标记,之后按照NK细胞:LOVO细胞=1:1进行混合共培养2h,PBS清洗-固定-PBS清洗后再用激光共聚焦进行成像。
结果显示,7D12-NK92MI与LOVO的结合率要显著高于NK92MI与LOVO的结合率(图6),达到了48%左右。表明7D12能显著增强NK92MI结合肿瘤的能力,经功能化纳米抗体修饰的免疫细胞有更好的肿瘤细胞黏附能力。
实施例5:功能化纳米抗体的免疫细胞的体外抗肿瘤活性检测
本实施例是对实施例2得到的7D12-NK92MI功能化纳米抗体的免疫细胞进行体外抗肿瘤活性分析,具体方法为:将未修饰纳米抗体的NK细胞和修饰纳米抗体的NK细胞分别按照不同的效靶比与肿瘤靶细胞共培养,收集培养上清通过LDH测定NK细胞对肿瘤细胞的杀伤活性。
具体过程如下:将NK92MI或7D12-NK92MI分别按照1:5、1:1、5:1的效靶比与LOVO细胞共培养,之后用CytoTox 96Non-Radioactive Cytotoxicity Assay测定NK细胞的细胞杀伤能力,如图7A所示,随着效靶比的升高,肿瘤杀伤活性显著增强,且修饰有7D12的NK92MI细胞杀伤活性要显著高于未修饰7D12的NK92MI细胞。
之后利用高表达EGFR的LOVO细胞和低表达EGFR的SW620细胞为靶细胞,分析其杀伤活性的提高是否由纳米抗体7D12所致,如图7B所示,修饰有7D12的NK92MI和未修饰7D12的NK92MI对SW620的杀伤没有显著性差异,而7D12-NK92MI对LOVO的杀伤活性显著高于NK92MI。
另外图7B显示的通过竞争性分析(free 7D12),先用游离纳米抗体7D12处理LOVO,再加入7D12-NK92MI进行共培养,其杀伤活性显著降低。
另取几种高表达EGFR的肿瘤细胞,如MDA-MB-468(乳腺癌)、A431(表皮鳞癌)、A549(肺癌)作为靶细胞,分析7D12-NK92MI的抗癌谱,结果如图8所示,7D12工程化后的NK92MI能显著的杀伤上述几种癌细胞。
本实施例的结果显示,修饰纳米抗体的NK细胞对各种肿瘤细胞的杀伤活性要显著高于未修饰纳米抗体的NK细胞,同时具有广泛的抗癌谱。
实施例6:功能化纳米抗体的免疫细胞内抗肿瘤活性
本实施例是检测实施例2得到的7D12-NK92MI功能化纳米抗体的免疫细胞的体内抗肿瘤活性,具体方法如下:
选用NK背景缺失的NOD-SCID小鼠作为模型,给其进行皮下殖瘤,每只小鼠腋下注射300万LOVO细胞,待肿瘤体积为15mm3时开始进行治疗处理,用PBS作为空白对照、NK92MI作为阴性对照,每只小鼠尾静脉注射300万治疗细胞,通过尾静脉分别注射未修饰纳米抗体的NK细胞和修饰纳米抗体的NK细胞,注射量为殖瘤时肿瘤细胞等量或更高,共注射5次,每2天注射一次。
定期进行肿瘤大小及小鼠体重的测定(如图9C、F),连续治疗两周后,实验组的肿瘤大小为均值为410.5mm3,而对照组及PBS组肿瘤大小均值分别为647.5mm3和665.7mm3;在整个治疗过程中,各组小鼠体重均无明显变化;随后对小鼠进行安乐死然后取其肿瘤进行瘤重的测定(如图9B、D),通过计算,实验组肿瘤重量均值为232.8mg,而对照组及PBS组肿瘤重量均值分别为338.7mg和329.1mg;另外肿瘤生长抑制率的结果显示,未修饰纳米抗体7D12的NK92MI细胞的抑制率为7.4%,而修饰纳米抗体7D12的NK92MI细胞的抑制率达到了44%之多,暗示着工程化纳米抗体7D12的NK92MI细胞的治疗效果要更好(图9E)。因此修饰纳米抗体的NK细胞治疗组有更好的肿瘤抑制率,而且对小鼠体重无任何影响,暗示着修饰纳米抗体的NK细胞肿瘤治疗效果较佳,同时有很好的生物安全性。
本发明公开了一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞制备及其应用,属于生物医药工程领域。将修饰有生物正交反应基团(如:叠氮基团、酮基)的非天然糖添加到免疫细胞如NK细胞的培养基中,以获得修饰有生物正交反应基团的免疫细胞;然后将一端为可与上述生物正交反应基团匹配产生连接反应的生物正交反应配对基团(如:二苯并环辛炔、羟胺)的靶向配基如纳米抗体在生理条件下通过生物正交反应修饰到免疫细胞表面,其中所述靶向配基与生物正交反应配对基团的连接方式为本发明公开的转肽酶SrtA介导的化学酶法,所述靶向配基具有高特异性识别并结合到肿瘤细胞表面高表达受体的特性。本发明公开的靶向配基修饰的免疫细胞可特异性的靶向结合于癌细胞,进而修饰后的免疫细胞产生分泌大量的细胞因子使癌细胞表面发生穿膜孔洞或吞噬裂解癌细胞的效应,具有特异性杀伤癌细胞的作用。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,其特征在于:表面修饰有生物正交反应基团的免疫细胞与表面修饰有生物正交反应配对基团的靶向配基通过生物正交反应对应连接,形成表面偶联有特异性识别并结合癌细胞表面高表达受体的靶向配基的靶向免疫细胞。
