Engineered human IgG antibodies with longer serum half-lives in primates.

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.


The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies.
A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG 2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG 2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys ϳ2-fold longer than the wild-type antibody.
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 PROCEDURES
Molecular Modeling-Molecular models of the human Fc⅐FcRn complex were generated based on the crystal structures (18,19) and a model (20) of the rat Fc⅐FcRn complex. First, the rat ␤2m and FcRn ␣ chains of the complex were replaced, respectively, with the human ␤2m (21) and FcRn ␣ chains (22). Next, the rat Fc residues of the complex were replaced with the corresponding human IgG 1 Fc residues (23), and then energy minimization calculations were done using SEGMOD and ENCAD (24,25) to produce a model of the human IgG 1 Fc⅐FcRn complex. The process was repeated to produce a model of the complex of human FcRn and the M3 variant of human IgG 2 (IgG 2 M3) (26).
IgG Mutagenesis, Expression, and Purification-The light and heavy chain cDNAs from a trioma cell line expressing the human anti-hepatitis B virus (HBV) antibody OST577 (27) were cloned by PCR. The light and heavy chain V-genes were converted by PCR into mini-exons and subcloned, respectively, into pV2, a derivative of pVk (28) containing the human 2 constant region, and the M3 variant of pVg2.D.Tt (26). Overlap extension PCR (29) was used to generate random amino acid substitutions at positions 250, 314, or 428 (numbered according to the EU index (23)) in the heavy chain of OST577-IgG 2 M3, until each possible amino acid at these positions was obtained. The resulting PCR fragments were subcloned into pVAg2M3-OST577, a derivative of the M3 variant of pVg2.D.Tt containing the pUC18 replication origin (30).
Human kidney cell line 293-H (Invitrogen) was transiently cotransfected with the antibody expression plasmids using the LipofectAMINE 2000 reagent (Invitrogen). Culture supernatants were concentrated and buffer exchanged into PBS, pH 6.0, with Vivaspin centrifugal concentrators (Vivascience, Hannover, Germany). Mouse myeloma cell line Sp2/0 (American Type Culture Collection, Manassas, VA) was stably cotransfected by electroporation. OST577-IgG 2 M3 antibodies were pu-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
FcRn Cloning and Surface Expression-Human ␤2m and FcRn ␣ chain cDNAs were cloned by PCR from human peripheral blood mononuclear cells and subcloned into pDL172, a derivative of pVk containing a glycosylphosphatidylinositol linkage signal from human decay-accelerating factor (31) fused to the FcRn ␣ chain, resulting in pDL208. Mouse myeloma cell line NS0 (European Collection of Animal Cell Cultures, Salisbury, Wiltshire, UK) was stably transfected with pDL208 by electroporation, yielding cell line NS0-HuFcRn.
Competitive Binding Assays-Transiently expressed IgG 2 M3 wildtype and mutant antibodies were tested for binding to human FcRn in a single-point competitive binding assay. Briefly, 2 ϫ 10 5 NS0-HuFcRn cells/test were washed with FACS binding buffer (FBB) (PBS containing 0.5% bovine serum albumin, 0.1% NaN 3 ), pH 8.0, and then with FBB, pH 6.0, and resuspended in 120 l of biotinylated OST577-IgG 2 M3 (8.3 g/ml) and concentrated supernatant (containing 8.3 g/ml of competitor antibody) in FBB, pH 6.0. After 1 h on ice, the cells were washed with FBB, pH 6.0, and resuspended in 25 l of 2.5 g/ml streptavidin-conjugated R-PE (BioSource, Camarillo, CA) in FBB, pH 6.0. After 30 min on ice, the cells were washed with FBB, pH 6.0, resuspended in 1% formaldehyde in PBS, and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences).
Purified IgG 2 M3 wild-type and mutant antibodies were further tested in a competitive binding assay using purified competitor antibody (2-fold serial dilutions from 208 to 0.102 g/ml) as described above. IC 50 values were calculated using GraphPad Prism, version 3.02 (GraphPad Software, San Diego, CA).
