Cancer RNAi Therapeutics Moving On

As RNAi Therapeutics targeting genes expressed in the liver, especially for orphan indications, have captured much of the recent interest in the technology, it is time to once again pay closer attention to RNAi Therapeutics in the oncology space.

Atu027 and TKM-PLK1 Moving into Phase II

Silence Therapeutics have just announced that they got the green light from the German regulators to test Atu027 in combination with small molecule gemcitabine in pancreatic cancer.  Following a short phase Ib to establish the safety of the combination (note: the original phase I was a monotherapy trial), the plan is to rapidly move into phase II which will aim at establishing proof of concept for efficacy.  Depending on the funding situation, it is possible that the company will expand Atu027 into other indications in phase II, possibly in collaboration with private investigators in the UK.

Clearly, the strength of Atu027 is its safety profile so far, one that has surpassed my own expectation for this positively charged lipoplex formulation.  The clinical attraction meanwhile is that Atu027 aims at a quite novel anti-cancer mechanistic approach by inhibiting metastatic spread by fortifying the vascular endothelia. 

The challenge with Atu027, however, is that the gene target, PKN3, remains a relative black box.  This not only demands a certain leap of faith in mechanistic terms, but also complicates the clinical development as biomarkers and other tools are not readily available.

This is quite different from TKM-PLK1, the cancer RNAi Therapeutics by Tekmira.  Polo-like kinase 1 is a hot target, but specificity/safety issues have so far dogged drug development efforts with small molecules, most notably represented by Boehringer-Ingelheim.  By contrast, the SNALP RNAi delivery approach promises to be more cancer tissue targeted and consequently should avoid some of the dose-limiting toxicity observed so far, especially the hematologic ones.

TKM-PLK1 has flown a bit under the radar and the phase I update late last year did not capture much attention.  This is probably because the response rate did not seem too much to get excited about.  However, as Ian MacLachlan, the CSO of Tekmira, pointed out at the AsiaTides meeting last week, it was a small dose-escalating phase I trial (20-30 patients) in advanced solid cancer patients and only four patients actually received TKM-PLK1 for two or more cycles (6 infusions over 2 months) at 0.6 mg/kg or above, dose and durations you would think would be required for advanced cancer patients to get a chance to respond in the first place. Notably, two out of those four showed signs of efficacy: one partial response and one with stable disease after more than 6 months on study drug.

In light of that, the phase I profile of TKM-PLK1 supports my view that it could be the foundation for an exciting PLK1-related cancer franchise for Tekmira and I look forward to seeing the full results presented at the upcoming AACR meeting.  My attention will be particularly focused at any direct measures of target knockdown.  Beyond that, with the company’s healthy financial situation, I look forward to a rich phase II strategy involving a number of solid cancers and innovative study designs such as patient selection based on genetic criteria.  As good as Tekmira's science is, they now also need to be as proactive and innovative on the clinical front.

ALN-VSP02 and CALAA01 Falling Behind

As TKM-PLK1 and Atu027 are moving into phase II, fellow early movers in oncology RNAi ALN-VSP02 and CALAA01 are falling behind. 

The next step for ALN-VSP02 will be a phase I in hepatocellular carcinoma (‘real’ liver cancer) in China under a license by Alnylam to Ascletis.  As noted by Patrick Lu of Sirnaomics at AsiaTides though, it would be a mistake to believe that the Chinese regulatory environment is any less demanding than in the West.  Au contraire, it seems that much more paperwork is required to file an RNAi-realted IND as the authorities there are keen on building their expertise in this new technology.  Expect a delay of at least 6 months from IND application to the green light to go ahead, after all the paperwork has been submitted.  For Tekmira watchers, I expect the $5M milestone thus in 2014, not in 2013.

While ALN-VSP02 still has a pulse, despite of the fact that it lacks the long circulation thought to be necessary for cancer uptake (note: delivery to liver cancer is a very different animal from delivery to normal liver; TKM-PLK1 is a long circulating SNALP), RIP CALAA-01 by Arrowhead Research.  Having said that, the value of CALAA-01 to Arrowhead Research has not been lost entirely as the company has filed for a patent covering a low/high dose strategy of minimizing immune stimulation.  

