Melanoma cell lines expressing shared immunodominant melanoma antigens and methods of using same

The invention pertains to a method of treating or protecting against melanoma that comprises (a) obtaining a melanoma cell line that expresses one or more shared immunodominant melanoma antigens, (b) modifying the melanoma cell line to render it capable of producing an increased level of a cytokine relative to the unmodified cell line, and (c) administering the melanoma cell line to a mammalian host that has melanoma or is at risk for developing melanoma. Preferably the melanoma cell line is allogeneic and is not MHC-matched to the host.

TECHNICAL FIELD OF THE INVENTION
 The present invention pertains to a method of treating or protecting
 against melanoma using as a vaccine one or more melanoma tumor cell lines
 that express multiple immunodominantshared melanoma antigens. In
 particular, the invention pertains to the method of using an allogeneic
 melanoma cell line as a vaccine. The present invention also relates to a
 melanoma cell line that expresses shared immunodominant melanoma antigens,
 and to a composition comprising cells of the melanoma cell line.
 BACKGROUND OF THE INVENTION
 It generally is accepted that tumor cells contain multiple specific
 alterations in the cellular genome responsible for their cancerous
 phenotype. These alterations affect the expression or function of genes
 that control cell growth and differentiation. For instance, typically
 these mutations are observed in oncogenes, or positive effectors of
 cellular transformation, such as ras, and in tumor suppressor genes (or
 recessive oncogenes) encoding negative growth regulators, the loss of
 function of which results in expression of a transformed phenotype. Such
 recessive oncogenes include p53, p21, Rb1, DCC, MCC, NFI, and WTI.
 Immunotherapy is a potential therapeutic approach for the treatment of
 cancer. Immunotherapy is based on the premise that the failure of the
 immune system to reject spontaneously arising tumors is related to the
 failure of the immune system to appropriately respond to tumor antigens.
 In a functioning immune system, tumor antigens are processed and expressed
 on the cell surface in the context of major histocompatibility complex
 (MHC) class I and II molecules, which, in humans, also are termed "human
 leukocyte associated" (HLA) molecules. Complexes of MHC class I and II
 molecules with antigenic peptides are recognized by CD8.sup.+ and
 CD4.sup.+ T cells, respectively. This recognition generates a set of
 secondary cellular signals and the paracrine release of specific cytokines
 or soluble so-called "biological response modifiers", that mediate
 interactions between cells and stimulate host defenses to fight off
 disease. The release of cytokines then results in the proliferation of
 antigen-specific T cells.
 Thus, active immunotherapy involves the injection of tumor cells to
 generate either a novel or an enhanced systemic immune response. The
 ability of this immunotherapeutic approach to augment a systemic T cell
 response against a tumor has been previously disclosed, e.g., amongst
 others, see International Application WO 92/05262, Fearon et al., Cell,
 60, 397-403 (1990), and Dranoff et al., Proc. Natl. Acad. Sci., 90,
 3539-43 (1993). The injected tumor cells usually are altered to enhance
 their immunogenicity, such as by admixture with non-specific adjuvants, or
 by genetic modification of the cells to express cytokines, or other immune
 co-stimulatory molecules. The tumor cells employed can be autologous,
 i.e., derived from the same host as is being treated. Alternately, the
 tumor cells can be MHC-matched, or derived from another host having the
 same, or at least some of the same, MHC complex molecules.
 Most whole cell cancer vaccines are produced using the patient's own tumor
 cells. There are two reasons for the use of such autologous vaccines.
 First, based on the results with murine tumors, it previously had been
 postulated that each tumor expresses tumor-associated-antigens (TAA) that
 are unique to each patient's tumor. Second, because T cell recognition
 depends on both the MHC allele as well as the specific antigen, use of
 cells from a patient's own tumor circumvents any need for matching of
 tumor or MHC antigens.
 However, the in vitro expansion of fresh human tumor explants necessary for
 the production of autologous tumor cell vaccines is labor-intensive,
 technically demanding, and frequently impossible for most histologic types
 of human tumors, even with highly specialized research facilities.
 Moreover, the production of a vaccine from each patient's tumor is quite
 expensive. There also is a substantial likelihood that after extended
 passage of autologous cells in culture, the antigenic composition of such
 cells will change relative to the primary tumor from which the cell line
 originated, making the cells ineffective as a vaccine. While such change
 is frequent with all established cell lines, as regarding the use of
 autologous cells as a tumor vaccine, it potentially will require the
 maintenance of freezer stocks of each initially-isolated cell line for
 each patient being treated using this approach.
 The recent results of Huang et al., Science, 264, 961-65 (1994), are
 relevant to the treatment of cancer using vaccines. Namely, prior to the
 study of Huang et al., tumor vaccine strategies were based on the
 understanding that the vaccinating tumor cells function as the antigen
 presenting cells (APCs) that present the tumor antigens on their MHC class
 I and II molecules, and directly activate the T cell arm of the immune
 response. In contrast, the results of Huang et al. indicate that the
 professional APCs of the host rather than the vaccinating tumor cells
 prime the T cell arm of the immune response. In the study of Huang et al.,
 tumor vaccine cells secreting the cytokine GM-CSF recruit to the region of
 the tumor bone marrow-derived APCs. The bone marrow-derived-APCs take up
 the whole cellular protein of the tumor for processing, and then present
 the antigenic peptide(s) on their MHC class I and II molecules. In this
 fashion, the APCs prime both the CD4.sup.+ and the CD8.sup.+ T cell arms
 of the immune system, resulting in the generation of a systemic antitumor
 immune response that is specific for the antigenic epitopes of the host
 tumor. These results suggest that it may not be necessary to use
 autologous or MHC-matched tumor cells in cancer treatment.
 Also relevant to the use of tumor vaccines, it has been confirmed that T
 cells are the critical mediator of systemic antitumor immunity induced by
 tumor vaccines (reviewed by Pardoll, Trends in Pharmacological Sciences,
 14, 202-08 (1993)). Thus, the production of a universal tumor vaccine,
 i.e., a vaccine that is applicable to the majority of patients with a
 particular type of cancer, requires knowledge of the existence of shared
 immunodominant tumor antigens recognized by T cells. Currently, shared
 immunodominant tumor antigens recognized by T cells have been identified
 in only one human cancer, melanoma. Melanoma is a malignant neoplasm
 derived from cells that are capable of forming melanin, and may occur in
 the skin of any part of the body, in the eye, or, less commonly, in the
 mucous membranes of the genitalia, anus, oral cavity, or other sites.
 Melanomas frequently metastasize widely, and the regional lymph nodes,
 liver, lungs, and brain are likely to be involved. Primary malignant
 melanoma of the skin is the leading cause of death from all diseases
 arising in the skin. Metastatic melanoma is frequently thought of as
 resistant to treatment. In fact, the most effective single agent for
 treatment of disseminated melanoma, dacarbazine
 (dimethyltriazenoimidazolecarboxamide or DTIC), induces a partial
 remission in only 20 percent of cases, and a complete response in less
 than 5 percent of cases (Fitzpatrick et al., "Malignant Melanoma of the
 Skin", In Harrison's Principles of Internal Medicine, Braunwald et al.,
 eds., Eleventh Ed. (McGraw-Hill Book Company: NY, 1987) 1595-97)).
