Wednesday, September 18, 2013

Developments in understanding polyspecificity

Leading up to the Antibody Engineering & Therapeutics Event, podcast, we'll have interviews with a few of the speakers from the event.  Today we feature a portion our interview with James Crowe, Jr., M.D., Director, Vanderbilt Vaccine Center, Vanderbilt University Medical Center.

Today, he answers the question:
What are recent developments in understanding polyspecificity?

His answer:
Well, the recent development of repertoire sequencing technologies has allowed us to start getting a sense of how big antibody repertoires are in human beings. There is a theoretical number of something like ten to the eleventh antibodies that can be generated by stitching variable gene segments together in recombination and adding injunctional diversity. So, these are the normal mechanisms by which you get your naïve repertoire. But, when people look at the repertoires of sequencing they seem to be smaller than predicted.

This leaves us with a conceptual problem – how, with a relatively small repertoire, can you recognize all of the antigens that you have to deal with? In fact, in something like the flu there are many closely related variant antigens. Or for HIV there are variants occurring in each person. For Hepatitis C there is extreme variability with point mutations going on in the virus antigens and it’s not entirely clear that the antibody repertoires are large enough to commit individual clones in the repertoire in each antigen that’s encountered. So, just mathematically you have a problem and it appears that what the B-cell repertoire system is doing is reusing B-cell receptors – which are also later the secreted antibody. So, they are reusing specificities for more than one antigen and when an antibody binds multiple antigens, it is termed “polyspecificity,” generally.

So, we’re certain that this does occur and now that people are able to generate human monoclonal antibodies in great numbers, it’s increasingly apparent that many antiviral antibodies also bind to host proteins or some antiviral antibodies by more than one viral protein or at least by more than one variant. So, the question is how do they do this? We had this concept that monoclonal antibodies are very specific. So, how do they bind to two or more antigens? I think recent developments using these higher throughput sequencing and computational techniques have given us some insight into what’s going on.

In the past we thought that most of the interaction between an antibody and an antigen was driven by the residues that are touching on both sides of that protein/protein interaction. So, very close within several Ångström relationships of the surface of an antibody combining site with the epitope on the virus. But more recently we’ve realized that it’s not just the contact residues that drive the interaction, but rather the contact residues are typically on the tips of hypervariable loops called the “complementarity determining regions”. These loops arrayed on a scaffold, which is the framework of the heavy chain or light chain. So, the framework loops and residues are holding up the variable loops and when you think about it, it becomes fairly intuitive to realize that if you altered the scaffold at the base of the antibody structure dramatically, then the orientation or placement of the hypervariable loops could change very dramatically on the interface. This appears to be one of the very important physical principals in antibodies that underlie polyspecificity or the molecular basis for polyspecificity.

It looks like the framework residues that allow some flexibility confer to those antibodies the ability to move variable loops around in space and, therefore, the antibody combining site is not fixed and rigid, but rather it is moving around and dynamic and it’s able to achieve various conformations. This may be how a single antibody can bind to more than one thing because that antibody exists in sort of a fluid state of multiple conformations. While it is exploring those various states, it may have the capacity to bind antigen A and when it’s in an unbalanced state when it’s in a different conformation – sampling a different part of its conformation space – it may be able to bind antigen B. So, that’s kind of the most existing recent development. It looks like the framework residues, rather than contact residues, may be driving flexibility in the frameworks and thus confer to those antibodies a capability to bind very diverse antigens.

I think this is a new notion that most of us have thought about. The contact residues (are) driving it, but instead the framework residues may be very important. That’s the principal breakthrough in the last year or so, I think, in understanding polyspecificity.

Dr. Crowe will be presenting Deep Sequencing the Human Antibody Response to Viral Infections and Human Germline Antibody Gene Segments Encode Polyspecific Antibodies. For more information on these sessions and the rest of the program, download the agenda. The Antibody Engineering & Therapeutics Event will take place December 8-12, 2013 in Huntington Beach, California. If you'd like to join us, as a reader of this blog,when you register to join us and mention priority code XD13172BLOGJP to save 20% off the standard rate.

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