PT100568A - MONOCLONAL AND ANTIGENIC ANTIBODIES FOR HUMAN MELANOMA - Google Patents

Description

" ΜΕ20: MONOCLONAL AND ANTIGEN ANTIBODIES FOR HUMAN MELANOMA "

TECHNICAL FIELD OF THE INVENTION The present invention relates to the discovery of a novel cell surface protein designated by ME20 antigen (AgME20), which exists in human melanoma cells, and antibodies such as mAb ME20 and antibody fragments which have been developed and react specifically with this cell surface protein. The present invention also relates to novel peptides which contain regions in their amino acid residue sequences which correspond substantially to the AgME20 domains.

Background of the Invention

Tumor cells express some antigens that do not exist, or exist in small amounts, in their corresponding normal cell parts. Many of them are antigen differentiation genes shared by tumor cells by tumor cells and by some embryonic cells. Some of these antigens with sufficient selectivity in tumors may be expected to serve as possible therapeutic agents. This possibility has been considered in the case of malignant melanoma [Hellström and Hellström, in Accomplishment in Cancer Research - 1984 Prize Year, General Motors Cancer Research Foundation, J. G. Fornter & J. E. Rhoads, eds., J.B.

Lippincott Company, Philadelphia, (1985) pp. 216-240; Hellstròm and Hellström, in In Vitro Diagnosis of Human Tumors Using Monoclonal Antibodies, Immunology Series, Η. Z. Kupchick, ed. Mareei Dekker, Inc. New York / Basel, (1988) Vol 39, pps. 123-139] and also in the cases of other tumors [Burchell and Taylor-Papa-dimitrious, in R. W. Baldwin and V. S. Byers, eds., Monoclonal Antibodies for Tumor Detection and Drug Targeting, Academic Press (1985) p. 1-15; Kemshead, ibid, pp. 281-302]. Many antibodies have been prepared for the cell surface antigens that are expressed by human tumors in larger amounts than by normal tissues. It is also believed that some antibodies to cell surface antigens may be cytotoxic to tumor cells in the presence of a complementary agent (Hellström et al., (1962) Allergy 9: 158-245; Hellström and Hellström Human Melanoma, S. Ferrone, ed., Springer-Verlag, New York, Berlin, Heidelburg (1990) pp. 442-466) and that some antibodies may play the role of antibody dependent cell cytotoxicide mediators ([Perlmann et al., (1969) Adv. Immunol., 11: 117-193; MacLennan et al., (1969), Immunol 17: 896-910; Shurzak et al., (1972) Int. J. Cancer : 316-323].

In the former case, a suitable source of complementary agent (usually rabbit or guinea pig) is required and in the latter case a source of effector cells (usually from the mouse) is required.

Antibodies such as those directed to tumor associated antigens can kill human tumor cells in the presence of human effector cells [Hellström et al., (1981) Int. J. Cancer 27: 281-285] in the presence of human effector cells.

presence of human serum which constitutes a source of complementary agent [Hellström et al., (1985) Proc. Natl. Acad. Know. 82: 1499-1502; Hellström et al., (1985) Monoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular and Cellular Biology,

Vol. 27, pps. 149-164, Alan R. Liss, Inc., NY].

Anti-tumor antibodies have been used in immunohistology [Garrigues et al., (1982) Int. J. Cancer 29: 511-515; Natali et al., In Carcinogenesis - A Comprehensive Surveil, C. J. Conti, T. J. Slaga and A.J.P. Klein-Szanto, eds.

Raven Press, Ltd. New York, (1989) Vol. 11. pps. 133-164] and in immunotherapy [Hellström and Hellström (1988) Pigment Cell Res. S.1: 180-184; Neuwelt et al., (1987) Neurosurgery 20: 885--895], and still in imaging for diagnosis [Larson et al., (1983) J. Clin. Invest. 72: 2101-2114; Murray et al., (1988) in Malignant Melanoma: Biology, Diagnostics and Therapy, L. Nathanson, ed., Khiwer Academic Publishers, Boston, p. 123-149].

Some techniques for the preparation of anti-cancer agents involve the labeling of antibodies with radioactive isotopes [Larson et al., (1983) Clin. Invest. T2: 2101-2114;

Order, (1984). The R. 10: 9-18; Carrasquillo et al., (1984) Cancer Treatments Reports 68: 317-328; de Nardo et al., (1985) Int. J. Radiation Oncology Biol. Phys. 11 335-345, or the conjugation of antibodies to toxins [Jansen et al. (1982) Immunol. Rev. 6_2: 185-216; Vitetta and Uhr (1984) Trans-plant. 537: 535-538] or anti-cancer drugs [Ghose et al., (1972) Brit. Med. J. 3: 495-499; Hurwitz et al., (1975) Cancer Res. 35: 1175-1181; Rowland et al., (1985) Cancer Immunol. Immunother. 19: 1-7]. In these cases the antibody provides -4- γ. in the specificity of discriminating the agent for the appropriate tumor cell and the isotope or drug provides the ability to destroy the tumor. However, a disadvantage of this type of approach up to the present invention has been the lack of high specificity of the antibody which in many cases can also bind to non-cancerous tissue.

Since both anti-cancer drugs and radioisotopes possess a high level of toxicity to normal tissues, this non-specific incorporation into various organs such as the kidneys, liver or bone marrow may possibly lead to considerable side effects.

Another approach to cancer therapy is the development of specific immunogens, often presented as therapeutic vaccines [Hellström, K.E., Hel. lstrom, I., and Hu, S.-L. (1990) " Why Cancer Vaccines? &Quot; In: New Generation Vaccines: The Molecular Approach (Woodrow, G. C., and Levine, M., eds.) Mareei Dekker, Inc. pps 885-862], These immunogens are used to induce a state of active tumor immunity in patients. The level of shift specificity is crucial to this process and the present invention raises this question for the case of melanoma which is an absolutely malignant neoplasm.

