Melanoma antigens and methods of use

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of cancer. More specifically, the present invention relates to antigens specific for melanoma carcinomas.

[0005] 2. Description of the Related Art

[0006] Several methods have been employed to isolate and clone tumor-associated antigens, and in general, these methods have relied upon the ability of the antigens to stimulate cytolytic T cells (1-4). These methods involve demanding techniques, including extensive manipulation and expansion of cytolytic T cells.

[0007] Furthermore, it is becoming increasingly apparent that tumor-bearing individuals also develop serological immune responses to tumor antigens. Antibodies directed towards mutated cellular genes have been described, including those reactive with mutant p53 (5, 6) and ras (7). In addition, humoral immune responses to non-mutated, aberrantly expressed tumor antigens, such as erbB-2 (8) and cathepsin D (9), have been reported.

[0008] The presence of humoral immunity to many known tumor-associated antigens suggests its use for identification of novel tumor-associated antigens. The feasibility of this strategy was demonstrated in a study by Pfreundschuh and coworkers (10, 11) who screened tumor-derived cDNA libraries with autologous patient sera and identified two known tumor antigens as well as several novel, putative tumor antigens. This technology, termed SEREX, for serological identification of antigens by recombinant expression cloning, has since been applied by many groups and has led to the substantial expansion of known tumor antigens (12).

[0009] Thus, the prior art is deficient in additional novel antigens specific to melanomas. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

[0010] Herein, the SEREX approach was used to identify melanoma antigens in patients undergoing active immunotherapy. The primary goal of identifying novel melanoma antigens is to expand the potential targets for immunotherapy. In addition, characterization of these proteins has the potential to impact on diverse areas of melanoma research including detection, diagnosis and staging, characterization of the genetic changes associated with tumorigenesis, and the principles of immune activation and tumor cell rejection.

[0011] Novel melanoma tumor-associated antigens may be useful for detection, diagnosis, and staging of melanomas. Novel melanoma tumor-associated antigens may also be useful for monitoring to detect recurrence and metastatic disease and to monitor disease burden (e.g., proteins expressed on the cell surface may provide targets for monitoring, i.e., via detection and imaging of tumors). Novel tumor-associated antigens may additionally be useful as targets for immunotherapy and intervention strategies.

[0012] One object of the present invention is to provide melanoma tumor-associated antigens and methods of using the melanoma tumor-associated antigens.

[0013] In one embodiment of the present invention, there is provided DNA encoding a melanoma tumor-associated antigen selected from the group consisting of: (a) isolated DNA as shown in SEQ ID Nos. 1-12; (b) isolated DNA which is complementary to isolated DNA of (a) above; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code.

[0014] In another embodiment of the present invention, there is provided an isolated and purified melanoma tumor-associated antigen coded for by the DNA disclosed herein.

[0015] In another embodiment of the present invention, there is provided a method for detecting mRNA coding for a melanoma tumor-associated antigen in a sample, comprising the steps of: (a) contacting a sample with an oligonucleotide probe having a sequence such as SEQ ID Nos. 1-12; and (b) detecting binding of the probe to the mRNA coding for a melanoma tumor-associated antigen in the sample.

[0016] In yet another embodiment of the present invention, there is provided a kit for detecting mRNA coding for a melanoma tumor-associated antigen, comprising: an oligonucleotide probe having a nucleotide sequence shown in SEQ ID Nos. 1-12. The kit may further comprises: a label with which to label the probe; and means for detecting the label.

[0017] In still yet another embodiment of the present invention, there is provided a method of detecting a melanoma tumor-associated antigen in a sample, comprising the steps of: (a) contacting a sample with an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein; and (b) detecting binding of the antibody to the melanoma tumor-associated antigen in the sample.

[0018] In another embodiment of the present invention, there is provided a kit for detecting a melanoma tumor-associated antigen, comprising: an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein. The kit may further comprise means to detect the antibody.

[0019] In another embodiment of the present invention, there is provided an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein.

