Hi, everybody. I think we're going to get started today. Thanks for coming out. I know it's busy in Midtown today and tomorrow. We appreciate everybody making the trip. We're talking about our ARC-520 product today, and just want to remind everybody that we will be making forward-looking statements within the meaning of the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. These statements are based on our current expectations and speak only as of the date hereof. Our actual results may differ materially and adversely from those expressed in any forward-looking statements as a result of various factors and uncertainties, including our ability to finance our operations, the future success of our scientific studies, our ability to successfully develop drug candidates, the timing for starting and completing clinical trials, rapid technological change in our markets, and the enforcement of our intellectual property rights.
Arrowhead Research Corporation's most recent annual report on Form 10-K and subsequent quarterly reports on Form 10-Q discuss some of the important risk factors that may affect our business, results of operations, and financial condition. We assume no obligation to update or revise forward-looking statements to reflect new events or circumstances. That out of the way, I just want to introduce the panelists for today's talk. We have Robert Gish, who is a consultant professor at Stanford Hospital and Medical Center. We have Robert Lanford, who is the director of the Southwest National Primate Research Center. We have Stephen Locarnini, who's the head of research and molecular development at the Victorian Infectious Diseases Reference Laboratory. Thanks to all of you for joining us today. It's a great panel, so we should have a good discussion.
For management, we have Chris Anzalone, our President and CEO, Dave Lewis, our Chief Scientific Officer, and Bruce Given, our Chief Operating Officer. We'll have a short introduction from Chris today to go over the data that we've presented in the press release and what we'll be talking about today. Dr. Gish will talk about the current consensus on HBV, what the field has generally believed about the disease. Dr. Lanford will introduce the study in chimpanzees that we've been conducting for the last little over a year. Dave Lewis will give some of the key findings from that study. Bruce Given will talk about our clinical program, the Heparc-2001 study, and some of the data that we've had along the way. Dr. Locarnini will talk about what this means to the field.
How does some of these data that we've generated in our clinical program and in our chimpanzee study, how does that challenge what the field has believed for some time about HBV? We'll have some closing remarks, and then we'll open the floor up to questions from the floor. With that, I will turn it over to Chris.
Thanks, Vincent. Thank you all for coming today. Thank you, Dr. Lanford, for coming from Texas, Dr. Gish, coming from California, Arizona, Nevada, and various other airports, and of course, Dr. Locarnini, for coming all the way from Australia. I think he got in at 1:30 last night or this morning. We are really excited to be here and to talk to you about some of the work we've been doing. We have generated a ton of data, necessarily, what we will be doing today is talking about top-line summary of some of those results. Stay tuned. We expect to provide additional data at scientific conferences and publications.
We have chimp data that will be presented at AASLD in two presentations, one poster, one oral, and we expect the clinical data to be presented at various conferences, as well as published in various journals. Let's start with a quick reminder of the proposed mode of action of ARC-520. In an untreated situation, naturally, the hepatitis B virus will get into an hepatocyte and do what any virus does. It will hijack the machinery of that cell and enable or cause that cell to produce new virus. Interestingly, what it also does is produce high quantities of free antigens or free proteins, notably S antigen or surface antigen. It's thought that that surface antigen, free surface antigen, immunosuppresses the host, and therefore is a strategy for the virus to keep from being cleared or controlled by the immune system.
ARC-520 is designed to interfere with all of that. It's designed to silence the entire HBV genome that's expressed by the cccDNA, what that means for us potentially is two things, I think. One is if we can turn down the level of circulating free S antigen, the theory goes that we could enable the immune system to come up, reconstitute itself, and control the virus. Second, and maybe a bit more subtly, but potentially equally important, is that we are designed to silence all of the gene products produced by cccDNA. To the extent that we can do that, we think that we can disrupt the virus over time and also potentially contribute to a functional cure. That's what we're trying to do with ARC-520. The treatment groups we'll be talking about today are as follows.
First, we have a chimp study that consisted of nine chimps that were first suppressed on NUCs and then given monthly doses of ARC-520, 6 to 11 doses of ARC-520. These animals all had biweekly blood samples, and we had several biopsies. A ton of data came in through this study over time. We also have 7 cohorts of patients in our clinical study. Four of these you know about. We've talked about these a lot in the past. These are all patients that are suppressed on NUCs. They are all E negative patients, we were looking at single doses of ARC-520 at 1 to 4 mgs per kg. The new cohorts that we have not yet talked about until today are as follows. Cohort 5 is on a background of NUC therapy. It is E positive patients, it's four milligrams per kilogram, again, one dose.
We have six patients, two patients on drug and two placebos. Cohort six is also on NUC therapy. These are E positive patients. They receive two doses of ARC-520 at two mgs per kg each, and those doses are separated by two weeks. Then cohort seven, which is really two subgroups. One is E positive patients and one is E negative patients, and these are not on a background of NUCs. These are NUC-naïve patients. If I were sitting in your seat, I think that I would have five questions for Arrowhead. One is ARC-520 well-tolerated? Is it safe? Second, what do you learn from the chimp study? Third, does the DPC platform work? Fourth, does ARC-520 work? Fifth, what are you going to do with ARC-520, and what's the outlook for 520? I'll go through all those briefly.
First is ARC-520 well-tolerated? We've now been in 84 humans. We've seen no AEs rated as serious or severe. We've seen no discontinuations due to AEs and no laboratory signs of end-organ tox. We also have data now from nine chimps, with between six and 11 monthly doses of ARC-520, we have seen no safety signals in any of the chimps. We think that ARC-520 has been well-tolerated, very well-tolerated in fact, to date. What did we learn from the chimp study? First, ARC-520 leads to deep S antigen knockdown. We have four E positive chimps. What we saw with them is a mean peak knockdown of 99% or two logs. We've got four E negative chimps, where we saw a mean peak knockdown of 81% or 0.7 log.
We've got one chimp that was a transitional chimp, was transitioning from E positive to E negative, that chimp had an 87.4% peak knockdown of S antigen, or about 0.9 logs. We've seen evidence of immune reactivation in one of our four E positive chimps. We look at the differences in S response of these chimps, we have concluded that the different responses are due to a decrease in cccDNA during the life cycle of the virus as well as a decrease in cccDNA during NUC therapy. That leaves significant S antigen production from integrated DNA. This is a new concept for the field, we think is important. We see deep knockdown with ARC-520 in chimps, we see a new paradigm for the life cycle of the virus. Does the platform work?
As I mentioned, our data suggests that integrated DNA becomes an increasingly important source of circulating S antigen as cccDNA is reduced. Therefore, to assess the platform activity, I think you really need to look at E antigen production or E antigen in circulation. E antigen is produced only by cccDNA, not by integrated DNA, ARC-520 is designed to silence expression of cccDNA. What did we find in humans? In a cohort of E positive patients receiving four mgs per kg of ARC-520, we saw a good knockdown of 92% mean max knockdown, or 1.2 logs, a maximum knockdown of 98%, or 1.7 logs of E antigen in these patients. Interestingly, we did not only knockdown E antigen, but we also knocked down core-related antigen, not only in E positive patients, but also in E negative patients.
We've got good data to suggest the platform is working well and consistently. We think that de-risks ARC-520 and also de-risks future candidates using the same DPC. Does ARC-520 work? Well, we reached 99% max knockdown, or 1.9 logs of S antigen after a single dose in a patient in cohort seven. We think that's the highest knockdown ever reported in a human using RNAi. Here's what we saw in cohort seven. Remember, these are e positive patients who are NUC-naïve, and we saw a mean max knockdown of about a log, and a maximum knockdown at 99%, or just under 2 logs. Now, keep in mind that this is just through day 15 of this cohort. We think that it's quite possible that this will continue to decrease, and we saw really good results in this cohort of patients.
Also keep in mind that we're not just knocking down S antigen. Remember the e antigen data and the core data, we should be disrupting this virus substantially. ARC-520, we think, is quite potent at silencing cccDNA expression. What's the outlook for ARC-520? Well, as we expected, I think that we will see a number of subpopulations within HBV that will respond differently to different treatments. We identified cccDNA and integrated DNA, or the ratio of those, as an important determinant of a subgroup. ARC-520 has been shown to be well-tolerated and deeply silences cccDNA, and we see NUC-naïve e positive patients as potentially the richest population of cccDNA. We think that's a very important target population for ARC-520. But the question is that a small slice of a very large market? We think the answer is no.
It's actually a very large market unto itself. For instance, in the U.S., roughly 95% of those thought to be chronically infected with hepatitis B are untreated, and 95% are NUC-naïve. Of those, it's thought that about half of them are e positive. Within Europe, it's thought that about 90% of chronic infections are untreated and therefore NUC-naïve, and about a third of those are e positive. Even so, we still think that ARC-520 is going to be powerful in other populations. We think it could be a good antiviral for both high and low cccDNA level patients for a few reasons. First, ARC-520 appears to deeply silence all cccDNA expression. As we talked about, we see good knockdown in e antigen and core antigen, and we think that will disrupt the virus, whether or not we are completely suppressing S antigen.
