||Description of our technology
iQur is concentrating its therapeutic research and development on a vaccine platform based on exploiting the immune stimulating properties of Hepatitis B core using patented Tandem Core® constructs. Whilst this is a platform technology, and is thus capable of carrying many different target antigens, iQur is developing a combined HAV and HBV vaccine within a single recombinant protein as its lead compound. Although a combined vaccine currently exists for both HAV and HBV, it has some issues with efficacy, most likely due to the poor immunogenicity of the HBV component of the vaccine, and is also more expensive to make than a single Tandem Core® since it is a mixture of two very different processes.
Tandem core based vaccines
Tandem core utilises the unique properties of the HBV core (HBc) protein. It is well documented that HBc is highly immunogenic in its own right, most likely because it has been shown to bind directly to the Toll-like receptor 2 (TLR2). HBc spontaneously forms dimers, which are in turn the building blocks of a virus like particle (VLP). HBc induces very strong antibody responses with both T-independent and T-dependent elements. Thus, HBc may be described as an adjuvant (immune stimulator) as well as a vaccine platform. VLP based vaccines are particularly attractive since they are made entirely from protein and their lack of viral nucleic acid is an advantage with respect to safety. Indeed, many recent “blockbuster” vaccines are based on VLP technology, for example the HPV vaccines Gardasil™ & Cervarix™.
The structure of HBc has been solved and is characterised by two core proteins which form the basic dimer associating to form a “spike”. At the tip of this spike is the major insertion region (MIR), into which third party antigens may be inserted. Although antigenic insertions have also been carried out at both the N and C-termini of the proteins, the MIR has been shown to be the optimal site of insertion. Conjugation of a third party antigen to the MIR confers antigenicity to hitherto immunologically silent inserts.
However, despite comprehensive investigation of this system, significant problems with this technology led to the cessation of its commercial development. The most serious of these issues was the discovery that large or hydrophobic inserts caused HBc dimers to disassociate, leading to the subsequent disruption of VLP folding. In the absence of VLP formation, the immunogenicity of HBc is greatly depressed. Hence, despite the undoubted ability to make non-immunogenic inserts immunogenic, this technology was not commercialised.
iQur’s technology presents a solution
IQur has overcome these limitations by developing the tandem core construct, in which two HBc proteins are linked in a genetic construct forming a single protein comprised of twin spikes. Thus, the resulting protein is already dimerised but is sufficiently flexible to accommodate large and/or hydrophobic inserts. This protein still forms VLPs which are indistinguishable from wild-type monomeric core proteins (see electron micrograph).
Each tandem core has two antigen insertion sites and thus a single recombinant protein may express two different antigens simultaneously. We have already shown that our system can accommodate large and hydrophobic antigens such as HAV-P1 (88kD) alongside HBV surface antigen (sAg). Thus, the combination of HAV and HBV inserts makes a chimeric dual vaccine which forms a VLP. These inserted antigens are structurally very similar to the wild-type proteins and thus give rise to comparable antibody levels. Furthermore, VLPs have been shown to be processed by antigen presenting cells and thus also give rise to cellular immune responses at the same time. This technology is not limited just to hepatitis and can be used to generate immune responses against other infectious pathogens including viruses, bacteria and parasites such as hepatitis and malaria, agents used in biodefence, animal pathogens and cancer associated antigens.
Tandem core® is a breakthrough with a number of unique features:
Proof of concept data
Initial experiments used Green Fluorescent Protein (GFP) inserted into the first core of a tandem construct, principally because GFP allows simple detection. This demonstrated a number of the fundamental principles behind tandem core;
(i) GFP is a fairly large insert (27kD) and was easily accommodated onto the core
(ii) electron microscopy showed that VLP were readily formed
(iii) Tandem Core expressing GFP protein gave off green fluorescence thus proving that the whole construct assembled in the appropriate conformation since it only fluoresces when it is correctly folded.
Immunisation against a single antigen
This construct was then tested in vivo using three groups of mice which were vaccinated with either (a) saline (b) “empty” tandem core (c) tandem-core expressing one GFP molecule. After 14 days strong antibody responses were detected to core protein in (b) and (c) and to GFP alone in (c). This was as predicted and proved that a tandem-core construct was capable of generating an antibody response to both the vector (core) and insert (GFP).