 The shared immunodominant melanoma antigens recognized by T cells fall into
 two main categories. One category of antigens encompasses proteins that
 are produced in melanoma cells, and are not produced in any other adult
 tissues with the exception of testis. These so-called tumor-specific
 shared antigens include the MAGE family antigens MAGE-1 and MAGE-3. Of
 these two antigens, MAGE-3 appears to be more widely produced and
 immunodominant than MAGE-1. MAGE-3 also is produced in other nonmelanotic
 tumors such as small cell lung cell carcinoma (SCLC), non-small cell lung
 cell carcinoma (non-SCLC), squamous cell carcinoma of the head and neck
 (SCCHN), colon cancer, and breast cancer. Similarly, MAGE-1 also is
 produced in breast cancer, glioblastoma, neuroblastoma, SCLC, and
 medullary cancer of the thyroid. The other category of shared melanoma
 antigens encompasses melanocyte lineage-specific differentiation antigens.
 These lineage-specific differentiation antigens are produced in
 melanocytes and their malignant counterpart, melanoma, and are produced in
 no other cells or tissues identified to date. These differentiation
 antigens include MART-1/Melan-A, tyrosinase, GP75, and GP100. These
 melanoma antigens, as well as other antigens (e.g., recently identified
 tumor-specific mutated antigens that may or may not prove to be shared),
 are further described in Table 1. It also is likely that further shared
 immunodominant melanoma antigens will be identified.
 Table 1. Melanoma Antigens Recognized by T Cells
 I. Melanocyte lineage-specific differentiation antigens
 gp100
 MART-1/Melan-A
 TRP1 (gp75)
 tyrosinase
 II. Tumor-specific shared antigens
 MAGE-1
 MAGE-3
 BAGE
 GAGE-1,2
 GnT-V
 p15
 III. Tumor-specific mutated antigens
 b-catenin
 MUM-1
 CDK4
 Knowledge of these shared melanoma antigens would provide the potential to
 identify either a single melanoma cell line that expresses all, or a
 majority of, the shared melanoma antigens, or a set of melanoma cell lines
 which collectively express all, or a majority of, these antigens. If such
 melanoma cell lines could be identified, these cell lines when employed as
 a vaccine would share at least one, and in most cases would share
 multiple, antigens with melanomas from virtually every patient with
 melanoma. The present invention provides a method of treating cancer using
 such cell lines, and, in particular, provides a method of treating
 melanoma, which does not rely on use of autologous or MHC-matched tumor
 cells, and that avoids the difficulties and shortcomings associated with
 such use. These and other objects and advantages of the present invention,
 as well as additional inventive features, will be apparent from the
 description of the invention set forth herein.
 BRIEF SUMMARY OF THE INVENTION
 The present invention provides a method of treating or protecting against
 melanoma that comprises the steps of obtaining a melanoma cell line that
 expresses one or more shared immunodominant melanoma antigens, modifying
 the melanoma cell line to render it capable of producing an increased
 level of a cytokine relative to the unmodified cell line, and
 administering the melanoma cell line to a mammalian host that has melanoma
 or is at risk for developing melanoma. Preferably the melanoma cell line
 is allogeneic and is not necessarily MHC-matched to the host.
 DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The method of the present invention of treating or protecting against
 melanoma comprises the steps of (a) obtaining a melanoma cell line that
 expresses one or more shared immunodominant melanoma antigens, (b)
 modifying the melanoma cell line to render it capable of producing an
 increased level of a cytokine relative to the unmodified melanoma cell
 line, and (c) administering the melanoma cell line to a mammalian host
 that has melanoma or is at risk for developing melanoma. Preferably, the
 administered melanoma cell line is allogeneic and is not necessarily
 MHC-matched to the host.
 Melanoma
 The method of the invention can be employed to treat or protect against
 melanoma. "Treating melanoma" according to the invention comprises
 administering to a host the melanoma cell lines set forth herein for the
 purpose of effecting a therapeutic response. Such treatment can be done in
 conjunction with other means for treatment of melanoma (e.g., surgical
 excision of a primary lesion). In particular, a therapeutic response is a
 systemic immune response (e.g., a T cell response) to melanoma antigens as
 further described herein. Such a response can be assessed by monitoring
 the attenuation of melanoma growth and/or melanoma regression. "Melanoma
 growth" includes an increase in melanoma size and/or the number of
 melanomas. "Melanoma regression" includes a reduction in melanoma mass.
 "Protecting against melanoma" according to the invention comprises
 administering to a susceptible host (e.g., hosts with poor tolerance to
 sunlight, patients with dysplastic nevi or large congenital melanocytic
 nevi, patients who have undergone resection of a primary melanoma lesion,
 etc.) the melanoma cell lines set forth herein for the purpose of
 preventing new melanoma from forming.
 "Melanoma" according to the invention includes malignant tumors arising
 from melanocytes in the skin or other sites, and which may contain dark
 pigment. The term encompasses such cancers as are localized in primary
 tumors, as well as melanoma cells not localized in tumors, for instance,
 which expand from a tumor locally by invasion of adjacent tissue, or which
 have metastasized.
 The method of treating or protecting against melanoma can be effectively
 carried out using a wide variety of different hosts. For instance, the
 method can be employed with various animalian hosts, but preferably is
 employed with mammalian hosts including, but not limited to, rodent, ape,
 chimpanzee, feline, canine, ungulate (such as ruminant or swine), as well
 as, in particular, human, hosts.
 Melanoma Cell Line
 As described herein, a "melanoma cell line" comprises cells that initially
 were derived from a melanoma. A melanoma cell line can be derived from any
 melanoma. Such cells typically have undergone some change such that they
 theoretically have indefinite growth in culture, i.e., unlike noncancerous
 cells, which can be cultured only for a finite period of time.
 A melanoma cell line employed in a method of treating cancer can be
 obtained by any suitable means but preferably is obtained by a method
 comprising the steps of (a) obtaining a sample of a melanoma from a
 mammalian host, (b) forming a single cell suspension from the melanoma
 sample, (c) pelleting the melanoma cells, (d) transferring the melanoma
 cells into tissue culture using standard sterile culture technique, and
 (e) maintaining the melanoma cells in tissue culture under conditions that
 allow the growth of the melanoma cells, as further described herein.
 More specifically, the sample of a melanoma typically is obtained at the
 time of surgery. The melanoma sample subsequently is handled and
 manipulated using sterile techniques, and in such a fashion so as to
 minimize tissue damage. The tissue sample preferably is placed on ice in a
 sterile container and moved to a laboratory laminar flow hood. The portion
 of the melanoma to be employed for isolation of a melanoma cell line is
 excised from the sample, and the remainder of the melanoma preferably is
 stored at a suitable temperature, e.g., -70.degree. C.
