Bye-bye Bunny

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Research antibodies are commonly made with the use of animals. But perhaps that can change, some say. 

Here's a podcast to accompany my recent story in Nature Methods called Change-makers bring on recombinant antibodies that is about  research antibodies, polyclonals, monoclonals, recombinant antibodies, animal-derived and non-animal derived antibodies, 

The podcast is with:

  • Alison Gray from Afability and the University of Nottingham,
  • Katie Crosby from Cell Signaling Technology,
  • Alejandra Solache from Abcam,
  • Carl Ascoli from Rockland Immunochemicals,
  • Andrew Bradbury from Specifica,
  • Achim Knappik from Bio-Rad,
  • Aled Edwards from the University of Toronto,
  • Fridtjof Lund-Johansen from Oslo University Hospital, and
  • Peter McPherson from McGill University.

A transcript is pasted below. 

Note: These podcasts are produced to be heard. If you can, please tune in. Transcripts are generated using speech recognition software and there’s a human editor. But a transcript may contain errors. Please check the corresponding audio before quoting.

Bye-Bye Bunny

Vivien

Hi welcome to Conversations with scientists. I’m Vivien Marx.   

This episode is called Bye-bye bunny. This music is called Coffee Pot by the Band Split Phase  This episode is about research into diseases such as COVID-19. It’s about research antibodies. And seeing how possible it might be to avoid gathering research antibodies from animals, such as rabbits. Is it possible to say bye-bye bunny? I asked around. There is certainly some change happening related to research antibodies and producing non-animal derived antibodies. First off, a bit about the antibodies in our bodies.  Antibodies are the body’s big helpers. When there’s an infection, the immune system is called to action by an invader, such as a virus. B cells, those are a kind of white blood cell, make antibodies--a diverse set of antibodies. We have many B cells and each B cell can make a different antibody as a reaction to an invading virus. 

Aled Edwards
Your body is armed with about 10 billion of them, different ones, different Velcro sticking out, waiting. 

Vivien

That’s Aled Edwards from the University of Toronto.  

Aled Edwards
Waiting for something they can recognize and they have different shapes, and if an antigen happens to come along that binds tightly to one of these B cells, it starts exploding and making more of it and pumping out antibodies. And but so, for example, with the SARS-Cov-2, when you get infected with that, I'm making up the number. But let's say A thousand B cells get activated. So your body makes a thousand different types of antibodies. One to this, one to here one to here one to here hear so you can sort of picture it. Right.

A lot of them is binding all over. And that is because there's many of them, it's poly polyclonals. So if you inject SARS into you, your body of the 10 billion B cells, a thousand will make an antibody. Each one is different. So you have a thousand different antibodies. And if you inject into a rabbit, the rabbit would make a thousand different. So those are called polyclonals. What we want parenthetically for the. Neutralizing antibody for this infection is the antibody that stops the virus from working, an antibody can stick to the virus, but that doesn't mean it will stop the virus from working.

Vivien
An antibody can stick to the virus but that doesn’t mean it will stop the virus from working. So labs need to find what Edwards calls the ‘business end’ of the virus. They want to find ways to intervene at that business end. Which is one aspect labs are now feverishly working on. 

Aled Edwards
And it's impossible to know now without results from people which are the real neutralizing antibodies, and so all the tests that are being developed, you know, that we hear about where people give blood and have a, they can say, say, first of all. The old versions of the test that you have been infected by a coronavirus, not the coronavirus, then they realized. We got rid of those tests, the new ones come out and say they're more selective for the SARS-COV-2. And they can say you were infected, but they can't tell you yet if you can get reinfected or not, because they there's we hope it works because they can take those antibodies in a test tube and say, hey, it blocks the virus in the test tube. But that doesn't say it's going to block the virus in a person. And so that's one of the interesting. And mysteries of this new virus, this and the immune responses, we don't know enough yet because ultimately the data has to come from people.

It's only been around for five months. I think that people are hoping, you know, now they've invented a new test. They they hope that the virus, imagine that the virus binds to this protein ACE-2. And then that's how it gets into cells and they can make a test that monitors that interaction, you know, so you'd have ACE-2, you have the antibody and then you get and see if it gets popped off by another. There's lots of different variations how you can make that kind of a test.

But it still doesn't show in a person you can that still shows, yeah, like Vivien you've made an antibody that can block the virus from binding ACE-2 in a test tube. We know that. Whether that anybody can protect you, because let's pretend it's got to get your airway epithelia and the antibodies don't get there. So you have them in your blood, but they never get to the right place and the virus can infect you anyway, it can only be learned in people.

Vivien
These are among the reasons why finding ways to stop COVID-19 is so hard. Part of working on diseases like COVID-19 and many other diseases is studying proteins that play a role in these diseases. Many companies are developing tests to detect antibodies to COVID-19. Antibodies are large Y-shaped proteins in our blood. And to detect them in a test you use a blood sample and research antibodies specific to the protein you’re hunting for. Carl Ascoli is chief science officer at the antibody company Rockland Immunochemicals and talks about the test his company is involved with.  

