WO2010098682A1

WO2010098682A1 - Cell marker for melanocyte lineage and cancer cells and uses thereof

Info

Publication number: WO2010098682A1
Application number: PCT/NZ2010/000034
Authority: WO
Inventors: Peter Roderick Dunbar; Oliver Horlacher; Vaughan John Feisst
Original assignee: Auckland Uniservices Limited
Priority date: 2009-02-27
Filing date: 2010-02-26
Publication date: 2010-09-02

Abstract

The present invention relates to novel proteins as cell markers and in particular to cell markers of melanocyte lineage and cancer cells. The invention also relates to novel targets for treatment and/or diagnosis of disorders of cells of the melanocyte lineage, and to compositions and methods for treatment and/or diagnosis of such disorders.

Description

CELL MARKER FOR MELANOCYTE LINEAGE AND CANCER CELLS AND USES THEREOF

TECHNICAL FIELD

The invention has been developed primarily for the diagnosis and/or treatment of melanocyte disorders such as cancer, and more specifically melanoma. However, it will be appreciated that the invention is not limited to this particular field of use. BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Melanoma is the most serious and aggressive form of skin cancer. However, if recognized and treated early, it is nearly 100 percent curable. If not captured early, the cancer can advance and spread to other parts of the body, where it becomes hard to treat and can be fatal. While it is not the most common skin cancer, it causes the most deaths.

Melanoma is a malignant tumor that originates in melanocytes. Melanocytes manufacture the pigment melanin within melanosomes, which are transferred to keratinocytes to pigment the skin. All melanocyte specific genes identified to date are in some way involved in the melanosome production pathway. Well known melanocyte- specifϊc genes include melanA, tyrosinase, trp-1, trp-2 and gp 100. MelanA, gplOO and tyrosinase are genes that are currently used as targets for immunotherapy.

Treatment at the early stages of melanoma is promising. Most people with thin, localized melanomas are cured by appropriate surgery. Early detection still remains the best weapon in fighting skin cancer and the cure rate continues to rise.

More treatments are becoming available for more advanced disease. Research has produced a greater understanding of melanoma, leading to the development of new drugs. A number of drugs that are active in fighting cancer cells are being used to treat melanoma, and are either administered alone or in combinations. Currently, dacarbazine (DTIC), given by injection, is the only chemotherapy approved by the FDA. DTIC may be combined with carmustin (BCNU) and tamoxifen, or with cisplatin and vinblastine. Another drug, temozolomide, can be given orally. Unfortunately, to date, the response of melanomas to chemotherapy has been limited, but a great deal of research into new drugs and new approaches is being carried out.

There is therefore a need not only for novel treatments but also for novel targets that may be used to develop alternative approaches to treatment of cancers such as melanoma, and other skin conditions which involve melanocytes, such as skin, mole, freckle and pigmentation disorders, and further modulation of melanocyte growth, localisation, survival and function. Further, there is a need for treatments and diagnostic reagents that target melanocytes and conditions associated with abnormal melanocyte growth, localisation and/or function.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

The present invention is concerned with novel genes and their expression products that appear to be expressed primarily in melanocytes. The present invention is also concerned with methods of modulating the expression or function of the genes and/or their products. Further, the present invention is concerned with novel recombinant proteins expressed in prokaryotic or eukaryotic cells and antibodies reactive with said proteins or parts thereof. In a first aspect, there is provided an isolated nucleic acid molecule that encodes a melanocyte-specific protein, comprising a nucleic acid sequence set out in SEQ. ID. No. 10 or functional fragments thereof.

Preferably, the nucleic acid molecule encodes a protein having SEQ. ID. No.l 1, or a functional fragment thereof. In a second aspect the present invention provides a recombinant protein selected from SEQ ID NO. 3 or SEQ ID NO. 11, or parts thereof. The recombinant proteins may be expressed in prokaryotic or eukaryotic cells. Hence, expression vectors and recombinant cells used for expression of the novel proteins of the present invention are also within the scope of the present invention. In a third aspect there is provided, an isolated nucleic acid molecule that encodes a melanocyte-specific . protein, wherein the nucleic acid molecule has at least 75% sequence identity with SEQ. ID. No. 10. Preferably the nucleic acid molecule has from 80% to 95% sequence identity with SEQ. ID. No. 10. Nucleic acid molecules that have higher sequence identities with SEQ. ID. NO. 10, for example 80%, 85%, 90% or 95% are even more preferred.

