MABS 2016, VOL. 8, NO. 6, 1098–1106 http://dx.doi.org/10.1080/19420862.2016.1189049

REPORT

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation Qian Gonga, Meredith Hazenb, Brett Marshallc, Susan R. Crowelld, Qinglin Oua, Athena W. Wonge, Wilson Phungf, Jean-Michel Vernesg, Y. Gloria Mengg, Max Tejadac, Dana Andersenh, and Robert F. Kelleyi a Department of Immunology, Genentech Inc., South San Francisco, CA, USA; bDepartment of Antibody Engineering, Genentech Inc., South San Francisco, CA, USA; cDepartment of Biological Technologies, Genentech Inc., South San Francisco, CA, USA; dDepartment of Preclinical and Translational Pharmacokinetics, Genentech Inc., South San Francisco, CA, USA; eDepartment of Early Stage Cell Culture, Genentech Inc., South San Francisco, CA, USA; f Department of Protein Chemistry, Genentech Inc., South San Francisco CA, USA; gDepartment of Biochemical and Cellular Pharmacology, Genentech Inc., South San Francisco, CA, USA; hDepartment of Pharmaceutical Development, Genentech Inc., South San Francisco, CA, USA; iDepartment of Drug Delivery, Genentech Inc., South San Francisco, CA, USA

ABSTRACT

ARTICLE HISTORY

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal (“afucosylation”). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. 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) 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 FcgRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.

Received 4 March 2016 Revised 26 April 2016 Accepted 7 May 2016

Introduction The crystallizable fragment (Fc) imparts effector function to full-length antibodies. These activities include long serum halflife due to binding of the Fc portion to the recycling FcRn receptor,1 activation of complement-dependent cytotoxicity (CDC) initiated through c1q binding to the Fc,2 and antibodydependent cell-mediated cytotoxicity (ADCC), which occurs through engagement of Fcg receptors on effector cells.3 Notably, binding of antibodies to FcgRIIIa on natural killer (NK) cells enables the immune system to target foreign cells for destruction. ADCC can be employed in therapeutic agents to remove pathologic cells including malignant tumor cells. For example, non-Hodgkin’s lymphoma (NHL) is effectively treated with a combination of a chemotherapy regimen and administration of the anti-CD20 antibody rituximab.4 Rituximab binds CD20 on normal and cancerous B cells, targeting them for destruction. Response to rituximab therapy is linked to a polymorphism in the FcgRIIIa receptor whereby patients having the higher Fc-affinity allele Val158, as compared with Phe158, show a more robust clinical response.5 These observations suggest that ADCC activity is required for the full effectiveness of rituximab therapy in patients afflicted with NHL. These results have prompted the development of antibodies CONTACT Robert F. Kelley [email protected] Supplemental data for this article can be accessed on the publisher’s website. © 2016 Genentech Inc.

KEYWORDS

ADCC; B cell depletion; CD20; IgG subclass

such as obinutuzumab (GazyvaÒ ) that are engineered to have increased ADCC activity.6 Both ADCC and CDC require glycosylation of the Fc residue Asn297 whereas FcRn interaction does not, and thus aglycosylated antibodies retain long serum half-life.7 ADCC activity is modulated by glycan structure and removal of the penultimate fucose (“afucosylation”) can increase FcgRIIIa affinity and ADCC activity.8 In addition to glycan structure, FcgRIIIa affinity and ADCC activity are sensitive to the subclass of the IgG heavy chain. Human IgG1 and IgG3 isotypes show good binding and activity whereas IgG2 and IgG4 isotypes have reduced binding and activity.2,9 For some therapeutic applications, decreased or abolished ADCC activity is required. In cases where the antibody binds a cell surface receptor to inhibit a signaling pathway, it may be undesirable to cause depletion of cells that express the receptor. Decreased ADCC activity may be achieved by producing an IgG1 antibody in aglycosylated form or by changing the isotype to IgG2 or IgG4. Aglycosylation is accomplished through expression of the antibody in E. coli 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

MABS

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 antihuman CD20 IgG4 isotype antibody An 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 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 has not been tested. Binding of x-huCD20.IgG4 to a panel of human Fcg receptors was reduced relative to the IgG1 isotype (Table 2). As shown in Fig. 1, binding of x-huCD20.IgG4 to human FcgRIIIa-V158 was very weak and did not approach saturation Table 1. Fucose levels on mAbs. mAb x-huCD20.IgG1 (CHO) x-huCD20.IgG4 (CHO) x-huCD20.IgG4 (Fut8KO) 5D2.mIgG2a (CHO) 5D2.hIgG4 (CHO) 5D2.hIgG4 (Fut8KO)

% Afucosylated Glycan 6 11 § 0.9 100 § 0.2 4 § 0.8 29 § 2.2 100 § 0.4

IgG4 antibodies were produced by transient transfection of CHO cells; IgG1 and mIgG2a antibodies were expressed by stable transfection of CHO cells. Values for % afucosylated glycan measured using mAb-Glyco chip are shown § standard error.

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at the highest concentrations tested; however, increased binding was observed for the material isolated from the Fut8-KO host. Complete afucosylation increased binding to the F158 allotype was 3.8-fold and the increase for Val158 was > 15-fold (Table 2). As observed for IgG1 antibodies,19 the increase in binding upon afucosylation was specific to FcgRIIIa. ADCC activity of afucosylated x-huCD20.IgG4 ADCC activity of the various x-huCD20 antibodies on WIL2S tumor cells was assessed using a NK cell line as effector (Fig. 2). x-huCD20.IgG4 (CHO) showed measurable ADCC activity, albeit much reduced relative to x-huCD20.IgG1 (CHO). A comparison with x-huCD20.IgG4 (Fut8KO) indicates that complete afucosylation increases the activity by nearly 50-fold, calculated from the ratio of EC50 values (Table 3), to within about 5-fold of the activity measured for x-huCD20.IgG1 (CHO). We further probed the effect of IgG4 afucosylation on ADCC activity by testing preparations of varied fucose levels. It is difficult to produce preparations of defined but varied fucose levels unless procedures are used to modulate the activity of the fucosyl transferase. This approach generates a distribution of molecules with one or both heavy chains of the assembled antibody being afucosylated. Instead, we produced these materials by blending of CHO-produced and Fut8KO-CHO-produced antibodies in various ratios. In these blends, and since the heavy chains do not exchange, assembled molecules have essentially neither or both chains afucosylated. A similar approach has been used to establish a quantitative relationship between fucose levels and ADCC activity for the IgG1 version of xhuCD20.16 As shown in Fig. 3A, the ADCC activity of these samples increases as the fraction of Fut8KO-CHO-derived antibody is increased. ADCC activity is barely detectable if the afucosylation level is

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation.

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with dep...
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