CN117025530B - 用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞(til)的方法 - Google Patents
用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞(til)的方法 Download PDFInfo
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Abstract
本发明提供了一种用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞(TIL)的方法,使用具有特定经过的抗OX40抗体和抗4‑1BB抗体处理TIL细胞,可提高其增殖活性,所述抗OX40抗体和抗4‑1BB抗体与相应的细胞因子配合使用,可避免对高剂量IL‑2的依赖,所述方法制备的TIL具有较高的成熟程度和增殖能力,可有效杀死肿瘤细胞,抑制动物模型中肿瘤组织生长,延长动物生存时间。
Description
技术领域
本发明属于生物技术研发领域,具体提供了一种用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞(TIL)的方法。
背景技术
有理论认为癌症是一种基因组疾病,其特点是基因组的不稳定性,在肿瘤进展过程中会积累大量点突变并发生结构改变。这种基因组变异可能会产生肿瘤抗原,该抗原可以被免疫系统识别为异体并引发细胞免疫反应。免疫系统在免疫监视中发挥着重要作用,适应性和先天免疫系统的免疫细胞渗透到肿瘤微环境中并有助于调节肿瘤进展。先天免疫细胞由自然杀伤细胞、嗜酸性粒细胞、嗜碱性粒细胞和吞噬细胞(包括肥大细胞、中性粒细胞、单核细胞、巨噬细胞和树突状细胞)组成,通过直接杀死肿瘤细胞或触发适应性免疫反应而抑制肿瘤形成。适应性免疫系统通过淋巴细胞发挥作用,包括B细胞和T细胞,其中B细胞在体液免疫反应中起主要作用,而T细胞则参与细胞介导的免疫反应。
正因如此,基于免疫系统的免疫疗法彻底改变了癌症治疗手段,并重振了肿瘤免疫学领域,包括过继细胞移植(adoptive cell transfer,ACT)和免疫检查点抑制剂(immune checkpoint inhibitors,ICIs)在内的几种类型的免疫疗法已获得持久的临床反应,但其疗效各不相同,并且只有部分癌症患者可以从中受益。肿瘤微环境(tumormicroenvironment,TME)中的免疫浸润已被证明在肿瘤发展中发挥关键作用,并将影响癌症患者的临床结果。对肿瘤浸润免疫细胞的全面分析将揭示癌症免疫逃避的机制,从而为开发新的治疗策略提供机会。肿瘤浸润淋巴细胞(Tumor-infiltrating lymphocyte,TIL)疗法是一种过继性细胞疗法,在TIL疗法中,通过活检或手术从肿瘤部位分离TIL,用白细胞介素2(IL-2)在体外刺激并扩增至大量,然后回输到患者体内。1982年,该领域的先驱美国国立卫生研究院的Steven Rosenberg博士及其同事首次从多种小鼠肿瘤模型中分离出TIL(参见Eberlein TJ, Rosenstein M, Rosenberg SA. Regression of a disseminatedsyngeneic solid tumor by systemic transfer of lymphoid cells expanded ininterleukin 2. J Exp Med. 1982;156(2):385–397),随后证明结合环磷酰胺治疗,在MC38 结肠腺癌模型中,TIL 和同时给予 IL-2 治愈了 100% 的肝转移小鼠和 50% 的肺转移小鼠(参见Rosenberg SA, Spiess P, Lafreniere R. A new approach to theadoptive immunotherapy of cancer with tumor-infiltrating lymphocytes.Science. 1986;233(4770):1318–1321),为TIL在人类晚期癌症治疗中的应用奠定了基础。TIL疗法在临床的最早尝试可以追溯到1988年,在转移性黑色素瘤中取得了60%的客观缓解率(参见Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, ToyST, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in theimmunotherapy of patients with metastatic melanoma. A preliminary report. NEngl J Med. 1988;319(25):1676–1680)。
