WO2021184448A1 - 一种抗肿瘤巨噬细胞的制备方法 - Google Patents

一种抗肿瘤巨噬细胞的制备方法 Download PDF

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WO2021184448A1
WO2021184448A1 PCT/CN2020/083530 CN2020083530W WO2021184448A1 WO 2021184448 A1 WO2021184448 A1 WO 2021184448A1 CN 2020083530 W CN2020083530 W CN 2020083530W WO 2021184448 A1 WO2021184448 A1 WO 2021184448A1
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macrophages
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郑俊武
金红林
万超
黄浩
郑成武
卫路
洪磊
金秀妍
刘群
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武汉圣润生物科技有限公司
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  • Figure 2 is a 40-fold microscopic view of macrophages cultured in vitro of the present invention after microparticle treatment;
  • Figure 3 is an electron micrograph of the microparticles derived from radiotherapy of the present invention.
  • Figure 4 is an analysis diagram of surface molecular changes after macrophages are treated with antigen-carrying microparticles of the present invention
  • Figure 5 is a flow cytometry result chart showing that anti-tumor macrophages of the present invention have a stronger ability to swallow tumor cells;
  • Fig. 6 is a statistical diagram of the volume of the tumor body during the treatment of solid tumors by intraperitoneal injection of macrophages according to the present invention
  • the invention relates to a method for reinfusion of anti-tumor macrophages, which mainly comprises a kind of autologous macrophages that are processed in vitro and transformed into anti-tumor macrophages.
  • the alleged anti-tumor macrophages were obtained by treating autologous macrophages with anti-tumor microparticles.
  • the anti-tumor macrophages have specific anti-tumor activity and a stronger ability to swallow tumor cells. Can inhibit the growth of solid tumors.
  • the macrophages are derived from the patient itself, and the microparticles are derived from the tumor cells themselves, which has good biological safety and biocompatibility.
  • the claimed anti-tumor macrophages can also be modified to have a stronger migration ability, and they can also be treated with immunomodulatory drugs.
  • macrophages can be colonized in mice for one year after the infusion of macrophages and remain active, and pro-inflammatory macrophages play an important role in anti-tumor immunity.
  • transfecting M-CSF and IFN- ⁇ into macrophages and injecting them can achieve good anti-tumor effects.
  • Pro-inflammatory macrophages can exert an anti-tumor effect, while macrophages also have the effect of antigen presentation, and microparticles carry tumor cell antigens, and microparticles processing macrophages can promote the polarization of macrophages into anti-inflammatory macrophages.
  • Phage cells can also allow macrophages to process tumor cell antigens to further activate T cells to exert anti-tumor effects.
  • a method for preparing anti-tumor macrophages includes the following steps:
  • Step 1 Obtain primary bone marrow stem cells or separate mononuclear cells from peripheral blood for primary cell culture, and 7 days after adding colony stimulating factor to obtain macrophages;
  • Step 5 Treat the microparticles obtained in step 4 to the primary macrophages obtained in step 1 to obtain the anti-tumor macrophages.
  • the tumor antigen-carrying microparticles are derived from autologous tumor cells or tumor cells produced by tumor cell lines.
  • the isolated tumor cells are derived from solid tumors.
  • An anti-tumor macrophage prepared by a method for preparing anti-tumor macrophages, wherein the anti-tumor macrophages are M1 pro-inflammatory macrophages; CD86 and MHC II are highly expressed on the surface of the anti-tumor macrophages. CD206 is low in expression; the reported anti-tumor macrophages have a stronger ability to swallow tumor cells.
  • anti-tumor micro-macrophages prepared by a method for preparing anti-tumor macrophages in anti-tumor therapy.
  • FIG. 1 The light microscope picture of the anti-tumor macrophages of the present invention is shown in FIG. 1.
  • the total amount collected is about 0.5ml; the vortexer slightly vortex to ensure that the microparticles are in suspension, perform the dyeing step; use 2% phosphotungstic acid solution After dyeing for 5 minutes, drip it on the copper net. After drying, observe the size and shape of MVs under the transmission electron microscope. As shown in Figure 3, the microparticles are round under the electron microscope.
