DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously

Beckmann, Roland; Jensen, Kristian; Fenn, Sebastian; Speck, Janina; Krause, Katrin; Meier, Anastasia; Röth, Melanie; Fauser, Sascha; Kimbung, Raymond; Logan, D.T.; Steegmaier, Martin; Kettenberger, Hubert

doi:10.1038/s41467-021-20949-3

Cited by 17 publications

(17 citation statements)

References 41 publications

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“…Additionally, we included three Fab fragments which were part of a study redesigning the Fab interfaces. Furthermore, we also investigated two λ light chain antibodies and two recently published DutaFab structures, which are characterized by their high stability and their ability to recognize two different antigens (Beckmann et al, 2021). Dual targeting (Duta) Fab molecules contain two independent and spatially separated binding sites within the CDR loops (H-side paratope and L-side paratope) that simultaneously allow to bind two target molecules at the same Fv.…”

Section: Datasetmentioning

confidence: 99%

Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

Fernández‐Quintero

Quoika

Wedl

et al. 2022

Front. Mol. Biosci.

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As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.

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Section: Datasetmentioning

confidence: 99%

Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

Fernández‐Quintero

Quoika

Wedl

et al. 2022

Front. Mol. Biosci.

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“…The H-side paratope consists of CDR H1, H3 and L2, while the L-side paratope comprises CDR L1, L3 and H2. Therefore, these Fabs are able to target two antigens simultaneously with the same Fv region, however the design of DutaFabs is comparatively complex ( 18 ). Furthermore, tetravalent IgG-like bispecific constructs were described that do not consist of regular Fab arms but rather of engineered arms in which one VH domain is attached to each of the constant CH 1 and CL domains ( 19 ).…”

Section: Introductionmentioning

confidence: 99%

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization

Harwardt

Bogen²,

Carrara³

et al. 2022

Front. Immunol.

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Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two‐in‐One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death‐ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

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“…Genentech Inc. disclosed two co-crystal structure of VEGF-A 8-109 in complex with VEGFR-1 D2 (Figure 4, PDB code: 1FLT and 1QTY) in 1997 and 1999 [61,69]. The epitopes of VEGF-A in contact with the VEGFR-1 D2 include residues from one monomer: (1) the N-terminal helix α1 (16-27), (2) the loop 2 connecting β3 to β4 (61-66), (3) the strand β7 (103-106), and residues from the other monomer: (4) the strand β2 (46-48) and ( 5) strands β5 and β6 together with the connecting loop 3 (79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91). These authors also conducted a domain deletion study of the extracellular domains of VEGFR-1.…”

Section: Receptors and Receptor Fragmentsmentioning

confidence: 99%

“…Recently, dual targeting antibody Fab fragments (DutaFabs) have been developed by Roche Inc. Two separate fragments of the CDR of one human Fv region bind specifically to two distinct targets: VEGF-A and PDGF-BB (human PDGF consituted of two B subunits) [84]. Interestingly, the two targets can be bound simultaneously and with high affinity (Table 1).…”

Section: Antibodies and Antibody Fragmentsmentioning

confidence: 99%

A Structural Overview of Vascular Endothelial Growth Factors Pharmacological Ligands: From Macromolecules to Designed Peptidomimetics

Gaucher

Vidal

et al. 2021

Molecules

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The vascular endothelial growth factor (VEGF) family of cytokines plays a key role in vasculogenesis, angiogenesis, and lymphangiogenesis. VEGF-A is the main member of this family, alongside placental growth factor (PlGF), VEGF-B/C/D in mammals, and VEGF-E/F in other organisms. To study the activities of these growth factors under physiological and pathological conditions, resulting in therapeutic applications in cancer and age-related macular degeneration, blocking ligands have been developed. These have mostly been large biomolecules like antibodies. Ligands with high affinities, at least in the nanomolar range, and accurate structural data from X-ray crystallography and NMR spectroscopy have been described. They constitute the main focus of this overview, which evidences similarities and differences in their binding modes. For VEGF-A ligands, and to a limited extent also for PlGF, a transition is now observed towards developing smaller ligands like nanobodies and peptides. These include unnatural amino acids and chemical modifications for designed and improved properties, such as serum stability and greater affinity. However, this review also highlights the scarcity of such small molecular entities and the striking lack of small organic molecule ligands. It also shows the gap between the rather large array of ligands targeting VEGF-A and the general absence of ligands binding other VEGF members, besides some antibodies. Future developments in these directions are expected in the upcoming years, and the study of these growth factors and their promising therapeutic applications will be welcomed.

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DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously

Cited by 17 publications

References 41 publications

Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization

A Structural Overview of Vascular Endothelial Growth Factors Pharmacological Ligands: From Macromolecules to Designed Peptidomimetics

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