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* MedImmune, Gaithersburg, MD 20878; and
Center for Immunology and Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-globulins, are mediated by the MHC
class I-related neonatal FcR (FcRn). A correlation exists in mice
between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and
their serum t1/2. To expand this
observation, phage display of mutagenized Fc fragments derived from a
human IgG1 was used to increase their affinity to both murine and human
FcRn. Ten variants were identified that have a higher affinity toward
murine and human FcRn at pH 6.0, with 
G (Gwild
type - Gmutant) from 1.0 to 2.0
kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants
exhibit a parallel increase in binding at pH 7.4 to murine, but not
human, FcRn. Although not degraded in blood in vitro, accumulated in
tissues, nor excreted in urine, their serum concentration in mice is
decreased. We propose that higher affinity to FcRn at pH 7.4 adversely
affects release into the serum and offsets the benefit of the enhanced
binding at pH 6.0. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FcRn is a heterodimer formed by the association of an L
(
2-microglobulin) and H (
-chain)
(13) chain. The -chain, composed of three domains
(1, 2, and 3), is anchored to the membrane via a transmembrane
region. The rat, murine, and human H chain sequences (7, 13, 14) as well as the rat and human FcRn structures
(15, 16, 17) are homologous to the class I major
histocompatibility complex molecules. Crystallographic studies of the
extracellular domain of unliganded human FcRn (17) and of
the extracellular domain of rat FcRn free and bound to rat IgG
(15, 16) have indicated that human and rat FcRn are
structurally similar. The most notable feature of the interaction of
murine and human FcRn with murine and human IgG is its pH dependency:
the Fc portion of IgGs binds FcRn with a high affinity at pH 6.0 and is
released at pH 7.2 (2, 18). This suggests, along with
colocalization studies (19), that for cells bathed in near
neutral pH, the IgG/FcRn complex forms within acidified endosomes. More
precisely, FcRn acts as a salvage receptor, binding and transporting
pinocytosed IgGs in intact form both within and across cells, and
rescuing them from a default degradative pathway. Although the
molecular mechanisms responsible for salvaging IgGs are still largely
unknown, it is thought that unbound IgGs are directed toward
proteolysis in lysosomes, whereas bound Igs are recycled to the surface
of the cells and released. This control takes place within the
endothelial cells located diffusely throughout adult tissues (5, 20).
Fc residues critical to the mouse Fc-mouse FcRn interaction have been
identified by site-directed mutagenesis. In particular, I253, H310,
H433, N434, and H435 (EU numbering) (21) are major
components of the functional epitope (3, 8, 22) in which
H310 and H435 are responsible for the pH dependence of binding
(23). I253, H310, and H435 were found to be critical for
the interaction of human Fc with murine FcRn (12).
Likewise, studies of the human Fc-human FcRn complex have demonstrated
the crucial role played by I253, S254, H435, and Y436 (8, 24). Functional epitopes on the rat FcRn have been also
identified by site-directed mutagenesis in both the -chain and
2-microglobulin chain (25, 26),
demonstrating which residues on the FcRn are critical for the
interaction with both mouse and rat IgG.
There seems to be a correlation between decrease (3) and
increase (11) in the affinity of murine hinge-Fc fragments
for murine FcRn at pH 6.0 and their serum
t1/2 in mice. The same correlation
exists when one considers the interaction of human hinge-Fc fragments
with mouse FcRn (8, 12). This observation has obvious
relevance for the generation of therapeutic Abs with increased serum
persistence. Although it has been proposed that a tighter IgG-FcRn
interaction at pH 7.5 can result in a decreased IgG serum persistence
(27), this phenomenon has not been investigated in detail.
Furthermore, most of the studies published to date have been limited to
the analysis of the interaction of rodent FcRn with rodent or human
IgGs (3, 11, 12, 16, 22, 23, 26, 28, 29, 30). Recent studies
involving the binding of human IgGs to human FcRn have provided
important information, such as the role of the receptor in the
maternofetal transfer of -globulin (8), the chemical
nature of the interface, and general characteristics of the interaction
(17), as well as the relative contribution of individual
residues to the energetics of complex formation (24).
Human Fc mutants with all solvent-exposed residues along the
CH2 and CH3 domains
substituted by alanine were produced (24). This led to the
identification of >12 variants with increased affinity to the human
receptor, most by a factor of 1.5- to 2-fold. Two combination mutants
showed increased affinity by up to 12-fold (24).
