Factor XI Data by ISIS Pharmaceuticals Revolutionizes Anti-Clotting Field

Yesterday, ISIS Pharmaceuticals disclosed in-depth data from their phase II anti-clotting study in about 300 patients undergoing total knee arthroplasty (TKA).  This took place at the American Society of Hematology (ASH) meeting alongside a publication in the New England Journal of Medicine (Bueller et al. 2014).  The data for the first time provide striking evidence that it is possible to dissociate anti-clotting activity from a commensurate increase in the risk of bleeding.

Accordingly, while the 200mg per week dose (moderate 59% FXI knockdown) achieved a rate of venous thromboembolism (VTE) similar to the enoxaparin standard-of-care comparator in the trial (27% and 30%, respectively), at the 300mg per week dose (much more robust 78% FXI knockdown) the VTE rate dropped by 7-fold to just 4%.  

At the same time, bleeding risk remained the same, if not better for ISIS-FXIRx vs enoxaparin: 3% bleeding events for both FXI cohorts vs 8% for enoxaparin, albeit this was not a statistically significant difference.

In terms of safety and tolerability, ISIS-FXIRx resulted in mild local skin reactions in 6.6% of the injections, but leading to no discontinuations.  Moreoever, no flu-like symptoms were recorded.  This is a stark departure from ISIS’ legacy drug mipomersen which suffered from frequent flu-like symptoms and injection site reactions resulting in relatively frequent drug discontinuations.

Challenging the paradigm

Until now, it has been widely thought that whenever you develop a new, more powerful anticoagulant, you will pay the price of more bleeding.  This has meant not only much development money wasted, but also created important market needs such as in patients which require anticoagulants with lower bleeding risks.

It is these markets, including patients with end-stage renal disease and atrial fibrillation, that ISIS Pharmaceuticals will address first as it seeks a partner more familiar with the complexities of the anti-clotting market.

What seems to underlie the surprising results is that with Factor XI you are targeting the intrinsic branch of the clotting cascade which prevents the formation of large clots that may travel around the body often with fatal consequences, but without impeding the ability to form small clots when healing tissue damage following trauma caused by external factors. 

By contrast, conventional anticoagulants such as enoxaparin (a heparin derivative), Factor Xa and thrombin inhibitors interfere with both processes.

Challenging the establishment

The strategy of addressing niches of high unmet need in the anticlotting market first is not only explained by potentially faster orphan-type development timelines, but also by the fact that by presenting such disruptive data in a multi-billion market, ISIS can be expected to encounter stiff resistance from the anti-clotting establishment.  This refers not only to competing pharmaceutical companies, but also their associated key opinion leaders from academia that enjoy a gate-keeper function partly due to the regulators commonly seeking their advice.   

So given that the efficacy results are undoubtedly impressive, expect over the coming days, months, if not years to hear the message that the ISIS-FXIRx data ‘might’ be a proof-of-principle for dissociating clotting from bleeding for anticoagulant therapy, but that this 300-patient study needs to be replicated in a larger population and that there are ‘concerns’ around the tolerability and convenience of ISIS-FXIRx, all the while the same groups are busy catching up developing antibody- and small molecule-based versions of anti-Factor XI drugs.

Progress in Predicting the Immune Response to an RNAi Therapeutics

When in 2010 Tekmira decided to prematurely terminate its first clinical RNAi Therapeutics candidate, TKM-ApoB, due to unexpected immune stimulation in a phase I, it was disappointing in a number of ways.  Not only did it destroy TKM-ApoB right there, it also cast doubts on how predictive our preclinical immune stimulation tests really were and whether as a result, other candidates may suffer similar fates.  Apparently, using rodents or even non-human primates for that purpose had not been all that useful either. It is also said that the event served as a red flag to some regulators which wanted the technology being developed more slowly.

Tekmira, however, soon claimed to have discovered the reason why their PBMC-based assay did not pick up on the immunostimulatory potential of TKM-ApoB and reported that they had have found a more predictive test tube assay instead.  Apparently, using heparin during the preparation of PBMCs (sample enriched for certain immune cells from blood) was the culprit and a whole blood-based assay (including things like red cells and albumin) would reflect much more reliably the situation in people.

A nice paper by Coch et al. from the Hartmann group in Bonn, Germany, now provides comprehensive insight into possible causes for false positives and false negatives when using in vitro immune stimulation assays.

Role of serum proteins  

It is known that phosphorothioate oligonucleotides, a particularly widely used chemistry in the antisense field, rapidly bind to proteins in serum.  Given its abundance and sticky nature itself, albumin is thought to be an important target of such oligos.  The group thus found that their binding in whole blood sequestered them from the immune cells such that the immune response was significantly lessened.  By contrast, in the PBMC-based assay, these phosphorothioates stimulated a robust immune response.

This insight is obviously particularly important if the interest was in developing oligonucleotides that are immunostimulatory on purpose, especially for cancer and viral applications (false positive in PBMC).  Whether the finding that in whole blood assay phosphorothioate oligos may not exhibit immune stimulation can be used to conclude that they will be safe is another question as the blood-based assays may not be informative on what is going on in the tissues.   

Role of anticoagulants

Closer to the TKM-ApoB story, another important insight was that the nature of the anticoagulant can have a big impact on the outcome of the assay.  When drawing blood, and especially when employing a whole blood assay, it is important to use an anticoagulant as otherwise standing blood would start to clot. 

EDTA and heparin are standard reagents for this purpose.  EDTA has been recognized that it can distort immune stimulation assays and thus is not used.  The surprise (well not that surprising in hindsight) is that heparin, due to its negative charge, could displace oligos such as RNAi triggers from their delivery vehicles, including liposomal nanoparticles (LNPs).  It is often the delivery agents that bring the oligonucleotides to the immune receptors.  Moreover, nanoparticle formulations such as LNPs can facilitate multivalent interactions as they hold together and present a number of oligonucleotides simultaneously.  This in turn can potentiate the immune signaling.

Accordingly, what probably caused the immunostimulation of TKM-ApoB to be missed is that heparin at some point displaced the ApoB siRNA from the liposomes thus destroying the potentiator effect of SNALPs.  Importantly, replacing heparin with hirudin (think leeches) did not suffer from the same limitation.  Therefore, human whole blood assays with hirudin as the anticoagulants appear to be the simplest and most reliable preclinical innate immune stimulation assay for Oligonucleotide Therapeutics development.

Growing the database

Of course, these insights are only a first, albeit critical step.  The next step is to understand what degree of immune stimulation in the test tube relates to a likely adverse event in the clinic.  For this, it is necessary to go back and forth between the test tube and clinical observations and correlate the clinical phenotype (cytokine production, fever/chill symptoms) with the test tube response. Complicating matters, different persons have individual innate immunostimulatory sensitivities, and depending on the state of the immune system (e.g. existing infection) there will also be intra-patient variability.

   

On a more positive note, not all delivery/RNAi trigger formats are equally prone to stimulating the immune system with LNPs probably being one of the more challenged formats with regard to innate immune stimulation.  Even so, the clinical track record with SNALPs after TKM-ApoB (lowered doses of steroids with ALN-TTR02, no steroids apparently used in the TKM-EBOLA trial) suggests that the new assays are having a positive impact already.