Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

Cambay, Florian; Raymond, Céline; Brochu, Denis; Gilbert, Michel; Cantin, Christiane; Lenferink, Anne E.G.; Grail, Maxime; Henry, Olivier; Crescenzo, Grégory De; Durocher, Yves

doi:10.1016/j.crimmu.2020.06.001

Cited by 10 publications

(19 citation statements)

References 66 publications

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“…CD16a for chicken immunization was produced in-house as two variants. The first one was a bivalent N-terminal Fc-fusion, where the Fc exhibited the P329G LALA and the N297A mutation to circumvent Fc-binding by the CD16a moiety, which would have led to aggregation ( 57 , 58 ). The second variant was a monomeric CD16a protein with an N-terminal His-tag and a C-terminal TwinStrep-tag.…”

Section: Resultsmentioning

confidence: 99%

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

et al. 2021

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Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.

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Section: Resultsmentioning

confidence: 99%

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

et al. 2021

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“…EndoS deglycosylation of IgG1 leads to decreased binding to human FCGR2a/b and FCGR3a, whereas binding to FCGR1 is not affected. Interestingly, reduced fucosylation leads to increased FCGR3a binding, as shown by Cambay et al [ 27 , 29 , 30 , 31 , 32 ].…”

Section: Introductionmentioning

confidence: 88%

Impact of Glycosylation and Species Origin on the Uptake and Permeation of IgGs through the Nasal Airway Mucosa

et al. 2020

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Although we have recently reported the involvement of neonatal Fc receptor (FcRn) in intranasal transport, the transport mechanisms are far from being elucidated. Ex vivo porcine olfactory tissue, primary cells from porcine olfactory epithelium (OEPC) and the human cell line RPMI 2650 were used to evaluate the permeation of porcine and human IgG antibodies through the nasal mucosa. IgGs were used in their wild type and deglycosylated form to investigate the impact of glycosylation. Further, the expression of FcRn and Fc-gamma receptor (FCGR) and their interaction with IgG were analyzed. Comparable permeation rates for human and porcine IgG were observed in OEPC, which display the highest expression of FcRn. Only traces of porcine IgGs could be recovered at the basolateral compartment in ex vivo olfactory tissue, while human IgGs reached far higher levels. Deglycosylated human IgG showed significantly higher permeation in comparison to the wild type in RPMI 2650 and OEPC, but insignificantly elevated in the ex vivo model. An immunoprecipitation with porcine primary cells and tissue identified FCGR2 as a potential interaction partner in the nasal mucosa. Glycosylation sensitive receptors appear to be involved in the uptake, transport, but also degradation of therapeutic IgGs in the airway epithelial layer.

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“…Minor glycan forms include afucosylated (≤1.7%), high mannose (≤1.3%), and sialylated (<0.2%) . Several studies reported N-glycans’ effect on the FcγR–IgG interactions. ,,,− Lack of core fucose (afucosylation) in the IgG structure was indicated as a main inducer for the ADCC activity, and it led to enhanced binding affinity to FcγRIIIa. , Most therapeutic monoclonal antibodies include less than 15% afucosylation. The efficacy of ADCC or a CDC-based mechanism could be altered with engineered afucosylation levels …”

Section: Resultsmentioning

confidence: 99%

“… 40 Several studies reported N-glycans’ effect on the FcγR–IgG interactions. 2 , 9 , 57 , 59 − 61 Lack of core fucose (afucosylation) in the IgG structure was indicated as a main inducer for the ADCC activity, and it led to enhanced binding affinity to FcγRIIIa. 57 , 60 Most therapeutic monoclonal antibodies include less than 15% afucosylation.…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, the IgG binding capacity of FcγRIa and protein A varied for all tested antibodies, indicating a glycosylation-dependent binding variation, as previously reported by research groups. 61 Increased concentrations of FcγRIa displayed a fast association profile in the sensorgram The kinetic parameters were calculated by Biacore Evaluation Software using a 1:1 Langmuir binding model. over the monoclonal antibody-captured surface (Figure 4C).…”

Section: Igg1 Binding Capacity Analysis Withmentioning

confidence: 99%

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Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

et al. 2022

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Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin–biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (k a 50–80 × 105 M–1 s–1) increased in comparison to His capture method (1.9–2.4 × 105 M–1 s–1). In addition, the dissociation rate (k d 10–5 s–1) seemed slower over the His capture method (10–4 s–1) and provided stability on the chip surface during the dissociation phase. The K D values for FcγRIa were found in the picomolar range (2.1–10.33 pM from steady-state affinity analysis and 37.5–46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

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Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

Cited by 10 publications

References 66 publications

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

Impact of Glycosylation and Species Origin on the Uptake and Permeation of IgGs through the Nasal Airway Mucosa

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

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