[0020] In still yet another embodiment of the present invention, there is provided a method of screening for compounds that inhibit the activity of a melanoma tumor-associated antigen, comprising the steps of: (a) contacting a sample with a compound, wherein the sample comprises a melanoma tumor-associated antigen encoded by the DNA disclosed herein; and (b) assaying for activity of the melanoma tumor-associated antigen. Generally, a decrease in the melanoma tumor-associated antigen activity in the presence of the compound relative to the melanoma tumor-associated antigen activity in the absence of the compound is indicative of a compound that inhibits the activity of the melanoma tumor-associated antigen.

[0021] In another embodiment of the present invention, there is provided a method of inhibiting the growth of a melanoma tumor in an individual, comprising the steps of: (a) treating an individual with a therapeutic compound, wherein the therapeutic compound comprises a thereapeutic moiety and a targeting moiety, wherein the targeting moiety recognizes a melanoma tumor-associated antigen encoded by the DNA disclosed herein; wherein the therapeutic compound inhibits the growth of the melanoma tumor in the individual.

[0022] In another embodiment of the present invention, there is provided a cancer vaccine composition, comprising a vector capable of expressing a DNA molecule such as SEQ ID Nos. 1-12, and an appropriate adjuvant.

[0023] In another embodiment of the present invention, there is provided a method of vaccinating an individual against cancer, comprising the step of: (a) administering to the individual a vector capable of expressing a DNA molecule such as SEQ ID Nos. 1-12, wherein said expression elicits an immune response specific towards a melonoma-specific antigen, thereby inducing immune-mediated destruction of melanoma cells.

[0024] In another embodiment of the present invention, there is provided a method of inhibiting the growth of a melanoma tumor, comprising the steps of: (a) administering to an individual a cancer vaccine comprising a vector expressing a DNA such as SEQ ID Nos. 1-12, wherein administration of said vaccine induces an immune response, thereby inhibiting the growth of a melanoma tumor.

[0025] Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

[0027]
FIG. 1 shows an example of a colony lift assay with a purified positive clone. Clone 3.1 was plated in a 50:50 mix with a negative control phage. Filters were lifted from the plates, incubated with serum from patient 1, and reactive plaques were detected with a labeled secondary antibody. Dark circles represent the positive plaques for clone 3.1, while negative plaques are not detected.

[0028]
FIG. 2 shows the 5′ and 3′ sequences of clone 3.1 (FIG. 2A); clone 3.14 (FIG. 2B); clone 3.3T (FIG. 2C); clone 5.17 (FIG. 2D); clone 5.31 (FIG. 2G); and complete sequence of clone 5.23 (FIG. 2E); clone 5.28 (FIG. 2F).

DETAILED DESCRIPTION OF THE INVENTION

[0029] The primary goal in identifying novel melanoma antigens is to expand the potential targets for immunotherapy. In addition, characterization of tumor antigens may impact diverse areas of melanoma research, including detection, diagnosis and staging, characterization of the genetic changes associated with tumorigenesis, and the principles of immune activation and tumor cell rejection.

[0030] The tumor-associated antigens of the present invention may be useful for detection, diagnosis, and staging of melanoma. Detection and diagnosis of melanoma is currently based on visual identification of melanoma lesions, while staging is based on depth of the lesion at the time of diagnosis. While these visual guidelines have proven useful, the use of additional marker proteins and molecular characterization of melanoma lesions may provide information useful in more accurately defining the clinical course of the disease.

[0031] The tumor-associated antigens of the present invention may also be useful for disease monitoring. Metastatic melanoma can spread to a variety of sites. The identification of the tumor-associated antigens of the present invention may allow recurrence and metastatic disease to be detected and disease burden monitored (e.g., by imaging an antigen-targeted melanoma cell).

[0032] The tumor-associated antigens of the present invention may further be useful as targets in immunotherapy. Several immunotherapy approaches directed towards melanoma cancers are currently under development. The tumor-associated antigens of the present invention will provide additional and specific therapeutic targets for intervention.

[0033] The SEREX approach has been used herein to identify novel melanoma antigens in patients undergoing active immunotherapy.

[0034] The present invention is directed towards a DNA encoding a melanoma tumor-associated antigen selected from the group consisting of: (a) isolated DNA having a sequence shown in SEQ ID Nos. 1-12; (b) isolated DNA which is complementary to the isolated DNA of (a) above; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code.