The nucleic acid sequence of the present invention may vary from that provided in SEQ. ID. No.10 as a result of codon degeneracy (i.e. alternative codons for the same amino acid residue) or preferential codon usage if the sequence is to be expressed in . different expression systems (e.g. prokaryotic, eukaryotic systems etc.).

In a fourth aspect, there is provided a nucleic acid molecule that is complementary to a nucleic acid sequence of SEQ. ID. No. 10, or fragments thereof.

In a fifth aspect, there is provided a recombinant protein comprising a polypeptide sequence encoded by a nucleic acid molecule selected from SEQ ID NO 2 or SEQ ID NO 10.

In a sixth aspect, there is provided a cell marker comprising a peptide or polypeptide sequence encoded by an isolated nucleic acid molecule of aspect one or aspect two, or part thereof. In a seventh aspect, there is provided a cell marker comprising a peptide or polypeptide sequence comprising SEQ ID No. 11. Preferably, the cell marker is a skin cell marker such as, for example, a melanocyte cell marker. Preferably the skin cell is a melanocyte. Even more preferred is that the cell marker is a cell marker for skin-related neoplastic conditions such as skin cancer. More preferably the neoplastic condition is melanoma. However, the condition may also be a disorder of pigmentation resulting from, for example, abnormal localisation, growth, or function of melanocytes.

In a eighth aspect, there is provided an antibody that binds to the peptide or polypeptide sequence encoded by a nucleic acid molecule of aspect one or aspect two, or part thereof, or to a protein comprising SEQ ID No. 11, or part thereof, or to a recombinant protein comprising SEQ ID NO 3 or part thereof

Preferably, the antibody is a monoclonal antibody or a functional fragment thereof.

Alternatively, the antibody is a polyclonal antibody or a functional fragment thereof. It will be understood that the term "antibody", as used in the context of the present invention, includes functional fragments of antibodies, derived by enzymatic means or recombinant expression.

In an ninth aspect, there is provided a pharmaceutical composition comprising an antibody according to the seventh aspect, optionally in combination with a pharmaceutically acceptable carrier. In a tenth aspect, there is provided a vector comprising a nucleic acid sequence set forth in any one of aspects one to three, or a part thereof, operatively linked to a promoter.

Preferably, the vector can be selected from an expression vector commonly used for in vitro protein expression in prokaryotic or eukaryotic cells, or vectors for introducing nucleic acid sequences into mammalian cells or vectors for mammalian gene therapy or vaccination.

In a eleventh aspect, there is provided a prokaryotic or eukaryotic cell comprising the expression vector according to the ninth aspect. Preferably, the prokaryotic cell is a bacterial cell or a fungal cell.

Preferably, the eukaryotic cell is selected from a mammalian cell, an avian cell, an amphibian cell, a reptilian cell or an insect cell.

In another aspect, there is provided the use of a nucleic acid molecule of aspect one or aspect two, a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or part thereof, or a protein comprising said peptide or polypeptide, as a marker of a melanocyte cell lineage.

In yet another aspect, there is provided use of a protein having SEQ ID No.11, or part thereof, as a marker of a melanocyte cell lineage.

In another aspect, there is provided the use of an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or part thereof, or a protein comprising said peptide or polypeptide, for identifying melanocyte cell lineage.

The identification of cell lineage can be conducted by a number of well known techniques, including for example FACS, ELISA, RIA, Western blot, cytometric bead array, SISCARPA , and the like.

In another aspect, there is provided a use of a nucleic acid molecule comprising a nucleic acid molecule of aspect one or aspect two, or part thereof, or a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or part thereof, or a protein comprising said peptide or polypeptide, or a protein having SEQ ID No.11, or part thereof, or an antibody that specifically binds to said peptide or polypeptide and/or said protein, in the diagnosis and/or detection of a skin cancer.

