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

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

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

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

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