Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines.

Nonclinical reproductive and developmental safety evaluation of the MAGE-A3 Cancer Immunotherapeutic, a therapeutic vaccine for cancer treatment.

We assessed potential toxic effects of the MAGE-A3 Cancer Immunotherapeutic on female fertility and embryo-fetal, pre- and post-natal development in rats and on male fertility in rats and monkeys. Three groups of 48 female (Study 1) or 22 male (Study 2) CD rats received 5 or 3 injections of 100µL of saline, AS15 immunostimulant, or MAGE-A3 Cancer Immunotherapeutic (MAGE-A3 recombinant protein combined with AS15) at various timepoints pre- or post-mating. Male Cynomolgus monkeys (Study 3) received 8 injections of 500µL of saline (n=2) or the MAGE-A3 Cancer Immunotherapeutic (n=6) every 2 weeks. Rats were sacrificed on gestation day 20 or lactation day 25 (Study 1) or 9 weeks after first injection (Study 2) and monkeys, 3 days or 8 weeks after last injection. Injections were well tolerated. Female rat mating performance or fertility, pre- and post-natal survival, offspring development up to 25 days of age, and male mating performance (rats) or fertility parameters (rats and monkeys) were unaffected.

Therapeutic potential of protein and adjuvant vaccinations on tumour growth.

Over 90% of cervical cancers are associated with HPV infection, the commonest being the HPV-16 subtype. Two early viral genes, E6 and 7, play major roles in the development and maintenance of the malignant phenotype. The vaccine potential of a recombinant HPV16 E7 protein was examined in two murine models of E7-expressing tumours. Formulations including the immunostimulants MPL and QS21 induced therapeutically active immune responses leading to regression of pre-established TC1 tumour lesions, associated with induction of IgG antibodies, lymphoproliferation and CTL. Our data provide a clear incentive to investigate the clinical application of this approach in cancer immunotherapy.

Liposomes that provide T-dependent help to weak antigens (T-independent antigens).

The design of an adjuvant for eliciting a thymus-dependent response to LPS, a well-defined thymus-independent antigen, is presented. Hybrid liposomes containing LPS and HA2 peptide from the hemagglutinin protein of influenza virus within the liposome bilayer were prepared (LPS/HA2 liposomes). The HA2 polypeptide contains epitopes recognized by T-helper lymphocytes and T-cytotoxic lymphocytes. Outbred mice immunized with LPS/HA2 liposomes produced anti-LPS-specific IgG responses. IgG subclass analysis indicated that IgG1, IgG2, and IgG3 antibodies were produced by these animals. LPS liposomes (liposomes without HA2) stimulated a T-independent response only. This was demonstrated by the detection of IgG3 but not IgG1 or IgG2 in serum of mice immunized with LPS liposomes. These results support the concept that the simultaneous incorporation into liposomes of a polypeptide with T-cell recognition sites along with a T-independent antigen can lead to the generation of cognate T-cell help for the T-independent antigen. The synthesis and characterization of a neo-lipopolysaccharide T-independent antigen for incorporation in hybrid HA2 liposomes are also presented. Findings are discussed relative to the liposome model used and implications for development of vaccines for use in humans.

Mannose-mediated targeted immunoadjuvant action of liposomes.

The effect of the ligand mannosylated albumin, covalently coupled to the surface of tetanus toxoid-containing dehydration-rehydration vesicles (DRV), on their adjuvanticity was investigated. Toxoid-containing DRV coated with mannosylated albumin bound in vitro to mouse (BALB/c) peritoneal macrophages selectively and to a greater extent than non-mannosylated DRV. ELISA assays of anti-toxoid IgG1 and IgG2b in the sera of BALB/c mice immunized with various toxoid-containing DRV preparations suggest that mannosylated liposomes are superior in immunoadjuvant action to conventional ones. Such targeted adjuvanticity appears to be influenced by the number of ligand molecules available on the surface of liposomes rather than the number of mannose residues on albumin.