In another aspect, there is provided the use of a nucleic acid molecule of aspect one or aspect two or part thereof, or an inhibitory or interfering RNA or DNA sense or antisense sequence, or an antibody that specifically binds to a polypeptide encoded by a nucleic acid of aspect one or aspect two, or part thereof, or a protein comprising said polypeptide in the preparation or manufacture of a medicament for the treatment of a skin cancer or disorders associated with melanoma and/or disorders of pigmentation, and/or abnormal function, localisation or growth of melanocytes.

In another aspect, there is provided a method of diagnosing and/or treating a skin cancer comprising the step of administering to a subject in need of said treatment an effective amount of an inhibitory or interfering RNA or DNA sense or antisense sequence capable of blocking or inhibiting expression of a nucleic acid molecule of aspect one or aspect two, or part thereof, or an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or part thereof, or a protein comprising said peptide or polypeptide, or a protein having SEQ. ID No.l l.

Preferably, the skin cancer is melanoma. In another aspect, there is provided a method of screening for the presence of a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, in a sample, comprising: a) contacting a biological sample with a nucleic acid having a sequence that is complementary to nucleic acid sequence of SEQ. ID. No. 10 or part thereof; and b) determining whether the nucleic acid binds to a nucleic acid molecule in the sample.

In another aspect, there is provided a method of screening for the presence of a peptide or polypeptide or part thereof, encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, in a sample, comprising: a) contacting a biological sample with an antibody that specifically binds to the peptide or polypeptide, or part thereof; and b) determining whether the antibody binds to the peptide or polypeptide, or part thereof, in the sample.

In another aspect, there is provided a method of screening for the presence of a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, and at least one other nucleic acid molecule which codes for a cell marker commonly used to identify cells of melanocyte lineage in a sample, comprising: a) contacting a biological sample with i) a nucleic acid molecule having a sequence that is complementary to nucleic acid sequence of SEQ. ID. No. 10 or part thereof, and ii) a nucleic acid molecule having a sequence that is complementary to the at least one other nucleic acid molecule or part thereof; and b) determining whether the nucleic acid molecules bind to the nucleic acid molecules in the sample.

In another aspect, there is provided a method of screening for the presence of a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, and at least one other polypeptide which is a cell marker commonly used to identify cells of melanocyte lineage in a sample, comprising: a) contacting a biological sample with i) an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, and ii) an antibody that specifically binds to the at least one other polypeptide; and b) determining whether the antibodies bind to their corresponding polypeptides in the sample.

The cell marker commonly used to identify cells of melanocyte lineage may be selected from for example, melanA, gplOO, tyrosinase, trp-1 and trp-2. It will be understood however, that any other markers that identify melanocyte cell origin can also be used in conjunction with the present invention.

The use of the cell marker encoded by SEQ. ID. No. 10 in a panel of other markers is advantageous since not all cancerous cells express traditional markers such as gplOO or melanA, and the like, which are typically used in methods for screening for melanoma. Inclusion of an additional marker such as that of the present invention significantly enhances the diagnostic power of the diagnostic methods used.

In another aspect, there is provided a method of diagnosing and/or detecting skin cancer or skin condition comprising: a) contacting a biological sample with a nucleic acid having a complementary sequence to nucleic acid sequence of SEQ. ID. No. 10 or part thereof; and b) detecting localisation of the nucleic acid as an indicator of the presence (and/or type) of skin cancer or skin condition.

In yet another aspect, there is provided a method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof; and b) detecting localisation of the antibody as an indicator of the presence (and/or type) of skin cancer or skin condition. In another aspect, there is provided a method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a nucleic acid having a complementary sequence to nucleic acid sequence of SEQ. ID. No. 10 or part thereof, and one or more other nucleic acids complementary to nucleic acid molecules selected from the group comprising, but not limited to melanA, gplOO, tyrosinase, trp-1 and trp-2, or parts thereof; and b) determining localisation of the nucleic acids as an indicator of the presence (and/or type) of skin cancer or skin condition.

In yet another aspect, there is provided a method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, and at least one other antibody that binds to a polypeptide encoded by melanA, gplOO, tyrosinase, trp-1 and trp-2, or parts thereof, and b) determining localisation of the antibodies as an indicator of the presence (and/or type) of skin cancer or skin condition.

