Name: potentiation of anticancer antibody efficacy by antineoplastic drugs: detection of antibody-drug synergism using the combination index equation
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Description: Watch this Scientific Journal Video about Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation at JoVE.com

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。

法国政府批准了动物住房和实验程序 (#C44 278号和 #APAFIS 03479.01 的协定)。根据欧盟 201/63/欧盟和法国 #2013----118号关于保护用于科学目的的动物的法律----进行了动物照料和程序。
1. mab 8b6 与 topotecan 体外药物相互作用的评价
<ol><li>96孔样品制备 注意: 请咨询机构的健康和安全委员会, 并遵守与实验室安全相关的当地法规规则。在使用任何介质、细胞系或试?...

<ol>
	<li>Chou, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+basis,+experimental+design,+and+computerized+simulation+of+synergism+and+antagonism+in+drug+combination+studies.">Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies.</a> Pharmacological Reviews. 58 (3), 621-681 (2006).</li><li>Bayat Mokhtari, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+in+combating+cancer.">Combination therapy in combating cancer.</a> Oncotarget. 8 (23), 38022-38043 (2017).</li><li>Zahreddine, H., Borden, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+insights+into+drug+resistance+in+cancer.">Mechanisms and insights into drug resistance in cancer.</a> Frontiers in Pharmacology. 4, 28 (2013).</li><li>Martin, T. P., Baguley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re:+"Postoperative+validation+of+bone-anchored+implants+in+the+single-sided+deafness+population."+Snapp+et+al.+Otol+Neurotol+2012:+33;291-6.">Re: "Postoperative validation of bone-anchored implants in the single-sided deafness population." Snapp et al. Otol Neurotol 2012: 33;291-6.</a> Otol Neurotol. 34 (4), 777-778 (2013).</li><li>Choi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+lung+cancer+cells+by+altered+dimerization+of+HSP27.">Sensitization of lung cancer cells by altered dimerization of HSP27.</a> Oncotarget. 8 (62), 105372-105382 (2017).</li><li>Weiner, L. M., Surana, R., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies:+versatile+platforms+for+cancer+immunotherapy.">Monoclonal antibodies: versatile platforms for cancer immunotherapy.</a> Nature Reviews Immunology. 10 (5), 317-327 (2010).</li><li>Mellor, J. D., Brown, M. P., Irving, H. R., Zalcberg, J. R., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+the+role+of+Fc+gamma+receptor+polymorphisms+in+the+response+to+monoclonal+antibodies+in+cancer.">A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer.</a> Journal of Hematology &amp; Oncology. 6, 1 (2013).</li><li>Kowalczyk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GD2-specific+14G2a+monoclonal+antibody+induces+apoptosis+and+enhances+cytotoxicity+of+chemotherapeutic+drugs+in+IMR-32+human+neuroblastoma+cells.">The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells.</a> Cancer Letters. 281 (2), 171-182 (2009).</li><li>Retter, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+proapoptotic+antiganglioside+GM2+monoclonal+antibody+and+evaluation+of+its+therapeutic+effect+on+melanoma+and+small+cell+lung+carcinoma+xenografts.">Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.</a> Cancer Research. 65 (14), 6425-6434 (2005).</li><li>Nakamura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induction+of+human+lung+cancer+cell+line+in+multicellular+heterospheroids+with+humanized+antiganglioside+GM2+monoclonal+antibody.">Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.</a> Cancer Research. 59 (20), 5323-5330 (1999).</li><li>Cochonneau, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+arrest+and+apoptosis+induced+by+O-acetyl-GD2-specific+monoclonal+antibody+8B6+inhibits+tumor+growth+in+vitro+and+in+vivo.">Cell cycle arrest and apoptosis induced by O-acetyl-GD2-specific monoclonal antibody 8B6 inhibits tumor growth in vitro and in vivo.</a> Cancer Letters. 333 (2), 194-204 (2013).</li><li>Chou, T. C., Martin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CompuSyn for drug combinations: PC software and user&#8217;s guide: a computer program for quantitation of synergism and antagonism in drug combinations, and the determination of IC50 and ED50 and LD50 values. , (2005).</li><li>Alvarez-Rueda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monoclonal+antibody+to+O-acetyl-GD2+ganglioside+and+not+to+GD2+shows+potent+anti-tumor+activity+without+peripheral+nervous+system+cross-reactivity.">A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity.</a> PLoS One. 6 (9), e25220 (2011).</li><li>Faraj, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblastoma+chemotherapy+can+be+augmented+by+immunotargeting+O-acetyl-GD2+tumor-associated+ganglioside.">Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside.</a> Oncoimmunology. 7 (1), e1373232 (2017).</li><li>Ishikawa, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+functional+human+blood+and+immune+systems+in+NOD/SCID/IL2+receptor+{gamma}+chain(null)+mice.">Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.