Second, the clinical data that we're talking about here is after a single dose. What we've seen in the chimps is an increase in knockdown over time, and we also believe that long-term disruption of the virus will have positive effects towards a functional cure. Third, it's unclear how much S antigen needs to be knocked down to achieve that functional cure, particularly when you're disrupting the virus. Even so, we have also expanded our HBV portfolio. We've nominated an additional candidate called ARC-521, and that now gives us 2 shots on goal. ARC-521 uses the same DPC as ARC-520, so we expect the same safety profile as we see in ARC-520. It's now been optimized to include integrant knockdown. It has been validated in chimps. You'll see later that we have seen multi-log knockdown with the integrated siRNAs.
We think it's a good complement for ARC-520, and we expect an IND or equivalent by the middle of 2016. We think that what we will be showing today will de-risk our entire program because of the tolerability we have seen in ARC-520 and because of the activity we've seen in ARC-520, not only for S antigen but for the other antigens produced by cccDNA, and we have just increased our ammunition to attack this difficult virus. With that, I will step aside and turn the mic over to Dr. Gish.
Morning, everybody. I'm here really wearing probably three hats. One of those is as a clinician. I practice, as you heard from Chris, in California. I have a number of clinics in Northern California, also in San Diego. I work with a group in San Jose that's got a large Vietnamese and Chinese practice. I also work in Arizona and Nevada. I have a very broad picture about liver disease in general and seeing hepatitis B in a variety of different communities. Another really important point is how do I think about hepatitis B today, and one of the things I think about every day is that the number two cause of cancer death in the world is liver cancer, and the number one cause of liver cancer is hepatitis B.
Come back to the U.S., the number one cause of cancer death in Vietnamese men is liver cancer. In my practice in San Jose, I'm seeing that every day. We're also thinking about hepatitis B in the U.S. differently now. We just had a paper published that estimates there's about 2 million infected individuals in the U.S. That was published in Hepatology just a few weeks ago. We really know this is a very important disease in our hepatology community. I also wear a hat of advocacy. I was in Glasgow about a week and a half ago for the World Hepatitis Summit. This is a collection of advocates, physicians, policymakers, government, WHO, and at that meeting, they are focused on eliminating viral hepatitis from the world by 2030. Now, eliminate is slightly different than eradicate.
Eliminate means we're going to take this down to a rare disease, and there's really two focuses. One is vaccine for hepatitis C, obviously treating and curing hepatitis C, but hepatitis B is testing and linkage to care. In my practice today, or in practice globally, what we really discuss with patients is taking a pill every day indefinitely. It's not quite forever because I'm an optimist. I'm from Kansas, thinking that we're going to have new treatments. We have a whole library of new drugs that are in development today. I know of at least 20 companies with about 30 new products that are in development that I think will lead to this next step, functional cure, which is S clearance.
I guess because I'm an optimist, I'm even thinking that the word real cure is probably down the road, maybe a decade away, but we may actually get there. What you're seeing on this slide is how we think in the clinic today. This first phase that you see on the left used to be called immune exhaustion, but we've changed the word slightly to immune trained. We think that the T and B cells that are key to clearing and controlling hepatitis B aren't tired. They're just distracted. They're trained to go in a different direction. They're trained not to attack those cells. We think that this patient population is now becoming quite small because of vaccine and aging population, and the majority of our patients are in this immune-active or immune clearance stage or this reactivation stage, which is really an e-negative disease.
It's a small subset that sit in that inactive carrier state. The reasoning is pretty simple. What we thought was a normal liver enzyme or normal ALT has changed dramatically. For a woman, an ALT over 20 is abnormal, and for a man, an ALT over 30 is abnormal. If you find elevated enzymes and a DNA level in the virus of over 2,000, we're talking about treating those individuals. That instead of 40% or maybe 50% of our practice is now probably 80% of our practice. Small subset are in this immune trained stage with high DNA and normal ALT, not yet thinking about that. The rest of these individuals are immune active or reactivation or e-negative disease.
We're also thinking e-negative is very different than e-positive because at least in e-positive, we can get to S antigen clearance at least in maybe 10% of patients with NUCs and maybe slightly higher with interferon therapy. Interferon's not being used much. In the U.S., it's a few percent of patients or providers that are using interferon for a couple of reasons, side effects, and we're missing a key test in the U.S. called quantitative surface antigen. In Europe, they have access to this test, quantitative surface antigen, which helps guide therapy either as a positive or negative predictor. Even in Europe, probably less than one in seven patients are getting interferon today, probably, again, because of side effects, and we're really only helping maybe one out of seven or one out of six patients with interferon long term. What do we need?
S clearance and DNA control. At least with a NUC, entecavir or tenofovir, you can take a pill every day, rare side effects, and you get DNA suppression, and we're changing outcomes. We're seeing much less liver failure. We think we're seeing less liver cancer, but those people are going to be taking those oral medications indefinitely. You're going to hear some very important information, which Chris has already hinted at. Our paradigm is changing. What you're going to hear today, we think, is going to have a huge impact on patient care and drug development. We think this data is quite interesting and provocative. That's today, and actually I'll even say yesterday because today is now the future with all this information. Drug development is going to go beyond NUCs and interferon.
We think, as with hepatitis C, you can attack this, control this virus, there's a huge shift towards hepatitis B now. WHO has made this a top priority finally. They have people focused on hepatitis B and policy. They have new guidelines that are focused heavily on hepatitis B. We think that hepatitis B is going to yield to an attack of combination therapy. Just like hepatitis C, one drug wasn't going to make it, right? One drug, suppression, you get resistance, you get treatment failure. Hepatitis B, we think, is even more complex than hepatitis C. Combination therapy is really going to be our key. This chronicity is a failure of the immune system to exert control, so we can either go after the immune system directly or indirectly by suppressing these viral infections.
Resolve acute infection is clearing S antigen. Those patients have a very active immune response. Near term, S clearance, DNA negativity, surface antibody positivity is our ideal world. Sterilizing cure, which means no DNA, I still think is a realistic possibility. There's some very special cases out there with hepatitis B where we actually think patients have cured themselves through some type, we think, of a dominant immune response. These antigens play a dominant role in immune suppression or immune control, this immune training that takes place. Yes, you can come in through the immune system, through the side door, or you suppress viral proteins and allow that immune system to be detrained or retrained to attack those viral infected cells. cccDNA is central but may not be the ultimate target.
We think that we've been a little distracted recently, thinking that that is the Holy Grail. We think that hepatitis B replication cycle and locations is multifactorial on how it trains. You're going to hear some very interesting information. Just to wrap things up, we're targeting hepatitis B through this immune system directed by, one, reducing antigenemia directly through a number of new technologies and methodologies. You can say, I'm going to go after the immune system with TLR7s, like with one drug that's in development, or checkpoint inhibitors. Super hot topic in cancer, including liver cancer, also hot topic in hepatitis B. There are some risks of even side effects like autoimmune disease with these, so we have to be very cautious. Then we may be able to change how we're killing cells or controlling cells or decreasing virus.
The cccDNA directed agents are logical. We're also thinking about entry inhibitors blocking uptake of virus, stopping capsid formation. Capsid masks the messenger RNA inside the liver cells and probably modulates the immune system in the hepatocyte itself. Then there may be some epigenetic control we can do with cccDNA as well. With those comments, where we are today, or now I'm going to say where we were yesterday, because today we're looking at a new future. I'm going to introduce Dr. Robert Lanford, who's the director of the SNPRC, and he'll be talking about the chimp facilities. Thank you very much.
Thank you, Bob, and good morning. I'm going to provide a fairly brief introduction to the research facility that conducted the chimpanzee trials that we're discussing today. Texas Biomedical Research Institute is a private, nonprofit research institute. I've been there for over 30 years performing basic research on hepatitis B and hepatitis C, as well as pre-clinical studies. The Southwest National Primate Research Center is part of TBRI. I'm the director of SNPRC. SNPRC is one of the seven NIH-funded national primate research centers. We work on a large diversity of human diseases and non-human primate models. To give you a brief background of the Texas Biomedical Research Institute, I mentioned that we're an independent, nonprofit research institute. We were founded in 1941. We began primate studies in 1957. Currently, we have about 350 employees.
70 of them are doctoral-level investigators, PhDs, MDs, and DVMs, and we average about 200 research projects ongoing at any time. We're on a 330-acre campus that has over 600,000 square feet of lab, animal, and support space. We operate under a $50 million a year budget that is primarily from federal grants and contracts, as well as support from pharmaceutical companies. We have about $120 million in our endowment, although recently the market's probably pushing that down a little bit. The major focus today would be on one of our four large primate colonies. We have baboon colonies, macaque colonies, marmoset colonies, but today, I want to focus on our chimpanzee colony. There's just several photographs here. The one on the left shows you an area that the chimpanzees are housed in called the playgrounds.
