Evolving sharply focused antibodies with complimentary surfaces

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There have been some papers describing heterotrimeric Env like immunogens for HIV, which have shown increased breadth of neutralization, but which can in principle generate diverse responses. Here we describe a method based on a heterodimer designed to elicit a single focused response. Immunogens with complimentary surfaces take advantage of the fact that to activate a B-cell, a key event in the immune system's affinity maturation feedback loop where the results of somatic hypermutation are selected, the immunogen is required to be at least a dimer. We arrange for the two surfaces of a dimeric immunogen to have the conserved binding site repeated on both elements of the dimer, and to have the periphery surrounding the conserved binding site on both surfaces be "complimentary." What we mean by this is if a mutation to the B-cell receptors improves the affinity to the conserved binding site, the affinity should be improved on both surfaces and this result is fed back to the system at full strength in the form of improved binding. If a mutation improves the affinity to a region in the periphery of the binding site on one element of the dimer, then if the surfaces are perfectly complimentary, the affinity to the same region on the complimentary surface will be reduced. The feedback signal from these mutations would thus be attenuated and more likely to be ignored. This should tend to focus binding on the conserved binding site with binding to the periphery remaining relatively unimproved.

This is not a perfect idea. In practice, if the heavy chain gets a good grip on the binding site of interest, there is enough flexibility for the light chain to bind in multiple locations. Something that has been done previously to sort broadly neutralizing antibodies is to resurface the periphery of the binding site of interest. One might like to try residues that are less sticky like alanines or amino acids with non-polar sidechains. The problem is, if you get too carried away with this kind of thing, you might end up with a protein that will no longer fold. One could try to arrange for polar or charged residues on the periphery of the binding site to be complimentary in a rough kind of way, but it wouldn't be perfect. It might be better than nothing though. Simple sequence variation in the unconserved regions might also be better than nothing.

Our assumption that a dimer is enough to activate a B-cell is not always true. In that study the ligands were bivalent anti-IgD or bivalent anti-IgM antibodies (m.w. >100 kDa). I don't know why these bivalent antibodies can't always activate a B-cell, but our dimers will likely be made from smaller and much simpler proteins, and when they bind a B-cell receptor, they may be more likely to bind the variable regions which could have an effect on activation. A recent paper is informative. They tested a small molecule hapten NIP (m.w. 323.04 Da) and hen egg lysozyme (m.w. 14.4 kDa) as antigens. In neither case could monomers activate either IgD or IgM B-cell receptors. An NIP dimer could activate an IgM receptor but not IgD. A hen egg lysozyme dimer could activate both. For NIP to activate IgD required a trimer. Hen egg lysozyme is a nice small protein that has a size that is along the lines of the kinds of things we would likely be using.