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.

Tekmira Delays Hypercholesterolemia Candidate: 2nd Gen Growing Pains or is it ApoB after all?

Tekmira’s ambition to grow a substantial clinical pipeline suffered a setback when it announced today that the IND for its candidate for the treatment of hypercholesterolemia, TKM-ApoB, has been delayed following a review of pre-clinical data. These data indicated that the ‘performance characteristics of the specific lipid nanoparticle formulation [...] have not met the Company’s expectations for the intended application'. This is the second substantial delay of Tekmira's pioneering ApoB program, and comes after the abandonment of a first ApoB candidate earlier this year.

The press release makes it very difficult to understand what the reason for the delay could have been. Given that LNP-siRNA delivery has been successfully demonstrated in hundreds of non-human primates, I have a hard time believing that they suddenly could not find a formulation that was able to potently knock down ApoB in the IND-enabling large animal studies. Even if they had some problems, it should not have led to an open-ended delay. I also don’t think that they could not get rid of the innate immunostimulatory potential of the siRNA they have been working with, because Tekmira had said that new modification patterns for this siRNA were able to abrogate immune stimulation also in the more sensitive ‘whole blood assay’ that they had developed. On the other hand, a clinically desirable profile for a given indication does consist of many more aspects besides potency and immune issues. Given that 2^nd gen formulations are relatively recent, whereas DLinDMA-based 1^st gen formulations have been quite thoroughly studied, some 2nd gen-related growing pains might be expected.

There have been questions raised about ApoB as a safe target for the treatment of hypercholesterolemia. Data in the RNAi literature itself and from the clinical program of ApoB-antisense molecule mipomersen (ISIS/Genzyme) show that inhibiting ApoB causes some, and sometimes indeed very significant accumulation of fat in the liver. While the medical importance is not clear yet, fatty liver and associated elevations in liver enzymes have raised some concerns. If Tekmira indeed sees the same toxicities that Mirus/Roche and Alnylam have seen (Marina Biotech and RXi also seem to have given up on ApoB), then it may be in the Company’s best interest to abandon the program. Accordingly, the press release was quick to highlight that the development of the company’s other 2 development candidates, one for solid cancers (TKM-PLK1) and one for Ebola infection (TKM-EBOLA) would be accelerated.

TKM-ApoB is also a particularly challenging indication as hypercholesterolemia is a chronic condition where you would have to safely administer a drug for sustained periods of time. Moreover, the regulatory bar for cardiovascular drug candidates has been raised quite a bit in recent years, especially in the US (outcome studies for most populations and increased safety scrutiny). The acute cancer and infectious disease indications may therefore be clinically more straightforward candidates to develop and, with Ebola, also offer earlier financial returns.

What speaks against the abandonment of ApoB as a target were references in the press release that the company would continue to study other formulations with ApoB. This, however, could also be interpreted as boilerplate or even a political move given that ApoB represents one of the five target picks from Alnylam and for that reason alone Tekmira may want to keep it alive at least on paper.

Overall, I am disappointed that the opportunity for TKM-ApoB to provide unequivocal clinical proof-of-concept data of potent gene knockdown with LNP-siRNA in the near-term will be missed. This now falls on the shoulder of Alnylam’s TTR program.

While clearly a bearish development, today’s news also provide some reason for optimism. One is that the strategic re-organization should be slightly positive for Tekmira’s finances as the new lead programs will be partly (PLK1- probably Alnylam and possible NCI support newly mentioned) and fully (Ebola) funded by others. Moreover, the company understands the importance of credible public relations by admitting to difficulties and calling disappointments what they are. It is also not pushing programs into the clinic because it is attractive to do so in the short-term: bonuses for the achievement of milestones; a relatively strong share price especially good if management does not intend to be there in the long run and a sale of the company was intended, etc- we’ve seen it all.

Just as RNAi Therapeutics was heating up, today is a humble reminder that in drug development setbacks can crop up when they are least expected. It will be important for the company to provide more clarity on today's development in the coming weeks, at least to the extent that it does not compromise their strategic position in RNAi triggers.