TIL 在治疗实体瘤方面可能具有一些独特的优势。首先,TIL由具有多个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,因此与其他过继性细胞疗法(例如嵌合抗原受体T)相比,TIL在解决肿瘤异质性方面可能更胜一筹。TIL在黑色素瘤等含有高突变负荷的实体瘤中表现出比CAR-T更好的临床疗效。其次,在体内受到肿瘤抗原刺激后,TIL往往主要由效应记忆T细胞组成,其表面表达趋化因子受体,如CCR5和CXCR3,TIL 在转移到患者体内后可以轻松归巢到抗原不同的组织。最后,TIL 治疗中很少报道脱靶毒性,这可能是由于 TIL 的 TCR 在 T 细胞免疫早期发展过程中的负选择所致。相反,CAR-T中的工程化肿瘤靶向单链可变片段(scFv)或TCR-T产品中的亲和力增强TCR如果与正常组织上的抗原发生交叉反应,则可能会导致毒性。
产生TIL的过程通常从预快速扩增阶段(pre-REP)开始,其中TIL从肿瘤碎片中解离或迁移出并进行初步扩增。然后,TIL 在快速扩增阶段 (REP) 响应刺激物(例如 IL-2和/或饲养细胞)进一步扩增。TIL 生产的传统程序是针对特定肿瘤识别进行分析,通常需要 4-6周。然而,TIL在体外长时间培养后容易衰竭,不能在患者体内长期维持。此外,体外生长自体肿瘤的成功率较低,导致接受TIL治疗的患者退出率超过50%(参见Dudley ME,Wunderlich JR, Shelton TE, Even J, Rosenberg SA. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy formelanoma patients. J Immunother. 2003;26(4):332–342),很大程度上限制了其临床应用。
为此,Rosenberg博士等人开发了一种“Young TIL”方法,无需体外选择肿瘤反应性即可快速扩增TIL,显着提高了TIL生产的时效性及其在体内的存活率和疗效(参见TranKQ, Zhou J, Durflinger KH, Langhan MM, Shelton TE, Wunderlich JR, et al.Minimally cultured tumor-infiltrating lymphocytes display optimalcharacteristics for adoptive cell therapy. J Immunother. 2008;31(8):742–751)。有研究表明,抗 PD-1、抗 4-1BB 或抗 CTLA-4 可以增加 TIL 扩增(参见Hall M, Liu H,Malafa M, Centeno B, Hodul PJ, Pimiento J, et al. Expansion of tumor-infiltrating lymphocytes (TIL) from human pancreatic tumors. J ImmunotherCancer. 2016;4:61);与单独使用 IL-2 相比,IL-2/15/21 的组合可以增强肺癌和结直肠癌中 TIL 的扩增,并提高 CD8+ T 细胞百分比以及 TCR 克隆多样性(参见Frank L,Simpson AM, Lotze M, Ritthipichai K, Mosychuk C. The T-cell Growth FactorCocktail IL-2/IL-15/IL-21 Enhances Expansion and Effector Function of Tumor-Infiltrating T cells in a Novel Process Developed by Iovance. Society forImmunotherapy of Cancer. 2017)。为了减少与高剂量 IL-2 相关的毒性并提高传统 TIL疗法的体内存活率和功能,下一代 TIL 产品正在积极研究中。下一代 TIL 是经过基因改造的 TIL,可通过病毒转导过度表达感兴趣的基因,或使用 CRISPR 或 TALEN 等技术敲除目标基因。然而,下一代TIL的开发可能面临一些重大挑战,在 TIL 中进行基因编辑在技术上可能很难实现,这可能是因为与 PBMC 相比,TIL 的细胞组成和生长速率不同。
