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Aglycosylated Igg Variants Expressed in Bacteria That Selectively Bind Fcγri Potentiate Tumor Cell Killing by Monocyte-Dendritic Cells

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Aglycosylated Igg Variants Expressed in Bacteria That Selectively Bind Fcγri Potentiate Tumor Cell Killing by Monocyte-Dendritic Cells Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells Sang Taek Junga, Sai T. Reddya, Tae Hyun Kangb, M. Jack Borroka, Inger Sandliec, Philip W. Tuckerb,d, and George Georgioua,b,d,e,1 aDepartment of Chemical Engineering, bInstitute for Cellular and Molecular Biology, dSection of Molecular Genetics and Microbiology, and eDepartment of Biomedical Engineering, University of Texas, Austin, TX 78712; and cDepartment of Molecular Biosciences and Center for Immune Regulation, University of Oslo, 0316 Oslo, Norway Edited by Frances H Arnold, California Institute of Technology, Pasadena, CA, and approved November 12, 2009 (received for review July 31, 2009) The N-linked glycan of immunoglobulin G (IgG) is indispensable The effector FcγRs interact with the upper CH2 and hinge re- for the interaction of the Fc domain with Fcγ receptors on effector gion of the IgG-Fc domain (Fig. 1A) in a manner that is critically cells and the clearance of target cells via antibody dependent dependent on the presence and composition of the single cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, N-linked glycan attached to N297. In contrast, FcRn, which binds aglycosylated Fc domains bind effector FcγRs poorly and cannot to the interface of CH2 and CH3 (5), is not significantly affected elicit ADCC. Using a novel bacterial display/flow cytometric library by the glycosylation status of the antibody constant region. The screening system we isolated Fc variants that bind to FcγRI (CD64) glycosylation state of IgG determines its affinity for effector with nanomolar affinity. Binding was critically dependent on ami- FcγRs and thus, modulates the activation of cytotoxic immune no acid substitutions (E382V, and to a lesser extent, M428I) distal to cells and their ability to participate in antibody-dependent cell- the putative FcγRI binding epitope within the CH3 domain. These mediated cytotoxicity (ADCC) (6, 7). ADCC is important for mutations did not adversely affect its pH-dependent interaction the clinical efficacy and outcomes of many important antibody with FcRn in vitro nor its serum persistence in vivo. Remarkably, therapeutics (8). Glycan composition and glycoform heterogene- the anti-Her2 IgG trastuzumab containing the E382V, M428I substi- ity, which is intrinsic to the expression of antibodies in mamma- tutions and expressed in E. coli exhibited highly selective binding lian cells, has a well-documented effect on the response to widely to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) used therapeutics such as Rituxan (rituximab, anti-CD20) and nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated Herceptin (trastuzumab, anti-Her2) (8, 9). This has led to intense version of trastuzumab (E382V, M428I) purified from HEK293T cells efforts to generate improved antibodies via glycoengineering (7). bound to all Fcγ receptors in a manner similar to that of clinical Wild-type aglycosylated antibodies can be expressed at a high grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), yield in bacterial cells and exhibit normal serum persistence but not glycosylated trastuzumab (E382V, M428I) or clinical grade and binding to FcRn (10). However, their lack of glycosylation trastuzumab, was capable of potentiating the killing of Her2 renders them completely unable to bind to FcγRs, and therefore overexpressing tumor cells with dendritic cells (DCs) as effectors. they do not induce FcγR-mediated effector functions (9). Re- These results indicate that aglycosylated IgGs can be engineered cently, structure-based mutagenesis and, in a separate study to display unique FcγR selectivity profiles that, in turn, mediate screening of small saturation libraries by yeast display, were used ADCC via mechanisms that are not normally displayed by glycosyl- to isolate aglycosylated IgG1 variants lacking N297 and carrying ated monoclonal antibodies. mutations in residues S298 and T299 that conferred binding to FcγRIIIa or FcγRIIa, respectively (11, 12). However, the respec- antibody engineering ∣ bacterial display ∣ bacterial expression ∣ tive antibody variants, following expression in HEK293 cells, directed evolution ∣ effector function exhibited significant binding to the inhibitory FcγRIIb receptor (12). This result was not surprising given the high degree of γ γ c receptors (FcRs) play a crucial role in linking the adaptive homology between Fc RIIa and Fc RIIb. Fimmune system to the effector functions of innate cells such as We have developed a system that relies on bacterial expression antigen presentation and antibody homeostasis (1). Human and flow cytometric screening of very large libraries of Fc mutants immune cells express six immunoglobulin (IgG) binding FcγRs for the isolation of variants that display a high degree of selec- B (FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb) that tivity and receptor affinity (Fig. 1 ). In this