Summary of the Invention The present invention is directed to providing a monoclonal antibody designated mAB ME20 which is immunospecific for a cell surface protein present in human melanoma cells and also in cells of a nevus fraction. .. · Lf, dysplastic but not significantly in other cells. It has been assumed that both melanoma and nevi are derived embryologically from the neural Christian. The present invention also encompasses ME20 mAb fragments such as the Fab, Fv, Fab 'and (Fab1) 2 fragments and still other antibodies and fragments of similar specificity. The monoclonal antibody ME20 (and its fractions) may be functionally linked, or conjugated to another compound such as a drug, a toxin, a marker agent, a radionuclide, a growth factor or an enzyme. The present invention also relates to diagnostic and therapeutic methods using that antibody and further describes the methods and compositions for the use of such monoclonal antibodies.

On the other hand, the present invention further relates to a novel cell surface antigen called AgME20, which is characteristic of human melanoma cells, and also targets a substantially pure peptide containing a region corresponding to a cell surface antigen domain of melanoma such as AgME20 antigen. The present invention also describes diagnostic and therapeutic methods that utilize that peptide and further describes methods and compositions using such a novel peptide.

Description of the Preferred Aspects The present invention relates to compounds and methods useful in the detection and treatment of human melanoma.

Specifically, the present invention relates to a monoclonal antibody monoclonal mAb ME20 and to fragments thereof such as Fab, Fv, Fab 'and Fabab, and also relates to the polypeptide chains synthesized which react immunity specifically with a human melanoma surface protein,

AgME20, â € ƒâ € ƒâ € ƒwherein the mAb ME20 antibody is as described herein. The presence of this existing protein on the cell surface is only detectable in melanoma cells and in Λ cells of a fraction of dysplastic nevi (2 in 6 tested). Both melanoma and nevi are thought to be derived embryologically from neural Christian cells. The mAb ME20 antibody, fragments thereof and other antibodies and fragments which are identical in shape can be used, inter alia, in the detection and / or treatment of melanoma, either alone or in combination with another compound. According to the preferred aspect, an antibody or fragment of the present invention functionally binds to a compound such as a drug, a toxin, a marker agent, a growth factor, a radionuclide or a radioisotope or an enzyme.

As used herein, the term " reacts immune " refers to the production of a specific binding interaction of a similar nature to the immunological binding that occurs between an antigen and an antibody directed to that antigen. According to a preferred aspect of the present invention, a polypeptide such as the monoclonal antibody mAb ME20-, reacts immune with a cell surface antigen, such as AgME20, in human melanoma cells. The polypeptides of the present invention bind to normal human adult cells and tissues to a much lower extent than to melanoma cells.

As used herein the term " bind to a much lesser extent " means that the binding will not be entirely detectable when standard immunohistological techniques are used. In contrast, the antibody mAb ME20 binds with a high degree of specificity to a novel antigen characteristic; of human melanoma. The ME20 mAb antibody and identical polypeptides and fragments of the present invention as described above, may functionally bind to other compounds and may be used for diagnostic, locational or therapeutic purposes.

As used herein, the terms " polypeptide " and " antibody " are interchangeable and refer to a molecule of natural or synthetic origin consisting of amino acid residues. of those coupled by peptide bonds that can specifically react with a human melanoma cell surface protein, encompassing the glycosylated proteins designated as immunoglobulins (antibodies), fragments of anthyodies and synthetic proteins, and fragments of proteins that are capable of combining or immunoreact specifically with a melanoma surface protein.

As used herein, the term " antigen 1 " refers to an entity which is capable of specifically binding to an antibody. In the event that an antigen is capable of inducing production, antibodies are also referred to as " immunogen ".

As used herein the term " functionally linked " refers to a bond that does not interfere with the ability of any of the attached groups to function as described. According to a preferred aspect, the mAb ME20 antibody is functionally linked to a biologically active compound such as a drug so as to allow the polypeptide to bind to a human melanoma surface protein and without · / Any interference with the biological activity of the drug.

According to a particularly preferred aspect the antibody mAb ME20 is functionally linked to a chemotherapeutic drug such as dacarbazine, adriamycin or mitomycin C.

The polypeptides of the present invention that specifically react with the ME20 antigen include antibodies such as the monoclonal antibody mAb ME20. The biological activity of the antibodies is multifaceted. The Fc region of the molecule largely determines the ability of antibody molecules to activate the complementary agent and mediates antibody-dependent cellular cytotoxicity (CCDA) [Uanue and Benacerraf (1984)] Textbook of Immunology, 2nd Edition, WiJL liams and Wilkins , Chap. 12, pps. 218-238].

Antibodies are divided into several clachs and subclasses. Preferred monoclonal antibodies of the present invention belong to the subclasses IgG1, IgG2a and IgG3. In general, antibodies of the IgG2a and IgG3 subclasses can often mediate CCDA and activate complement in serum.

The polypeptides of the present invention are preferably present in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers.

As used herein the term " pharmaceutically acceptable carrier " refers to a compound capable of being administered to a patient and which does not produce toxic or undesired effects upon such administration. As illustrative examples of pharmaceutically acceptable carriers are phosphate buffered saline solutions, the solution of -9-

Ringer, oils, gels and microspheres and also lipo sums. Other pharmaceutically acceptable carriers are well known in the pharmaceutical field and are considered to be globular by the present invention.

