Link:  Pharm/Biotech Resources

Title:  Antibodies against the MUC18 antigen

United States Patent:  6,924,360

Issued:  August 2, 2005

Inventors:  Green; Larry L. (San Francisco, CA); Bar-Eli; Menashe (Houston, TX)

Assignee:  Abgenix, Inc. (Freemont, CA)

Appl. No.:  330613

Filed:  December 26, 2002

The present invention relates generally to the generation and characterization of anti-MUC18 monoclonal antibodies. The invention further relates to the use of such anti-MUC18 antibodies in the diagnosis and treatment of disorders associated with increased activity of MUC18, in particular, tumors, such as melanomas.

SUMMARY OF THE INVENTION

Embodiments of the invention relate to monoclonal antibodies that were found to bind to the MUC18 antigen and affect MUC18 function. This application describes human anti-MUC18 antibodies and anti-MUC18 antibody preparations with desirable properties from a therapeutic perspective, including strong binding affinity for MUC18. in vitro.

In one aspect, the human anti-MUC18 antibody has a heavy and light chain variable domain.

In a further aspect, the present invention provides an anti-human MUC18 monoclonal antibody heavy chain, or a fragment thereof, having an amino acid sequence selected from the group consisting of: c3.19.1 (SEQ ID NO: 1), c6.11.3 (SEQ ID NO: 5), C3.10 (SEQ ID NO: 9), C3.22 (SEQ ID NO: 13), C3.27 (SEQ ID NO: 17), C3.45 (SEQ ID NO: 21), C3.65 (SEQ ID NO: 25), C6.1 (SEQ ID NO: 29), C6.9 (SEQ ID NO: 33) or C6.2 (SEQ ID NO: 37).

In an even further aspect, the present invention also provides an anti-human MUC18 monoclonal antibody light chain, or a fragment thereof, having an amino acid sequence selected from the group consisting of: 3.19.1 (SEQ ID NO: 2), 6.11.3 (SEQ ID NO: 6), C3.10 (SEQ ID NO: 10), C3.22 (SEQ ID NO: 14), C3.27 (SEQ ID NO: 18), C3.45 (SEQ ID NO: 22), C3.65 (SEQ ID NO: 26), C6.1 (SEQ ID NO: 30), C6.9 (SEQ ID NO: 34), or C6.2 (SEQ ID NO: 38).

In one embodiment, the light chain, or a fragment thereof, may be combined with the above-identified heavy chain or a fragment thereof or with other heavy chain sequences, provided that the antibody so produced retains the ability to bind to human MUC18.

In another aspect, the invention provides an anti-human MUC18 monoclonal antibody comprising: A) at least one light chain or a fragment thereof and (B) at least one heavy chain or a fragment thereof.

In a further aspect, the anti-MUC18 antibody is c3.19.1. Specifically, c3.19.1 is also referred to as ABX-MA1.

In another aspect, the anti-MUC18 antibody is c6.11.3, c3.10, c3.22, c3.27, c3.45, c3.65, c6.1, c6.12, c6.2 or c6.9.

Various forms of the antibody are contemplated herein. For example, the anti-MUC18 antibody may be full length antibody (e.g. having an intact human Fc region) or an antibody fragment (e.g. a Fab, Fab′ or F(ab′)₂). Futhermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (such as a cytotoxic agent). In one aspect, the invention provides an antibody of the invention linked to a radioisotype. In another aspect, the invention provides an antibody of the invention linked to a toxin, preferably ricin toxin or a toxin composed of a chemotherapeutic agent. In a further aspect, such antibodies of the invention may be used for the treatment of diseases such as tumors.

In one aspect, the invention provides an anti-human MUC18 monoclonal antibody which binds to and neutralizes a biological activity of at least human MUC18 or stimulates the internalization and down-regulation of the protein. The antibody can significantly reduce or eliminate a biological activity of the human MUC18 in question.

The biological activity of the subject human MUC18 may be cell proliferation. Further, the biological activity may include angiogenesis and cell proliferation important for primary tumor growth and metastasis, cell invasion and/or migration, and activation of metalloproteinase MMP-2. Even further, the biological activity may include growth and metastasis of tumor cells in patients with tumors, for example, melanoma.

