CD20, a transmembrane calcium channel required for B cell activation, proliferation, and differentiation, has emerged as a key therapeutic target for B-cell malignancies and autoimmune disorders.
Its selective expression on B-cells—from the late pre-B cell stage to memory cells, but not on early pre-B cells or plasma cells—makes it an ideal target for monoclonal antibodies. This specificity allows for effective B-cell depletion while preserving long-term immunological memory and supporting B-cell reconstitution after treatment.
Anti-CD20 mAbs were originally created to treat B cell proliferative diseases such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia, but they have since been used to treat autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.
These therapies' clinical benefits go beyond basic B cell elimination, perhaps resulting in the loss of dysregulated B cell activities, inflammatory cytokine production, and T cell activation.
Rituximab, the first approved anti-CD20 monoclonal antibody, was introduced in 1997, setting the stage for subsequent generations designed to enhance efficacy and reduce immunogenicity. While significant progress has been made in developing anti-CD20 therapies, a deeper understanding of their mechanisms of action and therapeutic implications remains essential.
This study uses the sophisticated SeromYx Fc effector function platform to profile the Fc effector functions of authorized anti-CD20 mAbs.
Using a well-characterized recombinant human full-length CD20 VLP from ACROBiosystems as an antigen, the study compares the biophysical binding and immune cellular effector capabilities of the first three approved anti-CD20 mAbs.
It also tries to uncover novel Fc effector activities that could improve treatment efficacy while potentially reducing safety hazards.
Through this extensive analysis, the study hopes to provide significant insights for optimizing existing anti-CD20 therapies and better guiding the development of next generation mAbs with better clinical results.
Results
Characterization of anti-CD20 mAb binding to CD20-VLP
The researchers compared Rituximab (RTX), a Type I first-generation mouse/human chimeric monoclonal antibody, to Ofatumumab (OFA), a Type I second-generation fully human monoclonal antibody, and Obinutuzumab (OBZ), a Type II third-generation fully humanized monoclonal antibody with a non-fucosylated modification.
To guarantee a fair comparison, all antibodies were procured from a single vendor, which meant they followed the same standard operating procedures for production, release, and handling. For the antigen, high-quality full-length human CD20 virus-like particles (VLPs) were used (Figure 1).
These VLPs, created with ACROBiosystems' platform based on the HEK293 expression system, provide a versatile approach for producing and investigating transmembrane proteins in a near-native environment.
The CD20 VLPs were tested for bioactivity and dynamic light scattering (DLS) data to ensure the antigen's structural integrity, functionality, and homogeneity.
This VLP format has various advantages, including appropriate protein orientation, density, and the retention of post-translational modifications, which are critical for retaining conformational epitopes and matching CD20's natural presentation on B cells.
The use of VLP-based CD20 allows for a variety of assays, such as antibody screening, bioactivity assessment, and flow cytometry, bridging the gap between in vitro research and cellular systems for more physiologically appropriate comparisons of anti-CD20 antibody levels.
Initially, an on-bead ELISA was used to assess the binding affinity of anti-CD20 antibodies and CD20-VLP. The results showed that Type I anti-CD20 mAbs RTX and OFA bound nearly twice as much to CD20-VLP as Type II mAb OBZ (Figure 2).
This finding is consistent with Type I mAbs' known behavior, which shows increased binding due to their capacity to translocate CD20 into lipid rafts as compared to Type II mAbs.
Figure 1. Virus-like particle (VLP) technology platform expresses transmembrane proteins on the host cell surface. Image Credit: ACROBiosystems
Figure 2. On-bead CD20-VLP ELISA. Image Credit: ACROBiosystems
Fc receptor binding profiles of anti-CD20 mAbs in the presence of CD20
Monoclonal antibody Fc characterization frequently focuses on the bipartite complex of Fc receptor and antibody. Physiologically, immune complexes involve the simultaneous interaction of antigen, antibody, and Fc receptor, and this higher-order complex elicits Fc-mediated immune activities that influence product safety and efficacy.
Therefore, bipartite binding assays using only antibodies and Fc receptors without antigens are insufficient for characterizing Fc-mediated biological reactions. To better predict the involvement of different immune cell types, a series of tripartite binding experiments incorporating Fc receptors and C1q were conducted.
This research examined the binding interactions between CD20-VLP, mAbs, and Fc receptors or C1q in the presence of antigen, providing a baseline understanding of immune complex interactions.
The three mAbs showed high binding to the activating FcγRIIA H131 and R131 variants, suggesting possible interaction with immune cells such as monocytes, neutrophils, dendritic cells, eosinophils, and basophils (Figure 3A & 3B).
Additionally, the mAbs bind to inhibitory FcγRIIB, potentially counterbalancing engagement with the same immune cell types (Figure 3C).
All three mAbs showed substantial binding to FcγRIIIA V158 and F158 variants, indicating ADCC activity via NK cell interaction (Figure 3E & 3F).
All mAbs demonstrated binding to FcγRIIIB, indicating contact with neutrophils (Figure 3D). In Fcγ receptor tripartite binding experiments, Type I mAbs (RTX and OFA) consistently showed stronger binding than Type II mAb (OBZ).
Figure 3. Tripartite Fc receptor binding predicts engagement of immune effectors. CD20-VLP conjugated fluorescent microspheres were used to assess the interaction of anti-CD20 mAbs with Fc receptors: A) FcγRIIA H131; B) FcγRIIA R131; C) FcγRIIB; D) FcγRIIIA V158 E) FcγRIIIA F158; F) FcγRIIIB. Image Credit: ACROBiosystems
RTX has shown strong complement-dependent cytotoxicity (CDC), and OFA was created to improve this activity, but OBZ was engineered to elicit substantially lesser complement activity through afucosylation.
