Discovery and Development of Tysabri (Natalizumab) for the Treatment of Multiple Sclerosis

Part 1: Immune Cell Migration to the CNS

00:00:07.02 Hi.
00:00:07.22 I'm Ted Yednock and I'm an immunologist trained at the University of California in San Francisco.
00:00:11.17 And for a number of years, I worked for a company called Elan pharmaceuticals.
00:00:15.09 At Elan, and we developed a drug called Tysabri for the treatment of multiple sclerosis.
00:00:20.09 So, I'm going to talk about the identification and development of Tysabri in two lectures.
00:00:26.04 Today, I'm going to talk about the scientific underpinnings of Tysabri and how we think
00:00:31.02 it works by inhibiting immune cell infiltration of the central nervous system,
00:00:35.15 preventing the damage associated with the autoimmunity in MS.
00:00:40.11 I'll also talk about the preclinical work and the clinical studies that we did in order
00:00:44.10 to demonstrate that the drug was safe and efficacious.
00:00:48.03 In the second lecture, I'll talk about how we believe the selective mechanism of this drug
00:00:52.20 allowed for the emergence of a very rare but serious brain infection called
00:00:57.27 progressive multifocal leukoencephalopathy, or PML.
00:01:01.19 And I'll also talk about the complexity that that caused for patients, and physicians,
00:01:06.25 and the FDA, as well as the companies, in trying to understand how PML was associated
00:01:12.07 with Tysabri.
00:01:13.22 So, at first, I...
00:01:15.05 I will say a few words about multiple sclerosis.
00:01:22.00 So, MS is an autoimmune disease, of the CNS, as I mentioned.
00:01:26.02 It's involved in the destruction of myelin.
00:01:27.25 So, there's a very strong T and B cell response against myelin.
00:01:31.27 And once myelin is lost, you have impaired nerve conduction and neuronal loss.
00:01:36.11 Now, MS is... can happen anywhere in the central nervous system, so it can affect any part
00:01:40.26 of your body.
00:01:41.27 A patient may feel tingling in an arm or hand, or they may have loss of vision, or issues
00:01:49.15 with control of bladder function, or... or not be able to walk.
00:01:54.06 It affects about 300,000 patients in the United States.
00:01:57.06 It's more common in women than men.
00:01:59.12 And, as I've alluded to, it's a devastating disease and it affects people in the
00:02:02.25 prime of life, between the ages of 20 and 40.
00:02:06.04 But it can be seen at any age.
00:02:08.23 Now, usually, MS is a relapsing... it begins as a relapsing-remitting disease.
00:02:14.05 So, the attacks can come along very quickly, you know, vision loss can happen very quickly,
00:02:19.17 and then, over a period of days to months, there will be a degree of recovery to varying levels.
00:02:25.08 But after 15 or 20 years, the disease often becomes progressive.
00:02:30.18 And this happens in about 65% of patients.
00:02:33.23 And now, it's more of a chronic neurodegenerative disease, without the acute inflammatory attacks.
00:02:40.12 So, MRI is actually a very useful diagnostic and monitoring tool for multiple sclerosis.
00:02:46.14 So, this is an MRI image.
00:02:48.14 And what... what is often done in MS is that patients are given a contrasting agent called
00:02:53.22 gadolinium.
00:02:54.22 It's injected intravenously, and normally it's excluded from the brain.
00:02:58.27 However, in MS, when there's an active lesion, the blood-brain barrier is leaky and so gadolinium
00:03:04.03 will enter the brain and show up very prominently in an MRI.
00:03:08.03 Now, if you were to look at this lesion through a microscope, you would see this.
00:03:13.17 This is a... a blood vessel in multiple sclerosis.
00:03:17.09 And what's happened here is there are immune cells traveling throughout the body in the bloodstream
00:03:22.27 and... now, normally, they just go right through the brain.
00:03:25.10 But in the case of MS, there's something happening to the blood vessel wall, and the immune cells
00:03:30.12 will adhere to the vessel wall, migrate in, and accumulate in these large clusters around
00:03:36.15 the blood vessel.
00:03:37.15 And this basically is ground zero for demyelination and neuronal damage.
00:03:41.18 So, in 1990, we had a very simple hypothesis, and that was that if we could inhibit immune
00:03:48.00 cell attachment to the blood vessel wall we could prevent their migration into the brain
00:03:51.19 and all the damage associated with that.
00:03:53.26 And so... when I say adhesion molecule, picture Velcro.
00:03:57.22 So, we're trying to identify the little hooks on the... on the immune cell that allow it
00:04:01.22 to attach to the blood vessel wall, specifically at times of inflammation.
00:04:05.28 Now, since this is a brain disease, it's really important to be able to do these kind of studies
00:04:11.09 using an... an animal model.
00:04:12.23 So, the best animal model for multiple sclerosis is experimental autoimmune encephalomyelitis,
00:04:18.24 or EAE.
00:04:20.20 Now, in EAE... this is a... a... a model in the guinea pig, but we also use models in
00:04:25.19 mice and rats.
00:04:26.26 So, in this model, this is a brain... a slice through the brain of a guinea pig with EAE.
00:04:32.03 You can see a large blood vessel.
00:04:34.05 And in the blood vessel there are infiltrating immune cells, which are the blue cells, here.
00:04:39.23 And the section is stained with a red dye for myelin.
00:04:42.18 So, you can see that around the blood vessel there's a huge loss of myelin.
00:04:47.22 And then, also, the... the little green dots are axons, or nerve fibers, that are... are...
00:04:54.02 you're looking at them in cross-section.
00:04:56.07 And normally, over here, you can see that the nerve fibers are surrounded by red myelin,
00:05:01.12 so they're quite well insulated from each other.
00:05:03.27 But when the myelin is lost, the nerve cells or the nerve fibers are pushed around and clustered,
00:05:07.24 and the spacing between is occupied by immune cells and by edema.
00:05:11.15 And so you can see, with this kind of damage, how nerve conduction would be impaired.
00:05:18.18 This is the way the model progresses in animals.
00:05:21.03 So, in the guinea pig, we immunize the animals with spinal cord homogenate.
00:05:26.17 This causes a very strong response, again, of B cells and T cells to myelin.
00:05:31.19 And so that... by day 9 or 10, the immune cells will begin to infiltrate the
00:05:35.14 central nervous system, and you'll begin to see a degree of hind limb paralysis in the... in
00:05:40.01 the acute phase.
00:05:41.01 And then the animals begin to get a little bit better.
00:05:43.21 But then, by day 15 or so, the disease begins to, well, progress, and enters the chronic phase.
00:05:50.18 And the... the difference here is this is when demyelination begins.
00:05:53.20 So, once demyelination begins, the nerve fibers are pushed around, the immune cells are even
00:05:58.15 more activated, and the disease progresses with just more and more demyelination.
00:06:03.27 When we were first doing these experiments, we were a very small lab in a...
00:06:07.05 in a small company and we really didn't have a good model of EAE.
00:06:11.19 In fact, we weren't working in this area at all.
00:06:14.17 We... we were trying to develop therapeutics for Alzheimer's disease.
00:06:18.14 And we were trying to develop an animal model of Alzheimer's.
00:06:21.06 It would have been the world's first model, which... which we eventually did, but
00:06:25.15 our initial attempts weren't so good.
00:06:27.07 And in fact, we really were inducing a model in which there was a chronic inflammation.
00:06:33.21 And it's that kind of inflammation that initially got us interested in this area to begin with.
00:06:37.26 So, we took inflamed brains from these animals in this strange model of Alzheimer's disease,
00:06:43.24 and did a very simple experiment.
00:06:45.27 We took these sections and overlaid them or exposed them to a suspension of human lymphocytes.
00:06:51.20 And we found that the lymphocytes would adhere selectively to these inflamed vessels.
00:06:55.26 So, you can see these very dark blue, large circles are the human lymphocytes, and they're
00:07:02.06 adhering right in the center of these blood vessels, right where you would expect to see
00:07:06.06 blood cells in the circulation.
00:07:08.13 They don't bind elsewhere in the section and they didn't bind to non-inflamed vessels.
00:07:12.16 So, the fact that we were getting physical adhesion of these cells to inflamed vessels
00:07:18.21 suggests that this is probably a physiologically relevant... relevant interaction.
00:07:22.26 Because these ligands -- whatever these adhesive ligands... whatever they are -- were being
00:07:26.07 induced in the context of a whole brain.
00:07:29.04 We also isolated endothelial cells from rat brains and put them into culture,
00:07:34.25 stimulated them with TNF, and we found, there,
00:07:37.17 that lymphocytes would adhere here as well.
00:07:39.17 So, that was great.
00:07:41.00 We now had two different assays by which we could look at lymphocyte interaction with
00:07:44.21 inflamed endothelium, so we could screen for inhibitors of that interaction.
00:07:49.12 What we did is made thousands of antibodies against the lymphocyte surface, at random,
00:07:54.22 and screened for antibodies that would inhibit this.
00:07:56.25 So, in this assay, we could screen thousands of antibodies.
00:08:00.15 And in this one, we could screen umm... perhaps in dozens.
00:08:03.07 It was a much more cumbersome assay.
00:08:06.00 But, nonetheless, both assays gave us exactly the same answer.
00:08:09.22 And that is that alpha 4 beta 1 integrin is very important for lymphocytes and monocytes
00:08:15.28 to adhere to the... to the inflamed brain vessel.