The photograph from the center is just a close-up of a few animals in the playground area. There are three adjacent playgrounds, and they have 24/7 indoor/outdoor access. The next on the right is what's called a Primadome. The size of this is misleading. If you look at that small dark spot at the bottom, that's a large chimpanzee. This is actually a very large geodesic dome. There are 12 of them in the complex. Each pair of two is interconnected so the chimpanzees can run from one dome to the next and interact between social groups. Our chimpanzee colony was initiated in 1967, and we immediately began translational research with pharmaceutical companies. Most notably, in the beginning, it was with Merck Laboratories developing the hepatitis B vaccine. The first vaccine was actually from contaminated human plasma that had been inactivated.
That was FDA approved, but surely, there were some concerns about the safety of this product, so the next vaccine that followed shortly after that was a recombinant product. It was one of the first recombinant products used in man. The first transmission of HIV to an animal model occurred in our chimpanzee colony when blood from an AIDS patient was transmitted to a chimpanzee. For many years, the chimpanzee was the only model for us to help understand what was going on with HIV and AIDS, but soon SIV was discovered, and the macaque model became the new model of the future. We only use chimpanzees when there is no other animal model available for that research.
The first transmission of non-A, non-B hepatitis to an animal model occurred in our chimpanzee colony as well as one other at about the same time, and this was from a transfusion patient. We now call non-A, non-B hepatitis hepatitis C. About 10 years after that first transmission, it was cloned from chimpanzee serum by Chiron Corporation, bringing about the era of HCV. We were the home for two of the NIH cooperative hepatitis C centers for over 15 years, conducting basic research on the mechanism of chronicity. Why do some patients resolve the illness, whereas others become persistent for life? What is a protective immune response? How does the virus interact with the host? During that same period of time, I was developing a program to look at preclinical trials for hepatitis C and hepatitis B therapeutics. We had over 20 sponsors over a 10-year period.
Multiple of our candidates entered into phase I and phase II clinical trials, and at least one of those, multiple components of one of those, became one of the FDA-approved cocktails for HCV cures. I want to mention our veterinary care program. We have a very high-quality animal care program to maintain healthy colonies. Healthy colonies are essential for good research. We have a high-quality veterinary and technical support program for research. There are eight veterinarians. Two are board-certified in experimental medicine, two are board-certified in pathology. We have an outstanding clinical and anatomical pathology program. We have a behavioral staff of nine individuals whose only job is to provide the enrichment and training for these animals. Three of them are dedicated to the chimpanzee area, and they are also involved in its assessment and intervention if there are any signs of abnormal behavior. AAALAC accreditation was renewed in 2015.
That is the highest standard for any animal care program, and it is a voluntary program that we submit to. I want to briefly show the design of this study. Nine chimps were used, as mentioned. Five of them were e antigen positive, four were e antigen negative. These animals had been chronically infected for many years with HBV, some for decades. Deep sequencing of the viral sequences and phylogenetic analysis points to variants in the virus. It is a human HBV with some variations that are typical of isolates that come from chimpanzees. We do not suspect that this has a clinical significance. We know that human HBV causes the same infection in chimpanzees. The reverse has never been tested, whether the chimpanzee virus does that in humans. Tissue and blood samples were assayed in my laboratory at Texas Biomedical Research, or some samples were sent out to specialized laboratories.
The efficacy readouts were the serum viral DNA levels by quantitative PCR assay, quantitative assays for the surface antigen and the e antigen by immunoassay or ELISA. Total liver HBV DNA was compared to cccDNA levels, again, by quantitative PCR assays. HBV RNA and host transcripts were quantified by RT-PCR assays, and there were additional readouts. Safety labs included clinical safety parameters that are standard, complete blood chemistries, complete blood counts and blood chemistry standardized. We have a hospital that has the same equipment running in human hospitals for these analyses. Most importantly, there are daily observations by the veterinary and technical and behavioral staff to make sure that animals on study are exhibiting normal behavior and normal feeding patterns. They are closely observed several times a day. With that, I want to turn the stage over to Dr. David Lewis, who will give some details of these studies.
Thank you, Robert. Good morning, everybody. As you said, I'm going to be presenting some of the results we had from these chimpanzee studies. I'm only going to be presenting some of the key data because, we're going to be presenting more details at the liver meeting in November with a poster as well as an oral presentation, as Chris previously mentioned. This is a slide showing the study design and the dosing and sampling schedule that we used in this study. We really based the design of this study on that which we used in the Heparc-2001 clinical trial in humans, in which patients enrolled had been on long-term NUC therapy. In this study, we had a lead-in period with NUCs that was only eight to 24 weeks to suppress viral replication.
We gave monthly doses of ARC-520 to these chimps, either at two, three, or four mg/kg. At the pre-study time points health check and regularly throughout the study, we took blood samples to monitor safety and efficacy, outlined by Dr. Lanford. We also took periodic liver biopsies. We took one at the pre-study time point again, and also periodically throughout the study in order to monitor different virological, immunological, and histological parameters. Let me tell you about the safety of ARC-520 in this multiple dose studies in chimps. We saw no sign of end organ toxicity. We saw no adverse changes in behavior, body weight, or food consumption in these animals, saying that ARC-520 was indeed very well tolerated in monthly dosing.
Interestingly, we did see some animals at the pre-study health check that had high or moderately high levels of transaminases at the baseline. These generally normalized under treatment, suggesting that the treatment actually moderated any viral induced liver damage. Another thing that we saw in one chimp was a ALT increase, which coincided with e antigen seroconversions, as well as signs of immune reaction. This is very interesting. e antigen seroconversion is considered a medical milestone in treating humans. We were able to capture a lot of data around this event in our chimps, which I think will shed light on how that process occurs in humans. We plan to present that data at a future conference.
This is the first data slide and shows you kind of at the beginning of the study, what the viral DNA levels were in the serum, in the graph on the left, and the S antigen levels in the serum in the graph on the right. We've divided both of these data sets according to the e antigen status of the chimps, because, as Chris mentioned, we saw differential responses to ARC-520 in these two populations. At the beginning of the study, we saw very high DNA levels in the e positive chimps, about 10 to the eighth or 10 to the ninth copies per ml. Much lower amount in the e antigen-negative chimps, where the levels were around the lower limit of quantification, or the LLOQ, as shown there.
After 8 to 24 weeks of NUC therapy, we saw a dramatic multi-log decrease in the viral DNA levels in the e antigen positive chimps, and also a drop to almost undetectable levels in the e antigen-negative chimps. This data that shows what happens during the NUC-only lead-in period is similar to what's seen in humans taking this type of NUC therapy. On the right, we have our S antigen levels. Again, at pre-study, we saw increased S antigen in e positive chimps compared to that in the e negative chimps, although the difference wasn't quite as large as we saw in the DNA titers. Similar to the experience in humans, under NUC therapy, these S antigen levels didn't really change. You can see that in ARC-520 day one.
NUCs, although they have a very large effect on lowering DNA levels in the serum, they really don't affect S antigen levels at all. This data shows you what happens to S antigen levels over the course of the study. This lead-in period that I've just been talking about with the nucleotide-only therapy, day minus 57 here on this graph to day one, which represents the first injection of ARC-520, you can see there's not much difference in S antigen levels. NUCs, again, don't affect S antigen levels in the blood. What you can see, even after the first dose of ARC-520, there's a dramatic drop in S antigen levels in these two groups of chimps, and that knockdown is sustained with subsequent monthly doses of ARC-520, and even gradually reduced even further.
At nadir, the knockdown that we saw in the e antigen-positive chimps was about 99%, or about 2.1 logs, and in e antigen-negative chimps, about 81%, or 0.7 logs. We were very excited about this data because it showed that ARC-520 could indeed dramatically lower the levels of S antigen in these animals. What we also saw, though, was, again, this kind of differential response between e antigen positive, which were highly responsive to ARC-520, and e antigen-negative chimps, which were still responses, but less so than in the e antigen-positive animals. We think we know the reasons for that now, and it has to do with the source DNA of the S antigen. I'll show you exactly what I mean by that and the evidence we have for that in the next few slides.
First, let me go over again the HBV life cycle, and mainly how its genome is replicated, because it's important to understand these different sources of DNA. Once HBV infects a hepatocyte here, it deposits its genome in the form of this relaxed circular, or RC DNA, into the nucleus, shown here. This RC DNA is converted into cccDNA, and the cccDNA, as we've heard, is really the template for all the viral mRNA production. It also makes the pregenomic RNA, which is used in viral DNA replication. This pregenomic RNA not only encodes some viral proteins, it can be translated, but it's also packaged in this viral capsid, and it's within this viral capsid that the RNA is reverse transcribed to DNA, and eventually forming this RC DNA in the capsid.
There's a separate replication product that's called double-stranded linear DNA, or DSL DNA, that's also produced. This DSL DNA is less than full genome length. It's replication defective because of that, it actually makes up about 10% of the replication products of HBV DNA replication. That's also packaged in this capsid. It's produced in this capsid. It can also be enveloped by S protein and exported from the hepatocyte into the bloodstream to make new viral particles. These capsids can also go directly back into the nucleus where they, again, deposit this RC DNA and this other separate replication product called DSL DNA. This DSL DNA, there's a fairly large body of literature out there that shows that this DSL DNA is actually the HBV DNA that integrates into the host genome. The DSL itself doesn't contribute to HBV replication.