We have now further characterized the human Fc-human FcRn and human Fc-mouse FcRn interactions. Using rationally designed libraries and phage display, we provide evidence that a human IgG1 can be engineered for large (>30 times) increases in affinity toward both human and murine FcRn. As a step toward a better understanding of these interactions, we investigated the binding of different IgG1 variants at neutral pH. We show that IgG1 mutants exhibiting an increase in affinity toward murine FcRn by a factor of >5-fold at pH 6.0 also show significantly better binding at pH 7.4 when compared with the wild-type molecule. In contrast, IgG1 mutants exhibiting an affinity increase toward human FcRn by a factor of up to 10-fold at pH 6.0 all bind very poorly at pH 7.4. When injected in equal amounts into different BALB/c mice, three IgG1 variants with a 1.4- to 19-fold affinity increase to murine FcRn at pH 6.0 exhibit lower than wild-type serum concentrations (reported in ngvariant/wild type/mlserum). Those mutants are not proteolyzed more efficiently than a wild-type control in serum or whole blood, nor are they seen to accumulate in different tissues or be excreted at a detectable level in urine. We hypothesize that retention of binding at neutral pH offsets the advantage of improved binding at pH 6.0, leading to the intracellular sequestration of the corresponding IgG molecules in FcRn-containing tissues.
Our results suggest that the pH dependency of binding of IgG1 to FcRn plays a critical role in determining the biological effect of an affinity-improving substitution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals were of analytical grade. Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA). TG1 and CJ236 Escherichia coli were obtained from APBiotech (Piscataway, NJ) and Bio-Rad (Richmond, CA), respectively.
Expression and purification of murine and human FcRn
Recombinant mouse and human FcRn were produced as described
(8, 29). Human FcRn was also obtained following isolation
from human placenta cDNA (Clontech, Palo Alto, CA) of the genes for
human 2-microglobulin (21) and
codons -23–267 of the human -chain (7) using standard
PCR protocols. L and H chains along with their native signal sequence
(7, 21) were cloned in pFastBac DUAL and pFastBac1
bacmids, respectively, and viral stocks produced in Spodoptera
frugiperda cells (Sf9) according to the manufacturer’s
instructions (Invitrogen, Carlsbad, CA). High-Five cells were infected
at a multiplicity of infection of 3 with the baculoviruses encoding
- and 2-chains using commercially available
protocols (Invitrogen). Supernatant of infected insect cells was then
adjusted to pH 6.0 with hydrochloric acid and applied to a 10 ml IgG
Sepharose 6 Fast Flow column (APBiotech). The resin was washed with 200
ml 50 mM MES, pH 6.0, and FcRn eluted with 0.1 M Tris-Cl, pH 8.0.
Purified FcRn (>95% homogeneity, as judged by SDS-PAGE) was dialyzed
against PBS, flash frozen, and stored at -70°C.
Construction of IgG1/Fc libraries
A human hinge-Fc gene spanning aa residues 226–478 (Kabat numbering) (21), derived from MEDI-493 human IgG1 (31), was cloned into the pCANTAB5E phagemid vector (APBiotech) as an SfiI/NotI fragment. Four libraries were generated by introducing random mutations at positions 251, 252, 254, 255, 256 (library 1); 308, 309, 311, 312, 314 (library 2); 385, 386, 387, 389 (library 3); and 428, 433, 434, 436 (library 4), using a Kunkel-based strategy (32). Briefly, four distinct hinge-Fc templates were generated using PCR by overlap extension (33), each containing one TAA stop codon at position 252 (library 1), 310 (library 2), 384 (library 3), or 429 (library 4), so that only mutagenized phagemids will give rise to Fc-displaying phage. Each TAA-containing ssDNA (TAAssDNA) was then prepared as follows: a single CJ236 E. coli colony harboring one of the four relevant TAA-containing phagemid was grown in 10 ml 2x YT medium supplemented with 10 µg/ml chloramphenicol and 100 µg/ml ampicillin. At OD600 = 1, VCSM13 helper phage (Stratagene, La Jolla, CA) was added to a final concentration of 1010 PFU/ml. After 2 h, the culture was transferred to 500 ml of 2x YT medium supplemented with 0.25 µg/ml uridine, 10 µg/ml chloramphenicol, 30 µg/ml kanamycin, and 100 µg/ml ampicillin, and grown overnight at 37°C. Phage were precipitated with PEG6000 using standard protocols (34) and purified using the Qiaprep Spin M13 Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. A total of 10–30 µg of each uracil-containg TAAssDNA template was then combined with 0.6 µg of the following phosphorylated oligonucleotides (MWG Biotech, High Point, NC) in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5, in a final volume of 250 µl (randomized regions are underlined): library 1, 5'-CATGTGACCTCAGGSNNSNNSNNGATSNNSNNGGTGTCCTTGGGTTTTGGGGGG-3'; library 2, 5'-GCACTTGTACTCCTTGCCATTSNNCCASNNSNNGTGSNNSNNGGTGAGGACGC-3'; library 3, 5'-GGTCTTGTAGTTSNNCTCSNNSNNSNNATTGCTCTCCC-3'; library 4, 5'-GGCTCTTCTGCGTSNNGTGSNNSNNCAGAGCCTCATGSNNCACGGAGCATGAG-3'.