Also provided is an isolated nucleic acid molecule encoding any of the antibodies described herein, a vector comprising the isolated nucleic acid molecule, a host cell transformed with the nucleic acid molecule, and a method of producing the antibody comprising culturing the host cell under conditions wherein the nucleic acid molecule is expressed to produce the antibody and optionally recovering the antibody from the host cell. The antibody may be of the IgG class. The isolated nucleic acid molecule preferably comprises a nucleotide sequence encoding a heavy chain variable domain of a monoclonal antibody, wherein the nucleotide sequence is selected from the group consisting of: c3.19.1 (SEQ ID NO: 3), c6.11.3 (SEQ ID NO: 7), C3.10 (SEQ ID NO: 11), C3.22 (SEQ ID NO: 15), C3.27 (SEQ ID NO: 19), C3.45 (SEQ ID NO: 23), C3.65 (SEQ ID NO: 27), C6.1 (SEQ ID NO: 31), C6.9 (SEQ ID NO: 35) or C6.2 (SEQ ID NO: 39), or a nucleotide sequence encoding a light chain variable domain of a monoclonal antibody, wherein said nucleotide sequence is selected from the group consisting of: 3.19.1 (SEQ ID NO: 4), 6.11.3 (SEQ ID NO: 8), C3.10 (SEQ ID NO: 12), C3.22 (SEQ ID NO: 16), C3.27 (SEQ ID NO: 20), C3.45 (SEQ ID NO: 24), C3.65 (SEQ ID NO: 28), C6.1 (SEQ ID NO: 32), C6.9 (SEQ ID NO: 36), or C6.2 (SEQ ID NO: 40).

In a different aspect, the invention provides a method for the treatment of a disease or condition associated with the expression of MUC18 in a patient, comprising administering to the patient an effective amount of an anti-MUC18 antibody. The patient is a mammalian patient, preferably a human patient. The disease is a tumor, such as melanoma.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Methods for Carrying Out One Embodiment of the Invention

1. Generation of Anti-MUC18 Antibodies

A description follows as to exemplary techniques for the production of the antibodies used in accordance with the present invention.

(a) Monoclonal Antibodies

Monoclonal Antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as herein above described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, cells expressing the antigen of interest may be used for immunization. Further alternatively, lymphocytes may be immunized in vitro. Animals are immunized against the immunogenic conjugates or derivatives by combing 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of Freud's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/0 the original amount of conjugate in Freud's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later the animals are bled and the serum is assayed for anti-MUC18 antibody titer. Antibodies are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same MUC18 antigen, but conjugated to a different protein and/or through a different cross-linking agent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.

Lymphocytes or more preferably, lymphocytes enriched for B cells isolated from such immunized animals are then fused with myeloma cells by an electrocell fusion process or by using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-109, [Academic Press, 1996]).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC.-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, [1987]).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem. 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the cells may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an anti-MUC18 monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an MUC18 and another antigen-combining site having specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

(b) Human Antibodies

Attempts to use the same technology for generating human mAbs have been hampered by the lack of a suitable human myeloma cell line. The best results were obtained using heteromyelomas (mouse×human hybrid myelomas) as fusion partners (Kozbor, J. Immunol. 133: 3001 (1984); Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). Alternatively, human antibody-secreting cells can be immortalized by infection with the Epstein-Barr virus (EBV). However, EBV-infected cells are difficult to clone and usually produce only relatively low yields of immunoglobulin (James and Bell, J. Immunol. Methods 100: 5-40 [1987]). In the future, the immortalization of human B cells might possibly be achieved by introducing a defined combination of transforming genes. Such a possibility is highlighted by a recent demonstration that the expression of the telomerase catalytic subunit together with the SV40 large T oncoprotein and an oncogenic allele of H-ras resulted in the tumorigenic conversion of normal human epithelial and fibroblast cells (Hahn et al., Nature 400: 464-468 [1999]).

It is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production (Jakobovits et al., Nature 362: 255-258 [1993]; Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 [1995]; Fishwild et al., Nat. Biotechnol. 14: 845-851 [1996]; Mendez et al., Nat. Genet. 15: 146-156 [1997]; Green, J. Immunol. Methods 231: 11-23 [1999]; Tomizuka et al., Proc. Natl. Acad. Sci. USA 97: 722-727 [2000]; reviewed in Little et al., Immunol. Today 21: 364-370 [2000]). For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J_H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production (Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-2555 [1993]). Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (Jakobovits et al., Nature 362: 255-258 [1993]).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have generated a line of transgenic mice designated as "XenoMouse® II" that, when challenged with an antigen, generates high affinity fully human antibodies. This was achieved by germ-line integration of megabase human heavy chain and light chain loci into mice with deletion into endogenous J_H segment as described above. The XenoMouse® II harbors 1,020 kb of human heavy chain locus containing approximately 66 V_H genes, complete D_H and J_H regions and three different constant regions (μ, δ and γ), and also harbors 800 kb of human κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. The antibodies produced in these mice closely resemble that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. The human antibodies are preferentially expressed over endogenous antibodies due to deletion in endogenous J_H segment that prevents gene rearrangement in the murine locus.