[0035] The present invention is also directed towards a vector comprising the DNA disclosed herein and regulatory elements necessary for expression of the DNA in a cell, wherein the DNA encodes a melanoma tumor-associated antigen. Also included in the present invention is a vector in which the DNA is positioned in reverse orientation relative to the regulatory elements such that a melanoma tumor-associated antigen antisense mRNA is produced. Further provided are host cells transfected with the above-described vector expressing a melanoma tumor-associated antigen. Representative host cells are bacterial cells, mammalian cells, plant cells and insect cells, more preferably, the bacterial cell is E. coli.

[0036] The present invention is additionally directed towards an isolated and purified melanoma tumor-associated antigen coded for by DNA selected from the group consisting of: (a) isolated DNA selected from the group consisting of SEQ ID Nos. 1-12; (b) isolated DNA which is complementary to the isolated DNA of (a) above; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code.

[0037] The present invention is further directed towards a method for detecting mRNA coding for a melanoma tumor-associated antigen in a sample, comprising the steps of: (a) contacting a sample with an oligonucleotide probe having a nucleotide sequence shown in SEQ ID Nos. 1-12; and (b) detecting binding of the probe to the mRNA coding for a melanoma tumor-associated antigen in the sample.

[0038] The present invention is also directed towards a kit for detecting mRNA coding for a melanoma tumor-associated antigen, comprising: an oligonucleotide probe having a nucleotide sequence such as SEQ ID Nos. 1-12. The above-described kit may further comprise a label with which to label the probe; and means for detecting the label.

[0039] The present invention is still further directed towards a method of detecting a melanoma tumor-associated antigen in a sample, comprising the steps of: (a) contacting a sample with an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein; and (b) detecting binding of the antibody to the melanoma tumor-associated antigen in the sample.

[0040] The present invention is additionally directed towards a kit for detecting a melanoma tumor-associated antigen, comprising: an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein. The above-described kit may further comprise means to detect the antibody.

[0041] The present invention is further directed towards an antibody specific for a melanoma tumor-associated antigen or a fragment thereof encoded by the DNA disclosed herein.

[0042] The present invention is also directed towards a method of screening for compounds that inhibit the activity of a melanoma tumor-associated antigen, comprising the steps of: (a) contacting a sample with a compound, wherein the sample comprises a melanoma tumor-associated antigen encoded by the DNA disclosed herein; and (b) assaying for activity of the melanoma tumor-associated antigen. Typically, a decrease in the melanoma tumor-associated antigen activity in the presence of the compound relative to the melanoma tumor-associated antigen activity in the absence of the compound is indicative of a compound that inhibits the activity of the melanoma tumor-associated antigen.

[0043] The antigens reported herein may play a role in signaling growth, activating the cell cycle, down-regulating inhibitors of growth and/or promoting metastatic spread. Alternatively, the antigens reported herein may normally be expressed in melanocytes and may or may not have a direct role in tumorigenesis.

[0044] The present invention is also directed towards a method of inhibiting the growth of a melanoma tumor in an individual, comprising the steps of: (a) treating an individual with a therapeutic compound, wherein the therapeutic compound comprises a thereapeutic moiety and a targeting moiety, wherein the targeting moiety recognizes a melanoma tumor-associated antigen encoded by the DNA disclosed herein; wherein the therapeutic compound inhibits the growth of the melanoma tumor in the individual. Preferred targeting moieties are an antibody or fragment thereof, or a ligand, while preferred therapeutic moieties are a therapeutic gene or protein, a toxin, a radiolabel or a virus.

[0045] The present invention is also directed toward a cancer vaccine composition, comprising a vector capable of expressing a DNA molecule having a sequence shown in SEQ ID Nos. 1-12, and an appropriate adjuvant.

[0046] The present invention is further directed toward a method of vaccinating an individual against cancer, comprising the steps of: (a) administering to the individual a vector capable of expressing a DNA molecule shown in SEQ ID Nos. 1-12, wherein expression elicits an immune response which is specific towards a melonoma-specific antigen, thereby inducing immune-mediated destruction of melanoma cells. Typically, the individual is at risk of getting cancer, suspected of having cancer or has cancer.