Each mixture was incubated at 90°C for 2 min, 50°C for 3 min, and
20°C for 5 min. Synthesis of the heteroduplex DNA was conducted by
adding 30 U of both T4 DNA ligase and T7 DNA polymerase in presence of
0.4 mM ATP, 1 mM dNTPs, and 6 mM DTT. The mixture was incubated for
4 h at 20°C. Heteroduplex DNA was affinity purified and desalted
using the Qiaquick DNA purification kit (Qiagen). A total of 1–5 µg
of heteroduplex DNA was then electroporated into 300 µl of
electrocompetent TG1 E. coli cells in a 2.5 kV field using
200 
resistance and 25 µF capacitance until a library size of
1 x 108 (library 1 and 2) or 1 x
107 (library 3 and 4) was reached. Cells were
grown in 500 ml of 2x YT medium containing 100 µg/ml ampicillin and
1010 PFU/ml of VCSM13 helper phage overnight at
37°C. Phage were precipitated with PEG6000, as previously described
(34).
Selection of the Fc libraries
Phage were resuspended in 5 ml 20 mM MES, pH 6.0/5% skimmed milk/0.05% Tween 20 and added (100 µl of 5 x 1012 PFU/ml/well) to 20 wells of a Maxisorp immunoplate (Nunc, Rochester, NY) previously coated with 1 µg of murine FcRn and blocked with 5% skimmed milk. After incubation for 2 h at 37°C, wells were washed 10–30 times with 20 mM MES, pH 6.0/0.2% Tween 20/0.3 M NaCl and phage eluted by incubation in 100 µl PBS, pH 7.4/well for 30 min at 37°C. Phage were used to reinfect exponentially growing E. coli TG1, as described (35). Three to six rounds of panning were conducted. Typically, 20–40 clones were characterized after three (library 1, 2, and 4) and six (library 3) rounds of panning by dideoxynucleotide sequencing (36) using a ABI3000 genomic analyzer (Applied Biosystems, Foster City, CA).
Construction, production, and purification of IgG1 variants
Representative Fc mutations were incorporated into the human
IgG1 MEDI-493 (31). The H chain was subjected to
site-directed mutagenesis using a Quick Change Mutagenesis kit
(Stratagene), according to the manufacturer’s instructions, and
sequences were verified using a ABI3000 sequencer. The different
constructs were expressed transiently in human embryonic kidney 293
cells using a CMV immediate-early promoter and dicistronic operon in
which the 1 chain is cosecreted with the 
-chain
(31). IgG1s were purified from the conditioned medium
directly on 1 ml HiTrap protein A columns, according to the
manufacturer’s instructions (APBiotech).
Surface plasmon resonance (SPR) measurements
The interaction of soluble murine and human FcRn with immobilized human IgG1 variants was monitored by SPR detection using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden). No aggregated FcRn that could interfere with affinity measurements (37, 38) was detected by gel filtration. Protein concentrations were calculated by the bicinchoninic acid method for both human and murine FcRn or using the 1% extinction coefficient at 280 nm of 1.5 for IgG1 wild type and variants. IgGs were coupled to the dextran matrix of a CM5 sensor chip (Pharmacia Biosensor) using an amine coupling kit, as described (39). The protein concentrations ranged from 3 to 5 µg/ml in 10 mM sodium acetate, pH 5.0. The activation period was set for 7 min at a flow rate of 10 µl/min, and the immobilization period was set to between 10 and 20 min at a flow rate of 10 µl/min. Excess reactive esters were quenched by injection of 70 µl of 1.0 M ethanolamine hydrochloride, pH 8.5. This typically resulted in the immobilization of between 500 and 4000 resonance units (RU). Human and murine FcRn were buffer exchanged against 50 mM PBS, pH 6.0, 7.4, or 8.5, containing 0.05% Tween 20. Dilutions were made in the same buffers. All binding experiments were performed at 25°C, with concentrations ranging from 2.86 µM to 1 nM at a flow rate of 5–10 µl/min; data were collected for 25–50 min, and three 1-min pulses of PBS, pH 7.4, were used to regenerate the surfaces. FcRn was also flowed over an uncoated cell, and the sensorgrams from these blank runs were subtracted from those obtained with IgG1-coupled chips. Runs were analyzed using the software BIAevaluation 3.1 (Pharmacia Biosensor). Association constants (Ka) were determined from Scatchard analysis by measuring the concentration of free reactants and complex at equilibrium after correction for nonspecific binding (40). Errors were estimated as the SD for two or three independent determinations and were <20%.
