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.

Data from Nucleonics HepB DNA-directed RNAi Therapeutics Study Published

Following major IP battles with Benitec and tensions high within the company, DNA-directed RNAi Therapeutics company Nucleonics became the first major RNAi Therapeutics company to go out of business in 2008. During the liquidation process, Alnylam surprised observers by scooping up Nucleonics’ IP assets. Adding to the confusion, Nucleonics had just initiated dosing in a phase I study of NucB1000, a candidate for the treatment of Hepatitis B Viral infection. The fate of this trial has been a mystery.

Now, after three years, scientists and clinicians involved in the study broke their silence and reported data from the study in the journal Antiviral Therapy (Gish et al., 2011). The results from the prematurely terminated study that had enrolled only three patients in the first dose cohort were largely in line with (at least my) expectations. More surprising, however, were some disclosures related to Alnylam’s apparent interest in NucB1000 and DNA-directed RNAi Therapeutics.

NucB1000 Background

The active ingredient of NucB1000 is a plasmid DNA that encodes for 4 shRNAi triggers targeting various regions of hepatitis B virus. The ability to easily accommodate a number of RNAi trigger sequences in one clinical candidate is one of the advantages of ddRNAi Therapeutics and particularly useful for antiviral applications as it should minimize viral escape. Using ddRNAi as opposed to a synthetic siRNA for HepB infection is also reasonable given the chronic nature of the disease.

This, however, is about it in terms of the positive attributes of NucB1000. The reason why I never thought this candidate had even a remote chance of clinical success is the fact that it uses a cationic lipid formulation to deliver the DNA to affected hepatocytes. Unlike the efficient liposomal delivery of small, synthetic siRNAs to the cytoplasm of hepatocytes, the field of gene therapy is still far away from using non-viral means to efficiently deliver large plasmid DNA- about 100-200x the molecular weight of siRNAs- to the nuclei of hepatocytes where the DNA can serve as the template for shRNAi trigger transcription. It is not just the size that complicates the cellular uptake and release of the nucleic acid, but the requirement for getting the DNA from the cytoplasm to the nucleus is the major rate-limiting step differentiating it from synthetic siRNA delivery.

The authors addressed this deficiency by stating that even if uptake efficiency was limited per given plasmid infusion, the long-lived expression from the plasmids means that it may be possible to achieve ddRNAi activity in sufficient numbers of hepatocytes simultaneously by multiple administrations. While I can follow the logic, in the absence of convincing pre-clinical data of this concept, it remains just that, a concept.

Of equal concern is that cationic lipid-formulated large plasmid DNA carries a high risk of triggering innate immune stimulations following intravenous administration. This has already been a challenge for lipid-formulated small siRNAi triggers, but it is less of a fundamental challenge there because of the limited sequence information in a given siRNA. This means that any inherent innate immune stimulatory potential can be relatively easily controlled by chemistry. The much larger plasmid DNA, which typically is of bacterial origin, does not allow for such control.

I should mention that in light of these concerns the authors state that no apparent immune stimulations were observed in the mouse and dog studies at much higher dosages than were anticipated for the trial.

Nevertheless, it is surprising to me that the FDA allowed this trial to go ahead based on pre-clinical co-transfection efficacy results that appeared to me to be glorified in vitro, and not in vivo models of HepB infections. It may be for this reason that the study authors made the point that they saw antiviral efficacy in a model that did not involve co-transfection:

‘Preliminary data showed that 1% of mouse hepatocytes were transfected. Subsequent experiments in which spike recovery was performed demonstrated that nearly 10% of cells were transfected; actual rates varied between 6% and 9% (Nucleonics, Inc. and Alnylam, unpublished data). A number of explanations for the discrepancy between this rate and the 20% knockdown of HBsAg have been postulated.’

‘A 20% knockdown of HBsAg’ to me does not sound like a robust pre-clinical efficacy result in support of clinical studies. I doubt that the FDA would have let the IND application pass solely based on this animal efficacy model.

I know, a non-viral ddRNAi approach sounds great on paper, and would you believe it, not just the VC investors and Nucleonics management were fooled, but also a company like J&J was about to contribute a fair amount to a planned $25M round C capital raise.

NucB1000 phase I results

Altogether only 3 of the planned 15 patients received NucB1000 in the phase I study. This was the first dose cohort which involved 5mg of plasmid DNA, ~0.06mg/kg. As expected, immune stimulation was observed in all 3 patients: a transient fever, accompanied by cytokine elevations, which quickly resolved following antipyretic treatment. This was described by the authors as a mild event and not a reason why patients would have dropped out of the trial- definitely not ‘life-threatening’.

Needless to say, given the nature of NucB1000 and the size of the trial, no signs of antiviral efficacy was seen.

To this day, it is not clear to me why exactly the trial was terminated. Did Nucleonics and their investors attempt a Hail Mary with the first dose cohort before cutting their loss on an increasingly improbable investment, or did they suddenly get cold feet as allegations of scientific misconduct relating to the pre-clinical data were raised? Given that money and lawsuits were involved, I favor the former with the latter serving as justification.