[0047] The present invention is also directed toward a method of inhibiting the growth of a melanoma tumor, comprising the steps of: (a) administering a cancer vaccine to an individual comprising a vector expressing a DNA such as SEQ ID Nos. 1-12, wherein administration of the vaccine induces an immune response, thereby inhibiting the growth of a melanoma tumor.

[0048] It will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0049] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, “Molecular Cloning: A Laboratory Manual (2nd Ed.)”, (1989); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

[0050] A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine,. guanine, thymine, or cytosine) in its either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. DNA structure is discussed herein according to the normal convention of showing only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0051] A “vector” is a replicon, such as plasmid, phage viral genome or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An “origin of replication” refers to those DNA sequences that participate in and/or regulate DNA synthesis. An “expression control sequence” or “regulatory elements necessary for expression” are DNA sequence(s) that control and regulate the transcription and translation of another DNA sequence. A coding sequence is “operably linked” and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[0052] In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

[0053] A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNAs from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A coding sequence may alternatively be transcribed in the opposite orientation (i.e., the nontranscribed strand is used as the template) to produce an antisense RNA molecule. An antisense RNA is complementary to an mRNA molecule produced from the transcribed strand.

[0054] A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence. A “cDNA” is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript. An “exon” is an expressed sequence transcribed from the gene locus, whereas an “intron” is a non-expressed, usually spliced-out, sequence.

[0055] Transcriptional and translational regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, provide for expression of a coding sequence in a host cell. A “cis-element” is a nucleotide sequence, also sometimes termed a “consensus sequence” or “motif”, that interacts with proteins that regulate expression of a specific gene locus. A “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates with the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0056] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as consensus sequences responsible for binding of RNA polymerase. Eukaryotic promoters often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters typically contain the −10 and −35 consensus sequences, as well as Shine-Dalgarno sequences for ribosome binding.

[0057] The term “oligonucleotide” is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally (as in a purified restriction digest) or produced synthetically, which is capable of acting as a point of initiation when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and a polymerizing agent such as a DNA polymerase, and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and the method used. For example, for diagnostic applications, depending upon the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0058] Primers are selected to be “substantially” complementary to a strand of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands under the appropriate conditions. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence to hybridize therewith and thereby initiate synthesis of the extension product.

[0059] As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes which cut double-stranded DNA at or near a specific nucleotide sequence.

[0060] “Recombinant DNA technology” refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include “gene splicing”, “molecular cloning”, “cloning” and “genetic engineering”. The product of these manipulations results in a “recombinant” or “recombinant molecule”.

[0061] A cell has been “transformed” or “transfected” with exogenous or heterologous DNA when such DNA has been introduced into the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an replicative episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived (by mitosis) from a single cell or ancestor. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed “transgenic”.

[0062] As used herein, the term “host” is meant to include not only prokaryotes, but also eukaryotes, such as yeast cells, plant cells and animal cells. A recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

[0063] As used herein, “fragment,” as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion/cleavage of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. The ability of a candidate fragment to exhibit an enzyme characteristic (e.g., binding to a specific antibody, or exhibiting enzymatic or catalytic activity) can be assessed by methods described herein. Purified fragments or antigenic fragments can be used to generate new regulatory enzymes using multiple functional fragments from different enzymes, as well as to generate antibodies, by employing standard protocols known to those skilled in the art.

[0064] Generally speaking, antibodies for use in these aspects of the present invention will preferably recognize antigens that are preferentially, or specifically, expressed by melanoma tumor cells. Such antibodies will also preferably exhibit properties of high affinity, such as exhibiting a Kd of <200 nM, and preferably, of <100 nM, and will not show significant reactivity with normal tissues, such as tissues from heart, kidney, brain, liver, bone marrow, colon, breast, prostate, thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or other life-sustaining organ or tissue in the human body. These tissues are important for the purposes of the present invention from the standpoint of low reactivity with the antibody. The term “reactivity,” as used herein, refers to an antibody or antibody fragment that, when applied to the particular tissue under conditions suitable for immunohistochemistry, will elicit staining only in positive cells and not negative cells. Particularly promising antibodies contemplated for use in the present invention are those having high reactivity specific to the melanoma tumor.