As the recent high-profile pulling from the market of a pegylated peptide (Omontys by Affymax) due to hypersensitivity, even anaphylactic reactions demonstrate (and also the infusion reactions reported by Alnylam in their SNALP RNAi Therapeutics trials), administration-related hypersensitivity reactions are an issue for the entire drug development arena.  In order to rescue the value of a number of blockbuster franchises, I expect that changing administration schedules could be one area attracting increased attention.  A patent here could have unexpected value.   

A New Crop of Cancer RNAi Therapeutics

Behind TKM-PLK1 and Atu027, a new crop of cancer RNAi Therapeutics is emerging.   Dicerna has signaled that it will finally move into clinical development (Q1 2014) with a liposomal Dicer-substrate formulation with a focus on liver cancer.  Of note, the liposomal formulation process is quite distinct from that of SNALP involving sequential core and envelope formation steps, potentially amenable to ligand-directed targeted delivery.

Another program worth following is another one for liver cancer, the subject of a collaboration of Korean company Bioneer andSanofi-Aventis.  The attraction of that program is that the nanoparticle here, SAMiRNA, comprises a single molecule, even to the extent that the RNAi trigger and delivery components can be manufactured on a single column.  Although I’m opposed to the fundamental notion that the ease of route of administration and the manufacturing process should drive drug development, don’t underestimate the attraction of meeting idealistic product profile when it comes to partnering with Big Pharma.    

The efforts above are a reminder that cancer could be an even bigger commercial opportunity for RNAi Therapeutics than liver-related indications.  The genome-wide targeting opportunity with RNAi Therapeutics has not been lost.  The pharmaceutical industry is watching.

Register for the GTC RNAi Research and Therapeutics meeting in San Francisco today (June 20-21).  Get a free RNAi Therapeutics blog T-shirt and 20% discount on registration by entering discount code 'RNABLG13'.

Tekmira and Arrowhead Advance Cancer RNAi Therapeutics

This week, important developments for two of the most advanced cancer RNAi Therapeutic candidates were announced. Arrowhead reported that it has now completed enrolment of the phase I CALAA-01 study and is initiating a dose-optimizing phase Ib study, while Tekmira disclosed that it has received the green light from the FDA to go ahead with an additional phase I study of TKM-PLK1, this time using Hepatic Arterial Infusion to complement an ongoing effort involving intravenous administration. This study will be conducted in collaboration with the National Cancer Institute (NCI).

CALAA-01: Optimizing dosing schedule

CALAA-01 is a targeted cyclodextrin-based nanoparticle formulation (RONDEL) of unmodified siRNA against RRM2, a gene involved in DNA synthesis (more background on CALAA-01 here). Enrolment in the phase I clinical trial commenced 3 years ago. Interim data were presented last year in a Nature paper and the ASCO annual meeting. At that time, about 15 patients with various forms of advanced solid cancers had been treated. Encouraging qualitative biopsy results showed that at least some of the RNAi Therapeutic reached its physical target and was able to mediate RNAi target cleavage there.

Until then, no dose-limiting toxicities had been seen. The ASCO presentation, however, revealed that there were fairly widespread, albeit low-grade (g1-2) immune stimulations (‘flu-like symptoms’). The reason why immune stimulation with this candidate was fairly common is likely related to the fact that unmodified siRNAs were used. Judging from Arrowhead’s quarterly conference call this week, it seems as if the immune stimulation warranted a discontinuation of further dose escalation. The company stated, and I certainly agree, that it should use modified siRNAs in the future to mitigate this side effect.

Interestingly, what the company apparently found in the study is that in some patients the immune stimulation subsided following repeat-administration of the drug, as if they became desensitized to the immunostimulatory potential. It is therefore the goal of the newly announced phase Ib study to find dosing regimes that take advantage of this preliminary finding to achieve higher doses, e.g. by first giving a lower dose in order to desensitize before continuing with the predicted higher, hopefully efficacious dose. If this hypothesis can be confirmed, it would not only be positive for CALAA-01, but a valuable insight for the entire field of RNAi Therapeutics.

