IMMUNOGLOBULINS FROM VITILIGO PLASMA FOR MELANOMA THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from US Provisional Application Number 60/773,319, filed February 15, 2006 , which is incorporated herein by reference in its entirety

FIELD OF INVENTION

[0002] This invention is directed to compositions and methods of treating melanoma in a subject. Specifically, the invention is directed to the use of immunoglobulins and their functional fragments isolated from individuals with vitiligo or melanoma patients for the treatment of melanoma.

BACKGROUND OF THE INVENTION

[0003] Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells ("Cellular and Molecular Immunology" (1991) (eds) Abbas A. K., Lechtman, A. H., Pober, J. S.; W. B. Saunders Company, Philadelphia: pages 340-341). Melanomas make up approximately three percent of all skin cancers and the worldwide increase in melanoma is unsurpassed by any other neoplasm ' with the exception of lung cancer in women ("Cellular and Molecular Immunology" (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.; W. B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7:1-16). Even when melanoma is apparently localized to the skin, up to 30% of the patients will develop systemic metastasis and the majority will die (Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7:1-16). Classic modalities of treating melanoma include surgery, radiation and chemotherapy. In the past decade immunotherapy and gene therapy have emerged as new and promising methods for treating melanoma .

[0004] Strong evidence that an immune response to cancer exists in humans is provided by the existence of lymphocytes within melanoma deposits. These lymphocytes, when isolated, are capable of recognizing specific tumor antigens on autologous and allogeneic melanomas in an MHC restricted fashion. (Itoh, K. et al. (1986), Cancer Res. 46: 3011-3017; Muul, L. M., et al. (1987), J. Immunol. 138:989-995); Topalian, S. L., et a!., (1989) 7. Immunol. 142: 3714-3725; Darrow, T. L., et al., (1989) 7.

Immunol. 142: 3329-3335; Horn, S. S., et al., (1991) J. Immunother. 10:153-164; Kawakami, Y., et al., (1992) J. Immunol 148: 638-643; Horn, S. S., et al, (1993) J. Immunother. 13:18-30; O ¹ NeU, B. H., et al., (1993) J. Immunol. 151: 1410-1418). TIL from patients with metastatic melanoma recognize shared antigens including melanocyte-melanoma lineage specific tissue antigens in vitro (Kawakami, Y., et al., (1993) J. Immunother. 14: 88-93; Anichini, A. et al., (1993) et al., J. Exp. Med. 177: 989-998). Anti- melanoma T cells appear to be enriched in TIL probably as a consequence of clonal expansion and accumulation at the tumor site in vivo (Sensi, M., et al., (1993) J. Exp. Med 178:1231-1246). The fact that many melanoma patients mount cellular and humoral responses against these tumors and that melanomas express both MHC antigens and tumor associated antigens (TAA) suggests that identification and characterization of additional melanoma antigens will be important for immunotherapy of patients with melanoma.

[0005] There is a strong relationship between vitiligo and melanoma [Horning R, Cut J, Bystryn JC. Relation between the incidence and level of pigment cell antibodies and disease activity in vitiligo. J Invest Dermatol 97: 1078-80, 1991; Fishman P, Azizi E, Shoenfeld Y, et al. Vitiligo autoantibodies are effective against melanoma. Cancer 72: 2365-2369, 1993. ; Gilhar A, Zelickson B, Ulman Y, Etzioni A. In vivo destruction of melanocytes by the IgG fraction of serum from patients with vitiligo. J Invest Dermatol 105: 683-686, 1995.; Rigel DS, Rogers GS, Friedman RJ. Prognosis in malignant melanoma. Dermatol Clin North Am 3:309-14, 1985. Bystryn JC, Rigel D, Friedman RJ, Kopf A. Prognostic significance of hyperpigmentation in malignant melanoma. Arch Dermatol 123: 1053-5, 1987; D'aelio R, Frati C, Fattarossi A, Aiuti F. Peripheral T cell subset imbalance inpatients with vitiligo and in their apparently healthy first degree relatives. Ann Allergy 65: 143-5, 1990.; Bystryn JC, Naughton GK. Immunity to pigmented cells in vitiligo and melanoma. Fed Proc 43:1664-5, 1984.; Lerner AB, Nordlund JJ. Should vitiligo be induced in patients after resection of primary melanoma? Editorial. Arch Dermatol 113: 421, 1977; Donaldson RC, Canaan SA Jr, McLean RB, Ackerman LV. Uveitis and vitiligo associated with BCG treatment for malignant melanoma. Surgery 76:771-8, 1974; Naughton GK, Eising M, Bystryn JC. Detection of autoantibodies to melanocytes in vitiligo by specific immunoprecipitation. Arch Dermatol 81:540-2, 1983; Norris DA, Kissinger RM, Naughton GK , Bystryn JC. Evidence for immunologic mechanisms in human vitiligo: patients ' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody dependent cellular cytotoxicity (ADCC). J Invest Dermatol 90:783-9, 1988) and in some patients with malignant melanoma, circulating antibodies against melanoma cells may be detected. Because these antibodies also destroy normal, non-malignant melanocytes, vitiligo tends to develop in malignant .melanoma patients. Furthermore, it has been found that the prognosis in melanoma patients is better when hypo-

pigmentation develops [Koh HK, Sober AJ, Nakagawa H, et al. Malignanat melanoma and vitiligo-like leukoderma: An electron microscopic study. J Am Acad Dermatol 9:696-708, 1983, Nordlund JJ, KirkwoodJM, Forget BM, et al. Vitiligo inpatients with metastatic melanoma: A good prognostic sign. J Am Acad Dermatol 9:689-96, 1983], Therefore the selective destruction of pigmented cells occurring in vitiligo could be viewed as a kind of "natural immunotherapy" for melanoma.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the invention provides a method for treating melanoma in a subject, comprising the step of administering to the subject a preparation of immunoglobulins or fragments thereof, in an amount sufficient to treat melanoma, wherein the preparation is isolated from the plasma or sera of a preselected donor or donors.

