Rouwendal Gerard Ja, van der Lee Miranda M, Meyer Saskia, Reiding Karli R, Schouten Jan, de Roo Guy, Egging David F, Leusen Jeanette Hw, Boross Peter, Wuhrer Manfred, Verheijden Gijs F, Dokter Wim H, Timmers Marco, Ubink Ruud
a Synthon Biopharmaceuticals B.V. ; Nijmegen ; The Netherlands.
b Laboratory for Translational Immunology; University Medical Center Utrecht ; Utrecht ; The Netherlands.
MAbs. 2016;8(1):74-86. doi: 10.1080/19420862.2015.1102812. Epub 2015 Oct 6.
Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.
单体IgA已被提议作为癌症治疗的一种替代抗体形式。在此，我们展示了关于抗HER2 IgA抗体作为抗癌药物的生产、纯化和功能评估的研究，并将其与抗HER2 IgG1曲妥珠单抗进行了比较。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析显示，在所使用的IgA同种型和细胞系中，糖基化特征存在显著差异，包括唾液酸化及其连接方式、岩藻糖基化（核心和天线状）以及高甘露糖型糖链种类的丰度。唾液酸化的增加被证明与体内血浆半衰期呈正相关。抗HER2 IgA2m2的聚合倾向可通过重链尾段18个氨基酸的缺失（与高甘露糖型N-聚糖种类的丧失一致）以及320位和480位的2个半胱氨酸到丝氨酸的突变来抑制。IgA2m1和IgA2m2亚型的HER2 F(ab')2介导的抗增殖作用与IgG1相似，而IgA1同种型的效力和功效则显著较低。使用人全血抗体依赖的细胞介导的细胞毒性（ADCC）试验进行的Fc介导的ADCC诱导，并未显示出IgA同种型之间有如此明显的差异。然而，在这些ADCC试验中，抗HER2 IgA抗体的效力被发现显著低于曲妥珠单抗。在BT-474乳腺癌异种移植模型中，将抗HER2 IgA抗体的体内抗肿瘤活性与曲妥珠单抗进行了比较。与单剂量的IgG1相比，IgA抗体的多次给药和唾液酸化弥补了其在小鼠体内天然IgA抗体较短的体内半衰期。就IgA2m2抗体而言，由此产生的高血浆暴露水平足以导致明显的肿瘤停滞，与在低得多的血浆暴露水平下观察到的曲妥珠单抗的情况相当。