00:08:18.16 So, in the presence of anti-alpha 4 or anti-beta 1, you can see that there was no longer any
00:08:24.00 human lymphocyte attachment to the vessels in this section.
00:08:29.09 So, this is what we thought was going on.
00:08:33.10 Lymphocytes and monocytes expressed alpha 4 beta 1 integrin and they were binding to
00:08:37.01 some ligand expressed by inflamed brain endothelial cells.
00:08:41.19 So, we also knew in 1990... and, in fact, I was...
00:08:45.01 I was kind of disappointed with this observation.
00:08:48.19 Because we knew that alpha 4 integrin was expressed by many cells in the circulation
00:08:53.13 -- lymphocytes, monocytes, eosinophils, basophils.
00:08:56.22 Unfortunately, it was low or negative on neutrophils, red blood cells, and platelets.
00:09:02.06 However, we had been hoping to find sort of a brain-specific adhesion molecule that was
00:09:06.15 expressed by a small subset of... of pathology-causing immune cells.
00:09:10.27 But, in fact, it was expressed very broadly.
00:09:13.20 Nonetheless, as you'll see, I believe that alpha 4 beta 1 integrin turned out to be
00:09:17.23 a pretty selective adhesion molecule for the CNS.
00:09:22.00 The molecule, alpha 4, had been cloned the year before by Yoshi Takada and Martin Hemler's group.
00:09:27.06 And a number of groups were also identifying the ligands for alpha 4.
00:09:32.13 For example, Elizabeth Wayner had found that an alternatively spliced form of fibronectin
00:09:36.20 was a ligand for alpha 4, and also Roy Lobb at Biogen had identified VCAM-1,
00:09:43.14 expressed by human umbilical vein endothelial cells in culture.
00:09:47.18 So... but no one really knew how alpha 4 beta 1 worked in vivo and how it contributed to
00:09:52.16 disease pathology.
00:09:54.00 And that's when we go back to umm... the need for a good animal model of EAE.
00:09:58.15 So, the model that we had with our Alzheimer's-inflamed brain wasn't really going to be good enough
00:10:03.21 for that.
00:10:04.21 So, we went down the street and visited Larry Steinman at Stanford.
00:10:09.19 And Larry Steinman is a world expert in multiple sclerosis and in EAE.
00:10:14.10 And we took... we went to him and showed him our results with alpha 4 beta 1 integrin,
00:10:18.17 and we had antibodies against alpha 4, and we asked him if he could help us test it in
00:10:23.05 a... in a really good model of EAE.
00:10:25.09 And that's exactly what he did.
00:10:27.03 Ni...
00:10:28.03 Nathan Karin, who was a postdoc in his lab at the time, was working with a rat model
00:10:32.21 of EAE.
00:10:33.21 So, the first thing we did was confirm that using brain... sections of inflamed brain
00:10:38.11 from animals with... rats with EAE, that we had the same sort of lymphocyte adhesion,
00:10:43.04 and that it was completely dependent upon alpha 4 integrin.
00:10:46.14 Nathan then tested one of our antibodies against alpha 4 and found that it was very protective
00:10:52.00 in preventing disease in these... in this animal model.
00:10:54.21 So, that was great.
00:10:56.09 Based on that, we... we started bringing in a number of different EAE models into our...
00:11:01.07 our little lab.
00:11:02.26 This was one, again, in the guinea pig.
00:11:04.25 And here we wanted to characterize more thoroughly what... how alpha 4 integrin works, how quickly
00:11:10.12 it works, and the impact that it can have on the disease.
00:11:13.15 So, in this study, we labeled lymphocytes with indium-111, which is a very high gamma emitter.
00:11:20.19 And we found that if you inject the cells back into the bloodstream they will migrate
00:11:25.18 to the brain.
00:11:26.18 So, you can just take out the brain and do a gamma count and see how many cells there.
00:11:30.07 But, in the presence of anti-alpha 4, inhibition was 80 or 90%.
00:11:35.11 So, in this short-term migration assay, lasting about 18 hours, alpha 4 integrin was clearly
00:11:41.09 affecting the ability of cells to migrate into the brain.
00:11:45.17 And then we treated guinea pigs with the antibody, just at the onset of disease.
00:11:51.06 This was like day 10 or 11, just when paralysis was beginning but before demyelination had happened.
00:11:57.28 And what we found is that by day 20 we had a very strong impact in preventing demyelination.
00:12:02.28 So, in the control-treated animal, you can see that they're... we have stained these
00:12:07.19 sections of spinal cord with a blue dye for myelin, and there's an extensive loss of myelin
00:12:14.01 by day 20 in the control animals.
00:12:16.01 However, in the presence of anti-alpha 4, the myelin is almost completely intact.
00:12:20.05 So, there was very strong protection against the loss of myelin.
00:12:26.22 We next... we next treated animals at day 20.
00:12:30.15 So, this is after demyelination has already occurred.
00:12:34.02 And we treated them for a long time -- 56 days -- because you wanted to see if it would
00:12:38.06 impact the disease and if that impact would last.
00:12:42.04 And what we saw was this: a very strong reversal of the paralysis and... that lasted for
00:12:47.17 the entire time of the treatment.
00:12:50.19 Okay, so we did this experiment a number of times, looking at different time points to
00:12:54.19 see what impact that we would have on myelin.
00:12:58.07 So, in this study, we treated the animals after demyelination had already occurred,
00:13:05.03 and then we inhibited alpha 4 integrin for various periods of time.
00:13:09.17 We found that after 20 or 40 days of treatment that we actually would allow myelin to repair.
00:13:15.26 So, the way we knew this was that under control conditions myelin is either present or it's not.
00:13:22.21 So, over here, you can see that... here is myelin, this is an area where the myelin
00:13:27.08 has been eliminated, and the... the line between the two is very, very stark.
00:13:31.25 So, it's either there or it's not.
00:13:34.17 However, in animals that had been treated with an inhibitor of alpha 4 integrin,
00:13:37.23 we saw this very pale blue pallor, which we confirmed by electron microscopy to be regeneration
00:13:44.16 of myelin.
00:13:45.16 So, we believe that what was happening is that by inhibiting immune cell infiltration
00:13:49.14 into the CNS we were dampening down this inflammatory response and basically creating an environment
00:13:55.22 that was permissive for remyelination.
00:13:57.22 So, these are the studies that led to the development of Tysabri, which is a... we took
00:14:05.20 our best antibody against alpha 4 integrin and humanized it by CDR grafting.
00:14:10.16 And this was done in collaboration with the MRCC in London.
00:14:14.19 And fortunately, the antibody retained the full potency of its murine parent.
00:14:22.00 Just to give a perspective on scale, this is a model of an antibody next to a model
00:14:26.26 of an integrin.
00:14:28.10 Umm... the... the antibody actually binds to the head group, up here, which is the
00:14:33.02 business end of the integrin.
00:14:34.02 This is where it binds to ligands.
00:14:35.22 Now, recently, the crystal structure of natalizumab bound to the head groups of alpha 4 integrin
00:14:42.04 has actually been solved, by Yamel Yu in Timothy Spring... in Tim Springer's group.
00:14:48.11 So, this is looking at the crystal structure, just at the head groups of the integrin umm...
00:14:54.23 and the blue is beta 1 and the green is alpha 4.
00:14:58.18 And the ligand binding site -- this is for VCAM-1 -- is right in between the two.
00:15:03.03 So, there are critical contact residues on both sides of the line.
00:15:06.12 Now, natalizumab, he found, bounds right next to this.
00:15:10.11 So, the binding actually doesn't overlap.
00:15:12.26 And, in fact, if you take the business end of VCAM, the first domain, it will bind just
00:15:18.16 fine in the presence of... of Tysabri.
00:15:21.20 However, the second domain of VCAM causes steric hindrance, so that, when natalizumab...
00:15:29.10 when natalizumab is bound in the presence of the full-length ligand, its binding energy
00:15:34.10 is greatly decreased because of the steric hindrance.
00:15:37.06 What they also noticed in the lab was three critical contact residues within the natalizumab
00:15:43.14 binding site that help to explain observations that we had made a number of years earlier.
00:15:48.12 So, when you're developing a drug, it's important to have animal species for efficacy testing,
00:15:53.05 but you also need to have two different animal species for toxicology.
00:15:56.27 You need to be able to show that it's safe in at least two species.
00:16:00.28 So, we were disappointed because our... our best model for EAE at the time was in the rat.
00:16:06.22 However, the rat has a mutation in one of these critical residues, and we found that
00:16:10.16 the antibody didn't react.
00:16:12.28 There also was an EAE model in the marmoset monkey, which was a model that was set up
00:16:18.05 at UCSF.
00:16:19.05 And, again, we found that our antibody did not react with the monkey, which... we were
00:16:22.11 really surprised, because it's very closely related to the human.
00:16:26.12 But now it makes sense with... with the finding that there's a mutation in one of these critical
00:16:31.02 amino acids.
00:16:32.12 So, we did find, however, that the... the antibody reacted with cynomolgus monkey and
00:16:38.20 with guinea pig.
00:16:39.20 And, as you can see, that the sequence of these... of the three critical contact residues
00:16:44.03 in these species are identical to those in human.
00:16:46.27 And these are the species that we had chosen for our toxicology.
00:16:52.21 So, in order to develop a drug, you need to do a lot of toxicology studies.
00:16:58.21 This is just a subset of the things that we did.
00:17:00.21 And I'm not going to go through all these, other than to mention a couple findings.
