9 research outputs found
Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody
Engineered monoclonal antibody with novel antigen-sweeping activity in vivo.
Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody
Inhibitory FcγRIIb-Mediated Soluble Antigen Clearance from Plasma by a pH-Dependent Antigen-Binding Antibody and Its Enhancement by Fc Engineering
Effect of sweeping antibody on high plasma concentration antigen.
<p>Effect of NPH-IgG1, PH-IgG1 and PH-v6 on a hFcRn-Tgm steady-state model with high hsIL-6R concentration of approximately 250 ng/mL in the presence of human IgG. NPH-IgG1, PH-IgG1 and PH-v6 were intravenously administered as multiple doses of 0.01 mg/kg every other day. Molar baseline hsIL-6R concentration (6.6 nM) is 5-fold higher than antibody concentration at 15 min (1.3 nM). Time profiles of total hsIL-6R plasma concentration (A) and free hsIL-6R percentage over control (B) are shown. Each data point represents the mean ± s.d. for total hsIL-6R concentration (n = 3–5 each). Free hsIL-6R percentage over control is determined from the pooled plasma sample of n = 3–5 each.</p
Mutations and FcRn binding affinity of hIgG1 Fc variants.
<p>Binding affinity (K<sub>D</sub>) of IgG1 and v1 to mFcRn at pH 7.0 and pH 6.0, binding affinity (K<sub>D</sub>) of IgG1, v2-v6 and v0 to hFcRn at pH 7.0 and pH 6.0, and mutations introduced in the Fc region are shown. Mutation sites in the Fc region are described in EU numbering. NT, not tested.</p
Antigen sweeping by pH-dependent antigen binding antibody with increased FcRn binding at neutral pH.
<p><i>In vivo</i> study of NPH-IgG1, PH-IgG1 and PH-v1 in normal mice. Effect of antibodies on the total hsIL-6R plasma concentration was evaluated in a co-injection model and a steady-state model. In the co-injection model, hsIL-6R, hsIL-6R+NPH-IgG1, hsIL-6R+PH-IgG1 and hsIL-6R+PH-v1 were intravenously administered as single doses of 50 µg/kg for hsIL-6R and 1 mg/kg for antibody and a time profile of total hsIL-6R plasma concentration (A) is shown. Each data point represents the mean ± s.d. (n = 3 each). In the steady-state model, steady-state plasma concentration of approximately 20 ng/mL hsIL-6R was maintained using an infusion pump filled with hsIL-6R solution, and NPH-IgG1, PH-IgG1 and PH-v1 were intravenously administered as single doses of 1 mg/kg and a time profile of total hsIL-6R plasma concentration (B) is shown. Each data point represents the mean ± s.d. (n = 3 each).</p
Characterization of sweeping antibody in hFcRn-Tgm.
<p>(A) <i>In vivo</i> study of NPH-IgG1, PH-IgG1, PH-YTE and PH-v2 in hFcRn-Tgm. Effect of antibodies on the total hsIL-6R plasma concentration was evaluated in a co-injection model. hsIL-6R, hsIL-6R+NPH-IgG1, hsIL-6R+PH-IgG1, hsIL-6R+PH-YTE and hsIL-6R+PH-v2 were intravenously administered as single doses of 50 µg/kg for hsIL-6R and 1 mg/kg for antibody and a time profile of total hsIL-6R plasma concentration is shown. Each data point represents the mean ± s.d. (n = 3 each). (B) Effect of pH-dependent antigen binding and increased binding affinity to FcRn at neutral pH on antigen sweeping in hFcRn-Tgm steady-state model with hsIL-6R plasma concentration of approximately 20 ng/mL. NPH-IgG1, NPH-v2, PH-IgG1, PH-v2 and PH-v0 were intravenously administered as single doses of 1 mg/kg. Time profile of total hsIL-6R plasma concentration is shown. Each data point represents the mean ± s.d. (n = 3 each).</p
<i>In vivo</i> study of sweeping antibodies in a normal mice hsIL-6R trans-signaling model.
<p>Effect of antibodies on the total hsIL-6R plasma concentration and SAA plasma concentration (as a marker for hsIL-6R antagonism) were evaluated. hsIL-6R was intravenously administered as a single dose of 250 µg/kg. At 2 h, non-neutralizing antibodies PHX-IgG1 and PHX-v1 were intravenously administered as single doses of 30 mg/kg (A, B), and neutralizing antibodies NPH-IgG1, PH-IgG1 and PH-v1 were intravenously administered as single doses of 0.03 mg/kg (C, D). At 24 h, hIL-6 was intravenously administered as a single dose of 8 µg/kg. Total hsIL-6R plasma concentration (A, C) and SAA plasma concentration (B, D) at 30 h is shown. Each data represents the mean ± s.d. for total hsIL-6R plasma concentration and the mean ± s.e. for SAA plasma concentration (n = 3–7 each). ND, not detected (below 0.195 ng/mL). Statistical significance was determined by t-test (*) or Tukey’s multiple comparison test (#) for total hsIL-6R and SAA plasma concentration.</p
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The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line
Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process