What's a good biomarker for HBV clinical trials?

by Robert Kruse

The clinical success of HCV cocktail drugs catapulting Gilead's and Abvie's stock the past year has led to increasing investor focus on potential HBV blockbuster drugs. The current therapies, reverse transcriptase inhibitors like entecavir, inhibits viral DNA production in the serum. The multi-log reduction in viral load, however doesn't lead to SVR in most patients. The viral genomes (cccDNA) inside the liver are stable, and viral antigen production is thought to continue suppresses the immune system.

For the investor, scientist, and physician, there are three principal biomarkers that should be noted in evaluating any preclinical or clinical HBV data. They are HBV surface antigen (HBsAg), HBV DNA (or titer), and HBV e antigen (HBeAg). These can all be drawn from the bloodstream of patients and evaluated by ELISA and qPCR. Liver HBV markers would be ideal but require patient biopsy, which is outside of the normal physician treating practices for HBV and is an expensive procedure compared to routine blood draws. Ideally though, they could be used to assess that the viral genome is reduced or inactivated in the liver, which is necessary for cure. Note that I said inactivation, because it has been shown that HBV cccDNA (circular HBV genome) in patients with resolved HBV infection from an acute scenario still possess remnants of HBV DNA that is identifiable by PCR. This suggests that epigenetic inactivation of cccDNA in addition to direct degradation mechanisms are at play during resolution of HBV infection.

HBsAg has received the most attention in clinical trials, particularly for HBV knockdown efforts with oligonucleotides (Isis with antisense and Arrowhead/Tekmira/Alnylam with siRNA) or with polymers (Replicor). The loss of HBsAg has been hypothesized to relieve immune suppression, that would then activate anti-HBV immunity. This hypothesis largely comes from the observation that HBsAg decreases when a person spontaneously clears infection in rare events. The problem is the chicken or the egg in this deduction. Did some type of seroconversion and antibody production lead to HBsAg clearance (as mimicked by these therapy strategies), or did a T cell response against a different HBV antigen or even a non-related liver disease, lead to these cases of resolved HBV.

One issue with current biomarkers is that HBsAg levels vary widely among patients, depending on disease status and serotype. Indeed, it is unknown what is a sufficient level of HBsAg production in order for the virus to suppress the immune system and HBsAg fulfill its other roles. What is known is that HBsAg can be reduced greatly without any impact on DNA levels. HBsAg is the envelope protein for the DNA virus, but is also secreted in the form of empty particles as well, which outnumber DNA containing particles by 1000 to 1. Therefore, a reduction in envelope won't remarkably change virion production. Without a reduction in DNA titer, then the levels of virus in the liver are never going to change and virus can continue to spread.

It seems then, that looking at DNA levels could be a better biomarker, since it is driven by a combination of viral genes and might offer a more accurate readout of the levels and/or suppression of the HBV genome. For example, while HBsAg levels might fluctuate for secretion inefficiencies and/or promoter mutations, the DNA levels are more accurately driven by the core promoter, which is sensitive to interferon suppression by an ongoing immune response. This is key since the majority HBV therapies rely on the activation of an immune response, directly or indirectly. If HBV DNA is suppressed in the serum dropping multiple logs, it means that genome amplification in hepatocytes is decreasing and no new hepatocytes can be infected.

The HBeAg marker could also be useful, being driven by the same core promoter region. However, since HBeAg seroconversion happens in a good fraction of patients, and they continue to have chronic infection, it might not be relevant for people looking for signs of viral cure. That said, the consensus among ID doctors is that HBeAg seroconversion is a good clinical sign, and it is correlated with lower HBV DNA levels, emphasizing the importance of that marker. Various epidemiological studies have shown that higher HBV DNA levels are associated with higher risk of HCC and cirrhosis. While similar reports have high HBsAg levels as a risk factor in certain settings, this is somewhat inconsistent since other forms of HBV has little HBsAg expression and still see signs of HCC and cirrhosis development, notably occult HBV.

Regardless of marker, a definitive cure for HBV should show significant multi-log reduction in all HBV markers, that is sustained off treatment. The first few patients will be examined by liver biopsy for HBV, just like the Berlin patient was biopsied in various sites for the presence of HIV. That said, in these early clinical trial reports, its much more opaque looking at viral readouts to see potential signs of response, particularly when dosing hasn't been optimized. The same is true for any surrogate studies in woodchuck or duck models. The therapeutic play for HBV won't be avoidance of clinical morbidities like HCC, since that takes many years to manifest, but rather a feast or famine for viral cure. I am personally wary of any studies that show drops in HBV levels in chimpanzees, and don't extend it out more months to see what happens, since it is during that time that the immune response could manifest and should be reported. If efficacious, one expectation could be an intermittent drop in HBV DNA caused by therapy, followed by another multi-log dropped precipitated by the immune response. An effective immune response against HBV is actually largely non-cytopathic, making ALT readouts for immune activity potentially confounding. However, HBV DNA levels are very sensitive to immune responses, making those readouts better.

This analysis can't address the validity of the HBsAg reduction hypothesis (ie how much to reduce in order to see the immune system reactivate?), but it does suggest that HBV DNA levels should be monitored more closely as a sign of treatment efficacy for all modalities. Of course, the confounder is that we have an effective treatment in reverse transcriptase inhibitors, meaning many patients in trials might be on entecavir, for example, and have undetectable DNA levels. This valuable marker is lost then. It is suggested here that unless necessary, trials with drugs as a monotherapy could provide a cleaner readout for HBV antiviral potency.

With the advent of new players (see OnCore and Tekmira merger) in the HBV therapeutic space, more attention will need to be assessed on phase I and II clinical data, and we will continue revisiting this topic of relevant HBV biomarkers here at Biotechr going forward.