Pharmacokinetic analyses
Analyses were conducted in BALB/c mice (Harlan, Indianapolis,
IN) for MEDI-493 wild type, G385D/Q386P/N389S, M252Y/S254T/T256E, and
H433K/N434F/Y436H, and were approved by MedImmune’s Review Board. Each
animal (10/group) was injected i.m. with 2.5 µg of protein in a
volume of 100 µl of PBS. Mice were bled from the retro-orbital sinus
for each time point. Data were collected every 24 h until day 5
postinjection and every 48 h thereafter until day 12. Urine and
serum were also collected at 6 and 24 h post-i.m. injection of 30
µg of each IgG1 (two animals/group). Concentrations were determined
by an anti-human IgG ELISA. Briefly, individual wells of a 96-well
Maxisorp Immunoplate (Nunc) were coated with 50 ng of a goat
anti-MEDI-493 (anti-idiotype) Ab (MedImmune). The plates were
blocked with 0.5% (w/v) BSA (Sigma-Aldrich, St. Louis, MO), incubated
with samples or standards (0.1–20 ng/ml), then with a HRP conjugate of
a goat anti-human Fc polyclonal Ab (Sigma-Aldrich). Peroxidase
activity was detected with 3,3',5,5'-tetramethylbenzidine (Kirkegaard &
Perry Laboratories, Gaithersburg, MD), and the reaction was quenched
with 0.18 M H2SO4. The
absorbance at 450 nm was measured with a
Vmax kinetic microplate reader running
SoftMaxPro 3.1.1 software (Molecular Devices, Sunnyvale, CA). The
t1/2 of the elimination phase (
phase), which takes place after the rapid redistribution phase, were
determined using a one-phase exponential decay model provided by
GraphPad Prism (GraphPad Software, San Diego, CA), using data points
between day 1 and 12 postinjection.
Immunoprecipitation and immunohistochemistry
Analyses were conducted in BALB/c mice for MEDI-493 wild type, M252Y/S254T/T256E, and H433K/N434F/Y436H. Using two mice per group, each animal was injected i.v. or i.m. with 30 µg of Ig in a volume of 100 µl of PBS or with 100 µl of PBS. After 24 h, animals were killed. Lungs, spleen, liver, intestine, heart, kidneys, bladder, as well as muscle at the site of i.m. injection were harvested.
Immunoprecipitation
Organs were removed into 1 ml PBS, ground, and lysed by sonication, and lysates were cleared by centrifugation. Immunoprecipitation was performed for 2 h at room temperature using 50 µl of a 50% suspension of goat anti-human IgG-Sepharose 4B conjugate (Zymed, South San Francisco, CA) previously washed three times with 1 ml triethylamine 100 mM, pH 11.0. Beads were washed three times with 1 ml PBS/0.1% Tween 20, pH 7.2, and elution was conducted in 50 µl 100 mM triethylamine, pH 11.0. Eluates were boiled for 5 min and loaded on a SDS-PAGE (4–20% gels; Invitrogen) in presence of 50 mM DTT. Electrotransfer to nitrocellulose filters was made as described (34). Detection was conducted with a HRP conjugate of a goat anti-human IgG (anti-Fab; Sigma-Aldrich) and the ECL Western blotting detection reagents (APBiotech).
Immunohistochemistry
Organs were removed into neutral buffered 10% Formalin solution (Sigma-Aldrich). Immunohistochemistry experiments were conducted at Molecular Histology (Gaithersburg, MD) on paraffin-embedded samples using a HRP conjugate of a goat anti-human IgG (anti-whole molecule; Sigma-Aldrich) and standard protocols (DAKO Handbook on Immunochemical Staining Methods; DAKO, Carpenteria, CA).
Phosphor imaging
Analyses were conducted in BALB/c mice. Briefly, MEDI-493 wild
type, M252Y/S254T/T256E, and H433K/N434F/Y436H were radiolabeled with
Na125I by the chloramine-T procedure
(41) at Lofstrand Labs (Gaithersburg, MD) to a specific
radioactivity of 
5 x 106 dpm/µg. Using
two mice per group, each animal was injected i.m. with 2.5 µg of
iodinated Ig in a volume of 100 µl of PBS. After 24 h, animals
were killed. Lungs, spleen, liver, intestine, and heart were harvested
and transferred into neutral buffered 10% Formalin solution
(Sigma-Aldrich). The radioactivity present was quantitated by phosphor
imaging analysis after 3-wk exposure (42) using a Fuji BAS
5000 running Image Gauge V3.45.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Libraries of human hinge-Fc fragments (residues 226–478, Kabat numbering) (21) derived from the human IgG1 MEDI-493 (31) were expressed using the pCANTAB5E phagemid vector. As previously noted (11), functional Fc-hinge homodimers linked to the gene III coat protein are formed and displayed at the surface of M13 by virtue of the leakiness of the amber stop codon located between hinge-Fc:E-Tag and gene III.
