A comprehensive set of analytical methods, including bioassays, is essential for accurately characterizing monoclonal antibody (mAb) candidates and assessing their functional performance in terms of efficacy and safety. Characterizing antibody-dependent cellular cytotoxicity (ADCC) that is elicited by the Fc region of mAbs requires more than just demonstrating the ability to target cancer cells; it necessitates the demonstration of effective binding to immune cell receptors and subsequent activation of immune responses. Collaborating with an experienced contract development and manufacturing organization (CDMO) like Tanvex CDMO that can leverage years of extensive expertise in biosimilar development provides access to a robust and validated toolkit, meeting rigorous regulatory standards and ensuring thorough antibody evaluation.
Introduction to ADCC
Antibody-dependent cellular cytotoxicity (ADCC) is an immune response mechanism that plays a critical role in mediating the destruction of cancer cells. This process involves the binding of the Fc (fragment crystallizable) region of immunoglobulin G (IgG) monoclonal antibodies (mAbs) to specific Fcγ receptors — predominantly CD16a or FcγRIIIa — on the surface of immune effector cells. This interaction triggers cellular signaling cascades within these immune cells, leading to the release of cytotoxic agents that lyse and ultimately kill the target cancer cells.1
Various immune cells, including natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells, are known to participate in ADCC, contributing to its effectiveness as a cancer therapy mechanism.2–4 Additionally, other types of antibodies, such as IgA and IgE, engage in similar mechanisms by binding to their respective receptors, Fcα and Fcε.
To enhance the therapeutic potency of mAbs, drug developers focus on increasing their binding affinity to these immune effector cell receptors. Much of this work involves the modification of glycosylation patterns, particularly the targeted elimination of fucose residues within the Fc region. These modifications aim to improve the efficacy of mAbs in eliciting a stronger ADCC response, thereby enhancing their overall anticancer activity.
Case Study: Development of a Biosimilar Monoclonal Antibody for the Treatment of Breast Cancer
The development of biosimilars requires a rigorous demonstration of biosimilarity to the approved reference product in terms of structural characteristics, pharmacokinetic behavior, and functional performance, encompassing both efficacy and safety.
In this example, a biosimilar mAb for the treatment of breast cancer was under development. The mAb comprises a Fab region, which specifically targets antigens on breast cancer cells, and an Fc region that promotes ADCC by engaging the FcγRIIIa receptor located on immune cells. This dual mechanism of action — direct targeting by the Fab and immune activation by the Fc — contributes to its therapeutic function.
To align with stringent regulatory standards, extensive analytical methodologies are required to accurately and precisely analyze the structural and functional integrity of both the Fab and Fc regions. Of note, comprehensive bioassays were developed to validate the biosimilar’s functionality, replicating both the primary targeting and secondary immune activation mechanisms. The bioassay team developed and qualified 15 distinct methods, ensuring thorough coverage of all necessary bioanalytical aspects to confirm biosimilarity.
Detailed Glycan Analysis in Biosimilar Development
Orthogonal methods, especially focusing on afucosylation — the most impactful factor influencing the antibody's ability to mediate ADCC — were utilized to ensure a comprehensive assessment of glycan profiles.5
Initially, the total afucosylation level was determined using hydrophilic interaction liquid chromatography (HILIC). This technique involves separating glycans released from the mAb, which are then detected using a fluorescence detector. This method is particularly effective for providing a detailed glycan profile, offering insights into the diversity and abundance of glycan structures present.
Subsequently, to further validate and refine the findings, glycopeptide analysis was performed. This method follows the tryptic digestion of the mAb, allowing for the examination of glycosylation at specific sites on the protein. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was then employed to analyze these glycopeptides, providing precise information about the location and structure of the glycans.5
Enhanced Assay Development for Functional Evaluation of Biosimilars
In addition to demonstrating equivalent glycan levels, regulatory authorities require biosimilars that rely partly on ADCC to prove both mechanisms of action (MOAs) through rigorous demonstrations of their biological activity. Not only must the mAb bind effectively to cancer cells, but its Fc region must also engage with immune cells with sufficient affinity to trigger an immune response.