Curious publication

Overall, the Gish et al. paper is a most unusual one. The introduction already states:

'The proprietary nature of these data imposes limitations; however, the authors felt the results merited presentation and discussion as they involve a first-in-human study with potentially important clinical implications.'

I agree that the data was valuable, if not somewhat predictable, and am glad the authors had the courage to publish them. At the same time, the publication can also be read as a justification for why the trial went ahead.

It is also a curious publication because it suggests that Alnylam did not just acquire the Nucleonics IP estate to bolster its synthetic RNAi IP, as Barry Greene stated at the time, but that it was quite a bit more interested in DNA-directed RNAi Therapeutics in general, and NucB1000 in particular than one would have thought. While it cannot be determined conclusively how much NucB1000-related work was actually performed on Alnylam’s dime, the following passages suggest that it was not insignificant:

‘Exhaustive cell culture studies have demonstrated specific down-regulation of all HBV RNAs, including pregenomic RNA as compared with mismatch controls (Nucleonics, Inc.; Alnylam Pharmaceuticals; CS and CP, unpublished data; Recombinant DNA Advisory Committee).’

‘Additionally, significant inhibition of HBV antigen expression and viral replication is also observed, and is consistent with mRNA knockdown (Nucleonics Inc. and Alnylam Pharmaceuticals; CS and CP, unpublished data).’

'When administered intravenously, the nanoparticle been shown in preclinical models to transfect hepatocytes and delivers the eiRNA plasmid to hepatocytes and to the cells of some other tissues, such as the skin (Nucleonics, Inc. and Alnylam Pharmaceuticals, unpublished data).’

Etc, etc, I think you get the idea. But before Benitec supporters get excited about this, I would caution that Alnylam’s situation 3 years ago was much different from what it is today. With a dwindling cash reserve and after Tuschl, Tekmira, and Bass, Alnylam’s appetite for confrontation and lawsuits should have cooled considerably.

This story is yet another example of how concern about appearances (virus = scary) and short-term profit thinking (questionable model systems and lack of scientific rigor) in RNAi Therapeutics caused precious capital to flow into undeserving projects and lawsuits.

Alnylam Says Adverse Event in Tekmira ApoB Trial Was ‘Life-Threatening’

One apparent tactic employed by Alnylam in the ongoing litigation with Tekmira is not to win on arguments, but by coming out with 'revelations' aimed at unsettling Tekmira investors and potential collaborators. A weakened share price and financial position would supposedly make Tekmira more amenable to a light settlement or lowball takeover offer. Much of this has been discussed on this blog before, but today I would like to bring to the attention of the readers here one particularly grave and damaging remark that Alnylam made in their Response to the Amended Complaint, namely that the one case of immunostimulation in the phase I ApoB study that caused Tekmira to stop the trial was ‘life-threatening’:

From the Response:

‘Further, Alnylam invited Tekmira to join a joint development committee that Alnylam had formed with one of its pharmaceutical partners with the goal of assisting Tekmira and the whole field in advancing LNP technology. Moreover, it is through clinical trials conducted, mainly by Alnylam, that critical elements of Tekmira’s siRNA delivery technology have been validated in the clinic, and Alnylam has provided critical advice and counsel to Tekmira related to their pre-clinical and clinical development activities for their own products. For example, Alnylam’s chief medical officer provided critical and urgent counsel to clinicians attending to a patient in a Tekmira clinical trial that experienced a serious, life-threatening adverse reaction to Tekmira’s drug [emphasis mine].'

Not only does Alnylam make it sound like they are holding little Tekmira’s hands, have the new Chief Medical Officer rush to inept Tekmira's help, and more or less run the trials for them, this statement essentially accuses Tekmira of lying to investors and in front of regulators about the adverse event. These are very serious allegations, some of which by the way were part of the rumor mill even before Alnylam made these statements now in public (!).

These characterizations are in sharp contrast to the way Tekmira used to describe them: serious enough to be taken into consideration for future SNALP development, but not a show-stopper and certainly not life-threatening. Here is how:

Disclosure of adverse event in the phase I ApoB press release:

‘The primary endpoints of the ApoB SNALP Phase 1 clinical trial were measures of safety and tolerability. ApoB SNALP was well tolerated overall in this study with no evidence of liver toxicity, which was the anticipated dose-limiting toxicity observed in preclinical studies. Of the two subjects treated at the highest dose level, one subject experienced flu-like symptoms consistent with stimulation of the immune system caused by the ApoB siRNA payload. The other subject treated at the highest dose level experienced no side effects. Based on the potential for the immune stimulation to interfere with further dose escalation, Tekmira decided to conclude the trial [emphasis mine].’

If the adverse event had been ‘life-threatening’, then not disclosing this in the press release and subsequent investor discussions would obviously have been misleading.

