CN115433713B - 一种自体肿瘤引流淋巴结淋巴细胞的制备方法及其应用 - Google Patents
一种自体肿瘤引流淋巴结淋巴细胞的制备方法及其应用 Download PDFInfo
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Abstract
本发明提供一种自体肿瘤引流淋巴结淋巴细胞的制备方法及其应用,制备方法包括步骤:A1、从患者中获取引流淋巴结组织,消化、分离,制备引流淋巴结单个核细胞;A2、对步骤A1获得的细胞进行培养,并激活、扩增其中包含的T细胞,获得自体肿瘤引流淋巴结淋巴细胞。通过本申请的制备方法,能够获得自体肿瘤引流淋巴结淋巴细胞(LNL);LNL相较于肿瘤浸润淋巴细胞,其作为过继回输细胞而言,便于体外的培养扩增,提高培养成功率,当过继转移至体内时,也能提供相应的杀伤效果,并具有优秀的体内扩增潜力以及体内作用的持久性、持续活性,克服现有技术中采用肿瘤浸润T细胞作为来源而可能导致的终末分化、功能失调、持久性差等缺陷。
Description
技术领域
本发明涉及细胞培养技术领域,更具体地,涉及一种自体肿瘤引流淋巴结淋巴细胞的制备方法及其应用。
背景技术
实体瘤与具有谱系标志物的血液系统恶性肿瘤不同,实体瘤具有高度异质性,因此,很难找到实体瘤中针对所有肿瘤细胞的理想靶点。过继性细胞免疫治疗是目前癌症治疗领域的热点研究之一,过往研究显示其在血液系统恶性肿瘤、实体瘤中均有应用以获得显著疗效的潜力,其中嵌合抗原受体T、TIL等疗法在肿瘤治疗方面也取得了多样、显著的进展。
对于实体瘤而言,嵌合抗原受体T疗法靶向单一肿瘤抗原通常会产生抗原丢失或导致更具恶性的肿瘤克隆复发,该类疗法还存在部分不足。TIL疗法与嵌合抗原受体T疗法同属于细胞免疫疗法,但TIL疗法在治疗实体瘤方面则具有一些显著的优势。过继细胞治疗(Adoptive cell therapy,ACT)最先由SteveA.Rosenberg教授和美国国立卫生院(NIH)的同事提出,首次从多个小鼠肿瘤模型中分离出TIL,是一种利用患者自身免疫系统治疗肿瘤的过继细胞疗法。该疗法通过从患者肿瘤中收集浸润的淋巴细胞,在体外进行培养和扩增,最后回输治疗患者。其作用原理是利用肿瘤浸润的淋巴细胞靶向多个肿瘤特异性抗原,且为MHC限制性,杀伤能力强,副作用小。TIL疗法的有效性已在许多实体瘤中得到验证,包括乳腺癌、卵巢癌、黑色素瘤肾细胞癌、非小细胞肺癌、前列腺癌、膀胱癌、头颈癌、肉瘤和胰腺癌。相较于其他过继细胞疗法(如嵌合抗原受体T和TCR-T疗法),TIL由具有多样化T细胞受体(TCR)克隆的T细胞组成,能够识别一系列肿瘤抗原,优越的肿瘤归巢能力和低脱靶毒性赋予其在治疗实体瘤方面的独特优势。
但是,TIL的获得具有挑战性,体外培养自体肿瘤的成功率较低,且经体外长期培养及大量扩增后,T细胞体内持久性较差、会产生T细胞终末分化、功能障碍或衰竭等。目前TIL获取方法皆为从患者手术切除获得的肿瘤剪碎处理或经酶消化,从肿瘤组织中培养获得细胞毒性T细胞,并通过连续传代扩增细胞直至达到回输数量级1010~1011。但体内TIL的抗肿瘤活性高度依赖于过继转移细胞的扩增、持久性和持续活性,虽然以效应型细胞为主导的T细胞产物具有更强的肿瘤细胞毒性,但这些细胞也倾向于终末分化和功能失调。虽有研究者优化了上述培养方案,用T细胞激活1、2和3信号(OKT3抗体、激动性CD137/4-1BB和IL-2)组合从肿瘤组织中培养TIL,提高了培养的成功率,但皆为外周组织水平获取TIL,仍然无法避免回输后终末分化、功能失调的倾向,过继转移细胞的扩增、持久性和持续活性问题仍无法解决。
