This was SUPPOSED to open with a report from Medicago Inc, on ‘Developing plant-made influenza vaccines: From discovery to commercial scale production’ – but didn’t, because they were all shaken up (in a good way) by having been effectively bought by Mitsubishi Tanabe Pharma Corporation, and no-one came.
This is a success story in its own right, however, as their recent and highly successful activities in the areas of making influenza vaccines and human rotavirus VLP-based vaccines in plants marked them out as a target for acquisition by Big(gish) Pharma – for which we commend them.
It is sad, however, that their only presence at the conference was on the back of my windbreaker B-)
Konstantin Musiychuk (Fraunhofer USA) was the first up, then, speaking on ‘Preclinical evaluation of VLP-based malaria transmission blocking vaccine’. He described how there are 3 types of intervention that may work with malaria: these are at the pre-erythrocytic, blood stage, and transmission blocking stages of infection. Antibodies to Pfs48/45, Pfs230 proteins block the fertility of or destroy the macrogamete. Pfs25 and 28 Abs block the ookinete to oocyst developmental phase; all potentially block transmission. Accordingly, they expressed these as fusions with the alfalfa mosaic virus (AMV) CP with mutation(s) to prevent glycosylation. The Pfs25 protein was the best candidate; they cloned a mutated version (glyc-), fused at the N-terminus to AMV CP, and expressed via their TMV-based “launch vector” after vacuum infiltration. He noted that the fusions have full-length and proteolysed products – which is needed for VLP formation as native CP is needed to avoid steric hindrance in assembly. They obtained nice particles as shown by EM, showing surface decoration. Dynamic light scattering [Ed: must get me one of those…] results show a nice tight range of 17nm particles.
They used the products with/out Alhydrogel as adjuvant, IM in mice: they got good titres maintained >170 days, with 2x inoculation. They diluted test sera with naive human serum and used this to membrane-feed mosquitoes, then after 1 week dissected them and assayed for parasites: oocyte counts in mid-gut reflected efficient blocking of acquisition. The adjuvant+ doses worked well down to 0.1 ug (100%). Single doses of 1, 5 or 25 worked 100% as well. After 6 months, 5 and 25 ug doses still gave 90%+ blocking.
They made GMP lots, very pure: 2 doses at 0 and 21 days resulted in complete blocking down to 0.3 ug, with >99% blocking after 40+ days. Tox studies were fine, although the Alhydrogel apparently causes some side effects. Scaleup from 1-50 kg showed no changes in the Ag. The Phase 1 trial is expected in Q3 2013.
This was most impressive: it is to be hoped that the promise is maintained!
Yoseph Shaaltiel (Protalix Biotherapeutics, Israel) spoke on Protalix’s new product: this was alpha galactosidase-A, for the treatment of Fabry disease. This is an X-linked lysosomal storage disease that results in massive storage of glycolipid Gb3, in cells, in the vascular system and elswhere, which impairs the tissue of the heart and affects kidney and other organ function. There were worse consequences than with Gaucher disease, while it was less obvious. The current therapy was seen as being bad, and patients had reduced life expectancy. There were 2 therapeutic enzymes on the market: these were Agalsidase Alfa and Beta; these were very inefficient and expensive, so cost benefit was very limited. 1/2 life in blood was normally just a few minutes, and the proteins were very immunogenic.
Protalix aimed at making a biobetter: this was made in tobacco cells cultured in bags (they used Icon vectors, so could not work in their favoured carrot), by cocultivation with Agrobacterium and then killing the bacteria. The protein subunits were PEGylated to reduce immunogenicity and x-linked using bis-NHS-PEG. This gave improved stability, longer circulatory 1/2 life, enhanced activity in target organs with similar to improved kinetics, so lower dosing and longer intervals between doses were possible. Yields were good too, and they could make the enzyme very pure. The product had the same kinetics as the commercial products with better activity over a wide pH range.
As far as glycosylation was concerned, the commercial product had very complex glycosylation, while the plant-made product’s profile was very consistent and simple. It had an enhanced circulatory 1/2 life, of 581 vs 13 min, and also had higher activity in target cells – heart, kidney – over time. Yoseph noted that the patents on the enzyme(s) were limited to CHO cell production, meaning they had a useful window to exploit.
A comment from Jim Carrick was that the FDA was not interested in PEGylated products, as this could lead to vacuolation of kidneys in the long term. Yoseph said their product was not the same, as normally PEGylation added 20-40 kDa, whereas theirs was a much shorter x-linker. Their product was, moreover, already in clinics, as the FDA had said they should move straight to patients rather than testing it in healthy people.
Lydia Meador (Arizona State University) reported on their lab’s HIV vaccine candidate, made in plants and also vectored by NYVAC-KC delB19 poxvirus. They had previously shown that a CTB-HIV membrane proximal region (MPR) fusion vaccine resulted in Ab that stops transcytosis of HIV by Ab; she noted that live vectors enhance T-cell responses compared to subunit vaccines, so a combination would be a good idea.
Accordingly, they had cleverly produced whole HIV Gag and a deconstructed gp41 – stable Gag transgenics, and transiently-produced dgp41 – in the same plants, to make 100nm VLPs. While VLPs are highly immunogenic alone, they wanted to prime with the NYVAC and boost with plant-made antigen. They obtained good p24 Ab responses with NYVAC and the VLP boost; gp41 less so. In terms of mucosal immunity, they saw the IgA response against gp41 was significantly higher in the NYVAC+VLP combination, as were CD8+ T-cells. She noted that the anti-NYVAC titre was high after 3x doses. In response to my question, she did not know if the NYVAC vaccine made VLPs in mice – which it may not do, even if it works in plants, due to different protein requirements for budding in mouse vs plant cells.