00:17:05.18 First of all, in the first study up here, it was a six-month study in cynomolgus monkey
00:17:10.15 involving very high doses of... of natalizumab.
00:17:13.08 Umm... natalizumab, by the way, is the generic name for Tysabri.
00:17:17.12 So, we did 60 milligrams per kilogram dosing every week.
00:17:22.12 And, in patients, we find that 3 milligrams per kilogram dosed every month
00:17:27.06 is what it takes for efficacy.
00:17:29.15 So, in fact, in the monkeys, we were achieving blood levels more than a thousand-fold that
00:17:33.28 you would find in humans.
00:17:35.10 So, at the end of 6 months, we looked to see what impact it would have, particularly on
00:17:40.02 the immune system.
00:17:41.03 Remember, alpha 4 integrin is expressed by almost all circulating mononuclear cells.
00:17:46.00 And so we were quite interested to see what would happen in the immune system.
00:17:50.17 And much to our relief, we found that hematopoiesis... bone marrow was... was very much intact, so
00:17:57.10 lymphocytes and red blood cells were being made normally.
00:18:00.05 And this was important because alpha 4 integrin had been shown to be involved in hematopoiesis.
00:18:04.11 We also did immunotoxics... immunotox... immunotoxicity profiling and found that if you vaccinated
00:18:13.20 animals in the presence of Tysabri the... they responded just... just fine to the vaccine.
00:18:18.24 There was a slight delay in the initial antibody response, but the animals quickly caught up.
00:18:25.01 And other than that, in these studies we also looked at the impact of natalizumab on implantation,
00:18:31.03 in development, and... as well as on development of the fetus during the full pregnancy,
00:18:37.16 and found very little impact there.
00:18:39.25 So, to summarize our... our preclinical and our toxicology studies, functional screens
00:18:47.09 are... are really important when trying to understand a disease process.
00:18:50.10 And this may be an obvious thing to say, but I'm just so impressed with the simplicity
00:18:54.26 of the tissue assay that we had performed, looking at the endogenous induction of ligands
00:19:01.13 involved in this inflammatory process.
00:19:03.19 In fact, this tissue assay is something that was established by Stamper and Woodruff in the 70s,
00:19:08.22 and had... has been used successfully for identifying a number of immune adhesion receptors.
00:19:14.20 Our other findings were that anti-alpha 4 was effective in multiple models of CNS inflammation,
00:19:19.19 whether it was a very funny Alzheimer's model or EAE.
00:19:22.18 And, in fact, in different labs around the world, it's been shown to be effective in
00:19:27.10 rat, mouse, guinea pig, and primate EAE, as well as
00:19:31.03 two different models of CNS viral inflammation.
00:19:34.14 So, these are models in which there's a CNS infection by a virus, the immune system goes
00:19:39.26 in to fight the virus and causes damage, and we found that anti-alpha 4 was actually protective there.
00:19:46.04 It protected against the damage and the... the body was still able to effectively fight
00:19:51.00 the virus.
00:19:52.00 So, again, it was suggest... suggesting that... that natalizu... natalizumab would be safe.
00:19:57.20 There was a lot of criticism of EAE models, because it doesn't exactly mimic the processes
00:20:04.07 of multiple sclerosis.
00:20:05.26 But in a case like this, where we're focusing on the immune infiltration aspect, I think
00:20:10.25 it's actually a very good model.
00:20:12.20 Because immune cells need to enter the brain to effectively induce EAE, and they also need
00:20:17.05 to go into the brain to cause MS.
00:20:19.24 So, by studying mechanisms involved in this infiltration process, I think that EAE reflects
00:20:25.22 what's happening in humans.
00:20:28.08 And then, finally, natalizumab appears to be safe and well tolerated in preclinical safety studies.
00:20:33.17 Again, this was important because... because of alpha 4 integrin's broad distribution.
00:20:38.00 It just speaks to the fact that toxicology is an empirical science.
00:20:42.12 You really don't know until you test it.
00:20:45.15 And then, even then, it's important to remember that you always need to be vigilant.
00:20:50.19 So, these are the studies that led to the clinical program for natalizumab.
00:20:55.19 In the first case, we looked at a Phase I study in healthy volunteers and found that
00:21:00.14 the antibody was well tolerated with low immunogenicity.
00:21:03.07 And this is just with a single dose.
00:21:05.11 And we also were able to determine that it has a 12 day half-life, which is sufficient
00:21:10.02 for once a month dosing at 3 milligrams per kilogram.
00:21:13.26 So, reasonable dosing.
00:21:16.15 As we saw in the animal models, there was a two-fold increase in the number of circulating lymphocytes.
00:21:22.14 This is reflec... this is consistent with the drug's mechanism of action.
00:21:26.11 It's making it a bit more difficult for cells to get out of the blood stream into tissues.
00:21:30.02 And so you quickly establish a new equilibrium, which involves more lymphocytes in the blood.
00:21:36.13 But this two-fold increase really represents just the higher end of normal range.
00:21:41.24 Based on those studies, it went into a Phase II study.
00:21:44.02 And... and initially we... our idea was to treat acute flares in MS.
00:21:49.26 So, a patient would have a flare, they would come in to the doctor, get a dose of natalizumab,
00:21:54.21 and hopefully we would quickly dampen down that flare and prevent the damage associated
00:21:59.02 with it.
00:22:00.02 In fact, what we found... and this is with two doses covering two months... we found
00:22:05.18 that there was no impact at all on the time of resolution or the outcome of that relapse.
00:22:12.02 So apparently, by the time a patient is feeling the onset of a relapse, the immune cells are
00:22:17.05 already in the brain, and by inhibiting more it really does not have an impact.
00:22:22.00 However, what we did notice in the study was that by the end of the second month the new
00:22:28.12 lesion development in MRI... by MRI had basically stopped.
00:22:31.28 So, this is looking at the cumulative number of new gadolinium-enhancing lesions by MRI.
00:22:38.11 And you can see that in the placebo group, in red, that there's a pretty consistent accumulation
00:22:43.02 of new lesions.
00:22:44.05 But, by the fourth week on natalizumab, these lines are beginning to diverge.
00:22:49.02 And so that... by the eighth week, the second month, when the drug is still on board,
00:22:53.11 you can see that new lesion activity had... had almost stopped.
00:22:57.05 At the next time point, when the drug was gone, MRI activity began to return.
00:23:01.14 So, this result led us to the idea that, since the drug appeared to be safe, perhaps we could
00:23:06.08 take it into MS for chronic treatment -- prevent the development of new lesions and hopefully
00:23:11.07 have an impact on the progression of disease.
00:23:14.12 So, this led to the our Phase IIb study, and this was a six-month study involving
00:23:21.18 monthly dosing as well as MRIs, which are indicated by the Ms, and then we follow the patients
00:23:27.27 for six months after that.
00:23:29.24 210 patients, looking at placebo and two different doses of drug.
00:23:34.11 And the primary endpoint would be looking... was looking at lesion burden, as well as relapse rate.
00:23:40.23 This is what we found.
00:23:41.27 So, in the months before treatment began, and at the time of treatment, all three groups
00:23:46.17 had sim... very similar levels of MRI activity.
00:23:51.00 And then, looking at the placebo group, they maintain this level of activity pretty consistently
00:23:55.11 over the six-month period.
00:23:57.03 However, in the groups that were treated with natalizumab,
00:24:00.06 and this is both for the 3 and the 6 milligram group,
00:24:03.12 you can see that MRI activity almost completely stopped.
00:24:06.19 So, again, by the second month, activity was very, very low.
00:24:10.08 Now, I have to say I...
00:24:11.15 I was sitting in the audience of the company when we first saw these results and it...
00:24:19.07 it really took my breath away.
00:24:20.15 This is... this was a day that I will remember in my career.
00:24:23.07 And I think that it... it really changed the way I look at things from then on out,
00:24:28.07 because as a scientist working in the bench it's really hard to believe that you can have an impact
00:24:32.20 on human disease.
00:24:33.22 And so this...this really made me believe that and, as I said, has had a long-lasting impact.
00:24:41.08 The drug also affected the number of relapses.
00:24:43.28 So, there was about a 50% reduction in the number of relapses, as well as a number of
00:24:48.02 patients who had a relapse.
00:24:50.00 And also decreased the need for steroid rescue.
00:24:52.25 So, these patients, if they had a flare, could... had the option of having steroid and on natalizumab
00:24:59.11 they did not feel the need to do that.
00:25:02.25 This is looking at the same data, the MRI data.
00:25:07.06 So, the blue line is 6 milligrams per kilogram, the red line is 3 milligrams per kilogram.
00:25:13.23 And so... you can see that... you can see that the drug is... for 3 milligrams per kilogram,
00:25:20.26 when it begins to go away, MRI activity returns.
00:25:24.25 And with 6 milligrams per kilogram, it takes a little bit longer for... for activity to resume.
00:25:29.07 You can overlay lymphocyte counts on top of this.
00:25:33.06 So, this is looking at the number of lymphocytes in the bloodstream.
00:25:36.22 And at a very beginning, you can see that there's this... this increase to lymphocyte
