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"Phase IIa trial in HIV/AIDS patients with drug candidate ABX464"....treatment/cure
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Download the PDF here
Download the PDF here
"We are pleased to announce the start of this next clinical trial with ABX464," said Prof. Hartmut Ehrlich, M.D., CEO of ABIVAX. "This Phase IIa clinical trial is designed to test the promising preclinical data observed in an animal model, which showed evidence of long lasting control of viral load following cessation of ABX464 treatment. If confirmed in HIV patients in this study, such a long-lasting effect would differentiate ABX464 from all existing HIV therapies." .....Prof. Bonaventura Clotet, Director of the IrsiCaixa AIDS Research Institute at the University Hospital Germans Trias i Pujol in Barcelona, Spain commented: "We are very excited to enroll the first patient into this important clinical trial. The outcome of this study aims to provide a proof of concept that ABX464 could potentially be part of a functional cure for HIV."......It works by inhibiting HIV replication through a novel mechanism (i.e. the modulation of RNA splicing) that may not be vulnerable to the development of resistance by the HIV virus, and may have a sustained effect in patients........http://www.businesswire.com/news/home/20160530005192/en/Ongoing-Clinical-Development-ABX464-ABIVAX-Launches-ABX464-004
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Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis......authors say: "ABX464 was able to significantly reduce the viral load.....prevented depletion of CD4+ cells.....described to neutralize the expression of the HIV-1 proviral genome and it is therefore a promising drug towards HIV cure.....We propose that ABX464 could be used in combination with current drugs or as a simpler and more effective replacement"
Retrovirology 2015
see article below following commentary
Noelie Campos1, Renier Myburgh3, Aude Garcel1, Audrey Vautrin2, Laure Lapasset2, Erika Schlapfer Nadal3,
Florence Mahuteau-Betzer4, Romain Najman1, Pauline Fornarelli1, Katjana Tantale2, Eugenia Basyuk2,
Martial Seveno7, Julian P Venables2, Bernard Pau5, Edouard Bertrand2, Mark A Wainberg6, Roberto F Speck3,
Didier Scherrer1 and Jamal Tazi2*
"In this report we present ABX464 first trials. Interestingly, this drug presents a new mechanism of action for HIV treatment and improves current therapies, hampering appearance of virus resistance. More importantly, it leads to a long lasting control of the viral levels. We propose that ABX464 could be used in combination with current drugs or as a simpler and more effective replacement."
"In our attempt to identify new more potent and non toxic HIV RNA splicing modulators inspired by the structure of IDC16, we present ABX464 which we show targets Rev function and therefore is susceptible to alter the viral expression in the reservoirs......no therapies are clinically available based on this mechanism.....ABX464 reduces the viral load over a period of 30 days of treatment (Figure 5d left panel) but more importantly, the viral load remains low for at least 52 days after treatment termination (Figure 5d right panel)."
"Figure 1b, shows dose dependent inhibition of HIV-1 replication by soluble ABX464 in stimulated PBMCs from 5 different donors with an IC50 ranging between 0.1 μM and 0.5 μM........Mice were treated twice a day (b.i.d) for 15 days by oral gavage with 20 mg/kg of ABX464. Measures of viral RNA showed that the oral treatment with ABX464 was able to significantly reduce the viral load over a period of 15 days of treatment (Figure 5a). FACS analysis of blood samples showed that treatment with ABX464 prevented depletion of CD4+ cells following infection of reconstituted mice and thereby restored the CD8+/CD4+ ratio back to that of non-infected mice (Figure 5b).
......Here, we describe ABX464, an enhancer of viral RNA splicing with validated efficacy in humanized mouse models of HIV-1 infection. ABX464 is the first therapeutic molecule described to neutralize the expression of the HIV-1 proviral genome and it is therefore a promising drug towards HIV cure.....