Daniel Tusé (Intrucept Biomedicine, Kentucky) – a company founded with Kenneth Palmer – spoke on ‘Safety and efficacy of plant-produced Griffithsin for antiviral indications’. He noted that while griffithsin was an excellent anti-HIV microbicide, it was also a reasonably broad-spectrum antiviral lectin, as it was effective against the recently-emerged MERS CoV and influenza viruses.
The protein was hard to make from seaweed, and E coli was useless for production; however, they got g/kg in tobacco via conventional rTMV vectors, and now even better with Icon and Nomad vectors. KBP had manufactured it to near-GMP production standards, again at g/kg yields, with product recovery at 30% from leaves and 50% from leaves + stems, to a final purity of 99.8%. The potency was the same as the alga-derived product, and they had 100s of gm of product.
As griffithsin binds HIV with very high affinity, its primary use would be as a topical microbicide, to prevent transmission of HIV and HSV; to prevent coronavirus infections, and to act on chronic virus infections. The protein is not mitogenic on PBMC and does not activate T cells; it does not produce inflammatory cytokines in human PBMC, unlike cyanovirin, which had a much worse proinflammatory profile. The epithelial toxicity was also very low, which was in contrast to some well-publicised agents which had disastrously resulted in increases of HIV acquisition in women using them.
A carbopol-based gel was found to have the best drug-release kinetics, so was adopted for formulating the product for use. This protects mice against genital herpes: herpes has 2x the risk of infection per exposure compared to HIV infection. The gel has broad specific activity against coronaviruses too, to a wide spectrum of viruses from human, cow, chicken and pig. It could protect mice against SARS CoV, if given intranasally at 2 doses/day.
The protein also has uses in prevention of infection in the organ transplant area, eg against hepatitis C virus (HCV): it prevents infection of Huh-7 cells by cell-culture derived HCV, and partially protects hepatocytes from viral spread in vivo. If injected in animals it persists, and maintains an anti-HIV activity. It is immunogenic, but only weakly so, and Ab to it don’t neutralize its effects. Their lab was using rational design to take out T-cell epitopes without affecting antiviral activity.
Daniel stressed that this is a new drug, which can be preferentially be made in plants at high yield, with very low cost of goods; that it was effective and safe.
Hugh Haydon (KBP) mentioned that the cost of goods was “pennies/dose”.
This was an interactive discussion session, addressing the topic ‘Commercialisation of molecular pharming products – objectives and targets for the next 5 years’.
Hugh Haydon of Kentucky BioProcessing (KBP), , speaking on behalf of the new MAPP, KBP and Icon collaboration, addressed product selection. He noted that MAPP was responsible for product development, Icon for technology development and purification, and KBP for large-scale manufacture. They had spun out Solmab as a collaborative vehicle for production of MAbs for infectious disease therapy.
He described their product selection rationale: this was based on
- proof of concept data
- platform suitability
- capacity for dual use of product
- availability of capital
- speed of the regulatory process
- regulatory success rate
- scalability of existing infrastructure
Accordingly, they had selected a “biobetter” of Synagis, and an Ebola MAb cocktail. The Synagis equivalent was better due platform parameters, known clinical parameters, the fact there were established markets which can grow, government and NGO humanitarian interest, and potential adaptation to other viruses. For Ebola, they had a 3 MAb cocktail that was known to work, strong government interest (for a stockpile), a more rapid regulatory pathway, and a tropical disease voucher from the FDA. He pointed out that these products won’t make blockbuster status, but are appropriate for small companies like theirs.
Kevin Whaley (MAPP) spoke on how we needed therapeutics that were multipurpose (disease, indication) as well as multi-vaccines. The attributes of the new biologics were multi-use, speed of production, scale of production, and cost advantage – especially for global health products costing <$US10/g, at scales of >10K kgs, with increased efficacy (pathology, cancer), increased acceptability and access. He noted that all modern paediatric vaccines are multi – this saves visits to clinics, especially in developing countries.
Scott Deeter (InVitria) noted that the biologics market was edging up to being worth $US125 billion – and reckons progress with plant-produced products is excellent.
John Butler (Bayer) thinks we are still looking for suitable products! He was of the opinion that initial targets were too difficult (eg NHL – and flu??!), and that improved product characteristics must benefit from being plant-made. He was adamant that PMP must not compete on price with other platforms – because there was no such thing as a bottleneck in fermentation capacity world-wide, and established industry could just cut prices if they wanted to. He spoke of real and perceived hurdles:
- regulatory pathway isn’t a hurdle
- plant vs human glycosylation is not either, as plant-specific glycans were not more immunogenic than human
Real risks were that:
- there were well-established alternatives
- the plant-made product industry was overstretched in terms of resources
Einat Brill (Protalix) addressed their future strategy:
- new biologics for orphan indications (clinical trials were smaller, one needed only several 10s kg a year for an entire disease cohort)
- recombinant vaccines
- hard to express proteins that were best expressed in plants
- Biobetters of commercial products
- They would continue to establish PMP regulatory environment as a viable route for biologic drugs development
- Biobetter efficacy: longer circulatory half life for favourable clinical outcome
- regimen frequency: longer treatment intervals due to increased drug stability, with lower dosing
- Changing administration route (eg: oral vs injectable): helps to improve patient compliance
This was an excellent session, if only to hear how people who have been involved in getting PMPs to the market viewed the prospects for the industry – and it appeared favourable, despite John Butler’s caveats.