As used herein the term " marker agent " refers to a single atom or molecule which, when functionally linked to a product of the present invention, allows detection of its presence in a test method. Illustrative are fluorescent dyes, radioisotopes, enzymes and antibodies which can be detected independently or which can be detected together with the addition of a substrate or other molecule which reacts with them as marker substances. The monoclonal antibody mAb ME20 exists in two isotype forms, respectively IgG1 and IgG2a, these forms being produced from hybridomas that are deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, in full compliance with the deposition requirements of the United States Patent and Trademark Office and the Budapest Treaty on June 4, 1991, respectively, having the accession numbers HB10764 and HB10763.

Antibodies of the present invention are specifically immunoreactive with sites determining in a glycoprotein antigen known as AgME20 associated with human melanoma cells and not found in other human tumors. Such immunoreactive specificity for such antibodies greatly diminishes the likelihood that the antibodies of the present invention will bind to breast, lung, colon, and the like carcinomas.

In particular it is noted that it was not observed which. either binding of mAb ME20 antibody to normal adult human cells and that tissues were detected by immunohistological assay.

Antibodies of the present invention are useful either alone or in combination with other agents in tumor and / or tumoricidal methods for the treatment of melanoma. Such antibodies may be prepared by the hybridoma, recombinant or synthetic methods and encompass the fragments, multimers and derivatives thereof. As used herein the term " multimer " refers to a molecule that contains several repeating units of a portion of the molecule. A multimeric peptide of the present invention preferably contains between 2 and about 10 repeating regions of a portion of a peptide amino acid residue sequence functionally linked together. According to one aspect of the present invention there is provided a multimeric antibody corresponding to various molecules of an antibody, such as mAb antibody ME20, functionally linked to each other to provide a molecule having several functional binding sites each of which can react immunologically specifically with a human melanoma cell surface protein such as the AgME20 antigen. The antibodies of the present invention may also be used in methods and tests for the diagnosis, detection and treatment of human melanoma. The present invention also encompasses a substantially purified peptide which contains a region that emits or practically-

corresponds to a domain of a human melanoma cell surface protein. The peptides of the present invention are capable of specifically immune-reacting as an antibody of the present invention such as mAb ME20 antibody.

As used herein the term " substantially " refers to a high level of similarity. A substantially purified peptide of the present invention means a preparation having less than ten percent of other different peptides. A substantially identical sequence according to the present invention exhibits a variation of less than ten percent with respect to the reference sequence.

According to a preferred aspect, a peptide of the present invention encompasses molecules of substantially purified natural or synthetic origin containing sequences of amino acid residues and / or glycosylation. Which may react immunity with the monoclonal antibody ME20.

Such peptides may be prepared by standard methods of purification of proteins and by chemical or recombinant synthesis and encompass fragments, multimers and derivatives thereof. which retain the ability to react specifically with the antibodies of the present invention. The human melanoma cell surface protein, ME20, designated AgME20, is a glycoprotein having a molecular weight of approximately 80 kD to 120 kD. According to a preferred aspect the cell surface protein of ME20 has a molecular weight, determined according to the SDS-PAGE protocol, comprised between approximately 100 kD and 116 kD. -12-

The antibodies of the present invention may be used in methods of treatment and for the detection of human melanoma.

According to one aspect, an antibody of the present invention capable of binding to a human melanoma cell surface protein can be used to detect the presence of human melanoma cells in assays performed in vitro and in vivo. According to this method the human cells of a patient are treated with an effective amount of the antibody so as to allow binding of the antibody to any melanoma cells that are present. The presence of the bound antibody is then detected by any of several methods known in the art. By way of illustration, the methods of detection encompass radiographic detection of the presence of a radioisotope functionally linked to the antibody, the use of a second identifiable antibody which is detectable and which reacts immune to the exposed region of the antibody and also the use of indicator means, such as a substrate that is converted into a fluorescent molecule by an enzyme functionally bound to the antibody.

According to another aspect a polypeptide of the present invention, such as mAb ME20 antibody, is administered according to a therapeutically effective dose to a given patient, together with a chemotherapeutic agent for a period considered sufficient to cause the inhibition of further proliferation of any melanoma cells present. By way of illustration, it is to be understood that the chemotherapeutic agents are preferably the drugs and the commonly used toxins used in the treatment of melanoma. The chemotherapeutic agent may be added separately from the polypeptide of the present invention, together with the polypeptide or functionally linked to the polypeptide as in the case of a conjugate between a drug and a polypeptide. The present invention also encompasses an immunogenic composition capable of generating an immune response when administered to a patient. The immunogenic compositions have a substantially purified peptide of the present invention corresponding to a region of a human melanoma cell surface protein and also contain an immune response enhancing agent such as a hapten or an adjuvant, such as alum. This type of cell surface protein, such as AgME20, can also be administered to a patient in several types of " vaccines ", for example, AgME20 can be expressed on a virus from a recombinant vaccine [Estin et al., ( 1988) Proc. Natl. Acad. Know. USA 85: 1052-1056] containing the gene encoding melanoma-associated peptide of 80-120 kD or a part thereof. Other specific immunogens associated with the AgME20 antigen are also claimed as part of the present invention.

According to one aspect of the present invention an immunogenic composition containing a peptide of the present invention is administered to a given patient in a dose sufficient to generate a targeted immune response to human melanoma cells. According to the present invention it is noted that the immunogenic peptidic composition can generate an immune response in a patient being specifically directed against an epitope present on the cell surface protein 14 of human melanoma such as AgME20 antigen. The present invention also encompasses kits containing antibodies and / or peptides, said kits further containing instructions suitable for their use. According to a specific aspect, each kit according to the present invention contains the monoclonal antibody ME20.