Having said that, in light of the clinical findings so far and reservations about the chosen gene target, I do not find CALAA-01 to be a compelling candidate. While I can agree with the phase Ib study, it is more because it will allow the company to gather further information about the RONDEL delivery system without much further delay as it continues its efforts to partner and monetize the delivery technology.

Situations like these highlight the risk of RNAi Therapeutics platform companies like Arrowhead/Calando and Benitec that refuse to have in-house R&D. In this case, in-house R&D could have already readied a modified RRM2 siRNA formulation, or even a formulation targeting a more exciting cancer gene target, without too much additional effort. Instead, more needs to be invested in a candidate that is difficult to partner already and may never make it into phase II, leaving Arrowhead to hope that their argument that the CALAA-01 studies are a way of de-risking the RONDEL delivery platform will convince potential partners and investors.

In my opinion, virtual drug development may be possible for one-in-a-kind therapeutic candidates, but is not compatible with building a platform.

Tekmira and NCI to Begin Hepatic Arterial Infusion (HAI) Study of PLK-1

The clinical development of TKM-PLK1 began earlier this year with a phase I dose escalation study using intravenous administration. Similar to ALN-VSP02, TKM-PLK1 is a SNALP-based formulation, but should be more widely applicable because its pharmacology was tailored so that it can also reach tumor tissues outside the liver. What makes TKM-PLK1 particularly exciting is that its gene target is probably the most potent one in the cancer field, having emerged many times as a top hit in genome- and kinome-wide RNAi screens for rapidly killing cancer cells without affecting normal cells.

The new phase I study of TKM-PLK1 that was announced this week will not only allow the company to more quickly gather important pharmacologic information on the drug, but should also be a viable alternative route of administering TKM-PLK1 for treating primary liver cancer and cancers with liver involvement by using Hepatic Artery Infusion to expose tumors to very high concentrations of the drug which would otherwise not be possible by simple intravenous administration.

Black box less black

One challenge in the development of novel cancer therapeutics is that it can take a long time before their true potential becomes clear. Biomarkers are therefore highly sought after, with RECIST tumor response criteria being a popular way of determining anti-tumor efficacy. Unfortunately, merely measuring immediate tumor responses is not always predictive of therapeutic success as sometimes you see such responses, but without concomitant increases in life expectancies (e.g. Avastin in advanced breast cancer, and many chemotherapeutic settings), and sometimes you don’t see them, yet you do get increases in life expectancies, an issue particularly relevant for targeted therapeutics (e.g. Provenge in prostate cancer).

Access to tumor biopsies is therefore very valuable in determining at an early stage whether your drug functions as expected on a molecular level and what tissue concentrations you need to achieve to trigger these responses. It is therefore an attraction of the new study that it will allow for biopsy collection. Through this, Tekmira should further learn more about which tumors types may be most suitable (e.g. primary/metastatic, genotype, pH).

Aside from the scientific value of the study, this study has the added benefit in that it could help validate Tekmira's platform technology for cancer in a controlled and timely manner, at a time when Alnylam evaluates Tekmira’s technology mainly for knocking down genes in ‘normal’ liver. Clinical data from both these approaches are expected in coming months and should serve as catalysts.

A treatment in its own right

Liver tumors almost exclusively derive their blood supply from the arterial circulation, whereas 75% of the blood flow in normal liver comes from the portal vein. Because of SNALP pharmacokinetics, this means that directly infusing TKM-PLK1 should have the benefit of increasing the drug exposure of liver tumors while at the same time greatly reducing potentially dose limiting exposures of the normal liver(***see correction and clarification below), the predicted dose-limiting organ for SNALP technology. This therefore should make it possible to achieve higher maximally tolerated doses, an important goal in most cancer drug developments.