 With use of a single cell suspension, the suspension is formed by
 enzymatically digesting the cells, preferably overnight. For instance, the
 sample is suspended in a solution that contains collagenase. The solution
 also can contain DNAse and/or hyaluronidase. Cell culture medium can be
 employed to carry out the digestion. The resultant single cell suspension
 is pelleted, and the pellets are resuspended in a small volume of tissue
 culture medium. The resuspended cells preferably then are inoculated into
 tissue culture medium appropriate for the growth of the cells in culture
 at a density of about 5.times.10.sup.5 tumor cells/ml.
 Alternately, the fresh tumor sample is minced into small pieces which are
 placed into culture directly. This other preferred method of isolating a
 melanoma cell line comprises the steps of (a) obtaining a sample of
 melanoma from a mammalian host, (b) mincing the sample to obtain fragments
 thereof, (c) transferring the fragments of fresh tumor into tissue
 culture, and (d) maintaining the melanoma cells in tissue culture under
 conditions that allow the growth of the cells.
 Regardless of the means used to transfer the melanoma cells into tissue
 culture (and any means can be employed, such as is known to one of
 ordinary skill in the art), once transferred, the cultures can be
 maintained at about 35-40.degree. C. in the presence of about 5-8%
 CO.sub.2. Preferably the medium employed for cell growth is one that has
 wide applicability for supporting growth of many types of cell culture,
 e.g., a medium that utilizes a bicarbonate buffering system and various
 amino acids and vitamins. Optimally the medium is RPMI 1640 medium, which
 desirably has been supplemented with bovine serum (e.g., fetal bovine
 serum), preferably at a concentration of from about 5 to about 20%. The
 medium can contain various additional factors as necessary, e.g., when
 required for the growth of the melanoma cells, or for maintenance of the
 melanoma cells in an undifferentiated state. The medium and medium
 components are readily available, and can be obtained, for instance, from
 commercial suppliers. The tumor cell cultures can be fed and recultured as
 necessary, e.g., typically every 1 to 10 days. The tumor cells also can be
 subjected to differential trypsinization to remove other cells (e.g.,
 stromal cells) that can overgrow the primary tumor cultures. Also,
 suppression of fibroblast overgrowth can be achieved by supplementing the
 culture medium with cholera toxin (e.g., 10 ng/ml).
 When it appears that a substantially purified culture of the melanoma cells
 has been obtained (e.g., as judged by the appearance or growth behavior of
 the cultures), various tests can be carried out as necessary or desirable
 to confirm the purity of the cultures. For instance, this can be confirmed
 by flow cytometry or immunocytology to validate expression of
 melanoma-associated proteins or gangliosides. This is done using
 antibodies that are readily available, and as known to one of skill in the
 art.
 Shared Immunodominant Melanoma Antigens
 Tests can be carried out on cells of the melanoma cell line to confirm that
 the melanoma cell line produces (i.e., "expresses") shared immunodominant
 antigens. Preferably according to the invention the melanoma cell line
 expresses one or more shared immunodominant melanoma antigens as described
 herein, or as identified in the future. The term "shared" refers to the
 fact that antigens unique to a particular individual's own tumor will not
 be useful for a generally applicable vaccine; rather, antigens that are
 shared by multiple (i.e., more than one) cases of a particular tumor type
 are required. The term "immunodominant" refers to the fact that for
 reasons of processing, binding to MHC or otherwise, certain antigens are
 capable of being more efficiently recognized by T cells from the
 vaccinated host.
 In particular, preferably according to the invention, it is confirmed that
 tumor infiltrating lymphocytes from more than one patient, and, optimally,
 more than three patients, recognize the melanoma cell line. Moreover, RNA
 expression and/or protein production of shared immunodominant melanoma
 antigens desirably are assessed using standard techniques that are known
 in the art and are further described herein (e.g., PCR-based assays,
 Northern and Western assays, other immunological assays, and the like).
 According to the invention, preferably the shared immunodominant melanoma
 antigens are selected from the group consisting of melanocyte-specific
 differentiation antigens and tumor-specific shared antigens, as defined
 herein, or which are identified at some point in the future. In
 particular, desirably the shared immunodominant melanoma antigens comprise
 one or more melanocyte-specific differentiation antigens and one or more
 tumor-specific shared antigens. Optimally the melanoma cell line expresses
 at least three shared immunodominant melanoma antigens. In a preferred
 embodiment, the shared immunodominant melanoma antigens are selected from
 the group consisting of MAGE-1, MAGE-3, MART-1/Melan-A, tyrosinase, gp75,
 gp100, BAGE, GAGE-1, GAGE-2, GnT-V, and p15 antigens.
 The melanoma cell lines also can comprise tumor-specific mutated antigens.
 For instance, the melanoma cell lines preferably can comprise the
 b-catenin tumor-specific mutated antigen. Similarly, the melanoma cell
 lines preferably can comprise the MUM-1 tumor-specific mutated antigen.
 Also, the melanoma cell lines can comprise the CDK4 tumor-specific mutated
 antigen.
 Preferably the melanoma cell line employed as a vaccine in the method of
 the invention exhibits stable production of shared immunodominant melanoma
 antigens with continued passage. In particular, preferably the melanoma
 cell line expresses the MAGE-3 antigen along with another shared
 immunodominant melanoma antigen. Desirably the melanoma cell line
 expresses two shared immunodominant antigens selected from the group
 consisting of the MAGE-3, tyrosinase, MART-1/Melan-A, gp75, and gp100
 antigens. Even more preferably, the melanoma cell line expresses three
 shared immunodominant antigens selected from the group consisting of the
 MAGE-3, tyrosinase, MART-1/Melan-A, gp75, and gp 100 antigens. Desirably,
 the melanoma cell line expresses four shared immunodominant antigens
 selected from the group consisting of the MAGE-3, tyrosinase,
 MART-1/Melan-A, gp75, and gp100 antigens. Optimally,the melanomacell line
 expresses the MAGE-3, tyrosinase, MART-1/Melan-A, gp75, and gp100
 antigens. In particular, the melanoma cell line applied in the method of
 the invention preferably is 526-MEL or 624-MEL.
 Cytokine
 In the present inventive method of treating cancer, preferably the melanoma
 cell line has been modified to render it capable of producing an increased
 level of a cytokine relative to the unmodified melanoma cell line. A
 "cytokine" is, as that term is understood by one skilled in the art, any
 immunomodulating protein (including a modified protein such as a
 glycoprotein) that enhances the responsiveness of a host immune system to
 a melanoma present in the host. Preferably the cytokine is not itself
 immunogenic to the host, and potentiates immunity by activating or
 enhancing the activity of cells of the immune system.