Mutagenesis and selection
Four libraries (1, 2, 3, 4) were constructed by Kunkel-based
mutagenesis, which contained Fc variants with residues randomized at
contact positions between Fc and murine or human FcRn. Library 1
targeted positions 251, 252, and 254–256 (EU numbering)
(21); library 2 positions 308, 309, 311, 312, and 314;
library 3 positions 385, 386, 387, and 389; and library 4 positions
428, 433, 434, and 436. D312 and N389 are not predicted to contact
murine or human FcRn, but due to their solvent-exposed nature as well
as close proximity to the FcRn binding site, these residues were also
randomized. Residues I253, H310, and H435 are highly conserved across
species (21), play a major role in the energetics and pH
specificity of binding to murine and human FcRn (8, 24, 29), and therefore were fixed during mutagenesis to retain the
crucial functional epitope. To limit the size of the libraries and to
avoid cumbersome sampling procedures, W313, E388, H429, E430, A431, and
L432, which are not predicted to contact rat or human FcRn (16, 23), were also fixed. Libraries 1 and 2 consist of 1 x
108 clones each, representing a significant
proportion of the 3.4 x 107
(325) possible codon permutations from the NNS
randomization strategy. Likewise, libraries 3 and 4 of 1 x
107 clones each represented a significant
proportion of the 1.0 x 106
(324) possible codon permutations. After three
rounds of panning, a strong consensus sequence had emerged for
libraries 1, 2, and 4 (Fig. 1
), but not
library 3. After six rounds of panning, a consensus sequence had
appeared for library 3 (Fig. 1). Clones isolated from library 1 have
predominantly L at position 251, Y at 252, T or S at 254, R at 255, and
broader variability at 256 (Fig. 1B). Clones isolated from
library 2 have predominantly T at position 308, P at 309, S at 311, D
at 312, and L at 314 (Fig. 1B). Clones isolated from library
3 have more variability at every position, but predominantly R at
position 385, T at 386, R at 387, and P at 389 (Fig. 1B).
Clones isolated from library 4 have predominantly M at position 428, Y
at 434, and broader specificity at other positions, with H, R, K, and S
being frequently found at 433, and H, Y, R, and T at 436 (Fig. 1B).
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The ability of murine and human FcRn to bind the Fc variants was
investigated using SPR (BIAcore). Representative mutations identified
after panning libraries 1 through 4 (Fig. 1
A) were
introduced into the Fc portion of a human IgG1 (Table I). Injection of different concentrations
of human or murine FcRn over the immobilized IgG1 variants gave
concentration-dependent binding at pH 6.0. A typical resonance profile
for equilibrium binding is shown for the M252Y/S254T/T256E mutant (Fig. 2, A and B). In all
cases, equilibrium (or near-equilibrium) binding levels were reached
within 50 min. The Scatchard plots for the binding of the
M252Y/S254T/T256E mutant to murine and human FcRn are shown in Fig. 2, C and D. The plots were all linear, and apparent
Ka were calculated from the relevant
slopes. Measurements were conducted in duplicate or triplicate and
confirmed that the immobilized IgGs retained their original binding
activity.
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Binding of the Fc mutants
The Kd for the interaction of
wild-type human IgG1 with murine and human FcRn at pH 6.0 (269 and 2527
nM, respectively) agree well with the values determined by others
(8). Our data (Table I) indicate that the I253A mutation
virtually abolishes binding to human and murine FcRn, as reported
previously (12, 24). This is not the result of misfolding
of the Ab, as this mutant retains the same sp. act. as the wild-type
molecule (MEDI-493) in a microneutralization assay (31)
(data not shown).
We generated human IgG1 mutants with increased binding affinity toward
both murine and human FcRn at pH 6.0 (Table I). Overall, improvements
in complex stability were less marked for the human IgG1-human FcRn
pair than for the human IgG1-murine FcRn pair. The largest observed
increases in affinity for single Fc region mutants toward murine and
human FcRn compared with wild-type IgG1 were 30 (N434F/Y436H)- and 11
(M252Y/S254T/T256E)-fold, respectively. However, ranking of the most
critical positions remains unchanged when comparing human and murine
FcRn: the largest increases in IgG1-murine FcRn complex stability
(G 
1.3 kcal/mol) occurred for mutations at positions 252,
254, and 256 (M252Y/S254T/T256E and M252W) and 433, 434, and 436
(H433K/N434F/Y436H and N434F/Y436H). Likewise, the same mutations were
found to have the most profound impact on the IgG1-human FcRn
interaction and also resulted in the largest increases in complex
stability (G 1.0 kcal/mol). Substitutions at
positions 308, 309, 311, 385, 386, 387, and 389 had little or no effect
on the stability of the complexes involving human or murine FcRn
(G 
0.4 kcal/mol). Therefore, residues at the center of
the Fc-FcRn combining site contribute significantly more to improvement
in complex stability than residues at the periphery (Fig. 3). Two combination variants were tested
in which two Fc regions were simultaneously substituted. When
tested against human FcRn, M252Y/S254T/T256E/H433K/N434F/Y436H shows an
additive effect and exhibits binding that is better than
M252Y/S254T/T256E and H433K/N434F/Y436H, but does not bind
significantly better than H433K/N434F/Y436H to murine FcRn.