It is for this reason that Hepatic Artery Infusion (HAI) has become an accepted and frequently used drug delivery modality in the treatment of unresectable liver tumors. Therefore, should the results suggest therapeutic efficacy in this setting, it could well pave the way towards studying HAI of TKM-PLK1 directly in phase III studies.

****Correction and clarification: I have received justified criticism of my statement that HAI would ‘greatly reduce potentially dose limiting exposures of the normal liver’.

The criticism was based on the notion that the normal liver in patients with liver tumors would still go on and be supplied by blood from the hepatic artery in addition to the blood supplied by the portal vein. Consequently, a good chunk of what you provide by HAI would also end up in the normal liver parenchyma. On the other hand, the critique was based on the assumption that the liver tumors would not compete for blood from the hepatic artery. My take on that is that this is probably a good assumption especially for smaller tumors, but in cases where liver tumors make up the bulk of the total liver mass, at least based on high-school physics (pressure and diameter of a tube determining the flow of liquids), there should be at least some quantitative shunting of hepatic arterial blood away from the normal liver.

Moreover, the criticism raised another factor determining whether direct HAI of SNALP will enhance SNALP uptake by cancer cells relative to intravenous infusion, namely whether the SNALP particles get taken up efficiently during the first pass of SNALP through the liver tumor. SNALP uptake is determined by various factors such as time, ionization behavior, active targeting, LDL-receptor status, so that’s probably a more complicated discussion.

Overall, the point is well taken, and while SNALP administration by HAI should have a positive effect on the ratio of drug concentration in Tumor relative to Normal Liver and therefore should increase the therapeutic index, I will have to partially retract my statement in that this is mainly due to optimized exposure of liver tumors during first pass and may vary significantly from tumor to tumor, and that another reason for choosing the hepatic artery vs portal vein may be just the relative surgical ease of accessing these structures.

Targeted Delivery Strategies Coming to the Fore

Hand in hand with a rapidly expanding understanding of the biological mechanisms of RNAi Therapeutics delivery, we are hearing more and more about ligand-guided targeted delivery. Just today, mdRNA announced notice of allowance for a patent application on the identification of a peptide specifically binding to the cell surface of hepatocellular carcinoma cells, and Alnylam has recently started to talk about their discoveries on ApoE-dependent and Apo-E independent SNALP cellular uptake pathways paving the way towards new targeted SNALP delivery strategies (Systemic RNAi Delivery Roundtable). As it increasingly looks like Calando’s transferrin-targeted CALAA-01 will not remain the only targeted RNAi Therapeutics formulation in the clinic for long as targeted delivery is set to provide the next push towards more potent and safer RNAi Therapeutics delivery, I will try and briefly explain the rationale behind targeted delivery and some of the challenges that need to be overcome.

Success in targeted delivery is measured by either preferential uptake of the siRNA in the target tissue compared to non-target tissues or achieving lower efficacious dosages by taking advantage of particularly productive receptor-mediated uptake pathways, preferably both. The detailed pharmacological effects are not only determined by the ligand, but also by where it is attached to. Steps that may be affected can include biodistribution, cellular uptake once at the target tissue, or the avoidance of certain cells and tissues.

SNALP delivery for example may benefit from targeting ligands by reducing uptake by macrophages or relying on non-specific charge-charge interactions for cellular uptake, both of which can be safety liabilities. For the liver, de-targeting from macrophages may be even more important than active targeting. The DPCs by Mirus (now Roche) were particularly exciting here in that the data suggested that the right presentation of ligands (simple sugars in this case) on the particle surface may eliminate the unspecific uptake of a delivery platform and instead re-target it to new cell types. Equally exciting data by Alnylam suggests that it should be possible to greatly limit the ‘non-specific’, mainly ApoE-mediated uptake of ionizable SNALPs by shielding their surface and then re-direct them by adding new ligands on their surface. By then further increasing their circulation times through creating very stable particles (e.g. by increasing the stability of the stealth shield), a delivery platform may then also be applicable to new therapeutic application fields by increasing the chances that a ligand recognizes receptors in distal tissues.