[0007] In another embodiment, the invention provides a composition for the treatment of melanoma in a subject, comprising: a preparation of immunoglobulins or a fragments thereof, isolated from plasma or sera of a preselected donor or donors.

DETAILEP DESCRIPTION OF THE INVENTION

This invention relates in one embodiment to compositions and methods of treating melanoma in a subject. Specifically, the invention is directed to the use of immunoglobulins and their functional fragments isolated from individuals with vitiligo or melanoma patients in the treatment of melanoma, In another embodiment his invention relates to the use of immunoglobulins and their functional fragments isolated from individuals with vitiligo or pooled from a group of individuals with vitiligo. In yet another embodiment this invention relates to immunoglobulins and their functional fragments from an individual with melanoma or pooled from a group of melanoma patients.

[0008] In one embodiment non-specific immunoglobulins, their fragments, derivatives and metabolites, manufactured from the plasma of healthy donors has anti-cancer properties. These properties appear to be due to both the immunomodulatory effects as well as direct effects of certain sub-classes of antibody populations present within the donor population.

[0009] The use of pooled immunoglobulins, their fractions, derivatives and metabolites, isolated from individuals with vitiligo as described herein, offers a number of advantages when compared with

monoclonal antibodies (mAbs). In one embodiment, immunoglobulins, their fractions, derivatives and metabolites are not monoclonal, and thereby may permit better (e.g. more avid, greater aggregation on the cell surface) immunologic interaction with the target cell when compared to mAbs. In another embodiment, there may be better mobilization of immunological cofactors (e.g. complement) with polyvalent immunoglobulins, their fractions, derivatives and metabolites In one embodiment, immunoglobulins, pooled or derived from indivduals, their fractions, derivatives and metabolites have more than one specificity which might enhance interaction with the target cell.

[00010] In one embodiment, the invention provides a method for treating melanoma in a subject, comprising the step of administering to the subject a preparation of polyvalent immunoglobulins, their fractions, derivatives and metabolites In one embodiment, immunoglobulins, pooled or derived from indivduals, their fractions, derivatives and metabolites in an amount sufficient to treat melanoma, wherein the immunoglobulin preparation or fragments thereof is isolated from the plasma or sera of a preselected donor or donors.

[00011] In one embodiment, the term "immunoglobulins" or "IgG" refers to proteins secreted by cells of the immune system known as B-cells or plasma cells. In one embodiment, each immunoglobulin consists of a complex of protein chains known as the heavy and light chains. Each heavy chain is linked to a single light chain via disulfide bonds. The resulting complex is in turn linked by additional disulfide bonds to an identical heavy-light chain complex. This basic unit can be assembled by the cell into several specialized forms by varying the structure and number of heavy chains. Different heavy chain structures produce differing molecules, known as "classes" of immunoglobulins. These classes may also have different numbers of the basic units described above.

[00012] In one embodiment, the term "immunoglobulin" is used synonymously with the term "antibody", and encompasses all classes of immunoglobulins including, without limitation, IgG, IgM, IgA, IgD and IgE and all subclasses of immunoglobulins such as the IgG subclasses IgGl, IgG2, IgG3, and IgG4 found in or produced by cells or animals including humans. The term "immunoglobulin" encompasses both membrane immunoglobulins and secreted immunoglobulins. Membrane immunoglobulins are transmembrane proteins of B cells, and act as the B cells' antigen receptor. Secreted immunoglobulins are structurally identical to their membrane counterparts except that they lack the trans-membrane region of amino acids at the C-terminus of membrane immunoglobulins. Secreted immunoglobulins are present in extracellular fluids and secretions. Antibodies include in another embodiment complete antibodies (e.g., bivalent IgG, pentavalent IgM) or fragments of antibodies in other embodiments, which

contain an antigen binding site. Such fragment include in one embodiment Fab, F(ab') ₂ , Fv and single chain Fv (scFv) fragments. In one embodiment, such fragments may or may not include antibody constant domains. In another embodiment, F(ab)'s lack constant domains which are required for complement fixation. scFvs are composed of an antibody variable light chain (V _L ) linked to a variable heavy chain (V _H ) by a flexible linker. scFvs are able to bind antigen and can be rapidly produced in bacteria. The invention includes antibodies and antibody fragments which are produced in bacteria and in mammalian cell culture. An antibody obtained from a bacteriophage library can be a complete antibody or an antibody fragment. In one embodiment, the domains present in such a library are heavy chain variable domains (V _H ) and light chain variable domains (VL) which together comprise Fv or scFv, with the addition, in another embodiment, of a heavy chain constant domain (C _HI ) and a light chain constant domain (CL). The four domains (i.e., VH - C _H I and VL - CL) comprise an Fab. Complete antibodies are obtained in one embodiment, from such a library by replacing missing constant domains once a desired V _H - V _L combination has been identified.

[00013] Antibodies of the invention can be monoclonal antibodies (mAb) in one embodiment, or polyclonal antibodies in another embodiment. Antibodies of the invention which are useful for the compositions, methods and kits of the invention can be from any source, and in addition may be chimeric. In one embodiment, sources of antibodies can be from a mouse, or a rat, a plant, or a human in other embodiments. Antibodies of the invention which are useful for the compositions, and methods of the invention have reduced antigenicity in humans (to reduce or eliminate the risk of formation of anti- human andtibodies), and in another embodiment, are not antigenic in humans. Chimeric antibodies for use the invention contain in one embodiment, human amino acid sequences and include humanized antibodies which are non-human antibodies substituted with sequences of human origin to reduce or eliminate immunogenicity, but which retain the antigen binding characteristics of the non-human antibody.