因此,现有技术亟需一种可作为ACT中T细胞来源的过继转移细胞,以克服现有技术中回输后倾向终末分化、功能失调,以提高过继转移后扩增、持久性、持续活性效果。
发明内容
本发明旨在克服上述现有技术的至少一种不足,提供一种自体肿瘤引流淋巴结淋巴细胞的制备方法及其应用,通过该制备方法能获得可应用为过继转移细胞的自体肿瘤引流淋巴结淋巴细胞,以提高过继转移细胞的培养成功率以及回输后的疗效和作用持久性。
本发明的一个目的在于提供一种自体肿瘤引流淋巴结淋巴细胞的制备方法,包括步骤:
A1、从患者中获取引流淋巴结组织,消化、分离,制备引流淋巴结单个核细胞;
A2、对步骤A1获得的细胞进行培养,并激活、扩增其中包含的T细胞,获得自体肿瘤引流淋巴结淋巴细胞。
过继的T细胞治疗效果与T细胞的分化程度息息相关,对于分化程度低的记忆T细胞,其体内存续时间长,有更强的抑瘤效果。而接触过抗原不同程度刺激的CD8+T细胞在体外细胞扩增期间和体内过继转移治疗后直接影响体内细胞存续、分化和治疗效果。与效应记忆T细胞(TEM)和效应T细胞(TEFF)相比,T记忆干细胞(TSCM)和中枢记忆T细胞(TCM)具有更好的持久性和抗肿瘤免疫力,但如果经常受抗原和炎症信号刺激,TSCM将表现为多种抑制受体,如PD-1、TIM-3和LAG-3的表达,以及从氧化磷酸化到糖酵解的代谢变化,这些变化会导致T细胞耗竭。而具有和维持较少分化表型的T细胞的过继转移,对抗肿瘤疗效和患者预后至关重要。
淋巴结是次级淋巴器官,次级淋巴器官又是成熟淋巴细胞(T淋巴细胞、B淋巴细胞)定居的场所,在胸腺发育成熟后经血循环至淋巴结,健康人的淋巴结中T细胞占淋巴细胞的75%。引流淋巴结是原发肿瘤发生淋巴结转移必然会经过的第一批淋巴结,淋巴结中的T细胞可以分为未接触过抗原的初始T细胞(TN)、经抗原不同程度刺激的T记忆干细胞(TSCM)、中枢记忆T细胞(TCM)、效应记忆T细胞(TEM)和处于耗竭的T细胞(TEX)。已知PD-1既是激活的标志又是耗竭的标志,但多种抑制性受体的共表达才是耗竭的主要特征,故可将耗竭的T细胞标志物设置为CD3+PD-1+Tim3+LAG3+。慢性感染或长期肿瘤抗原刺激下,T淋巴细胞会扩增并分化为清除病原体的效应细胞和记忆细胞。记忆细胞可以存活较长时间,以保障机体在受到相同抗原再次攻击时快速反应起保护作用,且TSCM细胞已显示出其强大的肿瘤治疗潜力。而肿瘤浸润的T细胞则大多数为趋于终末分化的效应T细胞,增殖能力弱,倾向耗竭,培养成功率低,在此基础上,本发明人提出从引流淋巴结中获取淋巴细胞,且作为ACT中T细胞来源可能更优于肿瘤组织T细胞。为此,本发明人提出了制备自体肿瘤引流淋巴结淋巴细胞的方法,并获得相应的自体肿瘤引流淋巴结淋巴细胞(LNL细胞)。该制备方法简单,易于操作,且制备获得的LNL细胞易于培养,能提高细胞培养成功率,作为过继转移细胞(包括TIL)在过继回输后能提供有效的肿瘤杀伤效果,相比于其他来源过继转移细胞,具有显著疗效和作用持久性。该制备方法能从淋巴器官淋巴结中获取较低分化水平且接触过肿瘤抗原的T细胞,以提高细胞制品中记忆T细胞的比例,延长体内治疗的存续时间,减少T细胞耗竭。且本申请提供的制备方法也作为一种培养方法而存在,能提高过继转移细胞LNL细胞的培养成功率,克服现有技术中过继转移细胞的培养难度大等缺陷。
