Creative Biolabs provides professional service of FcγRIIIa binding assay to facilitate characterization of FcγRIIIa binding properties for custom antibody candidates.
FcγRIIIa, or CD16a, is commonly expressed on mononuclear phagocytes and NK cells. It is among the stimulatory FcγR classes, which can trigger cellular activation such as respiratory burst and release of inflammatory cytokines, proteases, and prostaglandins. Typical FcγRIIIa has a multi-chain structure associated with immunoreceptor tyrosine-based activation motif (ITAM)-bearing γ- or ζ-chain dimers to elicit positive signal transduction, in a similar way to BCRs and TCRs. One of the major functions of FcγRIIIa is interacting with the Fc domain of certain IgG subclasses (IgG1 and IgG3), thereby mediating antibody-dependent cellular cytotoxicity (ADCC). However, as FcγRIIIa exhibits intermediate affinity toward antibody Fc regions, effector cell activation can only occur when a sufficient number of FcγRIIIa are clustered at the cell surface by multivalent antigen-antibody complexes. Of note, FcgRIIIa displays polymorphism due to the valine-to-phenylalanine (V-to-F) substitution at position 176. The resulting three allelic variants FF/VV/FV differs in Fc binding affinities, which further leads to varied sensitivities toward antibody agents in vivo.
It has been proven that the binding characteristics between antibody Fc regions and FcγRs acts as a pivotal indicator for antibody cytotoxic potential, which plays a key part in determining the therapeutic efficacy of antibody treatments. Particularly, for anti-cancer or anti-autoimmune diseases, such potencies must be strictly modulated and validated to obtain optimal in vivo pharmacodynamical behaviors.
Fig 1. Allelic variants of human FcγRIIIa. (Salmon JE, Pricop L. 2001)
Given these critical features of FcγRIIIa, Creative Biolabs has launched a full range of techniques to provide comprehensive analysis of the interacting mode between FcγRIIIa and antibody Fc region. Our approaches including but not limited to:
Case Study
Fig.2 Binding and fitting curves between sample-1 with FcγRIIIa/CD16a (F176) at different concentrations. (Creative Biolabs)
Fig.3 Binding and fitting curves between sample-1 with FcγRIIIa/CD16a (V176) at different concentrations. (Creative Biolabs)
Table.1 SPR Result Summary between sample-1 with FcγRIIIa/CD16a 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 FcγRIIIa/CD16a (F176) | 42.7 | Sample-1 | 4.883-1250 nM | 1.50E+05 | 3.66E-02 | 2.43E-07 | 117.3 | 2.58 | 1:1 binding |
| His Capture | Human FcγRIIIa/CD16a (V176) | 44.2 | Sample-1 | 4.883-312.5 nM | 2.15E+05 | 8.51E-03 | 3.96E-08 | 112.7 | 6.66 | 1:1 binding |
Our state-of-the-art platform can guarantee high precision, sensitivity, and fidelity, which is also well compatible to automated high-throughput studies. Clinical-grade positive controls will be applied as reference. This assay can serve as a further complement for cell-based ADCC test, to in-depth understand the mechanism of action of lead antibody candidates. Yet there might be a substantial discrepancy between in vitro assessments and in vivo potency, mainly due to the disturbance of endogenous human serum IgGs, which need to be correctly interpreted. Moreover, based on multiple leading-edge strategies, Creative Biolabs also offers Fc engineering service to optimize ADCC activities through altering Fc binding properties. For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
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