United States Patent:   6,020,309

Assignee:  Cancer Research Campaign Technology Limited (London, GB)

Filed:  January 24, 1996

The invention provides pharmaceutical formulations for Immunizing against papillomavirus tumors or lesions. Formulations may be selected from the group consisting of: (i) a formulation which comprises a papillomavirus (PV) L2 protein or fragment thereof that is effective to provide a protective effect to lessen the occurrence and severity of lesions or tumors caused by PV infection and an aluminum compound; (ii) a formulation which comprises a bovine papillomavirus (BPV) L2 protein or said fragment thereof and an aluminum compound: (iii) a formulation which comprises a BPV4 L2 protein or said fragment thereof and an aluminum compound; (iv) a formulation which comprises a BPV-4 protein or said fragment thereof and an adjuvant; (v) a formulation which comprises a BPV-4 L2 protein or said fragment thereof and an adjuvant; and (vi) a formulation which comprises a BPV4 protein or said fragment thereof and an aluminum compound.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceuticals based on papillomaviruses.

Papillomaviruses induce a variety of lesions both in humans and in animals. Some papillomas, albeit benign, are themselves a clinical problem, such as laryngeal papillomas of children or penile papillomas of bulls and others are known to be a risk factor in the pathogenesis of cancer, as in the case of flat lesions of the cervix or penile condylomata in humans. Therefore both in human and veterinary medicine, antiviral pharmaceuticals against papillomaviruses e.g. for prophylactic use to protect against the establishment of a serious infection would be of major advantage.

Vaccination studies in humans present several problems. First of all experimentation is ethically unacceptable. Secondly, very limited amounts of virus are available as some lesions, in particular those of the cervix, do not produce viral progeny, and no in vitro system is yet available which allows vegetative replication of virus.

The production of viral proteins in bacteria and the use of synthetic peptides have circumvented this last problem and have allowed the ongoing analysis of the immune response to papillomavirus infection (see for instance Jenison et al, 1988 J. Virol, 62 p. 2115 and Tindle et al, 1990 J. Gen. Virol, 71 p. 1347.

Effective prophylactic vaccines, both natural (Jarrett et al, 1990 The Vet. Record, 126 p. 449) and genetically engineered (Pilachinski et al, 1986 Ciba Foundation Symposium Vol. 120 p. 136) have already been produced against bovine papillomaviruses, and regression of Shope papillomas has been achieved by vaccinating rabbits with tumour tissue extracts (Evans et al, 1962 J. Nat. Cancer Inst. 29 p. 277). The bovine system is an excellent model for papillomavirus induced diseases in the human and for medicaments against such diseases in humans given the several similarities between the bovine and human disease. Namely multiple virus types with high lesion specificity (Jarrett et al, 1984 Virol. 136 p. 255), homology of genetic structure, and progression of some lesions to malignancy. The bovine system also presents several advantages in that cofactors in oncogenesis are known (Campo and Jarrett, 1986 In Papillomaviruses, Ciba Foundation Symposium 120, John Wiley and Sons p. 117) and, above all, direct experimentation is possible (Jarrett, 1985 In advances in Viral Oncology (Ed. G. Klein) 5 p.83).

Vaccination has been traditionally regarded as a prophylactic measure. Hosts are immunised against a pathogen and on subsequent exposure to the same organism, the infection is aborted at a preclinical stage. This is usually accomplished in virus infections by a neutralizing event directed at a superficial epitope on a structural protein. Virus-induced tumours such as papillomas often persist for long periods and are then, in a proportion of cases, rejected. The host may then be immune to reinfection (Jarrett, 1985 supra). The nature of the rejection mechanism and its mediators is unknown, but it is advantageous to induce the rejection mechanism early in a tumour life cycle. In veterinary practice, it has been known for a long time that crude extracts of papillomas could sometimes, but by no means always, cause the rejection of homologous papillomas (Olson et al, 1959 Am. J. Vet. Res. 21 p. 233, Evans et al, 1962 supra). These experiments were carried out before it was known that there were different types of papillomavirus in a species (Jarrett et al, 1984 supra; de Villier, 1989 J. Virol. 63 p. 4898), and that these are probably all immunologically type-specific (Jarrett et al. 1990 The Vet. Record 126 p. 473; Jenison et al, 1988 supra).

Papillomaviruses can infect and cause tumours in both cutaneous and mucous epithelia. In man and animals mucosal papillomas are often serious lesions which tend to run a prolonged course, as in the case of human laryngeal, genital and cervical papillomas (Steinberg, 1987 In the Papovaviride Vol. 2 (ed. N. P. Salzman and P. M. Howley) p. 265; zur Hausen, 1991 Virol. 184 p. 9). These may be causal factors in the subsequent development of malignancies, as in the case of human papillomavirus (HPV) type 16-associated cervical carcinoma in women (zur Hausen, 1991 supra), and bovine papillomavirus (BPV) type 4-associated alimentary cancer in cattle (Campo and Jarrett, 1986 supra). Clearly, there is an urgent need for pharmaceuticals against papillomavirus diseases.