00:25:39.22 counts that's maintained very stably throughout the treatment period.
00:25:43.01 But then, in the 3 milligram per kilogram due... dose group, you can see that as the
00:25:47.12 drug goes away the lymphocyte counts drop, and the MRI activity goes up.
00:25:52.07 And with the 6 milligram per kilogram group, you can see this is... this is basically shifted
00:25:56.11 by a month.
00:25:57.11 So, the cell counts drop and the MRI activity comes up a little bit later.
00:26:01.07 So, this is very consistent.
00:26:02.16 This finding is very consistent with the mechanism of action that we're thinking.
00:26:06.07 As blood cells leave the... as immune cells leave the bloodstream, the MRI activity would
00:26:12.14 resume.
00:26:13.14 Alright, so our... for our Phase III studies in relapsing-remitting multiple sclerosis,
00:26:19.08 in order to get approval for a drug you actually have to have two separate Phase III studies.
00:26:24.03 And so both of these were about a thousand patients.
00:26:26.15 Umm... the first one involved monotherapy, so it was just Tysabri versus placebo.
00:26:31.11 The second one was on... with patients who were already on interferon beta, which was
00:26:36.05 the standard of care for MS patients.
00:26:38.28 And in this case, these patients were having disease breakthrough even on interferon.
00:26:42.27 So, the trial was natalizumab... natalizumab plus interferon versus interferon by itself.
00:26:49.05 So, this was a randomized double-blind study, as were the Phase II, and involved patients
00:26:55.08 who had to have at least one relapse in the prior year to the study.
00:26:58.14 It was a two-year treatment with an early assessment at one year.
00:27:04.24 And the findings were very consistent with what we saw in Phase II.
00:27:07.13 The number of gadolinium-enhancing lesions, both in first year... in the first year,
00:27:12.28 as well as the second year, were inhibited by 92%.
00:27:15.17 So, very strong inhibition of... of this apparently acute inflammatory reaction.
00:27:21.15 And this translated to a 68% reduction in relapse rate, again, both in the first year
00:27:26.26 and the second year, as well as in the third year.
00:27:30.02 This is an open label extension study at this point, so there isn't a placebo control because
00:27:34.02 everyone's on drug.
00:27:35.13 But, in all three years, the disease activity or the relapse activity was reduced to the
00:27:40.22 same basal level.
00:27:45.21 And this is an interesting finding.
00:27:47.05 This is a subset analysis looking at how different levels of activity would impact the efficacy
00:27:54.03 of the drug.
00:27:55.03 So, this is comparing patients who had had one relapse at the onset of when they were
00:27:59.14 coming into the trial in the previous year versus three relapses in the previous year.
00:28:04.22 And so you can see that patients who had high levels of disease activity, relapse activity
00:28:09.11 was inhibited down to the same level as everyone else.
00:28:12.07 So, it appears as though natalizumab has a very strong effect on patients with
00:28:16.23 highly active disease, inhibiting the... the relapse rate down to the same level.
00:28:24.14 And then, finally, this is the critical primary endpoint, at the second year of analysis.
00:28:29.04 And this was, how does it affect the overall progression of the disease in patients,
00:28:33.04 as measured by the standard EDSS scale?
00:28:36.24 And you can see that there's about a 54% reduction when looking at a sustained response over
00:28:41.28 six months.
00:28:42.28 Again, another subset analysis, looking at patients with highly active disease.
00:28:48.05 Again, there's an even stronger reduction, a 64% reduction, in the progression of EDS scores
00:28:54.22 in patients with... with more than two relapses and gadolinium lesions before they
00:28:59.15 came into the study.
00:29:02.28 And then, finally, this was not a point... this was not a formal endpoint in the study,
00:29:07.07 but nonetheless was a measure that was done.
00:29:09.23 And this is the MSFC scale.
00:29:12.22 And here you can see that the placebo patients... well, actually, what's interesting about this
00:29:16.08 scale is that it involves ability to walk, ability to have dexterity in the hands,
00:29:22.09 as well as cognitive function, a measure of cognitive function.
00:29:26.21 You can see that the placebo group maintained fairly stably over the two-year period, whereas
00:29:31.21 patients on natalizumab actually showed a level of improvement.
00:29:36.18 Importantly, these findings have been consistent across a number of studies.
00:29:42.04 These are actually looking at registrations and observational studies done by MS investigators
00:29:47.09 across the world, and very similar levels of efficacy with respect to relapse rate.
00:29:52.15 So, in summary, two years of treatment with natalizumab decreases disease activity, reduces
00:29:58.24 the risk of relapse by 68%, and reduces the risk of sustained disability progression by
00:30:05.04 42-54%.
00:30:06.04 To summarize the drug's safety in these clinical trials, these two-year clinical trials,
00:30:13.06 common adverse events were headache, fatigue, and arthralgias,
00:30:15.25 which are commonly seen in clinical trials.
00:30:18.22 There also were a few more infusion site reactions in natalizumab versus placebo.
00:30:24.21 Natalizumab actually causes a persistent anti... anti-natalizumab response in about 6% of patients.
00:30:32.04 And so, in these patients, actually, the drug loses this efficacy because the antibody response
00:30:36.26 causes it to be cleared very, very quickly, which is consistent with the infusion site reactions.
00:30:43.19 Importantly, there was a similar incidence of infections and malignancies between the
00:30:48.13 two groups.
00:30:49.13 In fact, if you look at this more closely, this is looking at the risk of all infections,
00:30:54.17 and this is for both studies combined.
00:30:57.05 So, both the monotherapy as well as the interferon therapy trial.
00:31:03.10 And you can see that there is no measurable difference in the rate of overall infection
00:31:08.16 between natali... natalizumab and placebo.
00:31:14.05 So, this is just the timeline of development, looking back in the beginning at 1990,
00:31:20.27 when the target was... of alpha 4 integrin was first identified for multiple sclerosis.
00:31:25.13 The antibody was humanized.
00:31:27.28 About five years later we started the clinical trials.
00:31:31.14 Biogen came in with Elan in about the year 2000.
00:31:36.01 And when developing a drug like this for multiple sclerosis, it's very expensive, because the
00:31:41.01 trials are large, there's a lot of MRI measurements, and so it's really important to have a partner.
00:31:45.18 And, in fact, Biogen was a great partner, because they already had a highly effective
00:31:51.03 drug for multiple sclerosis in the clinic and they knew a lot about... about the disease.
00:31:55.25 Shortly after the partnership was when we had the readout of the Phase II data,
00:32:00.16 you know, the day that... that changed my career.
00:32:03.01 So, I think that Biogen was very happy about that as well.
00:32:06.24 The phase III study was then shortly initiated.
00:32:09.20 The drug was approved in... in November of 2004.
00:32:13.14 The Phase... the second year of the Phase III study completed and read out shortly thereafter.
00:32:19.01 So, all said, 15 years from conception, 10 years of clinical experience involving 4,000 patients.
00:32:28.02 Now, in the second part of my talk, I'm going to discuss what happens next.
00:32:32.13 And this was just three months after the drug was approved.
00:32:35.15 This was in February of 2005.
00:32:37.23 The entire world for Tysabri changed.
00:32:41.03 So, in February of that year, we found our first two cases of PML.
00:32:45.21 And PML is a viral infection in the central nervous system that usually results in death
00:32:50.04 or severe disability.
00:32:51.04 So, it's a very serious infection.
00:32:55.01 Two patients in the MS trial had developed PML.
00:32:58.22 And amazingly, they had developed PML almost within a week of each other.
00:33:02.12 So, after ten years of clinical experience, two patients developed PML within a week.
00:33:06.23 And so this was alarming to everyone, because it... it suggested, well, maybe this is
00:33:11.04 just the tip of the iceberg, and half the patients could have PML.
00:33:14.13 So, dosing of the drug was suspended.
00:33:17.21 And we undertook a comprehensive safety evaluation at that point.
00:33:21.11 This was extremely complicated, because there were 3,000 patients involved or already
00:33:26.05 are still... are still in clinical trials, as well a number of patients who were just beginning
00:33:30.24 to take the drug from its approval.
00:33:34.04 So, what I'll talk about in my second part, in the second lecture, is how the medical
00:33:39.15 and regulatory communities working together with the companies established a path to allow
00:33:44.04 natalizumab to return, as well as the work that we've done since then to better understand
00:33:48.17 the safety of natalizumab and how PML can be monitored.
00:33:51.25 So, at this point, I just want to end by thanking all of the collaborators in the... both the
00:33:58.17 preclinical and clinical studies.
00:34:00.05 I...
00:34:01.05 I will at the end of my second lecture actually give a more detailed list of some of those contributors.
00:34:06.10 But needless to say, there were a ton of very talented people involved in the development
00:34:11.10 of this drug.
00:34:12.11 And also I want to thank the many patients who participated in the clinical trials.
00:34:17.22 Without their participation, it would be impossible to dev... to develop therapies for... for disease.