[00014] The term "immunoglobulin" encompasses in one embodiment, fragments of immunoglobulins such as, for example, fragments F(ab') ₂ , Fab', Fab, Fc, Facb, pFc', and Fd, as well as immunoglobulin derivatives and metabolites in another embodiment. In one embodiment, metabolites of immunoglobulin are products resulting from the metabolism of immunoglobulins by a living organism. A wide variety of derivatives of immunoglobulins may be prepared in other embodiments by known methods, which in one embodiment, involve breaking peptide or disulfide bonds in the immunoglobulin. Immunoglobulins may also be derivatized in one embodiment to include modified or synthetic or unnatural amino acids. In one embodiment derivatives of immunoglobulins comprise immunoglobulins conjugated to a moiety

such as a toxin (e.g. cellular toxin, cisplatin), a labelling molecule (e.g. fluorescin, Texas Red), a radioactive atom or molecule (e.g. . ¹²⁵ I) for therapeutic or diagnostic use, an enzyme (e.g. avidin, horseradish peroxidase, alkaline phosphatase), et cetera. Immunoglobulins may include in other embodiments, post-translational modifications such as phosphorylation, glyocsylation, myristilation, prenylation, ADP-ribosylation, methylation, acetylation, hydroxylation, carboxylation, and oxidation- reduction, or may be catϊonized or anionized to alter the overall charge of the immunoglobulin In one embodiment, the immunoglobulins fragments used in the methods and compositions of the invention are Fab, Fab', Fabl, Fab2, Fc, or monomeric IgG. Additional analogs and derivatives of the immunoglobulins, or their fragments, which would be expected to retain or prolong their activity in whole or in part, and which are expected to be useful in the methods described herein, may also be easily made by one of skill in the art. One such modification may be the attachment of polyethylene glycol (PEG)onto moieties that can react with immunoglobulins or PEG derivatives into the analogues or derivatives of the immunoglobulins, by conventional techniques to enable the attachment of PEG moieties.

[00015] In one embodiment, preparation of immunoglobulins comprises compositions consisting solely of immunoglobulins (such as monoclonal or polyclonal immunoglobulins of a single specificity or combinations of monoclonal or polyclonal immunoglobulins of different specificities and/or different classes) and which may contain impurities (including the naturally-occuring components of a tissue or plasma into which the antibody was produced by transgenic means); or compositions comprising immunoglobulins, pharmaceutically acceptable diluents, carriers, adjuvants, liposomes and other therapeutic agents, et cetera; partially-purified in-process intermediate preparations of immunoglobulins; and articles comprising immunoglobulins or having immunoglobulins immobilized upon them or otherwise disposed thereon. In other embodiments, the compositions comprising the immunoglobulins as described herein, similarly comprise fragments, derivatives or metabolites of the immunoglobulins.

[00016] In one embodiment, immunoglobulins, their fractions, derivatives or metabolites are combined with buffer or salt solutions. The immunoglobulin its fraction, derivative or metabolite may also be combined with another therapeutic agent. The immunoglobulin, its fraction, derivative or metabolite and the other therapeutic agent may be co-packaged for administration by intravenous injection in one embodiment, or by topical administration in another embodiment. This may take the form of simple inclusion into a single external package, or they may be provided in a single container. An example of a more advanced form of packaging would be the use of immunoglobulins, their fractions, derivatives or metabolites in liposome preparations. Typically, the immunoglobulin, its fraction, derivative or

metabolite is embedded in at least the outer layer of the liposome where it can act as a targeting agent by binding to structures on or in the desired cells or tissues. The drug, which is contained within the liposome, is then released at this specific site, such as a melanoma tumor site in one embodiment, providing a more concentrated drug therapy with a larger therapeutic index than achievable by ordinary systemic therapy.

[00017] In one embodiment, the immunoglobulins, their fragments, derivatives or metabolites isolated from plasma or sera of a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, is administered in a therapeutically effective amount, The actual amount administered, and the rate and time-course of administration, will depend in one embodiment, on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences

The dosage of the immunoglobulins, their fragments, derivatives or metabolites and the method of administration will vary in certain embodiments with the severity and nature of the particular melanoma condition being treated, the. duration of treatment, the adjunct therapy used, the age and physical condition of the subject of treatment and like factors within the specific knowledge and expertise of the treating physician. Tn one embodiment, single dosages for intravenous and intracavitary administration can typically range from 250 mg to 2 g per kilogram body weight, preferably 2 g/kg (unless otherwise indicated, the unit designated "mg/kg" or "g/kg", as used herein, refers to milligrams or grams per kilogram of body weight). In one embodiment, the immunoglobulins, their fragments, derivatives or metabolites as described herein, are effective in inhibiting metastasis when administered by intravenous or intraperitoneal injection.

[00018] In one embodiment, the treatment described herein may also be used either preceding, concurrent or subsequent to a surgical procedure to remove the primary tumor. Frequently, metastasis of tumor cells will occur as a result of the physical manipulation of the tumor during surgery. However, the use of the treatment described herein in conjunction with surgery reduces in one embodiment, the risk of metastasis and consequently this combination of methods would be a more attractive treatment option for the complete elimination of cancerous tumors such as melanoma in one embodiment.

[00019] Other treatment modalities such as chemotherapy, vaccine administration, radiation therapy, immunotherapy, cell therapy or their combination may also be used in conjunction with the methods of the present invention.

