Antibody-RNAi Trigger Conjugates Show Signs of Life

Despite jettisoning RNAi Therapeutics 4 years ago, the more innovative arm of Roche, Genentech, has continued to dabble in the technology.  In particular, it has been interested in applying its monoclonal antibody know-how, including antibody-drug conjugates (ADCs) to the delivery of RNAi Therapeutics.  A recent publication by Cuellar and colleagues provides insights into these efforts.

THIOMABs for the creation of drug-like conjugates

While antibodies have a relatively long history in the delivery of RNAi Therapeutics, some of the early findings were generally quite difficult to replicate.  Part of the problem may have been the fact the early efforts involved structurally ill-defined non-covalent protein-nucleic acid complexes held together by charge-charge interactions.

To get around this issue, Genentech applied their THIOMAB platform which allows for the covalent addition of therapeutic payloads at defined cysteine residues.  This process yielded THIOMAB-siRNA conjugates with one, or more often two RNAi triggers per monoclonal antibody.  To help visualize them, also for pharmacokinetic considerations, think of the monoclonal antibody part being ~13x bigger/heavier than each RNAi trigger.

Of note, the RNAi triggers were partially (~50% of residues) modified with 2’-O-methyls and 2’-F for stability (siSTABLE from Dharmacon).

Not all receptors are created equal

One of the reasons why I am picking out this paper for discussion is the thoroughness of the research presented.  For example, the Genentech researchers selected not just one, but seven distinct cell surface receptors for which various THIOMAB-siRNA were created which in turn were be tested in a number of settings.  The receptors were partly chosen to capture a range of cellular trafficking behaviors such as rapid lysosomal uptake, recycling receptors, and slow-turnover receptors.

Importantly, the research validated a critical rationale for the use of antibody-RNAi trigger conjugates namely uptake of the RNAi triggers that is dependent on the presence of the cognate receptor and covalent linkage to the antibody.

For all the conjugates (ARCs), the bulk was shown to accumulate in lysosomes regardless of presumed uptake kinetics.  Interestingly, despite the seemingly shared uptake pathway, if not dynamics, only two of the seven receptors were associated with gene silencing (TENB2 and NaPi2b, but not e.g. Her2). 

The degree of silencing was much weaker compared to when the same conjugates were lipofected with ~50% silencing starting to be seen at 10nM.  This was followed by a shallow dose-response plateauing at 70-80% silencing around 500nM.  Based on the data presented, it seems that limiting amounts of receptors were responsible for this. 

Selective accumulation in mouse tumor model

A highlight of the publication to me was the investigation of THIOMAB-siRNAs in a mouse tumor model.  Although you may consider a ~30% silencing (i.e. 70% expression of normal remaining) when you cherrypick tumor areas of most efficient delivery a somewhat disappointing outcome as was reported in this case, important lessons can be learned from that.

Firstly, THIOMAB-siRNAs only accumulated in the tumor when the tumor expressed the cognate receptor.  This is unlike nanoparticulate RNAi delivery to tumors which relies on a passive process of accumulation (the EPR effect).  This opens up the prospect of RNAi drugs with ‘cleaner’ delivery profiles with an increased margin of safety.

Selective accumulation for these conjugates also suggests that the ~180kDa macromolecules were able to relatively rapidly exchange between tumor interstitium and blood circulation despite their size.  Consequently, other conjugates in a similar size range or below, including Arrowhead’s DPCs, should be amenable to such ‘active targeting’ as well.

Nevertheless, despite this apparent agility, imaging techniques showed that tumor delivery was largely restricted to areas next to the vasculature.  This limitation in fact is what is also seen with nanoparticles which rely on the EPR effect.  So while the active targeting capability is an important step forward, tumor penetration issues remain to be solved.

Path forward

Despite the arguably underwhelming in vitro and in vivo knockdown results if you just look at the numbers, I am optimistic that the studies have further supported that systemic RNAi delivery can be applied beyond the liver, vascular endothelial cells, certain cells in the kidney and phagocytes.

What is missing in that particular piece of research making the conjugates that make it into the cells count.  Given the lysosomal accumulation, an obvious strategy to unlocking the true potential of ARCs would be to apply endosomal escape chemistries, especially masked chemistries such as in the DPCs by Arrowhead Research (actually specifically referenced by the authors).

In almost the same vein, the sparse chemical modification used mean that potency improvements will be gained if heavier modification is applied even without adding endosomal escape chemistries.  This is also because some of the data are consistent with a model whereby the THIOMAB-siRNA conjugates get broken apart in late endolysosomal compartments and it is from that population of freed RNAi triggers that escape into the cytoplasm may occur (note: this does not exclude spontaneous endolysosomal rupture as an additional escape pathway).  If RNAi triggers were made metabolically more stable through more extensive modification, the amount of RNAi triggers available for such escape would obviously be larger.

All that is lacking then is the issue of tumor penetration.  I could imagine, however, that some self-delivering and/or lipophilic strategies could be quite useful here.  The Genentech researchers actually speculate that the RNAi trigger may have been the culprit in limiting the claimed natural ability of monoclonal antibodies to more deeply penetrate tumors.  This suggests that charge masking may overcome this limitation.  