 As used herein, a cytokine includes, but is not restricted to, such
 proteins as interferons, interleukins (e.g., IL-1 to IL-17), tumor
 necrosis factor (TNF), erythropoietin (EPO), macrophage colony stimulating
 factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and
 granulocyte-macrophagecolony stimulating factor (GM-CSF). Preferably the
 cytokine is GM-CSF.
 "Modifying" a melanoma cell line according to the invention comprises the
 transfer of genetic material capable of imparting increased expression of
 a cytokine of interest. The genetic material can be in the form of naked
 DNA or a "vector" encompassing a DNA molecule such as a plasmid, virus or
 other vehicle, which contains one or more heterologous or recombinant DNA
 sequences, e.g., a cytokine gene or cytokine coding sequence of interest
 under the control of a functional promoter and possibly also an enhancer,
 and that is capable of functioning as a vector as that term is understood
 by those of ordinary skill in the art. Appropriate viral vectors include,
 but are not limited to simian virus 40, bovine papilloma virus,
 Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney
 murine leukemia virus, Harvey murine sarcoma virus, and Rous sarcoma
 virus.
 Reference to a vector or other DNA sequences as "recombinant" merely
 acknowledges the linkage of DNA sequences which typically are not
 conjoined as isolated from nature. A "gene" is any nucleic acid sequence
 coding for a protein or a nascent mRNA molecule. Whereas a gene comprises
 coding sequences plus any non-coding (e.g., regulatory sequences), a
 "coding sequence" does not include any non-coding DNA. A "promoter" is a
 DNA sequence that directs the binding of RNA polymerase and thereby
 promotes RNA synthesis. "Enhancers" are cis-acting elements of DNA that
 stimulate or inhibit transcription of adjacent genes. An enhancer that
 inhibits transcription also is termed a "silencer". Enhancers differ from
 DNA-binding sites for sequence-specific DNA binding proteins found only in
 the promoter (which also are termed "promoter elements") in that enhancers
 can function in either orientation, and over distances of up to several
 kilobase pairs (kb), even from a position downstream of a transcribed
 region.
 Any suitable vector can be employed that is appropriate for introduction of
 nucleic acids into eukaryotic melanoma cells, or more particularly animal
 melanoma cells, such as mammalian, e.g., human, melanoma cells. Preferably
 the vector is compatible with the melanoma cell, e.g., is capable of
 imparting expression of the cytokine gene or coding sequence, and is
 stably maintained or relatively stably maintained in the melanoma cell.
 Desirably the vector comprises an origin of replication. Preferably the
 vector also comprises a so-called "marker" function by which the vector
 can be identified and selected (e.g., an antibiotic resistance gene). When
 a cytokine coding sequence (as opposed to a cytokine gene having its own
 promoter) is transferred, optimally the vector also contains a promoter
 that is capable of driving expression of the coding sequence and that is
 operably linked to the coding sequence. A coding sequence is "operably
 linked" to a promoter (e.g., when both the coding sequence and the
 promoter together constitute a native or recombinant cytokine gene) when
 the promoter is capable of directing transcription of the coding sequence.
 As used herein, cytokine "gene" or "coding sequence" includes cytokine
 genomic or cDNA sequences, greater and lesser sequences and mutations
 thereof, whether isolated from nature or synthesized in whole or in part,
 as long as the gene or coding sequence is capable of expressing or capable
 of being expressed into a protein having the characteristic function of
 the cytokine, i.e., the ability to stimulate the host immune response. The
 means of modifying genes or coding sequences are well known in the art,
 and also can be accomplished by means of commercially available kits
 (e.g., New England Biolabs, Inc., Beverly, Mass.; Clontech, Palo Alto,
 Calif.). The cytokine gene or coding sequence can be of any suitable
 source, for example, isolated from any mammalian species such as human.
 Preferably, however, the cytokine gene or coding sequence comprises a
 GM-CSF sequence, particularly a human or murine GM-CSF gene or coding
 sequence including a human or murine GM-CSF cDNA sequence (e.g., as
 described by Cantrell et al., Proc. Natl. Acad. Sci., 82, 6250-54 (1985)).
 In the recombinant vectors of the present invention, preferably all the
 proper transcription, translation and processing signals (e.g., splicing
 and polyadenylation signals) are correctly arranged on the vector such
 that the cytokine gene or coding sequence will be appropriately
 transcribed and translated in the melanoma cells into which it is
 introduced. The manipulation of such signals to ensure appropriate
 expression in host cells is well within the knowledge and expertise of the
 ordinary skilled artisan. Whereas a cytokine gene is controlled by (i.e.,
 operably linked to) its own promoter, another promoter, including a
 constitutive promoter, such as, for instance the adenoviral type 2 (Ad2)
 or type 5 (Ad5) major late promoter (MLP) and tripartite leader, the
 cytomegalovirus (CMV) immediate early promoter/enhancer, the Rous sarcoma
 virus long terminal repeat (RSV-LTR), and others, can be employed to
 command expression of the cytokine coding sequence.
 Alternately, a tissue-specific promoter (i.e., a promoter that is
 preferentially activated in a given tissue and results in expression of a
 gene product in the tissue where activated) can be used in the vector.
 Such promoters include but are not limited to the elastase I gene control
 region which is active in pancreatic acinar cells as described by Swift et
 al., Cell, 38, 639-46 (1984) and MacDonald, Hepatology, 7, 425-515 (1987);
 the insulin gene control region which is active in pancreatic beta cells
 as described by Hanahan, Nature, 315, 115-22 (1985); the
 hepatocyte-specific promoter for albumin or alpha-1 antitrypsin described
 by Frain et al., Mol. Cell. Biol., 10, 991-99 (1990) and Ciliberto et al.,
 Cell, 41, 531-40 (1985); and the albumin and alpha-1 antitrypsin gene
 control regions which both are active in liver as described by Pinkert et
 al., Genes and Devel., 1, 268-76 (1987) and Kelsey et al., Genes and
 Devel., 1, 161-71 (1987).
 Similarly, a melanoma-specificpromoter, akin to the carcinoembryonic
 antigen for colon carcinoma described by Schrewe et al., Mol. Cell Biol.,
 10, 2738-48 (1990), can be used in the vector. Along the same cell lines,
 promoters that are selectively activated at different developmental stages
 (e.g., globin genes are differentially transcribed in embryos and adults)
 can be employed for gene therapy of certain types of cancer.
 Another option is to use an inducible promoter, such as the IL-8 promoter,
 which is responsive to TNF, or the 6-16 promoter, which is responsive to
 interferons, or to use other similar promoters responsive to other
 cytokines or other factors present in a host or that can be administered
 exogenously. Use of a cytokine-inducible promoter has the added advantage
 of allowing for auto-inducible expression of a cytokine gene. According to
 the invention, any promoter can be altered by mutagenesis, so long as it
 has the desired binding capability and promoter strength.
