David L. Dunn, MD, Ph.D: Molecular Biology of Endotoxin Antagonism

[ Title Slide ]   Molecular Biology of Endotoxin Antagonism

Now what I would like to discuss in the next ten minutes is an area that I've been very interested in but I've really built on a tradition, I think, probably going back 50 years in surgery. Surgeons have always been interested in why their patients develop infection; moreover, why they die of infection. And all of us know whether it's a mixed infection or a bloodstream infection, that endotoxin has played a key role. And some of my role models at the University of Michigan when I was in medical school were surgeons and surgery residents who were studying large animal models, using endotoxin injection as the challenge. And I remember wondering at that time, what was known about that and subsequently I had the good fortune to work with one of the people at the University of Minnesota, Dennis Watson, who was the chairman of microbiology there when I was doing my graduate school work, who literally put this field on a firm footing in the 1940s and '50s and defined with some German chemists the biology, pathophysiology and in fact the molecular biology of the endotoxin molecule.

Now my interests stemmed from a surgical problem. Just as Chuck mentioned, I think that that's where things should start. Why do patients who are in the intensive care unit who develop multiple system organ failure, who have gram negative rods in the bloodstream, why is their mortality 40 percent? We had a pretty good idea that it was because of endotoxemia, although that has not been as well substantiated in the clinical setting as we would like perhaps. But it became very clear, that this molecule, which is very, very complex clearly was a target of opportunity, if you will, for inhibition, for antagonism. That's where this work started. We knew that in animal models we could readily reproduce endotoxemia by direct endotoxin injection or by injecting gram negative bacteria. And in fact, based upon the work that I mentioned of previous investigators, a lot was known about the endotoxin molecule. Not much was known about endotoxin antagonism. About the only molecule that was available was Polymixin B, and I think that all of you know that it's highly nephrotoxic. It never really saw clinical utility.

[ Slide 1 ]   Gram Negative Bacterial Lipopolysaccharide (LPS, Endotoxin)

Now the endotoxin molecule, found embedded in the wall of gram negative bacteria, as I mentioned, is complex. It has three different regions. This O-antigen region, which is different for every strain of bacteria and not a good target for a magic bullet.

[ Slide 2 ]   Anti-Endotoxin mAbs

The core lipid A region, though, is more interesting because many, many types of bacteria have this region in common. And in fact, the lipid A molecule is the toxic moiety. That had been pretty well-defined. So where much of my work delving into molecular biology started was developing hybridomas, monoclonal antibodies derived from hybridomal cell lines, directed against this so-called DCLA, deep core lipid A region, and the reason we were interested in this is that this did represent the toxic moiety and it was possible to develop cross reactive antibodies directed against this region. Now what's interesting about endotoxin is that it is highly complex, as I've already mentioned.

[ Slide 3 ]   Gram Negative Bacterial Lipopolysaccharide (LPS, Endotoxin)

It consists of a series of polysaccharide residues, but intriguingly these are all linked to lipid A, which is a complex lipid structure with very long chain fatty acids that are found virtually nowhere else in nature. And this represented a conundrum, right to begin with, because developing antagonists against this lipid A region was thought to be highly difficult because we're working with proteins, antibodies, in fact. And as I think that all of the investigators who are presenting probably know, collaborations are very important and I just wanted to bring that up for all the residents in the room. If you can find good collaborators in your department, in other departments at the Baylor College of Medicine or certainly in the basic sciences, cultivate those collaborators.

[ Slide 4 ]   Bactericidal/Permeability- Increasing Protein (BPI/BP55)

This slide talks about BPI, which in fact was initially termed BP 55. One of the microbiologists who sat on my Ph.D. committee was Buelah Gray. She was a friend of mine. We occasionally played tennis together. And she saw me one day in the hall and asked me if I could find a paper in Surgery. It turned out that the reason she wanted this was because it mentioned BPI and she in fact was the investigator who had described this protein to begin with. And I said to Beulah, "That's interesting, I didn't even realize you were working in this area currently".

Why is BPI or BP 55 interesting? It's a host defense protein and it binds to endotoxin. It's a naturally occurring endotoxin antagonist and it has three properties, an amphipathic loop, a beta sheet, and it's cationic. It is a potent endotoxin antagonist. Well, that collaboration took off, and the next thing we began asking - we were just working with antibodies at the time and there were a number of superb surgery residents getting their Ph.D.s in surgery or biomedical sciences - they in fact began asking questions that I hadn't asked previously, and the questions were "were there any other similar compounds?" And the answer was "yes".

[ Slide 5 ]   Limulus Anti-LPS factor (LALF)

I think that many of you may be familiar with this. The horseshoe crab has a host defense protein as well. And LALF binds very similarly to endotoxin as BPI did and interestingly, is 40 percent homologous in terms of its genetic sequence. Three similar factors, identical to BPI and the same sort of activity. In fact, in some of our models it's been more active than BPI.

[ Slide 6 ]   Sequence Homology of BPI, LALF, and LBP

Tim Billiar and his group at Pittsburgh know more about lipopolysaccharide binding protein better than our group. Interestingly, this is the third host defense protein that binds to endotoxin. So we had three lined up there and when we began looking at genetic sequences we could tell that based upon the amino acid sequence in these proteins they all had a homologous binding region and some of the other investigators working in this area were clearly defining similar properties for all of these compounds.

