Structural characterization of a human Fc fragment engineered for lack of effector functions

Human Fc fragments containing the L234F/L235E/P331S triple mutation exhibit a dramatic decrease in their binding to several effector molecules (CD64, CD32A, CD16 and C1q). The three-dimensional structure of such a mutated fragment reveals that these broad-ranging functional effects are not caused by major structural rearrangements in the Fc moiety

Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon α-2A

Crystals of the complex between the Fab fragment of a human anti-interferon α therapeutic antibody and human interferon α-2A have been obtained and diffracted to 3.0 Å resolution

EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.

<p>When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10⁻⁴ s⁻¹).</p

Pairing mAbs 3B10 and 1C1 results in enhanced EphA2 detection sensitivity in conditioned media.

<p>(A) Binding of detection antibody mAb 3B2 plotted against EphA2 concentrations. (B) Logarithmic scale display with binding signals in ∼0.3–30 RU (or ∼0.03–3 ng/cm²) range. The bi-epitope 3B10-1C1 surface detected the lowest EphA2 concentration (15.6 pM at a binding signal of 6 RU or 0.6 ng/cm²), an ∼100- and 200-fold improvement in detection limits when compared with the corresponding 3B10 (1.3 nM) and 1C1 (3.1 nM), respectively, single-epitope surfaces.</p

Binding and epitope characterization of various anti-EphA2 mAbs.

<p>(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm²) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10⁻²−10⁻³ s⁻¹ range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.</p

Generation and characterization of high density bi-epitope SPR sensor surfaces.

<p>(A) Immobilization sensorgrams of mAbs 3B10, 1C1 and 3B10-1C1 mixture. The immobilization profiles are comparable and yielded a high density surface (∼5,000–5,500 RU or ∼500–550 ng/cm²). (B) Confirmation of the co-existence of functional antibodies on the bi-epitope surfaces. Excess of mAbs 3B10 or 1C1 (1 µM) inhibited EphA2 binding to the single-epitope 3B10 or 1C1 surfaces, respectively, but not to the bi-epitope 3B10-1C1 surface.</p

Binding affinity of IgG to target antigens.

<p>Binding affinity of IgG to target antigens.</p