Checkpoints Drugs Promise to Unlock RNA Therapeutics Cancer Potential

If you are involved in cancer drug development, you are probably thinking of ways to exploit checkpoint inhibitors for your purposes.  Checkpoints are the mechanisms whereby cancer cells avoid being recognized by the immune system as foreign and the initial clinical results of inhibiting them, e.g. by targeting CTLA4 and PD-1/PD-L1, has caused immune-oncology as a whole to take the cancer drug development space by storm.

Suddenly, every scientist and their technician believe that by taking checkpoints out of the equation, their immune approach to cancer, which in many cases was marked by failure after failure in the past, will work. 

Take e.g. cancer vaccines and the idea of grinding up tumors and use the cell mash to train dendritic cells.  Sounds compelling to me, but I also know that I lost a bunch much money on the very same idea 10 or so years ago with nothing, but some early promising data from the clinical Wild East to support it. 

Others meanwhile believe that their technologies may enable immune-oncology by e.g. being able to modulate the tumor microenvironment so as to permit better access of the tumor killing cells.

RNA Therapeutics are no exception.  RNA Therapeutics, due to its endless targeting opportunities, has always been considered promising for oncology, but has struggled to show clear-cut success either due to drug delivery issues or due to the difficulties of predicting immune activities based on rodent studies.

Emblematic of this reversal of fortunes is the toll-like receptor (TLR) field of activating the innate immune system with oligonucleotide stimulants.  The idea here is to provide an environment that is more conducive to tumor cell killing and/or to directly impact tumor cell survival (e.g. interferon stimulation).  Art Krieg, of TLR9 (CpG) fame, but who has been erring as its lost son in the (blooming) deserts of RNAi, RNaseH antisense, splice modulation, and RNA activation, is apparently re-energized enough to go back to TLR Therapeutics and has started Checkmate Pharmaceuticals which will likely try and harness TLR agonists for cancer immunotherapy.

Nanoparticle delivery may also see a revival.  This is because they have a propensity, usually undesired, to be taken up by phagocytic cells, some of which may be effective in antigen presentation (e.g. dendritic cells).  I have thus noticed that a number of mRNA cancer vaccine approaches involve nanoparticle delivery with the aim of expressing tumor antigens in the training camps of the immune system, the lymph nodes.  

Nanoparticles may also be a way to knock down the gate-keepers in the tumor microenvironment which inhibit tumor infiltration by cytotoxic T (incl. CAR T-cells) and other helpful immune cells.  Since gate-keepers should be most useful when positioned at the entrance, this might actually take advantage of another limitation of many nanoparticle delivery technologies, namely getting stuck close to the vasculature instead of penetrating deep into the tumor.

Once deeper into the tumor, the struggle may not be over for cytotoxic T cells and tumor eating cells due to potentially immunosuppressive activities in the tumor microenvironment.  This is the new positioning for the phosphorothioate-based antisense molecule ISIS-STAT3 by Isis Pharmaceuticals and AstraZeneca for which, like for other phosphorothioate-based approaches, the demonstration of robust uptake and gene knockdown activity in tumor cells themselves is lacking, but functional uptake in cells of the tumor microenvironment has been reported.

Finally, RNA Therapeutics such as CRISPR genome editing or straightforward self-delivering RNAi (see recent license of MirImmune from RXi Pharmaceuticals) can be tools for the ex vivo preparation of T- and dendritic cells.  A self-delivering approach may be advantageous here as it may function in normal cell culture media and thus not confound cell signaling pathways in the maturation of these cells.

Investor, tread carefully

Overall, I’m convinced that there are synergistic potentials to be exploited and checkpoint inhibition may open the door to certain that have failed in the past.  Nevertheless, one should be mindful that most immune oncology drugs only work in a fraction (maybe 25%) of patients and the current hype around immune oncology guarantees that there will be many bad apples for investors to avoid.  Trust me, I've seen it when RNAi was indiscrimately hyped and abused for short-term financial gain in 2006-8.  Just because you can contrive a link to immune oncology or because a desperate Big Pharma does a deal, does not mean that all the biological problems will dissolve.  The bubble will burst after which mostly only well-financed quality plays will recover.

Disclosure: I am fishing for short opportunities in the immune oncology space as a hedge against a (hopefully temporary) correction to what seems to be an in a number of areas (e.g. gene therapies, immune oncology, one-drug orphan wonders such as Alexion etc) overheated biotech space.