由此可见,虽然TIL疗法是一种有效的肿瘤免疫治疗手段,但是其制备过程中却存在诸多困难,传统的依赖于高剂量IL-2的方法,容易产生细胞毒性,而基因改造方式又面临操作复杂,成本高昂的困境。为此,本发明提供了一种用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞的方法,能够避免使用单一的高剂量的IL-2,同时可简单快捷的获得符合要求的TIL细胞。
发明内容
本发明中的第一方面提供了一种利用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞的方法,其特征在于,所述肿瘤坏死因子受体超家族激动剂为抗OX40抗体和抗4-1BB抗体,所述方法包括以下步骤:
(1)将肿瘤组织用消化液消化,过滤后收集单细胞悬液,通过离心法获得淋巴细胞;
(2)将淋巴细胞接种至培养瓶中,加入IL-2、IL-10、OKT3和抗OX40抗体,所述抗OX40抗体的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ IDNO.2所示;
(3)培养1周后,更换新鲜细胞培养基,同时加入IL-15、IL-21、IFN-γ和抗4-1BB抗体,所述抗4-1BB抗体的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示;
(4)继续培养2-3周后,收获肿瘤浸润淋巴细胞。
肿瘤浸润淋巴细胞虽早已被研究人员所重视,并应用到肿瘤的免疫治疗中,但该类细胞的制备方法却一直差强人意,传统方法为采用高剂量的IL-2进行培养,但这种方法易产生不良反应,且所制备的细胞在分化程度和增殖能力上难以满足抗肿瘤的药效需要。尽管现有技术中,已经公开了多种改进方案,如采用IFN-γ、TNF-α、IL-6、IL-10、IL-15、IL-21等细胞因子替代IL-2,以及使用抗OX40、CD133、CD137、PD-1抗体进行孵育,但是所获得的肿瘤浸润淋巴细胞仍面临增殖缓慢、分化程度低、抗肿瘤活性低等困境。为此,本发明提出以具有特定氨基酸结构的抗OX40抗体和抗4-1BB抗体,并配合其他细胞因子,顺序孵育肿瘤浸润淋巴细胞的方式培养细胞,进而改善所述肿瘤浸润淋巴细胞性能。
进一步的,所述步骤(1)中消化液为胶原酶Ⅰ、胶原酶Ⅱ、胶原酶IV、透明质酸酶按照质量比5:5:3:1构成的混合溶液。
进一步的,所述步骤(1)中离心法包括将单细胞悬液1500rpm离心5min,PBS洗涤3遍;将100%的Percoll悬液加入离心管底部,然后加入等量用RPMI1640培养液稀释的75%Percoll分离液,最上面一层加入等量细胞悬液,3000rpm离心20-30min;离心结束后,用吸管抽取分离液之间的灰白色细胞层。
进一步的,所述步骤(2)中加入终浓度为1000-2000IU/ml的IL-2、300-600IU/ml的IL-10、10-50ng/ml的OKT3和5-10μg/ml抗OX40抗体。
进一步的,所述步骤(2)中加入终浓度为1500IU/ml的IL-2,500IU/ml的IL-10、20ng/ml的OKT3和5μg/ml抗OX40抗体。
进一步的,所述步骤(3)中加入终浓度为500-1000IU/ml的IL-15,500-800IU/ml的IL-21、5-20ng/ml的IFN-γ和10-50μg/ml抗4-1BB抗体。
进一步的,所述步骤(3)中加入终浓度为1000IU/ml的IL-15,600IU/ml的IL-21、20ng/ml的IFN-γ和40μg/ml抗4-1BB抗体。
本发明中的第二方面提供了一种所述方法制备的肿瘤浸润淋巴细胞在制备抗肿瘤药物中的应用。
进一步的,所述肿瘤选自肺癌、鼻咽癌、乳腺癌、结直肠癌、胃癌、肝癌、黑色素瘤、肾细胞癌、霍奇金淋巴瘤中的至少一种。
进一步的,其特征在于所述的肿瘤为肝癌。
有益效果
本发明提供了一种用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞(TIL) 的方法,具有以下优势:
(1)使用具有特定经过的抗OX40抗体和抗4-1BB抗体处理TIL细胞,可提高其增殖活性;
(2)所述抗OX40抗体和抗4-1BB抗体与相应的细胞因子配合使用,可避免对高剂量IL-2的依赖;
(3)所述方法制备的TIL具有较高的成熟程度和增殖能力,可有效杀死肿瘤细胞。
附图说明
图1:抗OX40抗体促进肿瘤淋巴浸润细胞增殖图;
图2:抗OX40抗体和抗4-1BB抗体促进肿瘤淋巴浸润细胞增殖图;
图3:肿瘤模型小鼠生存周期曲线图;
图4:模型动物血清中IFN-γ表达水平变化图;