Carl Ascoli
In 2004, when SARS coronavirus was an issue, we generated an antibody to SARS coronavirus in such a way that it was pan-reactive with various clades of coronavirus at that time. And as a consequence, we had in our system already to go an antibody against coronavirus that was more than 95 percent reactive with this current coronavirus. And so we had an antibody in the United States already produced. And as a consequence, that particular reagent has been taken up by non-exclusively by at least 25 diagnostic companies, three or four global major diagnostic companies that have gone through EU way and have begun manufacturing with it.

So we're actually the 'chip inside', you know, the old saying for the computer, we don't make the computer, we make the chip inside. We make the antibody inside the kit that are just being deployed now for antigen detection

Vivien
Antibodies are used inside kits of various kinds. For COVID-19, companies distributing tests in the United States need what is called an Emergency Use Authorizaition from the Food and Drug Administration, an EUA. And in the case of Rockland Immunochemicals, the antibody they use is a particular kind.  

Carl Ascoli
Vivien, it's a polyclonal antibody. And it works in serologic assays, it works in a ELISAs, it works in lateral flow assays, it works in flow cytometry, it works in immunofluorescence microscopy. It works on paraffin, embedded formalin, fixed tissue taken from cadavers by immunohistochemistry, and it neutralizes the virus. And that's actually one of the strengths of a polyclonal antibody in that it it mimics the natural immune system. So touché 

Vivien
Touché? Why touché? Because not everyone likes polyclonals as research antibodies. Polyclonal antibodies are used on many lab benches, in many papers. To make polyclonals, companies start by immunizing an animal, usually a rabbit and gathering antibodies from the blood of that animal. 

Aled Edwards
Polyclonals. Okay, I do believe that we should be getting rid of those in large part because it is impossible to assure lots, a lot variability. And we are scientists after all, and we should have trust in the reagents that we're using, as it were. And because polyclonals can vary so much from rabbit to rabbit to rabbit.

Vivien

Besides this inherent variability, an issue with polyclonals is that they are non-renewable. Fridtjof Lund-Johansen from Oslo University Hospital explains.

Fridtjof Lund-Johansen
Polyclonals that are made by many companies are non-renewable, right? Because you immunize a goat or a rabbit. When the rabbit dies. You don't have all this discussion. We've had lots of these discussions about the old fashioned, if you like, polyclonals. They are not renewable because unless you have DNA for the B-cells, which most people don't have, you just immunized, you get a serum. So it's non-renewable.

Alejandra Solache

Yeah, I think polyclonals have had their time. 

Vivien

That’s Alejandra Solache vice president of new product development at the antibody company Abcam. 

Alejandra Solache
I think, you know, like any any technology that was great at that time, like tapes, probably people don't even know right now, CDs all of that now this new technology improved technologies come up, and who really needs a CD or player player anymore figure out how to play it. And if the subject in nature, nature, methods and trends in biological sciences and elsewhere, I see the polyclonals that the way that that basically is like a like a tape that that could break and you have to fix it. And if you couldn't fix it, then you just can't you can't have that music anymore. 

Vivien

Some companies and academics, but not all, are done with polyclonals. Some of the issues that can happen with experiments using polyclonals are due to the fact that batches of polyclonals can vary from one lot to another. After all, the antibodies might have been generated with different rabbits. But there are plenty of people who still find polyclonals work well for their purposes. And companies have ways to minimize batch-to-batch variability. Am just going to set that polyclonal discussion aside for a moment to get to some other kinds of commercial antibodies beyond polyclonals.

There are monoclonals, there are recombinants, there are animal free-antibodies. And the list goes on. Scientists can buy antibodies from any number of companies. And they have around 2-3 million antibodies to choose from. 2-3 million, that’s quite a selection. The first step I mentioned, the one that starts with immunizing an animal, is one Alison Gray from the University of Nottingham would like to change. She founded a non-profit called Afability, all about advocating for animal-free antibody production. She, along with colleagues, has published papers on this subject, in Nature, in Nature Methods, in Science, in Trends in Biotechnology and elsewhere.

And recently there’s been a European Commission report with input from a panel of experts on the subject of animal-free antibodies. It’s called EU Reference Laboratory for alternatives to animal testing Recommendation on non-animal-derived antibodies. That report builds on a directive about the need to use methods that avoid the use of animals. Here’s Alison Gray.

Alison Gray
And recently, there's been a European Commission report with input from a panel of experts on the subject of animal free antibodies. It's called EU Reference Laboratory for Alternatives to Animal Testing recommendation on non animal derived antibodies. That report builds on a directive about the need to use methods that avoid the use of animals. Here's Alison Gray.

The EU directive has been there for a number of years in its present form and in the EU directive, it's clear. So that is already there. That regulation states that if an alternative exists you're obliged to use it, obviously it has to be a An alternative that is at least as good as, you know, the method it's looking to replace. 