RNAi Therapeutics and Innate Immunity- Keeping the Field Honest

As part of the RNAi Therapeutics review series in Human Gene Therapy earlier this year, former Protiva scientists Adam Judge and Ian MacLachlan (both now with Tekmira following the Protiva-Tekmira reunion) made some rather bold statements with regards to the interpretations of a number of pre-clinical RNAi Therapeutics validation papers (‘Overcoming the innate immune response to small interfering RNA’). As part of the same reviews series, in the risk section of “The Business of RNAi Therapeutics”, I also cautioned that some of the first RNAi Therapeutics candidates may show clinical efficacy, but not necessarily for all the right reasons.

The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.

Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.

Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.

The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.

But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).

As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.

Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.

The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.

Developing Multi-Functional RNAi Therapeutics

The great attraction of developing therapeutics based on RNAi is its inherent specificity and applicability to virtually any gene for the rational design of drugs. The specificity of gene silencing is not just a theory, but very powerfully attested by the many successes of applying RNAi even to genome-wide screens to uncover gene function (and also more targets for RNAi). Delivery of the RNAi inducer to the cells of interest would further add to such specificity and consequently safety.

However, as has long been known, certain nucleic acids elicit immune responses, and siRNAs are no exception to this. Although this does not apply to appropriately designed and vetted siRNAs, when it does, it may well interfere with the interpretation and predictability of the knockdown phenotype. However, instead of describing once again methods whereby such responses can be avoided and phenotypes more consistently obtained (siRNA length, structure, and modifications; bioinformatics etc), I would like to take the opportunity here to point out the potential for RNAi Therapeutics that include a immune-regulatory element. Moreover, as this is typically related to the uptake of the RNAi formulation in cells other than the primary target cells, I would also like to make us consider the potential for RNAi Therapeutics exploiting the entire biodistribution of a particular RNAi drug delivery system.

When we think about indications such as cancer and viral infections, the importance of the immune system in eliminating the disease cannot be underestimated. Cytokine therapies e.g. are well known to these areas of medicine and it is no coincidence that there have been long-standing efforts in harnessing the ability of nucleic acids to induce TLR and other immune signaling pathways to improve both cellular and humoral immune responses.

It is therefore conceivable that an immuno-stimulatory siRNA is not necessarily screened out during the siRNA selection process, but is deliberately packaged into a nanoparticle which in addition to the primary target cells (cancer cell, virus infected cell, etc.) would also be taken up by phagocytic cells where the cytokine stimulation would lead to enhanced antigen presentation or the augmentation of monoclonal antibody therapies. To further take full advantage of the biodistribution of the RNAi formulation, the RNAi drug could also contain two or more different siRNAs, each one designed to knock down a suitable gene in the various cell types that the nanoparticle is taken up in (e.g. in the case of a liver delivery system that enters both Kupffer cells and hepatocytes, an siRNA against a immuno-regulatory gene expressed in the Kupffer cells and maybe other phagocytes and one siRNA for a hepatocyte-specific gene).

In addition to immune-stimulation, certain siRNA formulations could be used for concomitant gene knockdown and immune suppression. As work by Protiva (now Tekmira) has shown, siRNA modifications may not only be used to avoid unwanted TLR signaling through siRNAs, but to inhibit these TLR responses in trans. A single modified siRNA could thus be employed in a two-pronged gene knockdown/TLR signaling inhibition strategy for treating autoimmune disorders.

Based on the acquisition of prior TLR company Coley by Pfizer, Alnylam’s vaccine spin-off intentions, and Tekmira’s IP and know-how on the immunological properties of nucleic acids, I would not be surprised if we should be hearing relatively soon more about such multi-functional RNAi Therapeutics.

Rather than considering innate immune responses and imperfect biodistribution as nuisances, it may well turn out that a number of RNAi Therapeutics may get the extra bit of efficacy out of simultaneously modulating immune responses and knocking down genes in multiple cell types. In my opinion, the medical and commercial opportunities for that are currently underappreciated.

….and the Winner Is: Pfizer and Coley Pharmaceuticals

While at the Keystone meeting, I was reminded that we may already have a winner for the RNAi Therapeutics Deal contest highlighted on this blog. As this contest as to who will be involved in the next large transaction of the RNAi Therapeutics space was still ongoing, Pfizer quietly bought Coley Pharmaceuticals. Although I noted this in one of my entries
and wildly speculated that this $164M acquisition may serve as a stepping stone towards grander RNAi Therapeutics ambitions, it was the 2008 Pfizer Analyst Day that made it clear that the major focus of Coley together with the RTC in Boston would be the development of RNAi Therapeutics.