In another aspect, there is provided a method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a panel of nucleic acid molecules or parts thereof, comprising melanA, gplOO, tyrosinase, trp-1 and trp-2 and a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof; and b) determining localisation of the nucleic acid molecules as an indicator of the presence (and/or type) of skin cancer or skin condition.

In yet another aspect, there is provided a method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a panel of antibodies that bind specifically to melanA, gplOO, tyrosinase, trp-1 and trp-2 and a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof; and b) determining localisation of the antibodies as indicator of the presence (and/or type) of skin cancer or skin condition.

Preferably, the biological sample used in the method of diagnosing skin cancer is selected from the group consisting of a tissue biopsy, a biological fluid and a surgical specimen. It will be understood, however, that the diagnosis and/or detection of skin cancer with nucleic acid molecules and antibodies of the present invention may also be conducted in vivo or ex vivo. Preferably, the nucleic acid molecules or antibodies used in the method of screening or diagnosis are tagged with a visible or detectable marker to permit visual inspection or measurement/detection. The tag is preferably selected from a group consisting of a fluorescent dye, an isotope, an enzyme (e.g. peroxidase or alkaline phosphatase), a chemical moiety (e.g. biotin) that binds another labelled ligand (e.g. avidin) with high affinity. For example, tagged sequences SEQ ID NOs 12 and 13 described herein are useful for such a purpose.

Preferably, the skin cancer to be screened, diagnosed or treated is melanoma. Additionally the melanoma can be in the form of a cutaneous melanoma, a melanoma of the nail bed, a melanoma of the eye or a mucosal melanoma. Alternatively, the skin cancer to be screened, diagnosed or treated may be of metastatic origin.

The skin condition may be a disorder of pigmentation, and/or abnormal function, localisation or growth of melanocytes.

In another aspect, there is provided a method of modulating expression of genes associated with skin cancer or skin condition comprising contacting a cell with a short interfering nucleic acid molecule directed against a nucleic acid sequence of SEQ. ID. No. 10 or part thereof.

Preferably, the short interfering nucleic acid molecule is selected from a group consisting of short interfering RNA (siRNA), double stranded RNA, micro RNA and short hairpin DNA.

In another aspect, there is provided a method of modulating expression of genes associated with skin cancer or skin condition comprising contacting the cells with an antisense nucleic acid molecule directed against a nucleic acid sequence of SEQ. ID. No. 10 or part thereof. Preferably, the skin cancer is melanoma.

In another aspect there is provided the use of a nucleic acid molecule comprising a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, or an inhibitory or interfering RNA or DNA sense or antisense sequence, or an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of aspect one or aspect two, or a part or variant thereof, or a protein comprising said peptide or polypeptide, in the treatment of disorders associated with melanoma and/or disorders of pigmentation, and/or abnormal function, localisation or growth of melanocytes. Preferably the disorders of pigmentation, whether or not in association with melanoma or other skin cancer, include hyperpigmentation disorders such as melasma, cafe au lait macules, drug-induced hyper-pigmentation and phototoxic reactions or hypo- pigmentation disorders such as vitiligo. Further, blockade of immune recognition of a peptide encoded by SEQ. ID. No. 10 or part thereof, or a protein comprising said peptide, can also be used to treat skin disorders caused by immune attack on melanocytes, for example autoimmune vitiligo (Ogg GS, Dunbar PR, Romero P, Chen JL, Cerundolo V (1998) High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. Journal of Experimental Medicine 188:1203-8). More preferably, the disorders of pigmentation is selected from hypo-pigmentation (and/or hyper-pigmentation.

In another aspect, there is provided a method of modulating expression of genes associated with changes in skin pigmentation or cosmetic lightening or darkening of skin tone, comprising contacting a cell with an inhibitory or interfering RNA or DNA, sense or antisense, sequence directed against a nucleic acid molecule of aspect one or aspect two.

The nucleic acid molecules and antibodies of present invention may be formulated into pharmaceutical preparations by any number of processes well known in the art and may include injectable, topical, oral, slow release and other suitable dosage forms and preparations.