Vivien

The idea behind the new report has been to assess non-animal derived antibodies to assess and document if they can replace animal-derived ones.

Alison Gray
Where we are now is how we address that question is what do we do?First of all. You know, let's not take your word for it. Let's find out what really is the situation, which is why it's why we have to create all this work aroundage, you know, the work, the working group and everything to look at all the literature, you know. Don't just just take it from me, will bring in a panel of experts. Well, experts at all, you know, to make it. unbiased. People that cover all aspects of antibody development and let's really work at what the situation is, and then we know we're in a strong position to address this and say confidently there are alternatives and we're ready to use them. Then it's up to the member states to implement the recommendations because they're already aware of what the EU directive says.

Vivien

The report is circulating through the EU member states and they will individually decide what they’re going to do. Oh and by the way, Alison Gray will be pursuing the goal of animal-free antibodies irrespective of Brexit. Of course, it’s not easy to create change,. 

Alison Gray
I think that's the problem with antibodies is that they're always put aside slightly because they're not the main focus of their experiment. They are a tool to make, you know, to prove their hypothesis. And they just want to be able to buy it, use it, forget about it. 

Vivien

Other types of antibodies, too, beyond polyclonals start with immunizing an animal. Traditional monoclonal antibodies for research use, for example. Traditionally, monoclonals are made by injecting an antigen into an animal, which might be a rabbit or a mouse. B cells are gathered from those animals and those cells are fused to cancer cells, myeloma cells in particular.

This creates a so-called hybridoma, which secretes antibodies. A hybridoma has often been cultured by injecting it into a mouse’s abdomen. The hybridoma grows in the ascites, the fluid that collects in the abdominal cavity. After injection, the mice can in some cases start breathing rapidly, hunch over and not move much. These are all indications the animal is in pain from this ascites method of producing monoclonal antibodies for research use.

Alison Gray
In Europe, the ascites method shouldn't be used anymore. That's clear. And yet we it's only recently this year that the new statistics for the European use of animals in scientific procedures was published. And that's when I became aware of the number of animals that are still being used in Europe to produce antibodies by the ascites method. And that's shocking, really shocking that there can be no justification for that. And, you know, member states have been spoken to, the competent authorities in each member state have been spoken to. They are aware of this so that the fact that that's still being authorized is just shocking. I would expect that to change now. But now that it's being flagged up and exposed like that, I would expect this to change.

Vivien

For now, the ascites method is still used to produce monoclonal antibodies for research use. I say ‘research use’ because there are therapeutic monoclonal antibodies given to people as drugs. Those are not made in mice. But with antibodies for research use, the ascites method is often used.

Alison Gray

There's no scientific reason to carry on using the ascites method. You can put that down on paper if you want to. It it is. I mean, you could say that's a lot of methods, not just the ascites methods. People become experts in their technology and they want to keep using it. So I think that's useful. It's about. You know, we now have methods where even if you do continue to immunize animals, yeah, you can use cell culture to produce to mass produce antibodies. The ascites woudl offer very little benefit. I suppose in the beginning you will probably get higher yields. But that's not the case anymore.

Vivien
Although a number of antibody companies still use the assay, this method to produce monoclonal antibodies for research use, some companies are phasing out this method. Here's Alejandra Solache from Abcam.

Alejandra Solache    
That ascites production method really has been for us, something that we didn't really utilize, that we have to go through the tissue culture methodology for our internal development. But we had in the past sourced antibodies from other suppliers and and we have accepted antibodies that have been being produced through this type of methodology. But that's something we have now changed and and we have made significant progress now in terms of eliminating ascites-derived antibodies from our catalog.

Vivien

The ascites method is not being abandoned everywhere.  

Alejandra Solache

Particularly companies in the US are still producing quite a lot that way.

Vivien

Moving away from the ascites method, some antibody companies have chosen to grow hybridomas in cell culture instead. But hybridomas can be unstable. So a different type of antibody generation and production has emerged. Antibodies made that way are called recombinant antibodies. Here’s Katie Crosby, who directs immunohistochemistry at the antibody company Cell Signaling Technology:

Katie Crosby

When one hears recombinants. There's two sort of ways that it's being used. One is in the actual generation of the antibody. And that's what you are talking about with respect to animal-free. So there are libraries that are created that include the cDNA or a combination of cDNAs and then those are expressed in yeast or phage. And then there goes through a whole set of panning experiments and various antibodies are identified and then they are I can't think of the right word, they're produced and then they are made available for use.

So that's a true sort of animal free generation of an antibody. And then that is recombinant both in its generation and in its manufacture. The other approach to recombinant and the one that's more, maybe more common in the research tool space is the actual production of the antibody. So animals are still used in the generation of the antibody, whether it's mice or rabbits, and they're immunized and antibodies are identified and then they are cloned into expression systems and then they are produced recombinants.