进一步地,自体肿瘤引流淋巴结淋巴细胞为DC诱导的自体肿瘤引流淋巴结淋巴细胞,还包括步骤:
A3、从同一患者外周血获得DC细胞,并使用肿瘤裂解物脉冲DC,使DC呈递肿瘤抗原,然后与步骤A2中自体肿瘤引流淋巴结淋巴细胞共培养,获得DC诱导的自体肿瘤引流淋巴结淋巴细胞。
本申请除了直接获得LNL细胞并利用其特性克服现有技术缺陷外,还能利用IL-2、DC递呈的肿瘤裂解物进一步刺激淋巴细胞特异性增殖,增大扩增倍数,并增加其中有利于过继回输后持久性、疗效的T细胞亚型比例。
进一步地,步骤A1具体包括:
A11、活检获取引流淋巴结,去除多余脂肪后洗涤,剪碎,再加入至含胶原酶I、胶原酶III和DNA酶的人淋巴细胞无血清培养基中孵育、消化;
A12、组织消化后用生理盐水稀释,使用细胞过滤器过滤细胞收集得到单细胞悬液;
A13、获得单细胞悬液后,用人淋巴细胞分离液Ficoll密度梯度离心,吸取离心后以单个核细胞为主的白膜层,分离制备单个核细胞悬液,洗涤,种于孔板,获得引流淋巴结单个核细胞。
进一步地,步骤A2具体包括:
A21、将步骤A1获得的引流淋巴结单个核细胞加至初始培养基中培养,所述初始培养基为人淋巴细胞无血清培养基、重组人IFN-γ及人AB血清或自体血浆或血清替代物混合形成;
A22、经初始培养后,使用CD3单抗、重组人IL-2刺激,使T细胞激活并扩增;
A23、待T细胞完全被活化后,定时更换一半的新鲜持续扩增培养基维持T细胞的增殖,获得自体肿瘤引流淋巴结淋巴细胞,所述持续扩增培养基为人淋巴细胞无血清培养基、重组人IL-2及人AB血清或自体血浆或血清替代物混合形成。
进一步地,步骤A3具体包括:
A31、静脉抽取同一患者的外周血,离心去上层血浆,同等体积生理盐水稀释后,用人淋巴细胞分离液Ficoll密度梯度离心,获得外周血单个核细胞;
A32、获得外周血单个核细胞后,贴壁培养,再更换DC培养基培养以获得DC细胞,所述DC培养基为人淋巴细胞无血清培养基、GM-CSF/IL-4混合形成;
A33、使用肿瘤裂解物脉冲至DC细胞,DC经成熟培养基培养成熟后与自体肿瘤引流淋巴结淋巴细胞共培养,获得DC诱导的自体肿瘤引流淋巴结淋巴细胞,所述DC成熟培养基为人淋巴细胞无血清培养基、TNFα/IL-1β/IL-6混合形成。
本发明的又一目的在于提供上述制备方法所得自体肿瘤引流淋巴结淋巴细胞在制备预防、治疗肿瘤药物中的用途。
进一步地,预防、治疗肿瘤药物包括过继性细胞免疫治疗药物。
本发明的再一目的在于提供上述制备方法所得自体肿瘤引流淋巴结淋巴细胞在制备ACT疗法细胞中的用途。ACT疗法细胞包括TIL细胞。
本发明的再一目的在于提供一种细胞制剂,含有上述自体肿瘤引流淋巴结淋巴细胞,及药学上可接受的载体或溶剂。
进一步地,溶剂为PBS溶液。
本发明的再一目的在于提供一种肿瘤治疗用过继性细胞,来源于自体肿瘤引流淋巴结淋巴细胞。在本发明的一个实施例中,通过过继回输LNL细胞,能够实现其杀伤肿瘤的效果,抑制肿瘤生长,其具有过继回输以治疗肿瘤的功能。基于该来源细胞,有望克服现有技术传统TIL细胞的不足。
与现有技术相比,本发明的有益效果为:通过本申请提供的制备方法,能够获得自体肿瘤引流淋巴结淋巴细胞(LNL),LNL相较于肿瘤浸润淋巴细胞而言,其作为过继回输细胞而言,便于实现体外的培养扩增,提高培养成功率,当过继转移至体内时,也能显著增强体内扩增效果以及体内作用的持久性、持续活性,克服现有技术中采用肿瘤浸润T细胞作为来源而可能导致的终末分化、功能失调、持久性差等缺陷。本申请制备方法操作简单,实验器材等要求低,便于广泛应用在医院等科研院所中,有望成为包括ACT疗法等过继性细胞免疫治疗方案上新的突破,实现有效治疗制剂、治疗方案等,使更多的患者得以有效的治疗。