图5:模型动物血清中IL-6表达水平变化图。
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂生物材料、检测试剂盒,如无特殊说明,均可从商业途径获得。
实施例1 肿瘤坏死因子受体超家族激动剂的筛选和获得
本发明中使用的肿瘤坏死因子受体超家族激动剂为抗OX40抗体和抗4-1BB抗体,所述抗体为发明人在早期使用中通过噬菌体展示技术筛选并获得,其中抗OX40抗体(记为anti-OX40)的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQID NO.2所示;抗4-1BB抗体(记为anti-4-1BB)的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示。
值得说明的是,所述抗OX40抗体与目标抗原具有中等亲和力,经测定其与目标抗原的KD值为3.15×10-8M,而抗4-1BB抗体则与目标抗原具有高亲和力,经测定其与目标抗原的KD值为2.64×10-9M,使用这种具有亲和力梯度的抗体顺序处理,更有利于促进肿瘤浸润淋巴细胞的增殖。
实施例2 肿瘤淋巴浸润细胞的提取
2.1 TIL细胞的传统培养方法
TIL细胞的传统培养方法主要依赖于高剂量的IL-2(参见Hannah M K et al,Modeling ex vivo tumor-infltrating lymphocyte expansion from establishedsolid malignancies, ONCOIMMUNOLOGY,2021, VOL. 10, NO. 1, e1959101),本发明中以肝癌组织为例,提取TIL细胞,按照如下步骤进行:
(1)取肝癌患者手术后的肿瘤组织样本,置于50mL 离心管内,并加入适量含有抗生素(10U/mL 青霉素、1mg/mL 链霉素)的 RPMI1640 培养基,在4℃环境中保存。
(2)将标本管消毒后移至超净工作台内,除去肿瘤样本中的脂肪组织、纤维组织和坏死区域。称取 1g肿瘤组织,用眼科剪在无菌条件下剪碎,将剪碎的组织块移至洁净离心管中,使用包含0.1mg/mL胶原酶Ⅰ、0.1mg/mL胶原酶Ⅱ、0.06mg/mL胶原酶IV和0.02mg/mL透明质酸酶的混合酶液进行消化,在37℃恒温箱内孵育1-2h。过100um滤网收集单细胞悬液,将单细胞悬液1500rpm离心5min,PBS洗涤3遍;用 9 份 Percoll 与 1 份 10×PBS 混合制成等渗的 100%的Percoll悬液(购自Solarbio公司),密度约 1.129 g/mL,将所述100%的Percoll悬液加入离心管底部,然后加入等量用RPMI1640培养液稀释的75%Percoll分离液,最上面一层加入等量细胞悬液,3000rpm离心20-30min;离心结束后,用吸管抽取分离液之间的灰白色细胞层,即 TIL细胞。
(3)将所述TIL细胞接种于含有10%FBS的RPMI1640完全培养基中,同时加入终浓度为6000IU/ml的IL-2,37℃ 5%CO2培养,每2-3天更换新鲜培养基,待细胞融合度达到80%以上时收获TIL细胞,记为IL-2-TIL细胞。
2.2 抗OX40抗体促进肿瘤淋巴浸润细胞增殖
使用高剂量的IL-2不仅生长缓慢,而且可能诱发严重的不良反应,因此本发明中试图使用不同细胞因子的组合策略,替代单一的IL-2,并且通过加入肿瘤坏死因子受体超家族激动剂刺激扩增,但是在早期实验中发现,使用针对OX40抗原具有高亲和力的抗体,如BMS-986178,其扩增效果并不理想,因此本发明中使用了具有中等亲和力的新型抗OX40抗体,具体步骤如下:
(1)取肝癌患者手术后的肿瘤组织样本,置于50mL 离心管内,并加入适量含有抗生素(10U/mL 青霉素、1mg/mL 链霉素)的 RPMI1640 培养基,在4℃环境中保存。将肿瘤组织样本运输到实验室后,
(2)将标本管消毒后移至超净工作台内,除去肿瘤样本中的脂肪组织、纤维组织和坏死区域。称取 1g肿瘤组织,用眼科剪在无菌条件下剪碎,将剪碎的组织块移至洁净离心管中,使用0.1mg/mL胶原酶Ⅰ、0.1mg/mL胶原酶Ⅱ、0.06mg/mL胶原酶IV和0.02mg/mL透明质酸酶的混合酶液进行消化,在37℃恒温箱内孵育1-2h。过100um滤网收集单细胞悬液,将单细胞悬液1500rpm离心5min,PBS洗涤3遍;用 9 份 Percoll 与 1 份 10×PBS 混合制成等渗的 100%的Percoll悬液,密度约 1.129 g/mL,将所述100%的Percoll悬液加入离心管底部,然后加入等量用RPMI1640培养液稀释的75%percoll分离液,最上面一层加入等量细胞悬液,3000rpm离心20-30min;离心结束后,用巴氏吸管抽取分离液之间的灰白色细胞层,即TIL细胞。
