Adjuvants: the players

Several Australian companies are trialling new 'natural' and synthetic adjuvants that combine potent immuno-stimulatory activity with low toxicity. Australia's biggest biotech, Melbourne's CSL (ASX:CSL), is boosting its experimental vaccines with its Iscomatrix adjuvant system, a phospholipid-cholesterol formulation containing a purified saponin extract from the bark of the South American tree Quillaja saponaria.

Canberra's Vaxine is also experimenting with a potent, plant-derived adjuvant, Advax, a modified version of a compound called inulin, extracted from dahlia tubers.

In its recent takeover of small US biopharmaceutical company Galenica, Sydney cancer therapeutics developer Australian Cancer Technologies, or AustCancer (ASX:ACU) acquired the intellectual rights for Galenica's GPI-0100, a potent immune-booster, that exhibits cancer-fighting properties in its own right.

Melbourne's VacTX, a subsidiary of Perth biotech investment company Eqitx (ASX:EQX), is developing novel vaccines for infectious diseases, in which the adjuvant is built into custom-designed lipopeptide antigens. The technology was developed by the Australian Vaccine Cooperative Research Centre.

CSIRO Animal Health spin-off Imugene (ASX:IMU) is another company whose experimental vaccines come with an in-built adjuvant.

Imugene's Adenoviral Delivery Vector System (ADVS) employs transgenic adenoviruses expressing antigens from viruses or bacteria that cause livestock disease. A transgene coding for the immune system's own, potent adjuvant, gamma interferon, delivers the immunogenic kick.

The key ingredient in the Canberra vaccine developer's adjuvant is a modified form of inulin, a polyfructose (plant sugar) extract from dahlia tubers. Biochemist Dr Peter Cooper, of the John Curtin School of Medical Research, discovered inulin's adjuvant properties 25 years ago while searching for an anti-cancer drug to activate the immune system's complement pathway to induce tumour necrosis.

Vaxine

Vaxine chairman Nikolai Petrovsky says that despite showing promising anti-cancer activity, inulin had yet to be developed as a cancer drug. But Cooper subsequently discovered inulin was a powerful adjuvant -- and very safe, because polyfructoses are non-toxic, even at high doses, and in the body they are metabolised to glucose or excreted unchanged by the kidneys.

Cooper traced the adjuvant activity to a particular isomer, gamma-inulin, but after his retirement, the discovery gathered dust until he drew it to Petrovsky's attention four years ago.

Petrovsky's research team at Canberra Hospital 'tweaked' the molecule to create a new, synthetic isomer, alpha-inulin, that is an even more potent adjuvant and activator of the complement pathway.

"We use it as a base, and add other things to it," Petrovsky says. "It's the first tailored adjuvant. It's not like alum, which either works, or doesn't -- we can fine-tune it for particular antigens, by adding alum, albumin or other substances.

"The beauty of it is that it's very good at stimulating both antibody and T-cell immunity, the bugbear of most adjuvants. It does this by stimulating the immune system through activating the complement pathway."

Petrovsky says low toxicity is paramount in an adjuvant -- the reality is that most adjuvants that elicit good T-cell immunity in mice are too toxic to be used in humans.

A Phase I trial of inulin as an adjuvant for the human papilloma virus (HPV) vaccine developed by University of Queensland researcher Dr Ian Frazer to protect against cervical cancer, confirmed it produced no adverse side-effects. (Merck and Co are now commercialising the HPV vaccine, using CSL's Iscomatrix adjuvant.)

Vaxine is working with several research groups in Australia and overseas on novel vaccines that will exploit its Advax adjuvant. Its current focus is on hepatitis B and influenza vaccines, but it has projects on type 1 diabetes, malaria, gastric and other cancers.

It is planning a Phase I/II trial of a prophylactic and therapeutic vaccine for hepatitis B in healthy human volunteers in the second quarter of 2005.

Vaxine is applying its Advax technology to developing vaccines for, among other things, cancer -- principally gastric cancer -- hepatitis B, influenza and malaria. It will soon begin Phase I trials of its experimental hepatitis B vaccine -- one aimed at establishing its capacity to prevent the disease, the other to explore its potential as a therapeutic to clear chronic, life-threatening hepatitis B infections.

AustCancer

With its takeover last year of Alabama-based Galenica Pharmaceuticals, Sydney cancer-therapy developer AustCancer acquired Galenica's promising GPI-0100 'immunity booster' -- an adjuvant technology, by another name.

Galenica has spent US$12 million developing its novel adjuvant, antigen and carrier technologies. GPI-0100 is being evaluated as a post-operative therapeutic for prostate cancer at America's largest cancer-research institute, the Memorial Sloan Kettering Cancer Centre. It is designed to be conjugated to antigens to boost their immunogenicity.

GPI-0100 relies on the same natural compound used in Australian vaccine manufacturer CSL's Iscomatrix adjuvant: a saponin extract from the bark of the Chilean soap bark tree Quillaria saponaria.

The beverage industry uses modified Quillaria saponin extracts as a foaming agent. As an adjuvant, the extract has multiple advantages, according to AustCancer's adjuvant program leader Dr Dante Marciani: it's non-toxic, extremely safe, stable, simple to formulate, and elicits strong antibody and CTL responses. It also induces high levels of interferon-gamma.