[0065] A standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue obtained from tissue in accordance with conventional Northern blot hybridization techniques known to those persons of ordinary skill in the art. Alternatively, a standard Southern blot assay may be used to confirm the presence and the copy number of a gene in accordance with conventional Southern blot hybridization techniques known to those of ordinary skill in the art. Both the Northern blot and Southern blot use a hybridization probe, e.g., radiolabelled cDNA, either containing the full-length, single stranded DNA or a fragment of the DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The oligonucleotide hybridization probe can be labelled by any of the many different methods known to those skilled in this art. Conditions for Northern and Southern hybridizations, i.e., stringency, can be determined for that particular system empirically and/or experimentally, and defining appropriate hybridization conditions is well within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

[0066] By “high stringency” is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42° C. in the presence of about 50% formamide; a first wash at about 65° C. with about 2×SSC containing 1% SDS; followed by a second wash at about 65° C. with about 0.1×SSC.

[0067] By “substantially pure DNA” is meant DNA that is not part of a milieu in which the DNA naturally occurs, by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in SEQ ID Nos. 1-12 or which encodes an alternative splice variant of SEQ ID Nos. 1-12.

[0068] The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re.

[0069] Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and routinely utilized. The preferred enzymes are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase, etc. U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

[0070] By “degeneracy of the genetic code” is meant that some amino acids are specified by more than one codon. Accordingly, a single protein sequence could be coded for by multiple DNA sequences due to the degeneracy of the genetic code.

[0071] As used herein, “cancer vaccine” refers to a therapeutic vaccine consisting of a vector encoding an antigenic protein or a peptide fragment thereof. Immunization of an individual with such a vaccine is meant to induce an immune response to the protein or peptide, and is directed towards a method of inhibiting growth or promoting destruction of the melanoma tumor in the individual.

[0072] As used herein, “immunotherapy”, as used in the context of cancer therapy, refers to a therapeutic method achieved by manipulation of an individual's immune system to inhibit growth or promote destruction of a tumor.

[0073] As used herein, “antigen” generally refers to a protein or polypeptide which can, in certain formulations or settings, be recognized by the immune system and elicit an immune response. Although carbohydrate moieties may also act as antigens, as used herein, antigens are defined as proteins or polypeptides which may or may not be modified post-translationally.

[0074] As used herein, “tumor-associated antigen” refers to an antigen which is associated with tumor cells. Such proteins need not be expressed exclusively in or on tumor cells. Generally, tumor-associated antigens are fetal proteins aberrantly expressed in tumor cells, mutated cellular proteins which are antigenic due to the mutation, viral proteins expressed in tumor cells, normal cellular proteins highly expressed in the tumor compared to normal tissue, or normal cellular proteins which are mislocalized.

[0075] As used herein, “melanoma antigen” refers to an antigen which is expressed in melanoma cells, however, expressed need not be limited to only melanoma cells.

[0076] As used herein, “polymerase chain reaction” or “PCR” refers to an enzymatic reaction using primers specific for a DNA or cDNA sequence which results in amplification of the specified sequence.

[0077] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:

EXAMPLE 1

Patient Population and Clinical Trial

[0078] Nine patients with advanced stage melanoma were enrolled in a Phase Ib clinical trial (NCI Protocol T97-0005) of intratumoral injection of a recombinant canarypox virus encoding the human interleukin-12 (IL-12) gene. The recombinant virus was provided by Pasteur Merieux Connaught through an agreement with the National Cancer Institute. Patients were injected with the recombinant virus encoding IL-12 on days 1, 4, 8, and 11 and serum was collected on days 0, 18 and 43.

