FcγR Binding Assays

Antibodies and antibody-based therapeutics have the potential to elicit immune functions through binding to Fc-γ receptors (FcγRs) on immune cells or fixing complement. With extensive experience in Fc function analysis & characterization, Creative Biolabs offers a versatile technology platform for providing better assistance on Fc receptor profiling of custom antibodies, especially for FcγR binding analysis.

Introduction to Fcγ Receptors

One major mechanism through which therapeutic antibodies deliver their clinical efficacy is to elicit indirectly cytotoxicity via activating immune effector components. This is achieved by antibody Fc regions, which can interact with specific Fc receptors (i.e. FcγR) or complements. FcγRs, which are primarily expressed on leukocyte surface, can generally be classified into three types: FcγRI, FcγRII (FcγRIIa, FcγRIIb, and FcγRIIc), and FcγRIII (FcγRIIIa and FcγRIIIb). They bind the same region on IgG Fc, yet with different affinities (FcγRI is the high-affinity receptor) and functions (FcγRIIb has an inhibitory motif while others are activating receptors with ITAM). Binding can be affected by genetic polymorphism of the receptors, as well as different glycosylation patterns in the Fc region of an antibody. Following Fc-FcγR binding, subsequent activation responses occur, including effector cell recruitment to the target sites, the release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. For instance, FcγRIIIa (CD16a) is believed to play an essential part in ADCC, and FcγRIIa (CD32a) can mediate ACDP activity.

Fig.1 Antibody binding to Fc receptors. (Jennewein, 2017)

As a consequence, comprehensive analysis of antibody Fc interactions with FcγRs, accompanied with cell-based cytotoxicity assessment (ADCC/ADCP), has become an integral part of therapeutic antibody development, which is strongly required by regulatory departments. Based on our state-of-the-art technologies and seasoned scientists, Creative Biolabs brings out a novel, reliable, and highly flexible platform for evaluating FcγR binding characteristics. We offer a full range of FcγR targets with corresponding positive controls, including:

• FcγR1/CD64
• FcγR2a/CD32a (R/H variant)
• FcγR2b/CD32b
• FcγR3a/CD16a (V/F variant)
• FcγR3b/CD16b

Creative Biolabs is capable of performing FcγR binding assays for various IgG antibody isotypes (IgG1, IgG2, IgG3, and IgG4) or Fc fusion proteins with different approaches, including but not limited to:

• ELISA
• Flow cytometry
• Biacore surface plasmon resonance (SPR)
• Octet bio-layer interferometry (BLI)

We take advantage of the cutting-edge surface plasmon resonance (SPR) technology to execute our FcγR binding assays. At Creative Biolabs, we provide a series of assays to evaluate FcγR binding capacity for humanized antibodies. In addition, we also deliver numerous FcγR binding assays of other species to screen non-humanized antibodies or examine the cross-reaction among species.

In combination with routine cell-based cytotoxicity assays like ADCC and ADCP, FcγR binding results can act as a further supplement, thus providing a full-scale illustration about the antibody mechanism of action. Moreover, we also provide professional services of FcRn binding assay and C1q binding assay.

Because of the competition of endogenous human serum IgG and therapeutic antibodies for binding effector FcγRs, there may be a substantial discrepancy between in vitro assessments and actual in vivo cytotoxicity potency. Hence, improved Fc avidity and affinity toward FcγRs can be extremely significant for enhancing in vivo clinical efficacy. Fully understanding this need, Creative Biolabs offers additional Fc engineering service, by genetic manipulation and glycoform modification, to up- or down-regulate the binding affinities towards specific FcγRs.

Scientists at Creative Biolabs have broad expertise in tailoring a variety of antibody functional assays and are pleased to apply the expertise in helping our worldwide customers with their leading-edge antibody projects. Our world-class assay platform ensures high accuracy, sensitivity, and well-compatible to automated high-throughput studies. For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

1. Jennewein, M. F.; Alter, G. The immunoregulatory roles of antibody glycosylation. Trends in immunology. 2017, 38(5): 358-372.

For Research Use Only.