How does GA101 work?

My old colleagues Tim Illidge, Mark Cragg and Martin Glennie have published their recent work on the new anti-CD20 monoclonal antibody, GA101, in a recent edition of Blood. http://bloodjournal.hematologylibrary.org/content/117/17/4519.abstract

GA101 is a fully humanized, type II, properly glycosolated monoclonal, which has been enginered to produce better killing than rituximab.

Anti-CD20 monoclonals invoke a wide range of killing methods, chief among them being antibody dependent cellular cytotoxicity (ADCC), but also phagocytosis mediated by macrophages and/or NK cells through the Fc gamma receptor link, complement dependent cytotoxicity (CDC) and sometimes direct induction of programmed cell death (PCD). PCD certainly does not play a significant part in rituximab killing, but is it important for other anti-CD20 monoclonals?

They have compared GA101 with rituximab against a panel of B-lymphoma cell lines. This shows that GA101 is much better at PCD than rituximab, even when the Fc part of teh molecule is removed. PCD seems to require what they call homotypic adhesion, which simply means that the lymphoma cells aggregate together when exposed to the antibody. The aggregation of the lymphoma cells causes alterations to the actin cytoskeleton of the cells. The actin molecules move to the points of cell to cell contact and if this is chemically inhibited then PCD is deiminished.

Next they showed that the actin-dependent death was independent of apoptosis, and could still work in apoptosis-resistent cells. Lysosymes are involved in PCD. It appears that GA101 induces lysosomal membrane permeabilization and cathepsin-mediated cell death. Cathepsin B is a classic lysosomal protease.

So how does GA101 fit into the pantheon of monoclonal anti-CD20s? It binds to a very sinilar site on the larger loop of the CD20 molecule (in contrast to ofatumumab which binds to the smaller loop). However, recent crystallographic evidence shows that it binds with a completely different orientation to rituximab. How exactly the aggregation of lymphoma cells in response to antibody coating occurs is not clear. It certainly requires bivalency, but the FC portion of the antibody is not required.

Another unexplained phenomnon is the fact that not all cells are killed by PCD, yet when the surviving cells are washed and retested they are killed in the same proportion; they have not acquired resistance.

GA101 is not the only monoclonal capable of triggering such severe actin relocalization; again it is not clear why it does so.

Lysosome function as cellular recycling and waste didposal units by degrading organelles and macromolecules delivered to the lysosomal compartment by autophagy, endocytosis and phagocytosis. They carry over 50 different degradation enzymes. If they leak they destroy the cell that conatins them in a non-apoptotic way. Again the link between actin repositioning and lysosomal permeabilization is missing.

All this work should not detract from the fact that GA101 has been glycoengineered to produce enhanced ADCC, but it does appear that it has a second string to its bow; one that should overcome the need for a TP53 pathway and therefore GA101 may prove to be effective in drug resistant CLL.