  • Lewis-luc subcutaneous tumor model 5*104 Lewis-LUC cells per mouse were inoculated subcutaneously into the root of the right thigh of C57, and the injection volume was 50 ⁇ L. After the tumor volume reached 20mm3, random grouping was carried out, and mice with too large or too small tumors were eliminated and divided into PBS group, microparticle polarization M1 group, and microparticle non-polarization M2 group. The amount of macrophages injected into the abdominal cavity is 5*104 per mouse. Inject once every two days and treat 3 times. The size of the tumor is measured every two days. As shown in Figure 6, the macrophages polarized by microparticles can better inhibit tumor growth.

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Abstract

一种抗肿瘤巨噬细胞的制备方法,包括以下步骤:步骤1、获得原代骨髓干细胞或分离外周血中单核细胞进行原代细胞培养,加入集落刺激因子7天后获得巨噬细胞;步骤2、获得离体肿瘤细胞进行原代肿瘤细胞培养,并获得原代肿瘤细胞;步骤3、将步骤2得到的原代肿瘤细胞及培养液进行放疗照射,收集放疗后的上清液,获得所需微颗粒和凋亡的肿瘤细胞碎片混合物;步骤4、将步骤3得到的混合物进行离心,即得到携带肿瘤抗原微颗粒;步骤5、将步骤4得到的微颗粒处理步骤1获得的巨噬细胞,即得到抗肿瘤巨噬细胞。

Description

一种抗肿瘤巨噬细胞的制备方法 技术领域
本发明涉及抗肿瘤领域,更特别地,涉及一种抗肿瘤巨噬方法的制备方法。
背景技术
巨噬细胞是免疫反应中的下游效应关键的执行者,肿瘤微环境中,巨噬占50%以上,更重要的是巨噬细胞的比例与肿瘤的预后呈负相关,进一步说明了巨噬细胞在肿瘤中的重要性。肿瘤组织浸润区的巨噬细胞(Tumor Associated Macrophages,TAMs)促进了肿瘤的进展,肿瘤细胞通过高表达CD47和巨噬细胞表面的SIRPα结合,直接导致肿瘤细胞不仅可以和肿瘤细胞和谐共处,而且还会通过促进肿瘤内血管的增殖,抑制效应T细胞等促进肿瘤的扩增和生长。以往针对巨噬细胞的应用集中在抑制单核细胞在肿瘤微环境分化为肿瘤相关巨噬细胞、杀伤肿瘤相关巨噬细胞以及促进微环境中TAMs重编程为M1等,但清除巨噬细胞会抑制全身免疫反应,使机体易感染。由此我们提出回输特异性抗肿瘤巨噬细胞从而发挥抑制肿瘤生长的效果。
微颗粒由肿瘤细胞所释放,经放疗、双氧水或过氧化氢处理的肿瘤细胞释放更多携带肿瘤抗原的微颗粒,此种微颗粒可以促进巨噬细胞向M1极化,增强巨噬细胞对肿瘤细胞的吞噬,也可协同效应T细胞发挥抗肿瘤效果。