The following examples further illustrate the present invention and are for illustrative and non-limiting purposes only. EXAMPLE 1 MONOCLONAL ANTIBODY ME20 A. Preparation of antibody ME20

Balb / C (approximately 2 months old) mice were given six inoculations both subcutaneously (sc) and intraperitoneally (ip) with human melanoma cells. The first four inoculations were carried out with a time lapse of about one month, the fifth inoculation being carried out one week later and the sixth inoculation being carried out three weeks after the fifth inoculation. Each inoculation contained about 10 H3614 and / or H3606 human melanoma cells (obtained from primary tumors); inoculations 1, 2 and 3 contained H3614 cells and inoculations 4, 5 and 6 contained both H3614 and H3606 cells.

Three days after the sixth inoculation the spleen was removed from a mouse and used in the fusion protocol. The spleen was destroyed so as to obtain cells scraping over various sieves and then rinsed with RPMI medium. The spleen cells were separated from the remaining tissue by centrifugation at 200æg for 10 minutes and with them was resuspended in RPMI medium and then mixed with Ag8-653 myeloma cells at a ratio between Ag8- 653 / spleen cells of the order of 1:10.

The mixture of Ag8-653 cells and immunized spleen cells was subjected to centrifugation at 200 x g for 10 minutes and the supernatant was then removed by keeping the cells at 37øC.

Polyethylene glycol (PEG, 1 ml) was added to the cells over a period of one minute with stirring for another minute.

Iscove-modified Dulbecco's medium (MDMI) without HAT (hypoxanthine, aminopterin, thymidine) was added (the added amount was 1 ml over the 1 minute period); then an additional 8 ml was added over 2 minutes. The cells were centrifuged for 10 minutes at 160 x g at ambient temperature. The supernatant was removed and the cells were resuspended in 25 ml of MDMI. This new cell suspension was mixed with 2 x 10 mouse thymocytes freshly obtained from Balb / C mice aged 3 to 4 weeks. The cell culture mixture of Ag8-653 cells, spleen cells and thymocytes was used to prepare a suspension in one volume of MDMI and 2 ml of 50 x HAT. The culture was maintained at 37øC under an atmosphere containing 7%

of C02 for a time interval varying between 12 and 16 hours, and then the cells were transferred to 96-well microtiter plates in the amount of

an atmosphere containing 7% CO 2. After 3 days the plates were examined microscopically for the determination of the melt efficiency.

The hybrid cells were then screened for antibody production assay, and the pooled hybrids were transferred to 48 well plates with additional thymocytes added.

The resulting antibodies produced by the hybrid clones were then screened in accordance with the enzyme-linked immunosorbent assay (ELI-SA protocol) in the immunization of melanoma cell lines and fibroblast (human skin fibroblasts). The ME20 antibody was isolated based on its binding property to melanoma cells but without any binding to the fibroblasts. Further better characterization of mAb ME20 is obtained by immunohistology and cellular analysis in SCAF.

Studies were performed using the fluorescence activated cell sorting method (SCAF) at 4Â ° C and 37Â ° C temperatures on H3606 melanoma cells. The results presented below demonstrate that antibody ME20 binds primarily to the melanoma cell surface at 4Â ° C and at 37Â ° C the antibody was detected essentially in the perinuclear region. The perinuclear location was further confirmed by microscopy.

Confocal sink. Internalization of the antibody occurs at a temperature of 37 ° C. B. Immunohistochemistry The binding of mAb ME20 antibody to various normal and tumor human tissues was observed according to the immunohistological peroxidase-anti-peroxidase (PAP) techniques described by Hellström et al., (1983) J. Immunol . 130: 1467. The results shown in Table 1 demonstrated that mAb ME20 antibody binds specifically to human melanoma and binds to about 1/3 of the measured diphasic nevi, but does not bind to normal human tissue. Both melanoma cells and nevi cells are believed to derive embryologically from neural Christian and may share or express some of the common surface antigens. The inexistence of any detectable binding of mAb ME20 antibody to normal tissue demonstrates that this antibody is highly specific and useful for the detection and treatment of human melanoma. -18-

TABLE 1

IMMUNO-HISTOLOGY TUMOR FABRIC NORMAL TISSUE Type Positive / Not tested Type Positive / No En outpatient Breast 0 10 Suprarenal 0 3 Colon 0 10 Intestine 0 1 Lung 0 15 Brain 0 1 Melanoma 18 19 Breast 0 3 Ovary 0 2 Colon 0 6 Nevos (di-plastics) 2 6 Esophagus 0 3 Heart 0 4 Kidney 0 10 Liver 0 8 Lung 0 8 Lymphatic Nevus 0 4 Optic Nerve 0 1 Ovaries 0 2 Pancreas 0 6 Skin 0 5 Spleen 0 9 Stomach 0 4 Testis 0 2 Thyroid 0 2 Tonsil 0-1 -19- C. Characterization of antibody ME20 with antibody mAb ME20

H360635 melanoma cells were metabolically labeled using S-methionine for 10 minutes followed by incubation in methionine-free RPMI-1640 medium enriched with 5% dialyzed fetal bovine serum for a time between 0 and 10 hours at 37 ° C. The cell pellet was then extracted with phosphate buffered saline at pH 7.4, alternatively containing 1% NP-40, PMSF (10æg / ml) and aprotinin (20æg / ml) or 0.4% Triton X-100 and proteinase inhibitors. Immunoprecipitation of the ME20 antigen was incubated by incubating the cell lysate with mAb ME20 antibody for 15 minutes at 4 ° C. The antigen-antibody complex precipitated with rabbit anti-mouse IgG and " Protein A-Sepharose " (Sigma Chemical Co., St.