[00020] Alternatively, targeting therapies may be used in another embodiment, to deliver the Immunoglobulins preparation, their fragments (such as Fab, Fab*, Fabl, Fab2, Fc, monomeric IgG, or thir combination in certain embodiments), derivatives or metabolites compositions isolated from plasma or sera described herein, more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable in one embodiment, for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

[00021] In one embodiment, plasma refers to the liquid medium in which blood cells are suspended and which contains salts, proteins and other organic compounds. In one embodiment, "fresh" blood plasma, frozen (stored) and subsequently thawed plasma or source plasma in other embodiments, is used for the separation purposes of the compositions and methods described herein. "Source plasma" is defined in one embodiment, as plasma collected by plasmapheresis and intended for further use such as fractionation into other materials in certain embodiments. In one embodiment, source plasma is used as a transfusion product in the compositions and methods described herein. Frozen (stored) plasma or serum is maintained in one embodiment, at sub-freezing temperatures, such as storage conditions of about -20 to about -80 degrees centigrade in one embodiment, until thawed and used. "Fresh" plasma or serum is refrigerated in another embodiment or maintained on ice until used, with isolation being performed as soon as possible.

[00022] In one embodiment, blood from a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, is drawn by standard methods into a collection bag, or a plastic collection bottle containing for example,, sodium citrate, ACD, CPD, CPD Al, heparin, or similar anticoagulants for preparation of plasma. In one embodiment, the plasma is separated from whole blood prior to being frozen. In another embodiment, fresh plasma is separated from whole blood by centrϊfugation. In one embodiment, the plasma is used for isolation and purification of immunoglobulins, their fragments derivatives or metabolites. In another embodiment, the fragments isolated and purified are Fab, Fab', Fabl, Fab2, Fc, monomeric IgG, or their combination in other embodiments.

[00023] Isolation of immunoglobulins is done in one embodiment by selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in the plasma or sera. In one embodiment, precipitation agents used are ethanol, polyethylene glycol, lyotropic (anti-chaotropϊc) salts such as ammonium sulfate and potassium phosphate, and caprylic acid. In another embodiment, ion exchange chromatography is used for isolation of immunoglobulins. In one embodiment, this processed is referred to as "plasma fractionation"

[00024] In another embodiment, Protein A and Protein G affinity chromatography are used for isolation and purification of immunoglobulins used in the methods and compositions of the invention, particularly for isolation of monoclonal antibodies, mainly due to the ease of use and the high purity obtained. In one embodiment, the same method is used for isolation and purification of polyclonal antibodies as well. In another embodiment, hydrophobic chromatography used for isolation of immunoglobulins, e.g in "Application Note 210, BioProcess Media" published by Pharmacia LKB Biotechnology, 1991. In this reference a state of the art product "Phenyl Sepharose High Performance" is described for the purpose of purifying monoclonal antibodies from cell culture supernatants.

[00025] In another embodiment, thiophilic adsorption chromatography is used for isolation of the immunoglobulins, their fragments, derivatives and metabolites. (J. Porath et al; FEBS Letters, vol. 1S5, p.306, 1985).In one embodiment, divinyl sulfone activated agarose coupled with various Iigands show specific binding of immunoglobulins in the presence of 0.5 M potassium sulfate, i.e. a lyotropic salt. In one embodiment, the sulfone group, from the vinyl sulfone spacer, and the resulting thio-ether in the ligand are structurally necessary to obtain the specificity and capacity for binding of antibodies.

[00026] In one embodiment, any isolation method capable of producing antibodies capable of binding to melanoma tumors or their metastases are appropriate for use in the methods and compositions of the invention.

[00027] In one embodiment, the plasma donor from which the immunoglobulins, their fragments, derivatives and metabolites are isolated for the compositions and methods described herein, is a Vitiligo patient, or a Vitiligo patient who has a diffuse vitiligo; a patient in which the Vitiligo developed in response to melanoma; a melanoma patient where anti-melanoma antibodies were identified; or a combination thereof in other embodiments. In one embodiment, the donor from which a given preparation is taken is a pool of donors. In one embodiment any vitiligo patient or pool of patients may be used as donors for the purposes of the methods and compositions described herein.

[00028] In one embodiment, the compositions described herein are used in the methods described herein. In one embodiment, the invention provides a composition for the treatment of melanoma in a subject, comprising: immunoglobulins preparation or a fragment, a derivative or metabolite thereof, isolated from plasma or sera of a preselected donor or donors.

[00029] In one embodiment, any donor used for the methods described herein, may be used for the compositions described herein. According to this aspect of the invention and in one embodiment, the invention provides a composition for the treatment of melanoma in a subject, comprising: immunoglobulins preparation or a fragment, a derivative or metabolite thereof, isolated from plasma or sera of a vitiligo patient while, in another embodiment, has diffuse vitiligo; or a melanoma patient who developed vitiligo in response to treatment of melanoma; a melanoma patient with identified circulating anti-melanoma antibodies, or a combination thereof in other embodiments.

[00030] Vitiligo, as used herein, refers in one embodiment to a cutaneous disease in which there is a complete loss of pigment in localized areas of the skin. This loss of pigment results in the effected areas being white. In other embodiments, this condition has a tendancy to occur around the mouth and the eyes. In one embodiment, the depigmented skin is sun sensitive, and thus is subject to sunburns and skin cancer such as melanomas in one embodiment.

[00031] In another embodiment, the term "vitiligo" may be referred to as leucoderma. In one embodiment, the depigmented areas are lacking in the skin pigment melanin, and in another embodiment, the disease is the result of the destruction or inhibition of the melanin secreting melanocytes in the affected areas. In yet other embodiments, there may be some hereditary component to the disease, since approximately 30% of the cases have a familial correlation. In one embodiment, the disease may be the result of an autoimmune or a specific metabolic defect may be involved, or, environmental factors to play a role in the aetiology of the disease in other mbodiment.