This rank order was maintained in the biophysical investigation, with OFA having the greatest C1q binding, followed by RTX and OBZ (Figure 4).
Figure 4. Tripartite C1q binding of anti-CD20 mAbs. CD20-VLP conjugated fluorescent microspheres were used to assess the interaction of anti-CD20 mAbs with C1q. Image Credit: ACROBiosystems
Comparing anti-CD20 mAbs in canonical Fc effector functional cell-based assays
To evaluate the classical Fc effector activities of anti-CD20 mAbs, four assays were used: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent complement deposition (ADCD), and CDC.
All three anti-CD20 mAbs, RTX, OFA, and OBZ, induced strong ADCP activity in undifferentiated monocytes (Figure 5A). In contrast, the ADCC experiment utilizing CD20-expressing Raji cells demonstrated that the next-generation mAbs, OFA and OBZ, outperformed RTX (Figure 5B).
OFA and OBZ have significant ADCC activity, indicating a superior ability to recruit and activate natural killer (NK) cells, resulting in more effective lysis and death of target cells. In the CDC experiment, the predicted hierarchy of complement-dependent cytotoxicity was observed among the mAbs against CD20 identified on Raji cell surfaces.
OFA had the highest CDC activity, closely followed by RTX, but OBZ had much lower CDC activity (Figure 5C). The data significantly support the mAbs' tripartite C1q binding and ADCD actions against CD20-VLP (Figure 5D).
Figure 5. Confirmation of known Fc effector functions of anti-CD20 mAbs. The ability of anti-CD20 mAbs to A) induce phagocytosis of antigen-functionalized fluorescent beads by undifferentiated monocytes was demonstrated in the ADCP assay, B) elicit NK-cell mediated specific lysis of Raji cells was evaluated in the ADCC assay, C) effect complement dependent cell death of Raji cells was shown in the CDC assay, and D) engage the complement system via C3b was shown in the ADCD assay. Image Credit: ACROBiosystems
Uncovering novel Fc effector functions of anti-CD20 mAbs
Additional cell-based functional experiments from the SeromYx platform were conducted to identify potential Fc effector activities of anti-CD20 monoclonal antibodies. The findings include insights into antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent eosinophil phagocytosis (ADEP).
In the ADNP experiment, all three anti-CD20 mAbs showed significant neutrophil-mediated phagocytosis activity (Figure 6A). This data shows that the tested antibodies engage and activate neutrophils, allowing them to phagocytose target cells.
Similarly, in the ADEP assay, the three anti-CD20 mAbs showed high eosinophil-mediated phagocytosis activity (Figure 6B). This finding suggests that the antibodies can efficiently activate eosinophils and cause eosinophil-mediated clearance of target cells.
Figure 6. Novel Fc effector functions of anti-CD20 mAbs. The ability of anti-CD20 mAbs to A) induce phagocytosis of antigen-functionalized fluorescent beads by neutrophils isolated
from healthy human volunteers was assessed in the ADNP assay and B) induce primary human eosinophil-mediated phagocytosis of antigen-functionalized fluorescent beads was assessed in the ADEP assay. Image Credit: ACROBiosystems
Summary
This article provides important insights into the profile of authorized anti-CD20 monoclonal antibodies (mAbs) with the SeromYx Fc effector function platform.
High-quality, full-length human CD20 virus-like particles (VLPs) were used from ACROBiosystems to conduct a physiologically appropriate assessment of antibody binding and effector functions.
This allowed for a detailed comparison of Type I (rituximab and ofatumumab) and Type II (obinutuzumab) anti-CD20 mAbs, revealing unique binding patterns and effector function capabilities.
The findings showed that Type I mAbs bind better to CD20-VLPs and Fc receptors in the presence of antigen than Type II mAbs, showing how structural variations may influence their modes of action.
This study found a strong agreement between biophysical tripartite binding assays and cellular effector function experiments, confirming the predictive value of tripartite binding assays for mAb effector activities.
Importantly, the identification of strong antibody-dependent neutrophil phagocytosis (ADNP) and eosinophil phagocytosis (ADEP) activity for anti-CD20 mAbs helped to expand our understanding of their potential in vivo processes.
The data implies that neutrophils and eosinophils may play a role in the efficacy and safety of these mAbs in a variety of disease states and tissue settings.
In addition, the separation of Type I and Type II mAbs across numerous assays emphasizes the significance of these distinctions in therapeutic applications and next-generation antibody creation.
To summarize, broadly profiling Fc effector function using the SeromYx Fc effector function platform not only recapitulated known Fc effector activities of anti-CD20 mAbs but also revealed additional possible mechanisms of action.
These findings have significant implications for improving current anti-CD20 therapy and creating new, more effective mAbs.
The CD20-VLP method offers the option to build and characterize mAbs with tailored effector function profiles for specific therapeutic applications, potentially leading to more personalized and effective treatments for a wide range of disorders.
Acknowledgments
Produced from materials originally authored by Peter Hsueh and Michael Friedman from ACROBiosystems and Shashi Jatiani from SeromYx Systems.
References
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- Alduaij, W. and Illidge, T.M. (2011). The future of anti-CD20 monoclonal antibodies: are we making progress? Blood, 117(11), pp.2993–3001. https://doi.org/10.1182/blood-2010-07-298356.
- Teeling, J.L. (2004). Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood, 104(6), pp.1793–1800. https://doi.org/10.1182/blood-2004-01-0039.
- SeromYx (2021) Understanding your product and avoiding nasty surprises in mAb development. https://www.seromyx.com/wp-content/uploads/2024/06/Seromyx-White-Paper-2024.pdf.
About ACROBiosystems
ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.
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