So there's two different ways to think about recombinant from with respect to that. CST right now is very heavily involved in recombinant antibody production, and that offers benefits to customers because they are very consistent from lot to lot. And it sort of avoids some of the pitfalls of the hybridoma technology where hybridoma cells can be unstable and can lose expression of the antibody, or they can express light other light chains and antibody preparations can be made more complicated.

And therefore, when one has a recombinant production, they're very consistent from lot a lot, which offers a benefit to customers. They're very scalable. So there can be large quantities of antibody produced, giving customers basically unlimited access to two antibody. And that can be an advantage when they're doing something like a very long term study where they want to be able to use the same batch of material for a very long period of time. 

Vivien

Abcam too is revving up recombinant antibody production. 

Alejandra Solache

Internally for our antibody development and all of our new antibody development is just exclusively recombinants now. So we don't develop any antibodies, And I think we will not be developing ascites-derived antibodies internally. 

Vivien

Recombinant antibodies are not automatically non-animal derived, they can involve immunizing an animal and gathering antibodies and capturing the antibody sequence. Recombinants are renewable and can be engineered. Katie Crosby talks about monoclonals and polyclonals. 

Katie Crosby

Monoclonal antibodies are certainly advantageous over polyclonals antibodies with respect to lot to lot consistency. But recombinants monoclonal antibodies are are even better still because they are highly consistent and and scalable. And that's an advantage. Typically hybridoma are put into mice and ascites is generated. And so to make large batches of material requires a large number of animals. So that's not ideal. Or they are. The cells are cultured in media and then the antibody is extracted or purified from that media.
And so that can be costly and take up a lot of space in order to manufacture large quantities. That recombinant production can be is very scalable. 

Vivien

Recombinant antibodies are scalable. They can be engineered. They’ve been derived from a gene. A monoclonal can begin the classic way and then be turned into a recombinant antibody. Fridtjof Lund-Johansen explains.  

Fridtjof Lund-Johansen

A regular monoclonal can be made into a recombinant antibody. That's that's a matter of that's a matter of cloning the Immunoglobulin genes and making it in, you know, in a recombinant way. That is really the definition of recombinant antibody. So, so many antibodies that are sold by major companies today, such as Abcam and Thermo, CST. They are recombinant, meaning that they are they have the DNA sequence of the gene and they can they can put modifications on it because they like to produce the exact same the same way. As long as you have a defined DNA sequence, you have a recombinant antibody . 

Vivien

The term recombinant doesn’t say anything about whether or not the antibody is non-animal-derived as in not derived from a rabbit or a goat or another animal. What the European Commission report and scientists like Alison Gray would like to see, is the use of approaches that avoid animal immunization. 

Fridtjof Lund-Johansen

Of course, if you could avoid the whole immunization it would be wonderful. 

Vivien

One way to avoid immunization of animals is to start out with donated B cells from people. 

Fridtjof Lund-Johansen
The idea is that you can make antibodies to anything if you get all B cells from an individual. Well, that's not necessarily all. But let's say a large number of what you often do is, immunoglobulins consist of two chains, the heavy and the light chain. What they do is that when you make these libraries, you shuffle those around so that they are randomly paired.

So let's say that you start with a million B cells and they're all different. They won't be, let's say you can do that. If you show the heavy and light chains, well, then you have the diversity of a million by a million. Right. So then suddenly you have 10 to the 12. Of course we don't. You're not going to get a million B-cells because that's probably as many B-cell as we have in total. But let's say you can start with 100,000.

So just by shuffling the heavy and light chains around, you're going to have the square of that. And then what people do, in addition, is that they insert mutations into CDR3s 

Vivien

The CDR3s are complementarity-determining region 3s, they’re diverse binding loops that one can use when generating recombinant antibodies.  For example, recombinant antibodies that do not involve animal immunization at all.

Andrew Bradbury was a researcher at Los Alamos National Laboratory when he cofounded a company, Specifica, that develops and sells antibody libraries. He has left Los Alamos and is at Specifica full-time as its chief scientific officer. The libraries the company makes do not involve immunizing animals. The antibodies are semi-synthetic and synthetic CDRs based on information from in-house antibody databases combined with with natural CDR3s from donated human B cells. One recent customer is Bayer, that company bought a library from Specifica.

Specifica also makes antibodies for clients who approach them with targets, which might be proteins involved in a disease. And Bradbury and his team use in-house developed libraries to make antibodies against those targets. Here’s Andrew Bradbury: 

Andrew Bradbury
All my career, I've wanted to make really good antibody libraries, and I felt like this is the future. I was never able to get funding in the academic world to do that.

Vivien
Huh.

Andrew Bradbury
And that just isn't there isn't the interest really. It's because people are interested in funding. What you do with libraries, but not the actual making it. It's completely different in the in the commercial world People are interested in really good libraries because they know where they can get really good antibodies from these libraries.

These libraries really are the culmination of decades of work on my part, and it's required is taken in some years of thought of how can we improve this, what can we do. That's resulted in I think these libraries are actually performing even better than I expected. I've been surprised. Which is great of course.

