Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation

Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) Rabbit Polyclonal to SFRS7 and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody. or through mutation of Asn297 to remove the glycosylation site for production in mammalian cells.7 However, aglycosylation can affect the conformation10 and stability11 of the Fc. Glycosylated IgG4 is an alternative with a preferred version having the hinge mutation S228P (Eu numbering) to stabilize the antibody against Fab arm exchange.12 Although in general IgG4 isotype antibodies have reduced capacity to support in vitro ADCC, in vivo target cell depletion activity in humans has been noted with an IgG4 version of an anti-CD52 antibody.13 As with IgG1 isotype antibodies produced in CHO cells,14 there could be variability in the fucose levels on IgG4 antibodies. Afucosylation has been demonstrated previously to increase the in vitro ADCC activity of an anti-CD20, IgG4 isotype antibody.9 Here, we show that afucosylation does increase the in vivo B cell depletion activity of an IgG4 isotype anti-CD20 antibody, suggesting that, for some IgG4 antibodies produced in CHO, it may be necessary to monitor fucosylation status in order to limit cell killing activity. Results Production and receptor-binding measurements on anti-human CD20 IgG4 KRas G12C inhibitor 3 isotype antibody An KRas G12C inhibitor 3 anti-human CD20 antibody (x-huCD20) was chosen for tests of IgG4 isotype switch on activity because an assay of ADCC activity on huCD20 expressing cell lines is readily available,15 the level of activity for the IgG1 subclass antibody is sensitive to fucosylation status,16 and in vivo activity can be measured in pre-clinical models.17,18 An x-huCD20-IgG4 heavy chain construct was expressed in both Chinese hamster ovary (CHO) and Fut8-knock-out (KO) CHO cells. Antibody was easily purified from both expression hosts using standard methods and shown to bind CD20 in a WIL2 cell-based assay (Fig.?S1). CD20-binding of the 2 2 preparations of x-huCD20.IgG4 was similar, but appeared to be slightly weaker than the binding of x-huCD20.IgG1 (CHO). The relative binding affinities compared to IgG1 were 0.5 and 0.75 for IgG4 produced in CHO and Fut8-KO CHO cells, respectively. As expected, 100% afucosylated glycan was observed for the antibody obtained from expression in the Fut8KO host. The percentage of afucosylated glycan on IgG4 antibody produced transiently in CHO was higher than for IgG1 or mIgG2a subclass antibodies purified from stably transfected CHO cells (Table?1). The reproducibility and generality of this effect KRas G12C inhibitor 3 has not been tested. Table 1. Fucose levels on mAbs. = 0.99; error bars represent corresponding standard error of the mean and in most cases are smaller than the data point symbol. Depletion of B cells in mouse model using anti-mouse CD20.IgG4 antibody Given that afucosylation increased the in vitro ADCC activity of IgG4 antibody, we tested whether this translated to in vivo activity in an animal model relevant to human disease. A model of B cell depletion in mice has been used previously to compare therapeutics intended for treatment of B cell malignancies.18 In mice, a change in B cell population in the blood and tissues upon antibody treatment is easily monitored using fluorescence-activated cell sorting (FACS) analysis. For our purposes, we used mice having the murine FcRIIIa knocked out and transgenic for human FcRIIIa-Val158. However, since these mice are not transgenic for human CD20, and because x-huCD20 doesn’t bind murine CD20, it was necessary to change to an antibody specific for murine CD20. Mouse monoclonal antibody 5D2.16.8 was chosen, reformatted as a mouse/human IgG4 chimera, and expressed in both CHO and Fut8KO cells. As shown by FACS analysis (Fig.?S2), the IgG4 antibody is functional for binding mCD20. The IgG4 version of 5D2 showed very weak binding across a panel of mouse FcR; however, afucosylation did increase apparent binding affinity to mFcRIV (Supplemental Table?1). Since the fucosylated antibody produced in CHO had very weak binding to mFcRIV, it was not possible to calculate the fold improvement in binding accompanying afucosylation. Administration of 5D2.mIgG2a at either 12.5?g or KRas G12C inhibitor 3 125?g led to a rapid depletion of peripheral blood B cells KRas G12C inhibitor 3 (Fig.?4A) relative to the isotype control antibody, anti-ragweed. 5D2.hIgG4 (CHO C 29% afucosylated) at either dose did not show significant activity for B cell depletion. In contrast, 5D2.hIgG4 (Fut8KO-CHO C 100% afucosylated) showed activity comparable to.