EXAMPLE 2

cDNA Library Construction

[0079] A cDNA expression library derived from the melanoma cell lines MEL888 and MEL624 (kindly provided by S. Rosenberg, National Cancer Institute) was synthesized in the 1-ZAP Express vector (Stratagene). Briefly, total RNA was isolated from the cells using the RNA-Stat60 reagent (TelTestB), according to the manufacturer's directions. mRNA was isolated using a 5′→3′ mRNA isolation kit. Five to seven micrograms of mRNA was reverse transcribed using an oligo dT primer with an internal XhoI site. After second strand synthesis, EcoRI adapters were added by ligation. The cDNA was passed through a size exclusion column (Pharmacia) which eliminates cDNAs smaller than 400 bp in size and the cDNA fragments cloned into the λZapExpress vector, packaged according to the manufacturer's instructions, and used to infect E. coli cells. As a preliminary characterization of the library, inserts from twenty-five randomly selected recombinant plaques were PCR amplified using T3 and T7 primers to determine insert size ranges (0.5 to 3.0 kb). The library contained 6.8×106 primary recombinants.

EXAMPLE 3

Immunoscreening

[0080] Table 1 shows isolated clones screened for reactivity with all patients from the study. A cDNA expression library was generated from the melanoma cell lines MEL888 and MEL624, and serum from two individuals (patients one and two) was diluted to 1:250, mixed in equal volume and used to screen the library. Recombinant plaques were plated at a density of approximately 25,000 plaques per 150 mm plate, and a total of approximately 200,000 plaques were screened. After a four hour incubation of the plates at 37° C., protein expression was induced by incubation of the plates with nitrocellulose filters saturated with isopropyl β-D-thiogalactoside (IPTG) overnight. Filters were blocked with 1% BSA in Tris-buffered saline (TBS; 20 mM Tris (pH 7.5), 150 mM NaCl) and screened with patient sera. Primary sera is preabsorped with E. coli phage lysate (Stratagene) and diluted 1:250 for screening. After incubating filters with diluted sera, the filters were washed with TBST (TBS with 0.05% Tween 20 [Sigma]) and incubated with alkaline phosphate-conjugated goat anti-human IgG (H+L) antibodies (Jackson Labs) at a dilution of 1:5,000 for 1 hour at room temperature. After washing, an NBT/BCIP colorimetric assay was used to identify positive clones. Positive plaques were purified to clonality for further study.

[0081] Isolated clones (FIG. 1) were then screened for reactivity against sera from nine patients with advanced stage melanoma who were intratumorally injected with a recombinant canarypox virus encoding the human interleukin-12 (IL-12) gene. Patients were injected with the recombinant virus encoding IL-12 on days 1, 4, 8, and 11 and serum was collected on days 0, 18 and 43. Confirmed immunoreactive plaques were evaluated for reactivity with serum from ten normal individuals. Only those plaques which were not reactive with ten normal sera were processed further.

1TABLE 1 Patients Day 43Patient 1123456789Day 1803.1+−−−−−−−−+−3.14+−−−−−−−−+−5.16+−−−−−−−−+−5.17++−−−−−−−+−5.23+−−−−−−−−+−5.28+−−−−−−−−+−5.31+−−−−−−−−+−3.3T−−−−+−−−+n/an/a

[0082] Isolated clones were screened for reactivity with all patients from the study.

EXAMPLE 4

Isotype Analysis

[0083] Upon plaque purification, the isotype of the reactive antibodies were determined using human isotype specific antibodies (Southern Biotech), according to the recommended procedure. Isotype analysis demonstrated the presence of predominantly IgG antibodies, consistent with a mature, Tn-dependent immune response.

EXAMPLE 5

Isolation of Plasmid DNA and DNA Sequence Analysis

[0084] Plasmid DNA containing cDNA inserts of interest were isolated from purified plaques by in vivo excision using a helper phage system (ExAssist, Stratagene). For single clone excision, approximately 105 phage particles were used to infect XL-1-Blue MRF cells in the presence of the helper phage and the cells were incubated for 3 hours at 37° C. To isolate the excised phagemid which are packaged as filamentous phage particles, the culture was heated to 70° C. for 20 min, spun at 1000×g for 15 min and the supernatant collected. These phage were used to infect XLOR cells (Stratagene) and the cells were plated on selective media. These cells do not permit growth of the helper phage and only allow propagation of the phagemid. Single colonies were grown in liquid culture and DNA isolated by standard miniprep procedures.

[0085] Partial DNA sequence of each insert was determined using an automated DNA sequencer and vector specific primers. Partial DNA sequences were used in BLAST searches through the National Center for Biotechnology Information database to identify sequences which matched previously described genes or expressed sequence tags (ESTs) (Table 2).