M252Y/S254T/T256E/G385R/Q386T/P387R/N389P does not show any additive
effect, as binding to both murine and human FcRn is not significantly
different from M252Y/S254T/T256E.
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G 1.0 kcal/mol)
showed a parallel significant increase in binding to the mouse receptor
at pH 7.4 (SPR signalpH
7.4/SPR signalpH
6.0 0.5 at equilibrium; Fig. 4, C and D). IgG1
mutants exhibiting a small increase in affinity toward murine FcRn at
pH 6.0 (G < 0.4 kcal/mol) bound poorly to the
mouse receptor at pH 7.4 (Fig. 4B). Raising the pH to 8.5
resulted in a dramatically lower binding of the mutants to murine FcRn
when compared with at pH 6.0 and 7.4 (Fig. 4, A–D).
In contrast, IgG1 mutants exhibiting a large affinity increase toward
human FcRn at pH 6.0 (G up to 1.4 kcal/mol) only showed minimal
binding to the human receptor at pH 7.4 (SPR signalpH
7.4/SPR signalpH
6.0 < 0.1 at equilibrium; Fig. 4, G and
H). Only M252Y/S254T/T256E/H433K/N434F/Y436H
(G = 2.4 kcal/mol at pH 6.0) bound
significantly to human FcRn at pH 7.4 (SPR signalpH
7.4/SPR signalpH
6.0 = 0.45 at equilibrium; unpublished observations). None
of the variants tested bound at a detectable level to human FcRn at
pH 8.5.
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To correlate magnitude of the increase in affinity of the Fc-FcRn
interaction with biological consequences, three IgG1 mutants were
selected for pharmacokinetic studies. Those display large
(M252Y/S254T/T256E and H433K/N434F/Y436H) to small (G385D/Q386P/N389S)
increases in affinity toward both murine and human FcRn. The
pharmacokinetics of MEDI-493 wild type, G385D/Q386P/N389S,
M252Y/S254T/T256E, and H433K/N434F/Y436H were analyzed in BALB/c mice.
Those molecules were injected in equal amounts into different mice, and
their serum concentration (reported in ngvariant/wild
type/mlserum) was determined by ELISA
(see Materials and Methods). We report an inverse
correlation between the binding affinity of the IgG1 variants to murine
FcRn at pH 6.0 and their serum concentration (Fig. 5). M252Y/S254T/T256E and
G385D/Q386P/N389S show significantly lower than wild-type serum
concentrations starting at day 1 post-i.m. injection of 2.5 µg/animal
of IgG1, whereas H433K/N434F/Y436H is virtually undetectable at the
same time point (Fig. 5). Similar clearance results were obtained after
i.v. injection of 2.5 µg/animal of IgG1 (data not shown). The same
trends are observed after i.v. or i.m. injection of a >10-fold higher
amount (30 µg/animal) of H433K/N434F/Y436H and M252Y/S254T/T256E as
early as 6 h postinjection (Fig. 6).
This is not the result of an enhanced degradation of the variants by
serum or blood cell-associated proteases or of denaturation, as they
have the same ex vivo stability in mouse serum and whole blood as the
wild-type IgG1 for 24 h at 37°C, as determined by an
anti-human IgG ELISA requiring the simultaneous presence of both a
functional idiotope and Fc region (see Materials and
Methods, data not shown). The same assay did not detect MEDI-493
wild type, M252Y/S254T/T256E, or H433K/N434F/Y436H in urine. Although
the IgG1 variants we tested exhibit lower serum concentration when
compared with wild type, phase
t1/2 are not significantly different
for MEDI-493 wild type, G385D/Q386P/N389S, and M252Y/S254T/T256E (Table II). The relationship, if any, between
serum concentrations and phase
t1/2 is at present unclear.
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Presence of MEDI-493 wild type, M252Y/S254T/T256E, and
H433K/N434F/Y436H was investigated by immunoprecipitation of different
organs and tissue (lungs, spleen, intestine, heart, kidney, bladder,
and site of i.m. injection) using an anti-human IgG conjugate.
Muscle tissue at the site of injection revealed two polypeptide chains
with apparent molecular mass 50 and 30 kDa, corresponding
to the H and L chains, respectively, of MEDI-493 wild type,
M252Y/S254T/T256E, and H433K/N434F/Y436H (Fig. 7). No significant difference could be
observed between the three IgGs. Those molecules could not be detected
in lungs, spleen, intestine, heart, kidney, or bladder after i.m. or
i.v. injection. Those results indicate that no enhanced retention or
degradation of the mutants is taking place in muscle tissue. In
agreement with those data, immunohistochemistry on lungs, spleen,
liver, intestine, heart, kidney, and bladder did not reveal any
significant amounts of M252Y/S254T/T256E and H433K/N434F/Y436H
(unpublished observations).
