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In the development of therapeutic antibodies, the engineering and functional characterization of the Fc domain are critical for ensuring efficacy, safety, and pharmacokinetics. The Fc domain mediates interactions with Fc receptors (FcRs), which are essential for triggering immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis(ADCP), and for modulating the half-life of the antibody.
FcRs can be classified into several types, including the activating receptors (e.g., FcγRI, FcγRIII) and inhibitory receptors (e.g., FcγRIIb), each of which plays a distinct role in immune responses. A systematic Fc functional assessment strategy enables researchers to optimize antibody effector functions, mitigate adverse effects, enhance pharmacokinetic stability, and identify potential safety risks at early stages. Half-life evaluation and in vitro functional validation are indispensable assessment steps in therapeutic antibody development, playing a decisive role in the optimization of antibody candidates and their successful clinical translation.
Overview of the FcR family. (DOI:/10.1084/jem.20151267)
The half-life of an antibody is one of the key parameters used to assess its pharmacokinetic properties. It directly influences both the efficacy of the drug and the frequency of administration. One of the primary factors determining the half-life of therapeutic antibodies is the interaction between the antibody’s Fc region and the neonatal Fc receptor (FcRn). FcRn plays a critical role in regulating the recycling of antibodies within the body, thereby prolonging their half-life (Roopenian & Akilesh, 2007).
The Fc fragment of antibodies binds to FcRn (composed of FCGRT and B2M), particularly under acidic conditions in endosomal compartments. This binding prevents the lysosomal degradation of antibodies, allowing them to be recycled back into circulation. The affinity between the Fc region of the antibody and FcRn is therefore a determinant of the antibody’s half-life, and high affinity is associated with prolonged circulation time. Evaluating FcRn binding affinity provides valuable insight into the pharmacokinetic properties of antibodies, serving as an essential tool for therapeutic antibody development. This interaction forms the theoretical basis for predicting the pharmacokinetics and optimizing the design of antibodies with improved half-life characteristics (Shire et al., 2006).
Therapeutic antibodies can exploit Fc-mediated effector functions to exert cytotoxic effects on target cells, such as tumor cells, through mechanisms like Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), and Antibody-Dependent Cellular Phagocytosis (ADCP). These mechanisms are essential for the therapeutic efficacy of monoclonal antibodies, especially in oncology.
• ADCC:In ADCC, the Fab portion of the antibody binds to specific antigens on the surface of target cells, such as tumor cells, while the Fc portion interacts with Fcγ receptors (FcγRs) on immune effector cells like Natural Killer (NK) cells. This interaction activates NK cells, leading to the release of cytotoxic substances that induce apoptosis in the target cells (Msaouel et al., 2015).
• ADCP: In ADCP, the Fc portion of the antibody binds to FcγRs on macrophages, especially tumor-associated macrophages (TAMs), which facilitates the phagocytosis and subsequent degradation of the target cells. This mechanism is important for the clearance of cancer cells and contributes to the overall antitumor activity of therapeutic antibodies (Weiner, 2015).
• Good Stability: Unlike the LAL assay, recombinant Factor C assay is produced using genetic engineering, ensuring strict quality control and minimal batch-to-batch variation. This guarantees stable and reliable results across different time periods and batches, providing strong support for quality control in pharmaceutical manufacturing.
• CDC: Complement activation through the Fc region of the antibody also plays a significant role in the immune-mediated destruction of target cells. The binding of antibodies to antigens on target cells triggers the classical complement cascade, leading to cell lysis and death (Nimmerjahn & Ravetch, 2008).
Fc-mediated effector functions: the schematic depicts the variety of cells expressing Fc receptors (FcRs) and the different types of Fc-mediated effector functions. (DOI:10.1080/19420862.2025.2453515)
The use of in vitro models to validate ADCC, ADCP, and CDC is vital for screening therapeutic antibodies in preclinical stages. These models provide quantitative data on the potency of the antibody to trigger immune responses and can predict the clinical success of therapeutic antibody candidates. Moreover, evaluating these functions helps optimize the therapeutic window and minimize off-target toxicity, ensuring better clinical outcomes for patients (Shi et al., 2018).
The assessment of antibody half-life, driven by the interaction between the Fc region and FcRn, is an essential step in the development of therapeutic antibodies. Additionally, the validation of Fc-mediated effector functions, such as ADCC, ADCP, and CDC, ensures the therapeutic efficacy of these antibodies, particularly in oncology. Both of these evaluation steps provide critical insights into the pharmacokinetics and immunological activity of antibody candidates, offering valuable information for optimizing their clinical development. The successful incorporation of these evaluations into the preclinical pipeline is paramount for the creation of effective and safe therapeutic antibodies.