 Accordingly, the present invention provides a vector that comprises a
 nucleic acid sequence encoding a cytokine as defined above, and that can
 be employed in the method of the present invention of treating cancer. In
 particular, the present invention provides a recombinant vector comprising
 a nucleic acid sequence encoding a human GM-CSF. Thus, preferably, the
 present invention provides the vector designated as pcDNA3/Neo-GM-CSF,
 which is further described herein.
 In the method of the present invention, the naked DNA or recombinant vector
 can be employed to transfer a cytokine gene or coding sequence to a cell
 in vitro, which preferably is a cell of an established melanoma cell line.
 Various methods can be employed for delivering new genetic material to
 cells in vitro. For instance, such methods include electroporation,
 membrane fusion with liposomes, high velocity bombardment with DNA-coated
 microprojectiles, incubation with calcium phosphate-DNA precipitate, DEAE
 dextran mediated transfection, infection with modified viral nucleic
 acids, direct microinjection into single cells, and the like. Other
 methods are available and are known to those skilled in the art. Thus, the
 present invention provides a substantially purified melanoma cell line
 wherein the cell line has been modified to render it capable of producing
 an increased level of a cytokine (preferably GM-CSF) relative to the
 unmodified melanoma cell line.
 The level of cytokine produced by the modified melanoma cell is important
 in the context of the present invention for the purpose of obtaining an
 immunostimulatory response. Preferably the modified (e.g., transfected or
 transformed) melanoma cell line produces a level of cytokine that is
 increased over that observed for the unmodified (i.e., parental) melanoma
 cell line. Even more preferably, the modified cell line produces a level
 of cytokine that results in cytokine secretion greater than 36 ng/10.sup.6
 cells/day.
 The present invention also encompasses a method of treating or protecting
 against melanoma wherein the cytokine is provided not by the administered
 melanoma cells, but is provided by some other means. This method comprises
 simply (a) obtaining a melanoma cell line that expresses one or more
 shared immunodominant melanoma antigens, and (b) administering the
 melanoma cell line to a mammalian host that has melanoma or is at risk for
 developing melanoma. In this method, the melanoma cell line is not
 modified prior to administration to render it capable of producing an
 increased level of a cytokine. Instead, cytokine is provided by some other
 means known in the art. For instance, cells of the melanoma cell line can
 be administered with cytokine encapsulated in microspheres (see, e.g.,
 Golumbek et al., Cancer Research, 53, 1-4 (1993)) or liposomes (see, e.g.,
 Nabel et al., Proc. Natl. Acad. Sci., 90, 11307-11 (1993)).
 Administering the Melanoma Cell Line
 "Administering" cells of the melanoma cell line to a mammalian host refers
 to the actual physical introduction of the melanoma cells, particularly
 the modified (i.e., cytokine-producing) melanoma cells, into the host. Any
 and all methods of introducing the melanoma cells into the host are
 contemplated according to the invention; the method is not dependent on
 any particular means of introduction and is not to be so construed. Means
 of introduction are well known to those skilled in the art, and several
 such introduction means are exemplified herein.
 While it is anticipated that the administered melanoma cell line may have
 some MHC antigens in common with the host melanoma, for the purpose of
 this invention, it is not necessary that the administered melanoma cell
 and the host have any MHC antigens in common. Accordingly, the present
 invention encompasses the administration of a melanoma cell line which is
 allogeneic (i.e., from a different individual) to the host, and which is
 not necessarily MHC-matched to the host. According to this invention a
 melanoma cell line is "not MHC-matched" to a host when it does not share
 any MHC antigens in common with the host, or when it does not share any of
 the MHC antigens with the host which typically are MHC-matched when using
 allogeneic melanoma cell vaccines (e.g., MHC class I antigens, especially
 HLA-A2).
 Also, preferably the melanoma cell line (e.g., the modified melanoma cell
 line) is irradiated prior to administration to prevent cell replication,
 and possible melanoma formation in vivo. For irradiation of melanoma
 cells, the melanoma cells typically are harvested, transferred to a test
 tube in liquid medium, and irradiated at room temperature using a .sup.137
 CS source. Preferably the cells are irradiated at a dose rate of from
 about 50 to about 200 rads/min, even more preferably, from about 120 to
 about 140 rads/min. Preferably the cells are irradiated with a total dose
 sufficient to inhibit the majority of cells, preferably about 100% of the
 cells, from proliferating in vitro. Thus, desirably the cells are
 irradiated with a total dose of from about 10,000 to 30,000 rads.
 Moreover, the melanoma cell line (e.g., the modified melanoma cell line)
 optimally is treated prior to administration to enhance its
 immunogenicity. Preferably this treatment comprises, as described herein,
 further genetic manipulation, such as, for instance, introduction of other
 cytokine or immune co-stimulatory functions, or, for example, admixture
 with nonspecific adjuvants including but not limited to Freund's complete
 or incomplete adjuvant, emulsions comprised of bacterial and mycobacterial
 cell wall components, and the like.
 Accordingly, the allogeneic melanoma cell lines can be used to vaccinate
 patients with melanomas for the purpose of generating a systemic
 antimelanoma immune response against the patient's own melanoma. To the
 extent that MAGE-3 also is produced in other nonmelanotic tumors such as
 SCLC, non-SCLC, SCCHN, colon cancer, and breast cancer, and that MAGE-1
 also is produced in breast cancer, glioblastoma, neuroblastoma, SCLC, and
 medullary cancer of the thyroid, allogeneic melanoma cell lines according
 to the invention that express MAGE-3 and/orMAGE-1 antigens also can be
 employed for the treatment of these other nonmelanotic tumors.
 To facilitate administration, an allogeneic melanoma cell line according to
 the invention, particularly a modified allogeneic melanoma cell line that
 has been treated prior to administration to enhance its immunogenicity,
 can be made into a pharmaceutical composition or implant appropriate for
 administration in vivo, with appropriate carriers or diluents, which
 further can be pharmaceutically acceptable. The means of making such a
 composition or an implant have been described in the art (see, for
 instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed.
 (1980)). Where appropriate, a melanoma cell line can be formulated into a
 preparation in semisolid or liquid form, such as a capsule, solution,
 injection, inhalant, or aerosol, in the usual ways for their respective
 route of administration. Means known in the art can be utilized to prevent
 or minimize release and absorption of the composition until it reaches the
 target tissue or organ, or to ensure timed-release of the composition.
 Preferably, however, a pharmaceutically acceptable form is employed which
 does not ineffectuate the compositions of the present invention. Thus,
 desirably the melanoma cell line can be made into a pharmaceutical
 composition comprising a balanced salt solution, preferably Hanks'
 balanced salt solution, or normal saline.
