Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life

Grevys, Algirdas; Frick, Rahel; Mester, Simone; Flem-Karlsen, Karine; Nilsen, Jeannette; Foss, Stian; Sand, Kine Marita Knudsen; Emrich, Thomas; Fischer, Jens A. A.; Greiff, Victor; Sandlie, Inger; Schlothauer, Tilman; Andersen, Jan Terje

doi:10.1016/j.isci.2022.103746

Cited by 37 publications

(34 citation statements)

References 85 publications

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“…Notably, efficient cellular uptake of Abs correlates with high net positive charge, facilitating interaction with the negatively charged cell membrane. This is exemplified by the anti-p40 IgG1 briakinumab, which has large positive charge patches in the variable domains of its Fab arms that delay its release from FcRn at neutral pH 45 and enhances its unspecific cellular uptake in endothelial cells 79 . This gives briakinumab a plasma half-life of only 8-9 days in humans 80,81 .…”

Section: Discussionmentioning

confidence: 99%

Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life

et al. 2022

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Antibody-based therapeutics (ABTs) are used to treat a range of diseases. Most ABTs are either full-length IgG1 antibodies or fusions between for instance antigen (Ag)-binding receptor domains and the IgG1 Fc fragment. Interestingly, their plasma half-life varies considerably, which may relate to how they engage the neonatal Fc receptor (FcRn). As such, there is a need for an in-depth understanding of how different features of ABTs affect FcRn-binding and transport behavior. Here, we report on how FcRn-engagement of the IgG1 Fc fragment compare to clinically relevant IgGs and receptor domain Fc fusions, binding to VEGF or TNF-α. The results reveal FcRn-dependent intracellular accumulation of the Fc, which is in line with shorter plasma half-life than that of full-length IgG1 in human FcRn-expressing mice. Receptor domain fusion to the Fc increases its half-life, but not to the extent of IgG1. This is mirrored by a reduced cellular recycling capacity of the Fc-fusions. In addition, binding of cognate Ag to ABTs show that complexes of similar size undergo cellular transport at different rates, which could be explained by the biophysical properties of each ABT. Thus, the study provides knowledge that should guide tailoring of ABTs regarding optimal cellular sorting and plasma half-life.

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Section: Discussionmentioning

confidence: 99%

Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life

et al. 2022

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“…Therefore, this multivalent binding protein concept presents a tunable and versatile technology platform for enhancing the potency of existing nanobodies simply by multimerization, allowing for the assembly of a large number of binding domains in a single molecule. Apart from increasing the molecular size and functional affinity, we proved that the Quads retained pH-dependent binding to hFcRn, which translated into cellular recycling, predictive for half-life extension in vivo . These parameters could potentially be used to further improve the efficacy and pharmacokinetic properties of nanobodies targeting snake toxins.…”

Section: Discussionmentioning

confidence: 99%

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes

Wade

Rimbault

Ali³

et al. 2022

Bioconjugate Chem.

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Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo.Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

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“…HID HSCT recipients routinely received immunoglobulin (400 mg/kg) on days 1, 11, 21, and 31 after transplantation at our center ( 27 ). In addition, serum IgG had a long half-life of approximately 3 weeks ( 45 ). The above two points may account for normal IgG levels at 1 and 2 months after HSCT in the present study.…”

Section: Discussionmentioning

confidence: 99%

Immune Reconstitution of Patients Who Recovered From Steroid-Refractory Acute Graft-Versus-Host Disease After Basiliximab Treatment

et al. 2022

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We aimed to identify the characteristics of immune reconstitution (IR) in patients who recovered from steroid-refractory acute graft-versus-host disease (SR-aGVHD) after basiliximab treatment. A total of 179, 124, 80, and 92 patients were included in the analysis for IR at 3, 6, 9, and 12 months, respectively, after haploidentical donor hematopoietic stem cell transplantation (HID HSCT). We observed that IR was fastest for monocytes and CD8+ T cells, followed by lymphocytes, CD3+ T cells, and CD19+ B cells and slowest for CD4+ T cells. Almost all immune cell subsets recovered comparably between patients receiving <5 doses and ≥5 doses of basiliximab. Most immune cell subsets recovered comparably between SR-aGVHD patients who recovered after basiliximab treatment and event-free HID HSCT recipients. Patients who recovered from SR-aGVHD after basiliximab treatment experienced satisfactory IR, which suggested that basiliximab may not have prolonged the negative impact on IR in these patients.

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Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life

Cited by 37 publications

References 85 publications

Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life

Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes

Immune Reconstitution of Patients Who Recovered From Steroid-Refractory Acute Graft-Versus-Host Disease After Basiliximab Treatment

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