The clinical details of the adverse event were then disclosed at the Drug Information Association '3rd Oligonucleotide-based Therapeutics Conference' held in Bethesda on March 23, 2010. An important function of these DIA meetings is to further the dialogue between industry and the US regulatory authorities and I assume that the FDA was in attendance and that Tekmira assumed the same when it reported the following:

‘Adverse Event in Suject 190:

- 53 Year old 103 kg male w/ hyperlipidemia

- Second in cohort to be treated with 0.6 mg/kg

- Tolerated infusion well

- ~2.5 hours felt ‘wobbly in legs’

- ~4 hours rigors, vomiting, fever, hypotension, hypoxia, HR 110 BPM

- Treated with Ibuprofin, O2, saline infusion

- Fever resolved in 3 hours

- ~5 hours BP reached nadir

- Patient continued to receive fluids

- IV methylprednisolone (120mg)

- BP, HR, O2 Sat improved

- Patient normal that night

- Returned to study unit the next morning

- Discharged the next day as per protocol

- Patient maintained perfect cogntion throughout episode

Event described as moderate in severity and related to study drug [emphasis mine].'

The account above is consistent with characterizing the event as ‘moderate’ and in no way suggests a ‘life-threatening’ event. While not exactly encouraging, such safety findings are common in clinical trials.

I’m confident that if this comes to trial, this damaging characterization of the ApoB trial will add another few millions to the damages awarded to Tekmira. I simply cannot believe that Tekmira’s management would risk getting in trouble with the SEC and FDA in such a way. There simply is no middle-ground. I know Tekmira shareholders are getting tired of hearing this, an attitude attesting to the success of Alnylam's tactics.

In related News: Alnylam guides down cash as ALN-VSP02 wraps up

Alnylam reported Q2 financial results after the close yesterday. Months after the departure of the former CFO and following the $150M shelf filing, the replacement had the pleasure of announcing that Alnylam is guiding down cash from ‘greater than $275M’ guided 3 and 6 months ago to now ‘greater than $250M’. No explanations were provided for the implied 50% increase in net burn. I hope that the new CFO has retained his ‘sense of humor’- apparently a job requirement at Alnylam [correction 5 August, 2011: in the conference call that followed the press release of the financial results the increase in expenditures were attributed largely to the 5x15TM efforts; not very illuminating really, but there was an explanation].

On the day of the financial results, Alnylam also disclosed that it has completed the phase I study in liver cancer with ALN-VSP02. There was no real new development in this study from the comprehensive data presented at this year’s ASCO (related blog here). What I noticed though is that this drug candidate has now been fully renamed ALN-VSP, dropping the '02' which may have been a painful reminder that Alnylam got all its clinically-relevant LNP delivery from Tekmira [hint: my strong impression based on the VSP conflicts that emerged 4 years ago with the remarks by David Bumcrot is that ‘01’ was a lipidoid formulation, and the ‘02’ formulation resulted from Alnylam giving up and seeking help from Protiva (now part of Tekmira, of course)].

Merck ‘Not Pulling Back in RNAi’ as Company Closes RNAi Trigger Research Unit

[Update: important clarification by Merck on the site closure, here]

As reported by the San Francisco Business Times and Xconomy, Merck will be closing its RNAi Trigger research unit in San Francisco as part of global restructuring efforts that include cutting ~12,000 employees by 2015 as Merck seeks to drive short-term bottom-line growth. While not exactly a move that will instill confidence in RNAi Therapeutics, a Merck spokesperson nevertheless was quoted as stating that ‘[Merck] is not pulling back in RNAi’ and that they have ‘still couple hundred employees working in RNAi’.

Global Pressures

Merck’s announcement is certain to be touted by some as representing a devastating verdict on the feasibility of RNAi Therapeutics. The news, however, comes at a time when confidence in innovative R&D in the pharmaceutical industry in general is at an all-time low to the degree that Big Pharma extols the licensing of phase III candidates (and beyond) as investments in innovation.

You’ve all heard it, too: Economic uncertainty and strained healthcare systems in the US and Europe, the patent cliff and declining R&D productivity worsened by a challenging, extremely risk-averse regulatory environment, all mean that the easiest way for Big Pharma executives to keep their jobs is to slash early-stage R&D and have their successors deal with the mother-of-all R&D productivity declines that undoubtedly will hit the industry 10 years down the line.

Merck actually has been one of the more vocal Big Pharma supporters of R&D. In February it withdrew its long-term financial guidance to preserve R&D flexibility, and Merck's CEO Ken Frazier as recently as in a July 13 opinion piece in the Wall Street Journal warned US politicians that their policies are killing pharmaceutical innovation and threaten US leadership: click here for the must-read.