For the ADCC MOA, the Tanvex Bioassay team developed and qualified methods to demonstrate the binding activity and binding kinetics of CD16a with the Fc region of the mAb, as well as to show target cell killing by immune effector cells.
The demonstration of binding activity between CD16a and the Fc region of the mAb is challenging because the binding affinity is typically weaker than the Fab’s binding to cancer cell receptors. Consequently, traditional assays such as ELISA (enzyme-linked immunosorbent assay), which depend on robust binding and procedurally require several wash steps, are inadequate. Instead, for this biosimilar, a homogeneous assay technology, AlphaLISA (now under the brand Revvity by Perkin Elmer), was adopted because it requires no wash steps, which allows for a sensitive analysis of the binding activity of such a weak, transient bond. This assay was successfully qualified and transferred to the Quality Control (QC) team and is now employed as a release assay for the biosimilar.
The assay was also part of a study to support process development to enhance the afucosylation of the biosimilar mAb to increase biosimilarity to the originator drug for this attribute. A comprehensive design of experiment (DOE) study was conducted in which three different process parameters were investigated to determine their impact on afucosylation: dissolved oxygen setpoint (DO%), pH range, and temperature during the upstream cell culture process. Temperature was identified as the main contributor to increasing afucosylation and ADCC, and therefore the new process incorporated this temperature adjustment (“post-change"). “Pre-change" lots measured around 100% (92.9% – 114.7%) relative binding activity against the pre-change reference standard, and the “post-change” lots measured on average about 25% higher (108.5% – 145.0%) against the same reference standard using the qualified AlphaLISA method (Figure 1).6
Figure 1. Comparison of CD16a (FcγRIIIa (158V)) AlphaLISA relative binding activity of four post-change lots and nine pre-change lots of the biosimilar mAb.
Furthermore, the binding kinetics were meticulously analyzed using surface plasmon resonance (SPR) technology by Biacore. '
Once sufficient binding of the Fc region to immune effector cells is confirmed, regulators require data showing recruitment of immune cells and killing of the cancer cells via ADCC following binding of mAb molecules to the target cancer cell receptors. Such bioassays are highly complex because they involve two different types of cells. For the mAb in this case study, those cells are breast cancer target cells (SKBR3 cell line) and immune effector cells.
An ADCC cytotoxicity assay was developed using natural killer (NK) cells engineered to overexpress the CD16a receptor (FcγRIIIa 158V variant) and using the DELFIA (dissociation-enhanced lanthanide fluorescent immunoassay) assay principle (Revvity) to enable easier detection during in vitro analysis. In short, the target cells are loaded with a pre-dye and then combined with the effector cells at certain concentrations and in a specified ratio and incubated. Next, varying doses of the biosimilar mAb candidate are added and incubated again. At this step, the Fab region of the biosimilar mAb binds to the specific receptor expressed in the target cancer cell and concurrently, the Fc region binds to the CD16a receptors in the plasma membrane of the NK effector cells. This leads to activation of signaling pathways in the NK cells and subsequent killing of the target cells by substances secreted by the NK cells, which releases the loaded pre-dye into the solution. After adding Europium solution, which binds to the pre-dye and forms a highly fluorescent and stable chelate, the level of cancer cell death is determined as a function of the measured fluorescence depending on the mAb dose.
This assay was also used to evaluate the process change mentioned above6 and demonstrated that the relative NK cytotoxicity level was significantly increased from 72.2% to 150.8% in the lots made with “pre-change” process conditions to 96.2% to 180.1% in lots made using the “post-change” process conditions, demonstrating the effect of increased afucosylated glycans in the “post-change” lots (Figure 2).
Figure 2. Primary NK cell ADCC activity increased in four post-change lots as compared with nine pre-change lots.
While it would be ideal to transfer this method into QC, it is too complicated and variable to meet the requirements for a release assay. It is, however, a highly valuable assay during the characterization phase for demonstrating biosimilarity or to confirm effectiveness of process changes, as shown above.