发明内容
为解决以上问题,本发明提供了一种抗肿瘤巨噬细胞的制备方法,该抗肿瘤巨噬细胞通过直接吞噬肿瘤和激活T细胞发挥抗肿瘤效应,解决常规化疗效果差、副作用大、易产生耐药性的技术问题并实现个体化治疗。
本发明是由以下技术手段实现的:
一种抗肿瘤巨噬细胞的制备方法,包括以下步骤:
步骤1、获得原代骨髓干细胞或分离外周血中单核细胞进行原代细胞培养,加入集落刺激因子7天后获得巨噬细胞;
步骤2、获得离体肿瘤细胞进行原代肿瘤细胞培养,并获得原代肿瘤细胞;
步骤3、将所述步骤2得到的原代肿瘤细胞及培养液进行放疗照射,收集放疗后的上清液,获得所需微颗粒和凋亡的肿瘤细胞碎片混合物;
步骤4、将所述步骤3得到的混合物进行离心,即得到所述携带肿瘤抗原微颗粒;
步骤5、将所述步骤4得到的微颗粒处理步骤1获得的原代巨噬细胞,即得到所述抗肿瘤巨噬细胞。
所述的巨噬细胞可来源于人体外周血分离所得淋巴细胞
所述携带肿瘤抗原的微颗粒来源于自体来源的肿瘤细胞或肿瘤细胞系产生的肿瘤细胞。
所述离体肿瘤细胞来自于实体瘤。
一种抗肿瘤巨噬细胞的制备方法制得的抗肿瘤巨噬细胞,所述抗肿瘤巨噬细胞为M1促炎巨噬细胞;所述抗肿瘤巨噬细胞表面CD86和MHC II高表达,胞内CD206低表达;所诉抗肿瘤巨噬细胞具有更强吞噬肿瘤细胞的能力。
一种抗肿瘤巨噬细胞的制备方法制得的抗肿瘤微巨噬细胞在抗肿瘤疗法中的应用。
本发明具有以下优点:
①实现个性化治疗,制备出可针对患者不同的肿瘤抗原的巨噬细胞;
②制备工艺简单,较易生产;
③生产所需周期短;
④所需制备成本低;
⑤功能可扩展:可对巨噬细胞进行基因编程,使其具有更强的激活免疫的作用。
附图说明
图1为本发明体外培养的巨噬细胞40倍显微镜下图;
图2为本发明体外培养的巨噬细胞在微颗粒处理后的40倍镜下图;
图3为本发明放疗来源微颗粒的电镜图;
图4为本发明携带抗原微颗粒处理巨噬细胞后表面分子变化分析图;
图5为本发明抗肿瘤巨噬细胞具有更强的吞噬肿瘤细胞能力的流式结果图;
图6为本发明巨噬细胞腹腔注射治疗实体瘤过程中瘤体的体积统计图;
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
本发明涉及一种抗肿瘤巨噬细胞的回输方法,其主要包括一种自体来源的巨噬细胞经过体外处理改造为抗肿瘤巨噬细胞。所诉抗肿瘤巨噬细胞由抗肿瘤微颗粒处理自体来源巨噬细胞获得。所述抗肿瘤巨噬细胞,具有较特异的抗肿瘤活性,具有更强的吞噬肿瘤细胞的能力。可抑制实体瘤的生长。所述巨噬细胞来自患者本身,微颗粒来源于肿瘤细胞自体,生物安全性和生物相容性好。所诉抗肿瘤巨噬细胞也可经过改造,具有更强迁移能力,也可联合免疫调节药物进行治疗。
本发明的具体原理如下:
Nature曾报导将巨噬细胞回输后巨噬细胞定植在小鼠体内可持续一年并保持活性,而促炎型巨噬细胞在抗肿瘤免疫中发挥了重要作用。也有文献将 M-CSF和IFN-γ转染进巨噬细胞瘤内注射可以获得良好的抗肿瘤效果。促炎型巨噬细胞可发挥抗肿瘤效果,同时巨噬细胞也有抗原递呈的作用,而微颗粒携带肿瘤细胞抗原,微颗粒处理巨噬细胞既可促进巨噬细胞极化为抗炎型巨噬细胞也可让巨噬细胞处理肿瘤细胞的抗原进一步激活T细胞从而发挥抗肿瘤效果。
一种抗肿瘤巨噬细胞的制备方法,包括以下步骤:
步骤1、获得原代骨髓干细胞或分离外周血中单核细胞进行原代细胞培养,加入集落刺激因子7天后获得巨噬细胞;