Merck’s agony of having to decide between Wall Street and Washington-mandated short-term bottom-line growth (mind you, London, Berlin, and Paris are no better when it comes to appreciating the benefits of biomedical innovation), has also been apparent in recent comments on RNAi Therapeutics. In a June 28 interview with FierceBiotech from the BIO International Convention, David Nicholson, Senior VP and Head of Worldwide Licensing and Knowledge Management, stated that Merck has ‘chosen to stick with RNAi’ drawing parallels to the monoclonal antibody space that had undergone similar moodswings before becoming a widely accepted new drug modality.

This interview by the way was also picked up and tweeted by Alnylam which must have been concerned how the SF site closure, a development certainly in the making and rumored then, would be interpreted.

Importantly, the revelation today that a ‘couple hundred’ employees will continue to work on RNAi is consistent with Merck being far from giving up on RNAi Therapeutics, just as Novartis’ decision last fall not to expand their relationship with Alnylam was more a verdict on that specific relationship rather than Novartis' general view of RNAi Therapeutics.

Closure of SF Unit as Focus Shifts Towards Products and Delivery

If you accept these statements and RNAi Therapeutics remains viable at Merck, then the move to close the RNAi Trigger research unit at San Francisco Mission Bay and eliminate ~50 jobs can be interpreted as a shift in Merck's strategic focus from RNAi triggers to products and delivery.

RNAi triggers and related IP, of course, remain important for RNAi Therapeutics development and it is inconceivable that Merck would write off the $1B investment in Sirna Therapeutics' RNAi trigger IP as it continues to consider RNAi Therapeutics. It therefore seems that as Merck puts less effort into RNAi triggers, it must have felt satisfied with its IP position and acquired expertise there so that it was prepared to sacrifice this part of RNAi Therapeutics at a time that every unit, but emerging markets and biosimilars it seems had to contribute their cost savings.

Going forward, much of the success of Merck's RNAi Therapeutics will probably rest on its West Point, Pennsylvania, formulation/delivery unit. Among the research conducted there have been detailed liposomal siRNA delivery studies with the goal of using RNAi as a therapeutic and not merely target validation tool. Still, as Jeremy Caldwell, a person with a target discovery/validation background takes over responsibility for RNAi at Merck, we will have to see how fast Merck will transition RNAi into clinical development.

In addition to progress in delivery, it is the adoption of RNAi Therapeutics by the global R&D organization that will determine the clinical success of the technology at Merck. It may be one of the ironies of Roche’s RNAi efforts that they tried to preserve RNAi innovation by isolating the Kulmbach unit as a Center of Excellence, but then failing to rally broad support for the technology from the disease area groups. Because of liposomal delivery and Merck’s product focus, acceptance of RNAi Therapeutics by its lipid management, metabolic disease, and, of course, oncology groups will be particularly important.

PS (8/2/2011): In a statement to Gene Silencing News, Merck confirmed that the closing of the San Francisco site does not represent a departure from its RNAi Therapeutics strategy and that it still considers the technology to be transformative for human disease treatment.

Preview: Phase II Study of AtuRNAi PF-04523655 for Wet AMD

PF-04523655, formerly known as RTP-801i, is the clinically most advanced RNAi Therapeutic candidate and has been in two phase II studies for diabetic macular edema (DME) and the exudative form of age-related macular degeneration (wet AMD). ‘655 is a 19bp blunt-end AtuRNAi trigger targeting the RTP801/REDD1 apoptotic stress response gene in the choroid. ‘655 was originally discovered by Silence Therapeutics and Quark Pharmaceuticals, licensed to Quark and eventually partnered by Pfizer which is largely in control of clinical development.

Data from the DME study were reported earlier this year (see related blog entry here). These showed that while ‘655 was well tolerated and efficacy was strongly suggestive of superiority versus laser photocoagulation, the old standard-of-care, Pfizer and Quark agreed to run a phase IIb study using higher dosages to take into account the emerging standard-of-care for DME, Lucentis, a monoclonal antibody against VEGF. Data from the phase II wet AMD study should be imminent.

Unlike the DME study, the ~150 patient wet AMD trial design for the wet AMD study, MONET, already took into account Lucentis as the new standard-of-care in that indication. As you can see from the treatment groups below, the goal is to either show superiority to Lucentis, or at least show a synergistic effect when used in combination with Lucentis (a positive outcome would be to show superiority of Arms 4 or 5 over Arm 1). Unlike many other wet AMD/DME-targeting agents in development, ‘655 should not act via the VEGF pathway and therefore has potential as an add-on to Lucentis.

Treatment arms in phase II wet AMD study:

Arm 1: 0.5 mg intravitreal injection of Lucentis given every 4 weeks from baseline to Week 12 (note: 0.5mg once a month is the recommended standard for Lucentis);

Arm 2: 0.5 mg of Lucentis given by intravitreal injection at baseline followed by 3 mg of PF-04523655 given every 2 weeks from Week 4 to Week 12;

Arm 3: 0.5 mg of Lucentis given by intravitreal injection at baseline followed by 1 mg of PF-04523655 given (weekly) from Week 4 to Week 12;

Arm 4: 0.5 mg of Lucentis given at baseline by intravitreal injection followed by 3 mg of PF-04523655 given every 4 weeks from Week 4 to Week 12;

Arm 5: 0.5 mg of Lucentis given by intravitreal injection at baseline followed by 1 mg of PF-04523655 (30 minutes later) given in combination every 4 weeks from baseline to Week 12.

