Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions

Bobkov, Vladimir; Zarca, Aurélien; Hout, Anneleen Van; Arimont, Marta; Doijen, Jordi; Bialkowska, Magdalena; Toffoli, Elisa C.; Klarenbeek, A.; Woning, Bas van der; Vliet, Hans J. van der; Loy, Tom Van; Haard, Hans de; Schols, Dominique; Heukers, Raimond; Smit, Martine J.

doi:10.1016/j.bcp.2018.10.014

Cited by 49 publications

(52 citation statements)

References 78 publications

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“…Nanobody binding to LgBiT-CXCR4 was subsequently detected using complemented luminescence. The resulting affinity of VUN400-HiBiT at LgBiT-CXCR4 (pK D 7.25) was in good agreement with previous data determining the affinity of VUN400 at the unmodified CXCR4 receptor with radioligand binding (pIC 50 7.3; Bobkov et al, 2018). These data are also in agreement with the general observation that C-terminal fusion to nanobodies does not affect their binding characteristics (Fang et al, 2016).…”

Section: Discussionsupporting

confidence: 90%

“…The improved selectivity and extended half-lives of antibodies compared to small molecules has meant there has been much interest in using antibody-based approaches to target CXCR4 therapeutically (Hutchings et al, 2017;Bobkov et al, 2019). This has included the recent development of a panel of single domain antibody fragments, called nanobodies, which are able to bind CXCR4 (Jahnichen et al, 2010;de Wit et al, 2017;Bobkov et al, 2018;Van Hout et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Nanobodies which that bind to the extracellular domains of GPCRs are able to modulate receptor activity and organization (De Groof et al, 2019a). Several studies have investigated the therapeutic potential of extracellular nanobodies which target chemokine GPCRs, including CXCR2 (Bradley et al, 2015), CXCR4 (Jahnichen et al, 2010;de Wit et al, 2017;Bobkov et al, 2018;Van Hout et al, 2018), ACKR3 (Maussang et al, 2013), and US28 (De Groof et al, 2019b). Given their relatively large N-terminus compared to the other Class A GPCRs, and the fact that their endogenous ligands are peptides, chemokine GPCRs are ideal candidates to target with extracellular nanobodies.…”

Section: Introductionmentioning

confidence: 99%

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Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Soave

Heukers

Kellam

et al. 2020

Preprint

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max 150 words)Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Soave

Heukers

Kellam

et al. 2020

Preprint

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“…It is for instance the case when nanobodies are produced as Fc-fusions to restore the ADCC and CDC effector functions in vivo and to increase their apparent binding affinity by the avidity effect provided by the resulting bivalent molecule. Anti-CXCR4 nanobodies fused to human IgG1 Fc that were expressed and purified from HEK293T cells in suspension specifically induced the ADCC-and CDC-mediate cell death of leukemia cells that overexpressed such receptor but did not affect CXCR4 negative cells [41]. Expi293F cells were used for producing the reconstituted anti-HER1 nanobody-Fc constructs necessary for the fabrication of immunotoxins by means of intein-mediated splicing and assembling with gelonin [42].…”

Section: How To Choose Between Mammalian and Bacterial Expression Sysmentioning

confidence: 99%

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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“…This paves the way for potential therapeutic use of HCMV-specific nanobodies. In addition to their intrinsic activity, the efficacy of such nanobodies in experimental and clinical settings could be further enhanced through coupling to effector molecules 8,40,41,42,43,44 .…”

Section: Hcmv Establishes a Latent Infection In Cd34 + Progenitor Celmentioning

confidence: 99%

Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies

Groof

Heukers

Lim

et al. 2020

Preprint

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Latent reservoirs of viral pathogens are significant barriers to eradication of these viruses. During latency, herpesviruses maintain their genome, with little gene expression, making latent infections refractory to current treatments targeting viral replication. In the case of human cytomegalovirus (HCMV), sporadic reactivation events are well controlled by the immune system. However, in immunocompromised or immunosuppressed individuals, HCMV reactivation often results in morbidity in solid organ and stem cell transplant patients. Clearance of the latent reservoir could lower the incidence and severity of HCMV-associated disease. Here, we develop a virus specific nanobody (VUN100b) that partially inhibits signaling of the viral receptor US28. VUN100b treatment partially reverses latency without fully reactivating the virus. Moreover, VUN100b treatment drives recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals. This study shows the potential of VUN100b as a therapy to clear the HCMV latent reservoir of transplant patients.

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Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions

Cited by 49 publications

References 78 publications

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies

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