[00032] In one embodiment, vitiligo is subdivided into two clinical types: vitiligo non segmentalis (type A) and vitiligo segmentalis (type B). Type A is more common, has a potential lifelong evolution and is associated with Koebner phenomenon and frequently with autoimmune diseases, such as Sutton nevus, thyroid disorders, juvenile diabetes mellitus, pernicious anemia and Addison's disease. Type B is rarer and has a dermatomal distribution , wherein following a rapid onset and evolution it usually exhibits a stable course. The natural course of the disease is unpredictable in one embodiment, but it is progressive

in other embodiments. In one embodiment some degree of spontaneous repigmentation occurs in 10- 20% of patients, occurring in a perifollicular pattern.

[00033] In one embodiment, there is a relationship between vitiligo and melanoma [Horning R, Cui J, Bystryn JC. Relation between the incidence and level of pigment ^Cβ tt antibodies and disease activity in vitiligo. J Invest Dermatol 97: 1078-80, 1991; Fishman P, Azizi E, Shoenfeld Y, et al Vitiligo autoantibodies are effective against melanoma. Cancer 72: 2365-2369, 1993. ; Gilhar A, Zelickson B, Ulman Y, EtzioniA. In vivo destruction of melanocytes by the IgG fraction of serum from patients with vitiligo. J Invest Dermatol 105: 683-686, 1995.; Rigel DS, Rogers GS, Friedman RJ. Prognosis in malignant melanoma. Dermatol Clin North Am 3:309-14, 1985. Bystryn JC, Rigel D, Friedman RJ, Kopf A. Prognostic significance of hyperpigmentation in malignant melanoma. Arch Dermatol 123: 1053-5, 1987; D'aelio R ₁ Frati C, Fattarossi A, Aiuti F. Peripheral T cell subset imbalance inpatients with vitiligo and in their apparently healthy first degree relatives. Ann Allergy 65: 143-5, 1990.; Bystryn JC, Naughton GK. Immunity to pigmented cells in vitiligo and melanoma. Fed Proc 43:1664-5, 1984.; Lerner AB, NordlundJJ. Should vitiligo be induced in patients after resection of primary melanoma? Editorial. Arch Dermatol 113: 421, 1977; Donaldson RC ₁ Canaan SA Jr, McLean RB, Ackerman LV. uveitis and vitiligo associated with BCG treatment for malignant melanoma. Surgery 76:771-8, 1974; Naughton GK, Eising M, Bystryn JC. Detection of autoantibodies to melanocytes in vitiligo by specific immunoprecipitation. Arch Dermatol 81:540-2, 1983; Norn's DA, Kissinger RM, Naughton GK , Bystryn JC. Evidence for immunologic mechanisms in human vitiligo: patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody dependent cellular cytotoxicity (ADCC). J Invest Dermatol 90:783-9, 1988] and in another embodiment, in patients with malignant melanoma, circulating antibodies against melanoma cells are present. Because these antibodies also destroy normal, non-malignant melanocytes in other embodiments, vitiligo develops in malignant melanoma patients. In one embodiment, the prognosis in melanoma patients is better when hypo-pigmentation develops [Koh HK, Sober AJ, Nakagawa H, et al. Malignanat melanoma and vitiligo-like leukoderma: An electron microscopic study. J Am Acad Dermatol 9:696-708, 1983, Nordlund JJ, Kirkwood JM, Forget BM, et al. Vitiligo in patients with metastatic melanoma: A good prognostic sign. J Am Acad Dermatol 9:689-96, 1983]. Therefore in one embodiment, the selective destruction of pigmented cells occurring in vitiligo is natural immunotherapy for melanoma, and immunoglobulins, their fragments, derivatives or metabolites used in the composition described herein, which is isolated from melanoma patients in which vitiligo developed, or in another embodiment, from vitiligo patients or pool of patients, is useful in the treatment of melanoma according to the methods described herein.

[00034] In one embodiment, the immunoglobulins, their fragments, derivatives or metabolites used in the compositions and methods of the invention, is isolated from the plasma or sera of vitiligo patients in which the vitiligo is characterized as diffuse vitiligo. The term "diffuse vitiligo" refers in one embodiment to "generalized vitiligo", or "vitiligo non segmentalis (type A)", wherein the patchy white areas are often symmetrical, flat, have definite borders, and may affect or spread to any part of the body.

[00035] In one embodiment, the immunoglobulins, their fragments, derivatives or metabolites used in the methods and compositions described herein, developed in, and were isolated from a subject suffering from melanoma, in which the vitiligo developed as a response to the melanoma treatment

[00036] In one embodiment - melanoma, or in another embodiment, metastatic melanoma responds to a variety of immunotherapies, including in one embodiment, the administration of IL-23 and the adoptive transfer of T cells along with 1L-2. In another embodiment, some melanoma patients develop vitiligo during or after immunotherapy, and T cells that are reactive to both melanoma cells and cultured melanocytes have been detected in these patients, indicating that one embodiment, T cells may be involved in in the destruction of melanoma and melanocytes. In one embodiment, T cells specific for melanocyte proteins play an important role in melanoma rejection and in the development of vitiligo in vivo. In one embodiment, the vitiligo developed in melanoma or metastatic melanom might not be caused by autoimmunity, or in another embodiment, the immune responses might not be strong enough to detect the antibodies specific to the melanoma cell phenotype or the metastatic melanoma cell phenotype, which, are different from on another. In one embodiment, the plasma used for the isolation of the immunoglobulins, their fragments, derivatives or metabolites, used for treatment of melanoma in a subject in the compositions and methods described herein, is isolated from a patient, or pool of patients, in which vitiligo is shown to have been autoimmune in nature.