study, we focused γ enable various functions, including phagocytosis, cytokine pro- on the isolation of aglycosylated IgG that bound to Fc RI, as duction, superoxide production, release of serotonin, and inhibi- its role in ADCC is far less understood than that of other acti- tion of B cell activation (1, 2). An additional IgG binding vating receptors (1, 2). It has been suggested that because of γ receptor, the neonatal Fc receptor (FcRn), plays a critical role its high affinity, Fc RI on effector cells is occupied by circulating in IgG homeostasis by mediating antibody recycling via intracel- IgG and thus is involved in ADCC only during the early stage of lular trafficking, thus preventing degradation in lysosomes (3). the immune response when antibody concentration is low (13). With the exception of FcγRIIIb, human FcγRs are type I trans- However, the avidity associated with the formation of immune γ membrane glycoproteins. Activating FcγRs such as FcγRI complexes must be capable of displacing soluble IgG from Fc RI (CD64), FcγRIIa (CD32a), and FcγRIIIa (CD16a) have an immunoreceptor tyrosine-based activation motif in their cyto- Author contributions: S.T.J. and G.G. designed research; S.T.J., S.T.R., T.H.K., and M.J.B. plasmic tail and trigger activation of immune responses. The inhib- performed research; I.S. contributed new reagents and analytic tools; S.T.J., S.T.R., itory receptor FcγRIIb (CD32b) processes an immunoreceptor T.H.K., M.J.B., P.W.T., and G.G. analyzed data; and S.T.J. and G.G. wrote the paper. tyrosine-based inhibitory motif in its cytoplasmic domain that The authors declare no conflict of interest. downregulates immune responses (4). The affinity of FcγRII This article is a PNAS Direct Submission. −6 and FcγRIII to monomeric IgG is very low (Kd ¼ ∼10 − 1To whom correspondence should be addressed. E-mail: [email protected]. 10−7 γ M), whereas Fc RI exhibits high affinity to monomeric This article contains supporting information online at www.pnas.org/cgi/content/full/ −8 −9 IgG (Kd ¼ ∼10 − 10 M). 0908590107/DCSupplemental. 604–609 ∣ PNAS ∣ January 12, 2010 ∣ vol. 107 ∣ no. 2 www.pnas.org/cgi/doi/10.1073/pnas.0908590107 Downloaded by guest on September 24, 2021 Fig. 1. Isolation of high-affinity aglycosylated IgG1-Fc to FcγR. (A) Binding epitopes of Fc receptors and C1q on IgG-Fc (8). (B) Schematic showing the display system for library screening. (C) Effect of trehalose on the periplasmic display of Fc. Cells expressing either IgG-Fc domain or the M18 scFv as a control were grown with or without 0.5 M trehalose, spheroplasted and labeled with 2.5 μg∕ml of Protein A-FITC for detection. (D) Fluorescence histogram showing spheroplasted cells expressing either wt IgG-Fc or highly fluorescent variants labeled with 30 nM of FcγRI-FITC (P < 0.001 using unpaired Student’s t-test relative to wt IgG-Fc) isolated from the fourth round. M: Mean fluorescence intensity. and consistent with this hypothesis there is increasing evidence protein retention following spheroplasting, even in the presence of that the activation of this receptor plays an important role in 0.5 M trehalose (Fig. S1C). the resolution of certain infections and in cancer (14–16). We report here the engineering of aglycosylated IgG anti- High Throughput Screening of Aglycosylated Fc Libraries for bodies that contain amino acid substitutions in the CH3 domain Increased FcγRI Binding. The retention of ligand-accessible Fc in that are distant from the FcγR-binding epitope located in the spheroplasted cells was exploited for the flow cytometric screen- CH2 domain yet confer low nanomolar affinity to FcγRI but ing of large libraries for FcγR binding. We initially constructed not to the inhibitory FcγRIIb nor to other effector FcγRs. libraries composed of (i) random mutants by error-prone PCR Engineered, bacterially expressed trastuzumab IgGs potentiated (∼9.2 × 108 transformants) containing 0.5% nt substitutions the lysis of Her2 overexpressing SkBr3 breast cancer cells by per gene (as determined by the sequencing of 20 random effector monocyte-derived dendritic cells (mDCs). Strikingly, clones) and (ii) 10-a.a. random insertions encoded by NNS glycosylated IgGs, clinical grade trastuzumab (Herceptin) or randomization scheme (where N ¼ equimolar A; T; C; G; mammalian cell-expressed trastuzumab that contained the same S ¼ equimolar C; G) (∼2.8 × 107 transformants representing a CH3 domain mutations failed to activate the cytotoxic ability of miniscule fraction of the theoretical diversity) between N297 mDCs. Our results indicate that aglycosylated IgGs engineered to and S298 with the intent of replacing the glycan that is normally display unique FcγR-binding characteristics may be useful both attached to N297 with a peptide epitope. Transformants from the for elucidating the function of FcγRI and for the selective recruit- two libraries were pooled and, following induction of protein ment of mDCs in cancer therapy. synthesis, spheroplasts were prepared and labeled with the appro- γ B priate Fc R conjugated to FITC (Fig. 1 ). Highly fluorescent SCIENCES Results clones were isolated by FACS. The respective genes were rescued Bacterial Display of Aglycosylated Fc Domains.
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