Creative Biolabs has established an advanced surface plasmon resonance (SPR) platform to determine the interaction between therapeutic antibodies (like IgGs) and fragment crystallizable γ receptors (FcγR) or neonatal Fc receptor (FcRn). Through our high-throughput system, clients could accelerate their research with the most comprehensive view of therapeutic antibodies antibody and with reducing project costs and timelines.
Different FcγR subclasses are known to be present on human effector cells: the high-affinity FcγRI (CD64) and the low-affinity receptors FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b). Within these five subclasses, different polymorphic variants exist, and in some cases, they can influence binding of IgG to these receptors. Therapeutic antibodies are one of the largest classes of modern biopharmaceuticals. Interactions of IgGs with effector cells through FcγRs are often considered a mode of action of therapeutic antibodies. FcγRs are cell surface receptors that can be found on innate immune effector cells such as natural killer cells and macrophages. A therapeutic IgG binds to a membrane-bound antigen on target cells by its complementary-determining regions (CDRs) in the variable domain, while the Fc region in the constant domain can bind to various FcγRs on effector cells, which could lead to effector function, like antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP). The binding kinetics and affinities of IgG-FcγR interactions are hence important parameters for understanding FcγR-mediated immune function. Furthermore, FcRn determines the half-life of IgGs in the bloodstream. Binding of Fc receptor to IgG takes place in the endosome at acidic pH, and the IgG is then recycled back into plasma at neutral pH, thereby preventing lysosomal degradation. Therefore, binding of therapeutic antibodies to FcγRs/FcRn should be evaluated as part of the critical quality attribute (CQA) assessment.
SPR is an extremely sensitive method to detect molecular interactions by tracking the change of signal via sensor chips. Aiming at rapid measurements of IgG samples for high-throughput screening purposes, scientists from Creative Biolabs have developed several SPR methods for FcγRn or FcRn binding to determine IgG-FcγRn/FcRn interaction. General binding response includes three phases: association phase, equilibrium phase, and dissociation phase. Monitoring the changes in the SPR signal over time results in a plot of the binding response versus time called sensorgram. Fitting of the sensorgram data allows calculation of binding parameters, such as the association (Ka) and dissociation (Kd) rate constants and ultimately determine the binding affinity (KD).
Creative Biolabs is pleased to share our cutting-edge technology and extensive expertise in the determination of IgG-FcγRn/FcRn interaction by SPR to facilitate our clients’ research and project development. We can offer high-quality customized SPR services by adjusting protocols to meet every unique requirement. Please feel free to contact us for more information.
Fig. 2 Affinities reported in the literature between FcγRs and IgGs. (Catherine Forest-Nault, 2021)
One of the key quality attributes of monoclonal antibodies is the N-glycosylation of their Fc regions. This modification not only provides stability to the antibody, but also affects the interaction between immunoglobulin G (IgG) and the Fcγ receptor (FcγR), thus regulating the immune response. Here, the researchers introduced in detail the use of different SPR biosensor methods to characterize IgG-FcγR interactions, and analyzed and emphasized their respective advantages and inherent experimental complexity. At the same time, they reviewed the recent results of SPR on the effect of N-glycosylation on the IgG-FcγR interaction.
Surface Plasmon Resonance (SPR) is a sensitive and real-time method used to measure interactions between biomolecules, including the binding of immunoglobulin G (IgG) antibodies to receptors like FcγR (Fc gamma receptors) and FcRn (neonatal Fc receptor). In this assay, one molecule (typically the receptor) is immobilized on a sensor chip, and the other (IgG) is flowed over the surface. The binding interaction causes a change in the refractive index near the surface, which is detected and measured by SPR, providing quantitative data on the affinity, kinetics, and concentration.
The key parameters measured include the association rate constant, which indicates how quickly the IgG binds to the receptor, and the dissociation rate constant, which shows how rapidly the complex disassociates. The equilibrium dissociation constant can also be calculated from these values, providing a measure of the binding affinity between the IgG and the receptor. These parameters help in understanding the strength and stability of the binding interaction.
Analyzing IgG binding to FcγR and FcRn is crucial for understanding and optimizing the therapeutic efficacy and pharmacokinetics of antibody-based drugs. FcγR interactions influence antibody-dependent cellular cytotoxicity (ADCC) and other immune responses, while FcRn interactions affect the distribution, half-life, and recycling of IgGs in the body. SPR provides a detailed characterization of these interactions, aiding in the design of antibodies with improved efficacy and safety profiles.
SPR is capable of analyzing the binding characteristics of different IgG subclasses (such as IgG1, IgG2, IgG3, and IgG4) to various FcγR isoforms and FcRn. This is important because different IgG subclasses have varying affinities for Fc receptors, which can significantly influence their biological activity and therapeutic application. SPR allows for the comparative analysis of these subclasses under the same experimental conditions, providing insights into their functional roles.
Preparing the sensor chip involves immobilizing the receptor (FcγR or FcRn) on the chip's surface. This can be done through direct covalent attachment or capturing the receptor via a tagged molecule or an antibody already bound to the chip. The immobilization must be stable and oriented to maintain the receptor's native conformation and functionality. Proper immobilization ensures that the receptor is active and can interact with IgGs flowing over the chip during the assay.
Several factors can influence the accuracy of SPR measurements, including the quality and purity of the IgG and receptor proteins, the flow rate of the IgG solution over the sensor chip, the density of the receptor immobilization, and the buffer conditions. Interactions can also be affected by nonspecific binding or mass transport limitations if the experimental conditions are not optimized. Careful control of these variables is essential for obtaining reliable and reproducible data.
SPR is highly sensitive and can differentiate between high-affinity and low-affinity interactions. This capability is crucial for antibody engineering, where modifications might be made to the Fc region of an IgG to alter its binding affinity to Fc receptors. SPR provides real-time, quantitative data that allow researchers to assess how changes in antibody structure influence binding dynamics and affinities.
SPR offers several advantages over other methods such as ELISA or radioligand binding assays. It provides real-time monitoring of binding events without the need for labeling the interacting partners, which can alter their properties. SPR can measure both the kinetics and the strength of the interaction, providing a comprehensive view of the binding behavior. Additionally, it requires relatively small amounts of sample and can be used to test a variety of experimental conditions in a high-throughput manner.
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