Immunity to multiple antigens
A next iteration of the development was to examine the immune response produced to a tandem core construct expressing two antigens simultaneously. Unfortunately, there is no acceptable model of hepatitis infection in mice, so a surrogate system was devised. This uses HBV sAg in core 1 and influenza M2 in core 2. The latter insertion can then be used to test for protection against a live viral (flu) infection. It is also noteworthy that M2 is often described as the “universal flu vaccine” because it is largely invariant, irrespective of the strain of influenza it is found in. This is in stark contrast to both hemagglutinin and neuraminidase which are commonly used in flu vaccines and which must be varied annually. However, M2 is also widely acknowledged as being largely non-immunogenic and thus also makes a good test of the tandem core system since conjugation to core should improve the strength of the immune response elicited.
Mice were immunised three times with 5ug of sAg-M2 tandem core at weekly intervals. Blood samples were taken at regular intervals and tested by ELISA for antibodies to (i) HBc (carrier) (ii) HBV sAg (antigen1) and (iii) M2 (antigen2). Clear evidence of antibody responses to all three elements was recorded. Furthermore, these were very long-lasting since they could be detected nearly six months later.
Interestingly, the effect of delivering tandem core on adjuvant showed variability. Neither antibody to core nor sAg were affected by the presence of alum. However, M2 responses were enhanced when presented on alum. One possible explanation for this may be that both core and sAg elicit predominantly antibody responses, whilst M2 may also have a cellular component which may need adjuvant assistance to develop. These data suggest that for some inserts adjuvant may not be necessary, indicating the potent effect of the tandem core vaccine, and for others, when adjuvant is required, the activity would be greatly improved.
These data clearly demonstrate that tandem core can indeed induce multiple strong immune responses simultaneously from a single recombinant protein.
Protection against lethal influenza challenge
Having established that a recombinant tandem core protein carrying two antigens does indeed elicit multiple immune responses, an influenza challenge was carried out. When sAg-M2 was delivered on alum 80% of animals were protected from lethal challenge. Similarly, when the vaccine was delivered in the absence of adjuvant protection was also seen, although this was reduced to 20%. These data provide clear evidence that tandem core based vaccines are capable of generating multiple immune responses and that these can lead to complete antiviral responses.
Clinical vaccine candidate
Work is now focused on the development of tandem core based clinical vaccine candidates. Two major issues are being examined;
Insert optimisation: As described above, iQur has chosen a combined HAV/HBV vaccine as its preferred target. The HBV element is based on sAg, as used in Engerix-B. The current HAV vaccine, Havrix, is an inactivated HAV virus. However, an alternative approach is to use the HAV-P1 gene which encodes for all four elements of the viral capsid. This is a large insert, but has been used previously to generate anti-HAV immune responses (Karayianis et al 2004).
Both inserts have been successfully cloned into tandem core and a series of refinements are currently being tested to enhance the level of immune response.
Expression system: Initial development of tandem core was carried out in an E.coli based expression system. However, when the product was grown to commercial scale, the levels of lipopolysaccharide (LPS) associated with this system were found to be unacceptable for clinical purposes. Hence, a decision was taken to re-derive the tandem core plasmid for expression in yeast. This has several advantages including (i) low levels of LPS (ii) good levels of protein expression (iii) regulatory compliance (Engerix B is made in yeast).
Much of this development has been co-funded by the UK government’s Technology Strategy Board (TSB) with an aim to develop a commercial expression system for a vaccine candidate. These awards aim to promote UK industry and enhance collaboration between partners. As part of the acceptance of this, iQur ensured that all intellectual property remained with the company. An initial grant funded the development of a good manufacturing process (GMP) in partnership with University of Leeds and biotech companies, Mologic and Arecor Ltd. The value of this consortium was £1,093,350, of which 50% was paid by TSB. GMP development was carried out by Eden Biodesign and has been fully documented ready for technology transfer. A second grant was recently secured which will co-fund a full GMP manufacturing run (consortium value £1,133,500). The partners in this are University College London (Bioengineering) and Mologic Ltd. More information can be found on the Hepatacore project website