Despite recent limited success (Kreider et al, 1986 J. Virol. 59 p. 369; Sterling et al, 1990 J. Virol. 64 p. 6305; Meyers et al, 1992 Science 257 p. 971; Dollard et al, 1992 Genes and Development 6 p. 1131), papillomaviruses are notoriously refractory to growth in cultured cells (Teichaman and LaPorta, 1987 In The Papovaviridae, Vol. 2 (ed. N. P. Salzman and P. M. Howley) p. 109) and the consequent lack of viral reagents has delayed the analysis of the immune response to the infection. The recent availability of recombinant technology has allowed the production of both early and late viral proteins in large amounts and in a purified form (Tindle et al, 1990 supra.; Jarrett et al, 1991 Virol. 184 p. 33; Ghim et al, 1992 Virology 190 p. 548; Stacey et al, 1992 J. Gen. Virol. 73 p. 2337) and thus has for the first time made it possible to study their immunising potential. It has already been shown that in cattle it is possible to use viruses and tumour extracts to induce both protection against, papillomavirus-induced cutaneous lesions (Jarrett et al, The Vet Record 1990 126 p. 473; The Vet Record 1990 126 p. 449) and to use recombinant proteins to induce both protection against and rejection of PV-induced cutaneous lesions (Virology 1991, 184 p. 33). The applicants now show that one can vaccinate in a practically significant fashion against papillomavirus for example the mucosal virus BPV-4, which in the field is a causative factor of squamous cell carcinoma of the upper alimentary canal (Campo and Jarrett, 1986 supra).

The present invention provides a pharmaceutical formulation for the prophylaxis of papillomavirus tumours or lesions selected from the group consisting of: (i) a formulation which comprises a papillomavirus (PV) L2 protein or prophylactically effective fragment thereof and an aluminium compound, (ii) a formulation which comprises a bovine papillomavirus (BPV) L2 protein or prophylactically effective fragment thereof and an aluminium compound; (iii) a formulation which comprises a BPV-4 L2 protein or prophylactically effective fragment thereof and an aluminium compound; (iv) a formulation which comprises a BPV-4 protein or prophylactically effective fragment thereof and an adjuvant; (v) a formulation which comprises a BPV-4 L2 protein or prophylactically effective fragment thereof and an adjuvant; (vi) a formulation which comprises a BPV-4 protein or prophylactically effective fragment thereof and an aluminium compound; (vii) a formulation which comprises a PV L2 protein or prophylactically effective fragment thereof and an adjuvant; (viii) a formulation which comprises an HPV L2 protein or a prophylactically effective fragment thereof and an adjuvant; (ix) a formulation which comprises an HPV L2 protein or a prophylactically effective fragment thereof and an aluminium compound; (x) a formulation which comprises an HPV-16 L2 protein or a prophylactically effective fragment thereof and ad adjuvant; (xi) a formulation which comprises an HPV-16 L2 protein or a prophylactically effective fragment thereof and an aluminium compound.

The pharmaceutical formulation may comprise a PV L2 protein or a prophylactically effective fragment thereof and an aluminium compound. The PV may be BPV. The BPV may be BPV-4.

The papillomavirus protein or prophylactically effective fragment thereof may be in the form of a fusion protein with a different co-protein. The co-protein may be glutathione-S-transferase.

The papillomavirus protein or prophylactically effective fragment thereof may be produced by recombinant DNA techniques.

The aluminium compound may comprise a mixture of aluminium hydroxide and aluminium phosphate. There may be 3% aluminium hydroxide and 2% aluminium phosphate. The aluminium compound may be aluminium gel.

Also provided is the use of a pharmaceutical formulation as above in medicine for the prophylaxis of papillomavirus tumours or lesions.

Also provided is use of (i) a PV L2 protein or prophylactically effective fragment thereof and an aluminium compound; or (ii) a BPV L2 protein or prophylactically effective fragment thereof and an aluminium compound; or (iii) a BPV-4 L2 protein or prophylactically effective fragment thereof and an aluminium compound; or (iv) a BPV-4 protein or prophylactically effective fragment thereof and an adjuvant; or (v) a BPV-4 L2 protein or prophylactically effective fragment thereof and an adjuvant; or (vi) a BPV-4 protein or prophylactically effective fragment thereof and an aluminium compound; or (vii) a PV L2 protein or prophylactically effective fragment thereof and an adjuvant; or (viii) an HPV L2 protein or a prophylactically effective fragment thereof and an adjuvant; or (ix) an HPV L2 protein or a prophylactically effective fragment thereof and an aluminium compound; or (x) an HPV-16 L2 protein or a prophylactically effective fragment thereof and all adjuvant; or (xi) an HPV-16 L2 protein or a prophylactically effective fragment thereof and an aluminium compound in the production of a vaccine for the prophylaxis of papillomavirus tumours or lesions.

Also provided is a method of immunising a mammal against papillomavirus tumours or lesions which comprises the administration of a pharmaceutical formulation as above in a therapeutically effective dosage.

Vaccines based upon the pharmaceutical formulations for prophylactic use as described above, can be prepared in accordance with methods well known in the art. Likewise, dosage rates can be determined according to known methods. Attention is directed to New Trends and Developments in Vaccines, Editors A. Voller and H. Friedman, University Park Press, Bultimore, 1978 and to Remington's Pharmaceutical Science by E. W. Martin.

Claim 1 of 7 Claims

1. A method of lessening the occurrence and severity of lesions or tumors caused by papillomavirus infection, comprising administering to a mammal post-infection with papillomavirus:

(i) a PV L2 protein or fragment thereof effective to provide a protective effect to lessen the occurrence and severity of lesions or tumors caused by PV infection and an aluminum compound; or

(ii) a bovine papillomavirus (BPV) L2 protein or a fragment thereof effective to provide a protective effect to lessen the occurrence and severity of lesions or tumors caused by BPV infection and an aluminum compound; or

(iii) a BPV-4 L2 protein or fragment thereof effective to provide a protective effect to lessen the occurrence and severity of lesions or tumors caused by BPV infection and an aluminum compound; or

(iv) a BPV-4 protein or fragment thereof effective to provide a protective effect to lessen the occurrence and severity of lesions or tumors caused by BPV infection and an aluminum compound.

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