Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding

Brain Shuttle Antibody for Alzheimer’s Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode

Summary: Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target. : Weber et al. show that a Brain Shuttle antibody against amyloid-β prevents plaque formation mediated by the effector function. Their study reveals that the effector function is hidden in the periphery, due to the unique binding mode of the Brain Shuttle, but becomes fully active in the brain. Keywords: blood-brain barrier, Alzheimer’s disease, antibody effector function, brain delivery, Brain Shuttle, antibody engineerin

Target binding and thermal stability.

<p>(A) Surface plasmon resonance sensogram of the Fab and CrossFab interacting with their target, hAng2. One out of three runs for the Fab and the CrossFab is shown. Coloured curves represent the measured data at various Fab or CrossFab concentrations, while the result of the global fit to a 1∶1 Langmuir model is illustrated by black curves. The table shows average values and standard deviations derived from triplicate measurements (k_a: association rate; k_d: dissociation rate; K_D: affinity). (B) Measurement of protein stability by temperature-dependent protein autofluorescence emission maximum wavelength.</p

Data collection and refinement statistics.

*<p>Values in parentheses are for the highest-resolution shell. , where is the scaled observed intensity of the <i>j</i>th observation of reflection <i>h</i>, and is the mean value of corresponding symmetry-related reflections.</p

Elbow angles and relative domain orientation.

<p>(A) Superposition of Fab and CrossFab with example structures covering the range of observed elbow angles. Example structures (shown as loops), their orientations and color coding are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Stanfield1" target="_blank">[24]</a>. All structures are superimposed via their V_L domains. The colors of Fab and CrossFab are analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a>. (B) Superposition of the CrossFab with its closest structural homolog (PDB code 3FMG). (C) Relative domain orientation of variable and constant domains in Fab and CrossFab. The pseudo-twofold axes of variable and constant domains are shown as light and dark purple dumbbells, respectively. Molecules are oriented so that the axes connecting the last Fv residues' C-alpha atoms, as well as the Fv pseudo-twofold axis are parallel to the paper plane. An asterisk marks the V_L-C_H1 junction, which is two amino acids longer than the corresponding V_L-C_L junction.</p

Crystal structures and electron density maps.

<p>(A) Comparison of the Fab and CrossFab structures side-by-side. Residues involved in hydrogen bonds between the constant and the variable domains are shown as sticks and hydrogen bonds are shown as red dashed lines. The color code used is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a>. (B) Superposition of variable domains in Fab and CrossFab. C-alpha atoms excluding CDR H3 were used. Fab and CrossFab variable domains superimpose with an rmsd of 0.43 Å. (C) Superposition of the constant domains in Fab and CrossFab using C-alpha atoms. Fab and CrossFab constant domains superimpose with an rmsd of 0.40 Å. (D) Composite omit maps around the V_HV_L-CH₁C_L interface of the CrossFab and the Fab, contoured at 1.0 sigma using a carve distance of 2.0 Å.</p

CrossMab design.

<p>(A) Schematic representation of the domain crossover leading to CrossFabs. Right: Combination of a Fab and a CrossFab to obtain a CrossMab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Schaefer1" target="_blank">[16]</a>. Antibody domains are symbolized as ovals. Light colors are used for LC domains; darker colors are used for HC domains. This color code is used throughout. (B) Sequence alignment of the elbow and adjacent regions of LC and HC in Fab and CrossFab.</p