It's dead for HBV replication, it can actually be carried into daughter cells of hepatocytes when they divide, which they do quite often in chronic HBV. It's propagated that way. We were interested in looking at these different forms of HBV DNA in the chimps, and perhaps they would shed light on some of this differential response that we're seeing between these two groups, these e positive and these e negative chimps. We did biopsies after this NUC lead-in period, prior to them receiving ARC-520. What we saw was pretty interesting. Most of the HBV DNA in the liver of e positive chimps was actually this normal cccDNA that we think of as being controlling for viral antigen production and making the pregenomic RNA in replication.
What we saw in the e antigen-negative chimps, though, was dramatically less of this cccDNA, about 500-fold times less than in the e positive chimps. I think what's even more interesting is that the cccDNA in this e-negative chimp population is just 5% of the HBV DNA. 95% of the HBV DNA in this chimp liver is not cccDNA. What is it? Well, we knew that on NUC therapy, the level of total HBV DNA in the liver didn't change. Whatever this form was, it wasn't dependent on viral replication. This immediately made us think that perhaps the greatest burden of HBV DNA in these e negative chimps is actually the integrated DNA. Here's a slide that kind of illustrates what we think a liver lobule might look in these two different population groups in these chimps.
We have e antigen-negative chimp hepatocytes on the left, and e antigen negative on the right. You see the hepatocytes here in yellow. I've drawn the nucleus here. You can see the cccDNA pointed out, and then integrated HBV DNA. In the e-positive chimps, we have a lot of cccDNA, and that is borne out by our data. Probably 95% or more of liver hepatocytes in these chimps are infected. We have very high viral titers, the cccDNA is always being replenished by this HBV replication. Not that there isn't integrated DNA in these chimps, it's just that it's a very small proportion in this particular population. On the other hand, in e-negative chimps, we have the reverse. We have very low amounts of cccDNA, and that makes sense because there's not much viral replication on there.
Viral DNA titers are very low, cccDNA is required for viral replication. We also have a large amount of total HBV DNA somewhere in there, and we think that's the integrated DNA. In e antigen negative, this shows that most of the DNA burden for HBV in e negative chimps is actually integrated DNA. Why is that important, and how does that relate to our differential response to ARC-520? We think is that this integrated DNA actually produces S antigen. Moreover, these transcripts that make S antigen from integrated DNA seem to be blind to ARC-520. Here's why. Once we get integration of this DSL DNA into the host chromosome, and that's shown here, you actually get deletions on the ends of the DSL DNA.
Those are shown by these dotted lines here at the end of this integrated HBV DNA. What that means is that some of the promoters for other viral antigens, shown here at the end, on the right end, can be missing in the DSL DNA. They're actually dislocated to the open reading frames that they control, except for S. S is in the middle of this integrated DNA. The reading frame is intact for S protein, and also the cis-regulatory elements that control transcription of the S mRNA are intact. Theoretically, you can make these transcripts, and you can make full-length S antigen protein. This goes against what was currently thought in the field that this integrated DNA is here, maybe it plays a role in cancer, liver cancer, but not really thought of as a source of viral antigens.
I think what we see is that, yes, it's theoretically possible. We have a high burden of integrated DNA in e antigen negative chimps. It's entirely possible that maybe this is actually a source of S antigen. Why when we give ARC-520 can't we knock down this source of S antigen? The answer is because where the ARC-520s lie. ARC-520 was designed against transcripts that are expressed from cccDNA. In the integrated DNA, those target sites are missing in the transcript because of the way that the integration occurs. This is why we see this lower level response to ARC-520 in e antigen negative chimps. e antigen negative chimps still have cccDNA, and we're able to knock that down just fine with ARC-520. We get deep knockdown in e antigen positive chimps, which have a high level of cccDNA.
In the e negatives, a greater proportion is actually produced from this integrated DNA, and we happen to be missing that with ARC-520. Another thing that we did to prove that we're actually getting expression from integrated DNA is to make another siRNA that targeted specifically these integrated transcripts and then inject e negative chimps with that, and the results are shown in this slide. Here we treated two e antigen negative chimps. Both of them had been on ARC-520 treatment for seven doses. At the end of that treatment, they were about 77% reduced in their S antigen production. After ARC-520, we gave them this siRNA that targeted integrated S protein. What we saw was all of a sudden, another further dramatic decrease in S antigen levels, down to 99.8% of baseline.
This actually represents going from 77% to 98%, about another two logs of knockdown by using this integrated targeted DNA. This, I think, is really good evidence, along with some of the other things that we have, that, yes, S antigen is produced from integrated DNA, which is previously thought never thought to occur, or if it did, in some very, very minor level. This really, I think, changed the way we interpreted a lot of things about our study, about our drug, and also about some of the clinical results. In conclusion, I want to reiterate that we did see robust, sustained, direct antiviral effect on S antigen production in all the chimps, e positive or e negative. e positives were the highest responders, and in our best chimp, we saw about 2.7 logs of knockdown after monthly dosing of ARC-520.
e antigen negative chimps, we also saw a good response, but not quite as big as we saw with the e positive chimps, up to about 0.9 logs. Importantly, all these chimps were very tolerant of ARC-520 injections. We saw no safety issues. I think from a biological, virological standpoint, I think what we really learned about HBV from this chimp study is that HBV integrated DNA is actually important. It can be important for the HBV life cycle. It may be important in maintaining chronicity. With that, I think I'll end and introduce Dr. Bruce Given, who's our Chief Operating Officer, and who will talk to you about some of the human clinical trial results that we've been getting.
Wants to talk to me for some reason here. Good morning, everybody. I am Bruce Given. I'm the Chief Operating Officer. I also head research and development and act as the Chief Medical Officer for the company as well, which sort of explains why I'm here speaking to you today. This is one of those rare opportunities in drug development where you get to get out on a frontier. The advantage of that is it's a lot of fun. You discover a lot of new things. The disadvantage is there's no road map, and there's just a wilderness. We've had a very interesting year as we've had the opportunity to combine information we had coming in from our clinical work with information coming from the chimp data, and it's been a bit of a travelogue.
We thought really the best way to present this today was to take you on that journey as we had experienced it. Chris has already said the main conclusions from the presentation, but there are a few things worth reiterating. Again, the trial design, the first four cohorts were in the e negative NUC-experienced patients. The fifth cohort was also NUC-experienced, but now e positive. We went ahead in these last three cohorts, essentially using four mgs per kg. The dose was well-tolerated. We thought we didn't want to miss anything by maybe taking a dose that was too low. We've just lost our projection here.
He's going to plug it in.
It's a bad time. A long cable.
You've done your airplane run. I might pull it out here.
For those of you at home, we're just plugging the projector back in. It'll be a moment here.
Make sure you put those disk packs back the right way. Now we're sort of playing airplanes.
Well, I'll go ahead and keep speaking while we wait for this to reboot. Cohort 5 was also a blinded cohort of six active and two placebo patients in E positives treated with 4 mgs per kg. NUC experienced, as I said. Many of you may remember that our chimpanzee that we presented at AASLD two years ago had two divided doses, a 2 mg per kg dose, and then two weeks later, a 3 mg per kg dose. We thought it might be worthwhile to go ahead and emulate that in patients as well. In this particular case, we felt that we had had enough placebo control data, we had seen enough safety and tolerability that we were comfortable going open label for cohort 6. Cohort 7 was our first foray into naive patients who had never been on NUCs or interferon.
Here we wanted to look at both E positives and E negatives, and we had six of each. If you go back where we were a year plus ago, DPCs had never before been in the clinic. It's easy to forget, but this was a new technology. We really did not know how to correlate all of our animal work with what we would expect in humans. We did have the experience with the one chimpanzee, which made us feel fairly comfortable that it was likely that 1 and 2 mgs per kg were going to be active doses, but we didn't know that. It's easy to forget how we were out on that frontier. We had good safety and tolerability in healthy volunteers through 2 mgs per kg.
Recognizing that we might want to go higher, we did keep the normal volunteer study open and active so that we could go higher if we thought that was worthwhile. You'll recognize this data on the slide from AASLD last year. It's a little bit different. Some of you will recall we had that strange data point in the placebo group at day 57, which now you no longer see. One of the things we learned in the first two cohorts was that in measuring surface antigen on a visit-by-visit basis, there was enough inter-assay, between-assay variability that it really, at times, created some issues for us in interpretation.
What we now do is we routinely, after we finish a complete cohort, we analyze all the samples in a single assay for all the patients, and that really smooths the data by taking away this inter-assay variability and gives us cleaner data like this. We had this data and our interpretation of that data, looking at it, there was a dose response between one and two mg per kg. Our experience in all of our animal data across multiple genes and actually dating back to before we acquired the technology from Roche, indicated to us that we thought we were at the bottom of the dose response curve. Because we have endosomal escape as a key feature of our platform, we expect a steep dose response curve. We thought three or four mg per kg were probably going to be at the top.
