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Discussion
NAP tolerability
NAPs are phosphorothioated oligonucleotides (PS-ONs) whose antiviral effect against HBV infection is independent of nucleotide sequence [11, 12]. Unlike standard antisense compounds, NAPs can be more easily engineered to remove the secondary pro-inflammatory / immunostimulatory effects of single stranded nucleic acids while maintaining their antiviral activity in vivo [13, 15]. The primary tolerability issue with REP 2055 in the REP 101 study was IV administration related side effects similar to those reported for other PS-ONs administered to humans by IV infusion [16, 17] which was well controlled with REP 2139-Ca in the REP 102 study. Additionally, short term combination therapy with REP 2139-Ca and thymosin or peg-IFN up to 26 weeks was well tolerated. The liver flares during monotherapy with either REP 2055 or REP 2139-Ca occurred following initial reductions of serum HBsAg and HBV DNA, were self-resolving with continued NAP therapy and were not accompanied by any symptoms or serological evidence of liver dysfunction. These liver flares are also routinely observed with treatment with interferons, and are thought to be evidence of re-activation of immune function in the liver [18]. These liver flares may be evidence of reactivation of the immune response in the liver with reduction of circulating HBsAg (discussed below) but additional analysis of T-cell response during transaminase flares concomitant with serum HBsAg reduction in future trials will be required to address this hypothesis.
PS-ONs increase mineral elimination in the urine [19] and the resulting compensatory response includes liberation of mineral stores (along with heavy metals if present) from the bones into the circulation. All study patients and untreated control subjects at the trial site were shown to have substantial pre-existing heavy metal loads (data not shown) as a result of chronic heavy metal exposure known to be endemic at the trial site [20–22]. The 2’ ribose modifications present in REP 2139 are absent in REP 2055 (Fig 2) and serve to reduce immunoreactivity [23, 24] but also block degradation by endonucleases [25], rendering REP 2139 substantially more stable than REP 2055 and leading to greater accumulation of REP 2139-Ca in pre-clinical models with chronic exposure (A. Vaillant, unpublished data). Thus chronic exposure to REP 2139-Ca likely establishes a greater mineral elimination (and heavy metal liberation) than REP 2055, consistent with development of hair loss, dysphagia and dysgeusia observed in patients with REP 2139-Ca therapy but not with REP 2055.
These symptoms have not been previously reported with the clinical use of PS-ONs, however, these studies are the first to be conducted in a locale where substantial heavy metal exposure is highly endemic in the population. Importantly, in a currently ongoing clinical trial with REP 2139-Ca in Caucasian patients (REP 301 study, NCT02233075) in a European locale where patients with heavy metal exposure were excluded from participation, none of these symptoms have been observed with comparable REP 2139-Ca exposure in combination with peg-IFN (A. Vaillant, unpublished data).
These clinical observations underscore the importance of being aware of the potential complications of the enhanced mineral elimination known to occur with PS-ON therapy in general and further suggest that mineral supplementation should accompany any oligonucleotide based therapy, regardless of the disease state in patients receiving therapy.
Therapeutic implications of NAPs
Although further controlled trials will be required to confirm the initial safety observations and findings of antiviral activity observed in the proof-of-concept studies presented here, NAP monotherapy (either REP 2055 or REP 2139-Ca) was clearly accompanied by substantial reductions or clearance of serum HBsAg and appearance of anti-HBs < 10 mIU / ml with concomitant reductions in serum HBV DNA in 16 of 20 patients with HBeAg+ chronic HBV infection. These effects are very similar to the antiviral effects of REP 2055 reported in Pekin ducks with previously established persistent DHBV infection [13]. No evidence of virologic breakthrough was observed during NAP monotherapy lasting as long as 54 weeks or during combined treatment with immunotherapy in these initial studies. Moreover, two patients receiving an initial course of NAP therapy (REP 2055 in patients 2 and 7 in the REP 101 protocol), who experienced viral rebound during follow-up off-treatment responded identically with a second course of NAP therapy (REP 101 patient 2 responded to a second course of REP 2055 following viral rebound and in REP 101 patient 7, who became REP 102 patient 9, viral rebound after withdrawal of REP 2055 responded to REP 2139-Ca) suggesting the development of resistance to NAP therapy may be unlikely, however larger clinical trials will be required to ascertain if viral resistance to NAP therapy can occur.
The absence of rebound of HBV infection after cessation of REP 2055 treatment in 3/7 patients for 52 weeks parallels the sustained control of DHBV infection observed in vivo after REP 2055 monotherapy in ducks, which was correlated with transcriptional inactivation and clearance of cccDNA [13]. The more durable and complete suppression of serum viremia achieved in 2 patients who received REP 2055 monotherapy (where serum HBV DNA is not detectable and HBsAg is not currently detectable or detectable at residual levels, see Table 4) may reflect the establishment of control of cccDNA and / or integrated HBV genomes in these patients but this remains to be proven. More frequent dosing with REP 2055 appeared to improve the clearance of HBsAg and allow the achievement of serum HBsAg < LLOQ however similar reductions in serum HBsAg were achieved with REP 2139-Ca in the absence of periods of more frequent dosing which may be related to the increased stability of REP 2139.