Louis, MO). After washing of the immunoprecipitate, the cells were subjected to SDS-PAGE analysis according to the protocol under reducing conditions and were displayed by fluorography after impregnation of the gel with " AMPLIFY " (Amersham, Arlington Heights , IL).

H3606 human melanoma cells were metabolically labeled with H-glucosamine by incubation in 50% ISCOVE DMEM, 50% RPMI-1640 glucose-free medium and 5% dialyzed fetal bovine serum. ME20 antigen was immunoprecipitated from the cell lysate with ME20 mAb, rabbit anti-mouse IgG and " Protein A-Sepharose ". The immunoprecipitate was subjected to analysis on the SDS-PAGE protocol under reduction conditions and autoradiography was performed.

A major band of Mr = 105,000 (as terminated by the SDS-PAGE protocol on 7.5% polyacrylamide gels) of the AgME20 antigen was produced after 10 minutes of labeling and remained the predominant band for about 1 hour, if very weak after about 3 hours. A high molecular weight component of Mr = 120000 emerged after 10 minutes and remained present for 2 hours. After longer test times (4 to 10 hours) the Mr = 97000 band was the most predominant component immuno-precipitated by mAb ME20 antibody. ✓ ME20 antibody specifically precipitated glycoprotein antigens with Mr = 120000, Mr = 105000 and Mr = 97000 as determined by the SDS-PAGE protocol on 7.5% polyacrylamide gels. The results demonstrate that the antigenic determinant recognized by mAb ME20 antibody is located on a new glycoprotein. The membrane bound antigen is released in conditioned medium from H3606 cells. The soluble form of the antigeneAgME20 has a Mr = 97000 value. Analysis of the amino terminal sequence indicates that the soluble form of the antigen AgME20 has the same amino terminal sequence of the membrane bound antigen MEL20 and which is also a single chain glycoprotein. D. Characterization of binding of monoclonal antibody ME20

The ability of mAb ME20 antibody to bind to and internalize in tumor cells was investigated by the indirect immunotoxin -21 method in order to detect the internalization of an antibody-toxin conjugate with subsequent killing of the cells that internalize the conjugate in according to the method of Till et al. (1988) Can cer Res. 4: 1119-1123. A FAB fragment of the mouse conjugated anti-mouse immunoglobulin was reacted with a ricin A chain with cells previously exported at a concentration of 10æg / ml ME20 mAb or BR96 mAb. The BR96 mAb antibody is a monoclonal antibody that specifically binds to breast, colon and lung cancer cells and is internalized by the antigen-positive cells BR96 [Hellstrom et al., (1990) Cancer Res. 50: 2183-2190] .

The percent survival inhibition values of the tested cells in conjunction with the binding ratio values as measured by the SCAF method are shown in TABLE 2 and demonstrate the internalization of the mAb antibody in human melanoma cells such as evidenced by the specific annihilation of melanoma cells in the case of mAb ME20 antibody. TABLE 2

FACS

Cell Line Antibody. % Inhibition Ratio < inhibition 3606 ME20 95 16 (melanoma) BR961 26 1 3774 ME 20 82 2 (melanoma) BR96 8 1 2669 ME20 94 5 (melanoma) BR96 5 1 HT29 ME20 0 1 (colon) BR96 6 1 2707 ME20 31 1 (lung) BR96 98 29 3396 ME20 17 1 (breast) BR96 99 54 1 BR96 mAb antibody binds to breast, colon and lung cancer cells and is internalized by antigen-positive cells SCAF analysis of antibody binding po mAb ME20, as shown in TABLE 2, measured the binding of a fluorescein isothiocyanate (FITC) -labeled secondary antibody to in vitro tumor cells that had been treated with the concentration of 10æg / ml BR96 mAb or mAb ME20. -23-

Further studies were also performed, as shown in TABLE 3, using a secondary antibody of a FITC-labeled goat-conjugated mouse antibody. The binding ratio represents the ratio of the brightness, termed linear fluorescence equivalent (ELF), of cells affected by the specific mAb ME2G antibody and the unaffected cell population.

ELF sample - = Control ratio ELF I η control

Confocal microscopy was performed on H3606 human melanoma cells in order to better illustrate the internalization of mAb ME20 antibody. Viable H3606 cells were stained by exposing, at 4Â ° C, the mAb ME20 antibody conjugated to phycoerythrin (mAb ME20-PE). The labeled antibody was associated with microvilli located on the cell membrane of the cells.

In the case where the identically labeled cells were maintained at 37øC for 2 hours, the ME20 mAb showed a cytoplasmic perinuclear localization and was virtually absent from the cell surface in that the antibody had been internalized.

These studies indicated the native apparent distribution of the antigen AgME20. Cells that had previously been permeabilized by detergent and fixed with paraformal-24-

were exposed to mAb ME20-PE. The cellular distribution of the binding of the observed antibody was identical to that observed above in the case of mAb ME20 antibody binding at 4 ° C. In addition there was also intense staining associated with a perinuclear acentric compartment, possibly representing the Golgi apparatus. These observations suggest that the newly synthesized AgME20 antigen is transported through the Golgi apparatus, exported to the surface of the plasma membrane and then internalized after binding by mAb ME20.