</a> Blood. 106 (5), 1565-1573 (2005).</li><li>Ullman-Cullere, M. H., Foltz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+condition+scoring:+a+rapid+and+accurate+method+for+assessing+health+status+in+mice.">Body condition scoring: a rapid and accurate method for assessing health status in mice.</a> Laboratory Animal Science. 49 (3), 319-323 (1999).</li><li>Teicher, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+in+vitro+and+in+vivo+synergy.">Assays for in vitro and in vivo synergy.</a> Methods in Molecular Medicine. 85, 297-321 (2003).</li><li>White, D. B., Slocum, H. K., Brun, Y., Wrzosek, C., Greco, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+nonlinear+mixture+response+surface+paradigm+for+the+study+of+synergism:+a+three+drug+example.">A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example.</a> Current Drug Metabolism. 4 (5), 399-409 (2003).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.</a> Journal of Immunological Methods. 65 (1-2), 55-63 (1983).</li><li>Huyck, L., Ampe, C., Van Troys, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+XTT+cell+proliferation+assay+applied+to+cell+layers+embedded+in+three-dimensional+matrix.">The XTT cell proliferation assay applied to cell layers embedded in three-dimensional matrix.</a> Assay and Drug Development Technologies. 10 (4), 382-392 (2012).</li><li>Thompson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+of+topotecan+in+combination+with+vincristine+for+treatment+of+pediatric+solid+tumor+xenografts.">Synergy of topotecan in combination with vincristine for treatment of pediatric solid tumor xenografts.</a> Clinical Cancer Research. 5 (11), 3617-3631 (1999).</li><li>Tan, M., Fang, H. B., Tian, G. L., Houghton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+design+and+sample+size+determination+for+testing+synergism+in+drug+combination+studies+based+on+uniform+measures.">Experimental design and sample size determination for testing synergism in drug combination studies based on uniform measures.</a> Statistic in Medicine. 22 (13), 2091-2100 (2003).</li><li>Tang, X. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+EPHB6,+EFNB2,+and+EFNB3+expressions+in+human+neuroblastoma.">Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.</a> Proceding of the National Academy of Sciences of the United States of America. 97 (20), 10936-10941 (2000).</li><li>Mehta, R. R., Graves, J. M., Hart, G. D., Shilkaitis, A., Das Gupta, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+metastasis+of+human+breast+carcinomas+with+Matrigel+in+athymic+mice.">Growth and metastasis of human breast carcinomas with Matrigel in athymic mice.</a> Breast Cancer Research and Treatment. 25 (1), 65-71 (1993).</li><li>Mullen, P., Ritchie, A., Langdon, S. P., Miller, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Matrigel+on+the+tumorigenicity+of+human+breast+and+ovarian+carcinoma+cell+lines.">Effect of Matrigel on the tumorigenicity of human breast and ovarian carcinoma cell lines.</a> International Journal of Cancer. 67 (6), 816-820 (1996).</li><li>Feng, C., Tang, S., Wang, J., Liu, Y., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topotecan+plus+cyclophosphamide+as+maintenance+chemotherapy+for+children+with+high-risk+neuroblastoma+in+complete+remission:+short-term+curative+effects+and+toxicity.">Topotecan plus cyclophosphamide as maintenance chemotherapy for children with high-risk neuroblastoma in complete remission: short-term curative effects and toxicity.</a> Nan Fang Yi Ke Da Xue Xue Bao. 33 (8), 1107-1110 (2013).</li><li>Cheung, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglioside+GD2+specific+monoclonal+antibody+3F8:+a+phase+I+study+in+patients+with+neuroblastoma+and+malignant+melanoma.">Ganglioside GD2 specific monoclonal antibody 3F8: a phase I study in patients with neuroblastoma and malignant melanoma.</a> Journal of Clininical Oncology. 5 (9), 1430-1440 (1987).</li><li>Nair, A. B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> Journal of Basic Clinical Pharmacy. 7 (2), 27-31 (2016).</li><li>Dayde, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+burden+influences+exposure+and+response+to+rituximab:+pharmacokinetic-pharmacodynamic+modeling+using+a+syngeneic+bioluminescent+murine+model+expressing+human+CD20.">Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20.</a> Blood. 113 (16), 3765-3772 (2009).</li><li>Racki, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD-scid+IL2rgamma(null)+mouse+model+of+human+skin+transplantation+and+allograft+rejection.">NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.</a> Transplantation. 89 (5), 527-536 (2010).</li><li>Sherif, A., Winerdal, M., Winqvist, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Responses+to+Neoadjuvant+Chemotherapy+in+Muscle+Invasive+Bladder+Cancer.">Immune Responses to Neoadjuvant Chemotherapy in Muscle Invasive Bladder Cancer.</a> Bladder Cancer. 4 (1), 1-7 (2018).</li></ol>