Lipoplexes such as Silence Therapeutics’ Atuplexes may also benefit from targeting ligands. Since the interaction of immune cells with blood endothelia is very well studied this may e.g. allow it to be targeted to specific endothelia such as the blood-brain-barrier.

Targeting ligands are already part of many siRNA-conjugate approaches. Achieving endosomal release in addition to cellular uptake is a big challenge for this area of delivery, and it will be interesting to see whether in fact those receptors that prove effective for nanoparticle delivery may be the types of receptors to be avoided for siRNA-conjugates in favor of channeling them into more non-specific pathways.

There are, of course, also challenges associated with targeted delivery. One is to identify suitable ligand-receptor interactions as endosomal maturation processes can differ greatly, e.g. in the degree and rate of acidification and receptor recycling, which imposes new types of pharmacokinetic demands on a delivery system.

A systematic effort to discover the best receptors may be to screen a panel of siRNA-nanoparticles containing (single-chain/nanobody-type) antibodies on their surface and that are targeted to a wide array of cell surface receptors and then select those with the best silencing results. The most promising receptors, hopefully patentable, may be pursued then either with the antibodies themselves or alternative, smaller ligands. New ligands to given receptors may be discovered through panning peptide display libraries against that receptor, something e.g. that mdRNA does with their trp-cage peptide libraries.

Avoiding adaptive immunity, especially to novel designer ligands is another added challenge for targeted delivery. And finally, when all these questions have been answered, the not-so-trivial task is to find formulation methods that allow for clinical and commercial scale-up of the more complex particles. At the end of the day, however, it is those platforms for which a detailed mechanistic basis has been established and those teams that have turned formulation into an art that will succeed. It can be done.

CALAA-01 Results Indicate RNAi Therapeutics to Revolutionize Solid Cancer Treatment

In yet another important proof point that RNAi Therapeutics is slowly but surely morphing into a therapeutic reality, Calando just reported in the highly prestigious journal Nature an interim analysis of their phase I clinical study of CALAA-01 demonstrating an RNAi mechanism of action in solid cancer patients. The results are the most direct demonstration of RNAi in Man thus far, and, at least equally significant, show that the Enhanced Permeability and Retention (EPR) effect is indeed about to turn into the Achilles' Heel of solid cancers in the era of nanoparticle siRNA delivery.

Solid cancers are considered by many the most important near-term value driver for RNAi Therapeutics. The fact that Calando’s RONDEL nanoparticle delivery technology has now been shown to accumulate in solid cancer tissues is not only an important de-risking event for CALAA-01 itself, but it immediately suggest that RONDEL, and also quite likely other nanoparticle-siRNA delivery technologies, can facilitate the targeting of essentially any gene in solid cancers. It is this flexibility of the platform that makes RNAi Therapeutics ideally suited to satisfy the demands of a genetically such heterogeneous disease group as cancer.

Nanoparticle-siRNA delivery such as RONDEL is currently the most promising approach for treating solid cancers with RNAi Therapeutics. All of these rely on the particles being able to circulate sufficiently long in the blood stream and being small enough so that they can passively accumulate through EPR in the solid tumors as these lack an effective way of draining them through lymphatics. While the absolute amount of RONDEL particles that accumulated in the tumors remains to be determined, the Nature study clearly showed that a decent amount of them does accumulate there at ~0.6mg/kg.

While particle stability and ability to accumulate through EPR is a critical first step where we should also expect to see important differentiation between the technologies, once there, the nanoparticles then have to be taken up by the cancer cells and moreover mediate the cytoplasmic release of the RNAi trigger into the cytoplasm. Once again, RONDEL meets this challenge as it was possible to detect cleaved target mRNA characteristic of an RNAi mechanism of action. Although the so called RACE assay used for this analysis is not quantitative, in my experience, you have to get sufficiently robust RNAi to cleanly detect such products. Consistent with this notion, well over 50% target knockdown was detected for CALAA-01, although the limited number of samples (3) and the reliance on often only historical negative controls leaves open the question of the exact knockdown potency of CALAA-01.