[ Slide 7 ]   Molecular Basis of Endotoxin Antagonism

Now what we did then was to go back to the drawing board and quite frankly I retooled my lab in many ways in terms of what was currently available in molecular biology. We began working with a biochemist so that we could develop peptides based upon that binding region, collaborations with Dr. Norman Wainwright out at the Scripps Institute who literally had defined how LALF binds to endotoxin, and then went back and began looking at our own anti-endotoxin antibodies and also, fourth point on this slide, developing anti-idiotypic antibodies.

[ Slide 8 ]   Endotoxin Binding Beta Sheet

What we were interested in was this beta sheet region of each of these molecules and

[ Slide 9 ]   Endotoxin Binding Domains

wondered if the anti-endotoxin molecules also might possess this loop that very, very clearly bound to endotoxin.

[ Slide 10 ]   Synthetic Anti-LPS Peptides

Well, the first thing we did was to develop small peptides based upon the consensus sequence of each of these proteins. In looking at this it was very clear that we needed some help. Our biochemist, Dr. Kevin Mayo, had been working in this area. And a series of peptides were developed. This one's an irrelevant one. But you can see that each one was a little bit different but based upon that beta sheet-binding region of each of these three host defense proteins.

[ Slide 11 ]   Experimental Gram-Negative Bacterial Sepsis

It's fairly simple to take this into an animal model, with an endotoxin challenge using D-galactosamine as a sensitizing agent. And what we found once we had established in vitro that these small peptides were capable of endotoxin antagonism, was in fact that they were highly capable of antagonizing endotoxin in vivo.

[ Slide 12 ]   Protective Capacity of Anti_LPS Pepetide BG 38t

As shown here, one of the most active ones, zero percent mortality compared to our irrelevant peptide in this animal model.

[ Slide 13 ]   Natural Endotoxin Antagonists

The next step, really, was to go back and look at our initial anti-DCLA monoclonal antibodies. Monoclonal antibodies are ideally suited for this analysis as opposed to polyclonal antibodies for a variety of reasons, but suffice it to say that the quantity that can be generated allows molecular characterization. One of the interesting things that we found in analyzing the binding site of each of these known endotoxin antagonists was that there were virtually no acidic amino acids. This is very unique in terms of protein analysis. They were analyzed according to the types of amino acids present and the binding region had no acidic amino acids.

[ Slide 14 ]   Analysis of Anti-DCLA mAb 7C5 CDR

When we looked at our anti-DCLA monoclonal antibodies, one of them shown here, the analysis that we did was essentially a back calculation based upon initially the genetic analysis of the variable regions of both the light and the heavy chain and then analyzing what amino acids had to be present. This is a simpler tool to use these days rather than sequencing the protein chains in these regions.

[ Slide 15 ]   Anti-DCLA mAbs

And what we found, when we analyzed this, was virtually the same thing in terms of very, very few acidic amino acids being present. In particular, in the light chain region you can see that there are virtually none. Both the heavy and the light variable portions together, a little bit more, but compared to control monoclonal antibodies you can see that there is a marked diminution there. Now this may be related to charge characteristics, but very clearly we have molecular tools to dissect some of these issues out.

[ Slide 16 ]   Anti-idiotypic Monoclonal Antibodies

And then finally taking these antibodies and generating anti-idiotypic monoclonals, where we can ostensibly study the mirror image of the binding region. We've done this because we want to study the receptor for endotoxin at the same time, possibly developing antagonists on this basis.

[ Slide 17 ]   Anti-Idiotypic Anti-LPS Antibody

Anti-idiotypic antibodies clearly might bind to this receptor but not stimulate it if we pick the right one.

[ Slide 18 ]   Inhibition of LPS-Induced TNF-alpha Secretion in Vitro

We went ahead and did that and this is work that Dr. Richard Batafarano, who is now training at Washington University, did. And basically this anti-idiotypic antibody compared to control antibody markedly inhibits endotoxin induced TNF secretion in this model.

[ Slide 19 ]   Bactericidal/Permeability-Increasing Protein (BPI/BP55)

Finally, just to return to where this work is going, it's pretty clear that we have a good idea of how endotoxin antagonism occurs. We know about the charge characteristics of several different molecules including the ones where the work started, the monoclonal antibodies. BPI is of particular interest because it's so potent as an endotoxin antagonist.

[ Slide 20 ]   Conclusions/Future Studies

And our future studies are really looking at dissecting this out even more clearly. We believe currently that this beta sheet confirmation plus charge characteristics is necessary and sufficient for endotoxin antagonism. What we're going to do at this point and what we've been working on over the last couple of years is ongoing synthesis of peptides that have even greater potential in these models. We want to compare the hypervariable regions of both additional anti-DCLA monoclonal antibodies as well as a battery of anti-idiotypic monoclonals. And actually, I think that the most interesting work has been to create fusion proteins based upon the binding region of BPI and the best part of some of our antibodies and we're developing very small single chain Fv fragments that at least in vitro have a great deal of capacity to inhibit endotoxin. Thank you very much for your attention.

Thank you.