Given the importance of considering TLR receptors for the design of RNA therapeutics, only highlighted by the recent Nature paper
on the class-related antiangiogenic potential of dsRNAs for the treatment of age-related macular degeneration, it now appears to have been a particularly shrewd acquisition bringing in TLR know-how as well as an established nucleic acids operation. For those hoping another Pfizer-related deal may be imminent, I would rather be skeptical as Pfizer probably has now their hands full organizing and evaluating the potential of this new platform.

Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 2

The second day of the Meeting was filled with cutting-edge science related to RNAi drug development with emphasis on RNAi mechanism, delivery, and cytokine responses triggered by various nucleic acid classes.

Tuschl kicked off the day by presenting data on the systemic identification of Argonaute interaction partners (mostly published data) and a new approach towards identifying microRNA targets. In contrast to microRNA target identification algorithms which heavily rely on sequence conservation, Tuschl and colleagues identified RNAs pulled down in Argonaute immuno-precipitations and then sequencing. Validating the approach, these RNAs were enriched for sequences with seed targets of the most abundantly expressed microRNAs in the tested cell line (1.8x enrichment over random). He finally reported on small molecule screens aimed at identifying inhibitors of microRNA maturation, however, with less success. It appears to me that he should stick to his guns and work at inhibiting microRNAs and their precursors instead by nucleic acids, such as antagomirs, which have proven to be much more potent and specific inhibitors of microRNA activity.

Ingo Roehl (Roche Gmbh, former Alnylam Europe) gave a nice presentation on progress in the development of analytical chemistries to support DMPK/PD (Drug Metabolism and Pharmacokinetics/dynamics) studies of RNAi Therapeutics. It was encouraging to see significant improvements that now allow siRNA and endogenous microRNA quantitations in the picomole-femtomole range in biological samples at high-throughput. In contrast to methods used by other groups, their coupled HPLC mass-spec setup allows for the discrimination between intact siRNAs and their degradation products. However, further improvements in sensitivity are desired as the potency of siRNAs means that biological activity can be observed long after siRNA levels drop below the level of quantitation.

Peter Linsley from Rosetta Inpharmatics (a subsidiary of Merck), which gained early “notoriety” for being the first group to point out the problem of off-targeting by siRNAs some years ago, highlighted the dilemma posed by the fact that the off-target signature of a given siRNA is extremely different in mouse and man. This means that mice will not function as a safety model for off-target toxicity studies. Furthermore, by limiting oneself to cross-species specific siRNAs for the sake confirming the treatment in the animal model, many siRNAs with better potencies and off-targeting signatures in humans may ultimately be missed. Also of interest is the fact that by leveraging its genomic expression profiling capabilities, Rosetta Inpharmatics has now established microRNA overexpression signatures for over 150 microRNAs, raising the question when Merck will officially announce its entry into the microRNA therapeutics arena.

Following on from his identification of SID-1 in being required for systemic RNAi in worms by acting as an siRNA channel, Craig Hunter (Harvard University) presented data to support a model in which siRNAs enter and exit cells via SID-1 by diffusion, whereas other proteins such as SID-2 are involved in binding RNAs so as to increase their concentration close to the siRNA channel. That this is relevant for the development of RNAi therapeutics is highlighted by the recent Nature Biotech paper by Dr. Stoffel from the ETH Zurich and Alnylam where data suggested a role for SID-1 in taking up siRNAs following docking of siRNA-studded lipoprotein particles to their cellular receptors. In addition to walking his audience the published data-heavy paper, Stoffel stated that he saw no reason why it should not be possible to create artificial lipoprotein particles containing siRNAs and targeting agents.

Instead of Dinah Sah or David Bumcrot presenting Alnylam’s progress on cancer RNAi therapeutics, Rachel Myers stood in to give a general overview of the approach Alnylam takes to RNAi-based drug development. Since little new primary data was presented, I thought it is noteworthy that both Ingo Roehl and Myers gave scientific credit for the development of the hotly contested SNALP development to Protiva Biotherapeutics and that the delivery technology used in last week's Nature paper on microRNA competition by siRNAs was called "Alnylam proprietary”. Also, it was a great relief for me to hear that Alnylam had reproduced all of Sailen Barik’s data on the RSV-RNAi treatment paradigm, including efficacy of siRNAs after RSV infection. Indeed, siRNAs reduce viral titers up to 3 days after infection in a mouse model where maximal viral burden is seen by day 4 of the infection. Moreover, illustrating that the upcoming proof-of-concept studies for RSV in the experimental infection model is not merely an academic RNAi therapeutics de-risking exercise, the patients that Alnylam expects to treat with ALN-RSV01 will have infection in the upper respiratory tract with little or no exposure in the lung. Consequently, ALN-RSV01 will be a combination of treating upper respiratory tract infection and prophylaxis for the lung. Everybody, of course, is quite restless now to learn about the outcome of the phase II experimental infection results which appear to be on track and from which will be presented in detail early next year without precluding a PR on the results in December.