Vivien

At Specifica, Bradbury has an in-house effort ongoing to develop potential therapeutic  antibodies to the virus Sars-COV-2, which attacks the ACE-2 receptor in cells. That leads to COVID-19. 

Andrew Bradbury
We have a pretty, pretty small effort on it. But we've now got antibodies that are able to inhibit the binding of this spike protein to the ACE-2 receptor. And we're moving forward, I would say more slowly than others, but at some point it would be nice to get a partner take on these these antibodies. 

Vivien

Bradbury was on the expert panel involved in the European Commission report on non-animal derived antibodies. / So recombinants can be made using antibody genes obtained through animal immunization, but can be entirely synthetic or they can be derived from human B cells, which is called a naïve library. And naïve libraries are the ones Specifica uses. 

Andrew Bradbury

Our libraries are a mixture of antibody scaffolds that are already in the clinic, so therefore that are very well behaved and then we take what you call diversity or binding loops from sequencing we've done of human genes, the antibody genes, and then we put those binding loops in the antibody scaffolds. And along the way, what we do is we eliminate all the all those binding loops that create potential problems. So whereas most people, what they've done is they've created libraries, they generate lots of antibodies.

After they've done the selection . They then look at all the antibodies that come out and they don't use that one, that one and that one because they've got problems. We try to eliminate all the problems up front. So the antibodies that come out of our libraries. And the ones we've tested so far are as good as the clinical candidates that we use as the basis for the scaffolds 

Vivien

To get to these libraries takes a process. Back in the 1990s when Achim Knappik was at a company called MorphoSys in Germany, he developed a fully synthetic antibody library. MorphoSys set up a division called Antibodies by Design that used this approach to develop antibodies for researchers. This division was bought by Bio-Rad and now Knappik directs Bio-Rad’s non-animal-derived antibodies division. These antibodies are in the Bio-Rad catalog alongside antibodies made in ways that do involve immunizing animals. Achim Knappik was also on the expert panel for the European Commission report.

Achim Knappik
I mean, this is all completely, fully synthetic. So there are antibody genes, that is the that is the library that has been made by gene synthesis. So there is not at any point any animal involved in the design and construction process. These antibody genes are then transformed or bacteria are transformed with these antibody genes. And this collection of bacterial clones, each containing a different antibody gene, is what we call the library.

Vivien

This library is where things start when making new antibodies, in this case non-animal derived recombinant antibodies. An important part of making such antibodies is the use of phage display technology. Fridtjof Lund-Johansen explains.

Fridtjof Lund-Johansen
Phage display is the technique to take to display DNA from immunoglobulins OK, you can. So you take B-cell and make DNA and you can isolate the Immunoglobulin genes and then you can display them on the surface of the phage , on the surface of yeast or something like that. It's all the same thing. This is display technology. Your starting material is what makes the difference. If you start by immunizing a mouse, you can isolate the Immunoglobulin genes from that and be phage display or yeast display or and that is still display. And it's just a different way of screening than traditional hybridoma.

Now, what people often think of when they hear phage display is to take B-cells from somebody who's not immunized. And so if you take my B-cell and make antibodies to mouse IgG. That is a sort of a naive library because I have not been immunized against that.

Most people who work with naive libraries will pull something out. They will do several rounds of panning and will end up with some antibodies And now they will do affinity maturation of those where they mutate the CDR3, then do selection, et cetera, so they will try to copy the process that goes on during an immune response, where this happens, somatic hyper mutation it's called in vivo.

And that's when the B cells mutate the CDR3 you the CD three, you get higher, more high affinity antibodies. So what is done? What often people think of when they talk about recombinant antibodies is the concept of copying the immune response in vitro. You start out with a very large number of genes from B-cells, which is your name repertoire, and then you do selections with the antigen and then you end up in many cases, doing affinity maturation. So it's sort of a copying the immune response, but it's done in vitro. 

Vivien

Phage display is not new. Nor is working with naïve libraries.  Generating antibody libraries without the use of animals and by essentially trying to copy the immune response in vitro is what is needed to generate non-animal-derived antibodies. Katie Crosby from Cell Signaling Technology comments on the prospects of this approach. 

Katie Crosby
That requires the generation of very high quality library of cDNA and the adoption of a new technology, basically the sort of display of phage display in order to generate antibodies, and that is not something that every company is set up to do. So there would be sort of the cost of implementing that and scaling that. And I myself have not worked in that world. I don't know exactly what's entailed and how doable that would be in terms of making an entire manufacturing operation, not using animals for immunization at all. I'm just not sure what that would entail.

Vivien

Other companies, too, are looking at the approaches to make animal free antibodies. Alejandra Solache at Abcam comments on the prospects for this technology at her company. 