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?...

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。
<img alt="Equation 1" src="/files/ftp_upload/58291/58291eq1.jpg">
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。
<img alt="Equation 2" src="/files/ftp_upload/58291/58291eq2.jpg" style="font-size: 14px;">
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。

<table border="0" cellpadding="0" cellspacing="0" style="width:1389px;" width="1390">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cell Proliferation kit (MTT)</td>
			<td style="width:161px;">Roche</td>
			<td style="width:132px;">11-465-007-001</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CompuSyn software</td>
			<td style="width:161px;">ComboSyn</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Combosyn can be downloaded for free at http://www.combosyn.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Electric shaver</td>
			<td style="width:161px;">Bioseb</td>
			<td style="width:132px;">BIO-1556</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fetal calf serum</td>
			<td style="width:161px;">Eurobio</td>
			<td style="width:132px;">CVFSVFF00-01</td>
			<td style="width:852px;">10% heat-inactivated fetal calf serum in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Firefox&#160;</td>
			<td style="width:161px;">Mozilla Corporation</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Firefox can be downloaded for free at http://www.mozilla.org/en-US/firefox/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heat lamp</td>
			<td style="width:161px;">Verre&#38;Quartz</td>
			<td style="width:132px;">4003/1R</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Human neuroblastoma IMR-5 cell line</td>
			<td style="width:161px;">Accegen Biotechnology</td>
			<td style="width:132px;">ABC-TC0450</td>
			<td style="width:852px;">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)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">L-glutamine</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">25030-024</td>
			<td style="width:852px;">2 mM in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Lysis solution&#160;</td>
			<td style="width:161px;">Roche&#160;</td>
			<td style="width:132px;">11-465-007-001</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">mAb 8B6</td>
			<td style="width:161px;">University of Nantes</td>
			<td style="width:132px;">N/A</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Matrigel</td>
			<td style="width:161px;">Corning</td>
			<td style="width:132px;">354248</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Multiskan FC</td>
			<td style="width:161px;">Thermofischer Scientific&#160;</td>
			<td style="width:132px;">N08625</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Needle 21G 1 &#189;&#160;</td>
			<td style="width:161px;">BD Microlance</td>
			<td style="width:132px;">304432</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Needle 25G 1</td>
			<td style="width:161px;">Terumo</td>
			<td style="width:132px;">NN-2525R</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">NSG mice</td>
			<td style="width:161px;">Charles River Laboratories</td>
			<td style="width:132px;">5557</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Nunc MicroWell 96-well microplates</td>
			<td style="width:161px;">Thermofisher</td>
			<td style="width:132px;">167008</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PBS</td>
			<td style="width:161px;">VWR</td>
			<td style="width:132px;">L182-10</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PBS, 0,05% EDTA</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td>E9884</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PC that runs windows 7</td>
			<td style="width:161px;">Microsoft</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Windows 7 can be purchased at http://www.microsoft.com/en-gb/software-download/windows7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Penicillin-Streptomycin</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">15140-122</td>
			<td style="width:852px;">100 units/mL penicillin and 100 mg/mL streptomycin in RPMI 1640</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Reagent reservoir</td>
			<td style="width:161px;">Thermofischer Scientific</td>
			<td style="width:132px;">8094</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Rodent restrainer</td>
			<td style="width:161px;">Bioseb</td>
			<td style="width:132px;">TV-150-SM</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI 1640</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">31870-025</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe 1 mL</td>
			<td style="width:161px;">Henke Sass Wolf</td>
			<td style="width:132px;">5010.200V0</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Topotecan</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:132px;">T2705</td>
			<td style="width:852px;"></td>
		</tr>
	</tbody>
</table>