Part 2: Response to the Emergence of Progressive Multifocal Leukoencephalopathy

00:00:07.13 Hi, I'm Ted Yednock.
00:00:08.17 And welcome to the second lecture on the discovery development of Tysabri.
00:00:12.17 So, multiple sclerosis is an autoimmune disease in which circulating immune cells infiltrate
00:00:17.15 the brain and form these zones of myelin destruction and neuronal loss.
00:00:23.14 And what we discussed in the first lecture is that circulating immune cells express
00:00:27.11 a receptor called alpha 4 beta 1 integrin that allows them to adhere to the blood vessel wall
00:00:32.04 and migrate into the tissue.
00:00:33.21 So, we developed a drug called Tysabri, which recognizes a... alpha 4 beta 1 integrin,
00:00:39.14 blocks the ability of cells to adhere to the blood vessel wall, and stops the ongoing inflammation.
00:00:46.22 Tysabri proved to be highly effective in a number of clinical trials.
00:00:51.00 So, from the Phase III studies we learned that Tysabri will inhibit the development
00:00:56.13 of new gadolinium... new gadolinium-enhancing lesions by more than 90%.
00:01:00.27 It reduces the risk of relapse by 68%.
00:01:04.03 And importantly, it reduces the progression of disability by about 50% as well.
00:01:11.08 So, this is the timeline of development for Tysabri.
00:01:14.26 We started... we first recognized the potential of alpha 4 integrin
00:01:18.15 as a target for multiple sclerosis in 1990.
00:01:21.19 And after 15 years and 10 years of clinical experience in 4,000 patients, the drug was
00:01:26.18 approved in 2004.
00:01:29.18 So, a few months after approval, everything changed for Tysabri.
00:01:34.23 In February of 2005, two... two patients in the MS trials were identified with
00:01:40.08 progressive multifocal leukoencephalopathy, or PML.
00:01:44.04 The FDA was notified immediately and natalizumab dosing was voluntarily suspended.
00:01:50.22 This was a big issue because there were 3,000 patients who were involved in clinical trials,
00:01:55.18 and also a number of patients were beginning to take the drug in the commercial setting.
00:02:00.07 At that point, we initiated a comprehensive safety evaluation, part of which then, in...
00:02:04.20 in March of 2005, a third case was identified in a Crohn's patient who originally was thought
00:02:09.24 to have had an astrocytoma.
00:02:11.20 But instead it was PML.
00:02:15.15 So, the questions we had in 2005 were many.
00:02:19.10 Umm... how is natalizumab associated with PML?
00:02:22.14 Who is at risk?
00:02:23.14 Can the risk be mitigated?
00:02:24.14 And how do we add... address the risk of death and disability in patients
00:02:29.07 who have developed PML?
00:02:30.28 So, this was the safety evaluation.
00:02:33.22 The primary objective was to determine if any patients had unrecognized PML or any other
00:02:40.08 atypical infections.
00:02:41.15 So, remember, we had seen these two patients develop PML only a week apart.
00:02:46.25 And this was after being in the clinic for ten years.
00:02:49.06 So, the concern was that this was just the tip of the iceberg, and that many patients
00:02:53.01 would... could potentially have this infection.
00:02:56.15 So, out of 3826 patients who were involved in the clinical trials, 91 of them...
00:03:02.24 91% of them voluntarily participated in this safety evaluation.
00:03:07.26 This involved about 3000 MRI scans, an analysis of almost 400 CSF samples trying to determine
00:03:15.08 the presence of JCV.
00:03:17.10 JC virus is the virus that causes PML, and so looking for its DNA in the CSF was a way
00:03:23.22 of determining if patients had PML.
00:03:27.13 All the data went to the independent adjudication committee and they determined that there were
00:03:32.20 no more additional cases of PML besides the three that had been previously identified.
00:03:38.13 So, we then submitted this information to the FDA.
00:03:42.22 And they held an advisory committee meeting.
00:03:45.01 So, an advisory committee is 12 independent experts from different fields.
00:03:50.11 So, there would be treating physicians, there would be medical ethicists there would be
00:03:54.23 people who were infectious disease experts, and also there was a patient who was...
00:03:59.17 a panelist who was an MS patient.
00:04:03.25 And so they heard the data.
00:04:06.01 And usually these... these meetings last for a day, but in this case it was extended to
00:04:11.28 two days, because there were so many MS patients who wanted to come and testify.
00:04:17.02 They had many messages that they wanted to deliver to the Advisory Committee,
00:04:21.01 but by and large the main message was, please let us make our own decision about what drugs
00:04:26.13 we take.
00:04:27.27 The idea was that the risk of developing multiple sclerosis was 1 in 1000.
00:04:32.06 The risk of developing PML at that time was also 1 in 1000.
00:04:35.19 And so they wanted to be able to make that choice.
00:04:38.17 The advisory committee listened very closely and they unanimously agreed to reintroduce
00:04:45.04 natalizumab.
00:04:46.04 However, it was reintroduced with a number of restrictions.
00:04:51.23 That month, the two clinical trials, the two Phase III clinical trials, were published
00:04:56.25 back-to-back, along with the results of the safety evaluation
00:05:00.03 in the New England Journal of Medicine.
00:05:02.10 So, Tysabri was put back on the market, but it came with a big black box warning in
00:05:07.16 its package insert.
00:05:09.05 And basically, it says that Tysabri is increased... is associated with an increased risk of PML,
00:05:16.05 which is an opportunistic viral infection in the brain that
00:05:18.22 usually leads to death or severe disability.
00:05:22.05 Also, risk factors associated with this could involve prior exposure to immunosuppressants,
00:05:27.19 so it was mandated that Tysabri be used in a monotherapy setting.
00:05:32.23 So, by itself.
00:05:34.24 Furthermore, patients were to be monitored very closely and Tysabri was to be withheld
00:05:39.22 at the... any idea or any indication that there was PML, which is particularly complicated
00:05:46.17 in multiple sclerosis patients, where you expect there to be neurological symptoms.
00:05:50.20 And so, distinguishing between MS and PML was one of the difficulties that was faced.
00:05:56.21 And also, most importantly was that Tysabri was only available through a highly restricted
00:06:01.16 program.
00:06:02.16 So, each vial of Tysabri was tracked to which patient received it.
00:06:08.04 And patients were only allowed to receive Tysabri if they had a confirmed diagnosis
00:06:12.24 of relapsing-remitting multiple sclerosis.
00:06:15.17 So, there was no off-label use of this drug, for example.
00:06:18.26 So, flash forward to 2013.
00:06:22.05 And this is the experience that we've had with Tysabri in the marketplace.
00:06:27.01 Overall, there's been 120,000 patients who have been exposed to Tysabri.
00:06:31.02 90,000 for more than a year, all the way down to 31,000 for four years.
00:06:38.02 And the overall incidence of PML is about 3.4 per 1000 patients.
00:06:43.24 Now, this is not distributed evenly throughout the... throughout the treatment period.
00:06:48.06 It really depends upon the duration of treatment.
00:06:50.14 So, in the first year, between effusions 1 and 12, almost no one gets PML.
00:06:55.23 It's a very, very low risk, or very, very low incidence.
00:06:59.00 However, in the second year, you begin to see PML come up -- so, 0.67 patients per 1000
00:07:04.27 will develop PML.
00:07:06.14 And then, by the third year, it comes out to about 2 patients per 1000.
00:07:10.07 And this is maintained in subsequent years.
00:07:13.11 Now, remember, this is a cumulative risk, however.
00:07:16.00 So, every year there's a risk of 2 in 1000 of developing PML.
00:07:19.22 And that's why the overall risk is 3.4.
00:07:24.11 So, we've learned that the risk of PML increases with longer duration of treatment.
00:07:29.15 There's also approximately a two-fold increase in patients who have been exposed to
00:07:33.28 prior immunosuppressant agents.
00:07:35.07 And as I said, the overall risk is 3.4 per 1000.
00:07:39.03 So, this is not good enough.
00:07:42.19 Patients having to face... patients who take Tysabri usually have very highly active disease.
00:07:49.15 And so they face the disability that will come with highly active disease or
00:07:56.03 they'll look at taking Tysabri, which has a significant benefit.
00:07:59.23 However, they also then face the risk of PML.
00:08:02.14 And having to go to bed every night thinking about, will I wake up with PML, is just not acceptable.
00:08:09.20 So, how can the risk of PML be mitigated?
00:08:14.19 Historically, PML was very rare.
00:08:17.20 And so there's a limited understanding.
00:08:19.21 And, in fact, before HIV, the only people who got PML were patients who were undergoing
00:08:26.14 bone marrow transplant or some other transplant, so they were profoundly immunosuppressed.
00:08:32.07 With HIV, however, it became much more common.
00:08:36.17 So, up to 50 per 1000...
00:08:38.07 50 patients per 1000 with HIV, or with AIDS, would develop PML.
00:08:47.08 Another issue with it is that there was no animal model, so it's very difficult to...
00:08:51.16 to track the natural history of how the virus leads to... to the disease, or how immunosuppression
00:08:58.12 can affect that.
00:08:59.19 It's also not easy to sample the brain, so there are... there are very few tissue banks
00:09:03.26 that would allow you to examine the virus in brain tissue.
00:09:07.10 There's no commercially available assay for JCV.
00:09:10.08 And, as I mentioned before, it's really difficult to be able to monitor PML and understand,
00:09:15.28 is it PML or is it MS?
00:09:18.17 So, Biogen and Elan undertook a number of initiatives to address this risk mitigation.
00:09:25.02 First of all, in terms of prediction and prevention, we looked at viral factors that could be contributing
00:09:30.14 to... to turning an infection into... into PML.
00:09:35.19 And this is something I'll talk about a bit later.
00:09:38.02 We considered patient factors such as genetics or their immune status.
00:09:42.12 It turns out it's very difficult to measure generic immune status in patients.
00:09:47.08 And then, in terms of managing patients who develop PML, it turns out with the HIV experience
00:09:53.09 that the best way to manage PML is to reconstitute the immune system.