......ABX464 also very efficiently inhibited the replication of viral strains harbouring mutations that confer resistance to different therapeutic agents in vitro (Figure 2b). While the antiviral drug 3TC was not highly active on K65R and M184V mutant strains, both strains were inhibited by ABX464......There were no resistance-inducing mutations detected after treatment with ABX464 for at least 24 weeks (Figure 2c).......The majority of low and high frequency mutations were equally present in treated and untreated samples, demonstrating that ABX464 does not select for specific mutations
In this study NOG hu mice were infected with the YU2 HIV-1 virus and fed daily for 30 days with 40 mg/kg of ABX464 (Figure 5d left panel) or with HAART (3TC-Tenofovir-Raltegravir) (Figure 5d middle panel) and viral loads were measured as explicated before. Most significantly, unlike HAART, ABX464 leads to a sustained reduction of viral levels in humanized mice after treatment cessation (Figure 5d right panel). Compared to HAART, ABX464 treatment induced a slower kinetic of viral load reduction (Figure 5d left panel). Whereas HAART was very efficient at reducing the viral load to undetectable level after 2-3 weeks of treatment in all infected mice (Figure 5d middle panel), with ABX464 only 2 out of 6 mice had undetectable levels of viral load, 2 mice were under 1000 copies and 2 mice are still have a viral load higher than 6000 copies after 1 month of treatment (Figure 5d left panel). While we cannot exclude the possibility that the slow kinetics of viral reduction could be inherent to ABX464's mode of action, it is also possible that in this mouse model ABX464 used as monotherapy, is inefficient at reducing the viral load to undetectable levels."
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"ABX464 is indeed the first small molecule drug that (indirectly) targets the Rev function, although early gene therapy approaches already focused on Rev inhibition [4]. ABX464 is therefore the first member of a new drug class that blocks viral gene expression in cells that were already infected and in which the integrated provirus has already been established. The ability to block the production of new viruses seems an important asset for attempts to silence viral reservoirs.....A Phase 2 study of ABX464 in patients with HIV was recently initiated. Although we would immediately agree that ABX464 remains an interesting drug candidate that could form a welcome addition to the current classes of antivirals, some caution is warranted because the sustained drug action may be an artifact of the pre-clinical model system. The search continues for anti-latency drugs, but one should entertain and exclude indirect effects when a magical sustained drug action is observed."
ABX464: a good drug candidate instead of a magic bullet
Ben Berkhout and Yme U van der Velden
Retrovirology 2015
Abstract
Despite the significant number of antiviral drugs that are currently available in the clinics of developed countries, none of these affect the production stage of HIV-1 replication, more specifically the process of viral gene expression. For instance, several early attempts failed to generate inhibitors of the viral Tat protein, the small activator of viral transcription from the long terminal repeat (LTR) promoter. A recent study published in Retrovirology by Campos et al. presents a new small molecule inhibitor, ABX464, that targets the other small viral protein essential for viral gene expression, the Rev protein (Retrovirology 12:30, 2015). Targeting of multiple virus replication steps and silencing the generation of new progeny may be of particular value for current attempts to develop novel therapeutic strategies that provide a cure or functional cure for HIV-1 infection (Nat Rev Immunol 12: 607-614, 2012). We will briefly review some of the unique antiviral properties of ABX464, with the focus on its surprising ability to exhibit a sustained antiviral effect in a humanized mouse model. Although ABX464 may remain an important new addition to the anti-HIV arsenal, we do present a sobering alternative explanation for the long-lasting reduction in viral load after treatment cessation.
In the Retrovirology study [1], ABX464 was shown to block HIV-1 replication by preventing the export of unspliced viral RNA from the nucleus to the cytoplasm, a process that is regulated by the viral Rev protein. No direct ABX464-Rev interaction is proposed, but instead the drug seems to interact with an important cellular RNA regulatory complex. Although this indole drug class was originally described as targeting the splicing factor SRSF1 [2], ABX464 was found to bind directly to the cap binding complex (CBC). CBC controls RNA export and RNA splicing and interacts directly with the Rev protein [3]. In the new report, ABX464 was proposed to bind CBC and to specifically prevent Rev-mediated export of viral RNA, without interfering with cap binding or export of cellular transcripts [1]. ABX464 is indeed the first small molecule drug that (indirectly) targets the Rev function, although early gene therapy approaches already focused on Rev inhibition [4]. ABX464 is therefore the first member of a new drug class that blocks viral gene expression in cells that were already infected and in which the integrated provirus has already been established. The ability to block the production of new viruses seems an important asset for attempts to silence viral reservoirs.