Dosing had been completed and preliminary data from the study were known already in November 2010. Unfortunately, while ‘655 as single agent or in combination with Lucentis did show improvements in mean visual acuity over the 3 month dosing period, 6 and 9 letters respectively, it failed to show superiority to Lucentis at any of the doses at the important 4 month primary endpoint. No remarkable safety events were seen in MONET (all this can be gleaned from reading Quark Pharmaceutical’s latest prospectus). By comparison, large studies with Lucentis (0.5mg, monthly) have shown 6.6-9.8 letter average improvements from baseline at two years.

Outlook for ‘655 in Wet AMD

Most likely due to missing the primary 4-month endpoint, Quark Pharmaceuticals already indicated that following full data review Pfizer is unlikely to directly enter phase III trials, but would either decide to run a phase IIb study or abandon the program altogether. It is disappointing that ‘655 has apparently not shown synergism when used together with Lucentis as one might have expected from the presumed mechanism of action of ‘655, but possibly not from an immunostimulatory antiangiogenic VEGF-related artefact. A synergistic effect, however, may be masked by the initial Lucentis activity and only emerge over time. What is also often forgotten when it comes to the competitive space for the novel therapeutics in ocular diseases is long-term safety, not just absolute efficacy and dosing frequency, so studies with longer dosing than just the 3 months in MONET may be worth the risk. Maybe such safety and efficacy signals will emerge from the upcoming full data presentation.

Finally, instead of combining '655 with an antibody like Lucentis, combining it with the approved anti-VEGF aptamer Macugen (an oligonucleotide) might have practical advantages. Macugen has been struggling in the marketplace following the introduction of Lucentis due to perceived potency disadvantages. Curiously though, it is Pfizer that holds the commercial rights to Macugen, and combining it with '655 to reinvigorate the competitiveness of Macugen must have crossed their mind.

On the Importance of Semple-Wheeler

The Semple-Wheeler (S-W) patents can be considered essential intellectual property (IP) for those liposomal siRNA formulations currently in development that have shown clinical promise. Because Alnylam obtained significant control over Semple-Wheeler (S-W) through a license from Tekmira, this patent estate has been important to Alnylam in controlling access to Tekmira’s SNALP delivery technology and, equally important, keeping Tekmira close to its chest.

Today, I will provide a brief re-cap of S-W and explain why its strategic importance is rapidly waning as patent expiration dates draw close.

What Semple-Wheeler Covers

Most critically, S-W cover LNPs comprising

a) ionizable lipids (e.g. DLinDMA, MC3), a modified lipid that prevents particle aggregation (e.g. PEG-lipid) in addition to the nucleic acid cargo (e.g. siRNA; Semple US6858225); and/or

b) a cationic lipid, a non-cationic lipid, a PEG-lipid conjugate in addition to the nucleic acid (i.e. the chemistry of a SNALP liposome; Wheeler US6815432).

While, until S-W has been tested in court, there will always been some uncertainty as to the real scope of the claims, e.g. in light of such newfangled terms like ‘lipidoids’ or other classes of lipids that may not have been covered by the examples in the specification, such strategies would seem to stand on weak scientific grounds.

Ownership and Control over Semple-Wheeler

Originally, Old Tekmira had an exclusive license to S-W from the University of British Columbia (UBC). Realizing the importance of liposomal delivery for the first wave of value creation in systemic RNAi Therapeutics, Alnylam obtained exclusive rights to Tekmira’s exclusive rights to S-W in January 2007. By this, Old Tekmira’s ability to further monetize this patent estate became greatly limited, and, in the absence of other significant RNAi assets at that time (i.e. lack of Protiva IP, know-how, and trade secrets), was widely regarded as a de facto satellite company of Alnylam.

Fair enough- not only did Alnylam pay $8M in upfront for these rights and is on the hook for milestones and royalties, Old Tekmira was in turn granted RNAi trigger IP and the use of S-W for the selected targets. Interestingly, the latter has been brought into question by Alnylam by stating in their recent Response to Tekmira’s Amended Complaint that Tekmira actually does not have rights to S-W and that Alnylam had given Tekmira such notice:

‘23. Consistent with this pattern, Tekmira has failed to adequately disclose the limitations of its licenses to investors. Despite the clear terms of the license agreements and notice from Alnylam that it lacked licenses to the Semple & Wheeler patent series and Isis patents, Tekmira made representation to the contrary in its public filings with the SEC and in other documents provided to investors.’