Alejandra Solache
If you have a great library and this is something I have been discussing with. So a lot of people about making recombinant antibodies animal-free, if if you have a great library, which is going to be hugely expensive, you can actually do it. I mean, we are actually developing antibodies through phage display as well, and we have made multiple antibodies through that technology. And it's a great technology for when you cannot utilize an animal, for example, developing antibodies to toxins or or when you are not able to get an antibody to an animal and get support for that if they are very high conserved proteins or or something like that.

I think it is a great tool. And what we are trying to do at Abcam is to have most of the multiple different tools in our arsenal to to be able to to generate recombinant antibodies. So for us, it is very important to to get to having as many recombinant antibodies as possible. We have already 20,000 recombinant antibodies in our catalog. And that's what we want to be concentrating on really throughout any technology. Having excellent libraries is an important part of developing the animals free recombinants. But at the same time we recognize that the technology not quite there to convert absolutely every every recombinant or every single new antibody to the animal-free.

Vivien

Generating polyclonals is easiest for companies and the least expensive, for now. Carl Ascoli from Rockland Immunochemicals speculates about how things might develop in the future 

Carl Ascoli
10 years from now, will the technology change in such a way that synthetically produced antibodies are more cost effective and more widely available? It's certainly possible, but let's just see what happens right now, I don't think that one tool in the toolbox is what you should have. And by that I mean just recombinant antibodies. I think there are there is a purpose for each of the forms polyclonal, conventional hybridoma-based monoclonal and recombinant monoclonal. I think there's a there's an appropriate use for all of those.

But you shouldn't start taking tools out of the toolbox and say, no, we shouldn't use polyclonal antibodies because they're derived in this way or because they don't have reproducibility that other forms have. I don't think we're there at that point right now where we should be taking tools out of the toolbox.  

Vivien

There has been and there remains prejudice against recombinant antibodies. This is apparently a kind of hangover from the early libraries. Achim Knappik from Bio-Rad explains how those prejudices may have gotten their start back in the 1990s.

Achim Knappik
I would imagine that the very first libraries that were developed in the 1990s and then also in the early 2000s, those libraries were rather small scale, like 10 to the 6, 10 to the 7 members. And of course, in this field, size matters. So it's if you have a large library, the chance to find an antibody with high specificity and affinity just increases. And therefore, you need you need a certain size in order to find decent antibodies. And this is could be could have been certainly the case that the antibodies from the original libraries there were certainly binding and were specific, but probably the affinity was not as high.

So this is one reason. Another reason which is often overlooked is that the the antibodies that are produced from these libraries of monovalent remember, this is a technology that works with bacteria and bacteria are not capable of producing full-length IgG antibodies, for instance. So this is y-shaped two-armed structure. This cannot be produced functionally in bacteria. And so what groups like us, what they are using is a fragment of the antibody. In our case, it's the Fab fragment. That first outcome of the library is an Fab.

So it's a monovalent antibody. It has only one of the two arms. And in many applications where, for instance, the antigen is is on a surface like a Western blot on cells, avidity is very important in terms of binding strength. If you can probably imagine if you have two arms and both can bind to an antigen because there are two antigens closely side by side of each other, that this binding will be stronger than if only one arm is able to bind.


And this this effect is called avidity. And if an antibody is used in its monovalent form, it doesn't have to the binding strength Bio-Rad antibody. So what in, most of the cases that we work on, antibody development, we convert it into a bivalent version and before we send it to the customer. Depends a little bit on the application. Sometimes the monovalency is also very, very good, but often bivalency is better. 

Vivien

Recombinant antibodies and non-animal derived antibodies may not be new. But there are hold-ups related to them. Andrew Bradbury offers his views on some of the factors that influence this hold-up.

 Andrew Bradbury
One hold up is that there has been a suspicion of these in vitro libraries, for the reasons that I mentioned that were mentioned in the report. It's a problem related to overpromising of the technology in the beginning, which I think happens with all technologies. And what's happened since those early, early libraries and early promises that people have been beavering away, generating better and better libraries. And I would say that now the libraries are really good and they can replace the use animals, as has been as was described in that report and in a number of publications.

So that's one problem. The second problem. So assuming that people were willing to accept antibodies that came from in vitro libraries, then the second problem is who's going to make them and who's going to test them? Who's going to characterize them? And I think that's the biggest problem at the moment. I guess if a company like Abcam or CST or Miltenyi they could take on a library and they could make a library themselves. But it's actually it's a really specialized art, I would say. And it's not something that if you haven't had any experience in it, you wouldn't want to do that because you'd end up with a library that may be far less functional than you think it is.

And in fact, first library I made, which we call generation one, was made 20 years ago. And we thought that had a diversity of about 100 million different antibodies. When next gen sequencing came along, we actually sequenced it.

It turned out it only had a diversity of three million antibodies in the heavy chain.. So in other words, it was 30 times less diverse than we thought, and because of that, I took on the use of next gen sequencing as a quality control in the production of our libraries. It was painful to see how bad some of these libraries were, but at the same time, it also provided us with the opportunity of improving libraries because we could we could find out where the bottlenecks are and we could fix them.