The new report in Retrovirology adds several important new insights on ABX464's mode of action. First and perhaps surprisingly given the proposed binding to the cellular CBC regulator, ABX464 does not seem to affect other RNA processes in human cells, thus avoiding an impact on the physiological state of the cell. Besides being non-toxic, ABX464 does not trigger the evolution of HIV-1 variants that are resistant to the drug action. A likely explanation is that the direct drug target is the cellular CBC and not a viral component, consistent with previous antiviral strategies [5]. It is therefore also likely that this drug will be active against various HIV-1 isolates and different subtypes, possibly even HIV-2 if it is similarly dependent on the CBC-Rev axis, but this was not tested. On top of this, a remarkable sustained antiviral effect was reported in a pre-clinical in vivo model system. Thus, ABX464 seems a drug with optimal properties: selective drug action, absence of drug-resistance, and a sustained antiviral effect that would be very welcome for cure attempts. However, we would like to present an alternative explanation for the sustained antiviral effect. To do so, we have to discuss in some detail the experimental conditions at which this surprising effect was observed.
Profound suppression of the HIV-1 YU2 strain was obtained by state of the art combined antiretroviral therapy (cART) in the humanized immune system (HIS) mouse model, but an immediate rebound of the viral load was observed after the treatment was stopped. ABX464 monotherapy also led to a significant reduction of the viral load, but virus suppression was slower and less complete than with cART. The perplexing result was that this antiviral effect lasted for several weeks after cessation of ABX464 treatment [1]. No such an effect has ever been described for other treatments. The explanation provided is that ABX464 prevents virus replication in macrophages, thereby blocking the establishment of a viral reservoir that will cause the rebound after stopping therapy.
There are several reasons why this interpretation is not likely. First of all, it seems quite unlikely that macrophages form the major cellular component of the HIV-1 reservoir [6, 7]. Second, ABX464 could never have blocked all reservoir formation as the drug was added only at day 52 after the experimental infection of the mice. In fact, all reservoirs will likely have been established already by that time. For instance, a recent macaque study indicated that reservoirs are seeded after just 3 days [8]. Although we certainly value the power of the HIS mouse model [9], this unique in vivo model remains rather variable and complex as each HIS mouse is generated individually and critical parameters such as engraftment and source of hematopoietic stem cells differ between experiments. We like to offer an alternative explanation for these striking results, also because such a sustained inhibitory effect after drug removal has never been described for antivirals, including ABX464, in more simple in vitro settings. Because ABX464 does only sub-optimally inhibit YU2 replication, it could be that most target cells have been depleted (by virus replication and/or immune reactions) after 100 days, the moment at which the therapy was stopped. The absence of sufficient target cells would provide a very simple explanation for the observed lack of viral rebound. Although the CCR5-tropic YU2 virus has been reported to sustain viral replication for 150-200 days in HIS mice without a noticeable decline in viral load [10, 11], another study [12] demonstrated an almost log10 reduction in viral load after 100 days of infection, which is in the range of the observed sustained reduction in viral load upon ABX464 withdrawal and which could be explained by a shortage of target cells. In fact, some of the additional data in the ABX464 study do support this alternative scenario. Close inspection of the results obtained for individual mice indicates that there is a correlation between the viral loads at cessation of therapy and the viral loads at the end of the experiment. This correlation may be imposed by the number of target cells available. One could obviously test this alternative explanation in many ways. The authors could for instance have refuted this alternative scenario by including an untreated YU2-infected group to monitor the CD4+ T cell dynamics throughout the experiment and at sacrifice. We believe that this commentary is relevant as the original study did not entertain any alternative explanations for the sustained drug action and the ABX464 antiviral was evaluated for the pharmacokinetic properties and biological safety in healthy volunteers by the ABIVAX biotech company in a Phase 1 study. No serious adverse events were scored and no clinically significant abnormal result was reported in physical examinations, laboratory tests, vital signs and ECG. A Phase 2 study of ABX464 in patients with HIV was recently initiated. Although we would immediately agree that ABX464 remains an interesting drug candidate that could form a welcome addition to the current classes of antivirals, some caution is warranted because the sustained drug action may be an artifact of the pre-clinical model system. The search continues for anti-latency drugs, but one should entertain and exclude indirect effects when a magical sustained drug action is observed.