potentiation of anticancer antibody efficacy by antineoplastic drugs: detection of antibody-drug synergism using the combination index equation

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。

代表性结果和数字根据允许适应从更加早期出版的工作14。
抗 oggd2 mab 8b6 协同增强托普替康对神经母细胞瘤细胞系生长的抑制作用:
为了建立用于评估 topotecan 和 mab 8b6 之间协同作用的药物和抗体浓度, 首先用 mAb 法测定了人 imr5 神经母细胞瘤细胞的药?...

Watch this Scientific Journal Video about Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation at JoVE.com

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

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。

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

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

免疫疗法与化疗相结合是开发癌症有效疗法的创新方法。主要目标是实现协同，治疗效果，剂量和毒性减少，并尽量减少或延迟诱导耐药性。然而，出于实际和伦理原因，无法确定患者的协同能力。因此，在临床试验中直接结合之前，应进行体外和/或动物结合研究，以获得临床研究的理由。一个简单的强健方法，以药物之间的协同作用量化是基于由S shouan Talale开发的组合指数方程，如本视频所示。利用这种方法及其计算机化模拟，我们在此证明了单克隆抗体8B6之间的协同作用，该协同性是O-乙酰-GD2刚利赛德神经母细胞瘤抗原和化疗药物托普托特在神经母细胞瘤细胞细胞上特有的。简言之，在确定人类IMO中每种化合物的有效剂量50值后，用两种化合物的公平比率对这些细胞进行插管，并使用自动计算机模拟计算组合指数值。我们接下来在Vivvo，在一个IMO，5肿瘤异种模型中确认了这些结果。我们在T75烧瓶中生长了 IMR-5 细胞，在显微镜下观察它们，以检查细胞汇合。从烧瓶中洗出细胞介质，用5 mL的PBS清洗。添加 3 mL 的 0.05%EDTA/PBS 溶液。将烧瓶返回孵化器 3 分钟。在显微镜下检查细胞培养，了解细胞分离。将10 mL的完整细胞介质加入烧瓶中，并将细胞悬浮液转移到无菌的15 mL锥形管中。使用血细胞计在300克下离心5分钟。取出并丢弃上一液。在"完全生长介质"中重新悬浮细胞。调整介质体积，获得10万细胞/mL.种子84孔的最终浓度，96孔培养板，每个孔有1万个细胞，即100微升细胞悬浮液。在细胞培养箱中孵育细胞18小时.在500微升完全生长介质中培养单克隆抗体，获得单克隆抗体浓度240微克/mL的抗体工作溶液。在500微升完全生长介质中稀释药物，获得最终浓度为120纳米摩尔的药物工作溶液。执行协议中所示的 5、双倍连续错觉。