 Thus, the present invention provides a pharmaceutical composition that
 comprises a pharmaceutically acceptable carrier and cells of a melanoma
 cell line according to the invention, or any other melanoma cell line
 expressing one or more shared immunodominant antigens, as described
 herein. Preferably, the invention provides a pharma-ceutical composition
 comprising a pharmaceutically acceptable carrier and a melanoma cell line,
 particularly wherein the melanoma cell line is 526-MEL or 624-MEL, which
 has been modified to produce an increased level of a cytokine, optimally
 GM-CSF. The invention also provides a pharmaceutical composition that
 preferably comprises a pharmaceutically acceptable carrier and cells of a
 multiplicity of the melanoma cell lines according to the invention. For
 instance, the composition preferably comprises cells of more than one cell
 line according to the invention, and optimally comprises cells of more
 than one cell line, e.g., comprises 526-MEL and 624-MEL cells, or
 comprises cells selected from the group consisting of 526-MEL, 624-MEL,
 and some other cell line.
 In pharmaceutical dosage form, a composition can be used alone or in
 appropriate association, as well as in combination, with other
 pharmaceutically active compounds and methods of treatment. For example,
 in applying a method of the present invention for the treatment of cancer,
 in particular, for the treatment of melanoma, such treatment can be
 employed in conjunction with other means of treatment of cancer,
 particularly melanoma, e.g., surgical ablation, irradiation, chemotherapy,
 and the like. In terms of chemotherapy, a composition according to the
 invention can be employed in addition to the use of dacarbazine,
 dactinomycin, carmustine, procarbazine, vinblastine, and interferon, as
 well as other drugs used to treat melanoma.
 A pharmaceutical composition of the present invention can be delivered via
 various routes and to various sites in a mammalian, particularly human,
 body to achieve a particular effect. One skilled in the art will recognize
 that, although more than one route can be used for administration, a
 particular route can provide a more immediate and more effective reaction
 than another route. Local or systemic delivery can be accomplished by
 administration comprising application or instillation of the formulation
 into body cavities, inhalation or insufflation of an aerosol, or by
 parenteral introduction, comprising intramuscular, intravenous,
 intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal
 administration. Preferably delivery is accomplished by subcutaneous or
 intradermal administration.
 A composition of the present invention can be provided in unit dosage form
 wherein each dosage unit, e.g., an injection, contains a predetermined
 amount of the composition, alone or in appropriate combination with other
 active agents. The term "unit dosage form" as used herein refers to
 physically discrete units suitable as unitary dosages for human and animal
 subjects, each unit containing a predetermined quantity of the composition
 of the present invention, alone or in combination with other active
 agents, calculated in an amount sufficient to produce the desired effect,
 in association with a pharmaceutically acceptable diluent, carrier, or
 vehicle, where appropriate. The specifications for the novel unit dosage
 forms of the present invention depend on the particular pharmacodynamics
 associated with the pharmaceutical composition in the particular host.
 Preferably a sufficient number of the modified melanoma cells are present
 in the composition and introduced into the host such that expression of
 cytokine by the host cell, and subsequent recruitment of APCs to the
 melanoma site, results in a greater immune response to the extant host
 melanoma than would otherwise result in the absence of such treatment, as
 further discussed herein. Accordingly, the amount of vaccine cells
 administered should take into account the route of administration and
 should be such that a sufficient number of the melanoma cells will be
 introduced so as to achieve the desired therapeutic (i.e.
 immunopotentiating)response. Furthermore, the amounts of each active agent
 included in the compositions described herein (e.g., the amount per each
 cell to be contacted or the amount per certain body weight) can vary in
 different applications. In general, the concentration of modified melanoma
 cells preferably should be sufficient to provide in the host being treated
 at least from about 1.times.10.sup.6 to about 1.times.10.sup.9 melanoma
 cells, even more preferably, from about 1.times.10.sup.7 to about
 5.times.10.sup.8 melanoma cells, although any suitable amount can be
 utilized either above, e.g., greater than 5.times.10.sup.8 cells, or
 below, e.g., less than 1.times.10.sup.7 cells.
 These values provide general guidance of the range of each component to be
 utilized by the practitioner upon optimizing the method of the present
 invention for practice of the invention. The recitation herein of such
 ranges by no means precludes the use of a higher or lower amount of a
 component, as might be warranted in a particular application. For example,
 the actual dose and schedule can vary depending on whether the
 compositions are administered in combination with other pharmaceutical
 compositions, or depending on interindividual differences in
 pharmacokinetics, drug disposition, and metabolism. One skilled in the art
 readily can make any necessary adjustments in accordance with the
 exigencies of the particular situation. Moreover, the effective amount of
 the compositions can be further approximated through analogy to other
 compounds known to inhibit the growth of cancer cells, in particular,
 melanoma cells.
 One skilled in the art also is aware of means to monitor a therapeutic
 (i.e., systemic immune) response upon administering a composition of the
 present invention. In particular, the therapeutic response can be assessed
 by monitoring attenuation of melanoma growth and/or melanoma regression.
 The attenuation of melanoma growth or melanoma regression in response to
 treatment can be monitored using several end-points known to those skilled
 in the art including, for instance, the number of melanomas, melanoma mass
 or size, or reduction/prevention of metastasis. These described methods by
 no means are all-inclusive, and further methods to suit the specific
 application will be apparent to the ordinary skilled artisan.

EXAMPLES
 The following examples further illustrate the present invention but, of
 course, should not be construed as in any way limiting its scope.
 Example 1
 This example illustrates the method of obtaining and culturing the melanoma
 cell lines that express one or more shared immunodominant melanoma
 antigens.
 Melanoma cell lines were established from surgical resection specimens.
 Standard means as previously described and as known to one of ordinary
 skill in the art were employed to isolate the cell lines (see, e.g.,
 Freshney, Culture of Animal Cells, (3d Ed.) Wiley-Liss, Inc., NY (1993)).
 In particular, the tumors were dispersed into single cell suspensions by
 overnight enzymatic digestion with collagenase, DNAse and hyaluronidase,
 and were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS).
 The cells were then propagated in culture using standard sterile tissue
 culture technique.
 Example 2
 This example describes a characterization of the melanoma cell lines that
 expresses one or more shared immunodominant melanoma antigens.
 The presence within a melanoma cell line of shared immunodominant antigens
 can be confirmed by showing that T cells from patients with melanoma that
 recognize the patient's own melanoma also will recognize the particular
 allogeneic melanoma cell line in question. In order to make this
 determination, there must be sharing of at least one MHC class I antigen
 between the patient from which the T cells are derived and the melanoma
 line being tested. One of the best MHC antigens to use for these purposes
 is HLA-A2 since it is expressed in roughly 50% of Caucasian individuals.
 Thus, fresh tumor suspensions were passed over Ficoll-Hypaque gradients
 (Lymphocyte Separation Medium, Organon Technical Corporation, Durham,
 N.C.) to isolate and grow T cell populations that recognize melanoma. The
 gradient interfaces containing viable tumor cells and lymphocytes were
 washed, adjusted to a total cell concentration of about 2.5 to about
 5.0.times.10.sup.5 cells per ml, and cultured in complete medium. Complete
 medium consisted of RPMI 1640 with 10% heat-inactivated type AB human
 serum, 50 IU/ml penicillin and 50 mg/ml streptomycin (Biofluids,
 Rockville, Md.), 50 mg/ml gentamicin (GIBCO Laboratories, Chagrin Falls,
 Ohio), 10 mM HEPES buffer (Biofluids), and 2 mM L-glutamine (MA
 Bioproducts, Walkersville, Md.). The medium was supplemented with 6000
 IU/ml IL-2 and the supernatant from LAK cell cultures. Cultures were
 maintained at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere in a
 variety of tissue culture vessels, including 24-well plates and 175
 cm.sup.2 flasks. Under these conditions, tumor-infiltrating T cells grow
 selectively. Tumor infiltrating lymphocyte (TIL) cultures were expanded in
 IL-2 for at least four weeks.