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To more accurately determine the concentrations of MEDI-493 wild
type, M252Y/S254T/T256E, and H433K/N434F/Y436H in different organs, a
more sensitive detection assay was used in which IgG1s were iodinated
and i.m. injected in BALB/c mice. Different tissues were then submitted
to a phosphor imaging analysis. It revealed that MEDI-493 wild type is
found at the highest levels in lungs, spleen, heart, and liver.
Intestine exhibits lower levels. M252Y/S254T/T256E and
H433K/N434F/Y436H follow the same trend,
but at significantly lower concentrations overall than MEDI-493 wild
type (Table III, Fig. 8). Those results
indicate that no significant enhanced retention of the mutants is
taking place in the organs tested. No conclusion pertaining to the
degradation rate of the mutants in those organs can be formulated
because the lower-than-wild-type organ concentrations might just
reflect the overall lower serum concentrations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1/2 domains of murine and human FcRn
(1, 15, 16, 23). These Fc libraries introduced
substitutions in three loops at the
CH2-CH3 junction (strands B
and E of CH2, and G of CH3)
and in one region between strands C and D of
CH3.
Engineering of the Fc-FcRn interaction indicates that major
improvements in murine FcRn-human IgG1 and human FcRn-human IgG1
complex stability (0.6 kcal/mol G 2.4
kcal/mol) occur on substituting residues located in a band across the
Fc-FcRn interface (M252, S254, T256, H433, N434, and Y436).
Substitutions of residues at the periphery (V308, L309, Q311, G385,
Q386, P387, and N389) resulted in little or no increase in the free
energy of complex stabilization (G 0.4 kcal/mol).
V308T/L309P/Q311S, G385R/Q386T/P387R/N389P, and, to a lesser extent,
G385D/Q386P/N389S were strongly selected for (Fig. 1, A and
B), but did not exhibit a marked increase in affinity toward
both murine and human FcRn (G 0.4 kcal/mol). This
suggests that the two peripheral FcRn-contacting regions are not
suitable targets for the engineering of IgGs with significant increases
in affinity for FcRn. This might reflect the hot spot nature of the
central region. In this situation, the energy of stabilization arises
primarily from a few residues located at the center of a
protein-protein interface (40). In fact, central residues
I253 and H435 have already been shown to be critical to formation of
the complex between human Fc and murine FcRn (12), and
I253, S254, H435, and Y436 play a crucial role in the interaction of
human IgG1 with human FcRn (8, 24). Thus, proximity of a
pre-existing hot spot might be an important criteria to consider when
engineering mutants for improved binding activity. However, H310 has
been shown to be part of the energetic epitope for both human and
murine FcRn, and yet mutations in its vicinity did not yield any
variant with improvement in affinity above 1.7-fold. More likely is
that additional factors around the substituted residue, such as the
local chemical microenvironment, protein conformational
changes, as well as solvent reorganization, have to be taken into
account. Alternatively, the observed pattern might reflect other
factors, such as library design, incomplete library sampling, and
inefficient selection of more favorable residues at the peripheral
positions due to a decrease in Fc stability or less efficient display
at the surface of M13.
Interestingly, the variant with the highest affinity to human FcRn was obtained by combining the M252Y/S254T/T256E and H433K/N434F/Y436H mutations and exhibited a 57-fold increase in affinity relative to the wild-type IgG1. In comparison, both single substitutions resulted in only an 11- and 6.5-fold improvement in binding to human FcRn, respectively, as compared with wild-type IgG1. In contrast, the combination of M252Y/S254T/T256E with G385R/Q386T/P387R/N389P did not result in further affinity improvement toward human FcRn relative to M252Y/S254T/T256E. This confirms the dominance of the central over the peripheral residues in the energetics of the complex formation.
Efficient binding of human Fc to murine FcRn at pH 6.0 requires the
presence of several wild-type Fc residues. For example, leucine is very
conserved at 251, arginine at 255, aspartic acid at 312, leucine at
314, and methionine at 428 (Fig. 1). Retention of those wild-type
residues indicates they are the most favorable for efficient binding to
murine FcRn. These amino acids are well conserved in human and murine
IgGs (21). In particular, D312 is not predicted to contact
human or murine FcRn, and conservation of this residue in the
phage-derived clones suggests that it has an important, but indirect
role in the energetics of association with murine FcRn. Alternative
explanations include biases in the library or incomplete sampling.
Another specificity trend is observed when one considers positions 308,
309, and 311 in which threonine, proline, and serine, respectively, are
very strongly favored over the corresponding wild-type residues (Fig. 1). Surprisingly, this does not correlate with the magnitude of
increase in affinity, as V308T/L309P/Q311S binds less than 2-fold
better than the wild-type IgG1 to both human and murine FcRn (Table I).