It is unclear on what grounds Alnylam makes these claims. Does it claim that Tekmira apparently was never granted such rights, or does it simply mean that Alnylam considers the lawsuit brought by Tekmira as grounds for terminating these rights? In any case, the irony is not lost as it is Tekmira through which Alnylam gained access to S-W and it is for Tekmira to terminate Alnylam's rights if at all.

Strategic Importance of Semple-Wheeler in Light of Upcoming Patent Expirations

In biotechnology, patent infringement typically only becomes ground for lawsuits either when drugs/devices allegedly covered by such claims are about to be commercialized or as generics want to muscle their way onto the markets before the expiration of patents covering an innovator drug. Because of the so called Research Exemption, it is very difficult to enforce patent rights before any commercialization of related drugs, i.e. during the period that patented technologies are being used for drug development pre-commercialization.

Certain licensing and collaboration agreements may be considered to fall between the Research Exemption and the commercialization of drugs, as they may involve technologies covered by 3^rd party patents, even if these patents can be assumed to have expired by the time drugs are ready to be commercialized. In other words, the LCAs may be interpreted as a way of commercializing patents. Although such actions are less common, emotions are running high, so Alnylam may use S-W as pre-text for throwing a wrench into any significant LCA that Tekmira may strike. Old Tekmira e.g. once sued Protiva for allegedly licensing S-W to Merck, and one can only speculate to what extent Alnylam instructed Old Tekmira to do so.

The reason though why such actions are unusual is that chances of enforcing such patent rights are slim if the parties involved in an LCA word their agreement appropriately (e.g. by simply excluding the appearance, implicitly or explicitly, that the partner gains rights to S-W; performing work in countries where S-W is not in force etc etc). As such, Tekmira has substantial freedom to strike deals as long as SNALP-based drugs are not being commercialized before the expiration of the fundamental S-W patents…which should be around 2015-7:

Semple US6858225 (ionizable LNPs):

Date of patent: Feb 22, 20005

Filing date: June 29, 2001

Priority date: May 14, 1997 (claims priority to No. 08/856474)

Approximate term of patent: Feb 22, 2005- May 14, 2017

Wheeler US6815432 (SNALP chemistry):

Date of patent: Nov 9, 2004

Filing date: Feb 24, 2003

Priority date: June 7, 1995 (claims priority to No. 08/5981501; note that although this is just before the famous June 1995 cut-off date, because the patent application of ‘432 was filed after that date, patent terms are according to the new regime)

Approximate term of patent: Nov 9, 2004- June 7, 2015

Tekmira business development prospects

Two factors largely controlled by Alnylam have long held back Tekmira’s full business development potential. In addition to granting Alnylam exclusive rights to S-W, it was particularly Alnylam’s RNAi trigger gate-keeper claim that tied SNALP technology to Alnylam.

Until the expiration of S-W, only those companies with access to S-W (Tekmira, Alnylam, Roche, Takeda, and supposedly Novartis) could commercialize SNALP-based drugs until 2017. Because Tekmira has 8 target picks for which it can use S-W, Tekmira is able to offer an acquirer or product-specific co-development partner more than enough access to S-W until then. 6 years is not a long time in drug development, about the time it would take from initiation of phase I to approval if all goes smoothly. The ~2017 expiration of S-W therefore means that its strategic importance for the development of SNALP-based RNAi Therapeutics is waning rapidly. 2007-8 was a different story.

In this light, a gate-keeping position of Alnylam in RNAi triggers, based on the Tuschl and Kreutzer-Limmer patents would be the real rate-limiting factor, as the terms for these patents may last into 2021-2. However, the RNAi trigger landscape has changed significantly over the last 2-3 years as the importance of Kreutzer-Limmer is rapidly diminishing in Europe and is nowhere to be seen in the US, Tuschl I turns out to be of no relevance for therapeutic applications, and even the validity of Tuschl II is now being questioned. Even if T-II can survive the Utah challenge, as it covers only certain RNAi triggers with certain 3’ overhangs, it is not the type of gate-keeping IP that KL or T-I may have become with a bit of luck.

Consequently, with the strategic importance of S-W waning and access to RNAi triggers ceasing to be a limiting factor, Tekmira’s business development is almost entirely out of Alnylam’s control. If, as a result of the litigation, Alnylam lost all rights to Tekmira’s technology (certainly a plausible outcome if it comes to a trial*), the attraction of partnering with or acquiring Tekmira would increase even more.

* As I am writing this, Alnylam has just filed a $150M shelf registration with the SEC. This typically happens in anticipation of selling stock to the public. The timing of this shelf registration is unusual though, because Alnylam has over $300M in cash/cash equivalents, meaning that with the current and anticipated burn, it should not have a need for raising funds any time soon- under normal circumstances. Given my assessment of the strength of Tekmira’s case and the existential risk to Alnylam’s business, it would not surprise me if Alnylam’s lawyers are advising the company to settle the case. A $150M capital raise may be in the right range to allow for Alnylam to survive and remain an independent company.