The binding avidity of mAb ME20 antibody to human melanoma H3606 cells was studied for two isotopes of mAb ME20 antibody. MAb ME20 ioda-125 mAb antibody (I-mAbME20) was subjected to an immunoreactivity assay against the antigen-positive cells and then used in H3606 cell binding studies. Scatchard was assayed and the results obtained translate a Ka value of approximately 10 M for both the IgG1 and IgG2 isotopes of mAb ME20 antibody. EXAMPLE 2 AMYGENEO. ME20 A. Purification

Antigen ME20 (AgME20) was isolated from human melanoma tumor cells (H3606 cells) obtained from a metastatic lesion defined by the inventors as a cell line and partially purified by immunoaffinity chromatography. A suspension of phosphate buffered saline (PBS) was prepared with H3606 cells. An equal volume of solubilization buffer was added until a final concentration of 0.4 M NaCl, 1% Triton X-100, 10 mM EDTA in 17 mM phosphate buffer, pH 7.6 was obtained. The buffer contained the following proteinase inhibitors: 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 mM BAEE (NÎ ± -benzoyl L-arginine ethyl ester), and 1 Âμg / ml each The cells were treated with solubilization buffer under gentle agitation for 30 minutes at 4Â ° C. The in soluble material was removed by centrifugation at 100,000 xg for 90 minutes and at the temperature of 4Â ° C. The antigen ME20 was purified from the supernatant by immunoaffinity chromatography. A solution of mAb ME20 (5 mg / ml) was prepared in 0.2 M sodium phosphate buffer containing 0.5 M NaCl, pH 8.2 and added to 1 g of wet tresyl-Sepharose (Sigma) .The coupling reaction was kept under gentle stirring for 16 hours at 4 ° C. The gel was treated with Tris 0.2 M HCl, pH 8.5 for 5 hours at 20 ° C and washed with: (1) 0.2 M sodium acetate buffer 0.5 mM NaCl, pH 3.5, (2) 17 mM phosphate buffer containing 0.4 M NaCl and 1% Triton X-100, pH 7.4; (3) phosphate buffered saline containing 1% Triton X-100, pH 7.4; (4) 100 mM diethylamine containing 0.2% Triton X-100, pH 11.5; and (5) phosphate buffered saline containing 1% Triton X-100. The cell lysate was applied to a 1 ml column of (mAb -26-

ΜΕ 20) -Sepharose and recirculated for 12 to 16 hours at 4 ° C. The affinity support was washed with PBS containing 0.2% Triton X-100 and eluted with 100 mM diethylamine antilene containing 0.2% Triton X-100, pH 11.5. The elution product was neutralized with 2M Tris-HCl buffer, pH 6.8. The partially purified AgME20 antigen was further purified according to the SDS-PAGE protocol. The eluate from the column was subjected to dialysis in the presence of 0.1% SDS. SDS-PAGE (10% acrylamide) according to the Laemmli method was performed using gel miniplates with 0.75 mm thick spacers (BioRad).

After electrophoresis, the SDS-polyacrylamide gel was stained with Coomassie brilliant blue and then discolored. The bands of the colored antigen ME20 (Mr = 116000 and 100000) were excised with a razor blade and then electroeluted. ✓

The ME20 antigen was also recovered from the SDS-polyacrylamide gels by electroblotting onto a membrane &quot; Problot &quot; (Applied Biosystems, Inc.) using &quot; Mini-Transblot Electrophoretic Transfer Cell &quot; (BioRad Laboratories, Richmond, CA), as described by Matsudaira, P., (1987), J. Biol. Chem. 261; 10035-10038. The membrane was stained with Coomassie brilliant blue, then decolorized and the AgME20 colored antigen bands (Mr = 116000 and 100000) were excised using a razor blade for further analysis of the amino terminal sequence. -27 B. Sequential analysis

Edman's automatic degradation of three preparations, 9.9 pmol, 7.7 pmol and 3.0 pmol, respectively, each of AgME20 antigen was carried out on a 475A pulsed liquid protein sequencer (Applied Bio systems, inc. .) equipped with a transverse vertical flow reaction charger, as described by Hewick et al. (Hewick, RM, et al., (1981) J. Biol. Chem. 256: 7990-7997].

Phenylthio-idantoine (PTH) derivatives with amino acids were analyzed by reverse phase high performance liquid chromatography (HPLC) using a 120A model of a real-time HPLC unit (Applied Biosystems, Inc.) with a column PTH Cl8 (2.1 x 220 mm, Applied Biosystems, Inc.) and using a gradient of sodium acetate / tetrahydrofuran / acetonitrile to elute. Reduction and quantification of results were performed using a Nelson 760 interface, a Hewlett Packard 9816 computer, and a 900A / model 475A computer application for chromatogram analysis (Applied Biosystems, Inc.).

Sequence studies indicated that 3 major membrane-bound ME20 antigens (Mr = 116000; 100000; and 8000 as determined by the SDS-PAGE protocol are 10% polyacrylamide gels) had identical amino-terminal amino acid sequences. The amino terminal sequence is designated as I.D. NQ 1 is as follows: KVPRNQD w LG Lys Go Pro Arg Asn Gin Asp Trp Leu Gly 1 5 10 VSRQLRTKAW Will be Arg Gin Leu Arg Thr Lys Ala Trp 15 20 NRQLYPEWTX Asn Arg Gin Leu Tyr Pro Glu Trp Thr X 25

(X: the amino acid has not been identified)

In addition, a soluble form of the AgME20 antigen was isolated from the conditioned medium of H3606 cells. The antigen was purified by affinity chromatography and recovered from the mAb ME20 antibody column by elution with 100 mM diethylamine, pH 11.5, in the absence of Triton X-100. The Mr value of the soluble antigen was 97,000.

