Creative Biolabs brings a comprehensive portfolio of elegant assays to help worldwide clients better understand the Fc binding properties of their lead antibodies against diverse FcγRs, particularly FcγRIIa.
FcγRs, a large group of membrane receptors that expresses on leukocytes, can mediate immune effector activities through interacting with antibody Fc regions. There exists extensive structure diversity among different FcγR subclasses, leading to different binding affinities, distinct signal transduction pathways, and specific cell type expression patterns. In general, they can be classified into stimulatory FcγRs and inhibitory FcγRs. Amongst them, FcγRIIa, or CD32a, is a stimulatory FcγR with relatively low Fc affinity. In contrast to FcγRIIIa, it is a single-chain receptor with an extracellular ligand-binding domain and ITAMs in the cytoplasmic domain. FcγRIIa is widely expressed on a broad spectrum of cells and participates in diverse cell type-specific functions. Once activated, FcγRIIa can mediate ADCP activities when located on macrophages; when located on neutrophils, it triggers respiratory burst and internalization; while on platelets, it induces aggregation and degranulation; for dendritic cells, it can help to promote antigen presenting process. Notably, co-clustering with other stimulatory FcγRs, e.g. FcγRIIIa, FcγRIIa can produce synergic effects and amply activation signals for immune effector cells.
It is strongly suggested that varied FcγR genotypes should be taken into consideration when applying antibody drugs with immune effector potencies. Like FcγRIIIa, FcgRIIa also has two co-dominantly expressed alleles at position 131 in the extracellular domain, H and R (histidine and arginine). This polymorphism causes significant differences in binding affinities toward antibody Fc regions among the three allelic variants (HH/RR/HR).
Fig 1. Allelic variants of human FcγRIIa. (Salmon JE, Pricop L. 2001)
In effort to in-depth study the binding properties of custom antibodies against FcγRIIa, Creative Biolabs has established a comprehensive testing platform based on our state-of-the-art technologies and extensive expertise in this field. We perform FcγRIIa binding assays using multiple approaches, including but not limited to:
Case Study
Fig.2 Binding and fitting curves between sample-1 with FcγRIIa/CD32a (R167) at different concentrations. (Creative Biolabs)
Fig.3 Binding and fitting curves between sample-1 with FcγRIIa/CD32a (H167) at different concentrations. (Creative Biolabs)
Table.1 SPR result summary between sample-1 with FcγRIIa/CD32a subtypes. (Creative Biolabs)
| Method | Ligand | Capture Level (RU) | Analyte | Analyte Conc. | ka (1/Ms) | kd (1/s) | KD (M) | Rmax (RU) | Chi² (RU²) | Fit method |
| His Capture | Human FcγRIIa/CD32a (R167) | 46.6 | Sample-1 | 156.25-20000 nM | NA | NA | 1.29E-05 | 78.6 | 0.33 | Steady-state affinity |
| His Capture | Human FcγRIIa/CD32a (H167) | 44.0 | Sample-1 | 78.125-20000 nM | NA | NA | 4.98E-06 | 147.8 | 5.51 | Steady-state affinity |
Clinical-grade reference antibodies will be used as positive controls. Our seasoned scientists in Creative Biolabs have vast experience in tailoring personalized assays with appropriate concentration design, endpoints, exposure methods, and other parameters to address specific custom issues. In combination with cell-based cytotoxicity assays like ADCC, ADCP, FcγRIIa binding assay can provide more mechanistic insight upon the in vivo potency of lead antibodies during the early development stage. However, there may be a substantial discrepancy between in vitro assessments and in vivo cytotoxicity potency, mainly due to competing endogenous human serum IgGs, so correct interpretation is quite essential. Moreover, Creative Biolabs also offers additional Fc engineering service for better modulating Fc characteristics regarding FcγRs. For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
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