I am open to this idea, but based on my understanding of the literature, tumor penetration is a challenging issue for basically all therapeutic modalities, monoclonal antibodies and 'even' small molecules, so let's not blame double-strandedness for all pharmacologic problems.

With interest in RNA Therapeutics growing by the day, expect lines of delivery research such as this one to be picked up and pushed to the next level.

PS: for antibody-mediated RNA delivery, Avidity NanoMedicines and the Roche-ISIS collaboration on CNS delivery should also be worth watching.

January RNA Therapeutics Deal Frenzy Kicked Off

As is now tradition for Januaries in RNA Therapeutics, expect this month to be filled with mouthwatering business development announcements.

Without a doubt, privately held messenger RNA Therapeutics company Moderna Therapeutics stole the show today by announcing a $450M financing involving 'Viking Global Investors LP, Invus, RA Capital Management, and Wellington Management Company, LLP, as well as existing investors AstraZeneca and Alexion Pharmaceuticals'.

Biggest private financing in biotech history EVER.

With $800M in cash, close to a billion USD raised since starting up 2 years ago or so, the biggest challenge will be how to efficiently put the capital to good use and move into the clinic in the not-too-distant future.  Before that happens, we should be seeing one of the hottest biotech IPOs in 2015.

Before Moderna announced their financing, ISIS Pharmaceuticals was the talk of the day with their GI-related autoimmune deal with Johnson&Johnson.  Not only does the early development deal provide ISIS with another nice junk of upfront cash, this time $35M, and a gazillion in potential biotech milestone payments and royalties, jn practical terms I expect the research to be particularly useful to ISIS as it develops oral delivery for systemic applications (think lipid franchise for which the new CEO will be officially enthroned tomorrow).  

I believe that the deal was primarily driven by JNJ's interest in the space following the whopping $710M upfront deal by Celgene last year when it acquired rights to an antisense compound developed by oligonucleotide therapeutics nobody Nogra Pharma for IBD applications.  The high upfront payment indicates that competitive bidding was involved and it is likely that JNJ lost out.

Remember, Big Pharma moves in herds.

The good news for JNJ is that if the first-generation phosphorothioate DNA is indeed working by an on-target mechanism (which I doubt), then a competing compound employing much more advanced chemistry and design by ISIS Pharmaceuticals will be infinitely more potent and beat Celgene in the market.  And for $35M and $800M in milestones if everything goes according to plan, it's a comparative steal.

For ISIS Pharmaceuticals it is just another step to broaden their expertise and applications of their platform.  Moreover, the upfront payment will help the company to remain cashflow neutral to slightly positive until 2017, at which time I expect drug sales to be finally taking off.

Leveraging New RNAi Trigger Chemistries for Gene Knockdown in Phagocytic (and Other) Cells

Much of the achievement of solid RNAi gene knockdowns in hepatocytes (liver) by non-LNP means (Arrowhead DPCs and Alnylam’s 2^nd gen GalNAc-siRNAs) has involved the use of heavy modifications that render the RNAi triggers highly stable. This is because nucleic acids that are not protected by the delivery chemistry itself would otherwise be subject to rapid degradation in extracellular body fluids.

In the case of Alnylam’s GalNAcs, they can even function in the absence of an explicit endosomal release chemistry.  Moreover, GalNAcs and DPCs have shown more sustained gene knockdowns than is achieved with LNP delivery which historically has relied on minimally modified RNAi triggers. 

All that is required is receptor-mediated uptake into the endosomal-lysosomal pathway which is an immensely degradative environment (esp. the lysosomes).

Considering the extended duration of silencing and degradative environment, it seems as if the RNAi triggers have to be able to survive for long enough in late endosomes/lysosomes so that when they get an opportunity to escape by as yet undefined mechanism(s), they are still there ready for gene silencing action.

Such chemistry progress may also be particularly useful for RNAi gene silencing in phagocytic cells of the immune system.  This is because there have been multiple reports, especially concerning the use of LNPs (e.g. Novobrantseva et al.)  where some, but not the very robust, hepatocyte-type of gene knockdown have been obtained.  It is the ability to confidently achieve robust knockdowns that opens the gate to a flood of therapeutic applications, and this is why pushing borderline-technologies over the edge is so tremendously valuable.

Uptake into phacocytes is usually not the problem as these have evolved to scavenge for foreign particles and macromolecules.  In fact, phagocytic uptake is often a nuisance in RNAi delivery both because it may cause off-target toxicity and because it can make pharmacology less predictable.

In the case of untargeted nanoparticles such as LNPs, phagocytosis is the likely uptake mechanism.  Similar to endosomal uptake, phagosomal uptake involves the fusion with lysosomal compartments meaning that phagosomal contents are also exposed to a highly hostile environment.  Since the normal endosomal escape chemistries and mechanisms do not appear to be very effective in phagosomes, one strategy besides of possibly tailoring existing mechanisms to the phagosomal environment (e.g. lipid pKas) is to simply use the same ultra-stable RNAi trigger chemistries that are showing promise in the liver.