It is in fact the pioneering work with RONDEL-siRNA delivery from which this pharmacokinetic model has been derived and that is now informing quite a bit our thinking of how to design siRNA-nanoparticles for solid cancer applications. For example, it was work on CALAA-01 that showed that the utility of the (transferrin) targeting ligand is in enhancing the cellular uptake of the nanoparticles, not so much in concentrating them in the tumors in the first place, and should be influential in the selection of targeting ligands being contemplated now for all types of delivery technologies, including SNALPs.


Summary of CALAA-01 results


CALAA-01 was also the first serious RNAi Therapeutics candidate for solid cancers that had entered the clinic in 2008. For a more thorough molecular background on this program, please refer to a previous entry (here) that was part of a 3-part collaborative series with Tobias Wolfram on the first RNAi Therapeutics solid cancer candidates. Briefly, CALAA-01 is a self-assembled nanoparticle system based on a cationic co-polymer containing cyclodextrin complexed with siRNA targeting RRM2, a gene involved in DNA replication, and that like most of the advanced nanoparticle-siRNA systems is stabilized through pegylation (some of these carry the transferrin targeting ligand). While Tobias and I had cautioned about a relative lack of preclinical in vivo characterization of CALAA-01, particularly not having directly demonstrated that CALAA-01 can knock down genes via RNAi in solid tumors following intravenous administration, it seems that the gamble of skipping some of the pre-clinical proof-of-concept has paid off for the investigators and Arrowhead Research (of which Calando is a subsidiary).

It was also a clever move that, amongst other tumor types, a number of melanoma patients were enrolled as such tumors can be easily accessed through biopsy and thus represent good material for molecular analysis of a clinical candidate. 3 of these volunteered to be biopsied. Despite the limited sample size, a number of conclusions can be drawn from the data: 1) RONDEL-siRNAs accumulated in the tumor tissues in a dose-dependent manner; 2) consistent with this, in the patient that received the highest dose, an RNAi mechanism of action was unambiguously confirmed by 5’ RACE; 3) this effect was long-lasting (>4 weeks) and accompanied by mRNA knockdown and disease stabilization; 4) RONDEL was safe and well tolerated in a study that involved repeat administrations and dose-limited toxicity has yet to be reached (right now at ~0.6mg/kg with dose escalation ongoing).

Clearly, these are highly encouraging results and call for the continuation of the phase I studies and further investigation of the modular RONDEL delivery platform. Importantly for shareholders, it should put Arrowhead in a better position to fulfill their stated goal of monetizing RONDEL either through licensing or outright sale. Part of the value would derive from the pioneering position of the RONDEL technology and from representing a complementary approach to lipid-based delivery which is currently dominating RNAi Therapeutics development programs.

Implications beyond CALAA-01 and RONDEL

Taking a step back, the CALAA-01 results add to the increasing amount of clinical data that first of all lay to rest concerns that there is a prohibitive fundamental (acute) toxicity issue for RNAi Therapeutics per se, and secondly provide evidence that therapeutic reality is within the reach of current systemic RNAi delivery technologies. They come on the heels of SNALP-ApoB results that showed preliminary signs of RNAi efficacy at a similar dose level while ongoing improvements in SNALP efficacy continue to widen its therapeutic index, and continuing dose escalations for both Alnylam’s VSP-02 and Silence Therapeutics’ Atu-027 solid cancer programs which can be interpreted as confirming the tolerability of some of the important delivery technologies in the space (SNALP and Atuplex).

With such progress, urgency to invest in RNAi Therapeutics, particularly in enabling delivery technologies should return. As the transition into the clinic is fraught with uncertainties for new chemistries (safety, efficacy, and manufacturing scale-up), it is particularly the less than a handful of delivery technologies that have made it there for which a land-grab might erupt as is also indicated by the fact that Tekmira now counts 8 RNAi-related pharmaceutical collaborators (both early and advanced stage), mdRNA at least 3 such early-stage collaborators, and a rumor-driven rise in the shares of RXi Pharmaceuticals possibly due to their mysterious claims on ‘self-delivering’ siRNAs. With the publication of the results that are about to garner widespread attention, Arrowhead Research/Calando should, of course, also get a few enquiries for RONDEL as it resumes its search for partners. Considering the generally weak share prices of RNAi Therapeutics companies, all the ingredients seem to be in place to see related deal activities, with a significant portion of the value being driven by the solid cancer opportunity for RNAi Therapeutics.