[00037] In one embodiment, the invention provides a composition for the treatment of melanoma in a subject, comprising: immunoglobulins preparation or a fragment thereof, isolated from plasma or sera of a preselected donor or donors and a pharmaceutically acceptable carrier, excipient, flow agent, processing aid, diluent or a combination thereof. In one embodiment, the carrier, excipient, lubricant, flow aid, processing aid or diluent is a gum, or a starch, a sugar, a cellulosic material, an acrylate, calcium carbonate, magnesium oxide, talc, lactose monohydrate, magnesium stearate, colloidal silicone dioxide or mixtures thereof in other embodiments.

[00038] The formulation may in one embodiment, be in the form of a bolus, or in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more pharmaceutically-acceptable carriers or diluents, or a binder such as gelatin or hydroxypropyl-methyl cellulose, together with one or more of a lubricant, anticoagulant, preservative, surface-active or dispersing agent or a combination thereof.

[00039] In another embodiment, the composition further comprises a binder, a disintegrant, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetner, a film forming agent, or any combination thereof.

[00040] In one embodiment, the composition is a particulate composition coated with a polymer (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. In one embodiment the pharmaceutical composition is administered topically, parenterally, paracancerally, transmucosally, transdermal^, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intratumorically, or intracranially.

[00041] In one embodiment, the compositions of this invention may be in the form of a pellet, a tablet, a capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a gel, an ointment, a cream, or a suppository.

[00042] In another embodiment, the composition is in a form suitable for oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical administration. In one embodiment the composition is a controlled release composition. In another embodiment, the composition is an immediate release composition. In one embodiment, the composition is a liquid dosage form. In another embodiment, the composition is a solid dosage form.

[00043] The compounds utilized in the methods and compositions of the present invention may be present in the form of free bases in one embodiment or pharmaceutically acceptable acid addition salts thereof in another embodiment. In one embodiment, the term "pharmaceutically-acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts of compounds of Formula I are prepared in another

embodiment, from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroϊodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesuIfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, b-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzyIethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound by reacting, in another embodiment, the appropriate acid or base with the compound.

[00044] In one embodiment, the term "pharmaceutically acceptable carriers" includes, but is not limited to, may refer to 0.01-0. IM and preferably 0.05M phosphate buffer, or in another embodiment 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be in another embodiment aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

[00045] In one embodiment, the compositions described herein, may include compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially, longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981; Newrnark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the jmmunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer- compound abducts less frequently or in lower doses than with the unmodified compound.

[00046] The compositions described herein, which may be υsed in the preparations used in the methods described herein, can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the active ingredients, or their physiologically tolerated derivatives in another embodiment, such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders such as acacia, cornstarch, gelatin, with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate.

[00047] Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection), the active ingredients or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[00048] In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

[00049] An active component which, in one embodiment is the plasma isolated from a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for

example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamϊno ethanol, histidine, procaine, and the like.

[00050] The compositions described herein, are formulated in one embodiment for oral delivery, wherein the active compounds which in one embodiment is plasma isolated from a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; an anticoagulant, such as sodium citrate or any of the whole blood anticoagulant-preservative solution such as ACD, CPD, CPD-Al, Adsol and the like; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain the active

^■ compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. In addition, the active compounds may be incorporated into sustained-release, pulsed release, controlled release or postponed release preparations and formulations.

[00051] Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[00052] In one embodiment, the composition described herein, or the preparation used in the methods described herein, can be delivered in a controlled release system. For example, the plasma isolated from a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, coHoϊdosomes, polymerosomes or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et a!., N. Engl. J. Med. 321:574 (1989). In

another embodiment, polymeric materials can be used. In another embodiment, a controlled release, system can be placed in proximity to the therapeutic target, i.e., the melanoma site, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).

[00053] Such compositions are in one embodiment liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g,, lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated . with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral.

[00054] In another embodiment, the compositions of this invention comprise one or more, pharmaceutically acceptable carrier materials.

[00055] In one embodiment, the carriers for use within such compositions are biocompatible, and in another embodiment, biodegradable. In other embodiments, the formulation may provide a relatively constant level of release of one active component. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. In other embodiments, release of, the plasma isolated from a vitiligo patient, or in another embodiment, from a melanoma patient in which antibodies to melanoma antigens are identified, may be event-triggered. The events triggering the release of , the plasma isolated from a vitiligo patient, or in another embodiment, from a melanoma patient in which

antibodies to melanoma antigens are identified, may be the same in one embodiment, or different in another embodiment Events triggering the release of the active components may be exposure to moisture in one embodiment, lower pH in another embodiment, or temperature threshold in another embodiment. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include micropartictes of poly(lactide- co-gϊycolide) _s polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative postponed- release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as phospholipids. The amount of active compound contained in one embodiment, within a sustained release formulation depends upon the site of administration, the rate and expected duration of release and the nature of the condition to be treated suppressed or inhibited.

[00056] In one embodiment, blood is taken using ordinary methods from a pool of patients exhibiting diffuse vitiligo in a container containing anticoagulant or anticoagulant-preservative solution, the plasma is spearated from the blood cellular components and then may be pooled with other plasmas treated in a similar manner. The separated plasma is then further processed to isolate immunoglobulins, their fragments, derivatives and metabolites, which, after isolation and purification are frozen for later use as an active agent in the compositions and methods as described herein. In one embodiment, the isolated immunoglobulins, their fragments, derivatives and metabolites are frozen at a rate that ensures the viability of the components. In another embodiment, the isolated immunoglobulins, their fragments, derivatives and metabolites are stored in the liquid state at temperatures and under conditions that ensure the viability of the components. In another embodiment, following the freezing of the immunoglobulins, their fragments, derivatives and metabolites, the preparation is further lyophilized.

