抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用
Summary
该方案介绍了如何利用周药和塔拉莱的组合指数方程来评价临床前模型中抗癌抗体与抗肿瘤药物之间的协同作用。
Abstract
化疗药物对敌对单克隆抗体 (mab) 的潜在作用是设计有效、更安全的癌症治疗方法的一个有价值的策略。在这里, 我们提供了一个协议, 以确定一个合理的组合在临床前的步骤。首先, 我们描述了一种基于细胞的检测方法, 利用周和塔拉莱1的组合指数方程来评价抗癌 mab 与细胞毒性药物之间的协同作用.这包括使用 mtt 检测肿瘤细胞药物和抗体敏感性, 然后进行自动计算机分析, 以计算组合指数 (ci) 值。ci 值和 lt;1 表明 mabs 与细胞毒性剂1之间的协同作用.为了证实体内的体外发现, 我们进一步描述了一种方法来评估在异种移植肿瘤模型中的组合方案的有效性。在这种模型中, 联合方案显著延缓了肿瘤的生长, 与单剂对照相比, 这将显著延长生存期。重要的是,体内实验表明, 组合方案是耐受性良好。该方案允许在临床前模型中有效评估抗癌药物组合, 并确定合理的组合, 以便在临床试验中进行评估。
Introduction
治疗大量不同类型癌症的常规方法是以单一疗法为基础的。即使它仍然在许多情况下使用, 这种方法遇到了几个障碍, 导致选择综合疗法2。特别是, 癌细胞在用单一药物治疗时更容易产生耐药性, 通过诱导替代生存机制3, 导致4例患者的治疗失败。此外, 在单一疗法中, 药物通常是在高剂量的。这种情况往往会导致强烈的剂量依赖性副作用的发生, 这可能是不能容忍的, 并迫使医生停止治疗2。由于这些原因, 抗癌分子的关联现在是首选的单一疗法。
理想的药物组合将是那些对肿瘤细胞协同作用的药物组合, 而不会增加对正常细胞的毒性。协同作用是指两种或两种以上药物的相互作用, 它们产生的治疗效果大于每种药物单独作用的总和。这种相互作用可能会提高临床治疗效果2。它限制了治疗的抵抗力, 提高了疗效, 也可以减少毒性2。事实上, 每种药物的剂量都可以通过针对不同的途径来降低副作用。此外, 其中一种分子还可以作为对抗癌细胞的增敏剂。第二种药物对致敏细胞的作用可以增强, 使用剂量较少, 可使用 5.
综合治疗可包括两种或两种以上的化疗药物和/或生物制剂, 如单克隆抗体6。这些 mabs 专门针对表达感兴趣的细胞表面抗原的细胞, 能够通过免疫途径杀死肿瘤细胞, 包括抗体依赖细胞介导的细胞毒性 (adcc), 并参与免疫效应细胞的作用7、和互补相关的细胞毒性 (cdc)6。它们还可以通过细胞凋亡8、9、10、11介导的非免疫机制发挥作用。在这种情况下, 诱导程序性细胞死亡过程可能会敏感癌细胞, 削弱其功能, 并使相关的化疗药物更有效, 在较低的剂量。因此, 原凋亡 mab 是设计抗肿瘤药物组合方案的理想选择。
不同的数学模型被描述了评估药物协同作用;其中之一是基于组合索引方法1。该方法以周一开发的中效应原理为基础.中效应方程将药物剂量与药物效应相关联, 如下所示。
在这里, d是药物剂量;dm是中效应剂量;法是受剂量影响的分数;m是表示剂量效应图1形状的指数.中效应剂量用于计算抑制或杀死 “x”% 细胞的药物的剂量dx .然后计算 ci 值, 以评估药物组合的加性效应, 如下所示。
ci 值为1表示加性效应, ci 值表示 lt;1 表示协同作用, 而 ci 值表示 gt;1 表示拮抗 1.计算机程序 “compusyn” 的可用性进一步促进了这种方法的应用, 该程序在自动模拟的所有剂量或效果水平下确定协同作用和拮抗作用。
本组开发了 o-乙酰-gd2 神经母细胞苷 (oacgd2) 神经母细胞瘤抗原 13的 mab 8b6 特异性 mab 8b6, 并进一步证明, 这种 mab 能够诱导细胞死亡, 具有凋亡的属性11。为了测试 mab 8b6 是否能对抗肿瘤药物 topotecan 敏感, 我们采用了周1开发的上述方法.首先, 我们确定了 mab 8b6 和 topotecan 的有效剂量 50 (ed50)。接下来, 利用上述模拟软件, 暴露基于 ed50值的两种化合物的等能比神经母细胞瘤细胞, 以确定 ci 值。这种方法使我们能够在体外证明 mab 8b6 和 topotecan 之间的协同作用。接下来, 我们描述一个协议, 以进一步评估这种组合方案在体内的效力和安全性。该方案可方便地应用于临床前研究中有效、安全的抗癌 mab 和化疗药物组合的选择。图 1提供了这项研究的示意图。
Protocol
Representative Results
Discussion
为了预测药物相互作用的影响, 可以采用三种方法: 等深线图方法 17、非线性混合模型18和组合指数1。组合索引分析是最常用的, 因为它的应用通过用户友好的计算机程序的可用性得到了简化。为此, 我们首先通过执行 mtt 检测19来描述单独或联合使用的每个制剂的剂量效应反应.这种方法依赖于活细胞的能力, 以减少四氮盐在紫?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