Techniques for generating antibodies using Abgenix's XenoMouse® technology include injection of a particular antigen of interest into such mice. Sera from such immunized animals may be screened for antibody-reactivity against the initial antigen. Lymphocytes may be isolated from lymph nodes or spleen cells and may further be selected for B cells by selecting for CD138-negative and CD19+ cells. The B cell cultures (BCCs) may be either fused to myeloma cells to generate hybridomas as detailed above or screened further for reactivity against the initial antigen. Such screening includes ELISA.

Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO₄ precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

In a preferred embodiment, the antibodies of the present invention comprise an anti-human MUC18 monoclonal antibody heavy chain or a fragment thereof, comprising the following CDR's (as defined by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols 1-3): (a) CDR1, (b) CDR2 and (c) CDR3.

The heavy chain of the antibodies in one embodiment of the present invention comprise of the following sequences: SEQ ID NO: 1; SEQ ID NO: 5; SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, OR SEQ ID NO: 37.

In yet another embodiment, the invention provides an anti-human MUC18 monoclonal antibody light chain or a fragment thereof, comprising the following CDRs: (a) CDR1, (b) CDR2 and (c) CDR3. The light chain of the antibodies in one embodiment of the present invention comprises one of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10; SEQ ID NO: 14, SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; or SEQ ID NO: 38.

In one aspect, the present invention includes anti-MUC18 antibodies such as c3.19.1 and c6.11.3. The heavy chain amino acid and nucleotide sequences of c3.19.1 are encoded by SEQ ID NO: 1 and 3, respectively, and the heavy chain amino acid and nucleotide sequence of c6.11.3 are encoded by 5 and 7, respectively. The light chain amino acid and nucleotide sequences of c3.19.1. are encoded by SEQ ID NO: 2 and 4, respectively, and the light chain amino acid and nucleotide sequences of c6.11.3 are encoded by 6 and 8, respectively.

2. Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One may further select antibodies with certain biological characteristics, as desired.

Binding to MUC18 Antigen

For example, to identify anti-MUC18 antibodies with high affinity for human MUC18, kinetic measurements and binding affinity of the anti-MUC18 antibodies were obtained from Biacore experiments. The Biacore experiments measured the affinity of MUC18 antibodies captured on a protein A surface for labeled MUC18 antigen and are further described in the examples below. Anti-MUC18 antibodies with a Kd of 6×10^-;10M were considered high affinity anti-MUC18 antibodies.

In a further example, to determine whether anti-MUC18 antibodies of the present invention were able to recognize denatured MUC18 in human melaroma cells, the antibodies were used for immunoblots of metastatic melanoma cells and non-metastatic melanoma cells (control). Those antibodies which were able to detect MUC18 in metastatic melanoma cells were selected as anti-MUC18 antibodies of interest.

Further, to identify anti-MUC18 antibodies that recognized the native form of the MUC18 protein on the surface of cells, flow cytometry analysis was performed. According to this assay, cells expressing the antigen of interest were detached from cell culture plates, incubated with either an isotype-matched control human antibody or the anti-MUC18 antibody for 20 minutes at 4° C. After washing, all samples were incubated with phycoerythrin-conjugated F(ab′)₂ fragments of Goat Anti-Human IgG (H+L) (Jackson) for 20 minutes at 4° C. in the dark. After several washings, the cells were resuspended in FACS buffer and analyzed by cytofluorometry. Those antibodies which shift the fluorescence intensity when compared to control antibodies were selected as anti-MUC18 antibodies of interest.

3. Therapeutic Compositions and Mode of Administration of Anti-MUC18 Antibodies

Therapeutic formulations of the anti-MUC18 antibodies of the invention are prepared for storage by mixing antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, Pa.: 1995)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The anti-MUC18 antibody to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The anti-MUC18 antibody ordinarily will be stored in lyophilized form or in solution.

Therapeutic anti-MUC18 antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of anti-MUC18 antibody administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional routes, or by sustained release systems as noted below. Preferably the antibody is given systemically.

Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D-(-;)-3-hydroxybutyric acid (EP 133,988). Sustained-release anti-MUC18 antibody compositions may also include liposomally entrapped antibody. Liposomes containing antibody are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal antibody therapy.

Anti-MUC18 antibody can also be administered by inhalation. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, anti-MUC18 antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

An "effective amount" of anti-MUC18 antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, the type of anti-MUC18 antibody employed, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the anti-MUC18 antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

Antibodies specific to tumor antigens such as anti-MUC18 are useful in targeting of tumor cells for destruction. For example, ricin, a cellular toxin derived from plants, is finding unique applications, especially in the fight against tumors and cancer. Implications are being discovered as to the use of ricin in the treatment of tumors. Ricin has been suggested to have a greater affinity for cancerous cells than normal cells (Montfort et al. 1987) and has been often termed as a "magic bullet" for targeting malignant tumors. Toxins such as ricin remain active even if the B chain of the toxin is removed. Accordingly, if the solitary A chain is coupled to a tumor-specific antibody, such as anti-MUC18 antibody, the toxin has a specific affinity for cancerous cells over normal cells (Taylorson 1996). For example, ricin immunotoxin has been developed to target the CD5 T-cell antigen often found in T-cell and B-cell malignancies (Kreitman et al. 1998). Further, the linking of such anti-MUC18 antibodies to radioisotopes provides advantages to tumor treatments. Unlike chemotherapy and other forms of cancer treatment, radioimmunotherapy or the administration of a radioisotope-antibody combination directly targets the cancer cells with minimal damage to surrounding normal, healthy tissue. With this "magic bullet," the patient can be treated with much smaller quantities of radioisotopes than other forms of treatment available today. Most commonly antibodies are conjugated with potent chemotherapeutic agents such as maytansine, geldanamycin or calichaemycin for delivery to tumors (Frankel et al., Cancer Biotherapy and Radiopharmaceuticals, 15:459-476 (2000); Knoll et al., Cancer Res., 60:6089-6094 (2000); Liu et al., Proc. Natl. Acad. Sci. USA, 93:8618-8623 (1996); Mandler et al., J. Natl. Cancer Inst., 92:1573-1581 (2000); and Ota et al., Int. J. Clin. Oncol., 4:236-240 (1999). These drugs are too toxic to be administered on their own. When conjugated to a therapeutic antibody such as MUC18, their biological activity can be directed specifically to the tumor cells. Accordingly, antibodies, such as MUC18 antibodies, can be modified to act as immunotoxins utilizing techniques that are well known in the art. See e.g., Vitetta et al., Immunol. Today, 14:252 (1993) and U.S. Pat. No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared utilizing techniques that are well known in the art. See e.g., Junghans et al., Cancer Chemotherapy and Biotherapy, pgs. 655-686 (second edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990, 5,648,471, and 5,697,901. The immunotoxins and radiolabeled molecules would be likely to kill cells expressing MUC18, and particularly those cells in which the antibodies of the invention are effective.

The patients to be treated with the anti-MUC18 antibody of the invention include patients with tumors, preferably melanoma and/or prostate or renal cancer. Other tumors include esophageal, pancreatic, colorectal tumors, carcinomas, such as renal cell carcinoma (RCC), cervical carcinomas and cervical intraepithelial squamous and glandular neoplasia, and cancers, such as colorectal cancer, breast cancer, lung cancer, and other malignancies. Patients are candidates for therapy in accord with this invention until such point as no healthy tissue remains to be protected from tumor progression. It is desirable to administer an anti-MUC18 antibody as early as possible in the development of the tumor, and to continue treatment for as long as is necessary.

In the treatment and prevention of tumor-associated disorder by an anti-MUC18 antibody, the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder, including treating chronic autoimmune conditions and immunosuppression maintenance in transplant recipients. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.

As a general proposition, the initial pharmaceutically effective amount of the antibody administered parenterally will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day. The desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of antibody, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.

As noted above, however, these suggested amounts of antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, as indicated above. For example, the antibody may be optionally formulated with one or more agents currently used to prevent or treat tumors such as standard- or high-dose chemotherapy and hematopoietic stem-cell transplantation. The effective amount of such other agents depends on the amount of anti-MUC18 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
 

Claim 1 of 57 Claims

1. An isolated monoclonal antibody comprising a heavy chain polypeptide, wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33 and 37, and wherein said monoclonal antibody binds to MUC18.

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