Rhesus Pharmacokinetics Study-A non-GLP ("good laboratory practice") pharmacokinetics (PK) study was approved by the Institutional Animal Care and Use Committee and conducted at the California National Primate Research Center (University of California, Davis). Twelve male rhesus macaques were randomized by weight and assigned to one of three study groups. Each animal received a single intravenous dose of wild-type or one of two mutants of OST577-IgG 2 M3 at 1 mg/kg by infusion over a span of 15 min. Blood samples were drawn prior to dosing on day 0, at 1 and 4 h after dosing, and at 1, 7, 14, 21, 28, 42, and 56 days. The concentrations of the OST577-IgG 2 M3 wildtype and mutant antibodies in rhesus serum samples were determined using a qualified enzyme-linked immunosorbent assay. Appropriately diluted serum samples and calibrators were captured with a mouse anti-OST577-IgG 1 idiotype monoclonal antibody (OST577-␥1 anti-id; Protein Design Labs, Inc.) and detected with horseradish peroxidaseconjugated goat anti-human light chain antibody (Southern Biotechnology Associates, Birmingham, AL). The serum antibody concentration data were fitted with a two-compartment model using WinNonlin Enterprise Edition, version 3.2 (Pharsight, Mountain View, CA).

Identification of IgG Mutants with Altered Binding to
FcRn-Molecular models of the human Fc⅐FcRn complex (Fig.  1) guided the selection of positions 250, 314, and 428 of the human IgG heavy chain for mutagenesis. Although the wildtype amino acids at these positions are located near the Fc⅐FcRn interface, it does not appear likely that they directly contribute to the pH-dependent interaction between Fc and FcRn. Inspection of the molecular models suggested that amino acid substitutions at these positions might increase or decrease the affinity of Fc for FcRn, without disrupting pH-dependent binding and release, by affecting the conformation of Fc amino acids that do interact with FcRn. Since the amino acids at and around these positions are conserved among all four human IgG subtypes (23), it is reasonable to expect that the FcRn binding phenotype resulting from an amino acid substitution in one IgG subtype could be transferred to the other three IgG subtypes. In this study, an IgG 2 M3 form (26) of the human anti-HBV monoclonal antibody OST577 (27) was chosen for mutagenesis because it would not be expected to bind either to antigen or Fc␥ receptors in HBV-free primates.
PCR mutagenesis was used to generate all 19 single amino acid substitutions at each position. Transiently expressed OST577-IgG 2 M3 mutants were screened for binding to NS0-HuFcRn cells in a single-point competitive binding assay (Fig.  2). Several of the mutants at positions 250 (e.g. Glu and Gln) ( Fig. 2A) and 428 (e.g. Phe and Leu) (Fig. 2C) appeared to be stronger competitors in this assay than the wild-type antibody, indicating that these mutants have increased binding to FcRn at pH 6.0. At position 250, it appears that the presence of a hydrogen bond acceptor in the appropriate geometry (e.g. Glu or Gln) is important for better FcRn binding, since smaller but chemically related residues (e.g. Asp or Asn) reduced binding to FcRn. At position 428, a large hydrophobic amino acid confers better FcRn binding. None of the mutations at position 314 (Fig. 2B) resulted in increased binding to FcRn.
In a previous study (13), human IgG 1 Fc position 428 was randomly mutated, and mutants were screened for binding to mouse FcRn by phage display; however, no mutants at this position with increased binding to mouse FcRn were obtained. This could be because of biases in the construction of the library, the loss of certain sequences during propagation in Escherichia coli, or incomplete sampling of the library during screening. Another explanation is that mouse rather than human FcRn was used to pan the human IgG 1 Fc mutant library in the previous study (13). Because the amino acids at and around position 428 are not conserved between the mouse and human Fc regions, it follows that an Fc mutant at position 428 may interact differently with mouse and human FcRn.
The best competitors among the mutants at positions 250 and 428 were stably expressed either alone (T250Q, M428L) or in combination (T250Q/M428L), and purified antibodies were compared in a competitive binding assay to human FcRn (Fig.   FIG. 1. Model of human IgG 2 M3 Fc⅐FcRn complex. The Fc chains are colored gray (proximal to FcRn) and yellow (distal to FcRn). For the receptor, ␤2m is dark blue, and the FcRn ␣ chain is light blue. Thr-250 is depicted in red and Met-428 in green.

Engineered Antibodies with Longer Serum Half-lives 6214
3). Comparison of the IC 50 values indicated that the single mutants T250Q and M428L showed an increase in binding to human FcRn at pH 6.0 of ϳ3and 7-fold, respectively, whereas the double mutant T250Q/M428L showed an increase in binding of ϳ28-fold. To confirm that binding was pH-dependent, the antibodies were allowed to bind to NS0-HuFcRn cells at pH 6.0 and were removed by washing the cells at pH 6.0, 6.5, 7.0, 7.5, or 8.0. As the pH value of the washes in successive samples was raised from pH 6.0 to 8.0, the binding of the wild-type and mutant antibodies to human FcRn was comparably diminished, with essentially no binding observed at pH 7.5 or above (data not shown). Similar results were obtained when these mutant antibodies were tested for binding to rhesus FcRn. The binding of the T250Q, M428L, and T250Q/M428L IgG 2 M3 mutants to rhesus FcRn at pH 6.0 was ϳ4-, 8-, and 27-fold better than the wild-type antibody, respectively, and binding to rhesus FcRn was pH-dependent (data not shown).