[00057] In one embodiment, the immunoglobulins, their fragments, derivatives and metabolites isolated from the plasma or sera of a melanoma patient in which antibodies to melanoma antigens such as HMW-MAA and tyrosinase are identified, is used in the methods and compositions of the invention. In one embodiment, the immunoglobulins, their fragments, derivatives and metabolites isolated from the plasma or sera of a melanoma patient in which the melanoma is immunogenic and can activate host immune responses capable of controlling the disease and causing tumor regression. In another embodiment, the immunoglobulins, their fragments, derivatives and metabolites isolated from the plasma or sera of a melanoma patient who is in remission or who have been "cured". In one embodiment, the term "cured" or "cure rate" used in reference to a melanoma patient, refers to the percent of patients that will be improved or alive over a given time period. In one embodiment, a cure is

a measure of the percentage of patients who are improved from a disease stage initially diagnosed, or are completely free or cured of cancer with respect to a certain interval of time. In one embodiment, cure rate is usually 100% minus the failure rate over that particular period of time. A cure rate does not always imply cure for cancer, but may be freedom from another type of disease, such as that resulting from metastasized melanoma. In one embodiment, cure rate might be that patients have an 85% to 95% chance of being cured or completely free from signs of returning melanoma of the most superficial level after five years. This is called the five-year cure rate. In one embodiment, surgery involving basal-cell carcinomas of "high risk" areas as a treatment may give only an 85% five-year cure rate. That means a 100% - 85% = 15% failure rate, or about one in six people have the tumor return to damage or harm them. In one embodiment, the immunoglobulins, their fragments, derivatives and metabolites, which are isolated from the plasma or sera of predetermined donors according to the methods and compositions described herein, increase the cure rate of melanoma, or reduce the failure rate of melanoma.

[00058} In one embodiment, the immunoglobulins, their fragments, derivatives and metabolites, which are isolated from a melanoma patient or pool of patients, in which antibodies to melanoma antigens such as HMW-MAA or tyrosinase are identified, is used in the methods and compositions of the invention. In one embodiment, melanoma is immunogenic and can activate host immune responses capable of controlling the disease and causing tumor regression.

[00059] In one embodiment, sera samples taken from the subject to be treated according to the methods described herein, is assayed for HMW-MAA antibodies, by Abl-Ab2 complex inhibition test, for B16 epitope antibodies, which are a heterogeneous group against various antigens presented on B 16 melanoma cells and for tyrosinase antibodies, which are specific against tyrosinase. In one embodiment dosage, or routes of administration or final composition in other embodiments, will be determined based on an assay directed towards the identification of antibodies to HMW-MAA or tyrosinase.

[00060] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in ^' another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[00061] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1: Pnrified Vitiligo Immunoglobulin for the Treatment of Melanoma in Mouse Models

[00062] These experiments are done to evaluate the anti tumor effect of purified immunoglobulins obtained from individuals with vitiligo, or pooled vitiligo plasma and control subjects in the BId-FlO experimental metastasis model in syngeneic mice.

Animals

[00063] Female C57BL/6J mice are purchased at 6-8 wks of age at study initiation. Administration of the purified immunoglobulin takes place following veterinary inspection and 1-Wk acclimatization, marking and randomization.

Experimental Procedures

[00064] Over a 4-wk experimental duration, purchase, propagation and preparation for injection of tumor cells takes place. Intravenous injection of tumor cells is carried out, while daily morbidity and mortality are checked. Body weight measurements are done twice weekly. Vitiligo sourced purified immunoglobulin is injected intraperitoneally once daily (including weekends). Mice are euthanized, their lungs excised, weighed and fixated, followed by metastasis count.

[00065] The following table describes the treatment grouping done:

[00066] Results show that pooled immunoglobulin from donors exhibiting diffuse vitiligo shows a marked decrease in the number and size of melanoma metastases, as well as at least a tumoristatic effect on the metastasized melanoma tumors.

Example 2: Vitiligo Purified Immunoglobulin for the Treatment of Human Melanoma in a Mouse

Model

[00067] These experiments are done to evaluate the anti tumor effect of purified immunoglobulin obtained from individual and pooled vitiligo and controls subjects, in a human melanoma experimental metastasis model in SCID mice.

Animals

[00068] Female CB17-SCID mice are purchased at 6-8 wks of age at study initiation. Administration of the purified immunoglobulin takes place following veterinary inspection and I-Wk acclimatization, marking and randomization.

Experimental Procedures

[00069] Over a 4-wk experimental duration, purchase, propagation and preparation for injection of tumor cells takes place. Intravenous injection of human melanoma A375 tumor cells is carried out, while daily morbidity and mortality are checked. Body weight measurements are done twice weekly. Vitiligo sourced purified immunoglobulin is injected intraperitoneally once daily (including weekends). Mice are euthanized once the first mouse dies, or for humane reasons, their lungs excised, weighed and fixated in formalin or Bulin Solution, followed by metastasis count.

[00070] The

[00071] The following table describes the Grouping Protocol

[00072] Results show that pooled purified immunoglobulin from donors exhibiting diffuse vitiligo shows a marked decrease in the number and size of melanoma metastases, as well as at least a tumoristatic effect on the metastasized melanoma tumors.

Example 3: Effect of Vitiligo Purified Immunoglobulin on Mouse Melanoma Cells

[00073] These experiments are done to evaluate the antϊ tumor metastases effect of purified immunoglobulin obtained from individual and pooled vitiligo and control subjects in the B16-F10 experimental metastasis model in syngeneic mice.

Animals

[00074] Female C57BL/6J mice are purchased at 6-8 wks of age at study initiation. Administration of the purified immunoglobulin takes place following veterinary Inspection and 1-Wk acclimatization marking and randomization.

