Type I anti-CD20 therapeutic antibodies

The binding of type I anti-CD20 mAbs leads to the formation of CD20 lipid rafts on the plasma membrane. This allows for binding of a higher number of antibodies to a single cell, that in turn induces more potent effector functions. X-ray crystallography and cryo–electron microscopy studies have made it possible to examine the 3D structures and differences in binding mechanisms between the two types. Type I antibodies show the ability to form “seeding complexes” resulting in concatenation of IgG:CD20 molecules (2:2 arrangement where CD20 dimer binds to 2 IgG) on the plasma membrane (Figure A). This promotes Fc clustering, which explains the higher degree of complement-dependant cytotoxicity (CDC). But, higher Fc clustering also means higher susceptibility to internalization and lysosomal degradation, that could hamper the clinical efficacy.

The first generation of CD20 therapeutic antibody includes the type I Rituximab (Genentech/Roche), which is a human-murine chimeric mAb that is still widely used for cancer and auto-immune diseases. Rituximab has truly revolutionized the field of mAb therapeutics as it was the first successful immunotherapeutic mAb drug to be approved for oncology. The addition of Rituximab in the treatment regimen along with chemotherapy has significantly increased overall survival rates of patients with B-cell malignancies. In-vitro thermodynamic analysis reveal a slower dissociation rate and higher opsonization rate for Rituximab contributing to its stability and efficacy. Apart from CDC, in-vitro assays and genetic-association studies suggest that Fcγ receptor (FcγR)-mediated ADCC (antibody-dependent cell cytotoxicity involving NK cells) and ADCP (antibody-dependent cell phagocytosis involving macrophages) are also important MOAs for Rituximab. Moreover, the high probability of internalization can reduce the cell surface levels of CD20, which could be one of the reasons contributing to Rituximab-resistance.

The second generation of type I unconjugated anti-CD20 therapeutic antibodies that are approved include Ofatumumab (Genmab/GlaxoSmithKline) and Ocrelizumab (Genentech/Roche). Ofatumumab, a fully human mAb, has been approved for CLL as it has shown in-vitro activity against Rituximab-resistant CLL cells, even at low CD20 expression levels. Compared to Rituximab, Ofatumumab binds closer to the B-cell plasma membrane, to an epitope spanning both the extracellular loops of CD20, resulting to an increased CDC. On the other hand, Ocrelizumab is a humanized mAb approved by FDA and EMA for multiple sclerosis. It shows an improved binding affinity to the low-affinity variants of the FcγRIIIa, thereby exhibiting a higher ADCC but lower CDC when compared to Rituximab.

Type II anti-CD20 therapeutic antibodies

Unlike type I, type II mAbs against CD20 causes no redistribution of CD20 within the plasma membrane into lipid rafts. This lack of redistribution attributes to a lower extent of complement activation, compared to type I mAbs. However, evidence from experiments conducted in B-cell lines as well as primary B-CLL cells indicates that type II antibodies cause a greater degree of directly induced, non-apoptotic cell death upon binding to target cells. Interestingly, studies show that type I and II mAbs bind an extremely similar epitope on the same loop of CD20 and it is plausible that only the orientation of binding differs between these mAbs but this has a significant effect on their activity. X-ray crystallography structures and tomography analysis show type II antibodies bind across the CD20 tetramer in a 1:2 fashion (one IgG binds to two CD20s) forming “terminal complexes”, thus preventing the binding of further mAbs (Figure B).

In order to address the requirement for new therapeutics along with increased efficacy, Obinutuzumab (Roche), a novel type II humanized CD20 mAb, was glycoengineered to treat patients with lymphocytic leukemia and lymphomas of B-cell origin. Obinutuzumab also qualified as the first FDA approved therapeutic antibody drug to receive a breakthrough therapy designation to treat the antecedently untreated CLL. Compared to Rituximab, Obinutuzumab induces stronger direct cell death and ADCC, thereby exhibiting superior activity in whole-blood B-cell depletion assays, in preclinical human lymphoma xenograft models, and in depleting B cells in nonhuman primates. This could be attributed to the strong homotypic adhesion and actin-dependent lysosome-mediated cell death produced by Obinutuzumab. Also, the enhanced FcγRIII binding, along with binding to CD20 in a different orientation at a wider elbow angle than type I mAb may be accountable for the different biologic characteristics. Obinutuzumab  therapeutic antibodies are designed for cancer targeting CD20.