步骤2、获得离体肿瘤细胞进行原代肿瘤细胞培养,并获得原代肿瘤细胞;
步骤3、将所述步骤2得到的原代肿瘤细胞及培养液进行放疗照射,收集放疗后的上清液,获得所需微颗粒和凋亡的肿瘤细胞碎片混合物;
步骤4、将所述步骤3得到的混合物进行离心,即得到所述携带肿瘤抗原微颗粒;
步骤5、将所述步骤4得到的微颗粒处理步骤1获得的原代巨噬细胞,即得到所述抗肿瘤巨噬细胞。
所述的巨噬细胞可来源于人体外周血分离所得淋巴细胞
所述携带肿瘤抗原的微颗粒来源于自体来源的肿瘤细胞或肿瘤细胞系产生的肿瘤细胞。
所述离体肿瘤细胞来自于实体瘤。
一种抗肿瘤巨噬细胞的制备方法制得的抗肿瘤巨噬细胞,所述抗肿瘤巨噬细胞为M1促炎巨噬细胞;所述抗肿瘤巨噬细胞表面CD86和MHC II高表达,胞内CD206低表达;所诉抗肿瘤巨噬细胞具有更强吞噬肿瘤细胞的能力。
一种抗肿瘤巨噬细胞的制备方法制得的抗肿瘤微巨噬细胞在抗肿瘤疗法中的应用。
所诉抗肿瘤巨噬细胞可腹腔和尾静脉注射。
本发明所述抗肿瘤巨噬细胞的光镜图如图1所示。
下文结合实施例对本发明的效果作进一步解释和验证。
1.骨髓来源巨噬细胞的培养
C57BL/6小鼠断颈处死后置于75%酒精中浸泡20min,取出小鼠放入超净工作台中。用眼科剪、眼科镊按顺序剥离小鼠的股骨及胫骨处皮肤与肌肉,剪断股骨/胫骨两端,用1ml无菌注射器吸取培养基由断端冲出骨髓,至肉眼可见骨质变白。收集含有骨髓的培养基,离心后弃去上清,加入含20ng/ml M-CSF的完全培养基,调整细胞浓度1*10 6ml于六孔板中,于铺板后第一、第四天更换新鲜含有20ng/ml M-CSF的完全培养基1ml/孔,第7天时即可分化成熟为巨噬细胞,认为此时巨噬细胞状态为M0,接下来可继续进行后续实验。若想得到M1/M2,则加入对应种属100ng/ml LPS+20ng/ml IFN-γ或20ng/ml IL-4+20ng/ml IL-1324h可诱导M0分化为M1或M2。微颗粒诱导前后巨噬细胞光镜下图如图1和图2所示。
2.微颗粒的提取
在10mm×10mm的培养皿中用10%FBS(胎牛血清)的培养基培养肿瘤细胞,待皿中细胞达到约5×10 6个时,以20GY的剂量进行放疗,放疗后的第一天换液,加入20ml含10%FBS的培养基,第3天收集培养皿内所有液体采用梯度离心法提取微颗粒。取放疗后的细胞培养基1000g离心10min后取上清,再将上清14000g,离心2min去除碎片后弃沉淀,最后将上清14000g、4℃离心60min,弃上清,沉淀即是微颗粒,将沉淀再用生理盐水洗两遍,1mlPBS(即磷酸缓冲盐)溶液重悬后4℃保存。
3.微颗粒的电镜鉴定
将收集的微颗粒重悬后取一部分至1ml离心管中,共计收集的量约0.5ml;涡旋仪轻微涡旋,以保证微颗粒处于悬浮状态,进行染色步骤;以2%磷钨酸溶液染色5min,滴于铜网上,干燥后透射电镜下观看MVs的大小及形态,如图3所示微颗粒电镜下呈圆形。
4.流式检测巨噬细胞表面及胞内标记物改变
将巨噬细胞消化成单细胞悬液离心并用1ml PBS(现配现用)重悬细胞并计数,调整细胞到合适浓度,转移至流式管中。向细胞悬液中加入FcR blocker(检测目的细胞为巨噬细胞故需要封闭),混匀,置于冰上放置5-10min。加入荧光抗体混合液,在冰上避光孵育30min(每10min混匀)后,加入3ml PBS,350g离心5min,此步骤重复2次。加入适量4%多聚甲醛,于室温避光固定30min后洗去,PBS重悬细胞等待检测。再用破膜工作液重悬固定后的细胞,350g离心5-10min,弃上清,并重复2次。用100ul破膜工作液重悬细胞,加入适量荧光抗体并混匀,在室温下避光孵育20min后,洗去抗体,PBS重悬细胞,等待上机检测。如图4所示,加入微颗粒之后,巨噬细胞表面的CD86相较于未加入微颗粒的巨噬细胞高表达,CD206低表达,提示微颗粒促进巨噬细胞向M1极化。
