A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Tu, Chao; Terraube, Virginie; Tam, Amy; Stochaj, Wayne R.; Fennell, Brian J.; Lin, Laura; Stahl, Mark; LaVallie, Edward R.; Somers, Will; Finlay, William J. J.; Mosyak, Lydia; Bard, Joel; Cunningham, Orla

doi:10.1074/jbc.m115.688010

Cited by 23 publications

(29 citation statements)

References 35 publications

(43 reference statements)

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“…• The β1-strand (25-HLKIL-29) presents a "path" of solvent-accessible residue backbones ( Fig 1I, black stroke), whereas the side chains have limited solvent accessibility due to secondary structure constraints ( Fig 1A and 1E); this arrangement favors backbone-backbone interactions with potential binding partners. In fact, the β1-strand of CXC chemokines mediates homodimerization [40] and may be "repurposed" for binding to the receptor (supported by modeling [41] and the recent NMR structure with the N-terminal peptide of CXCR4 [25]) or other proteins [42].…”

Section: A Structural Hypothesis Of the Cxcr4-cxcl12 Interaction Guidmentioning

confidence: 99%

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

et al. 2020

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Section: A Structural Hypothesis Of the Cxcr4-cxcl12 Interaction Guidmentioning

confidence: 99%

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

et al. 2020

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“…In fact, the β1-strand of CXC chemokines mediates their homodimerization [40] and may be "repurposed" for binding to the receptor (supported by modeling [41] and the recent NMR structure with the N-terminal peptide of CXCR4 [25]) or other proteins [42].…”

Section: A Structural Hypothesis Of the Cxcr4-cxcl12 Interaction Guidmentioning

confidence: 99%

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

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Stephens

Gustavsson

et al. 2020

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AbstractChemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide crosslinking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 β1-strand, while also recapitulating earlier findings from NMR, modeling and crystallography of homologous receptors. A crosslinking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N-terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor-targeting small molecules and biologics with novel pharmacology.

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“…Thus, creating better complementarity between the AAs of the same domain or at the interface between the variable domains may improve stability of scFv (16). Moreover, the scFv S1D4 exhibited a very high thermal stability [19,41,42] even without additional disulfide bridge [43], which could greatly increase long-term storage stability compared to other scFvs [30]. In terms of chemical stability, clusters II and III substitutions were deleterious.…”

Section: Discussionmentioning

confidence: 99%

“…When re-engineering variable domains, affinity maturation and antibody humanization are carefully considered and worked on [11][12][13][14][15][16]. In some cases, mutations in the antibody complementarity determining regions (CDRs) have shown to improve molecule stability [17,18] thus being able to repair intrinsic flaws in the packing between two V-domains [19]. More precisely, additional work has been carried out on the structural role of framework regions (FRs).…”

Section: Introductionmentioning

confidence: 99%

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

et al. 2020

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In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH-C-C loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 • C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

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A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Cited by 23 publications

References 35 publications

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

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