附图说明
图1显示流式结果:展示不同组织来源的T淋巴细胞占比及分型,其中含:TSCM:CD45RO-CD62L+CD95+;TCM:CD45RO+CD62L+;TEM:CD45RO+CD62L-;TEX:CD3+PD-1+Tim3+LAG3+;Th1:CD4+T-bet+;NK:CD3-CD56+。
图2显示细胞培养结果:展示LNL细胞培养形态及生长曲线。
图3显示流式细胞术检测LNL培养第30天的细胞亚型结果:T记忆细胞(TSCM,TCM,TEM),Th1,NK及T淋巴细胞耗竭情况(TEX)。
图4显示细胞水平LNL的功能评估:肿瘤裂解物或经肿瘤裂解物刺激的DC能够刺激培养的LNL活性标志物CD134,CD137表达上调。
图5显示实施例1中LNL对MDA-MB-231荷瘤鼠的疗效:A、治疗期间小鼠体重变化;B、肿瘤生长曲线;C、治疗结束后肿瘤抑制情况;D、治疗结束后肿瘤中T细胞浸润情况:IHC显示肿瘤中T细胞浸润情况;E、治疗结束后肿瘤细胞中存留T细胞占比。
图6显示实施例1中LNL对PDX模型鼠的疗效:A、治疗期间小鼠体重变化;B、肿瘤生长曲线;C、治疗结束后肿瘤抑制情况(LNL治疗组红色圆圈代表肿瘤完全消退)。
图7显示实施例1主要实验过程。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
现结合具体实例对本发明作进一步的说明,以下实施例仅是为了解释本发明,但不构成对本发明的限制。在以下实施例中所用到的试验样本及试验过程包括以下内容(如果实施例中未注明的实验具体条件,通常按照常规条件,或按照试剂公司所推荐的条件;下述实施例中所用的试剂、耗材等,如无特殊说明,均可从商业途径得到)。
实施例1
在本实施例中,以乳腺癌为例进行以下实验过程。
本实施例通过流式细胞术检测不同组织来源(相同体积的肿瘤和有肿瘤转移的引流淋巴结组织)中T淋巴细胞占比及分型,如图1所示,检测结果显示,不同组织来源T细胞的占比和分型具有显著差异,相比较肿瘤来源的T细胞,淋巴结中T细胞更多的处于更幼稚化的记忆细胞状态。在此基础上,本申请人进一步展开了自体肿瘤引流淋巴结淋巴细胞及其作为过继回输细胞来源的研究,以望克服现有技术其他过继转移细胞存在的部分不足。
具体地,展开有以下实验过程:
1、单个核细胞获取:
(1)活检获取引流淋巴结2-3个;
(2)利用解剖器械去除多余的脂肪后置于生理盐水中,重复洗3次;
(3)然后用无菌组织剪刀将肿瘤组织或淋巴结剪至约1-2mm3大小的颗粒,加入混合了1.5mg/ml胶原酶I、III和0.2mg/ml DNA酶的SuperCultureTML500人淋巴细胞无血清培养基中,37℃孵育半小时,每10min取出进行摇晃,消化组织块;进一步地,可采用全自动组织温和处理器实现该操作过程;
(4)组织消化后用生理盐水稀释,用无菌70μm细胞过滤器过滤细胞收集得到单细胞悬液,离心5min(2000rpm);
(5)用人淋巴细胞分离液Ficoll密度梯度离心,水平离心20min(室温、800g、加减速度调为1),吸取中间以单个核细胞为主的白膜层分离制备单个核细胞悬液,生理盐水洗涤细胞2次后,计数,以2~2.5×106个/mL种于24孔板;