The amino terminal amino acid sequence of the antigeneAgME20 was compared to the recently updated PIR database (edition 27) and to the integrated MIPSX database. Sequence comparison showed no significant similarities to any other known sequence. The specification and the previous examples are intended to illustrate the present invention but without limiting it. Various variations and modifications may be introduced without departing from the spirit and scope of the present invention. -29-

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Hellstrom, Ingegerd

Hellstrom, Karl Erik Marquardt, Hans

(ii) TITLE OF THE INVENTION: &quot; ME20: MONOCLONAL AND ANTIGENS ANTIBODIES FOR HUMAN MELANOMA &quot; (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESS: Bristol-Myers Squibb Company (B) STREET: 3005 First Avenue (C) USA (F) CP: 98121 (v) COMPUTER ITEMS: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM:

(Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) DEPOSIT DATE: (C) CLASSIFICATION:

C) INFORMATION ON REPRESENTATIVE / AGENT: (A) NAME: Sorrentino, Joseph (B) REGISTRATION NUMBER: 32,598 (C) REFERENCE NUMBER / PROCEDURE: ON0084- (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: ) 728-4800 (B) TELEFAX: (206) 448-4775 INFORMATION FOR SEQ. Amino acids (D) TOPOLOGY: Linear (li) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: None (iv) ANTI-BREAKING HYPHEN (8209) (F) TYPE OF FABRIC: Melanoma (H) CELL LINE: Human Melanoma H3606 cells -31- - NON-BREAKING HYPHEN (1) 31- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

Lys Go Pro Arg Asn Gin Asp Trp Leu Gly 15 10

Will be Arg Gin Leu Arg Thr Lys Ala Trp 15 20

Asn Arg Gin Leu Tyr Pro Glu Trp Thr 25

Claims (54)