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Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis
Retrovirology 2015
see article below following commentary
Noelie Campos1, Renier Myburgh3, Aude Garcel1, Audrey Vautrin2, Laure Lapasset2, Erika Schlapfer Nadal3,
Florence Mahuteau-Betzer4, Romain Najman1, Pauline Fornarelli1, Katjana Tantale2, Eugenia Basyuk2,
Martial Seveno7, Julian P Venables2, Bernard Pau5, Edouard Bertrand2, Mark A Wainberg6, Roberto F Speck3,
Didier Scherrer1 and Jamal Tazi2*
Abstract
Background
Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis.
Results
Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies.
Conclusions
ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.
Background
AIDS is a worldwide pandemic. Current therapies have succeeded in controlling the disease but long-term use of Anti-Retroviral Therapy (ART), is limited by issues of drug resistance and side effects [1-3]. Furthermore, the current ART drugs need to be taken for life time and only attenuate the disease without curing it [4]. Therefore, there is a continuous need for new drugs to HIV cure, in particular those acting through new and as yet unexplored mechanisms of action [5].
We took advantage of the unique feature of the viral genome once integrated in infected cells to require both activation and inhibition of splicing of precursor mRNAs to develop molecules capable of changing the balance between spliced and unspliced products. The HIV-1 DNA genome expresses a primary transcript of 9 kilobases (kb) that not only serves as genomic RNA for progeny virus but also as the mRNA that encodes the viral Gag and Gag-Pol proteins [6-8]. Alternative splicing is a key event for HIV replication.
Successful infection and production of new infectious viruses requires the balanced expression of seven additional viral proteins (Rev, Tat, Nef, Vif, Vpr, Vpu and Env) that are produced by splicing of the primary 9 kb transcripts among which the Tat and the Rev factors are absolutely required for viral gene expression at the transcriptional and post-transcriptional levels in infected cells [9,10]. While most cellular unspliced RNAs are retained in the nucleus where they are degraded, nuclear export of the unspliced viral RNAs is facilitated by the Rev protein, through binding to a viral sequence called the Rev responsive element (RRE) [9,11,12].
After screening a collection of chemical compounds, one indole derivative (IDC16) was discovered to interfere with splicing enhancer activity of the SR protein splicing factor SRSF1 [8,13,14]. This compound suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. However, IDC16 is a planar fused tetracyclic indole compound. Such molecules, and in particular those in which the polyaromatic nucleus is further substituted by a positively charged (protonated aminoalkyl) side chain, have been studied as potential anticancer agents [15,16]. The guiding principle is that they intercalate DNA and exhibit cytotoxic effects by interfering with the function of DNA processing enzymes such as topoisomerase I and II [17,18]. We thus designed alternative scaffolds which retain the structural characteristics of IDC16 but which lose the inherent affinity of the flat polycyclic molecules for DNA. We prepared molecules that are more flexible as they have fewer fused rings. This could circumvent potential side effects and allow a more optimal interaction with the protein target. A dedicated library of 1,000 compounds of potentially more potent and selective splicing modulators was synthesized.
Here, we describe ABX464, an enhancer of viral RNA splicing with validated efficacy in humanized mouse models of HIV-1 infection. ABX464 is the first therapeutic molecule described to neutralize the expression of the HIV-1 proviral genome and it is therefore a promising drug towards HIV cure.
Discussion
In our attempt to identify new more potent and non toxic HIV RNA splicing modulators inspired by the structure of IDC16, we present ABX464 which we show targets Rev function and therefore is susceptible to alter the viral expression in the reservoirs. The Rev protein facilitates the transport of unspliced and singly-spliced RNA to the cytoplasm in infected host cells by binding to RRE and competing with the major process of cellular mRNA export [11,27,38]. This Rev's essential role in HIV replication, its mode of action and specific interactions with its target RNA and cellular proteins enhance to the attractiveness of Rev as a target. Despite the fact that, a variety of approaches targeting Rev function, (including gene therapy [39-41]) have been developed to inhibit HIV-1 replication in cells cultured in vitro [42-49], no therapies are clinically available based on this mechanism. Many molecules selected this way have shown considerable toxicity in cells or have failed to specifically inhibit HIV replication [42,43,45].
