Impact of Antigen Density on Recognition by Monoclonal Antibodies

Abstract

Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and non-monotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition. Despite the continuous improvement of traditional chemotherapy, the use of monoclonal antibodies (mAbs) as drugs for the treatment of a variety of diseases has been growing steadily for the last two decades. 1 In this context, there is a strong interest in studying mAb recognition of cognate antigens. MAbs are used in oncology for many therapeutic targets including the CD20 antigen, the human epidermal growth factor receptor 2 (HER2), the vascular endothelial growth factor (VEGF), the epidermal growth factor receptor (EGFR) and the programmed cell death protein 1 (PD-1). 2 The hematopoietic differentiation antigens associated with cluster of differentiation (CD) represent the main targets for mAb in oncology. 3 In particular, the CD20 antigen is the target of several therapeutic mAbs and their derivatives (e.g. rituximab, ibritumomab, ofatumumab, obinutuzumab, ocrelizumab) that are successfully used to treat B-cell malignancies, including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis. 4 The antigen-binding activity of mAbs determines their biological efficacy and depends on several factors, including antigen density, association and dissociation rates. Several studies have suggested that the increase of CD20 antigen expression modulates the biological response of mAbs such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) that trigger cell death. 5,6,7 As the antigen distribution generally has a direct impact on the clinical efficacies of mAbs, it is of...

Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses

“…Additionally, understanding molecular recognition could pave the way for structure-based design of bioactive molecules. Very recently, it has been shown using cryo-electron microscopy (cryo-EM) that the native CD20 structure is a compact dimeric form allowing RTX cross-links that can induce complement recruitment. , This result corroborates other studies indicating that CD20s are not expressed in monomeric form, but organized into supramolecular protein complexes. , In parallel, our group has studied the effect of CD20 density on the recognition by RTX, which is known to be a critical point for the therapeutic response . An average critical inter-CD20 spacing of nearly 2 nm confers the best conditions for RTX binding.…”

“…7,8 In parallel, our group has studied the effect of CD20 density on the recognition by RTX, which is known to be a critical point for the therapeutic response. 9 An average critical inter-CD20 spacing of nearly 2 nm confers the best conditions for RTX binding. This value is in excellent agreement with cryo-EM structures.…”

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation

“…Most antibodies have K D values in the range of 10 -6 to 10 -9 . Antibody affinity can be measured using different methods such as label-free optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference, SPR, and ellipsometry-based (label-free) optical scanner [11] , [35] , [36] , [37] , [38] . K D calculated from the experimental results obtained after polyclonal anti-SCoV2-N interaction with covalently immobilized SCoV2-N falls in this range of K D values.…”

“…It was determined that the bivalent binding of monoclonal Abs to human immunodeficiency virus type 1 envelope glycoprotein trimer 2F5 epitopes results in greatly enhanced neutralization efficiency through an increase in binding avidity [10] . Additionally, the impact of CD20 epitopes surface density on the recognition and bivalent interaction with monoclonal Abs (rituximab) was analyzed using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) technique [11] . To study the affinity and avidity features of the anti-SCoV2-N, the noncontact, real time and sensitive methods are required.…”

Major Surface Antigens in Zoonotic Babesia

“…The binding of an antibody to its cognate antigen can be greatly affected by the physical state of the protein, either membrane-bound or soluble [54]. This is especially the case for GPI-anchored proteins, as these proteins could be packed in a membrane raft of potentially high density or homogeneously spread on the cell surface, which could have an impact on antibody binding (Figure 1C) [55]. For vesicle-bound antigens, it remains to be determined if the protein density and orientation are similar to the plasma membrane-bound proteins, and the interactions with antibodies would need to be characterized.…”