There appears to be a selection for residues with an aromatic side
chain at position 252 (Y, F, and W). Engineering of the interaction of
murine Fc fragments with murine FcRn has already revealed that a
leucine at this position is the most favorable residue
(11). No clones were found in our experiments that
contained a leucine at 252. This difference might reflect the distinct
local environment present in both murine and human Fc.
The three-dimensional structure of human and rodent FcRn is very similar (17). However, there are a number of differences at the interface of contact with Fc, the most notable of which is a substitution of L135 (human) for D137 (mouse). Our selection results indicate a strong trend toward conservation of H and selection of K and R at position 433 as well as toward selection of H and R at position 436. This underlines the importance of a positively charged residue at those locations. Interestingly, histidine at position 436 is highly conserved in murine IgG1 sequences, in which it is thought to interact through a salt bridge with murine FcRn D137 (23). In contrast, tyrosine at position 436 is conserved in human IgG1 sequences, in which it is believed to make hydrophobic interactions with human FcRn L135. Selection of H436 after selection of random human Fc libraries on murine FcRn probably recreates the important interaction with D137. This is supported by the 30-fold increase in the affinity of N434F/Y436H for murine FcRn compared with wild type. Elucidation of the molecular mechanisms by which human FcRn not only accommodates a histidine at position 436, but also has enhanced binding to N434F/Y436H will require a more detailed analysis, such as the construction of additional mutants and x-ray crystallography.
Surprisingly, efforts to use human FcRn for screening the libraries
were not successful due to the lack of consensus sequences after seven
rounds of panning. A likely explanation is that the selection
conditions we tested were detrimental to its stability and/or function.
As a result, libraries were panned against murine FcRn, with a binding
step at pH 6.0 and an elution step at pH 7.4. This strategy allowed
direct isolation of clones that bound more tightly at pH 6.0 than at pH
7.4. However, there was a marked difference in the effect of pH on the
binding of the IgG1 variants to either human or murine FcRn. Increases
in the stability of the complex with murine FcRn at pH 6.0 correlate
with increases in the stability of the complex at pH 7.4 (Fig. 4, A–D). In contrast, most IgG1 mutants bound very poorly to
human FcRn at pH 7.4 (Fig. 4E–H). In all cases, binding was
dramatically decreased when pH was raised to 8.5, strongly suggesting
the mutations we identified led to a change in the pKa of H310
and/or H435, which are responsible for the pH dependency of binding
(23). This has clear implications for the pharmacokinetics
of the IgG1 variants, as higher affinity to FcRn at pH 7.4 might
adversely affect release into the serum and offset the benefit of the
enhanced binding at pH 6.0. Our pharmacokinetic and
immunohistochemistry studies in mice support this hypothesis because
serum levels after i.m. or i.v. injection were consistently lower for
G385D/Q386P/N389S, M252Y/S254T/T256E, and H433K/N434F/Y436H when
compared with the wild-type molecule. Those mutants are also present at
lower levels in different tissues when compared with wild type. No
evidence of enhanced degradation in serum, blood, or muscle tissue at
the site of injection was found. Likewise, there was no detectable
enhanced excretion in urine. It is possible to hypothesize that because
of their more efficient binding at pH 7.4, the IgG1 mutants are
sequestered throughout the body in FcRn-containing tissues such as the
endothelium of small arterioles and capillaries (20).
Retention of the IgGs in cells may lead to their degradation. This may
not be an issue in humans because the majority of the IgG1 mutants we
tested do not bind well to human FcRn at pH 7.4 when compared at pH
6.0. If this hypothesis is correct, introduction of the mutations we
identified into the Fc fragment of therapeutic Abs may increase their
serum persistence.
Enhancements of the affinity of our human IgG variants for both human and murine FcRn are anticipated from further engineering, such as the randomization of new sets of residues and the selection of the best combinations of mutations by DNA shuffling (46). Those studies will yield important information pertaining to the molecular basis of protein-protein interactions. Provided retention of the pH dependency of binding can be conserved, it might be possible to generate IgG mutants exhibiting enhanced efficacy due to long serum t1/2. Those molecules could be a valuable addition to the therapeutic Ab field and have potential utility in domains as diverse as cancer, respiratory diseases, neonatal medicine, and diagnostic.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William F. Dall’Acqua, Department of Protein Engineering, MedImmune, Inc., 35 West Watkins Mill Road, Gaithersburg, MD 20878. E-mail address: dall'acquaw{at}medimmune.com
3 Abbreviations used in this paper: FcRn, neonatal FcR; RU, resonance unit; SPR, surface plasmon resonance.
Received for publication June 25, 2002. Accepted for publication September 3, 2002.
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W. Mi, S. Wanjie, S.-T. Lo, Z. Gan, B. Pickl-Herk, R. J. Ober, and E. S. Ward Targeting the Neonatal Fc Receptor for Antigen Delivery Using Engineered Fc Fragments J. Immunol., December 1, 2008; 181(11): 7550 - 7561. [Abstract] [Full Text] [PDF]
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