Next, an assay was developed using peripheral blood mononuclear cells (PBMCs) rather than engineered primary NK cells that overexpress the target receptor. This assay was qualified but not ultimately used for biosimilarity or QC release testing, as the U.S. Food and Drug Administration (FDA) did not require it.
Additionally, a novel reporter gene assay was developed, which bypasses traditional cell-mediated cytotoxicity measurements. Instead, it employs T cells (purchased from Promega) genetically engineered to express a reporter gene. These cells, which do not kill cancer cells, allow for the measurement of transcription factor activation — a surrogate marker of ADCC activation — when they bind to the mAb-treated cancer cells in the presence of target cancer cells. Albeit still requiring the two cell types, target (breast cancer cell line) and effector (surrogate reporter gene immune cell line), this method offers a more straightforward approach to confirming ADCC activity and has been successfully qualified, as well as used in the biosimilarity study and confirming process changes (data not shown).
Using this toolkit of ADCC-related biological assays, comparative studies involving the biosimilar and the branded reference biologic were conducted using multiple lots to ensure biosimilarity (with the exception of the PBMC assay). The robust data generated by these assays underpins the application for market authorization of this mAb biosimilar, with FDA approval expected by January 2025. Notably, a second Tanvex biosimilar has already been approved in the United States and Canada.
Comprehensive Platform Solution for Fc Binding Assays
The suite of biochemical methods and bioassays developed for the highlighted mAb biosimilar candidate now exist as a part of a comprehensive toolkit for mAb characterization and functionality determination that is part of the regulatory requirements for structural, pharmacokinetic, and functional evaluation of novel and biosimilar mAb candidates, including those with enhanced potency due to ADCC activity.
Beyond the specific assays outlined earlier, the Tanvex analytical platform encompasses a variety of methods tailored for different Fcγ receptors. These receptors play pivotal roles not only in ADCC but also in other MoAs, such as complement-dependent cytotoxicity (CDC) and C1q binding. For example, the platform includes seven distinct assays developed to analyze binding kinetics to Fcγ receptors Ia, IIa, IIb and IIIb, and the neonatal Fc receptor (FcRn), in addition to the aforementioned ADCC-related FcγRIIIa (158V and 158F form). This comprehensive set of assays is crucial for confirming the specific Fc binding characteristics of a mAb, even for determining the absence of binding or identifying non-functional binding interactions.
The versatility of this assay platform allows for minor adjustments and customization to meet the unique requirements of different mAb products. By leveraging these now well-established assays, pharmaceutical developers can significantly expedite the method development and qualification process. This not only saves time and resources but also ensures that the analytical methods employed are both robust and reliable. The availability of such comprehensive assay capabilities underscores Tanvex’s commitment to supporting the rapid and effective development of mAb therapies, thereby enhancing their therapeutic potential and ensuring regulatory compliance.
References
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2. MacLennan, I.C.M., G. Loewi, and B. Harding. “The role of immunoglobulins in lymphocyte-mediated cell damage, in vitro. I. Comparison of the effects of target cell specific antibody and normal serum factors on cellular damage by immune and non-immune lymphocytes.” Immunology. 18: 397–404 (1970).
3. Pudifin, D.J., B. Harding, and I.C.M. MacLennan. “The differential effect of γ irradiation on the sensitizing and effector stages of antibody dependent lymphocyte mediated cytotoxicity.” Immunology. 21: 853–60 (1971).
4. Nimmerjahn, F. and J. Ravetch. “Fcγ receptors as regulators of immune responses.” Nat. Rev. Immunol. 8: 34–47 (2008).
5. Rauniyar, N., Khetani, J., and Han, X. “Comparative analysis of Herceptin N-linked glycosylation by HILIC-FLD and LC-MS/MS methods.” J. Pharm. Biomed. Anal. 244 (2024)
6. Tao, Y., M. et al. “Enhancing ADCC Efficacy of an Anticancer mAb Therapeutic by Increasing % of Afucosylated Glycans through Cell Culture Process Optimization.” Antibody Engineering & Therapeutics, San Diego. 2023.