Enhanced IgG Hexamerization Mediates Efficient C1q Docking and Complement-Dependent Cytotoxicity; Preclinical Proof Of Concept On Primary CLL and Burkitt Lymphoma

We recently discovered that complement is activated by IgG hexamers that assemble on the surface of cells following antigen binding. The formation of hexamers is dependent on intermolecular interactions in the Fc interface between neighboring IgG molecules. This seminal finding allowed us to identify mutations that enhance the ability of IgG antibodies to bind C1 and induce complement-dependent lysis.

To study the role of Fc-mediated intermolecular contacts in complement-dependent cytotoxicity (CDC) of cancer cells by therapeutic IgG antibodies in biological relevant in vitro and in vivo model systems and to exploit this mechanism to improve CDC activity.

Based on an IgG hexamer crystal structure, we generated Fc mutations that were predicted to influence Fc:Fc interactions between IgG antibodies and we screened for improved C’ activation. The effect of these mutations on in vitro C1q binding and CDC-mediated killing of tumor B cell lines and patient-derived chronic lymphatic leukemia (CLL) cells was studied with therapeutic antibodies targeting different B cell antigens. The effect of a CDC-enhancing mutation was further studied in a Burkitt lymphoma xenograft model.

Here we show that the assembly of IgG hexamers on the cell surface represents an important step for efficient complement activation via the classical pathway by therapeutic antibodies against hematological targets. Critical amino acid residues in the Fc:Fc contacts between IgG molecules were identified and individual mutants with increased complement activation generated. Interestingly, introduction of an enhancing mutation was shown to significantly increase complement activation not only of antibodies capable of inducing CDC, but also of antibodies that were devoid of such activity. We provide a proof-of-concept for CD19, Type I and Type II CD20, CD38 and CD52 antibodies. Importantly, we were able to achieve or enable significant CDC of a panel of primary CLL cells by use of this novel approach. Finally, we show that the enhancing mutation resulted in a significant increase in the inhibition of in vivo tumor growth by a CD20 antibody in a subcutaneous Raji xenograft model in SCID mice.

The data obtained provide a novel insight into the mechanism of complement-dependent lysis. This mechanism is shown to be relevant for a panel of therapeutic antibodies against hematological targets. Mutations that improved the formation of Fc:Fc-mediated hexamerization resulted in strongly increased CDC of complement-resistant primary CLL cells and improved efficacy in an in vivo model with a Burkitt lymphoma cell line.

Beurskens:Genmab: Employment. de Jong:Genmab: Employment. Verploegen:Genmab: Employment. Voorhorst:Genmab: Employment. Strumane:Genmab: Employment. Lindorfer:Genmab: Research Funding. Taylor:Genmab: Research Funding. Schuurman:Genmab: Employment. Parren:Genmab: Employment.