00:09:57.12 So, with the advent of highly active antiretroviral therapy, patients were able to reconstitute
00:10:05.05 their immune system, HIV titers would go way down, and then, if they survived PML,
00:10:10.10 they could often then recover.
00:10:11.24 So, the same is true with Tysabri.
00:10:13.27 The idea is to take Tysabri off... to get Tysabri out of the bloodstream and allow
00:10:20.05 the immune system to reconstitute.
00:10:21.18 But then, another issue emerges, and that's a virus,
00:10:24.24 and this is immune reconstitution inflammatory syndrome,
00:10:28.12 where, once the immune system can get back into the brain, it recognizes
00:10:33.15 the virus and it responds very strongly, and actually causes a lot of CNS damage.
00:10:37.15 And this is actually a time when a lot of patients die because of the... the strong reaction.
00:10:43.16 So, it's important to manage IRIS.
00:10:46.12 So, PML is caused by the JC virus.
00:10:49.17 This is a polyomavirus or DNA virus.
00:10:53.08 Infection rates in the literature varied considerably, all the way from 35% to 85% of the population.
00:10:59.10 And once individuals are infected with JCV, they remain infected for life.
00:11:05.02 It can replicate in the kidney and is secreted in the urine.
00:11:08.24 And it can also cycle between active replication and periods of quiescence.
00:11:13.21 So, just looking for the virus in the urine isn't a good enough way of determining if
00:11:18.01 the patient really has been exposed.
00:11:21.23 It's thought that JCV undergoes mutation when it's replicating.
00:11:26.10 And... so that PML is always associated with mutations in the viral DNA regulatory regions.
00:11:33.08 And often, as I'll discuss, in the VP1 coat protein as well.
00:11:36.27 So, once the virus enters the brain, with immunosuppression it can then... the infection
00:11:42.16 can become meaningful, and it can... it can take off and cause PML.
00:11:47.26 In the case of Tysabri, this is probably because Tysabri is inhibiting immune cell infiltration
00:11:54.10 to... into the brain.
00:11:55.10 So, remember, Tysabri was designed to protect against the loss of myelin, which happens
00:12:01.01 in autoimmunity.
00:12:02.05 In this case, JC is infecting oligodendrocytes, which are the cells that make myelin.
00:12:07.20 So, the very cells that we're trying to protect with Tysabri in multiple sclerosis are the
00:12:12.09 ones that are vulnerable to infection with JC.
00:12:15.09 And... and I don't think that this is a coincidence.
00:12:18.28 So, this is the characteristics of JCV in healthy adults.
00:12:21.28 Umm... as I mentioned, it can replicate in the kidney and is excreted in the urine.
00:12:27.06 So, about 20-30% of adults will have JCV in their urine at any time.
00:12:32.14 It's rarely found the blood.
00:12:34.00 So, only 0.3-2% of adults, and even here it's not persistent -- it will go up and down.
00:12:40.18 In the case of PML, about 50% of patients have virus in the blood, but here it's always
00:12:45.15 wild-type virus.
00:12:46.17 There... there are never mutations that are found.
00:12:49.17 In contrast, in looking at viral isolates from the brain in PML, they always contain mutations,
00:12:55.23 particularly in the regulatory DNA regions of the virus.
00:12:58.17 Importantly, though, the... the strain of virus in the brain is always the same as that
00:13:03.24 that's found in the urine.
00:13:04.25 Now, there are many strains of JC virus throughout the world and they tend to have regional preferences.
00:13:11.03 However, again, in the brain it's always the same as in the urine.
00:13:14.11 And this is true also in the blood.
00:13:16.16 So, in PML, JCV is not always found there.
00:13:20.26 It may come up with prolonged infection.
00:13:23.13 But, when it is found, again, it's always the same strain as found in the brain as in
00:13:27.20 the urine.
00:13:28.20 So, these results suggest that the brain is not the primary site of infection for JCV.
00:13:33.10 It most likely is in the kidney or some other peripheral organ.
00:13:37.11 And then, with mutation, it somehow makes its way to the brain.
00:13:44.01 Now, this is work done by Leonid Gorelik at Biogen.
00:13:48.08 This is very nice work.
00:13:49.26 He's looking at the VP1 coat protein and found that, in looking at brain isolates of JCV,
00:13:57.25 about 80% of the viruses will contain a mutation in VP1.
00:14:02.06 VP1 is a sialic acid binding protein and it allows viruses to infect cells by binding
00:14:08.28 to sialic acid on the cell surface.
00:14:11.16 Looking in urine, none of the isolates had mutations in... in VP1.
00:14:17.03 And all of these mutations were involved in the sialic acid binding site.
00:14:21.00 So, it actually changes the ability of cells to infect cells... the ability... it changes
00:14:26.19 the ability of the virus to infect cells.
00:14:29.08 And therefore is changing cellular tropism.
00:14:33.08 This is an example of that.
00:14:34.23 Umm... so, what Leonid was able to do was to take mutated VP1 protein that had been
00:14:42.24 sequenced from viral isolates in the brain, and the... these proteins will self-assemble
00:14:47.13 into a viral-like particle.
00:14:49.09 So, it's a caspid... a capsid that looks umm... looks like a virus itself, but it has no DNA.
00:14:55.11 And then he was able to look at their ability to bind to the cell surface by FACS.
00:14:59.10 And what he found... that with wild-type sequence, you can see binding to the cell surface of
00:15:04.02 glial cells, which are from the brain, also binding to renal cells from the kidney,
00:15:08.06 and T cells.
00:15:09.22 However, looking at the viral isolate with the point mutation, it still binds normally
00:15:14.06 to the glial cells, but does not bind to renal cells or T cells or red blood cells.
00:15:20.13 So, this is a big difference.
00:15:22.24 And this has... this factors very heavily into a hypothesis of what may be happening
00:15:26.25 with PML infection.
00:15:28.20 So, the idea is that the virus, while it's replicating in the kidney, may mutate.
00:15:34.26 Now, the mutations have no selective advantage in the kidney, so when you look in the urine
00:15:40.05 there... there's a... they're.... if they're there, they're at very low levels and it's
00:15:43.24 impossible to detect.
00:15:45.08 However, if a... if a mutation does happen and it gets into the periphery, it no longer binds...
00:15:53.12 if it no longer binds red blood cells, then it may have greater access to tissues,
00:15:57.04 and this may be one way in which JCV is allowed to enter the brain.
00:16:00.18 And once in the brain, the virus can infect glial cells, and with immunosuppression or
00:16:06.11 with decreased immunosurveillance,
00:16:08.17 then the virus can take off and cause a full-blown infection.
00:16:13.15 Okay, so PML is likely caused by an interplay between a number of different factors.
00:16:18.15 If this square represents the total patient population, we already know that the
00:16:23.26 duration of treatment is an important risk factor.
00:16:25.16 So, the longer you've been on the drug, the more at risk you are for developing PML.
00:16:30.19 We also know that primary immunosuppression can play a role.
00:16:34.25 We suspect that other host factors can as well, such as the status of the immune system
00:16:40.07 or genetics.
00:16:41.07 But, by and large, the... the most important factor is clearly the presence of JCV itself.
00:16:46.27 If you don't have the JC virus, you cannot get PML.
00:16:50.17 So, that's very important.
00:16:52.22 We also know that viral mutations play a role in propagation of the disease.
00:16:57.22 And so, hopefully, by combining... by better understanding these different individual risk factors,
00:17:02.28 they could be combined into an algorithm to predict who is highest at risk for the
00:17:06.26 development of PML.
00:17:08.13 So, when looking at the virus, looking at the presence of the virus, I've already mentioned
00:17:13.08 that looking in the urine or the blood really is not very useful, because even in patients
00:17:17.21 with PML it's often not found there.
00:17:20.11 However, serum antibodies... a time-tested method for detecting infection.
00:17:26.06 HIV is a great example, for example.
00:17:29.10 People go in to umm... look at HIV... or, antibody status, to determine if they have
00:17:34.06 been infected with HIV.
00:17:35.18 So, it's good at detecting long-standing or recent infections.
00:17:39.10 It also is able to detect infections whether or not they are actively replicating, or they're
00:17:44.28 in quiescence.
00:17:46.12 Unfortunately, there are no commercial... or, there were none at the time,
00:17:50.01 commercialized standard... umm... standardized assays available.
00:17:54.00 Different labs have made their own assays and they all... basically, were not standardized
00:17:58.20 and gave very different results.
00:18:01.10 So, Biogen has a great department for developing assays, and they made an ELISA that was
00:18:09.03 very sensitive to detecting JCV.
00:18:11.01 So, it's pretty simple.
00:18:12.11 You take viral-like particles, that I discussed before.
00:18:16.03 Coat them on the bottom of a... of an assay well.
00:18:18.20 Then you expose the well to serum isolated from patients.
00:18:21.18 And if there are antibodies in the serum, they'll bind to the... to the viral-like particles
00:18:25.27 in the well.
00:18:26.27 And you can you detect the patient's antibodies with an anti-human IgG reagent.
00:18:31.12 They also then developed a secondary confirmation assay.
00:18:34.23 In this assay, the patient's serum is first incubated with virus in suspension.
00:18:39.05 And the idea is to try to compete... specifically compete any reactivity in the... than in the
00:18:44.18 primary ELISA.
00:18:46.08 And so, in this case, if you end up with a lower level of reactivity, you know that the
00:18:52.03 viral particles in suspension have competed antibodies and prevented their binding to
00:18:57.15 JCV viral-like particles in the plate.
00:19:00.13 However, if the signal has not changed, then that means that any binding that you're getting
00:19:04.20 is not specific.
00:19:05.22 And so it helps to determine the difference between being truly positive or negative.
00:19:11.05 So, when you apply the ELISA to a population, this is what you get.
00:19:16.09 Not a surprise.
00:19:17.09 You get a large spread.
00:19:18.12 And what does this really mean?
00:19:19.15 And how can you use this?