We went ahead and dosed in normal volunteers up to four mg per kg, and we're happy to see that it didn't really look like the safety profile changed at all to our eye. Because of that, we added the three and four mg per kg cohorts that the street was well aware of. Were still in e-negative patients that had been chronically NUC- exposed. To our surprise, the three and four mg per kg cohorts didn't look all that different from the two mg per kg cohort. The peak knockdown tended to be around 60%, which was not what we had expected. The other thing that we saw that surprised us a bit was we actually saw two patterns of response in S antigen.
The one pattern, and this patient is actually from cohort 5, but it's a particularly clean figure that shows you what I want to say here, that the first pattern is what we call primary responders. We know from animal studies, for instance, that if we give our product to an animal, within one to two hours we have hit the liver, we've gotten into the endosome, we've escaped the endosome, we are in the cytoplasm and starting to knock down the mRNAs of interest. It's very fast. Not surprising when you look at this, by day 3, we already have very substantial knockdown. This profile is, I think, emblematic of something else in the primary responders. You see that the peak effect is down around day 21, day 22, day 29. Three to four weeks with S antigen.
S antigen has fairly complex kinetics that we think we understand a bit. We could talk about if anybody cares, but that's usually where we peak. We start have a nice gradual increase back up. As we noted back at AASLD last year, one of the big surprises to us was how long the knockdown was. Again, you see that in this particular example. We also saw this late response and these late responders can look a little different. Not all of them have dropped this much. You see that for the first several weeks, the S antigen is bouncing up around basically baseline. It could be higher, it could be lower, but it's basically bouncing around baseline. After three to four weeks, we see this decline.
We've actually now seen this pattern in 25%-30% of the patients, and we've seen it in all of the cohorts. It seems to be a real phenomenon. That was one of the things that had surprised us. Meanwhile, we started to get the data from the chimp study. The chimp study took a while to get going. There's a lot of care taken in the planning of these and the institutional reviews are very strict and careful. The chimp studies took a while to get going, and we had the NUC lead-in period, but we started to see data right around the time that we were also looking at this clinical data from the first cohorts, and we saw this difference between the e positive and the e negative chimps.
That really pointed us toward wanting to test ARC-520 in e positive patients. That's where we went with the 4 mg per kg, either in a single dose or the divided dose cohorts 5 and 6. We're again on a background of chronic entecavir. This was very important for us because it allowed us to not only look at S antigen, but now we could look at e antigen. This was really before we quite understood the cccDNA part of this story, but it did give us a very different antigen to look at. This was really very exciting data when it came in because all of the patients that responded all had primary responses. They all peaked at day 8. e antigen has a different kinetic profile than S.
Each of the patients looked the same, and it was quite a deep response. The worst responder was 83%, which is 0.8 logs. The best responder was 1.7 logs or 98%. This was after a single dose. This was really powerful data to us because we confirmed all at once we were delivering to the hepatocyte, we were getting into the endosome, we were getting out of the endosome and into the cytoplasm. Those were all questions for us since this was our first time that we had been in humans and we weren't seeing what we had expected to see with respect to surface antigen up until this time. It validated the technology for us, which was very important. Subsequently, actually just in the last month, we had the opportunity to test a different antigen assay, and this is the core-related antigen assay.
It's not very commonly used. The site that the antibody sees is in core, but it also is in e antigen, and it also is in some e antigen metabolites. It's an assay that's not as specific as e antigen or S antigen. What's interesting about it is that it's something you can actually look at in e negative patients as well as e positive patients. We just got those results within the last 30 days or so, and core looks like e antigen. Everybody has the immediate response, and the reductions are around a mean nadir of around a log, et cetera. It really confirmed that even in the e antigen patients, we were getting, again, that deep knockdown. We were getting good delivery to the cytoplasm. It's very important to us.
We actually feel it generalizes in all likelihood to ARC-AAT and ARC-F12 as well, and any other RNAi programs that use this same delivery mechanism. Interestingly enough, the S antigen reductions in the e-positive NUC-experienced patients were somewhat better than we had seen in the e-negatives, but they weren't the dramatic difference that we had seen in the chimps. We saw a best knockdown in cohort 5 of about 80%, and the mean knockdown in the primary responders was the mean nadir was around 60%. Again, we had a couple of these late responders, which you see the average of these couple late responders here in green. We were faced with a conundrum here. We had the e antigen results that clearly validated the drug.
From the e antigen results, we knew that ARC-520 was doing what it was designed to do. The S antigen was not behaving the way we thought it would. We also had this issue of these two distinct patterns, which was really novel. Once again, the chimp data comes and gives us insights. The biopsy data was sort of the latest data that started to come out of the chimp program, and that's what started to really teach us about this question of cccDNA versus integrated DNA. In addition, right about this same time, we started to see some things in the academic space from human biopsy data in the entecavir world that was actually corroborating for us what we were seeing in the chimps and in our human studies.
What was found, as Dave just pointed out, is that e antigen loss itself is associated with sharp cccDNA reductions, but so too is NUC treatment. In fact, the notion that had been out there that cccDNA is dominating throughout the viral life cycle is starting to come under question. Integrated DNA really emerged as an unexpected source of S antigen production for us. As Dave mentioned, ARC-520 was optimized to silence expression from cccDNA. We had used the mouse plasmid model, which used a full-length DNA. Essentially, it was a model for cccDNA. As we started to think about integrated DNA, we knew that it was unlikely that we would silence most of those transcripts. What did this mean for us as we looked back on cohorts 1 and 6? Well, we knew how long our patients had been on entecavir.
The shortest had been on for a couple years, the longest for eight years, and the average was five years. C. Lai had actually put out a poster at last year's AASLD, where they had biopsied entecavir patients at baseline after something like five or six years of entecavir, then at 12 years, I think. Based on his poster, the prediction would be that if we had biopsied our patients, the cccDNA would either be undetectable or very low level. In fact, this data of C. Lai's that maybe had violated conventional wisdom that NUCs don't really affect cccDNA, we were finding data that sort of supported the notion that conventional wisdom was wrong. We developed a new hypothesis, and our hypothesis was that integrated DNA was placing a floor under S antigen.
In these chronically NUC-treated patients, especially in the e-negative patients, it was really quite a substantial part of the total surface antigen in these patients. As we thought about the chimps, and we tried to understand why the chimps had a much larger differential in their surface antigen, some of these chimps had occasionally seen a short-term exposure to another experimental agent. For the most part, they were more like naive patients than they would be like chronically treated patients. We actually viewed them as analogous to naive patients. This is what led us to do the naive cohort 7. We wanted to do both e-negative and e-positive because we really thought there was a good chance that they were going to look like the chimps.
We had further theorized that it was the e-positives that would give us the best result. As Chris said, this data is very fresh. The last patient in this cohort was treated last week. Most of these patients, we only have maybe 15 days of data. Some patients we don't even have any data yet. They received a single dose of four mgs per kg, and you could see here in the e-positive, we have quite a good reduction. We have a mean reduction at day 15 of one log. If these patients behave like we saw in the rest of the patients, the nadir is probably going to occur somewhere around day 22-day 29. We don't even think we're all the way at the nadir. One of our earliest patients has already achieved a maximum knockdown of 1.9 logs or 99%.
We find this data very exciting. The e-negative data is not up here yet because it's more complex. It looks like we have a couple of these late responders in the e-negative group. We have one responder that is better than any of the e-negative NUC-experienced patients. As we would expect, they're less impacted. That data, hopefully, we'll be able to see that at AASLD and people will be able to look at it. The e-positive data was very much confirmatory of what we expected. What all this has told us, first of all, it's easy to forget, but we now have experience in 84 humans. That's 48 patients and 36 normal volunteers who have received ARC-520. That's actually a pretty nice database. That's a single-dose database. We have these nine chimps that have received chronic ARC-520.
As Chris said, we've been really gratified to see that we really haven't encountered anything untoward yet that was concerning to us from a safety perspective. We now know that ARC-520 can produce very deep and sustained knockdown of cccDNA derived mRNAs and proteins. e antigen falls very nicely. Core antigen falls very nicely. Surface antigen is also deeply reduced in those patients that are cccDNA driven. The technology works. As Chris said, that 1.9 log reduction that we've seen in this e-positive patient, to our knowledge, is the highest reported single-dose knockdown ever seen with RNAi across any platform in any disease state. We're not just talking HBV. We don't think anybody has ever achieved that before. It says a lot about what this platform is capable of. Now a brief update on where that leaves us with our clinical program.
We've been talking about the MONARCH study over the last few months. This is our study that was designed after the Pharmasset work that gave us HCV cures with Sovaldi. It's an open architecture combination approach trying to find the best recipe or recipes to start giving us real functional cures in hepatitis B. Not surprisingly with all this, that study is now designed in its first few cohorts to enroll naive patients. Of course, we expect a deep knockdown of all the gene products, and we're really looking forward to that. We ought to be able to enroll this study because at this point in the major developed markets, only about 15% of diagnosed HBV, we're not even talking about prevalent, we're just talking about patients that are diagnosed, only about 15% are being treated.