An antibody, characterized in that it reacts immune and in a specific manner with a human melanoma cell surface protein, said surface protein having a molecular weight of about 120 kD to about 120 kD. is present in cells of human melanoma tumors but does not exist in other cells of human tumors. 2. The antibody of claim 1, having the binding characteristics of the monoclonal antibody ME20, having an IgG1 isotype such as produced by a hybridoma having ATCC Accession Number HB10764. 3. The antibody of claim 1, having the binding characteristics of the monoclonal antibody ME20, having an IgG2a isotype as produced by a hybridoma having the ATCC Accession Number HB10763. 2 ( 4. The antibody of claim 1, wherein the cell surface protein has a molecular weight of approximately 100 kDa. 5. The antibody of claim 1, wherein the cell surface protein is a glycosylated protein having a terminal sequence of amino acid residues substantially corresponding to Sequence I.D. No. 1. 6. The antibody of claim 5, wherein the surface protein has a molecular weight of about 116 kDa. 7. The antibody of claim 1, wherein the polypeptide is an antibody fragment capable of immune and speci fi cally reacting with the melanoma cell surface protein. 8. An antibody according to claim 7, wherein the antibody fragment incorporates a Fab, Fv, Fab 'or FCab' 9. The antibody of claim 1 which also incorporates a ligated compound functionally to the antibody. 10. An antibody according to claim 9, wherein the compound is selected from the group consisting of an identifier, a drug, a toxin, a growth factor, a radionuclide and an enzyme. 11. The antibody of claim 10, wherein said drug is dacarbazine. 12. An antibody fragment characterized in that it is capable of reacting in an immune and specific manner with the human melanoma cell surface protein and said cell surface protein has a molecular weight comprised between approximately 80 kDa and about 120 kDa. An antibody fragment according to claim 12, characterized in that the fragment incorporates a Fab, Fv, Fab1 or (Fab1? 14. Pharmaceutical composition, characterized in that it comprises incorporating an antibody which reacts immune and specifically to a human melanoma cell surface protein together with a pharmaceutically acceptable carrier. 15. The pharmaceutical composition according to claim 14, wherein the antibody is of the monoclonal type having the binding characteristics of the monoclonal antibody ME20, having an IgG1 isotype, which is produced by a hybridoma having the Accession Number of ATCC HB10764. 16. A pharmaceutical composition according to claim 14, wherein the antibody is of the monoclonal type having the binding characteristics of the monoclonal antibody ME20, having an IgG2a isotype, which is produced by a hybridoma having the Accession Number of ATCC HB10763. Pharmaceutical composition according to claim 14, characterized in that the antibody is an antibody fragment. 18. A pharmaceutical composition according to claim 17, wherein the antibody fragment incorporates a Fab, Fv, Fab 'or Fab' fragment. 19. A method for determining the presence of human melanoma cells, characterized in that: a) human cells are contacted with an effective amount of an antibody capable of reacting immune and in a specific manner with a cell surface protein of human melanoma so as to provide the binding of that antibody to any human melanoma cells present; and b) detecting binding of the antibody to the melanoma cell surface protein. 20. A method according to claim 19, wherein said antibody is an antibody or a fragment thereof, having monoclonal antibody binding characteristics ME20, having an IgG1 isotype which is produced by a hybridoma having the ATCC Accession Number HB10764. 21. The method of claim 19 wherein said antibody is an antibody or a fragment thereof having monoclonal antibody binding characteristics ME20, having an IgG2a isotype which is produced by a hybridoma having the Number ATCC Accession HB10763. 6 22. A method according to claim 19, wherein the binding of the antibody to the human melanoma cells is determined by the detection of the presence of antibody means functionally linked to the antibody. 23. The method of claim 22, wherein the identifying means is a radionuclide. 24. The method of claim 22, wherein the identifying means is a fluorescent label. 25. The method of claim 22, wherein the identifying means is an enzyme. 26. The method of claim 19, wherein the cells and the antibody contact in vitro. 27. A method according to claim 21, wherein the cells and the antibody contact in vivo. 28. A method for locating 7fp melanoma cells in a patient, wherein administering to said patient an effective amount of an antibody capable of reacting immune and in a specific manner with the cell surface protein of the cells of human melanoma for a sufficient time for the antibody to bind specifically to melanoma cells and then to detect the presence of any melanoma cells with the antibody bound to it. 29. The method according to claim 28, wherein the presence of the melanoma cells is determined by detecting the presence of the identifying medium functionally bound to the antibody. 30. The method of claim 28, wherein the presence of the melanoma cells is determined by administering to the patient a reagent capable of binding and indicating the presence of the melanoma cells attached to the antibody. 31. The method of claim 30, wherein the reagent is a second antibody capable of binding to the antibody bound to melanoma, said second antibody being functionally linked to the indicator medium. δ 32. The method of claim 29, wherein the identifying means is a radioisotope. 33. A method of treating melanoma in a patient characterized in that a therapeutically effective amount of an antibody capable of reacting in an immune and specific manner with a cell surface protein of melanoma cells is administered to said patient. melanoma, together with a chemotherapeutic agent for a sufficient time to inhibit the proliferation of melanoma cells. The method of claim 33, wherein the antibody functionally binds to a compound selected from the group consisting of a drug, a toxin, a growth factor, a radionuclide and an enzyme. A method according to claim 33, wherein the chemotherapeutic agent is a drug selected from the group consisting of doxorubicin, dacarbazine, mitomycin C, cis-platin and a nitrosourea. 36. A method as claimed in claim 34, characterized in that the compound is a radioisotope. 37. A substantially purified peptide comprising a region corresponding substantially to a domain of a cell surface protein of the human melanoma or a fragment thereof, wherein said region is immune reactive and of a specific mode with the monoclonal antibody ME20 as produced by the hi-bridoma having the ATCC Accession Number HB10764 or the ATCC Accession Number HB10763. 38. The peptide of claim 37 which is functionally linked to a compound selected from the group consisting of an identifier, a drug, a toxin, a growth factor, a radionuclide, a hapten, and a enzyme. 39. A peptide according to claim 37, wherein said melanoma cell surface protein has a molecular weight of from about 80 kD to about 116 kD and an amino acid residue terminal sequence corresponding to the sequence ID No. 1. 40. A peptide according to claim 39, which comprises a region substantially corresponding to a glycosylated domain of a 100 kD protein. 41. A peptide according to claim 39, characterized in that it contains a region substantially corresponding to a glycosylated domain of a 116 kD protein. 42. The peptide of claim 37, which is produced by recombinant expression. 43. A pharmaceutical composition comprising incorporating a substantially purified peptide containing a region corresponding substantially to a domain of a human melanoma cell surface protein or a fragment thereof, said region being capable of reacting immune and in a manner specific with the monoclonal antibody ME20 as produced by the hybridoma having the ATCC Accession Number HB10764 or the ATCC Accession Number HB10763 and a physiologically tolerable carrier. 44. An immunogenic composition, characterized in that it comprises incorporating a substantially purified peptide which contains a region corresponding substantially to a domain of a human melanoma cell surface protein or a which fragment is capable of immunoreacting with an antibody having the binding characteristics of the monoclonal antibody ME20 as produced by a hybridoma having the ATCC Accession Number HB10764 or the ATCC Accession Number HB10763 and an enhancing agent of the immune response. The composition of claim 44, wherein the immune response enhancing agent is functionally linked to the peptide. A composition according to claim 44, characterized in that the immune response enhancing agent is a hapten. The composition of claim 44, wherein the immune response enhancing agent is a physiologically tolerable adjuvant. Composition according to Claim 47, characterized in that the adjuvant is alum. 49. A method for inducing an immune response in a patient directed to a human melanoma, characterized in that 12 administering to said patient an immunogenic amount of a peptide which contains a region corresponding substantially to a domain of a human melanoma cell surface protein, or a fragment thereof, said region being capable of immunologically reacting with an antibody having the binding characteristics of the ME20 monoclonal antibody as produced by a hybridoma having the ATCC Accession Number HB10764 or the ATCC Accession Number HB10763. 50. A method according to claim 49, characterized in that an immune response enhancing agent is further administered to the patient. 51. A utility kit comprising: a) a package containing an antibody or a fragment thereof which specifically reacts immune to a human melanoma cell surface protein; and b) instructions for its use. 52. A kit according to claim 53, wherein the antibody has the binding characteristics of the monoclonal antibody ME20 produced by a hybridoma having the ATCC Accession Number HB10764 or 13 ATCC Accession Number HB10763. 53. A kit according to claim 51, further comprising a package containing a substantially purified peptide containing a region corresponding substantially to a domain of a human melanoma cell surface protein, or a carrier thereof. which region is capable of reacting immune and in a specific manner with the monoclonal antibody ME20 as produced by the hybridoma having ATCC Accession Number HB10764 or ATCC Accession Number HB10763. 54. A kit comprising: a) a package containing a substantially purified peptide containing a region corresponding substantially to a domain of a human melanoma cell surface protein, or a fragment thereof, said region capable of reacting in an immune and specific manner with the monoclonal antibody ME20 as produced by the hybridoma having the ATCC Accession Number HB10764 or the ATCC Accession Number HB10763; and b) instructions for its use. Oliciuí of the Industrial Property. 14 14 ABSTRACT &quot; ME20: MONOCLONAL AND ANTIGENS ANTIBODIES FOR HUMAN MELANOMA &quot; The present invention relates to a novel class of polypeptide molecules, including antibodies, that react in an immune and specific manner with a cell surface protein that is detectable in human melanoma tumor cells, together with specific peptides corresponding to the immunoreactive domains of the cell surface proteins. The invention also describes methods for detecting and treating human melanoma. 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