00:19:21.20 Well, typically, in assay development of this sort, you have a control negative population.
00:19:28.16 In this case, though, who isn't infected with JCV?
00:19:31.09 And that really is the question in the first place.
00:19:33.03 We don't know who does not carry the virus.
00:19:36.05 So, Biogen was very clever in their approach and they took patients who had JCV in their urine.
00:19:42.27 So, by definition, these people are clearly infected with the virus.
00:19:46.23 And so they could stand... they could serve as a positive control.
00:19:50.26 And so, when you look at the same data separated according to patients who had JCV in the urine
00:19:56.18 versus those that did not, you get a very clear separation in the populations.
00:20:02.17 A large spread that all... positive with... with... all could be considered positive with
00:20:09.27 virus in the urine.
00:20:11.03 And then, here, in... in viral-negative patients, again there's a big spread, and likely these
00:20:17.07 are patients who have been expected... who have been infected with the virus,
00:20:21.10 whereas these patients down here have not.
00:20:24.07 However, there's a lot in between.
00:20:26.17 And so, you can say that patients below this lower cut point, which is determined by
00:20:32.13 an algorithm is... are negative.
00:20:36.22 But the people who are here, right at the... at this level, they're indeterminate.
00:20:41.28 So, what can you say about their antibody status?
00:20:44.13 And this is where the two-step assay becomes of importance.
00:20:48.18 So, in the primary ELISA, 40% of patients scored as positive, 30% as negative,
00:20:54.16 and 30% were in that white circle as indeterminate.
00:20:58.11 With the confirmation test, then, it was found that 30%... these... these indeterminate about,
00:21:04.01 about 50/50 confirmed as positive -- in other words, they had specific antibodies against
00:21:08.18 JCV in their blood -- whereas 50% turned out as negative -- meaning that the reactivity
00:21:13.27 that was seen in the primary ELISA was nonspecific.
00:21:16.12 So, overall, that's about a 50/50 split in patients.
00:21:22.17 These are on MS patients who had been on natalizumab, so it's an appropriate demographic to be studying.
00:21:29.08 About 50% were positive and 50% were negative.
00:21:33.01 These results have held up over a number of different studies, both in the US and in Europe.
00:21:39.10 So, again, about a 50% positive rate -- somewhere between 50 and 60%.
00:21:44.22 However, you can also look at the same group of patients and follow them over time.
00:21:49.04 And what this graph shows is that being on natalizumab for zero all the way up to
00:21:55.02 four years, four or five years, does not increase the risk of being positive for the JC virus.
00:22:00.22 So, natalizumab is not causing JCV infection.
00:22:04.07 It's just allowing the emergence of PML when the virus makes its way to the brain.
00:22:08.15 So, we have this assay that looks robust and reproducible, but how do we know that it's meaningful?
00:22:15.01 The patient has PML.
00:22:16.01 In order for this assay to mean anything, it would have to be that this patient,
00:22:19.27 if they have been tested six months or two years before the onset of PML,
00:22:24.26 it would be important that they were positive in this test.
00:22:28.13 And so what we were able to do is identify 193 natalizumab patients who developed PML
00:22:36.18 and had blood samples that were stored 6 months to 15 years prior to the PML diagnosis.
00:22:42.19 And of these 193 patients, 191 were positive for the virus in this period of time before
00:22:50.26 6 months.
00:22:52.02 1 patient was negative at 9 months but positive at 6 and a half months.
00:22:57.00 And a second patient was negative at 15 months, but positive at 2 months.
00:23:01.21 So, it's great that these 2 patients ultimately were positive before the diagnosis of PML.
00:23:06.15 In fact, it's amazing that we even have these blood samples.
00:23:10.23 But it's... but it shows that there's a window of time, presumably between infection and
00:23:16.13 positivity, where patients are at risk.
00:23:19.11 And so, the best way of... and this is... is very much like HIV, where there's a
00:23:24.17 very dangerous window between infection with HIV and showing positive on a test.
00:23:29.17 So, there's precedent for it, but, in terms of using this as a... as a real, good
00:23:35.11 tool for risk stratification, it's important to have frequent testing somewhere between every
00:23:40.20 3 to 6 months.
00:23:42.23 So, as I said before, PML is caused by the interplay between a number of different factors.
00:23:48.26 And with the... an assay to determine the presence of JCV, that's a great foundation
00:23:54.17 for then building an algorithm to predict risk.
00:23:57.04 And this is what we've come up with so far.
00:23:59.15 So, a patient's antibody status is determined.
00:24:02.09 If they're positive and have been on the drug and... have had immunosuppressants and have
00:24:07.06 been on the drug for more than 2 years, then the risk of developing PML is 11 per 1000.
00:24:13.17 However, if a patient is negative, then regardless of how long they've been on the drug
00:24:19.11 or prior immunosuppressants, the risk is 0.1 in 1000.
00:24:22.15 And even that 0.1, as I mentioned before, may be due to this window between infection
00:24:28.07 and positivity.
00:24:31.05 So, what's next with Tysabri?
00:24:32.24 Well, obviously we want to continue to improve the JCV assay, so further refinement in this
00:24:39.21 and, for example, it... it... it may be possible that patients who have very low titers but
00:24:45.11 are positive could have a reduced risk until the virus levels change.
00:24:50.26 And that's just an idea at this point.
00:24:52.13 There's some preliminary data to support that, but that's something that is worth looking
00:24:56.16 into further.
00:24:58.17 Additional risk stratification tools, for example, if we could really understand who
00:25:01.26 was immunocompromised, that would be very, very helpful.
00:25:05.00 So, that's an ongoing area of interest.
00:25:08.02 And then, also, how to better manage PML once it occurs is also something that's very important.
00:25:15.22 With respect to other indications, these are other areas where Tysabri could be of benefit,
00:25:21.06 and this would be particularly true if we could better understand risk management.
00:25:25.08 So, Tysabri, for example, has been approved in the United States for treatment of Crohn's disease.
00:25:30.25 It isn't used very much for Crohn's because of the concern for PML and the fact that there
00:25:34.09 are other drugs available for Crohn's.
00:25:36.00 But, at... the better we understand risk management, the more of an option that that becomes.
00:25:40.21 There are also clinical trials underway for secondary progressive MS.
00:25:45.02 And we're also considering potential applications in stroke, acute cardiovascular indications,
00:25:52.28 head trauma, and even epilepsy, as well as others.
00:25:57.02 So, what have I learned with the many years of experience with Tysabri?
00:26:03.17 First of all, that drug discovery is hard.
00:26:05.25 Data really matters.
00:26:06.26 I mean, it always matters in science, but when you're developing a drug
00:26:10.12 data really matters.
00:26:12.07 And it has to be reproducible, it has to be robust, because it only gets more difficult
00:26:16.20 to prove efficacy in the clinic as you... as you advance there.
00:26:19.24 Umm... also, biology matters.
00:26:22.17 Throughout the entire development of a drug, it's important to maintain a focus on biology,
00:26:28.11 so that when side-effects or issues arise... the better you understand the mechanism of action,
00:26:33.11 the better that you can address what's going on.
00:26:36.26 A strong interface between R&D is critical.
00:26:40.27 So, when issues arise, level heads, robust discussion, appropriate egos and motivations
00:26:47.09 are... are critical.
00:26:49.00 So, always trying to remember that... you know, what is in the patients' best interests
00:26:54.02 should be the focus of the conversation.
00:26:56.03 Also, a connection with clinicians is just critical.
00:27:01.11 It helps... it helps me, people like me, who've spent their lives in the lab, to better understand
00:27:06.08 patient needs.
00:27:07.26 But an open dynamic research interface with clinicians is also very useful both ways.
00:27:14.07 I mean, we can... clinical samples from patients are so much more available than anything that
00:27:21.03 we would ever learn in an animal.
00:27:23.03 And likewise, data that we learned, that we can share back with the physician, is something
00:27:27.06 that is of value to him as well.
00:27:31.07 And then, last of all, science really can move from the bench to the bedside.
00:27:37.07 This, as I mentioned in my first talk, was something that really struck me the
00:27:41.14 first time that I saw a meaningful patient data.
00:27:44.12 And it's... it... it was never the same after that.
00:27:46.25 And so it's important to remember this on a day-to-day basis.
00:27:50.14 Because if it takes 15 years to develop a drug, you have to maintain focus on the fact
00:27:55.21 that this is a possibility.
00:27:57.22 So, there are many people involved in a project like Tysabri.
00:28:03.00 And there are many colleagues at Athena and Elan.
00:28:05.02 I'm just going to mention a few names.
00:28:07.03 Larry Fritz.
00:28:08.10 Allison Hulme, who led all the clinical trials for the early stage development of Tysabri.
00:28:14.05 Lars Ekman, who helped keep level heads, as I mentioned before.
00:28:18.05 And then in particular, I... at Biogen, I want to thank Al Sandrock, who helped me with
00:28:23.02 a number of the clinical slides.
00:28:26.05 And team members there who I interfaced with most directly, because they were involved
00:28:29.20 in research, were Susan Goelz and Leonid Gorelik.
00:28:32.05 Susan was important for making a strong interface with physicians,
00:28:36.11 and Leonid did all the viral work that I discussed.
00:28:38.28 Also, there were many, many clinical colleagues who helped us think through PML and provided
00:28:45.06 clinical samples.
00:28:46.21 And then, as I did in the first talk, I want to really give thanks to the many patients
00:28:51.06 who participated in these and other clinical studies.
00:28:53.22 So, thank you for your attention.