(6)PBMC:静脉抽取外周血,离心去上层血浆,采用步骤(5)相同的方法用人淋巴细胞分离液分离制备外周血单个核细胞;DC的获取是在获得PBMC后,贴壁2h,更换新鲜DC培养基(SuperCultureTML500人淋巴细胞无血清培养基+2U/ml GM-CSF/IL-4)培养获得。
2.T淋巴细胞初始培养
在初始培养基:SuperCultureTML500人淋巴细胞无血清培养基+10%人AB血清+1000U/mL的重组人IFN-γ中培养。
3.T淋巴细胞激活和快速扩增
(1)培养7天后,用100ng/mL的CD3单抗和1000IU/mL的重组人IL-2刺激步骤2中的细胞悬液,使T细胞激活并扩增;所述T细胞经CD3抗体刺激后被激活处于快速扩增期。
(2)每2~3天更换一半的新鲜培养基(新鲜培养基只添加重组人IL-2,未添加CD3单抗),并选择扩瓶,以保持细胞计数在2-2.5×106个/mL之间。
4.DC呈递肿瘤抗原刺激T细胞特异性扩增
T细胞经CD3刺激扩增7天后,通过负载肿瘤抗原的DC与之共培养以刺激T细胞特异性扩增。具体的,将同一患者肿瘤组织在液氮和37℃水浴锅之间反复冻融5次,离心去除细胞碎片,将含有肿瘤抗原的裂解物置于DC培养基中脉冲24h,经DC成熟因子刺激DC成熟24h后,将负载肿瘤抗原的DC与前述经CD3刺激扩增7天后所得T淋巴细胞进行共培养;
每2~3天更换一半的新鲜的扩增培养基(SuperCultureTML500人淋巴细胞无血清培养基+10%人AB血清+1000IU/mL的重组人IL-2)维持T细胞的增殖。
步骤3及步骤4关于分离出的LNL细胞培养状态如图2所示,其中Day为培养第2天的形态,REP-1W、REP-2W、REP-3W分别为培养值第1周、第2周、第3周的细胞培养形态代表图。图2右显示了相应的LNL细胞培养生长曲线,包括CD3单抗、DC诱导过程,如图2右所示,LNL细胞得到有效的培养扩增,且能持续较快的扩增。即LNL细胞相比于现有技术中其他过继细胞来源,其更易于培养、扩增,能解决现有技术中过继免疫细胞疗法中过继细胞难以培养、大量扩增的不足。
5.T细胞记忆亚群检测
(1)将LNL细胞连续培养至第30天取样,约105个/管,400g离心5min;
(2)弃上清,加入500μL PBS,重悬细胞,400g离心5min;
(3)弃上清,加入100μL PBS,实验管分别标记相应荧光CD3,CD4,CD8,CD45RO,CD62L,CD95,CD56、T-bet抗体,室温避光孵育20min,设置阴性对照组;
(4)加入500μL PBS,重悬细胞,400g离心5min,重复两次;
(5)弃上清,加入100μL PBS,流式上机检测;
(6)用Flowjo软件分析,以CD3+的细胞为分析对象设门,分析各亚群的比例(TSCM:CD45RO-CD62L+CD95+;TCM:CD45RO+CD62L+;TEM:CD45RO+CD62L-;
(7)检测肿瘤引流淋巴结中培养的LNL记忆T细胞比例,检测结果如图3所示。CD3+T细胞占细胞制剂的95%以上。具体地,中枢记忆T细胞、效应记忆T细胞占据较为显著的比例,表明了LNL细胞经培养至第30天,其仍然具有相应的分化、杀伤潜力。
6.T细胞耗竭相关表面分子的检测
(1)培养至第30天取样;
(2)按上述T细胞记忆亚群检测过程收集方法收集样本,标记荧光抗体CD3、CD4、CD8、PD1、TIM3、LAG3,洗涤后100μL PBS重悬,流式上机检测;
(3)用Flowjo软件分析,以CD3+的细胞为分析对象设门,T细胞耗竭情况以PD1、TIM3、LAG3阳性比例表示;