2.根据权利要求1所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,其特征在于:所述靶向配基为包括纳米抗体、适配体、单链抗体、糖配体、靶向肽在内的生物大分子。
3.根据权利要求1所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,其特征在于:所述免疫细胞包括NK细胞、T细胞、巨噬细胞,所述NK细胞包括但不限于NK92-MI细胞系、NK92细胞系及从人体中分离得到的原代NK细胞。
4.根据权利要求1所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,其特征在于:所述生物正交反应基团包括但不限于叠氮基团、酮/醛基、烯烃基、环炔基;所述生物正交反应配对基团为与非天然糖上的生物正交反应基团成对应用,对应的所述生物正交反应配对基团即为含炔基化合物、羟胺、含四氮唑化合物、含四嗪化合物,所述含炔基化合物包括但不限于用于无铜催化的二苯并环辛炔DBCO、二氟化环辛炔DIFO、双芳基环辛炔酮BERAC和用于铜催化的末端为炔基的化合物。
5.根据权利要求4所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞,其特征在于:所述非天然糖包括但不限于N-叠氮乙酰基乙酰化甘露糖胺Ac4ManNAz、N-叠氮乙酰基乙酰化半乳糖胺Ac4GalNAz、N-叠氮乙酰基乙酰化葡萄糖胺Ac4GlcNAz、N-叠氮乙酰基乙酰化甘露糖胺ManNAz、N-叠氮乙酰基乙酰化唾液酸SiaNAz、N-丙酰基乙酰化甘露糖胺ManLev、N-丙酰基乙酰化甘露糖胺Ac4ManLev。
6.一种基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法,其特征在于:将修饰有生物正交反应基团的非天然糖添加到免疫细胞培养基中,培养获得修饰有生物正交反应基团的免疫细胞;靶向配基的一端修饰生物正交反应配对基团形成功能化靶向配基,再通过生物正交反应修饰到免疫细胞表面,形成靶向免疫细胞。
7.根据权利要求6所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法,其特征在于:所述靶向配基的一端修饰生物正交反应配对基团形成功能化靶向配基是通过化学酶法合成,其过程为:先通过化学合成将生物正交反应配对基团的一端连接上聚乙二醇PEG链接子以及具有游离氨基的三甘氨酸肽,使其满足转肽酶Sortase A连接的条件,其中PEG包括但不限于四个聚合度的PEG4;原始的靶向配基通过生物表达或化学合成,使其一端含有LPXTG转肽酶Sortase A识别位点,X为任意氨基酸,在转肽酶Sortase A作用下,将连接有具有游离氨基的三甘氨酸肽、PEG链接子的生物正交反应配对基团与一端携带有氨基酸序列为LPXTG的原始的靶向配基进行连接,16℃反应5~9h,然后借助镍离子磁珠进行纯化,合成修饰有生物正交反应配对基团的靶向配基,记作功能化靶向配基。
8.根据权利要求7所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法,其特征在于:所述生物正交反应配对基团的一端与PEG链接子、具有游离氨基的三甘氨酸肽通过酰胺缩合反应相连。
9.根据权利要求6所述的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞的制备方法,其特征在于:所述生物正交反应条件为:修饰有生物正交反应基团的免疫细胞与功能化靶向配基混合,在包括但不限于PBS或生理盐水体系中进行生物正交反应。
10.根据权利要求1-5所述的或根据权利要求6-9任一方法制备的基于靶向配基细胞偶联技术的抗肿瘤免疫细胞应用于制备癌症治疗药物,其特征在于:所述癌症治疗药物所治疗的癌症含有所述靶向配基特异性识别的靶点,包括实体瘤、血液瘤以及淋巴瘤。
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