It is also possible that ligand-targeted conjugate approaches will be useful here, whether they enter the cells via phagocytic mechanisms or not.  In fact, Arrowhead Research (then Mirus Bio) in their seminal publication on DPCs (Rozema et al.) have demonstrated efficient uptake of DPC-conjugates into liver phagocytes (Kupffer cells) by using mannose as the targeting ligand (they haven’t tested and/or shown the corresponding RNAi knockdown though and this may relate to their using much less nucleic acid modification back in 2007). 

Since the mannose receptor is expressed on phagocytes throughout the body and not just in the liver, more extended circulation times promises the application of this conjugate-targeting strategy more generally.

Similar principles may apply to the targeting of other ‘frontier tissues’ for RNAi delivery.  However, given the degradative and differing nature of phagocytosis which means that other release mechanisms are not readily applied to this process, RNAi in phagocytes should particularly benefit from the new RNAi trigger chemistry developments.  I look forward to seeing the results.

Oligonucleotide Therapeutics Companies Crowding into GalNAc-Conjugation

Following Alnylam’s achievement of making subcutaneous administration work for RNAi gene knockdown in the liver by conjugating RNAi triggers to the GalNAc sugar, antisense companies have started to copy the approach.  As RNAi Therapeutics have made great progress in targeted delivery, antisense companies are starting to realize that in order to stay competitive and improve the safety of systemic uses of antisense, they need to get away from the notion of 'naked'/unformulated delivery that relies on swamping the body with phosphorothioated oligonucleotides.

This has become apparent at the AsiaTIDES meeting here in Tokyo where both Santaris and ISIS Pharmaceuticals disclosed their great interests in GalNAc conjugation.

In collaboration with Axolabs (the part of Roche RNAi Therapeutics that was not acquired by Arrowhead Research and that had familiarity with GalNAcs), Santaris presented phosphorothioate-LNA Factor VII (liver) knockdown data showing 80% gene knockdowns in mice following a single dose of 0.1mg/kg.  Even more potent knockdowns were seen at 0.25mg/kg and above. 

In another presentation by ISIS Pharmaceuticals on their cardiovascular franchise, the company noted that they will follow up on their initial Apo(A) (‘little A’) candidate with a GalNAc-conjugated version.  With this, they expect to increase potency by up to 10-fold, thus allowing for 10-30mg (~0.15-0.45mg/kg) dosing.

This illustrates the utility of the GalNAc receptor (ASGPR) and how the competing RNAi and antisense technologies are fertilizing each other.

Regulus Therapeutics, of course, is the first antisense (anti-miR) company of sorts that has adopted GalNAc conjugation for their liver-directed programs, most notably anti-miR122 for HCV that is about to enter clinical development.  Regulus obviously has a license to GalNAc from Alnylam.  Whether this also applies to ISIS Pharmaceuticals, remains to be seen.  The word on the lab floors is that GalNAcs per se are not patent-protected, so ISIS may use an entirely different linker strategy to Alnylam just as Arrowhead Research does with its GalNAc-targeted DPCs.

What it means for Tekmira and Marina Biotech

Since some of you are following my investment strategy, here is what I think this means for the liver-directed efforts by Tekmira and Marina Biotech (both of which I own). 

Marina Biotech could obviously follow the same path as ISIS Pharmaceuticals and Santaris in adopting GalNAc conjugation with its CRN technology.  As such, there should be no change in the competitive value of CRN compared to Santaris and ISIS antisense.  It could also attach GalNAcs to their liposome-based SMARTICLES for which delivery to the liver remains to be demonstrated.  To do all this, however, Marina Biotech needs to grow and establish in-house R&D.  

For Tekmira, it means that RNaseH antisense are getting close in potency for gene knockdown compared to the 2^nd gen MC3 SNALP LNPs (85-90% TTR gene knockdown in humans at 0.3mg/kg).  With the 3-fold more potent 3^rd generation SNALP LNPs which should enter the clinic this or early next year (TKM-ALDH2, TKM-HBV), Tekmira should stay well ahead of its competition with the most potent gene knockdown technology for the liver.  This means more addressable diseases and in most cases higher treatment effects as well.   And if it incorporated GalNAc-conjugated lipids into their liposomes, too, maybe that would extend that lead even further.

If gene knockdown in the liver is not a great example for why you need a competitive free market economy, then I don’t know what is. And, of course, there is no better example of why you need a lab ;).

Journal Club: Alnylam and Collaborators Make Progress in Understanding and Optimising siRNA Uptake In Vivo

In yet another elegant paper, Alnylam and collaborators from the ETH in Zurich and the Rockefeller report this week in the journal Nature Biotech on the mechanism of siRNA uptake in vivo (Wolfrum et al.). This is significant, since a systematic approach to understanding siRNA delivery in vivo should most adequately address the delivery challenge that is considered by many to be the main barrier to the broad application of RNAi as a therapy. It is also a sign of the maturity of the RNAi field in general that no only are there now a variety of innovative delivery systems evaluated almost by the day, systems that show promise serve as leads for a detailed investigation of the underlying biology.

The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.

Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.

Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.

In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.

Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.

Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.