Update May 29, 2010: More details about the study, including the safety profile was published recently in an abstract for the upcoming 2010 ASCO cancer conference:

Systemic delivery of siRNA via targeted nanoparticles in patients with cancer: Results from a first-in-class phase I clinical trial

Author(s): A. Ribas and colleagues

Background: Systemically delivered small interfering RNA (siRNA) would allow targeting oncogenic molecules beyond current approaches. We report on the first siRNA trial with a targeted nanoparticle delivery system. Methods: Open-label, dose- escalation trial in pts with solid refractory cancers receiving 4 i.v. infusions (30 min) on d 1, 3, 8, and 10 of 21-d cycles. CALAA-01 nanoparticles consisted of a cyclodextrin-based polymer, transferrin protein (hTf) targeting ligand, polyethylene glycol (PEG) for stability, and siRNA against ribonucleotide reductase M2 (RRM2). The 70 nm particles were designed to minimize renal clearance and allow tumor vasculature permeation with binding to tumor hTf receptors (TfR). Primary endpoints (safety, MTD determination) were based on the first cycle. Results: 15 pts accrued to 5 dose levels (3, 9, 18, 24, 30 mg/m²), median age 62 (range 53-85). Most common histologies: GI (4), melanoma (3). Dose escalation progressed with no DLTs. Most common treatment-related AEs: fatigue (47%), fever/chills (33%), allergic (33%), constipation (33%), nausea/vomiting (20%), all g1-2. 1 pt had g3 anemia and 2 pts had g2 thrombocytopenia, all shortly after CALAA-01 infusions with rapid recovery. 1 pt had possibly-related sinus bradycardia (g2). No objective tumor responses were seen; 1 pt at highest dose had stable metastatic melanoma for 4 mo, a change from prior course. Biopsies in 3 pts (melanoma) showed particles in tumors. At 30 mg/m² RRM2 knockdown (mRNA and protein) was seen with confirmation of mechanism by specific cleavage sequence (RACE-PCR). TfR was not downregulated in cancer cells.Conclusions: Systemic delivery of siRNA via targeted nanoparticles is safe and can induce specific, siRNA-mediated gene silencing. This approach could be expanded to any currently undruggable cancer therapy target.

Calando’s RONDEL RNAi Therapeutics Delivery Promising, but CALAA-01 Prematurely Entered into Clinic

[Part 1 of a 3-part collaborative series with Tobias Wolfram on the first notable attempts at RNAi Therapeutics for solid cancers that have entered the clinic]

When during the company's R&D day the CEO of Alnylam, John Maraganore, highlighted Calando’s cyclodextrin-based siRNA delivery technology (RONDEL) as one of the noteworthy non-SNALP systemic siRNA delivery technologies out there, it certainly piqued my interest. This is not least because any cash infusion and longer-term commitment by a partner like Alnylam would do wonders for the parent company of Calando, Arrowhead Research which is a conglomerate of early-stage, IP-focused business units and has just barely scraped by bankruptcy through a diet of cutbacks. While I always remembered the maturity of RONDEL, developed in the Mark Davis lab at Caltech, to be years behind SNALP, Tobias and I decided to to take a closer look at the development path of CALAA-01, the first clinical RONDEL delivery candidate and also investment focus of reorganized Arrowhead Research.

When Tobias first heard of the technology, it struck him as a very elegant, because simple, and modular method to formulate targeted nanoparticles. In fact, there are not many targeted nanoparticle siRNA delivery approaches where the components supposedly can be assembled by the pharmacist just before patient administration. RONDEL siRNA delivery consists of mixing together siRNA, a short cyclodextrin-containing polycation, and adamantane-coupled PEG stabilizers some of which carry a transferrin ligand, so as to create 60-80nm particles. These particles were rationally conceived to satisfy a range of pharmacologic and formulation considerations. Their suitability for solid cancer relies on the enhanced permeability and retention (EPR) effect of nanoparticles with reasonably long circulation times (here supposedly achieved by PEG stabilization), the ability of the particles to be taken up into cancer cells by transferrin receptor-mediated endocytosis and their subsequent release into the cytoplasm in a pH-dependent manner.