Vivien

Antibody companies, and I have only spoken to a few here, are taking different strategies related to polyclonals and monoclonals, recombinant antibodies and animal-free recombinant antibodies. Each company has plenty at stake. The path forward will depend on antibody consumers: scientists in labs around the world. Their behavior and the choices they make will shape what happens next. Here’s the University of Toronto’s Aled Edwards 

Aled Edwards

The consumer actually drives what the businesses do. with a recombinant antibody, people are like what the hxx is that, I’ve never  used one of those before. Professors like us, we’re so reluctant to change.

Vivien
Currently it isn’t easy for scientists to assess if a given antibody will work in their experiment, their application, or organism they study. That makes antibody shopping difficult. Fridtjof Lund-Johansen has an idea. 

Fridtjof Lund-Johansen
Let's get the antibodies, the best antibodies from both systems, from naive libraries and made by immunization against some very popular targets such as p53, EGF receptor, et cetera. Let's compare antibodies that have been made using naive libraries that have been made using immunization. And then if these antibodies are, if the ones that are made by naive libraries are equally good. Then I think it is, of course, interesting to discuss the possibility of replacing the traditional antibodies, but of course, you have to remember that there are there are three million antibodies on the market, right.

And replacing. Well, you don't want to replace all of them. Probably want to replace less than 10 percent of them because most of them are crap. But if you replace if you find the best ones and then say, OK, can we make can we make similar reagents without immunization? That would be great, but I still I think that the people who make antibodies from the naive libraries, they have to show and prove they can make antibodies that are as good as the best ones that have been made by immunization.

Vivien

The proof is hard to deliver. Such comparisons are difficult and expensive.

Fridtjof Lund-Johansen

What I want to do is that I want to compare very large numbers of antibodies to the same protein to find the best ones, and we can do that using the race in our array technology, and we can then test them for their ability to capture or immunoprecipitate, if you like, native or denatured proteins. That's something that I've been working on in the lab for many years.

I personally believe that this is this is a very good way of finding the best antibodies. What we what we have to prove is that this capture technique that we are using is also predictive for the performance of antibodies in other applications, such as western blotting, immunohistochemistry, immunofluorescence microscopy for example. We have not done that yet.

Vivien

The reason such assessments matter is that scientists spend too much time chasing down rabbit holes to figure out why an antibody that works in a published paper does not work in their experiment. And they know all too well the frustration of finding a study in the scientific literature and discovering that the antibody used in that experiment might not quite be showing what is accurate. 

Peter McPherson is a neuroscientist at McGill University’s Montreal Neurological Institute Hospital. Among the areas he studies are the currently incurable neurological diseases ALS or amyotrophic lateral sclerosis and FTD, frontotemporal dementia. C9ORF72 is a gene that plays a role in both of these diseases. Here’s Peter McPherson 

Peter McPherson
Discovered in 2011, this is the major disease gene in ALS and also in a disease called frontotemporal dementia. And in fact, the fact that this gene so there's families where they have this mutation in this gene C9ORF72 and it's a non coding mutation actually, it's not a coding sequence, it might affect expression levels of the protein it might affect, there might be some toxic gain of functions and the details as to how it causes pathology, still unknown it's debated.

Vivien

McPherson and his team, like other labs have focused on the C9ORF72 protein. They have been looking at a module this protein has, a so-called DENN domain. The domain offers hints about what this protein does and maybe the role it plays in disease. 

Peter McPherson

And so the fact that this ALS disease gene has this module suggests very clearly that the thing was involved in membrane trafficking,. The function of the protein: completely unknown. We start looking. Nothing in the literature. But there are some papers where they localize the protein. We look, I’ve got this little slide show and it’s kind of funny, my postdoc made it, we get laughs.

I press on the first one, reference above it, the nucleus lights up in green, then we click on the next one, the Golgi lights up in green, the endosomes light up in green, the lysosomes light up in green, the actin, the cytosol, by the time you’re done, the whole cell is full of green. Those were all published papers showing the localization of C9ORF72.

Vivien

Is it everywhere? 

Peter McPherson
It's possible. It's remotely possible. But we didn't really think that made much sense to us, because it turned out that almost all of those studies were done with a single antibody that simply did not recognize the gene in any of the tests. Those were the 15 papers that had been cited 3,500 times. So it just so what happened was one guy in the very first paper used an antibody from Santa Cruz that didn't work, polyclonal, and then it was published in an Neuron paper.

So so the next guy used it in, the next guy used it, and they all got different results, but they also didn't seem to really bother to care to and none of them validated the antibody. And it turns out that particular antibody simply does not recognize C9ORF72 in any application, period. So we were frustrated. You know, we're we're this is human health. This is real serious human health here, we're talking about. You know, part of what they use the antibodies for in some of these papers was there's a big debate. Do the levels of C9ORF72 protein go down in the disease? They use these antibodies to make conclusions about antibodies that didn't recognize the protein. 

