Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.
Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.
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DOI：
10.1002/bit.26545
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发表时间：
2018-05
影响因子：
3.8
通讯作者：
Ackerman ME
中科院分区：
文献类型：
--
作者：
Boesch AW;Kappel JH;Mahan AE;Chu TH;Crowley AR;Osei-Owusu NY;Alter G;Ackerman ME
随着抗体继续在药物发现和开发管道中占据主导地位，控制和优化其体内活性的努力已经成熟，可以结合复杂的能力来操纵特定Fc结合伙伴的参与。这些促进多种功能结果的努力包括调节IgG-Fc对FcγRs的亲和力，以交替增强或减少效应功能，如抗体依赖性细胞毒性和吞噬作用。虽然在体外和体内已经证明了许多能够引发可变效应功能的天然和工程Fc特征，但通过使用各种遗传，细胞和酶技术来阐明这些重要的功能关系需要付出巨大的努力。作为一种正交方法，我们证明了使用Fcγ r作为色谱亲和配体，可以从血清衍生的多克隆人IgG的复杂混合物中富集并同时识别出有利的结合种，这是一种包含Fc变体和翻译后修饰的负载材料。富含fc γ r的IgG被鉴定为亚类和糖型组成，这一生物分离步骤对抗体活性的影响在基于细胞的效应功能测定中被测量，包括自然杀伤细胞活化和单核细胞吞噬。这项工作展示了一种易于处理的方法，通过利用亲和层析，然后用高分辨率生物物理和功能分析进行二次分析，快速区分两种或多种相互作用的生物制剂之间的复杂功能关系，并强调了一个能够测量多种自然翻译后修饰的平台，这些修饰可能不容易用高纯度生产或容易用重组表达技术获得。
As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques.
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影响因子：
3.7
作者：
Dashivets T;Thomann M;Rueger P;Knaupp A;Buchner J;Schlothauer T
通讯作者：
Schlothauer T
影响因子：
4.1
作者：
Boesch AW;Alter G;Ackerman ME
通讯作者：
Ackerman ME
影响因子：
6.4
作者：
Drescher, B;Witte, T;Schmidt, RE
通讯作者：
Schmidt, RE
影响因子：
2.2
作者：
Al-Hubeshy, Z. B.;Coleman, A.;Goodier, M. R.
通讯作者：
Goodier, M. R.
影响因子：
15.9
作者：
Boruchov, AM;Heller, G;Young, JW
通讯作者：
Young, JW