(4)如图3所示,显示了肿瘤引流淋巴结中培养的LNL T细胞耗竭相关表面分子表达情况(TEX)。培养至第30天,其中TEX仅占据非常小的比例,不足3%,表明实施例提供的LNL细胞,其具有长期作用效力,能有效克服容易终末分化、衰竭的状况。
7.细胞水平LNL的功能评估
(1)LNL培养的第28天,按前述方法将同一患者肿瘤组织在液氮和37℃水浴锅之间反复冻融5次,离心去除细胞碎片,将含有肿瘤抗原的裂解物置于DC培养基中;
(2)24h后,添加DC成熟因子培养24h;
(3)LNL细胞培养的第30天,更换新鲜的培养基(SuperCultureTML500人淋巴细胞无血清培养基),以DC:LNL为1:10~1:100的比例置于DC培养瓶中,与成熟的DC共培养;或相同体积的肿瘤组织裂解物同一时间直接刺激同等计数的LNL;同时设置对照组:阴性对照组为相同计数的LNL更换新鲜培养基;阳性对照组为相同计数的LNL更换新鲜培养基并添加CD3抗体刺激;
(4)24h后观察DC形态及贴壁情况改变,按步骤5的方法收集样本,标记荧光抗体CD3,CD137,CD134;
(5)洗涤后重悬,流式上机检测。结果如图4所示,肿瘤裂解物或经肿瘤裂解物刺激的DC能够有效刺激培养的LNL活性标志物CD134,CD137表达上调。
8.动物实验体内评估LNL细胞疗效及持久性
(1)细胞制剂:取培养第14天的LNL细胞(经DC诱导),750g离心5min后,弃上清,用适量体积的溶媒(无菌PBS)将细胞重悬,计数并调整细胞浓度为1×107个/125μL,细胞计数时以活细胞为准。
(2)小鼠模型信息
(a)种属&品系:NOD/SCID
动物等级:SPF级
动物数量:15只
性别:雌
年龄:4周龄
体重范围:17~22g
造模:MDA-MB-231细胞植入皮下成瘤
随机分组:PBS组、IL-2组、LNL组(每组5只)
(b)种属&品系:NOD/SCID
动物等级:SPF级
动物数量:12只
性别:雌
年龄:4周龄
体重范围:17~22g
造模:原位肿瘤
随机分组:PBS组、IL-2组、LNL组(每组4只)
(3)治疗:细胞治疗组按1×107个/鼠尾静脉注射,体积为125μL,同时皮下辅以6000IU/125μL IL-2;其余对照组分别为尾静脉注射等体积的无菌PBS和皮下注射等体积的6000IU IL-2注射液。
(4)安全及疗效
a)监测小鼠体重变化、获得肿瘤生长曲线;
b)监测治疗3-14周后小鼠肿瘤抑制情况;
c)免疫组化检测PBS组、IL-2组、LNL组肿瘤淋巴细胞浸润情况(CD3)。
d)流式细胞术检测治疗结束后PBS组、IL-2组、LNL组淋巴细胞比例;
(5)安全及疗效结果
关于LNL对MDA-MB-231荷瘤鼠的安全和疗效(非特异),如图5A所示,治疗组小鼠的体重与IL-2组和PBS模型组比较均无统计学差异,表明LNL细胞制剂对实验鼠体重无明显影响,具有一定安全性。且肿瘤引流淋巴结中培养的LNL治疗组小鼠肿瘤明显抑制并逐渐消退(如图5B、5C所示),表明LNL细胞具有有效的肿瘤杀伤作用。治疗结束后,免疫组化结果显示LNL组肿瘤中淋巴细胞浸润情况,结果如图5D所示,LNL来源细胞得以显著浸润于肿瘤中;治疗结束后,流式细胞术检测LNL组治疗后小鼠肿瘤中T细胞不同亚型占比如图5E所示,相比于其他组近乎消失的CD3+、CD4+、CD8+亚型细胞,LNL组CD3+、CD4+、CD8+亚型细胞均有相当大比例留存,表明了其可长期作用的特性。
