Tae Hyun Kang

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

Bypassing glycosylation: engineering aglycosylated full-length IgG antibodies for human therapy

In recent years a number of aglycosylated therapeutic antibodies have entered the clinic. The clinical evaluation of these antibodies has served to dispel concerns that the absence of the ubiquitous N297 glycan in the Fc of IgG might result in immunogenicity, poor in vivo stability or unfavorable pharmacokinetics. Importantly, recent studies have now demonstrated that aglycosylated antibodies can be engineered to display novel effector functions and mechanisms of action that do not appear to be possible with their glycosylated counterparts. Moreover, the ability to manufacture aglycosylated antibodies in lower eukaryotes or in bacteria provides significant bioprocessing advantages in terms of shorter bioprocess development and running times and by completely bypassing the problems associated with the glycan heterogeneity of conventional antibodies. These advantages are poised to catapult aglycosylated antibodies to the forefront of protein therapeutics.

Effective Phagocytosis of Low Her2 Tumor Cell Lines with Engineered, Aglycosylated IgG Displaying High FcγRIIa Affinity and Selectivity

Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.

Influenza immunization elicits antibodies specific for an egg-adapted vaccine strain

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

IgG Fc domains that bind C1q but not effector Fc[gamma] receptors delineate the importance of complement-mediated effector functions

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Efficient expression and purification of human aglycosylated Fcγ receptors in Escherichia coli

Effector Fc gamma receptors (FcγRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcγRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcγRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcγR genes and optimization of sequences for N‐terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcγRI and FcγRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcγRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcγRs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21–30.

Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.