Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon.

Serum albumin-binding domain antibodies (AlbudAbs) have previously been shown to greatly extend the serum half-life of the interleukin-1 receptor antagonist IL-1ra. We have subsequently extended this approach to look at the in vitro activity, in vivo efficacy and pharmacokinetics of an agonist molecule, interferon (IFN)-alpha2b, fused to an AlbudAb. Here we describe this molecule and show that in this format AlbudAb half-life extension technology displays significant advantages in comparison with other methods of half-life extension, in particular genetic fusion to serum albumin. When compared directly IFN-alpha2b fused to an Albudab shows higher potency, increased serum half-life and greater efficacy than human serum albumin fused to IFN-alpha2b. AlbudAbs are therefore an ideal platform technology for creation of therapeutics with agonist activity and long serum half-lives.

Imiquimod and resiquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery.

Imiquimod, an immune response modifier and inducer of cytokines in vitro and in vivo, has been shown to have potent antiviral and antitumour activity and to act as an adjuvant for protein vaccination. We have undertaken studies in mice to investigate the potential of imiquimod and resiquimod to adjuvant DNA vaccination. These imidazoquinolines were administered by subcutaneous injection at the vaccination site immediately after particle-mediated immunotherapeutic delivery of plasmid DNA using a gene gun. Imiquimod was found to increase the number and maturation status of dendritic cells in draining lymph nodes, and to enhance antigen-specific CD4(+) and CD8(+) T cell responses, as assessed by analyses of clonal expansion, and the quantity and kinetics of cytokine production from these cells in lymph nodes and spleens collected after vaccination. A more substantial increase in IFN-gamma-producing, compared with IL-4-producing CD4(+) T cells suggested that imiquimod biased the immune response towards a predominance of Th1 cells. The analogue resiquimod was found to be to produce a similar Th1 biased immune response with a 10-fold reduced dose compared with imiquimod. Collectively, these studies suggest that both imiquimod and resiquimod may be suitable adjuvants for therapeutic DNA vaccines requiring induction of potent cytotoxic T cell responses.

Therapeutic immunisation with COPV early genes by epithelial DNA delivery.

Following challenge with COPV (canine oral papillomavirus), DNA plasmids encoding COPV L1, E1 or E2 protein were delivered into oral mucosal and cutaneous sites in beagles using particle-mediated immunotherapeutic delivery (PMID). Two weeks post-challenge, a priming dose of 8 microg DNA was delivered followed by a booster dose after a further two weeks. A group of control dogs were vaccinated using plasmid DNA encoding Hepatitis B virus surface (HBVs) gene. All of the control animals developed warts at the vast majority of sites (94%). All of the animals given wild type L1, E1, or E2 developed warts at most sites (88%, 75%, and 88%, respectively). The animals given codon optimised E2 however, were protected from wart growth with only one tiny lesion seen on a single animal that persisted for only a few days. The E1 codon optimised group was also significantly protected with a far lower number of smaller warts (48%) that persisted for a shorter duration. These data suggest that therapeutic immunisation by PMID with papillomavirus early genes is effective and emphasizes the importance of antigen load in the generation of protective responses to papillomavirus proteins.

Intraepithelial DNA immunisation with a plasmid encoding a codon optimised COPV E1 gene sequence, but not the wild-type gene sequence completely protects against mucosal challenge with infectious COPV in beagles.

DNA plasmids encoding the open reading frames of canine oral papillomavirus (COPV) nonstructural early genes E1, E2, or E7 protein were delivered into both oral mucosal and cutaneous epithelial sites in beagle dogs using particle-mediated immunotherapeutic delivery (PMID) technology. Control dogs were vaccinated with plasmid encoding either hepatitis B virus surface antigen (HBVs) or COPV L1. Using a prophylactic immunisation protocol, a priming dose of plasmid DNA was followed by a booster dose 6 weeks later. Four weeks after boost, all dogs were challenged with infectious COPV particles. Following viral challenge, as shown previously (M. A. Stanley et al., 2001, Vaccine 19, 2783-2792), mucosal papillomas developed in the negative-control HBVs vaccinated dogs, but all animals in the COPV L1 group were fully protected from disease development. In the early gene-vaccinated groups five of six in the E1-vaccinated dogs, two of six in E2-vaccinated dogs, and three of six in the E7-vaccinated beagles developed oral papillomas. Compared to the HBVs negative-control group the oral papillomas that did develop in the early-gene vaccinated beagles were significantly smaller, shorter in duration, and fewer in number. Taken together the disease burden was markedly reduced and this was statistically significant. In a second experiment one group of animals was vaccinated with plasmid encoding the wild-type COPV E1 gene, and a separate group was vaccinated with plasmid encoding a synthetic codon-optimised COPV E1 gene sequence. None of the codon-optimised E1-vaccinated animals developed papillomas at any challenge site. However, all animals vaccinated with wild-type E1 had papillomas. These data suggest that immunisation by PMID with papillomavirus early genes can significantly impact upon subsequent disease development and that full protection can be achieved using improved vectors encoding codon-optimised gene sequences perhaps emphasizing the importance of antigen load in the generation of protective responses to papillomavirus proteins.