• Antibody Screening and Validation
For the screening and validation of antibody drugs, we have developed a series of FcRn-related products, including:
>> FcRn Proteins
>> FcRn Binding Kits (TR-FRET)
>> Overexpressing Cell Lines
• Antibody Screening and Validation
To support Fc-mediated binding affinity studies, we have developed a comprehensive portfolio of Fc receptor-related products, including:
>> FcγRs, FcRn Proteins
>> FcR Binding Kits (TR-FRET)
>> Overexpressing Cell Lines
• In Vitro Functional Validation for ADCC/ADCP
For Determination of ADCP activity induced by antibodies,we have developed a a series of Jurkat Reporter Cell Lines, including:
>> Human CD32a / CD32b Jurkat Reporter Cell
>> Human CD64 Jurkat Reporter Cell
>> Human CD16a Jurkat Reporter Cell
| FcR Proteins |  Affinity verified by SPR & BLI: activity guaranteed, and protocols offered free of charge. |
High purity: SDS-PAGE verification purity>95%, SEC-MALS verification purity>90%. | |
Biotinylated Fc Receptor proteins labeled with AvitagTM offered: the labeling efficiency is high, and the labeling site is specific and clear. | |
Expressed by HEK293 Cells, Multiple species, High stability |
| Binding Kits (TR-FRET) | Simple and fast operation: No complicated washing steps & Results in just 1 hour. |
Comprehensive validation: Validated with various antibody subtypes and antibody drugs. | |
High throughput capability: Supports 500 tests, ideal for high-throughput screening. | |
High batch consistency:Strict control over raw materials and finished product quality, ensuring a stable supply. | |
Accurate and reliable results:High sensitivity with minimal matrix effects. |
| Overexpressing Cell Lines | Genetically modified cell lines best reflect MOA (Mechanism of Action) |
Higher activity and larger assay windowfor robust and reproducible cell-based bioassay | |
Comprehensive application data to support assay development and validation | |
Full tracible record, stringent quality control and validated cell passage stability | |
Parental cell line legally obtained from internationally recognized cell resource bank and commercially licensed | |
Global commercial license assistance whenever regulatory filing is required |
FcRn Proteins
FcRn High Homogeneity Structure Verified by SEC-MALS
The purity of Biotinylated Canine FCGRT&B2M Heterodimer Protein, His,Avitag&Tag Free (Cat. No. FCM-C82W9) is more than 95% and the molecular weight of this protein is around 43-53 kDa verified by SEC-MALS.
Binding Affinity of FcRn to Herceptin was Validated by SPR
Immobilized Biotinylated Human FCGRT&B2M Heterodimer Protein, His,Avitag (Cat. No. FCM-H82W7) on SA Chip can bind Herceptin® with an affinity constant of 0.267 μM as determined in a SPR assay (Biacore 8K) (QC tested).
FcRn Binding Kit（TR-FRET）
Application of antibody drug Half-Life Assessment
The half-lives of these 3 monoclonal antibodies currently in clinical use generally correlate with the binding affinity to FcRn. The kit has been used to detect 3 FDA approved antibody drugs of different binding affinity to FcRn, and the IC50 trends are consistent with affinity constant from SPR as well as the actual in vivo half-life published.
FcRn Overexpressing Cell Lines
Receptor Assay
FACS analysis of FcRn (FCGRT & B2M) on HEK293/Human FcRn (FCGRT & B2M) Stable Cell Line. FACS assay shows that FITC-Labeled Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F) Protein, His Tag (Cat. No. IG1-HF2H3) can bind to HEK293/Human FcRn (FCGRT & B2M) Stable Cell Line. HEK293/Human FcRn (FCGRT & B2M) Stable Cell Line was red line, negative control HEK293 cells was grey line(QC tested).
Roopenian DC, Akilesh S. FcRn: The neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7(9):715-725. doi:10.1038/nri2155.
Shire, S. J., et al. (2006). Development of Therapeutic Monoclonal Antibodies. Pharmaceutical Research, 23(10), 2121-2136.
Msaouel P, et al. Fc-Mediated Effector Functions in Therapeutic Monoclonal Antibodies. J Immunol. 2015;194(10):4862-4871.
Weiner, G. J. (2015). Monoclonal Antibodies in Cancer Therapy: Advances and Challenges. Nat Rev Cancer, 15, 87–100. doi:10.1038/nrc3803.
Nimmerjahn, F., & Ravetch, J. V. (2008). Fcgamma receptors as regulators of immune responses. Nat Rev Immunol, 8(1), 34-47.
Shi, J., et al. (2018). Recent advances in antibody-drug conjugates for cancer therapy. J Hematol Oncol, 11, 44.
Silvia Crescioli, Shashi Jatiani & Lenny Moise (2025) With great power, comes great responsibility: the importance of broadly measuring Fc-mediated effector function
early in the antibody development process, mAbs, 17:1, 2453515, DOI:10.1080/19420862.2025.2453515
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