A hostile takeover attempt as an alternative explanation? On paper, this would probably make most sense for Alnylam, but I don’t think so.

Solid Calimmune DNA-directed RNAi Therapeutics Candidate for HIV Nearing Clinical Development

With the backing of a $20M grant from the California Institute of Regenerative Medicines (CIRM), Calimmune has made progress in advancing a DNA-directed RNAi (ddRNAi) Therapeutics candidate for the treatment of HIV/AIDS towards clinical development in early 2012 (here a recent blurb in the Financial Times). Similar to an HIV candidate developed by City of Hope (CoH) and Benitec before it, the new treatment involves the modification of a patient’s own blood stem cells (hematopoietic stem cells, HSC) with a gene therapy comprising of an expressed small hairpin RNAi trigger. Although Calimmune is not prepared yet to share the details of this program, based on my review of the research conducted by groups associated with Calimmune, the likely candidate has the potential to become one of the most exciting ddRNAi Therapeutics product candidates to enter the clinic yet.

HIV therapy today and motivation for gene-based stem cell therapies

The treatment of HIV has made tremendous progress. Once a certain death sentence, for those with access it has instead largely become a chronic infection that can be kept in check with cocktails of small molecules targeting a variety of stages in the viral life-cycle (highly active antiretroviral therapies or hAART). Nevertheless, the need for taking daily pills for life comes at the cost of side effects, generally reduced quality of life, and the emergence of viral resistances. There is no cure yet for HIVAIDS.

Actually, there might be one example of a cure for HIV. In 2006, an AIDS leukemia patient, aka the Berlin patient, underwent a bone marrow transplant as a treatment for his leukemia. The doctors selected a bone marrow donor whose cells carried defects in the CCR5 gene on both chromosomes. After the transplantation, the patient was not immediately put back on antiretroviral therapy to allow for recovery of his new hematopoietic system. Surprisingly, despite the absence of drug treatment, the virus has not recurred to this day leading more and more experts to talk of the first functional cure of HIV/AIDS.

In hindsight, this result did not come totally as a surprise. CCR5 had been known to be an important entry receptor for the common CCR5-tropic HIV isolates. Epidemiologic evidence gathered in the mid 90s indicated that people with certain CCR5 deletions on both chromosomes were protected from HIV infection, and those with a CCR5 defect on only one chromosome had, on average, delayed disease progression and improved life expectancies. In fact, this research led to the development and recent approval of a class of drugs blocking the CCR5 protein (e.g. Maraviroc by Pfizer).

There remains, however, great interest in developing gene-based stem cell medicines against CCR5 (and other HIV viral and host targets) in the hope of generating HIV medicines with less side effects, reduced chance of viral resistance (one way of HIV resistance to drugs targeting the CCR5 protein is to bind to CCR5 in the presence of drug), and maybe even a cure. The Berlin patient indicates that CCR5 may be an ideal target for such gene-based stem cell therapies.

Two possible mechanisms by which such a strategy may succeed are based on eradication of HIV-permissive cells as they are killed off by the virus while the CCR5-impaired cells persist, or by improving the immune function of CCR5-impaired cells thereby allowing them to fight HIV infection in other places.

City of Hope/Benitec and the first DNA-directed RNAi Therapeutic for HIV

Calimmune’s ddRNAi candidate is not the first one for HIV. The City of Hope, with the financial backing of Benitec, already entered one into clinical development (rHIV-shl-TAR-CCR5RZ), results from which were reported last year in Science Translational Medicine. Recognizing the advantages, if not need, for targeting multiple stages of the HIV life-cycle at once, this candidate was not a pure ddRNAi therapeutics, but a triple RNA therapeutic that in addition to the shRNA RNAi trigger which targeted the viral tat/rev mRNA involved an expressed TAR RNA decoy and an expressed (RNA) ribozyme targeting CCR5. Notably, all three expression cassettes were driven by U6 promoters.

The expression cassettes were placed in a shared lentiviral vector and thus introduced ex vivo, i.e. outside the body, into hematopoietic stem cells isolated from the enrolled AIDS lymphoma patients. Because hematopoietic stem cell transplantation with full bone marrow ablation is associated with risks, but is standard second-line therapy for AIDS-related lymphoma, this patient population was chosen so that the trial participants would simultaneously receive a treatment benefit for their lymphoma while participating in this experimental trial. As an added measure of precaution, the majority of hematopoietic stem cells were left untreated and given together with the modified stem cells to ensure that the immune system would be reconstituted even if something went wrong with the gene therapy.

Four patients were treated per protocol in the phase I trial. Unfortunately, while there was no obvious significant adverse event as a result of the gene therapy, the molecular analyses indicated that rHIV-shl-TAR-CCR5RZ may not be the most promising RNA therapeutics candidate for HIV. Specifically, while the initial transduction efficiency was in line with what would have been expected for lentiviral delivery (~20%, see X-linked adrenoleukodystrophy trial here), the transduced cell population declined rapidly and the ones that persisted were just about detectable- too few to be therapeutically promising.