We think we have a pretty good population to go after to do this study. We've been talking for over a year about the 2002 and 2003 programs. 2002 are E negative on background NUC, 2003 E positives on chronic NUCs. These are more akin to the cohorts one through five. It's an interesting question of what's going to happen here, because if the S hypothesis was all that mattered, and if all that matters is S reduction, one might anticipate that we won't see functional cures here, or we won't see many. We just don't know if we can count on that because of all these effects we're having on the other viral proteins, pregenomic RNA, and everything else. We think it's important to do this and to carry out this test, and I don't want to predict one way or the other what may happen.
We have made one change, though, that is at the extension, we're going to enrich the extension. We're only going to take the patients into the extension that have at least a 70% reduction in surface antigen at the end of the four doses in 2002/2003, in the belief that we're certainly not hurting ourselves by doing that. We do want to understand whether functional cures are going to be seen in this population. We also added an additional product to the program, as Chris said. We took our very best trigger from ARC-520, and we combined it with our very best trigger against these integrated DNA sites to produce a product that we're calling ARC-521. This product is currently in GMP manufacturing. We expect to go into GLP tox this fall.
Assuming everything goes well, we expect to be filing to commence clinical studies around the middle of next year, in about nine months or so. We do expect ARC-521, if all of these theories are correct about what we've seen in our program to date, including the chimps, we expect ARC-521 to be more active against surface antigen that's derived from these integrated DNAs. It's a nice complementary program to ARC-520, and it's going to really be fascinating to see what happens over these next couple of years with 520 and 521 in the clinic. With that, I'll turn things over to Stephen Locarnini.
Thanks, Bruce. Good morning. Thanks, Bruce, and thanks, colleagues. It's a great pleasure to come up and share and discuss some of this exciting data that we're hearing this morning. My job is to, I guess, compare and contrast what we used to think about hepatitis B virus and what we now think about hepatitis B virus in the context of its replication, and therefore, in the context of its treatment. I'm actually from Melbourne, from the Victorian Infectious Diseases Reference Laboratory. I'm a medical virologist, and like Bob, also an advocate in hepatitis B. I now work, and I wish to thank America because if it wasn't for your world financial crisis some years ago, then my government would not have invested in the Doherty Institute.
To create infrastructure and a growing economy, the Doherty Institute, which was named after Peter Doherty, who won the Nobel Prize for immunology, is named and is actually a living patron in our center. It would not have been possible. I'm personally grateful. The Doherty Institute for Infection and Immunity is a partnership between the University of Melbourne, the premier university in Australia, and The Royal Melbourne Hospital, the premier clinical teaching hospital in Australia. It's located in the Parkville Healthcare Center, where the Walter and Eliza Hall Institute of Medical Research is also located, and as is the pharmacy and the center. It really is integrating infectious diseases research, teaching, and diagnostic and public health capability. We don't have a CDC, the Doherty's role is also to sort of provide resources and activities for outbreak investigation. My lab is the Victorian Infectious Diseases Reference Lab.
We have a clinical service, the VIDS. We have an infection control system with VICNISS, the DMI, the university department, and the microbiological diagnostic unit. We've been operational for just over 18 months. It's a very exciting time for us, and that's by way of background. As I said, I think the feeling that you're getting from the presentations this morning is that really, it's an exciting time in hepatitis B. The hepatitis B science has improved dramatically in the last 12 to 18 months, a significant reason for that is the impact of these studies that we're hearing this morning are having on how key opinion leaders are thinking in the field. I'll just see if this works. I won't touch that just in case something goes off.
This life cycle or the replication that's shown for you here, let's see if the button works. No, it doesn't. Is typically what we would always see in the textbooks, what I would write reviews on about the hepatitis B virus. It's e antigen positive. This is how it replicates. It infects the hepatocyte, comes in, its genomic DNA that you see on the left is converted into the so-called cccDNA, key replicative intermediate in the nucleus. From that, host cell enzymes make transcripts that are reverse transcribed, then you see the RC on the lower right corner. The critical pathway that was really planned for was that these minichromosomes or the cccDNA, which doesn't self-replicate, is replenished from this pool of replicating genomic DNA through the intracellular conversion pathway. That was what we thought was the typical replication process of hepatitis B.
Therefore, if we inhibited reverse transcription, we would dry up the intracellular conversion pathway, turn the newly replenished pool off, and eventually the mini chromosomes might just disappear. Well, that was what we hoped anyway. Really, that was pretty naive view in the '80s and '90s and heralded in what I call the nucleoside/nucleotide analog era. This was how we were thinking just a few years ago. The other key part of hepatitis B pathogenesis is surface antigen. Really, one of the things that took my interest with the ARC-520 approach was that they were prepared to address the issue of surface antigen. Now, just to give you an idea about what this thing is, I've got a little picture at the top there of what we see in the blood of a chronically infected individual, huge amounts of surface antigen.
The surface antigen is a subviral protein, as shown by that yellow circle, that really is produced in enormous excess of the virus itself. A thousandfold, sometimes 10,000-fold more. It circulates in the blood at concentrations that are just unbelievable. Almost 400 to 500 micrograms per mil. Now, if you do your sums, that's actually 1% of total serum protein. Now, because of that, Blumberg, who got the Nobel Prize for discovering hepatitis B surface antigen, used that as the first-generation vaccine, purified it out, and that was actually exploited by us or by biotechnology to make the first-generation vaccine. As you heard from Bob and others this morning, because of risks of AIDS and CJD and other factors, we moved over to recombinant very quickly. Let me just sort of linger a little bit on this surface protein. It's a unique protein in the database.
There is nothing like this protein that I have ever seen or any other biochemist has seen. It's got this unique conformational epitope, eight cysteines and eight prolines in the space of about 60 amino acids, four transmembrane domains. This guy is really different, and it's associated with an increased risk of liver cancer. Now, it obviously plays a key role of what we've learned in hepatitis B persistence. It suppresses both innate and adaptive responses. Really existing antiviral therapy, except for a small subset of patients treated with interferon, the surface antigens is basically not really affected. That's why I think the ARC-520 was really important in terms of trying to grapple with the surface antigen question. Now, the other question that we're going to think a little bit about this morning is the so-called cccDNA question. The question of CCC, what is cccDNA?
It's a very catchy little term. Some people call it 3C DNA, C3 DNA, or cccDNA. What in the hell is this thing? I spent a bit of my life on it some years ago and found out it's a bit like beads on a string. It's a transcriptional template of the virus, but it's not just a single entity, and this is what this slide is trying to tell you. It's not just a single target in a concept of actually what if we tried to eliminate or surgically strike, remove this molecule. It's not one but 21 different topoisomers. In the lower left-hand corner, I've got for you what we have been able to work out, what the structure of the mini chromosome is. You can see there from naught to 20, it's like a boomerang.
The boomerang is actually, we call these the boomerang gels, and it exists as an intense population or at the edge of the boomerang at 10, then at the lower right-hand version of that, another 20. It's clustered into these sort of beads-on-a-string structure, the closed compacted form, a low replication phenotype, and doesn't replicate much. The open form, B1, B2, these are half chromatinized. These are transcriptionally active and result in high viremia. I think that I know that there's a lot of discussion about getting a cccDNA magic bullet. The cccDNA magic bullet has got to not just address one particular topoisomer, but the other 20 as well. That's what we'll try and sort of, as we become more sophisticated in our understanding of hepatitis B science, what we need to get to.
As I think Bob has discussed this morning, and also Dave, we've realized that viral products are critical as well as host products in maintaining the integrity and the transcription ability of this cccDNA. On the left, I've got what we call a high replication phenotype, where we know the X protein of hepatitis B is a key component, drives transcription, blocks the methylation complex. We know that in both structures, in low and high replication phenotype, the core protein is a key component in terms of sort of maintaining the integrity of the minichromosome. A sort of corollary would be that if we knocked out X and knocked down core, this would result in a reduction in the transcriptional activity of the cccDNA or minichromosome. Indeed, that's been what we think is the case. Here we are in sort of September 2015.
We had a pretty good understanding of what hepatitis B virus was like and how it replicated. We had good clinical practice guidelines about how to treat hepatitis B and control it, but we had really no way in terms of actually moving beyond that, what I call a roadblock that we'd come into hepatitis B. We'd known about integrated DNA for quite a while, but we had really dismissed it. In my initial sort of replication life cycle that I presented to you, I did not talk about integrated DNA. If you look at the textbooks, we don't talk about it. We sort of don't, oh, well, we know it's associated with liver cancer. But we've now realized that it plays a key role not only in liver cancer but also in terms of another source of surface antigen.
