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. 2020 May 14:18:1210-1220.
doi: 10.1016/j.csbj.2020.05.007. eCollection 2020.

The Contorsbody, an antibody format for agonism: Design, structure, and function

Guy J Georges  1 , Stefan Dengl  1 , Alexander Bujotzek  1 , Friederike Hesse  1 , Jens A A Fischer  1 , Achim Gärtner  1 , Jörg Benz  2 , Matthias E Lauer  2 , Philippe Ringler  3 , Henning Stahlberg  3 , Friederike Plath  4 , Ulrich Brinkmann  1 , Sabine Imhof-Jung  1
Affiliations

The Contorsbody, an antibody format for agonism: Design, structure, and function

Guy J Georges et al. Comput Struct Biotechnol J. .

Abstract

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor 'cis'-dimerization without 'trans'-bridging of cells or mere receptor blockade.

Keywords: Antibody format; Electron microscopy; Molecular dynamic; Molecular modeling; Protein engineering; Receptor dimerization.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
A: Lego representation of the Contorsbody format. B: Lego representation of the heavy chain (dark blue and grey) and light chain (light blue). C: Representation of the linear sequence of the single chain Contorsbody with detailed composition of the linkers and the flanking amino acids. D: Contorsbody format molecular model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Time series plots (top) and histograms (bottom) of the paratope to paratope (center of geometry) distances for Contorsbodies with a) one G4S repeat (linker length 5), b) two G4S repeats (linker length 10) and c) three G4S repeats (linker length 15). The paratope to paratope distance at simulation start is indicated with a red line. The average paratope to paratope distance over the second half of the simulation is indicated with a yellow dashed line. Given the low distance variability observed for Contorsbody with linker length 5, the simulation was suspended after 100 ns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Exemplary molecular states extracted from the MD simulation of the Contorsbody with linker length 10 shown in front view (top row) and bottom-up view (bottom row) representing the a) maximal, b) minimal and c) average paratope to paratope distances. Paratope centers of geometry are indicated as purple spheres. The measured distances are indicated as green dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
NS-TEM representative 2D classes (23.6 × 23.6 nm) of the analog 2 Contorsbody. Top row: representative 2D pictures of the Contorsbody molecule alone. Bottom row: representative 2D pictures of the Contorsbody molecule in complex with an anti-Fc Fab showing that the Fc part of the Contorsbody is recognized by one anti-Fc Fab. In both rows, the Fc moiety is often assignable as the most blurry part of the assembly.
Fig. 5
Fig. 5
Cryo-EM procedure to obtain cryo-EM maps of Contorsbody analog 2, i.e. a 2D classification of all the selected particles followed by a 3D classification into 10 classes. Three prominent 3D classes are containing 17, 18, and 19% of the particles.
Fig. 6
Fig. 6
Cryo-EM reconstitution of the Contorsbody 3D envelopes at 10 Å. The grey envelopes depict an open and a closed conformation in each row. In each row, a second representation shows the VH-CH1 ribbon Fab half, colored in dark blue, and the partner VL-Ck ribbon Fab half, colored in light blue. Grey ribbons of the Fc portion complete the overall architecture. A: Front view showing the two Fabs, B: Bottom view showing the Fab CDRs and the dimeric CH3 domains. C: Back view with the Fc moiety in front. D: Top views with the hinge region in front. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
A: a schematic representation of the Contorsbody architecture with written sequences corresponding to linkers (red) and less structured regions of the Contorsbody (bold). B: top view depicting the flexible linkers (red for (G4S)2 and black for regular sequence as indicated in A). Disulfide bridges are indicated in yellow. C: bottom view depicting the CDRs and their assembly to form a paratope. The red (G4S)2 is the only flexible part that connects the C-terminus of the Fc with the N-terminus of the VL, located in the back to CDRL3. D: Model structure of the anti-Fab Fab recognizing the constant part of a Fab. E: Negative staining TEM representative 2D classes of the complex between the anti-Fab Fab and the analog 2 Contorsbody showing only a type of helical arrangement of two anti-Fab (orange) with one Contorsbody (red arrows point to the Fc moiety, scale bar represents 10 nm). F: Lego translation of the previous observation with the orange arrows symbolizing the anti-Fab Fab moiety in complex with the Contorsbody. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8
Fig. 8
BT474 cell proliferation upon treatment with Trastuzumab IgG (green curve) and Trastuzumab Contorsbody (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 9
Fig. 9
A. One possible conformation of a bis-Fab of Trastuzumab (heavy chain in dark blue and light chain in cyan) on two Her2 receptors (in violet) showing an on scale representation of 2 intra-cellular domains coming in contact to promote a signal transduction for growth. B: schematic representation showing the top of the Fab, i.e. the paratope, binding to the Her2 receptor. C: same view with an arrow colored by N- to C-terminus (blue to red) representing the Her2 ECD. D: the Contorsbody architecture brings two adjacent Fabs and, consequently, fixes two receptors side-by-side. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References

    1. Abraham M.J., Murtola T., Schulz R., Páll S., Smith J.C., Hess B. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1:19–25. doi: 10.1016/j.softx.2015.06.001. - DOI
    1. Atwell S., Ridgway J.B.B., Wells J.A., Carter P. Stable heterodimer from remodeling the domain interface of a homodimerusing a phage display library. J Mol Biol. 1997;270:26–35. doi: 10.1006/jmbi.1997.1116. - DOI - PubMed
    1. Bujotzek A., Dunbar J., Lipsmeier F., Schäfer W., Antes I., Deane C.M. Prediction of VH–VL domain orientation for antibody variable domain modeling. Proteins Struct Funct Bioinf. 2015;83:681–695. doi: 10.1002/prot.24756. - DOI - PubMed
    1. Brinkmann U., Kontermann R.E. The making of bispecific antibodies. mAbs. 2017;9:182–212. doi: 10.1080/19420862.2016.1268307. - DOI - PMC - PubMed
    1. Biyani N. Focus: the interface between data collection and data processing in cryo-EM. J Struct Biol. 2017;198:124–133. doi: 10.1016/j.jsb.2017.03.007. - DOI - PubMed

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