2TABLE 2Melanoma tumor-associated antigen homologyClone & IdentifierHomology with:Reference 3.1 (SEQ ID No. 1 & 2)KIAA066313(=3.8 = 3.16)(GenBank Accession No.AB014563)3.14 (SEQ ID No. 3 & 4)Drosophila disc large protein14(GenBank Accession No.U13896)3.3T (SEQ ID No. 5 & 6)Ubiquilin; DA4115(GenBank Accession No.AF176069, HRIHFB2157)5.17 (SEQ ID No. 7 & 8)KIAA055516(GenBank Accession No.AB011127)5.23 (SEQ ID No. 9)EST: qu76c08.x1;17NCI-CGAP-ES02(GenBank Accession No.AI354862)5.28 (SEQ ID No. 10)Various ESTs;Similarity with TR: G5812235.31 (SEQ ID No. 11 & 12)RING3*18(GenBank Accession No.X96670)*RING3 has been described by Matthew Scanlan at the Ludwig Institute for Cancer Research as a potential breast tumor antigen based on SEREX.

[0086] The positive clones, derived from plaques that bound sera from patients intratumorally injected with a recombinant virus expressing IL-12, represent putative antigens specific to melanoma tumors.

EXAMPLE 6

Uses

[0087] Novel tumor-associated antigens may be useful for detection, diagnosis, and staging of melanoma. Detection and diagnosis of melanoma is currently based on visual identification of melanoma lesions, while staging is based on depth of the lesion at the time of diagnosis. While these visual guidelines have proven quite useful, the use of additional marker proteins and the molecular characterization of melanoma lesions may prove more accurate in defining the clinical course of the disease.

[0088] Novel tumor-associated antigens may also be useful for disease monitoring. Metastatic melanoma can spread to a variety of sites. The identification of novel tumor antigens may allow recurrence and metastatic disease to be detected and disease burden monitored. Antigenic proteins expressed on the cell surface may provide targets for detection and imaging of metastatic disease.

[0089] Tumor-associated antigens may additionally be useful as novel targets for immunotherapy. Several immunotherapeutical approaches to melanoma are currently under development, and tumor-associated antigens may provide additional therapeutic targets for intervention.

[0090] The following references were cited herein:

[0091] 1. van der Bruggen, P et al. Science 254:1643-1647, 1991.

[0092] 2. Kawakami, Y et al. Proc. Natl. Acad. Sci. USA 91:3515-3519, 1994

[0093] 3. Toso, J F et al. Cancer Res. 56:16-20, 1996.

[0094] 4. Robbins, P F et al. Cancer Res. 54:3124-3126, 1994.

[0095] 5. Schlichtholz, B et al. Cancer Res. 52: 6380-6384, 1992.

[0096] 6. Lubin, R et al. Cancer Res. 53:5872-5876, 1993.

[0097] 7. Stauss, H J J Natl Cancer Inst. 87:820-821, 1995.

[0098] 8. Disis, M L et al. Cancer Res. 54:16-20, 1994.

[0099] 9. Chinni S et al. Clin Cancer Res. 3:1557-1564, 1997.

[0100] 10. Sahin, et al. Proc. Natl Acad Sci. USA 92:11810-11813, 1995.

[0101] 11. Tureci, O et al. Cancer Res 56:4766-4772, 1996.

[0102] 12. Old, L O & Chen, Y T. J. Exp. Med. 187:1163-1167, 1998.

[0103] 13. Ishikawa, K et al. DNA Res. 5:169-176, 1998.

[0104] 14. Lu, R A et al. Proc. Natl. Acad. Sci. USA 91:9818-22, 1994.

[0105] 15. Hanaoka, E et al. J. Human Genet. 45:188-191, 2000.

[0106] 16. Ishikawa, K et al. DNA Res. 5:31-19, 1998.

[0107] 17. Natl. Cancer Institute; Cancer Genome Anatomy Project.

[0108] 18. Thorpe, K L et al. Immunogenetics 44:391-6, 1996.

[0109] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0110] One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

	Number	Date	Country
Parent	09691538	Oct 2000	US
Child	10754820	Jan 2004	US

Melanoma antigens and methods of use

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