The present invention is suitable for the diagnosis and/or treatment of skin cancers, in particular melanoma.

In the context of the present invention the term "LOC 146481" is used for convenience to describe the genomic locus LOC146481. Novel nucleic acid sequences of the present invention in addition to being allocated SEQ ID NOs. may also be referred to as ORFs, for example ORFl or ORF2. The expression products of the ORF sequences, when used generically for any protein product, are commonly referred to herein as SHML (Specific for Human Melanocyte Lineage) proteins or sequences, SHML-ORF sequences, or for designating expression products of specific ORFs the terms SHML-ORFl or SHML-ORF2 protein sequences, or alternatively pSHML-ORFl or pSHML-ORF2 are used. It will be understood that the acronyms are merely convenient designations and should not be construed to limit the nucleic acids or the proteins to human origin only. The present invention encompasses both mammalian and non-mammalian species SHML proteins. The terms SHML mRNA, SHML gene, SHML transcript and SHML protein may be used interchangeably with specific SEQ ID NOs. and are intended to describe the nucleic acid, mRNA and protein sequences of the present invention. The terms "nucleotide", "nucleic acid", "polynucleotide", "nucleic acid sequence" and the like, as used in the context of the present invention, are intended to encompass a gene, DNA, ssDNA, cDNA, synthetic DNA, RNA, genomic sequences and derivatives thereof.

The terms "peptide" and "polypeptide" as used in the context of the present invention are intended to encompass peptides and fragments of polypeptides or proteins which comprise an amino acid sequence encoded by SEQ. ID. No. 10 or parts thereof. Such sequences may be detectable by an antibody raised against a peptide encoded by SEQ. ID. No. 10 or part thereof, such as for example SEQ ID No. 11 or part thereof.

The term "treatment" as used herein covers any treatment of a disease and/or condition in an animal, particularly a human, and includes:

(i) preventing a disease and/or condition from occurring in a subject which may be predisposed to the disease and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease and/or condition, i.e., arresting its development; or (iii) relieving the disease and/or condition, i.e., causing regression of the disease and/or condition.

Reference to an "antibody" or "antibodies" includes reference to all the various forms of antibodies, including but not limited to: full antibodies (e.g. having an intact Fc region), including, for example, monoclonal antibodies; antigen-binding antibody fragments, including, for example, Fv, Fab, Fab' and F(ab')₂ fragments; humanized antibodies; human antibodies (e.g., produced in transgenic animals or through phage display); single domain antibodies and immunoglobulin-derived polypeptides produced through genetic engineering techniques. Unless otherwise specified, the terms "antibody" or "antibodies" and as used herein encompasses both full antibodies and antigen-binding fragments thereof. It is anticipated that the treatments described herein can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical administration, and inhalation. For oral administration, the treatments will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the treatments will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0. 9%) saline.

For methods of preparing various types of formulations and choice of carriers, excipients and additives, see standard pharmacy texts and manuals, such as for example, Remington: The Science and Practice of Pharmacy, 19^th Ed., Mack Publishing Co., 1995, the Theory and Practice of Industrial Pharmacy, Lachman L., et al. Lea & Febiger, Philadelphia 3^rd Edition, Bentley's Textbook of Pharmaceutics Ed. EA Rawlings Ballilliere Tindall, London 8^th Edition, incorporated in its entirety herein by reference.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: The PCR products derived from human melanocyte cDNA encoding SHML Sequence 1. All sequences are shown 5'- 3'. A) PCR product 1 - SEQ ID No. 9 - shows 486bp of the sequence of the PCR product generated from human melanocyte cDNA with forward primer 12F (5'- TGAGCCAGGAGCTCCACCAT-3') and reverse primer 1145R (5'- GCACCC AGCTCAAGCCTCAT-3 ; B) PCR product 2 - SEQ ID No. 10 - shows 546bp of the sequence of the PCR product generated from human melanocyte cDNA with forward primer 527F (5'- TGGTCAGGATGGAGGGGAAG-3') and reverse primer 1145R (5'- GCACCCAGCTC AAGCCTC AT-3 '); C) Alignment of two PCR products in A) and B), which overlap by lObp, to make

SEQ. ID. No. 1. The protein-encoding open reading frame (ORF) is underlined - this is SEQ. ID. No. 2.