(3)将所述TIL细胞接种于含有10%FBS的RPMI1640完全培养基中,同时加入终浓度为1500IU/ml的IL-2,500IU/ml的IL-10、20ng/ml的OKT3(相关细胞因子购自美国R&D公司)和5ug/ml抗OX40抗体,37℃ 5%CO2培养,每2-3天更换新鲜培养基,待细胞融合度达到80%以上时收获TIL细胞,记为anti OX40-TIL。
以高亲和力抗OX40抗体BMS-986178(一种高亲和力的人源抗体,参见OX40Agonist BMS-986178 Alone or in Combination With Nivolumab and/or Ipilimumabin Patients With Advanced Solid Tumors,Martin Gutierrez et al, Clin CancerRes. 2021 Jan 15;27(2):460-472)为对照,按照上述方法制备获得TIL细胞,记为BMS-TIL。
采用MTT法检测TIL细胞的增殖能力,分别取106个IL-2-TIL细胞(为对照组)、antiOX40-TIL细胞和BMS- TIL细胞,接种于96孔板中,各组设置5个复孔,置于37℃、5%CO2恒温培养箱中培养12h后,弃去原培养液,每孔加20μL 5g/L MTT溶液,置于37℃、5%CO2恒温培养箱中4h后,每孔加150μLDMSO,振荡10 min,用酶标仪测量波长490 nm处吸光度值(OD),并按公式计算细胞相对增殖率(relative growth,RGR),公式为:RGR=实验组OD值/对照组OD值×100%。
结果如图1所示,使用抗OX40抗体和IL-2、IL-10、OKT3等因子共同处理TIL细胞,比单独使用IL-2,能够更加有效的促进TIL细胞增殖,而且使用具有中等亲和力的抗OX40抗体,似乎更具优势。
2.3后续使用抗4-1BB抗体的促TIL细胞增殖作用
4-1BB是另一种肿瘤坏死因子受体超家族的重要成员,已有报道称使用抗4-1BB抗体可促进TIL细胞增殖和分化,本发明中使用具有独特轻重链结构的抗4-1BB抗体,所述抗体与目标抗原具有较高的亲和力,在使用抗OX40抗体和相应的细胞因子处理后,再次使用抗4-1BB抗体和其他有益的细胞因子进行二次诱导培养,能够进一步促进TIL细胞增殖,从而利于免疫细胞的培养和抗肿瘤作用的发挥。具体的培养步骤为,在使用抗OX40抗体和IL-2、IL-10、OKT3等因子培养1周后,更换新鲜细胞培养基,加入终浓度为1000IU/ml的IL-15,600IU/ml的IL-21、20ng/ml的IFN-γ(相关细胞因子购自美国R&D公司)和40ug/ml抗4-1BB抗体,继续培养2-3周后,收获肿瘤浸润淋巴细胞,记为双抗-TIL细胞。
采用MTT法检测TIL细胞的增殖能力,IL-2-TIL细胞为对照组,方法同2.2节,结果如图2所示,经过二次诱导后,TIL细胞的增殖能力大幅度提高,相对于对照组增长超过1倍。
2.4肿瘤坏死因子受体超家族激动剂对细胞分化的影响
收集上述TIL细胞,300g离心5min重悬,用PBS洗涤3次,调整细胞密度至1×106个/mL,分别加入CD3、CD4、CD8、CD56等荧光标记的抗体(购自BioLegend公司)并混匀,室温孵育15 min,随后加入 2-5 mL 的 PBS 以 500 rpm,5 min 离心洗涤一次,用流式细胞仪进行检测。
结果如表1所示,经过本发明中所提供的抗体处理后,双抗-TIL细胞和anti OX40-TIL细胞中CD3+、CD4+比例较高,说明细胞增殖较快,且细胞成熟程度较高,临床应用前景好,而采用传统方式制备获得的IL-2-TIL细胞则CD56+比例较高,细胞增殖和发育较慢。
表1 TIL细胞鉴定
细胞种类 | CD3 | CD4 | CD8 | CD56 |
IL-2-TIL | 71.3% | 25.4% | 33.6% | 31.6% |
anti OX40-TIL | 85.3% | 32.6% | 46.8% | 15.3% |
BMS-TIL | 79.6% | 28.7% | 42.7% | 20.5% |
双抗-TIL | 94.6% | 35.4% | 63.7% | 9.8% |
实施例3 TIL体内抗肿瘤实验
3.1 TIL抑制动物体内肿瘤生长