Before the session on RNAi/oligonucleotide delivery, a number of speakers spoke about recent findings on the immunogenic properties of various forms of RNAs, including siRNAs. The importance of considering the expression pattern of the main oligonucleotide receptors (the endosomal TLR-3,7,8,9 and the cytosolic RIG-I and MDA-5), their localization, and their exact ligands became evident and will inform modification strategies aimed at avoiding the induction of unwanted cytokine responses following siRNA administration which is something that Rachel Myers noted Alnylam is still trying to get a better handle on (see the delay in the pandemic flu program). Other groups, however, such as Gunther Hartmann’s group from Bonn, embrace the immunogenic properties of some, particularly unmodified siRNAs and would like to combine it with their silencing activity for treating cancer and viral infections. Some encouraging data in that regard were presented.

Song Li (Pittsburgh) presented the use of neutral lipids for delivering a variety of oligonucleotides to the pulmonary circulation. Like others at the meeting, this approach illustrates a trend away from using cationic liposomes which have been associated with interacting with components of the blood and may trigger certain toxicities. Delivery to endothelial cells of larger vessels and capillaries was particularly efficient, and his group is currently collaborating with ISIS on targeting endothelin-1 for treating hypertension. Encouragingly, he was able to report on good gene knockdowns and quite impressive in vivo efficacy data, such as the reduction of hypoxia-induced right ventricular hypertrophy.

Similarly impressive in vivo efficacy data were then reported by Klaus Giese from Silence Therapeutics, mostly located in the host city of Berlin. Without showing all the controls, tumor burden, metastasis, cell proliferation were all strongly reduced following systemic delivery of siRNAs in a number of mouse tumor models. Although I cannot agree with his repeated claims on the uniqueness of their Atu-siRNAi design and related IP claims, the Atuplex delivery technology certainly deserves more credit. Unlike other lipid-based delivery methods, siRNAs here associate with the liposomes externally thus allowing liposomal charge to be modulated from cationic, via neutral, to anionic. Of practical importance, these particles can be lyophylised for storage and shipping and then resuspended without loss of silencing activity. Progress has also been made on lung delivery and a number of pre-clinical programs are being pushed forward into the clinic.

The founder and CEO of the nucleic acid delivery company Novosom, Steffen Panzner, continued the string of impressive in vivo efficacy data in a mouse model of RA (inflammation of the paws) following delivery of their proprietary siRNA-loaded Smarticles, a charge-reversible liposomal delivery technology. One problem of using the more desirable, in terms of safety, neutral and anionic lipids, is their reduced siRNA binding affinity. Novosome’s amphoteric liposomes circumvent this problem by binding siRNAs at low pHs, at which point they are positively charged, and rapidly shifting them to higher pHs for closing the liposomes. During this process, most of externally bound siRNAs are shaved off, with the additional benefit of consequently minimising endosomal exposure of the siRNAs which may trigger TLR7-mediated cytokine responses.

The scientific day was closed by a talk from Alan Sachs of Merck who set out to emphasise that despite a dearth of recent information on Sirna’s/Merck’s preclinical and clinical RNAi therapeutics pipeline, that they were more than ever committed at developing RNAi therapeutics. Unfortunately, this was not followed by a presentation of primary scientific data and specific examples, but rather how Merck thinks about RNAi drug development in general and repeated appeals of please partnering with Merck, particularly in the area of targeted siRNA delivery. It appears to me that Merck would like to rival Alnylam in accessing the best minds in the RNAi drug delivery field and is offering to send out their siRNAs for free so that others can formulate them and report back on their findings without any strings attached as to the use of these results. Sachs feels that Merck, through their expertise on the genomics and genetics of gene expression through Rosetta Inpharmatics, that it knows best which targets are most suitable to go after for drug development. Since siRNAs allow essentially any gene to be targeted, he like other feels that this is the natural drug development platform to be harnessed and thinks about 21 month development timelines from gene identification to preclinical proof-of-concept given the availability of suitable biomarkers. While searching for more targeted delivery solutions and collaborators, Merck is meanwhile establishing a platform for siRNA delivery to the liver and, encouraged by ISIS' 301012 results, is clearly motivated in targeting ApoB100 with siRNAs for the treatment of hypercholesterolemia. I would not be surprised at all, to see them join the PCSK9 frenzy as well. Being second in a $50-60B should not be that bad.
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