 For analysis of TIL recognition of the melanoma cell lines (Topalian et
 al., J. Immunol. 142, 3714 (1989)), the cytolytic activity of cultured TIL
 against these cell lines was assessed using standard 4 hour .sup.51 Cr
 release assays. Alternately, specific secretion of cytokines by TIL
 cocultured with tumor cells was monitored. The results of these
 experiments are presented in Table 2.
 TABLE 2
 LYSIS OF HLA-A2.sup.+ MELANOMAS BY HLA-A2-RESTRICTED MELANOMA
 TIL
 Targets (% Lysis, E:T = 40)
 526- 553- 624- 677- 697- 1102- 1011- 560- Daudi
 Effectors MEL MEL MEL MEL MEL MEL MEL fibro
 lymphoma
 Exp. A
 TIL 620 48 35 55 67 53 33 5 -1 -1
 TIL 1073 40 26 44 29 24 23 -1 -3 -6
 TIL 1143 33 41 54 54 34 29 0 -2 -5
 TIL 1235 24 15 34 37 27 8 0 0 -4
 LAK Cells 30 79 60 62 18 53 63 51 68
 Exp. B
 TIL 501 58 19 53 54 47 7 0 8 -2
 TIL 660 47 26 50 46 41 6 1 0 1
 TIL 1074 13 9 14 8 5 2 -2 0 -3
 TIL 1128 17 4 20 15 18 3 0 0 -2
 LAK Cells 53 77 55 72 38 52 62 69 63
 HLA-A2 + + + + + + - + -
 As shown in Table 2, TIL cultures from eight different HLA-A2.sup.+
 patients (i.e., TIL 620, TIL 1073, TIL 1143, TIL 1235, TIL 501, TIL 660,
 TIL 1074, and TIL 1128) were tested against seven different established
 melanoma cell lines (i.e., 526-MEL, 553-MEL, 624-MEL, 677-MEL, 697-MEL, 11
 02-MEL, and 1011-MEL) as well as an established fibroblast cell line
 (560-fibro) and the Daudi lymphoma cell line. Two of the tumor cell lines,
 i.e., 526-MEL and 624-MEL, were recognized by all of the TIL cultures as
 determined by the specific lysis in a chromium release assay of &gt;10% at an
 effect to target tumor cell ration (E:T) equal to 40. Other of the
 melanoma cell lines except for 1011-MEL (HLA-A2 negative) were recognized
 by the majority of, or at least one of, the TIL cultures.
 526-MEL and 624-MEL were tested by reverse-transcriptasepolymerase chain
 reaction (RT-PCR) or Northern blotting for expression of the MZ2-E (or
 MAGE-1), MZ2-D (or MGE-3), MART-1/Melan-A, GP100, and GP75 antigens. The
 cell lines were assessed for tyrosinase production via recognition by a
 tyrosinase specific helper T cell line. The cell lines also were assessed
 for expression of GD3 by staining with a specific monoclonal antibody. The
 results of these experiments are presented in Table 3.
 TABLE 3
 EXPRESSION OF SHARED MELANOMA ANTIGENS
 BY MELANOMA CELL CULTURES
 Antigen 526-MEL 624-MEL
 MAGE-1 (MZ2-E) - -
 MAGE-3 (MZ2-D) + +
 Tyrosinase + +
 MART-1/Melan-A + +
 GP100 + +
 GP75 + +
 GD3 + +
 Both cell lines expressed all the common shared antigens tested by these
 assays with the exception of the MAGE-1 antigen, as presented in Table 3.
 These results confirm that the 526-MEL and 624-MEL melanoma cell lines
 express the majority of the immunodominant shared melanoma antigens. The
 results further confirm that the methods described herein can be employed
 to obtain and/or identify a melanoma cell line that expresses one or more
 shared immunodominant melanoma antigens.
 Example 3
 This example illustrates the method of modifying a melanoma cell line that
 expresses one or more shared immunodominant melanoma antigens to produce
 an increased amount of a cytokine. The cytokine
 granulocyte-macrophagecolony stimulating factor (GM-CSF) is potentially
 more potent than other cytokines in generating a systemic antimelanoma
 response in preclinical melanoma models (see, e.g., Dranoffet al., Proc.
 Natl. Acad. Sci., 90, 3539-42(1993)). Accordingly,the melanoma cell lines
 were modified to secrete GM-CSF. The melanoma cell lines 526-MEL and
 624-MEL described in Example 1 were employed as representative of an
 allogeneic melanoma cell line that expresses one or more shared
 immunodominant melanoma antigens.
 To facilitate manipulation of the cell lines, a recombinant human GM-CSF
 gene was cloned into pcDNA3/Neo (Invitrogen). The resulting recombinant
 vector is henceforth designated pcDNA3/Neo-GM-CSF. All cloning reactions
 and DNA manipulations were carried out using methods that are well known
 to the ordinary skilled artisan, and which have been described in the art
 (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd
 ed. (Cold Spring Harbor Laboratory, NY, (1982))). Enzymes employed in
 these reactions were obtained from commercial suppliers (e.g., New England
 Biolabs, Inc., Beverly, Mass.; Clontech, Palo Alto, Calif.; Boehringer
 Mannheim, Inc., Indianapolis, Ind.; etc.), and were used according to the
 manufacturers' recommendations.
 The plasmid pcDNA3/Neo-GM-CSF contains the human GM-CSF cytokine coding
 sequence under the control of the cytomegalovirus (CMV) promoter, and also
 contains the neomycin resistance gene controlled by a separate CMV
 promoter. The CMV promoter was employed since it is able to drive a
 relatively high level of gene expression in most eukaryotic cells (Boshart
 et al., Cell, 41, 521-30 (1985)). Initial studies using this vector for
 gene transfer to a human melanoma cell line confirm that, following
 selection for neomycin resistance, secreted levels of GM-CSF greater than
 36 ng/10.sup.6 cells/day were achieved. These initial studies confirm that
 the pcDNA3/Neo-GM-CSF plasmid is functional in eukaryotic cells. Moreover,
 this is the dose of GM-CSF that is required to generate an adequate
 antimelanoma immune response in a mouse model. Dilution experiments using
 varying concentrations of melanoma cells that either were or were not
 transduced with a retroviral vector carrying a GM-CSF gene confirm that,
 in the B16-F10 melanoma system, GM-CSF secretion below 36 ng/10.sup.6
 cells/day fails to generate the potent antimelanoma immunity seen at
 levels of secretion above this threshold. These findings underscore the
 importance of delivering high and sustained levels of GM-CSF directly at
 the site of the vaccinating melanoma cells that are the source of the
 relevant melanoma antigen.
