Engineered Human IgG Antibodies with Longer Serum Half-lives in Primates

Abstract

Antibody therapy is coming of age, with 15 monoclonal antibodies approved for therapeutic use in the United States and many others currently undergoing clinical trials (1). The advent of antibody engineering over the past two decades has contributed to the recent clinical success of therapeutic antibodies. The development of chimeric (2) and humanized (3) antibodies not only reduced the potent immunogenicity of rodent antibodies in humans but also improved the serum halflives and efficacy of such therapeutics compared with rodent antibodies. Phage display (4) and other display technologies have led to the ability to increase the affinity of antibodies for their target antigens. More recently, antibody engineering has been used to modify the effector functions of antibodies by altering their binding to C1q (5) and various Fc␥ receptors (6).The neonatal Fc receptor (FcRn) 1 is a heterodimer that comprises a transmembrane ␣ chain with structural homology to the extracellular domains of the ␣ chain of major histocompatibility complex class I molecules, and a soluble light chain consisting of ␤2-microglubulin (␤2m) (7). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (8). Evidence for the latter role initially came from studies indicating an unusually short serum halflife for IgG antibodies in ␤2m-deficient mice (9 -11). This observation led to the generation of mutant mouse hinge-Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (12). Recently, several studies have identified human IgG 1 mutants with enhanced FcRn binding (6, 13), although no improvement in the serum half-lives of these mutants was observed in mice (13) or reported in primates.The binding of IgG to FcRn is sharply pH-dependent; IgG binds to FcRn under mildly acidic conditions and is released under slightly basic conditions (14). It has been hypothesized that pinocytosed IgG antibodies are captured by FcRn in acidified endosomes, rescued from degradation in lysosomes, recycled back to the cell surface, and returned to the circulation (8). Mutagenesis studies have identified both the mouse (15, 16) and human (17) Fc residues believed to be important in mediating pH-dependent binding. The results of the mutagenesis studies are consistent with the interpretation of a crystallographic study of the Fc⅐FcRn interaction (18). In the current study, molecular modeling was used to identify residues in the human IgG Fc near the FcRn binding site that, when mutated, might alter binding to FcRn without affecting the pH dependence of this interaction. Following exhaustive mutagenesis at these positions, several IgG 2 mutants were identified with improved binding to FcRn at pH 6.0 that retained the property of pH-dependent release. A pharmacokinetics study in rhesus monkeys showed that two mutant IgG 2 antibodies with increased FcRn binding affinity had considerably longer serum half-lives than the wild-type antibody. EXPERIMENTAL PROCEDURESMolecular Modeling-Molecular models o...

Extending human IgG half-life using structure-guided design

“…These include residues that make direct contact with FcRn, as well as peripheral and non-surface exposed residues that have the potential to modify the interaction surface and/or influence the dynamics of the 250-helix. Indeed, some of these identified positions have been previously investigated, such as within the half-life enhancing mutants YTE, 10 LS (M428L/N434S), 13 AAA (T307A/E380A/N434A), 27 QL (T250Q/M428L) 11 and V308P. 28 In our analysis, we first investigated the role of each residue in silico , and identified the network of interactions mediated by that residue (Figure 2).…”

“…10–13 The FcRn affinity-enhancing mutants in these studies were identified from phage display coupled with directed mutagenesis, 14–16 alanine scanning, 17 or, in select cases, from molecular modeling and rational design. 11,13 The prototypical example of an FcRn affinity-enhancing Fc mutant is the M252Y/S254T/T256E (YTE) mutation which, when incorporated into motavizumab IgG1, is able to extend serum half-life in humans by more than four-fold. 18 In these studies, the Fc was primarily optimized for FcRn binding.…”

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

“…TMDD effects may be ameliorated by engineering pH-dependent binding, 44 and nonspecific binding 32 and FcRn affinity [9][10][11][12][13][14] can be modulated by antibody engineering. The somewhat limited success of engineering efforts to improve antibody clearance PK by modulating FcRn affinity 5 may be in many instances attributable to dominant nonspecific tissue clearance mechanisms that operate independently of FcRn recycling, as demonstrated for the antibodies in this report.…”

“…5 The residues of the IgG1 Fc portion that interact with FcRn are well established, [6][7][8] and as such many efforts have focused on improving FcRn affinity to improve PK. [9][10][11][12][13][14] In addition to Fc engineering efforts, multiple studies have reported disparate PK parameters in antibodies with identical Fc regions but different variable regions. [15][16][17][18][19][20] In particular, clearance rates are increased in antibodies with polyreactive [21][22][23] or cross-reactive 24,25 profiles.…”

Inhibition of transferrin iron release increases in vitro drug carrier efficacy

“…For example, a mutated version of granulocyte colony stimulating factor (GCSF) which favors the cellular recycling pathway results in a significantly extended GCSF half-life [17]. In addition, IgG antibodies with mutated Fc regions resulting in greater binding affinities for the FcRn receptor in the endosome show an increase in cellular recycling, leading to longer half-lives in vivo [18].…”