Vivien

In his lab McPherson continues his research on C9ORF72 and many other aspects relevant to ALS, FTD and other diseases. But together with Aled Edwards, McPherson is starting a large-scale characterization of antibodies.

They want to run a series of validation experiments with as many antibodies as they can get their hands on. The data will be made publicly available. The project itself is called YcharOS. 

Peter McPherson
One of the things we want to do is we want to change the culture a little bit. We don't ever plan to rate the antibodies. All we plan to do is do the experiments where we do the characterization of the antibody. We make all of that data openly available and we present the raw data. So we don't we don't make decisions. We don't we don't give it a score. We don't give it a one through ten. We present the data. Any scientist can go look at the data, do the interpretation, say, OK. 

Vivien

YcharOS is a startup that has raised money for example from the National Institutes of Health and the Michael J Fox Foundation. For now YcharOS’s home is in the McPherson lab but over time the hope is to raise enough money to set things up elsewhere and keep the characterization pipeline running. 

Another large-scale antibody venture is at the non-profit Institute for Protein Innovation in Boston. It was set up by biotech entrepreneur Timothy Springer and the plan for the organization’s Antibody Initiative is to make synthetic antibodies to all of the human body’s proteins and give the antibody sequences to the scientific community for their use. The team has made a large library of synthetic human antigen-binding fragments using yeast display, then is using those sequences to make antibodies, expressing them in cells, then selecting and validating the antibodies. 

Back to YcharOS, the venture by Peter McPherson and Aled Edwards.   

Aled Edwards
In my day job, right, I want to help invent medicines as a human right and make them affordable for everybody. And so our model for doing that is like Paul Newman's salad dressing. So the Newman's Own salad dressing is a for profit company. That pays its taxes, pays its employees, does a good job, but the profits after all of that don't go to the shareholders, don't go to Paul Newman's family's pocket, but get given away.

So they've given away 580 million dollars over the course of , to kids camps to this, to that, they give it away. That's why I thought, well, why the f can't we create a pharmaceutical industry like that? So we created a charity called the Agora Open Science Trust. It's not patenting anything. And we created little companies underneath: the first company we created with M4K pharma: meds for kids, pharma, and we're making a pediatric brain drug. And our mission is to charge a lowest price as possible as humanly possible.

We don't want any money and we're sharing the science as we go. We're putting it on YouTube every month. This is what the chemistry we did. That's our IP strategy, because you can't patent anything if it's in the public domain. So we put everything in the public domain so no one can patent anything. And that model, we thought, why don't we create an antibody validation service under that model, Agora created, we call it YcharOS. Ad what it's going to do is going to get knock-out cells. It's going to get antibodies from everybody. Create a standard operating procedure, do the experiment and just put it online. Don't rank them. We're just going to do the experiment properly and put it out there and the data will speak for themselves. 

I think all the players would agree that polyclonals ideally must be eliminated from the face of the planet. And it's going to take a while, but we must do it because it's unscientific to have a heterogeneous mix of a thousand different antibodies in there, you don't know what it's doing. They're not purified. You don't, you know, it's just unscientific. It's what we had at the time. And there are many different ways to make renewable ones, including the stuff that Andrew does. And I'm agnostic now as to which one, you know, would be the the one. And we should let the experiments tell us.

Vivien

Many antibody companies do their own in-house characterization and validation experiments. Alejandra Solache comments on that kind of work and on YcharOS.

Alejandra Solache
We can only do as much as we can do by ourselves, having organizations or other companies doing this is this absolutely the right thing to do. And particularly with the YcharOS initiative, which is offering a service of validation for academics and other companies that may not have access to these reagents and doing it in a controlled way, that these they are actually developing the expertise to do it in the right way is something that I think will be invaluable.

Vivien

Alison Gray, a proponent of non-animal derived antibodies wishes the characterization ventures  well. 

Alison Gray

I completely support both of what they're doing, but here I think it's fantastic. That's always, always very important. Whether it's animal derived or non animal derived: validation is absolutely key.

Vivien

Traditional antibodies, new types of antibodies, recombinants, animal-derived and non-animal derived antibodies. Researchers in academia, at non-profits and at companies have different views on research antibodies but it looks like research culture and research practices will possibly change a little.

Aled Edwards
We’re trying to create a better world, and it’s really hard.

Vivien

That was Conversations with Scientists.

Today’s episode was Bye-bye Bunny. It included Dr. Alison Gray from Afability and the University of Nottingham, Dr. Katie Crosby from Cell Signaling Technology, Dr. Alejandra Solache from Abcam, Dr. Carl Ascoli from Rockland Immunochemicals, Dr Andrew Bradbury from Specifica, Dr. Achim Knappik from Bio-Rad, Dr Aled Edwards from the University of Toronto, Dr. Fridtjof Lund-Johansen from Oslo University Hospital, and Dr. Peter McPherson from McGill University.This music is called Coffee Pot by the Band Split Phase. I’m Vivien Marx, thanks for listening.

Vivien Marx

Journalist , Nature Research

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