Pharmacokinetics of IgG Wild-type and Mutant Antibodies in Rhesus Monkeys-The PK behavior of the OST577-IgG 2 M3 wild-type, M428L, and T250Q/M428L antibodies was examined in rhesus monkeys. The PK profiles of the two mutants are clearly distinct from that of the wild-type (Fig. 4). The mean serum antibody concentrations of the M428L and T250Q/ M428L mutants were maintained at higher levels than wildtype OST577-IgG 2 M3 at all time points. Because the mean maximum serum antibody concentration (C max , Table I) was very similar among the three test groups, indicating that the administered antibodies were distributed to the circulation in a similar manner, the higher concentrations of the mutant IgG 2 M3 antibodies thereafter are attributable to their increased persistence in the serum. Analysis of the mean clearance (CL) indicated that this was the case. The mean CL, the volume of serum antibody cleared per unit of time, was ϳ1.8fold lower for the M428L mutant (0.0811 Ϯ 0.0384 ml/h/kg; p ϭ 0.057), and ϳ2.8-fold lower for the T250Q/M428L mutant (0.0514 Ϯ 0.0075 ml/h/kg; p ϭ 0.029) compared with wild-type OST577-IgG 2 M3 (0.144 Ϯ 0.047 ml/h/kg) (Table I), indicating a significant decrease in the clearance of the OST577-IgG 2 M3 M428L and T250Q/M428L mutants.
The PK profiles of the OST577-IgG 2 M3 wild-type and mu-   Table I). The elimination half-life for wild-type OST577-IgG 2 M3 in this study is similar to that for OST577-IgG 1 (324 Ϯ 85 h) in a previous PK study in rhesus monkeys (27).
Although there is an indication from the CL and AUC parameters that the T250Q/M428L mutant may have increased serum persistence compared with the M428L mutant, this difference may not be significant. Since the elimination half-lives of the two mutants appear similar, it is possible that a maximal increase in serum persistence has been achieved in rhesus monkeys with these mutants. The T250Q mutant, which showed a modest increase in binding to rhesus and human FcRn, may be expected to show an intermediate increase in serum half-life in primates.
While the M428L and T250Q/M428L amino acid substitutions are presumed to account for the observed increase in the elimination half-lives of the IgG 2 M3 antibodies, it is possible that post-translational modifications (e.g. glycosylation, Met oxidation) might affect their PK properties. However, it is unlikely that glycosylation differences would account for the observed half-life differences because previous studies have indicated that glycosylated and aglycosylated IgG antibodies have similar half-lives in mice (32) and chimpanzees (33). Moreover, carbohydrate analysis of the OST577-IgG 2 M3 wildtype and mutant antibodies revealed only minor differences in their glycosylation patterns (data not shown).
Engineered antibodies with increased serum half-lives might prove valuable in antibody therapy. For example, it may be possible to reduce the frequency of administration of such antibodies. This will be a great benefit to patients undergoing long-term antibody therapy. Based on the results described in this study, it is reasonable to expect that human IgG 1 , IgG 3 , and IgG 4 antibodies with longer serum half-lives also may be engineered by transferring the M428L or T250Q/M428L mutations into these IgG subtypes. In addition, it should now also be possible to alter the serum half-lives of other IgG-related therapeutics such as IgG Fc fusion proteins using this approach. Provided that the mutations described in this study do not substantially increase the immunogenicity of these therapeutics in humans, IgG antibodies and Fc fusion proteins with longer serum half-lives should represent a potent new class of human therapeutics. Pharmacokinetic parameters were calculated using WinNonlin (Pharsight). The group mean Ϯ S.D. is shown for each parameter. Mann-Whitney tests were done using GraphPad Prism (GraphPad Software) to compare the statistical significance of differences in the pharmacokinetic parameters between the wild-type (WT) group and each mutant group. C max , maximum serum antibody concentration; t1 ⁄2 , elimination (␤-phase) half-life. *, indicates a significant difference (p Ͻ 0.060).