以肝癌细胞系Huh7构建小鼠肿瘤模型,研究本发明中所提供的TIL细胞的抗肿瘤能力。取5×106 个肿瘤细胞接种于裸鼠右前肢腋窝处皮下,每天观察成瘤情况并记录,肿瘤体积生长至100mm3以上时,表示造模成功。
取30只造模成功小鼠,随机分为3组,分别为:双抗-TIL组,尾静脉注射1×106个双抗-TIL细胞,每周给药一次;IL-2-TIL组,尾静脉注射1×106个IL-2-TIL细胞,每周给药一次;对照组,每周给药一次注射等体积生理盐水。各种共治疗30天。每日观察动物生存状态,并记录,实验结束后绘制生存曲线。结果如图3所示,双抗-TIL治疗可显著延长动物生存周期,而IL-2-TIL虽有一定的治疗作用,但治疗效果明显劣于双抗-TIL组。
3.2 TIL促进免疫因子分泌
治疗2周后取小鼠血液,放置于抗凝管中室温静置1h,4℃、2000 rpm离心15min,获得小鼠血清。使用ELISA试剂盒(购自美国Abcam公司)检测血清中的IL-6和IFN-γ含量,具体操作步骤按照试剂盒说明书进行。
IFN-γ是机体内的主要免疫调节因子,在肿瘤免疫反应中具有重要地位,既可以直接作用于肿瘤细胞,促进其凋亡,又能够有效调动其他免疫因子释放和免疫细胞发挥功能,产生协同肿瘤抑制作用。在本发明中,施加TIL细胞后,动物血清中的IFN-γ含量显著增加,结果如图4所示,并且双抗-TIL组的IFN-γ水平显著高于采用传统方法获得的TIL,展现了更强的抗肿瘤效果。
IL-6也是机体内一种常见的免疫因子,但是在本节实验中,施加TIL细胞似乎没有对IL-6的释放具有显著影响,结果如图5所示,尽管双抗-TIL组的IL-6表达水平有所升高,但是未见显著差异。
综上所述,本发明提供的经过双抗处理的TIL细胞,不仅具有更强的增殖活性,而且抗肿瘤作用也有所提高,为新型细胞免疫疗法的开发和应用提供了研究基础。以上所述技术方案,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例,仍属于本发明技术方案的范围内。
Claims (7)
1.一种利用肿瘤坏死因子受体超家族激动剂扩增肿瘤浸润淋巴细胞的方法,其特征在于,所述肿瘤坏死因子受体超家族激动剂为抗OX40抗体和抗4-1BB抗体,所述方法包括以下步骤:
(1)将肿瘤组织用消化液消化,过滤后收集单细胞悬液,通过离心法获得淋巴细胞;
(2)将淋巴细胞接种至培养瓶中,加入IL-2、IL-10、OKT3和抗OX40抗体,所述抗OX40抗体的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ ID NO.2所示;
(3)培养1周后,更换新鲜细胞培养基,同时加入IL-15、IL-21、IFN-γ和抗4-1BB抗体,所述抗4-1BB抗体的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示;
(4)继续培养2-3周后,收获肿瘤浸润淋巴细胞。
2.根据权利要求1所述的方法,其特征在于,所述步骤(1)中消化液为胶原酶Ⅰ、胶原酶Ⅱ、胶原酶IV、透明质酸酶按照质量比5:5:3:1构成的混合溶液。
3.根据权利要求2所述的方法,其特征在于,所述步骤(1)中离心法包括将单细胞悬液1500rpm离心5min,PBS洗涤3遍;将100%的Percoll悬液加入离心管底部,然后加入等量用RPMI1640培养液稀释的75%Percoll分离液,最上面一层加入等量单细胞悬液,3000rpm离心20-30min;离心结束后,用吸管抽取分离液之间的灰白色细胞层。
4.根据权利要求1所述的方法,其特征在于,所述步骤(2)中加入终浓度为1000-2000IU/ml的IL-2、300-600IU/ml的IL-10、10-50ng/ml的OKT3和5-10μg/ml抗OX40抗体。
5.根据权利要求4所述的方法,其特征在于,所述步骤(2)中加入终浓度为1500IU/ml的IL-2,500IU/ml的IL-10、20ng/ml的OKT3和5μg/ml抗OX40抗体。
6.根据权利要求1所述的方法,其特征在于,所述步骤(3)中加入终浓度为500-1000IU/ml的IL-15,500-800IU/ml的IL-21、5-20ng/ml的IFN-γ和10-50μg/ml抗4-1BB抗体。
7.根据权利要求6所述的方法,其特征在于,所述步骤(3)中加入终浓度为1000IU/ml的IL-15,600IU/ml的IL-21、20ng/ml的IFN-γ和40μg/ml抗4-1BB抗体。
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