There's been minimal attention paid to it as a source of messenger RNA transcription or a critical component of chronic persistence. Even at baseline, as you heard this morning from Dave on the e antigen-negative chimps, they were below the lower limit of detection for cccDNA in hepatocytes. We've tried to sort of use the detection of surface antigen as a surrogate measure of the amount of cccDNA in the liver. We can't go around biopsying humans every sort of six to 12 months just to get a feel for what's happening in their liver. We've tried to use surrogate markers in the peripheral compartment, like quantitative S, to give us a measure for that. As we also heard this morning, the dogma was that the nucleoside/nucleotide analogs did not really affect the cccDNA levels.
As we've gone out into year five and year seven and 12 of these long-term monotherapy treatment programs, we're now starting to see from certainly our Asian experience that there are indeed some reductions in cccDNA with the conventional therapy. This, as I said, really allows us to rethink the role of all of these various intermediates that we've known hung around and are important in our understanding. What does it really mean in terms of chronic hepatitis B? Bruce talked about what would the Asian experience now in terms of what happens with long-term nucleoside/nucleotide analog therapy. Here's my 2D boomerang gel showing the minichromosomes with 21 minichromosomes of HBV. On the upper left is that life cycle that I introduced to you early.
What we've realized is that when we introduce entecavir or tenofovir in our patients, they don't get resistance, and they take all their pills every day, that over time, the cccDNA pool drops. It drops by about 50%, and it drops, we believe, in that group of minichromosomes shown for you in that little elbow, those fully transcriptionally active molecules that are making indeed hepatitis B products. That's it. It still leaves for you on the, if you look at the 20, the 18, 19, and 20, they're still there. They're the guys that hang around. They're the guys that when we stop antiviral therapy, bang, back comes hepatitis B. If we measure the total amount of minichromosomes or cccDNA on long-term NUC-treated patients, yes, we do see a 50% drop.
What have we now learned and now started to appreciate about integrated HBV DNA? If I had to sit down and, say, update the textbook on hepatitis B virology in 2015, this is what we would do, is that there's been a big shift in our clinics. We see a much more significant number of e antigen negative patients. E antigen negative patients are shown for you by that black arrow in the middle of the diagram. This is the e antigen. The virus mutates and drops off the e antigen. It's an accessory protein. It's not essential for replication. The phenotype of the disease that's caused by e antigen negative viruses obviously is e antigen negativity.
What's different about this replication diagram compared to the previous one is that just below the intracellular conversion pathway, we're now seeing a failure to translocate replicative intermediates, and we see this DSL DNA. The DSL DNA is in your lower left-hand corner. This double-stranded linear DNA is a key replicative intermediate precursor for integration. As Dave pointed out, 10%-15% of the intrahepatic DNA is thought to be this particular replicative intermediate, not all of the relaxed circular DNA that we've traditionally regarded. This floats around, and as the cell divides, it's promotorily integrated into the host itself. Viral integration is now a key part of the whole process of HBV replication. Now in the top left-hand part of that figure, you see that we've now got not one, but two sources of surface antigen.
We've got surface antigen coming from the episome, from the cccDNA molecule, the traditional life cycle of the virus. We've now got hepatitis B surface antigen coming from viral integrants as a consequence of the DSL. I think that as we shift from e antigen positive to e antigen negative over the natural history of chronic hepatitis B that Bob Gish talked about earlier, we're seeing much more frequent, this big shift in replication strategy from a productive to a more restricted replication cycle. Now we have two sources of surface antigen to address, and this is really critical in terms of interfering in a positive way and blocking the adverse effects of the natural history of chronic hepatitis B. How does all that fit into RNAi?
In addition to surface antigen, we know that ARC-520 is expected to have other important antiviral effects, both on the e antigen, the core antigen, and the X antigen. Now, I showed in an earlier slide of the minichromosome how critical the core antigen and, in particular, the X antigen are on the epigenetic regulation of the viral minichromosome or the cccDNA. I regard ARC-520 as a selective and yet broadly active, broadly based antiviral agent. That is, I think, really a plus of the technology and the mechanism of action of the RNAi. I think the fact that the e antigen is going down so dramatically as well as what Bruce showed for you in the clinical studies is really breathtaking. To me, as a virologist, I was staggered by some of those sort of dose-response effects with the ARC-520.
I think that what the data is now sort of teasing us to understand is these late responders for surface antigen. I think that it may very well be that a reasonable hypothesis that the ARC-520 is affecting the core protein and the X antigen, which are the epigenetic regulators of the viral minichromosome. Sorry, I'm going the wrong way. The way that the minichromosome transcribes is directly knocked out by the ARC-520. This all raises some very interesting questions in terms of how we go forward in terms of our drug development in hepatitis B. Will the capsid inhibitors have an impact in the context of NUC effects on cccDNA?
If already the long-term entecavir tenofovir treatment patients have got their maximum reduction of 50% of cccDNA, what role is left for the capsid inhibitors that would be required to or as well as packaging would affect transcriptional activity of the minichromosome as well? As I said, we're trying to develop surrogate assays to measure the effect of cccDNA drops in the liver in relationship to e antigen negative patients especially, looking at the peripheral compartment for those biomarkers. Now we're asking the question, as a consequence of the ARC-520 data, do we also need to get rid of integrated DNA? If the answer is yes, how will we actually achieve that? Of course, like in the HIV space at the moment, the epigenetic manipulation is important. Are we able to do likewise with HBV?
I think for me, what I've learned from these studies is that we really do need to address the question about integrated DNA and how we will address that in terms of going forward in our clinical trial. My own personal thinking in terms of hepatitis B has shifted, and I'm now thinking in terms of e antigen positive and e antigen negative diseases as different diseases, which has been something that the clinical practice guidelines have not really addressed. Not only are they different diseases, but they are different diseases that require different treatment approaches. What do I mean by that? Again, using our general HBV replication cartoons that we've been looking at this morning, here is the e antigen positive initial model that I had. What has the RNAi data taught us?
It's taught us that probably in a combination of Nuc, shown for you in the far right-hand red cross, the process of reverse transcription by the nucleoside/nucleotide analog therapy, and in combination with the RNAi ARC-520, the existing compound that you've heard this morning at the transcriptional level, I think that would be a very effective way of inhibiting HBV replication. We've also heard this morning that in the setting of e antigen negative chronic hepatitis B, that ARC-520 and the Nuc are not enough and that what we need is a different approach. I think one of the exciting parts of this program has been the fact that they've been able to prove that the next generation RNAi inhibitor ARC-521, which was targeted really to specifically the surface antigen transcript, as Dave elegantly showed you this morning.
That in combination with the Nuc, you've now got three sites of action with two drugs. You've got the RNAi ARC-520 inhibiting the integration phase, ARC-521 RNAi inhibiting the transcriptional phase, in combination with the Nuc would give you a significant effect on the surface antigen. I think you can now see that by putting together 520 and a Nuc, it accounts for, I believe, the antiviral effects that Arrowhead have been able to show in the e antigen positive patients of greater than one to two logs. Now with the ARC-521, I believe that they will be successful in achieving a greater than one log and two log reductions in the e antigen negative patients because of their ARC-521 inhibitor. Are we there yet? Almost, I think. The question that the immunologist will ask is how can we achieve surface antigen seroclearance?
How can we reduce this 1% of the total serum burden of protein circulating in our blood? How can we eliminate that and reduce the risk of cirrhosis and liver cancer? The immunologists have proposed, there's two ways that we can get rid of surface antigen or cccDNA out of the liver. There's the cytolytic mechanism, which means that probably you need to promote active T cell killing through CD8 activation pathways to promote and selectively kill the hepatocytes, and that these hepatocytes will be replaced preferably by uninfected hepatocytes diluting the cccDNA content. The other mechanism that the immunologists have proposed is a noncytolytic mechanism where antiviral cytokines like interferon gamma and TNF alpha on the right of that slide will actually noncytolytically cure the hepatocytes themselves.
I think that when you think about what we've learned from the nucleoside/nucleotide analogues, what we've now learned from the ARC-520 and the ARC-521 data, and we combine that, I think, with the interferon approaches proposed in the MONARCH studies, I think that we've now got the best of the best of the best treatment possible for hepatitis B. We're really heading in the right direction. As a virologist, what do I think is very exciting about the ARC-520 data? This is the first drug that really has had a direct antiviral effect on serum surface antigen. Bang. If for nothing else, that's a fantastic achievement. Also, by the way, e antigen and the core-related antigen levels have also been reduced.
We've learned that e antigen positive and e antigen negative disease have very different viral pathobiologies and therefore very different approaches clinically to managing these heavy burden of chronic liver disease. And I think this has significant important therapeutic and prognostic significance. With that, thank you.
All right. We have gobbled up an hour and a half of your life, and I don't want to gobble up too much more. I have two slides to close it up, and then we can take some questions. What have we done today? What have we shown you today? I think we've demonstrated that the DPC platform works in humans. We've seen deep and consistent knockdown in that which we are targeting, and it's been very well-tolerated. We think that has dramatic de-risking effects on ARC-520, ARC-521, ARC-AAT, and ARC-F12. These all use the same DPC. Second, I think that we have demonstrated that ARC-520 works in humans. Think of that in two ways. First- I think in all patient classes, we should see deep knockdown of non-S antigens, non-S proteins. There's this core antigen, e antigen, X antigen.