D) SEQ. ID. No.2 - ORF encoding protein

E) SEQ. ID. No. 3 is the protein sequence encoded by the underlined portion of SEQ. ID. No. 2.

Figure 2: Excerpt of the GenBank entry for the human FRG2 gene sequence Figure 3: Alignment of SHML protein sequence (SEQ. ID. No. 3) with FRG2 protein sequence (by Needleman-Wunsch global alignment, Geneious software, www.geneious.com). Potential nuclear localisation signals are boxed, and a potential protein kinase C phosphoryation site is underlined.

Figure 4: Expression of SHML, tyrosinase & melanA relative to the house-keeping gene HPRT in cDNA from human tissues, cell lines, and primary cell cultures. Expression of these three genes was quantified with quantitative RT-PCR relative to the housekeeping gene HPRT, which was expressed in all samples. SHML expression was detected in human skin, which contain melanocytes, and also in cultured human melanocytes, but not cultured human dermal fiboblasts. The melanocyte-associated gene tyrosinase and melanA were also expressed in human skin and melanocytes, but not dermal fibroblasts. A screen of these genes' expression in cDNA derived from 18 normal adult human tissues (Clontech), revealed 15 of these tissues did not express any of these genes. However, normal .brain, spinal cord, and testes expressed all three genes. We conclude that expression of SHML in normal tissues is restricted to melanocytes, and to normal adult tissues that contain cells of melanocytic lineage, namely skin, neural tissue, and testes. Similar experiments carried out in human lymph node samples infiltrated with metastatic melanoma revealed one ("MH") of four samples that expressed tyrosinae and melanA also expressed SHML, confirming that SHML is expressed in some human melanoma samples freshly isolated from patients. None of the three genes were expressed in a single sample of human lymph node that was not infiltrated with melanoma cells. When similar experiments were conducted on six human melanoma cell lines, SHML was expressed in four of the six, including one (MZ2) which did not express either tyrosinase or melanA. Data shown are the average of triplicate measurements, with error bars representing the standard error of the mean. Figure 5: siRNA knockdown in melanoma cell lines DAGI The melanoma cell line DAGI that expresses SHML and tyrosinase was treated with siRNAs targeting SHML and tyrosinase mRNA. Expression of SHML and tyrosinase were then measured by quantitative RT-PCR, and cells treated with siRNAs were compared with untreated cells. 1OnM siRNA targeting SHML mRNA achieved approximately 70% knockdown of SHML expression, similar to the level of knockdown seen with previously- validated siRNAs targeting tyrosinase mRNA. 5OnM siRNA had similar effects, but InM siRNAs were less effective. These data indicate the siRNAs used were capable of significantly knocking down SHML mRNA expression at both the 1OnM and 5OnM doses. Data shown are the average of triplicate measurements, with error bars representing the standard error of the mean. Figure 6: siRNAs targeting SHML gene expression

The two siRNA sequences used to knock down SHML expression in Figure 6 are shown, along with the positions of the sequences they target in the SHML sequence (bold and underlined in SEQ. ID. No. 1 as shown) Figure 7: Alternate transcripts coding for SHML. A)-D) Four predicted alternate transcripts are shown that are capable of encoding SHML protein (SEQ. ID. No. 3), each full length from transcription initiation site (TCATTCTT, nucleotides 1-8 in each transcript) to poly-A tail start. The coding sequence is underlined in each sequence. Three polymorphic sites are highlighted in each sequence by use of a bold capitalised A or G. The first site is an A/C polymorphism at residue 376 (SNP_ID=3751731), although all transcripts observed to date have an A at this position. The second site is a G/A polymorphism at residue 416 (SNP_ID=^:238004), and although this is encoded as A in the human genome reference sequence, all transcripts observed to date have a G at this position, as shown. The third site is a G/T polymorphism (SNP_ID=11541272), and all transcripts to date have a G at this site, as in the human genome reference sequence (the precise position in each transcript changes due to alternate splicing upstream). E) Alignment of the 4 alternate transcripts to show the region of alternative splicing. The first 360bp of each sequence, and the sequences 3' to the regions shown here, have been omitted, since they are identical between the transcripts. Figure 8: Features of the SHML promoter region. 4192bp of genomic DNA (- strand) is shown immediately 5' to, and including, the SHML transcription start site at the beginning of the transcripts in Figure 8 (SEQ. ID. Nos. 4-7). Important features are shown according to the key below, using combinations of bold, upper case, and underlined text.

