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一场来自抗体药开发与免疫原性的爱恨情仇

2018-11-28 15:27

这个故事先从抗体药的现状开始吧:


1986-2017 年的 31 年间,共有 157 个治疗性生物大分子药获 FDA 批准上市,其中抗体药最多,共 81 种。甚至 2016、2017 年所有获批上市的生物大分子药均是抗体药,一个月前EMA批准诺菲纳米抗体药物成为首个上市的纳米抗体药物的文章刷遍生物人的朋友圈,国内方面,康宁杰瑞率先申报靶向PD-L1的纳米抗体KN035,并随后获得FDA批准临床研究。


有媒体这样描述:“整个抗体药产业正处于一个令人激动的Turning Point时期!“目前,抗体药市场一路高歌,是生物药中的王牌,也是各药企巨头的必争之地。


抗体药的发展的迅速有目共睹,随着市场规模的日渐扩大,政策的支持,资本的青睐,预示着整个抗体药产业将会有更好的发展。恒瑞医药、康弘药业、康宁杰瑞、百济神州以及众多企业也纷纷涌入抗体药市场。


一场来自抗体药开发和免疫原性的战争也与之拉开序幕。。。


从诺和诺德长效重组Factor Ⅶ在临床Ⅲ期被叫停,到辉瑞PCSK9抑制剂在临床Ⅲ期宣布放弃开发,免疫原性带来的问题一次次给抗体药物开发下了绊子。


如何发现免疫原性的问题呢,如何让免疫原性和抗体药物开发的问题被提早发现呢?


免疫原性分析作为免疫原性评价的重要手段是时候站出来了。


但是,免疫原性分析一直以来缺乏明确的可操作的指导原则。


FDA老大哥表示,路总是要走的,问题总是要解决的。于是继2009版免疫原性指导原则之后,2016版FDA免疫原性指导原则(试行)的发布给免疫原性分析指明了道路,把两个相隔7年的制度原则进行比较,我们可以发现以下变化:


免疫原性的评价方式主要采用抗药抗体(anti-drug antibody, ADA)分析的方法进行。分析技术包括ELISA、ECL、SPR、RIA、MSD等,可灵活采取一种或几种组合,目前最常用的技术是ELISA和MSD。试验设计为多步检测法,包括筛选、确证、滴度和中和试验:



由于不可能将受试者体内的所有抗药抗体作为阳性对照,无法准确反映样本中抗药抗体的真实情况,免疫原性分析只能采用半定量的方式进行,一般都是使用内部开发的单克隆抗体或多克隆抗体。


为了解决免疫原性分析的问题以缩短抗体开发时间,ACROBiosystems公司开发了一系列高亲和力、高特异性的抗独特型抗体,应用于免疫原性分析。我们根据开发人员的实际应用场景,对每一个抗独特性抗体开发相应的实验方案,希望最大程度上加速药物研发的进程。


以下为应用案例:


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应用案例一:Anti-Adalimumab Antibodies(ADB-Y19)


Anti-Adalimumab Antibodies bridging ELISA for Anti-Drug Antibody (ADA) assay development. Immobilized adalimumab at 1 µg/ml, added increasing concentrations of Anti-Adalimumab Antibodies (Cat. No. ADB-Y19, 10% human serum) and then added biotinylated adalimumab at 5 µg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 0.6 ng/mL.



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Measured by its neutralizing ability in a functional ELISA. Immobilized adalimumab at 0.5 μg/mL (100 μL/well) can bind pre-mixed Anti-Adalimumab Antibodies (Cat. No.ADB-Y19) and Biotinylated Human TNF-alpha (Cat. No. TNA-H82E3) with an inhibition rate of 100%.

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应用案例2- Anti-Rituximab Antibodies(RIB-Y35)


Anti-Rituximab Antibodies bridging ELISA for Anti-Drug Antibody (ADA) assay development. Immobilized rituximab at 5 µg/ml, added increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35, 10% human serum) and then added biotinylated rituximab at 5 µg/ml. Detection was performed using HRP-conjugated streptavidin with a sensitivity of 20 ng/mL.

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Measured by its neutralizing ability in FACS. The data shows that the binding of rituximab to 293F overexpressing CD20 was inhibited by increasing concentrations of Anti-Rituximab Antibodies (Cat. No. RIB-Y35). The concentration of rituximab used is 10 ng/ml. The IC50 is 0.013 μg/ml.

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同时,对于每一个产品,ACROBiosystems还做了一系列实验来保证产品的高亲和力、高特异性和高稳定性。目前产品已覆盖adalimumab, rituximab, cetuximab, trastuzumab, bevacizumab等多个已上市的热门抗体药。


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高亲和力


亲和力代表了抗原和抗体间的结合强度,用KD值表示,KD值越小,抗体的亲和力越强。我们用SPR方法检测了所有抗独特型抗体的亲和力,了解这一指标,有助于在方法学建立过程中预测检测方法的灵敏度。

Anti-Adalimumab Antibodies (mouse IgG1, Cat. No. ADB-Y19) captured on CM5 chip via anti-mouse antibodies surface, can bind human adalimumab with an affinity constant of 1.36 pM.


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高特异性


对于每一个产品,我们都进行了特异性检测,包括对应的抗体药、抗体药靶点、不同IgG亚型和同亚型其他抗体药,只有结合对应的抗体药,而不结合其他蛋白或抗体的产品才会被放行。


Demonstration of the specificity of Anti-Cetuximab Antibodies (Cat. No. CEB-Y28) to the cetuximab.


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高稳定性


为确保产品稳定性,ACROBiosystems会做加速实验和冻融实验来验证产品稳定性,只有经过测试证实质量和活性没有任何影响的产品才会被放行。

Accelerated Stability Test

Reconstituted Anti-Trastuzumab Antibodies were diluted to 0.4 mg/ml, aliquoted and placed at 37°C. Aliquots were removed from 37°C at every time point and placed at 4°C along with the control. No significant loss of activity was observed.


Freeze-thaw Test

Anti-Trastuzumab Antibodies were subjected to the indicated number of freeze-thaw cycles (FT). No significant loss of activity was observed.


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产品列表(不断更新中)


Cat. No.

Antigen

Neutralizing Activity

Affinity KD, nM

Application

ADB-Y19

Adalimumab F(ab’)2

Neutralizing Antibody

0.0013

ADA assay

Neutralizing assay

Indirect ELISA

CEB-Y28

Cetuximab F(ab’)2

Neutralizing Antibody

0.0015

ADA assay

Neutralizing assay

Indirect ELISA

RIB-Y35

Rituximab F(ab’)2

Neutralizing Antibody

0.03

ADA assay

Neutralizing assay

Indirect ELISA

BEB-Y12

Bevacizumab F(ab’)2

Neutralizing Antibody

0.0828

Neutralizing assay

Indirect ELISA

BEB-Y9

Bevacizumab F(ab’)2

Neutralizing Antibody

1.92

ADA assay

Indirect ELISA

更多产品问题可点击下方按钮咨询,或直接拨打400-682-2521进行咨询~

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