赠款支助: 南特大学基金会, les bagouz ‘ a manon, la ligue contre le confres comnitéde loire-atlantique, comitédu morbihan 和 comitéde vendée, une rose pour s. a. r. a. h, l ‘ etoile de martin and la sociétéfrançaise de lutte contre les les les les les les les lescancers et les leucémies de l ‘ enfant et de l ‘ 卫生组织 (sfce)。m. b. 和 j. f. 由 la ligue contre le trocr 提供支持。提交人感谢结构 fédéédéter of Recherche françois bonamy 的 ute 设施。提交人还感谢 s. suzin 博士 (巴黎 inserm) 提供了 imr5 细胞, 并感谢 h. estéphan 女士提供的技术援助。
Materials
| Cell Proliferation kit (MTT) | Roche | 11-465-007-001 | |
| CompuSyn software | ComboSyn | Combosyn can be downloaded for free at http://www.combosyn.com | |
| Electric shaver | Bioseb | BIO-1556 | |
| Fetal calf serum | Eurobio | CVFSVFF00-01 | 10% heat-inactivated fetal calf serum in RPMI 1640 |
| Firefox | Mozilla Corporation | Firefox can be downloaded for free at http://www.mozilla.org/en-US/firefox/ | |
| Heat lamp | Verre&Quartz | 4003/1R | |
| Human neuroblastoma IMR-5 cell line | Accegen Biotechnology | ABC-TC0450 | IMR-5 is a clone of the human neuroblastoma cell line IMR32 5459762. IMR-5 cells were generously provided by Dr. Santos Susin (U.872, Paris, France) |
| L-glutamine | Gibco | 25030-024 | 2 mM in RPMI 1640 |
| Lysis solution | Roche | 11-465-007-001 | |
| mAb 8B6 | University of Nantes | N/A | |
| Matrigel | Corning | 354248 | |
| Multiskan FC | Thermofischer Scientific | N08625 | |
| Needle 21G 1 ½ | BD Microlance | 304432 | |
| Needle 25G 1 | Terumo | NN-2525R | |
| NSG mice | Charles River Laboratories | 5557 | |
| Nunc MicroWell 96-well microplates | Thermofisher | 167008 | |
| PBS | VWR | L182-10 | |
| PBS, 0,05% EDTA | Sigma-Aldrich | E9884 | |
| PC that runs windows 7 | Microsoft | Windows 7 can be purchased at http://www.microsoft.com/en-gb/software-download/windows7 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | 100 units/mL penicillin and 100 mg/mL streptomycin in RPMI 1640 |
| Reagent reservoir | Thermofischer Scientific | 8094 | |
| Rodent restrainer | Bioseb | TV-150-SM | |
| RPMI 1640 | Gibco | 31870-025 | |
| Syringe 1 mL | Henke Sass Wolf | 5010.200V0 | |
| Topotecan | Sigma-Aldrich | T2705 |
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