115..122 cAMP Response Element (TTATGTAA) (BOLD, UPPER CASE) 477..484 cAMP Response Element (TGAGGTTA) (BOLD, UPPER CASE)

707..711 Putative CCAAT box (underlined only)

1231. .1235 Putative CCAAT box (underlined only)

1487. .1491 Putative CCAAT box (underlined only)

1957. .1961 Putative CCAAT box (underlined only)

3136. .3140 Putative CCAAT box (underlined only)

338..344 Putative TATA box (bold only) 1418..1424 Putative TATA box (bold only) 1729..1735 Putative TATA box (bold only) 3443..3449 Putative TATA box (bold only) 3474..3480 Putative TATA box (bold only) 3779..3785 Putative TATA box (bold only) 3904..3910 Putative TATA box (bold only) 3943..3949 Putative TATA box (bold only) 541..546 Putative Mitf binding site (CACATG) (UPPER CASE,

UNDERLINED)

843. .848 Putative Mitf binding site (CATGTG) (UPPER CASE,

UNDERLINED)

1657 ..1662 Putative Mitf binding site (CATGTG) (UPPER CASE,

UNDERLINED)

2067 ..2072 Putative Mitf binding site (CACATG) (UPPER CASE,

UNDERLINED)

3352 ..3357 Putative Mitf binding site (CATGTG) (UPPER CASE,

UNDERLINED)

3578 ..3583 Putative Mitf binding site (CACATG) (UPPER CASE,

UNDERLINED)

3209..3218 CpG island, a potential transcription initiation site and SPl Transcription Factor binding site (UPPER

CASE ONLY) 4185..4192 Transcription Start Site (TCATTCTT) (bold, underlined) 4018 Site of a Single-Nucleotide Polymorphism (C/T, SNP

#11647911) (BOLD, UPPER CASE, UNDERLINED)

Figure 9. ORFl and ORF2 amplification using PrimeStar HS DNA polymerase. Both ORFl (left) and ORF2 (right) were amplified using a gradient of annealing temperatures and all reactions showed the expected size of PCR product. l=50.6°C; 2= 55.8°C; 3= 58.5°C; 4= 64.2°C and 5= 69.9°C, M= molecular size markers. Figure 10. BsrGI digestion of ORFl and ORF2 BP clones. Correct-sized bands are present in all lanes. 1-3= ORFl, 4-6= ORF2 and M= molecular size markers.

Figure 11. BsrGI digestion of ORFl and ORF2 LR clones. Correct-sized bands are present in all lanes for both ORFl (A) and ORF2 (B). 1-3= pDEST17, 4-6= pDEST15, 7-9= pDEST566 and M= molecular size markers. Figure 12. Expression and solubility experiments of ORFl constructs. His- (A), GST- (B) and MBP-tagged (C) ORFl were expressed in E. coli BL21pRP cells. Lane 1= insoluble fraction; 2= soluble fraction and M= molecular weight markers. Soluble expression is shown by asterisks.

Figure 13. Expression and solubility experiments of ORF2 constructs. His- (A), GST- (B) and MBP-tagged (C) ORF2 were expressed in E. coli BL21pRP cells. Lane 1= insoluble fraction; 2= soluble fraction and M= molecular weight markers. Soluble expression is shown by asterisks.

Figure 14. Purification of MBP-SHML-ORF 1 protein. Lane 1= soluble fraction before loading on MBPTrap column; 2= soluble fraction after loading on MBPTrap column; 3= bound fraction eluted using maltose. M= molecular weight markers.