5.流式检测巨噬细胞对肿瘤细胞的吞噬
10μM CFSE孵育肿瘤细胞10分钟后用PBS洗3遍,消化肿瘤细胞并加入4倍细胞量的肿瘤细胞于巨噬细胞之中,6小时之后消化细胞,APC通道F4/80染巨噬细胞,操作步骤同4,重悬后等待上机检测。如图7所示,微颗粒极化后的巨噬细胞具有更强的吞噬肿瘤细胞的能力。
6.抗肿瘤巨噬细胞抗实体瘤的动物实验
建立Lewis-luc皮下瘤模型:以每只鼠5*104个Lewis-LUC细胞的接种量接种于C57右侧大腿根部皮下,注射体积为50μL。待瘤体体积达到20mm3后进行随机分组,剔除瘤体过大或过小的老鼠,分组为PBS组、微颗粒极化 M1组、微颗粒不极化M2组。腹腔回输巨噬细胞的量为每只老鼠5*104个。每两天注射一次,治疗3次。每两天测量一次瘤体体积大小。如图6所示,微颗粒极化的巨噬组可以更好地抑制肿瘤的生长。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。

Claims (6)

  1. 一种抗肿瘤巨噬细胞的制备方法,其特征在于,包括以下步骤:
    步骤1、获得原代骨髓干细胞或分离外周血中单核细胞进行原代细胞培养,加入集落刺激因子7天后获得巨噬细胞;
    步骤2、获得离体肿瘤细胞进行原代肿瘤细胞培养,并获得原代肿瘤细胞;
    步骤3、将所述步骤2得到的原代肿瘤细胞及培养液进行放疗照射,收集放疗后的上清液,获得所需微颗粒和凋亡的肿瘤细胞碎片混合物;
    步骤4、将所述步骤3得到的混合物进行离心,即得到所述携带肿瘤抗原微颗粒;
    步骤5、将所述步骤4得到的微颗粒处理步骤1获得的原代巨噬细胞,即得到所述抗肿瘤巨噬细胞。
  2. 根据权利要求1所述的一种抗肿瘤巨噬细胞的制备方法,其特征在于,步骤1中所述培养为含10ng/ml M-CSF(巨噬细胞集落刺激因子)、FBS(胎牛血清),并于第3天和第5天换液,第7天所得为巨噬细胞;0.04mg/ml步骤4)所得微颗粒处理50万巨噬细胞4小时所得即所述抗肿瘤巨噬细胞。
  3. 根据权利要求1所述的一种抗肿瘤巨噬细胞的制备方法,其特征在于,所述步骤3的放疗剂量为5-20Gray,所述上清液收集时间为放疗后的第2-7天;过氧化氢浓度为1-8mM,上清液收集时间为处理后6-24小时;氢氧化钠处理溶液PH为10-11,上清液收集时间为处理后24-48小时。
  4. 根据权利要求1所述的一种携带肿瘤抗原巨噬的制备方法,其特征在于,所述巨噬细胞为自体来源巨噬细胞,微颗粒为自体来源的肿瘤细胞或肿瘤细胞系产生。
  5. 权利要求1--5任意一项所述的一种抗肿瘤巨噬细胞的制备方法制得的抗肿瘤巨噬细胞,其特征在于,所述抗肿瘤巨噬细胞为促炎型巨噬细胞,形态如蛋花状,CD86和MHC II高表达、CD206低表达。
  6. 一种抗肿瘤巨噬细胞,其特征在于,由权利要求4中任一项所述的抗肿瘤微颗粒通过处理而获得。
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