Sensitization of Immune System Against Haptenized Melanoma Antigens

Abstract

The invention is based on the observation that certain phenols, monophenols or benzenediols, can be metabolized into reactive quinones, in particular ortho-quinones and related reactive intermediates, which is brought about by oxidation of monophenols and benzenediols by proteins exhibiting tyrosinase activity, such as human tyrosinase and the related proteins TRP1 and TRP2. Although the substances and the produced reactive intermediates are toxic and can induce cell death, it is more relevant according to this invention that they function as haptens that become covalently bound to the tyrosinase enzymes, in particular to histidine moieties, in or near the catalytic site of proteins exhibiting tyrosinase activity, such as tyrosinase, TRP1 and TRP2. An immune response is then to be mounted against these haptenized auto-antigens, in order to treat melanocytic malignancies, in particular melanomas. This is brought about by topical administration of the phenol compounds that can function as tyrosinase substrate analogues. The invention hence provides medicaments for and methods of treating melanomas.

Description

FIELD OF THE INVENTION[0001]The invention relates to the field of medicine, in particular to the fields of immunology, autoimmunity and autoantigens. The invention further relates to chemical modification of antigens and methods and means for treatment of neoplastic disease, in particular melanoma.BACKGROUND OF THE INVENTION[0002]Over the past several decades, the incidence of melanoma has increased at a faster rate than that of any other solid tumor (1). The highest have been observed in Australia and New Zealand (27.9 / 100,000 among males and 25.0 among females) and in North America (10.9 / 100,000 among males and 7.7 among females). In 2001, it was estimated that 51 400 cases of invasive melanoma would be diagnosed (2) Early recognition and surgical excision of the primary tumor provide the best opportunity for obtaining a cure. However, prognosis associated with more advanced melanoma remains poor. Patients presenting with thick primary lesions, American Joint Committee on Cancer (...

AI Technical Summary

Benefits of technology

[0014]Contrary to the high doses of tyrosinase surrogate substrates used in case of intra arterial infusions as a chemotherapy, a thousand fold lower systemic concentration is achieved in the current invention by local application of the active compounds on the lesions or injection in the melanoma lesion, to evoke sensitization of the immune system against melanoma cells. The intra arterial infusion of monophenols and benzenediols, in particular catechols, bypasses the melanoma cell in the skin or in the malignant lesion. The invention comprises the use of ‘haptenized’ proteins, in particular proteins exhibiting a tyrosinase activity and fragments of those proteins, which may be applied for eliciting immune responses in vivo or in vitro and for the manufacture of medicaments or vaccines. In particular embodiments of the invention, the reaction of the immune system of a subject to be treated for pigment cell malignancies such as melanoma, is further stimulated by immune modulators applied on or in the lesion, for instance compounds eliciting a local inflammatory response. In a most preferred embodiment, the method and medicaments of the invention are applied in conjunction with steps to decrease the presence or the activity of regulatory T cells. Regulatory T cells function to prevent autoimmunity and hamper attempts to elicit an immunogenic response against auto-antigens derived from melanocytes such as tyrosinase and tyrosinase related proteins TRP1 and TRP2. The invention provides different optional measures and steps to minimize the obstruction of regulatory T cells in the process of generating a cellular immunogenic response against the modified autoantigens of the invention.DETAILED DESCRIPTION

[0022]In case of the desired sensitization of the immune system of a melanoma patient with a composition according to the invention, comprising monophenol or benzenediol compounds, the T-cell mediated cytotoxicity will be directed toward cells displaying the modified autoantigens, such as melanocytes, especially when the compound is applied topically on the lesion or injected intralesionally at relatively low doses. It will be particularly advantageous to repeat the administration to provide a continuous exposure of modified antigen to the immune system and thereby boost the immune response. More preferably, a slow release formulation of the phenol or catechol may be applied, providing a prolonged and sustained exposure, while at the same time avoiding the toxicity of high peak doses of the compound to be used. During and after treatment, all cells having a melanin metabolism, including normal melanocytes in the skin and hair, will disappear, locally and / or even systemically. This depigmenting effect is an unwanted but in the case of malignant and metastasizing melanoma an acceptable side effect of the treatment.

[0027]In other embodiments two or more compounds of the group of monophenols and benzenediols may be used in combination, simultaneously in one composition or in separate compositions, simultaneously or sequentially applied to the lesion in situ. The use of several compounds has the advantage that the auto-antigen providing proteins that have tyrosinase activity, will be modified with several compounds and / or reactive intermediates. Thereby several different ‘haptens’ on the tyrosinase enzymes will provide immune systems of treated subjects with a wider range of potential antigens that can be taken up and displayed by HLA molecules. Since the ‘fit’ of an antigen is among other factors highly dependent on HLA isotypes, this broadened approach will boost the potential immune response significantly. The mounting of a systemic auto-immune reaction against all cells having tyrosinase activity provides an excellent means to combat also distant metastases, even micrometastases, that are not accessible to surgical methods or radiotherapy and which are not accessible for topical drug administration. The capability of melanomas to spread out and to form local and distant metastases is a common problem in treatment of patients suffering from malignant melanomas. This problem can be effectively eliminated with the methods and medicaments of this invention.

[0031]A pharmaceutically acceptable composition according to the invention comprises at least one monophenol or benzenediol compound that can function as a substrate analogue of tyrosine and is capable of reacting with proteins exhibiting tyrosinase activity; tyrosinase proteins or tyrosinase related proteins 1 and 2, or which can be activated by these enzymes into a reactive intermediate which can subsequently react with tyrosinase or other related proteins. Optionally the composition may comprise one or more compounds selected from immune modifying compounds, immunogenic adjuvants and pharmaceutical excipients. Pharmaceutical excipients may comprise any excipient known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21nd Edition 2005, University of Sciences in Philadelphia. Pharmaceutical compositions and medicaments of the invention may thus comprise binders such as lactose, cellulose and derivatives thereof, polyvinylpyrrolidone (PVP), humectants, disintegration promoters, lubricants, disintegrants, starch and derivatives thereof, sugar solubilizers, anti-oxidants, preservatives, immuno-stimulatory adjuvants or other excipients. The invention provides methods and means to formulate and manufacture new medicaments and / or pharmaceutical formulations for the treatment of melanomas by topical administration to sensitize the immune system against melanoma antigens. The composition is preferably a composition that is optimized for trans-epidermal delivery, and may comprise skin penetrants or permeators and skin-permeation enhancers such as organic solvents such as DMSO, ethanol, or propylene glycol, whereby the resulting medium (skin / solvent) may have an increased partition coefficient for the therapeutic compound(s). In another embodiment the composition is a so called slow release formulation, which are known per se in the art of pharmacy, and may for instance comprise a release controlling polymer, -gel or -matrix, forming a depository of the active compound(s) (i.e. the monophenols and / or benzenediols), optionally surrounded or coated with a release controlling coating or membrane or biodegradable polymer, providing a slow but continued administration and / or release of the active compounds. Topical delivery compositions comprise ointments, pastes, gels, medicated powders, creams, lotions, aerosols, sprays, foams and medicated adhesives. Medicated adhesives, such as depositories on patches, allow a sustained delivery of the drug over days in many cases at a constant rate. Alternatively, the composition may also comprise a pharmaceutically acceptable liquid formulation which may be injected directly into the lesion.

Problems solved by technology

Although 4-Hydroxyanisole was used in the treatment of metastatic melanoma, the intra arterial infusion of high doses of 4-Hydroxyanisole was proven not effective and lead to severe toxic events.

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