关于LNL对PDX模型鼠的安全和疗效(特异),如图6A所示,治疗组小鼠的体重与IL-2组和PBS模型组比较均无统计学差异,表明LNL细胞制剂对实验鼠体重无明显影响,具有一定安全性。且肿瘤引流淋巴结中培养的LNL治疗组小鼠肿瘤明显抑制并逐渐消退(如图6B、6C所示),表明LNL细胞具备特异性的且有效的肿瘤杀伤作用。
显然,本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例,而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (6)
1.一种自体肿瘤引流淋巴结淋巴细胞的制备方法,其特征在于,包括步骤:
A1、从患者中获取引流淋巴结组织,消化、分离,制备引流淋巴结单个核细胞;具体包括:
A11、活检获取引流淋巴结,去除多余脂肪后洗涤,剪碎,再加入至含胶原酶I、胶原酶III和DNA酶的人淋巴细胞无血清培养基中孵育、消化;
A12、组织消化后用生理盐水稀释,使用细胞过滤器过滤细胞收集得到单细胞悬液;
A13、获得单细胞悬液后,用人淋巴细胞分离液Ficoll密度梯度离心,吸取离心后以单个核细胞为主的白膜层,分离制备单个核细胞悬液,洗涤,种于孔板,获得引流淋巴结单个核细胞;
A2、对步骤A1获得的细胞进行培养,并激活、扩增其中包含的T细胞,获得自体肿瘤引流淋巴结淋巴细胞;具体包括:
A21、将步骤A1获得的引流淋巴结单个核细胞加至初始培养基中培养,所述初始培养基为人淋巴细胞无血清培养基、1000U/mL的重组人IFN-γ及10%人AB血清或自体血浆或血清替代物混合形成;
A22、经初始培养后,使用100ng/mL的CD3单抗、1000IU/mL的重组人IL-2刺激;
A23、待T细胞完全被活化后,定时更换一半的新鲜持续扩增培养基维持T细胞的增殖,获得自体肿瘤引流淋巴结淋巴细胞,所述持续扩增培养基为人淋巴细胞无血清培养基、1000IU/mL的重组人IL-2及10%人AB血清或自体血浆或血清替代物混合形成;
A3、从同一患者外周血获得DC细胞,并使用肿瘤裂解物脉冲DC,使DC呈递肿瘤抗原,然后与步骤A2中自体肿瘤引流淋巴结淋巴细胞共培养,获得DC诱导的自体肿瘤引流淋巴结淋巴细胞;具体包括:
A31、静脉抽取同一患者的外周血,离心去上层血浆,同等体积生理盐水稀释后,用人淋巴细胞分离液Ficoll密度梯度离心,获得外周血单个核细胞;
A32、获得外周血单个核细胞后,贴壁培养,再更换DC培养基培养以获得DC细胞,所述DC培养基为人淋巴细胞无血清培养基、2U/mL GM-CSF/IL-4混合形成;
A33、使用肿瘤裂解物脉冲至DC细胞,DC经成熟培养基培养成熟后与自体肿瘤引流淋巴结淋巴细胞共培养,获得DC诱导的自体肿瘤引流淋巴结淋巴细胞,所述DC成熟培养基为人淋巴细胞无血清培养基、TNFα/IL-1β/IL-6混合形成。
2.权利要求1所述制备方法所得自体肿瘤引流淋巴结淋巴细胞在制备治疗乳腺癌药物中的用途。
3.根据权利要求2所述的用途,其特征在于,治疗乳腺癌药物包括过继性细胞免疫治疗药物。
4.权利要求1所述制备方法所得自体肿瘤引流淋巴结淋巴细胞在制备过继细胞免疫疗法细胞中的用途。
5.一种细胞制剂,其特征在于,含有权利要求1所述制备方法获得的自体肿瘤引流淋巴结淋巴细胞,及药学上可接受的载体或溶剂。
6.一种肿瘤治疗用过继性细胞,其特征在于,来源于权利要求1所述制备方法获得的自体肿瘤引流淋巴结淋巴细胞。
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