Experimental Procedures

[00075] Over a 4-wk experimental duration, purchase, propagation and preparation for injection of tumor cells takes place. Intravenous injection of tumor cells is carried out, while daily morbidity and mortality are checked. Body weight measurements are done twice weekly. Vitiligo sourced purified immunoglobulin is injected 7 days following the injevtion of tujmor cells and is done intraperitoneal Iy once daily (including weekends). Mice are euthanized, their lungs excised, weighed and fixated, followed by metastasis count.

[00076] The following table describes the treatment grouping done:

[00077] Results show that pooled purified immunoglobulinfrom donors exhibiting diffuse vitiligo shows a marked decrease in the number and size of melanoma metastases, as well as at least a tumoristatic effect on the metastasized melanoma tumors.

Example 4; Vitiligo Purified immunoglobulin Effects on Human Melanoma Cells

[00078] These experiments are done to evaluate the anti tumor effect of purified immunoglobulin obtained from individual and pooled vitiligo and controls subjects, in a human melanoma experimental metastasis model in SCID mice.

Animals

[00079] Female CB17-SCID mice are purchased at 6-8 wks of age at study initiation. Administration of the purified immunoglobulin takes place following veterinary Inspection and 1-Wk acclimatization, marking and randomization.

Experimental Procedures

[00080] Over a 4-wk experimental duration, purchase, propagation and preparation for injection of tumor cells takes place. Intravenous injection of human melanoma A375 tumor cells is carried out, while daily morbidity and mortality are checked Body weight measurements are done twice weekly. Vitiligo souiced purified immunoglobulin is injected intraperitoneal Iy once daily (including weekends) starting 8 days after the tumor cell injection, and are continued for 17 days thereafter. Mice are euthanized at the

17 day, their lungs excised, weighed and fixated in formalin or Bulin Solution, followed by metastasis count.

[00081] The following table describes the Grouping Protocol

[00082] Results show that pooled purified immunoglobulin from donors exhibiting diffuse vitiligo shows a marked decrease in the number and size of melanoma metastases, as well as at least a tumoristatic effect on the metastasized melanoma tumors.

Example 5: Vitilgo Purified Immunoglobulin Administered for the Treatment of Patients with

Stage III or Stage IV Inoperable Melanoma

[00083] The purpose of the example is to evaluate whether purified immunoglobulin obtained from donors with Vitiligo, can be administered for the treatment of patients with stage III or IV inoperable melanoma. The phase 1 part of .the study is designed to determine whether the Vitiligo-derived purified immunoglobulin can be safely administered, and the phase 2 part of the study is designed to provide additional safety data and to gain an understanding of whether administrating Vitiligo purified immunoglobulin can improve the clinical outcome for melanoma patients versus the currently available data.

[00084] This Is a randomized, multicenter, study where Vitiligo purified immunoglobulin is administered as an intravenous infusion on consecutives days followed by a rest period for a treatment cycles of 28 days.

[00085] I ^s ' cohort - 5 patients are randomized to each of the 3 treatment doses (100/ 200/ 400 mg/kg bodyweight) — total of 15 patients. Patients are randomized to 1 of the 3 purified immunoglobulin dose levels. Each patient is infused twice every 28 eight days and is followed for 28 days post the last infusion to detect potential safety issues. The 2 ^nd cohort does not start accrual, prior to a satisfactory safety analysis from the 1 ^st cohort. Nevertheless, the 1 ^st cohort patients continue treatment for up to the copmetion of 6 cycles (each cycle being 28 days) unless the patient develops progressive disease or intolerable toxicity. When there is evidence of clinical benefit (stable disease or tumor response defined as partial or complete response), patients may continue treatment beyond 6 cycles so long as toxicity remains within acceptable limits; these patients are taken off study after cycle 6, but receive treatment under compassionate access while following the same study schedule. Patients are replaced if they do not complete at least treatment 2 cycles and have at least one post-treatment disease assessment performed (unless there is a clear evidence of clinical progression after two (2) cycles).

[00086] 2 ^nd cohort — Upon positive results from the safety analysis of the first cohort, 5 patients are randomized to each of the three above described treatment doses to a total of 15 patients. Patients' treatments are administered similarly as to the I ^st cohort. Patients are replaced under the same conditions as of the 1 ^st cohort. Although recruitment is not suspended until after enrollment is completed for this cohort, a safety analysis is done to include the first 30 patients, including two infusions each and a follow up period.

[00087] 3 ^rd cohort - Additional 9 patients are randomized to each of the 3 treatment doses (total of 27 patients). Patients* treatment is administered as described above. A Simon two-stage designs is used to evaluate the efficacy of the treatment, after 6 cycles at each of the above described doses. Patients are replaced if they do not complete at least 2 cycles of treatment and have at least 1 post-treatment disease assessment performed (unless there is clear evidence of clinical progression after two (2) cycles).

[00088] 4 ^lh cohort — Additional 21 patients (maximum of 63 patients) are randomized to those treatment arms (doses, as descibed above) which have shown at least one response (as desciribed above) at the 3 ^rd cohort stage and are treated as described above. At the completion of the 4 ^lh cohort, if four or more

responses are observed for any dose level, then it is concluded that this dose level is a candidate for further clinical study. Patients are replaced in a similar method as in previous cohorts.

[00089] Results indicate that vitilogo purified immunoglobulin is a safe and effective compound for the treatment of stage III or IV inoperable melanoma.

[00090J The descriptions of the foregoing embodiments of the invention have been presented for purpose of illustration and description. They are not intended.to be exhaustive or to limit the invention to the precise forms disclosed herein, and obviously many modifications and variations are possible-in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention to thereby enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention, be defined by the claims appended hereto.