If this candidate were to be further developed, an important goal would be to increase the fraction of stem cells that are modified. This could either be by improving the transduction efficiency, by only providing stem cells that were treated with lentivirus instead of providing the untreated stem cells as a backup, or by using a protocol that chemically selects for the modified stem cells after their re-infusion. Still, I am skeptical that this would solve the problem as in light of other lentiviral and retroviral clinical experiences the observed decline in transduced cells seemed to be specific to rHIV-shl-TAR-CCR5RZ. It is therefore possible that some inherent toxicity of the expression cassette itself, possibly due to the use of U6 promoters, accounted for the poor long-term persistence of modified stem cells.

The Calimmune approach: A non-toxic, H1-driven shRNA targeting CCR5

The reason why I feel that Calimmune’s approach may have better prospects is that it has fully accounted for the U6-related shRNA toxicities and selected an H1 promoter-based RNAi expression cassette that was shown to be both safe/stable and, equally important, highly efficient in CCR5 knockdown in human and rhesus HSC-derived cells. Also, I like the fact that it is an RNAi trigger, and not a ribozyme, that is targeting CCR5, as I believe this to be the more efficient knockdown modality.

While Calimmune has yet to fully disclose their eventual clinical candidate, the one reservation that I have about the putative candidate at this time is that they may have failed to take advantage of the combinatorial potential of RNAi Therapeutics. With combinatorial potential I do not necessarily mean here combining ddRNAi with other RNA (like CoH/Benitec) or protein expression modalities- in fact, it may be scientifically 'cleaner' to use just RNAi for now- but targeting at least two HIV-related genes instead of one to minimize the emergence of viral resistance.

The panels on the left depict what in my mind have been the most impressive dataset from the development program. It shows the results from a rhesus monkey model in which the ddRNAi trigger was introduced into blood stem cells from two monkeys (RQ3570 and RQ5427 for those with good eyes) which (panel A) led to solid, long-term (!) 6-20% cell marking in the various cell lineages of the blood. Moreover, when the cells were sorted into those that were transduced (black bars, panel B) versus those that were not (grey bars, panel B) and the CCR5 levels measured in the respective cell populations, the CCR5 was found to be down-regulated by 80-90% in the transduced cells. And since your experiment is only as good as your negative controls, data from a control animal that received a lentivirus without the RNAi trigger (2RC003) show no differences in CCR5 levels between the two cell populations.

While I have yet to see the obligatory HIV in vivo challenge studies with this putative candidate, based on CCR5 genetics, a candidate with such transduction levels and knockdown potencies should stand a good chance at improving CD4+ T-cell counts for enhanced immune system vigor and delaying or maybe even eradicating HIV over time.

It is debatable to what degree a full CCR5 knockout compared to a highly potent CCR5 knockdown would bring additional benefits. Sangamo Biosciences for example has made tremendous progress in increasing the efficiency of gene disruption using their Zinc Finger Nuclease technology. Not surprisingly, this company also has a CCR5 hematopoietic stem cell candidate in the early pipeline. In a 2010 Nature Biotechnology paper, Sangamo reported an estimated frequency of 5-7% homozygous CCR5 gene disruption in human hematopoietic stem cells, and another 10% heterozygous gene disruptions.

Simplistically, taking upper estimates, ddRNAi may provide for 90% CCR5 knockdown in 20% of cells whereas ZFN technology may delete CCR5 altogether in 7% of cells and knockdown CCR5 by half in another 10%. Because these numbers are close and a clean knockout in some cells may make up for the slightly decreased overall knockdown levels, I would be even more excited to see Calimmune's current lead candidate paired with at least another shRNAi trigger, thereby exploiting said combinatorial potential of ddRNAi Therapeutics which ZFNs cannot provide as easily.

Benitec license?

Benitec, of course, will follow Calimmune’s developments with great interest as the company has rights to critical ddRNAi trigger patents. Curiously, both companies are based in Australia, but have significant roots also in the US South-West, so it should be possible to come to an amicable agreement.

License or not, it will be good for the entire field of RNAi Therapeutics, and ddRNAi Therapeutics in particular, for this trial to get underway in 2012 as it should attract significant general interest to a what looks like a solid RNAi Therapeutics candidate.

Acknowledgement: The idea for this blog came from a reader that alerted me to this interesting RNAi Therapeutics candidate that had flown below my radar, and maybe also to placate another reader that complained that the Tekmira-Alnylam feud was taking up too much space and there were other interesting things happening, especially in ddRNAi Therapeutics. So if you know of exciting RNAi Therapeutics developments that you believe I may be missing, please let me know by email (first name dot last name at gmail dot com). In most cases, I won’t be able to write about it immediately, but it won’t be forgotten either.

Update: On March 5, 2012, Calimmune acquired a global, non-exclusive license from Benitec to use ddRNAi in HIV/AIDS.