The finding that ABX464 binds to and stabilizes the CBC complex (Figure 4 and Additional file 5: Figure S4), together with the recent demonstration that Rev protein specifies the viral RNA pathway by competing with TREX complex [27], suggest that ABX464 acts as an agonist to favour the normal pathway of export of spliced mRNAs. Consistent with this suggestion, we found that ABX464 enhances the expression of spliced viral RNA, while reducing the expression of unspliced RNA without having any effect on cellular splicing (Figure 3).
Previous studies have shown that the presence of the CBC complex on the 5′ RNA cap favours the recruitment of TREX on the opened DNA at the transcription site for efficient processing and export of the mRNP [50,51], competition between Rev and TREX complex may occur at the transcription site. Our data together with recent observations using the MS2 system which allows for visualization of events at the transcription site and export [24], further confirm the hypothesis that Rev and TREX compete with one another. The recruitment of export factors to the site of transcription represents a mechanism by which specific RNAs can be exported to the cytoplasm by alternative mechanisms. If unspliced viral RNAs are committed, to the CRM1 dependent export, by Rev [24], TREX-specific transport of multispliced will be diminished [27,50,52]. Whether this competition mechanism involves direct interaction between Rev and CBC complex has been shown recently; Rev interacted with the CBC80 subunit of the CBC complex and inhibited the recruitment of the TREX complex [27]. ABX464 stabilizes the binding of CBC at the cap structure and thus prevents Rev from seizing control of the TREX that binds to the cap-proximal region and determines the RNA export pathway and splicing [50-52].
Current therapies specifically target individual viral enzymatic activities and this confers relatively little risk of toxic effects to the host but the risk of developing resistant viruses is high [53]. ABX464, on the other hand, exploits a smaller windows of specificity by interfering with Rev-mediated export pathway though direct binding to the CBC complex. ABX464 is unlikely to induce the development of resistance since it targets a cellular, rather than a viral, component, (the CBC complex), ABX464 is also expected be active on different HIV clades and mutants viruses. Results presented here are consistent with this expectation (Figure 2). ABX464 is globally not toxic to infected cells because it has little effect on the export of bulk mRNAs and did not alter splicing of cellular genes nor the production of snRNAs nor of histones mRNAs whose biogenesis are dependent on the CBC complex [26]. By preventing Rev function, ABX464 restores the normal pathway of global RNA biogenesis that is disturbed by Rev expression in infected cells (with integrated proviral genome). ABX464 outperforms known drugs that target Rev in toxicological studies in all these ways and could be used in combination with current treatments or as a simpler and more effective replacement.
ABX464 reduces the viral load over a period of 30 days of treatment (Figure 5d left panel) but more importantly, the viral load remains low for at least 52 days after treatment termination (Figure 5d right panel). In contrast, rebound up to levels comparable to the initial infection is seen in the HAART group (Figure 5d right panel). ABX464 is thus the first anti-HIV drug able to suppress viral load sustainably after treatment arrest. The long lasting effect of ABX464 in mice could be explained by the finding that ABX464 prevents HIV replication in macrophages. The HIV-1 infected macrophages are of critical importance in the pathogenesis of HIV because macrophages have the ability to cross the blood-tissue barrier and deliver the virus to all tissues and organs, including the brain [37]. In addition, unlike T lymphocytes that are depleted by viral infection, macrophages are relatively less prone to the cytopathic effect of the virus and infected macrophages harbour and produce the virus for a longer period of time. Macrophages treated by ABX464 are unable to produce viral particles to transmit HIV-1 from macrophage to CD4+ T cells [36]. As macrophages act as the antigen presenting cells and present processed pathogen antigen peptides to the CD4+ T cells via MHC II pathway [54], one would assume that viral proteins produced from multispliced transcripts could serve as antigens to boost the immune anti-viral response. Given that the expression of these multispliced viral RNA will depend on the chromatin organization of the proviral DNA in infected macrophages [55,56] and the cross talk between chromatin and the splicing machinery [57-59], one would speculate that ABX464 acting on the CBC complex at the transcription site, might set up a chromatin structure favoring the splicing of the primary transcript and that this could thereby set up the long lasting effect of the drug.
Conclusions
In this report we present ABX464 first trials. Interestingly, this drug presents a new mechanism of action for HIV treatment and improves current therapies, hampering appearance of virus resistance. More importantly, it leads to a long lasting control of the viral levels. We propose that ABX464 could be used in combination with current drugs or as a simpler and more effective replacement.
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