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Session 7: Autoimmunity and Allergy

Transcript of Part 3: Discovery and Development of Natalizumab for the Treatment of Multiple Sclerosis

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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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