稀释同样的方式药物加上单克隆性一体阳极性溶液。为了最终浓度，在相应的井中转移50微升每个药物溶液，如本实验布局所示。在培养箱中孵育细胞72小时。在每个井中加入10微升的MTT试剂溶液。在37摄氏度下孵育4小时。使用多通道移液器将 100 微升的 lycine 溶液添加到每井中，然后通过移液彻底混合。在37摄氏度下在加湿室中孵育4小时。使用分光光度计将吸收器以 570 和 620 纳米的速度进行除流。按如下计算细胞生存能力。使用以下等式计算影响分数值。运行仿真软件，单击新的实验按钮以获取主窗口。键入实验的名称。单击新的单一药物。键入名称。键入缩写。键入药物浓度单位。输入数据 1 剂量和 FA 值。按 Enter.重复此步骤，直到输入所有数据点。单击"完成"，按照相同的步骤进入单克隆抗体数据点。单击新药组合.选择药物和单克隆抗体。选择"恒定比率".单击"确定"。键入名称。键入缩写。类型药物单克隆抗体比。输入数据 1 剂量。按 Enter.程序将自动计算单克隆抗体 8B6 和组合的剂量。输入数据 1 FA 值。按 Enter.重复此步骤，直到输入所有数据点。单击"完成"，然后单击"生成报告"，选择药物和单克隆抗体，然后单击"确定"。选择组合，然后单击"确定"。选择标题、CI 表和摘要表，然后单击"确定"。键入文件名和分析，然后单击"保存"以生成报告。报表会自动在默认 Web 浏览器中打开。报告包含一个汇总表部分，包括标题、日期、最终名称、说明注释、m、Dm 和 r 参数、用作单一疗法或组合的代理的有效剂量 50，以及有效剂量 50、75、90 和 95.组合指数值小于 1 的组合指数值的组合指数表表示协同。值等于 1 表示附加性。值大于 1 表示对抗。在使用前将小瓶淹没在4摄氏度的冰箱中的冰中，将地下室膜基质解冻过夜。所有培养磨损，或与地下室膜公制接触的介质应预先磨光。收获文化 IMR5 。将电池转移到15 mL锥形管中，以300克离心5分钟。丢弃上一杯。用 15 mL 冰冷 PBS 清洗细胞两次。在冰冷的PBS中，为每mL500万个细胞准备细胞悬浮液。将电池悬浮液转移到 1.5 mL 微管中。旋转地下室膜基质小瓶。加入1卷基底膜基质试剂，通过移液混合，获得每mL250万细胞的细胞悬浮液。剃光将进行注射的侧翼。混合细胞，小心地将细胞悬浮液绘制成装有21g针头的1 mL注射器。检查是否确保注射器中没有气泡。用防腐剂溶液对小鼠的接种区域进行消毒。轻轻挤压注射部位手指之间侧翼上的鼠标皮。将针头完全插入皮肤折叠中。不要将针头深入组织，以确保皮下注射。在皮下注射100微升的 IMR5 细胞悬浮液。旋转注射器以防止液体扩散并提取针头。监测小鼠的体重和肿瘤生长。用口径测量肿瘤的长度和宽度。使用此公式计算肿瘤体积。当肿瘤达到平均体积 50 至 60 mm 时开始治疗

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Detection of Protease Activity by Fluorescent Peptide Zymography

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

长距离反向PCR对热线1的逆变活性的检测

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...