Unfortunately, what we soon came to realize was that while the concept is a very nice one indeed, the particles, particularly CALAA-01, remain to be better characterized both physically (shape, uniformity, storage and biological stability etc) and for their RNAi knockdown ability in vivo. For example, knockdown of RRM2, the target of CALAA-01, and subsequent tumor inhibition have not been demonstrated in a convincing in vivo system. Instead, knockdown efficiencies have largely been limited to in vitro studies. Moreover, these involved siRNAs that were selected with what today would be considered outdated methods and probably as a result were not very potent. The in vivo studies were essentially limited to pharmacological investigations, with the combination of in vitro efficacy and in vivo pharmacology forming the rationale for moving CALAA-01 into the clinic. Moreover, even when considering only the pharmacology, measures such as biodistributions and circulation times did not fit the model which may be explained by nanoparticle instability in vivo, something that really needs to be investigated further. Also, since the siRNAs were unmodified it strikes me as rather strange that no innate immune induction and only moderate adaptive immunity were reported.

What I found to be a valuable take-home message from those studies, although according to Tobias’ liking resting too much on indirect evidence, but not necessarily data obtained with the CALAA-01 clinical candidate, was that the utility of the targeting ligand appeared to be in increasing the cellular uptake of particles with little positive surface charge, less so in skewing the biodistribution towards the solid cancers per se. This could also be because the particles were cleared relatively rapidly from circulation, mostly into the kidney and bladder, which raises further questions about the purity and integrity of the particles. In this light, the mention of nanoparticle assembly by a pharmacist may also be interpreted a necessity due to storage problems of fully formulated particles. Nanoparticle assembly is notoriously sensitive to even slight changes in parameters such as temperature, speed of mixing etc so that it would be preferable for the physician to just administer the drug without the need for prior handling. It is therefore unfortunate that results from long-term storage and robustness of the formulation method were not presented. Nevertheless, an enhanced cellular uptake through the addition of a ligand could critically increase the therapeutic index of a cancer RNAi Therapeutic, and the modular nature of the RONDEL system should easily facilitate such additions.

Our assessment that CALAA-01 was probably entered into the clinic too early naturally rests on the publicly available data only. The publication dates of the relevant data, however, strongly suggest that they indeed represent the relevant data points and considering the financial situation of Calando, it would not appear that much were to be gained by holding back on positive data. This is by no means to belittle what otherwise is very rich science. Unfortunately, it is here that the tension between corporate demands for advancing a pipeline and the need to sufficiently advance the science is most evident and in the end risks harming both objectives. Optimistically, completion and evaluation of the CALAA-01 phase I trial will allow for valuable insights into the performance of RONDEL delivery in man for the benefit of any follow-up programs. Without any such strong data, it is questionable however whether the promise of RONDEL as a differentiated and flexible platform for RNAi Therapeutics delivery alone will be enough to make Arrowhead Research a good RNAi Therapeutics investment.

PS: To expand on the latter point, Tobias and I also discussed that, in general, it is easy to caution against entering RNAi Therapeutics candidates into the clinic early and dismiss such as a move of desperation. On the other hand, the case can be made that, when it comes to RNAi Therapeutics as a broadly applicable platform, clinically evaluating candidates which for example would not be expected to effect large knockdowns can be justified in that the data coming out of these studies may provide timely data invaluable for follow-up programs using very similar delivery approaches. Although investors will rightly fear the costs of a failed trial particularly for small biotech companies, such data may be valued more highly by a potential Big Pharma partner. This argument receives added weight in an environment like now where it is very difficult to raise capital from the public markets, and partnering is the primary means for small biotech to obtain capital at acceptable terms.