Videos in this Talk

Part 1: Immune Cell Migration to the CNS
Audience:
- Researcher
Part 2: Response to the Emergence of Progressive Multifocal Leukoencephalopathy
Audience:
- Researcher

Speaker: Ted Yednock

Total Duration: 1:04:19

Recorded: January 2014

All Talks in Human Disease

Talk Overview

Yednock relates the discovery and development, over 15 years, of the drug Tysabri, an alpha4 integrin antibody, as a treatment for multiple sclerosis.

In the first of his two talks, Ted Yednock begins with an overview of multiple sclerosis. He describes how, in MS, immune cells are able to transverse the wall of blood vessels and infiltrate the brain and central nervous system resulting in damage to the myelin surrounding neurons. Yednock and his colleagues hypothesized that by blocking the infiltration of immune cells into the brain, the progression of the disease might be slowed. They went on to identify alpha4 integrin as the molecule that mediates adhesion of immune cells to the blood vessel wall, and they found that an alpha4 integrin antibody (Natalizumab or Tysabri) could block infiltration of the blood cells into the brain in a animal model of MS. Yednock then details the clinical development of Tysabri through to its approval by the FDA in 2004.

Just a few months after Tysabri was approved for MS treatment, two patients developed progressive multifocal leukoencephalopathy (PML), a fatal or seriously debilitating disease. In his second talk, Yednock describes the response of medical and regulatory groups and researchers to this discovery and its impact on the treatment of MS patients with Natalizumab/Tysabri.

Speaker Bio

Ted Yednock

Dr. Yednock recently joined the Drug Advisory Board of the Myelin Repair Foundation, a nonprofit organization focused on the discovery and development of therapeutics for multiple sclerosis (MS). Previously Dr. Yednock was the Executive Vice President and Head of Global Research for Elan Pharmaceuticals and a scientist at Athena Neurosciences. Through his scientific work at… Continue Reading