The GPI-0100 is simply mixed with the antigens, but is itself conjugated to a carrier molecule, KLH.

The Memorial Sloan-Kettering is experimenting with prostate-cancer vaccine for post-operative patients, containing GPI-0100 and four abnormal oligosaccharide antigens expressed on the surface of prostate-cancer cells.

The Baylor College of Medicine is experimenting with the adjuvant in a novel therapy for kidney cancer, which exploits the fact that cancerous kidney cells over-express folate receptors.

The Baylor therapy involves linking folate to a hapten, to make it immunogenic, then injecting it into the patient. It labels the cancerous cells by binding to the folate receptors.

Patients are injected with the GPI-0100/KLH mix, to stimulate the immune system, then injected with folate linked to a hapten that makes it immunogenic. The folate marks the cancerous cells for a combined antibody/T-cell attack by binding to their folate receptors.

VacTX

Melbourne vaccine developer VacTX, a subsidiary of Perth biotech investment company Eqitx, has the adjuvant game down to a 'T', with technology that integrates antigen and adjuvant in a T-shaped molecule.

The idea came from Dr David Jackson's research group in the Cooperative Research Centre for Vaccine Technology -- Jackson doubles as VacTX's chief scientist.

Adjuvants can be "pretty toxic things", says Jackson. It's why Freund's complete adjuvant, despite its formidable immunostimulatory capacity, has never been licensed for use in human or veterinary vaccines

Inducing the immune system to pay attention to single proteins or peptides is no simple matter, when natural selection has shaped it over several hundred million years to recognise antigenically complex viruses, bacteria and parasites.

Jackson's team decided to try mimicry, by attaching the antigen to a PAM2-Cys molecule -- a complex of palmitic acid, glycerol and cysteine, which occurs naturally in mycoplasmas. Many bacteria have similar complexes in their cell walls.

The PAM2-Cys complex binds to the Toll-like receptor, TLR2, expressed on the surface of dendritic cells. The dendritic cell processes the attached antigen, and proceeds to the lymph nodes to alert the immune system's "SWAT squad", as Jackson describes it.

The fundamental problem of the MHC system's heterogeneity is addressed by using a complete protein, and the individual's immune system processes it according to how the alleles fall in the MHC lottery.

Jackson says that, despite the individuality of MHC responses, a vaccine comprising a few, select peptide antigens will still confer immunity in a high percentage of individuals if it is biased towards the antibody response -- the main problem lies with the cytotoxic T cell response which is more heavily dependent on the MHC.

His team has used its novel adjuvant technology to develop a vaccine to control reproduction in all mammals -- its target is luteinising hormone releasing hormone (LHRH). The vaccine elicits a strong antibody response to the hormone, which is ultra-conserved across mammals -- human, lion, and giraffe LHRH are identical.

LHRH sits atop the endocrine cascade that regulates sex-hormone production in both sexes, so the vaccine can potentially control fertility in all mammals. The vaccine would not be used in humans, except as a potentially life-saving therapy for sex-hormone dependent cancers like oestrogen-dependent breast and ovarian cancers in women, and testosterone-dependent prostate cancer in men.

Jackson's team is using LHRH and the digestive enzyme gastrin, secreted by oesophageal and pancreatic cancers, as model antigens for its PAM2-Cys adjuvant system. It has found it can also elicit a good CTL (cytotoxic T-lymphocyte) response by using appropriate nine-residue peptides.

"But the lipopeptide part of the adjuvant is really the key to opening the front door of the dendritic cells," he says. "When we began working on peptide vaccines eight or nine years ago we always ran up against the same problem: they're so poorly immunogenic unless you mix them with Freund's adjuvant. So we started thinking about lipids.

"We chose the influenza virus as a model, and Nicholas Ede and I simply stuck palmitic acid to our influenza peptides. Lorena Brown, our immunologist, was able to show a moderate cytotoxic lymphocyte response.

"But it became obvious we needed something more potent. We now use PAM2-Cys. It's at least as good as Freund's. We found that adding it to the centre of the composite molecule, rather than the end, made it more soluble and also more effective as a vaccine."

The linear peptide chain forms the 'bar' of the T-shaped complex, and the palmitic forms the stem, which attaches to a side chain projecting from one of the antigen's amino acids.

Jackson's team has now demonstrated the unique structure's efficacy against influenza, the intracellular bacterium Listeria monocytogenes, two animal models of tumours, as well as host proteins and peptides involved in normal physiology, without a single failure.

But these successes have been in animal models. "It would be surprising if it didn't work in humans, and if we can convince the Therapeutic Goods Administration, we'd like to test the influenza vaccine in human volunteers," Jackson says.

"Most current influenza vaccines do no not induce a cytotoxic response. By inducing a CTL response with our vaccine, we might establish a population of memory T-cells, and that would be very useful in quickly limiting infection due to influenza.

"Our vaccine would be different, because the appearance of new virus strains is currently driven by the antibody profiles elicited by current vaccines."