 The 526-MEL and 624-MEL cell lines were transfected with pcDNA3/Neo-GM-CSF
 by the calcium phosphate procedure. For these experiments, 526-MEL was
 transfected at culture passage 32, and 624-MEL was transfected at culture
 passage 28. GM-CSF levels were determined by ELISA. The results of these
 experiments are presented in Table 4.
 TABLE 4
 GM-CSF SECRETION BY TRANSFECTED
 MELANOMA CELL LINES
 GM-CSF (ng/10.sup.6 melanoma
 Cell Line Passage # cells/day)
 526-MEL 40 4.9
 43 2.1
 44 8.2
 624-MEL 35 18.4
 37 37.0
 38 85.5
 The GM-CSF secretion level observed for the 526-MEL cell line was less than
 10 ng/10.sup.6 melanoma cells/day. It is possible that GM-CSF secretion
 for the 526-MEL cell line can be increased with use of a different
 expression vector for transfection, or by selecting melanoma cell lines
 with higher levels of expression. In comparison, the GM-CSF secretion
 level observed for the melanoma cell line 624-MEL was over 80 ng/10.sup.6
 melanoma cells/day. Nontransfected melanomas did not secrete measurable
 amounts of GM-CSF.
 The methods employed in this example also can be used to generate melanoma
 cell lines capable of producing increased amounts of other cytokines, and
 can be used with other melanoma cell lines, all of which similarly can be
 employed as vaccines.
 Example 4
 This example illustrates further studies regarding GM-CSF administration to
 a host.
 Further studies confirm that GM-CSF secretion needs to parallel the known
 paracrine physiology of this cytokine. In particular, secretion must be at
 the site of the relevant antigens (i.e., the melanoma cells), as described
 in the previous example, and high levels must be sustained for several
 days (see, e.g., Dranoffet al., supra; Golumbek et al., supra). However,
 it appears that the melanoma cell itself need not be the source of GM-CSF
 secretion (Golumbek et al., supra). Immunologic protection and histologic
 infiltrates similar to those seen with retrovirally-transduced
 cytokine-expressing melanoma cells can be generated when GM-CSF is slowly
 released from biodegradable polymers co-injected with the melanoma cell.
 In addition, if a second non-cross reacting tumor is co-injected with a
 GM-CSF secreting melanoma, immunologic protection against both tumors can
 be generated. Simple injection of soluble GM-CSF along with melanoma
 cells, however, does not provide sustained local levels of this cytokine
 and does not generate systemic immunity (Golumbek et al., supra). Thus,
 the effectiveness of using an allogeneic melanoma cell that was not
 MHC-matched to the host cell for delivery of cytokine in vivo was
 explored.
 In murine models, it was demonstrated that the antimelanoma immunity
 generated with the delivery of GM-CSF by bystander allogeneic melanoma
 cells is comparable to that achieved when GM-CSF is delivered by the
 target melanoma cell itself. Specifically, in these experiments, BALB/c
 mice were subcutaneously vaccinated with irradiated CT26 colon carcinoma
 cells, with GM-CSF delivered either by retrovirally-transduced CT26 cells,
 or by retrovirally-transducedallogeneic B16-F10 cells. Two weeks later,
 mice were rechallenged with injections of wild-type strain CT26. The CT26
 colon carcinoma cell line possesses some intrinsic immunogenicity;
 however, a greater degree of protection was seen when GM-CSF was secreted
 at the vaccination site, whether by the syngeneic or the allogeneic cells.
 While it is unclear to what degree, or by what mechanism, the allogeneic
 melanoma cells can augment anti-CT26 immunity, these data strongly suggest
 that allogeneic delivery of GM-CSF in the context of the present invention
 is likely to be at least as effective as autologous melanoma delivery.
 Example 5
 This example illustrates the method of treating cancer by administering to
 a host in accordance with the invention, a melanoma cell line that
 expresses one or more shared immunodominant melanoma antigens, and
 preferably is allogeneic and is not necessarily MHC-matched to the host.
 Melanoma cell lines that secrete GM-CSF, preferably at levels greater than
 36 ng/10.sup.6 melanoma cells/day, are obtained and employed. The modified
 melanoma cells are harvested from the tissue culture flasks by
 trypsinization. The cells are washed using normal saline, pelleted, and
 resuspended in Hanks' balanced salt solution, or some other salt solution
 appropriate for introduction in vivo. The cells are resuspended at a
 concentration of from about 1.times.10.sup.6 to about 1.times.10.sup.8
 melanoma cells/ml. From about 0.1 to about 0.5 ml of this resuspension
 mixture is employed as a vaccine. Thus, preferably from about
 1.times.10.sup.6 to about 1.times.10.sup.9 melanoma cells are injected,
 and, optimally, from about 1.times.10.sup.7 to about 5.times.10.sup.8
 melanoma cells are injected in toto. Whereas the modified melanoma cells
 are injected subcutaneously in the mouse, the cells preferably are
 injected intradermally in humans.
 Prior to injection, the modified melanoma cells can be irradiated, e.g.,
 using a .sup.137 Cs source. Such irradiation prevents the replication of
 the tumor cells, but allows the cells to secrete GM-CSF and to remain
 metabolically active for at least a week in culture. Preferably
 irradiation can be carried out using a .sup.137 Cs source at a dose rate
 of about 120-140 rads/min to deliver a total dose of about 15,000 rads.
 The modified melanoma cells also can be altered to enhance their
 immunogenicity. For instance, the cells further can be genetically
 manipulated (e.g., through insertion of other cytokine or other immune
 stimulatory nucleic acid sequences, e.g., a cytokine other than, or in
 addition to GM-CSF ), or can be admixed with non-specific adjuvants (e.g.,
 Freund's complete or incomplete adjuvant, emulsions comprised of bacterial
 and mycobacterial cell wall components, and the like).
 The invention can be used in mammals (particularly humans) with melanoma,
 or that are at risk for developing melanoma. It also is anticipated that
 the patient can be treated prior to, or in addition to (i.e., concurrently
 or immediately following), immunotherapy as described herein with any
 number of methods as are employed to treat cancer, for instance, surgical
 resection, irradiation, chemotherapy, and the like.
 All of the references cited herein, including patents, patent applications,
 and publications, are hereby incorporated in their entireties by
 reference.
 While this invention has been described with an emphasis upon preferred
 embodiments, it will be obvious to those of ordinary skill in the art that
 variations of the preferred embodiments can be used and that it is
 intended that the invention can be practiced otherwise than as
 specifically described herein. Accordingly, this invention includes all
 modifications encompassed within the spirit and scope of the invention as
 defined by the following claims.