What are the techniques used to better understand 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 the techniques that your group has used to better understand polyspecificity?

Dr. Crowe: In order to sample the interacting energies of a large number of antibodies and antigens, it’s currently not possible to express thousands or millions of antibodies, at least not in an economic manner to measure energies. So, we’ve been focusing on using computational modeling experiments to determine the predicted interacting energies. In particular, we’ve been using a computational suite called Rosetta that was developed in Seattle. They have a Baker Lab and he’ll be talking at the IBC Conferences and going over the impact of Rosetta and structural modeling.

We’ve used Rosetta to ask: “What are the ideal or the optimal framework residues that can make antibodies have the ability to bind more than one antigen?” So, within Rosetta you cannot only model the binding to one antigen, but you can do a process called “multi-state design” in which you are asking the antibody to have more than one state – more than one bound state – which is binding to more than one antigen. By doing this, we asked Rosetta at many residues in the framework that we saw mutated in naturally occurring antibodies to just --- of the 20 amino acids that are available to predict which amino acids would allow binding to more than one antigen. Very remarkably, Rosetta predicted amino acids that are the germline encoded residues at those positions, which it is highly unexpected that a computer could pick at multiple positions the correct amino acid of 20 – an ensemble that would allow binding to more than one antigen.

So, the computational modeling is done on supercomputing. It is very computationally intensive. It predicted what the optimal sequences are and lo and behold, the optimal sequences for binding more than one antigen turned out to be the very germline sequences that we’re using in our genome. So, it looks like germline sequences are already in a state. It’s as if they are designed perfectly to be polyspecific and it may be that many – if not most – antibodies are polyspecific when they start.

The process of somatic mutation is, in essence, a two-step process. One in which the framework residues are mutated to reduce flexibility and rigidify the framework to orient the loops in a proper configuration. Then somatic mutations occur in the tips of the loops to adjust the interface so that their shape is complementarity and other interactions. So, really reducing flexibility achieves specificity and then you get some optimization of surface by somatic mutation.

So, we’ve focused on computational modeling, but the validation of the results of those computational experiments was the observation that the residues that are predicted are, in fact, the ones that are encoded by the genome. Even though the computer program didn’t know what those sequences were, it predicted them and they are the naturally occurring ones. So, we’ve been using Rosetta, trying to develop new modeling techniques within Rosetta and furthermore developing statistics to ask how expected or unexpected are the findings. It’s really quite remarkable that the computer can pick out the naturally occurring residues.

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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What are the current challenges in antibody engineering the field?

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's Question:
What are the current challenges in the field?

Dr. Crowe's answer:

Well, the technology is very powerful in that the numbers of throughput that can be achieved in the sequencing are impressive. But, the technologies are evolving constantly. There are a number of commercial suppliers of instruments and chemistries and re-agents and these are changing every couple of months. So, currently people are grappling with what are the best chemistries and how to deal with errors. So, all of these technologies involve polymerases that create errors and how to tell the difference between a sequencing error and a naturally occurring somatic variation is a big challenge in the field.
Also, the high throughput technologies give very good throughput, but relatively short read lengths. Currently, if you can get both ends of DNA to read 300 nucleotides in and stitch together and achieve something like a 500 nucleotide read, that’s considered a good effort. But, we need longer reads to sequence entire antibodies.

It is also very expensive right now. The price undoubtedly will come down and has been coming down. But it is still a relatively expensive technology and because of that, that has led people to try and combine samples. So, multiple samples in a single lane for sequencing to reduce the cost. This is multiplexing and the results can be deconvoluted if each of the amplicons is labeled in some way with a bar code or an index that is put on either with an adapter or a PCR. Learning which adapters and indexes and bar codes are compatible with various PCR primers is a technical challenge and deconvoluting the multiplex data set is somewhat challenging.

Another major obstacle in the field is that the numbers of sequences that come back are greater than the capacity of the software programs that we have to analyze antibody sequences. So, there are some great programs in the field – Immunogenetics Database (IMGT). There is another program from the NIH – JOINSOLVER and another program SoDA. All of these are from academic sources that are open, but they do not have the capacity to deal with millions or billions of sequences, for instance. IMGT currently has a high throughput upload of about 150,000, which sounds like a large number. But if you have a billion sequences, that really isn’t adequate to do your analysis. So, I think many people are scrambling in their own institutions or companies to develop local, proprietary methods for analysis and it’s not clear the best way to do that. 

I think finally the biggest next step in this sequencing technology is to figure out a way to link the heavy and light chain sequences because antibodies are encoded by two different, major, recombined chains– heavy and light chain and, of course, the specificity antibody derived from both heavy and light chains together. But the sequencing separates heavy and light chains and if you don’t have the natural pairing, you lose a lot of information that is very important. So, I think an obstacle in the field is the technical difficult in molecularly linking heavy and light chains from single cells and then being able to sequence both of those chains together. There are very many challenges despite the exciting promises in technology.

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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Antibody Engineering Interview: James Crowe, Jr., M.D., Director, Vanderbilt Vaccine Center, Vanderbilt University Medical Center

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:
How is the recent explosion of DNA sequencing technologies impacting the field of antibody engineering?

Dr. Crowe's Answer:

Well, I think we’ve all be watching the genomic sequencing effort with the human genome coming out and now every species on the earth is being sequenced. The technologies are very exciting because of the extreme throughput of nucleotide sequencing – either DNA or RNA – that can be achieved. Increasingly, these technologies are leaking into all biomedical disciplines.

I think in the last two or three years DNA sequencing has had an increasing impact on antibody engineering. The major impact is that most of the technologies in our field have focused on deriving single clones or single antibodies, monoclonal antibodies or sometimes building large libraries, but still selecting out individual members of those libraries. The amount of information that we get from such approaches is fairly limited. We end up with panels of antibodies that we study. But now with the sequencing technologies that are available, we can literally obtain millions or billions of sequences from a single sample. So, this opens opportunities to create or describe or analyze larger and larger libraries in humans or in experimental animals. So, I think that is the most exciting thing that the scale detail with which we can look at antibody repertoires now is just orders of magnitude more than was available to us previously.

One of the early impacts of not just the technologies – but in terms of the science and our understanding – is that the size of the repertoires and the complexity that we’re seeing is really changing our notion of how the human or animal B-cell response actually occurs. So, I think in the past – since we would obtain relatively few antibodies from our lab studies – we thought that the germinal center reactions that were responding to antigens generated a large number of variant antibodies. But in the end, one or a few B-cells won out based on affinity selection and the survivors from vaccination or an antigen stimulation were relatively limited. But now that we are looking at whole repertoires, we see that responses are not small collections of monoclonal antibodies, but that natural immune responses are really swarms of antibodies – large collections of variants that are highly related, but differ from each other by point mutations. Probably, there’s a wide spectrum of affinity represented in these swarms. Remarkably, they are maintained in a repertoire. There’s not the winnowing down or narrowing that we thought was occurring because of our previous limited techniques.

So, I think those are the biggest impacts. One is the explosive scale that we can use and also a new sense of a difference in the biology that we’re seeing from what we expected.

Download Dr.Crowe's full podcast here.

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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