More importantly, I think our experts believe that long-term disruption of the virus in this way could have dramatic effects towards functional cure. Second, we've shown a good, very deep knockdown of S antigen in NUC-naïve, e positive patients, and we've also shown good knockdown of e antigen in NUC-treated patients. Third, ARC-520 is well-tolerated. We've seen a very attractive safety profile thus far. Fourth, I think we have established a new paradigm, or at least proposed a new paradigm, for the HBV life cycle. I think that has moved the field forward and certainly informed us as we develop ARC-520 and ARC-521. Finally, we've expanded our HBV pipeline to include ARC-521. We think we have an extremely powerful and complete approach to this very difficult virus. Finally, stay tuned.
We have an awful lot of data that will be coming toward you through scientific presentations at conferences and through publications in journals. For instance, we will present the chimp data in two presentations at the AASLD, one poster, one oral, and we expect clinical data to come through publications and presentations as well. We are conducting right now, as we speak, multiple dose phase IIb studies of ARC-520. We'll be starting MONARCH combination studies shortly, and we expect ARC-521 to be at IND or equivalent by the middle of next year. We are excited about where we are. We are very excited about what we've just proven and shown you today, and I think we're in a good place going forward for the rest of this year and of course 2016 as well. With that, I'll open the floor up to questions.
I think Ted's got one. Ted's got one. Ted.
Hold on. Hold on. Let me get a mic on that.
Great. Thank you very much for a thorough presentation and certainly some provocative findings. Firstly, when should we get data from cohort 7, and will it be upon initiation of MONARCH that you will disclose those cohorts?
For cohort 7, and actually for the entire 2001 study, we will see if we can get a late breaker at AASLD, if that's possible. You will see some stuff there. If we don't, we will find our earliest possibility for presentation. But it's open label, we don't have the constraints that we had for cohorts 3, 4, and 5, which we just broke the blind about 10 days ago. With cohorts 6 and 7, we get to follow them as they go, which gives us the ability to try to find good forums for presenting. Your second question was about MONARCH. Well, I think that those initial cohorts at the point that goes into clinicaltrials.gov, it probably will explain what those cohorts are.
I think, Chris, maybe you just answered my next question, which is why still conduct HEPARC-2004 in the e antigen-negative patients? Is really the goal there to see if multiple doses will actually have more of an effect against the integrated DNA? Is that the correct way to think about that?
Let me be clear. We think that ARC-520 has great promise, not just for NUC-naïve, e positive patients, but also for other classes of patients, because it is clear that we're getting good deep knockdown of, we believe, all non-S antigens. When you think about that, if you are disrupting the virus to that extent, showing log-plus reductions of all the other components associated with this virus, we want to see what that does to normal bio function over time. Oh, by the way, we are knocking down S antigen in those low cccDNA patients. We see good knockdown with those. We just happen to see phenomenal knockdown of S antigen for those NUC-naïve, e positive patients.
We are as excited as ever about ARC-520, and we think it is sort of a combination product unto itself because we're knocking out all these gene products. But then, of course, 521 is just more specialized for also knocking down the integrated DNA. Do we need that to get to a functional cure? I don't know, but it makes us more comfortable to have that extra shot on goal, that extra bullet in our gun, if you will.
I'm sorry to monopolize the microphone, with the second-gen DPCs, do they offer the potential to get to a subq dose, and will you ultimately explore ARC-520 DPC, ARC-521 DPC?
Both ARC-520 and 521 are IV administered. Could we someday have a subq candidate for HBV? It's not something that we're focused on right now. We think that a finite treatment window for either ARC-520 and/or 521 is a perfectly acceptable drug if we can get to functional cures.
Yep.
I'm just wondering, for the chimp two years ago, was that e positive or e negative?
Michelle two years ago, she was e positive.
Okay. For 521, does 521 equal 520 plus anti-DSL DNA, or is it just going to be more targeted towards DSL DNA? Eventually, you want to do 520 plus 521, or 521 is enough.
ARC-520 has two siRNAs, both of which target in a similar region toward the three prime end of the viral DNA. What we did for 521 was we took the best of those two, and we combined it with the best RNA targeting integrated.
Okay.
It's a two component. It's also just two. It's not three. It's two RNAs.
Got it. Okay. Lastly, is ALT flare necessary for immune reactivation? There's some controversy there. You had one e plus that is converted to one of the chimps. Did that chimp have a flare? What is the general consensus now? Do you have that ALT flare?
Maybe we should send that down to either Bob Gish or Stephen Locarnini.
Let me say one thing on the chimps. As we mentioned, we did see signs of immune reactivation in one of the four e positive chimps. We hope to present more of those data at a later scientific meeting. We want to not go too much more into that, but relating to whether or not we need a flare. Go, Dr. Gish.
From a hepatology perspective, we were treating hepatitis C, we thought we were going to need to see these flares of ALT for hep C to be cleared. What we really learned that those immunologic events are going on intracellular really weren't showing up in the blood as a rising ALT level, but probably intracellular, there's a flare or an immune event taking place. I think ALT flares will be seen in a minority of these, either chimps or humans, as we move forward with our data analysis and won't be required. If we don't see an ALT flare, that's not a negative signal. I think what's going on in the cell, what we're seeing with viral products is key.
Steve.
Yeah. I think what will be exciting in the next year will be the impact of the lowering the surface antigen will have on the endogenous immune response. I don't think we can predict which way it's going to go. We're just going to see and learn what will happen in the treated patients. I think the reduction of that antigen burden will have an enormous effect on the endogenous immune response. We're already beginning to see early signs of immune recovery in some of the treated patients, and I think we're watching very closely that space to see what will be the final outcome.
Actually, I want to make an announcement really quick. Once the presentation's over, a copy of all the slides will be available on our website within 30 minutes after the conclusion. I also had a question from Michael Yee at RBC that was emailed that I want to present to the panel. He asks, "cccDNA is pretty much non-existent in e antigen negative patients, and integrated DNA, for the most part, only produces S antigen. Yet measurable virus is still being produced. We don't get viral declines in these patients, so millions of infectious particles are still being made. If integrated DNA can't make virus, where is virus in these patients coming from?
Dr. Locarnini and Dr. Gish, do you want to-
I think our ability is to measure the level of the cccDNA in the liver is it's only the copy number is between 1 and 5 per cell across the whole liver lobule, and it's a reflection of technology. In e antigen positive disease, we see a lot, like 10 copies per cell. In e antigen negative, we see 2 to 3. That's the sort of level that we're talking about. The PCR assays that we use, we're right on the level of sensitivity of those particular assays. Obviously, in e antigen negative disease, it's making virus, they're infectious, but it's our ability to measure them in terms of a replicative intermediate or a biomarker is the fact that they're just so low. Bob, do you have any comments?
I don't want to expand too much on that. I agree with Stephen's comments. It is a technical aspect of how we measure these molecules. We are pushing the technology pretty close to what our limits are. We are able to see cccDNA in the liver. More importantly, we can see replicative intermediates. It is just at the threshold of detection, in contrast to e positive patients, where it's very abundant and easily detected. I think there is the possibility through multiple recombination events that you could reconstruct a full-length genome through integrated copies. I think that would be much more rare, and it would be impossible for us to actually detect that event.
Thank you. On the cohort of seven non-Nuc e antigen positive patients, the data we were seeing is only six patients?
Yeah, the sixth patient, I don't have any post-treatment data yet. The day 15 data was four patients. The day 8 was five patients. Basically, it's still young data.
Okay. If you presented, I might have missed it. Do we have data for cohorts five and six, the e antigen positive patients, but they are on NUC?
Yeah, I showed the cohort five data. I didn't show the cohort six data just because it doesn't differ in any material way. If we wind up getting the opportunity to present this at AASLD, we'll be able to show everything. Here, we really had to stick to just top-line data. Six is not materially different than five.
Okay. All right, thank you.
For Scavenna.
Does this change now your definition of a functional cure, the new information about integrated DNA and so on?
Do you want to address that, Dr. Gish?
We're looking at this in multiple steps, I would say that functional cure remains S-loss, DNA undetectable. There's still some controversy about surface antibody positivity, whether that's required or not. Then this sterilizing cure, I'm going to go one step further that we are going to need to eliminate cells that are producing S antigen or potentially other viral proteins from integrated sources, because those S antigen proteins are going to modulate the immune system and allow persistent of other hepatocytes that may have replicative intermediates.
From our perspective also, our definition of functional cure has not changed until the experts tell us we should change it. Keep in mind that this goal of S-loss is not intended to be directly the result of 520. We are not trying to take S down to zero with the drug itself. What we're trying to do is disrupt the virus enough to decrease S to a point that the immune system can then take over and do the rest of the job for us. Okay. Well, thank you all for coming. It was a pleasure to speak with you today.