Building a Single Donor Phage Antibody Library with NGS Validation
Christina Lingham:
Hi everyone I'm Christina Lingham for the PEGS Boston Summit. I'm really pleased to have the opportunity today to speak with Andrew Bradburyof Specifica to talk to us about his presentation, "Building a Single Donor Phage Antibody Library with NGS Validation." He'll be giving a talk at the Display Biologics Track this April 30th through May 1st in Boston. Andrew thank you for joining us. Tell us about yourself, and your work.
Andrew Bradbury:
So I've been working in antibody engineering, and phage display for about 30 years I suppose. And I worked first in Cambridge. I did my PhD with César Milstein the inventor of monoclonal antibodies then moved to do a postdoc in Italy. That was supposed to be a couple of years it ends up being 10. And then from there I moved to Los Alamos where I was a senior scientist, and group leader until last year. And last year I retired, and joined the company I founded the year before full-time as Chief Scientific Officer. And the company's Specifica, and we specialize in making antibody libraries of the highest quality.
Christina Lingham:
Can you outline what you consider to be some of the greatest obstacles to developing libraries?
Andrew Bradbury:
So I think the real problem with developing libraries is a challenge of ignorance in the sense that when you're making a library you often don't know what's going into the library, you don't know what's coming out at the other end. And I think if you have antibodies floating around in your lab for example there's always a danger that you might be amplifying that contaminant antibody instead of the diversity, that you think you're putting into your library. And to make a good library you really need very careful quality control at every step.
And I think what's really changed recently is the ability to harness next-gen sequencing as part of that quality control. So that's what we do extensively is quality control every step in our library to make sure that we have the diversity we think we have. So for example the first library that I made was back in 2000, and we thought we had a hundred million different clones in the heavy-chain, and when we did next-gen sequencing of that library it turned out that we probably had 30 times less than that.
So it was about only three million different clones in the heavy-chain. And there have been a couple of other applications indicating that many libraries are far less diverse than what the authors think they are. And I think this is the fundamental problem. If you can be sure of what your true diversity is then it means that your libraries are going to be much more functional than if it turns out that your library is much, much smaller than you think it is. It's only three million instead of a hundred million then it's clear that you're not going to be pulling out as good antibodies as you think you will.
Christina Lingham:
Tell us more about your approach, and what's unique about it.
Andrew Bradbury:
So what I'm going to be talking about at PEGS is one approach we're taking to making antibody libraries among others, which also includes synthetic libraries. But there was a very interesting paper published last year where they looked at the diversity of naïve B cells, and their hypothesis, or their conclusion from the next-gen sequencing they had done was that every naïve B cell essentially contains a different antibody gene, and so this led me to think well if that's true then the obsession of people to get as many donors as possible into a library may be a bit misguided, and perhaps you can get enough diversity in a library from a single donor.
And so that's what we did we took essentially all of the circulating lymphocytes from a single donor, and we made an antibody library from that, and doing quality control all the way along with next-gen sequencing. And then we've now tested that, and it functions really rather well. And so this means that what's important when you're making natural libraries is really the number of lymphocytes you use as opposed to the number of donors.
That said I think you can probably get better libraries with more donors, but there's certainly enough diversity in a single person to create a very functional antibody library. And then if you think about this from the point of view of each of us walking around trying to avoid infections you'd expect that there should be enough diversity in a single human being to respond to many targets because we're all able to fight infections.
Christina Lingham:
And then what advice do you have for people creating antibody libraries using NGS?
Andrew Bradbury:
Check everything that's what I would say. Check your PCR products, check your assemblies, check your final libraries, and in each case assess the diversity that you're getting. Now one of the challenges that we faced at Specifica was the informatic, and analytical tools to actually analyze the diversity of the next-gen sequencing data, that you're getting. And now we've now set that up in-house. But there are a few companies out there I believe that will do that work for you. And of course if you find it all too challenging just come to Specifica, and Specifica can make your library for you.
Christina Lingham:
What are some of the most exciting developments emerging in the display field, and how has your work changed as a result?
Andrew Bradbury:
So I would say some of the biggest, and most exciting stuff has been the introduction of the concept of developability into antibodies. So in the past people have always been selecting antibodies on the basis of their biological activities their binding activities. And what has been pioneered by Dane Wittrup, and colleagues at Adimab has been this concept of developability. So it's not enough just to get an antibody that actually functions as you want it to function. You also want to get an antibody that you can actually make, and you can produce as a pharmaceutical, and then sell.
And so I think this whole idea of modifying, or ensuring that the antibodies that you pull out at a very early stage of development you look at them, and you ask yourself the question are these developable? I think that's a new paradigm, and so that's certainly affecting our work. We're looking at making libraries now that there are more antibodies, that are developable in other ways.
Christina Lingham:
Andrew that's fantastic. Thank you for your time, and insight.
Andrew Bradbury:
That's my pleasure. Thanks very much.
Christina Lingham:
That was Andrew Bradburyof Specifica. He'll be speaking in the Display Biologics Track at the upcoming PEGS Boston Summit this April. If you'd like to hear him in person go to PEGS Summit dot com for registration information, and enter the key code podcast. I'm Christina Lingham. Thank you for listening.