When REP 2055 was used therapeutically in vivo against persistent DHBV infection, many animals maintained substantial titers of serum DHBV DNA with no detectable levels of serum DHBsAg, moreover at the end of REP 2055 treatment in many ducks, DHBsAg was readily detectable in the liver but not detectable in the serum, suggesting that NAPs selectively block the release of DHBV subviral particles from infected hepatocytes by REP 2055 [13]. The persistence of significant serum HBV DNA titers while serum HBsAg was maintained near or < LLOQ with REP 2055 or REP 2139-Ca monotherapy in human patients has striking similarity to these effects observed with REP 2055 in ducks. Given that subviral particles constitute the bulk of serum (D)HBsAg in both DHBV infected ducks and human HBV infection [2, 26], these correlative observations of the effects of NAP monotherapy in DHBV and HBV infection suggest that selective blockage of subviral particle secretion by NAPs may also be occurring in human patients.
The continued suppression of viremia after withdrawal of NAP therapy may be related to reduction or clearance of serum HBsAg during therapy. Serum HBsAg sequesters anti-HBs but importantly has also been shown to have direct immunoinhibitory properties in vitro and in vivo [27–32]. HBsAg is the most abundantly circulating viral antigen and it may exert direct inhibition of the immune response (both adaptive and innate) to HBV infection. Therefore, when HBsAg is removed or substantially lowered, some form of restoration of the host immune response may occur, allowing for a functional control of infection to be established which persists after NAP therapy is withdrawn.
REP 2139-Ca was safely combined with short term duration (12–26 weeks) of either thymosin or peg-IFN, which resulted in a rapid and robust increase in anti-HBs production in all patients. More importantly, even with short term exposure to these immunotherapies, serum viremia did not rebound after withdrawal of treatment in 8/9 patients and although variable in duration, remained suppressed for extended periods (> 2 years) in 3 / 9 patients, suggesting that restoration of immune control of infection with immunotherapy might be easier to achieve with this novel combination therapy approach. These effects suggest a potentially synergistic improvement in the antiviral effect of peg-IFN and thymosin when combined with REP 2139-Ca. This synergism may be related to the reduction or absence of serum HBsAg in these patients as sustained control of infection after peg-IFN monotherapy is highly correlated with HBsAg clearance [3, 4]. The HBsAg protein itself has been shown to have direct immunoinhibitory properties on both innate and adaptive immune function [27–32] which may also interfere with the downstream effects of immunotherapeutic agents.
The persistence of serum HBV DNA in the absence of detectable serum HBsAg and in the presence of anti-HBs > 10 mIU / ml is a highly unusual clinical observation that was nevertheless common with NAP treatment in both REP 101 and 102 protocols and raises some important theoretical considerations: the amount of HBsAg present in the titers of HBV DNA persisting when serum HBsAg is < LLOQ may be below the LLOQ of the HBsAg test or may indicate the presence of immune escape HBV variants, which have been previously documented in human HBV infection [33–36]. Evaluation of the population genotypes of the HBV present in these patients during NAP therapy by deep sequencing is currently underway and may help to answer some of these questions. Fluctuations in anti-HBs levels were observed in the REP 101 study during treatment while HBsAg and HBV DNA declines were stable. However, fluctuations in serum anti-HBs were not observed in the REP 102 study during monotherapy. REP 2139 is significantly more stable than REP 2055 in human plasma (as discussed above) and may provide a more uniform antiviral effect between weekly doses than REP 2055. This reduced stability of REP 2055 could be an underlying factor in the fluctuating antibody response observed in the REP 101 study.
Future prospects
Both the REP 101 and 102 trials presented in this article and a third trial (REP 201 –NCT02726789) have been previously disclosed [37]. The REP 201 trial was a very small exploratory trial where REP 2139-Ca treatment with a full course of peg-IFN was attempted in three treatment naïve patients and two patients concurrently receiving ETV. Although similar responses in serum HBsAg, anti-HBs and HBV DNA were observed during treatment as in the REP 102 protocol, the very small patient numbers employed prevent conclusions on improvements in antiviral performance relative to those seen in the REP 102 trial. However the REP 201 trial suggests that NAPs might be used safely in combination with peg-IFN and ETV or other nucleos(t)ide based HBV polymerase inhibitors. This triple combination approach is currently being evaluated in patients with HBeAg negative chronic HBV infection in the REP 401 protocol (NCT02565719, see below).
The results of the REP 101 and REP 102 studies describe the preliminary steps of the development of NAPs for treatment of chronic HBV infection. They indicate that continued clinical evaluation of the antiviral efficacy of NAP-based combination therapy in chronic HBV infection is warranted. Although control groups were not possible in these studies, effective clearance of HBsAg and HBV DNA and the appearance of anti-HBs is unusual. Of greater potential interest is the potential synergistic improvement in the antiviral effects of immunotherapeutic agents when combined with NAP therapy as these might potentially be related to HBsAg reduction. Even with a limited course of immunotherapy, 8/9 patients were able to maintain suppression of serum viremia off-treatment, with many of these maintaining suppression longer than 1 year.
The proportion of patients able to maintain suppression of viremia off-treatment and the duration of this suppression may be significantly improved if NAP therapy was combined with a full course of immunotherapy (i.e. 48 weeks) or if used in combination therapy in patients already seroconverted for HBeAg or currently receiving therapy with ETV or TDF, agents which have been shown to lower intrahepatic cccDNA levels [38, 39]. These concepts are currently being evaluated in the protocol REP 401 protocol (NCT02565719), a randomized, controlled trial assessing the safety and efficacy of triple combination antiviral therapy with NAPs, peg-IFN and TDF in Caucasian patients with HBeAg negative chronic HBV.
Finally, additional trials with REP 2139-Ca should be conducted in patients from diverse ethnic groups and harbouring different HBV genotypes to investigate if the antiviral responses with NAP therapy observed in the patients in these studies could also be achieved in patients from other geographic regions. |
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发表于 2016-6-5 12:05
