Article Figures & Data
Figures
- FIGURE 1.
A, Fc sequences of phage clones identified after three (library 1, 2, and 4) or six (library 3) rounds of panning using murine FcRn. Residues in bold indicate fixed residues. Sequences underlined and in italic correspond to clones with an occurrence frequency of 10–20 times and of 2–5 times, respectively. B, Summary of the occurrence of selected residues at particular positions. The chart was generated by calculating the appearance frequency of the most commonly occurring residues at each randomized position.
- FIGURE 2.
A, Binding of murine FcRn to immobilized M252Y/S254T/T256E. Murine FcRn was injected at 11 different concentrations ranging from 1 to 556 nM over a surface on which 4000 RU of IgG1 had been coupled. B, Binding of human FcRn to immobilized M252Y/S254T/T256E. Human FcRn was injected at eight different concentrations ranging from 71 nM to 2.86 μM over a surface on which 1000 RU of IgG1 had been coupled. C and D, Scatchard analysis of the data in A and B, respectively, after correction for nonspecific binding. Req is the corrected equilibrium response at a given concentration C. The plots are linear with correlation coefficients of 0.97 and 0.998, respectively. The apparent Kd are 24 and 225 nM, respectively.
- FIGURE 3.
Space-filling model of the surface of the Fc fragment of a human IgG1 based upon the structure of a human IgG1 (47 ). The Fc fragment is oriented such that its docking surface with FcRn is pointing toward the reader. Residues are color coded according to the gain of free energy of stabilization of the Fc-FcRn complex: red, substitutions at those positions resulted in at least a 2.5- and 5-fold affinity increase to human and mouse FcRn, respectively; blue, substitutions at those positions were found to increase affinity by a factor of less than 2-fold in both the Fc-human FcRn and Fc-mouse FcRn interaction. The figure was drawn using Swiss pdb viewer (48 ).
- FIGURE 4.
A–D, BIAcore analysis of the binding of murine FcRn at pH 6.0, 7.4, and 8.5 to wild-type human IgG1, G385D/Q386P/N389S, M252Y/S254T/T256E, and H433K/N434F/Y436H, respectively, after correction for nonspecific binding. Murine FcRn was injected at a concentration of 1 μM over a surface on which ∼1000 RU of IgGs had been coupled. E–H, BIAcore analysis of the binding of human FcRn at pH 6.0, 7.4, and 8.5 to wild-type human IgG1, G385D/Q386P/N389S, M252Y/S254T/T256E, and H433K/N434F/Y436H, respectively, after correction for nonspecific binding. Human FcRn was injected at a concentration of 1 μM over a surface on which ∼1000 RU of IgGs had been coupled.
- FIGURE 5.
Clearance curves of human IgG1 wild type, G385D/Q386P/N389S (1.4-fold affinity increase to mouse FcRn), M252Y/S254T/T256E (10-fold affinity increase to mouse FcRn), and H433K/N434F/Y436H (19-fold affinity increase to mouse FcRn) following i.m. injection of 2.5 μg IgG1 in BALB/c mice. Each time point represents the average serum concentration for 10 mice. SDs are indicated by error bars.
- FIGURE 6.
Serum concentrations of human IgG1 wild type, M252Y/S254T/T256E, and H433K/N434F/Y436H 6 and 24 h postinjection of 30 μg IgG1 in BALB/c mice. SDs are indicated by error bars.
- FIGURE 7.
Immunoprecipitation of human IgG1 wild type, M252Y/S254T/T256E, and H433K/N434F/Y436H in muscle 24 h post-i.m. injection of 30 μg IgG1 in BALB/c mice.
- FIGURE 8.
Phosphor imaging of 125I in lung, liver, spleen, intestine, and heart for human IgG1 wild type, M252Y/S254T/T256E, and H433K/N434F/Y436H 24 h post-i.m. injection of 2.5 μg iodinated IgG1 in BALB/c mice.
Tables
- Table I.
Dissociation constants and relative free energy changes for the binding of IgG1/Fc mutants to murine and human FcRna
Mutant Dissociation Constant Fc/Murine FcRn (nM) ΔΔG (kcal/mol) Dissociation Constant Fc/Human FcRn (nM) ΔΔG (kcal/mol) Wild type 269 ± 1 NA 2527 ± 117 NA I253A NB NA NB NA M252Y/S254T/T256E 27 ± 6 1.4 225 ± 10 1.4 M252W 30 ± 1 1.3 408 ± 24 1.1 M252Y 41 ± 7 1.1 532 ± 37 0.9 M252Y/T256Q 39 ± 8 1.1 560 ± 102 0.9 M252F/T256D 52 ± 9 1.0 933 ± 170 0.6 V308T/L309P/Q311S 153 ± 23 0.3 1964 ± 84 0.1 G385D/Q386P/N389S 187 ± 10 0.2 2164 ± 331 0.1 G385R/Q386T/P387R/N389P 147 ± 24 0.4 1620 ± 61 0.3 H433K/N434F/Y436H 14 ± 2 1.8 399 ± 47 1.1 N434F/Y436H 9 ± 1 2.0 493 ± 7 1.0 H433R/N434Y/Y436H 14 ± 1 1.8 472 ± 61 1.0 [M252Y/S254T/T256E-H433K/N434F/Y436H] 9 ± 1 2.0 44 ± 3 2.4 [M252Y/S254T/T256E-G385R/Q386T/P387R/N389P] 28 ± 3 1.3 243 ± 48 1.4 -
a Affinity measurements were carried out by BIAcore, as described in Materials and Methods. Residue numbering is according to EU (21 ). The free energy change of dissociation, ΔG, is calculated as follows: ΔG = −R.T.1n[Kd], in which R is the gas constant, T is the absolute (Kelvin) temperature, and Kd is the dissociation constant. Differences in free energy changes are calculated as the differences between the ΔGs of wild-type and mutant reactions (ΔΔG = ΔGwild type − ΔGmutant). NB, no binding; NA, nonapplicable.
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Human IgG1 No. of Mice β Phase t1/2 (d) Wild type 10 3.2 ± 0.5 G385D/Q386P/N389S 10 2.8 ± 0.4 M252Y/S254T/T256E 10 3.0 ± 0.5 -
a Measurements were carried out, as described in Materials and Methods. Error was estimated as the SD for two independent series of measurements using MEDI-493 (wild type) IgG1.
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- Table III.
Phosphor imaging of 125I for wild-type and mutant human IgG1 molecules in BALB/c micea
Mutant Liver PSL/mm2b Lung PSL/mm2 Heart PSL/mm2 Spleen PSL/mm2 Small Intestine PSL/mm2 Large Intestine PSL/mm2 Wild type 117 ± 36 121 ± 37 123 ± 10 114 ± 6 35 ± 7 51 ± 6 M252Y/S254T/T256E 35 ± 3 29 ± 5 30 ± 6 27 ± 1 ND ND H433K/N434F/Y436H 13 ± 1 12 ± 1 12 ± 1 12 ± 1 ND ND -
a Measurements were carried out, as described in Materials and Methods, at Molecular Histology. Error was estimated as the SD for two independent series of measurements.
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b Photo-stimulated luminescence (PSL) density. ND, not determined due to low signal. Background slide reading was 10.7 PSL/mm2. Background plate reading was 9.1 PSL/mm2.
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