Figure 15. rTEV cleavage of purified MBP-SHML-ORFl protein. Lane 1= MBP- SHML-ORFl sample before cut and 2= cleaved MBP-SHML-ORFl after overnight incubation with rTEV at 4°C. M= molecular weight markers.

Figure 16. Presence of DNA in the purified MBP-SHML-ORFl fractions as shown by DNA agarose gel electrophoresis. (A) Different fractions showing DNA fragments and

(B) DNase I digestion of the samples from fraction 1 in A confirms presence of DNA.

Lane 1= control DNA without DNase I; 2= control DNA digested with DNasel; 3= MBP-ORFl control without DNase I; 4-7= MBP-SHML-ORF 1 fractions digested with DNase I over 10, 20, 30 and 40 min, respectively.

Figures 17 and 18. Cellular distribution of SHML-ORF2 in HEK293 cells.

Figure 19: SHML reactive T cells can be observed using the IFNγ assay. DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based on a surprising observation that an uncharacterised gene in the human genome (herein referred to as "SHML" gene) encodes a protein that is present in cells of melanocyte lineage, but not other cells. Whereas one open reading frame (ORF) that encodes a hypothetical protein was described in an earlier co-pending, co-owned PCT application, (herein designated ORFl) it has now been surprisingly found that the uncharacterised gene mentioned above includes a second ORF (designated herein ORF-2) that also encodes a protein.

A protein product of an ORF will be designated generically herein as SHML (or Shml) protein or sequence. More specifically, and by way of an example, the designation SHML-ORFl or pSHML-ORFl will be used to describe a protein expression product of ORFl nucleotide sequence and, similarly, SHML-ORF2 or pSHML-ORF2 will be used to describe a protein expression product of ORF2. nucleotide sequence.

ORF2, herein described also as SEQ ID NO 10, encodes a protein different from pSHML-ORFl (SEQ ID NO 3), having a different amino acid sequence and a different cellular localisation, but which is also specific for the melanocyte cell lineage. This second protein is designated herein SHML-ORF2 or pSHML-ORF2. Discovery of the proteins encoded by the SHML gene

Based on bioinformatic analysis of Expressed Sequence Tags and mRNA sequences in public databases, it was predicted that mRNA encoded from a genomic locus LOC 146481 might be expressed exclusively by cells of melanocytic lineage. The mRNA obtained from human melanocytes was sequenced (SEQ. ID. No. 1, see also

Figure 1) and showed that it was expressed by human melanocytes.

SEQ. ID. No.1 ■ ATATGTCTCAATGGTGGATGTAGGGAAACTTCACAGGCTTTGATGGGACTGAGG

TGGTGTCTGTTGTGTGAAGACAGGAGCTGAATGCTCAGGATCTAGGCTGCTTTT

CTTGTGGTCAGTTGGGTTACCTGTTTGGGACCAAGTCCATGTTTACTAAGGAGG CTGAATAAATTCTACAGAGGAAAGTGGAGCTCCCAGGATGACCGAGGAAGGGTA AGAAAAGGGAACCAGAGTCGAGCTCACATGAGGGAAACTCCAAGAAAAGGAAAA TCGGTTCCAAGGACAGCTGCCAAGACAGAGCTGGTAGAATTTTGCCTTTTTCCC CCAAAGGGAAAATAAGCAGTTGACTTTTATAGAGGGTTTGAGCAGCAAGGTGAC TGAAAAAATTTTTGAGATCCTGGCACTCAGAGAATATTTGGGGACTCAGAAGGT GTTAAATTTCACTTCATTTAAGTACTAATATGGTCAGGATGGAGGGGAAGATTC AAATGTTCACCTGAAGGTGCATGGGAGGTTTTGGAAATGAGGGTCGGTGGGAAC TGTGCTGGCGAGTATCTCACTCACATGCTTCTTTTGCGGGGATCTGTCCAGGAG AGGAGCACAGCTTGATGTTGAAAAAGAAACTAAAGTCCTCCACTTTGCACAGCA ATAAACTCAAGGAGACCTGTGATGCCCACCACAGAGGGCCTTTCATGACTCACA CTGGGTGCACCAAGCAGCACAGGTCTCAGACCCTAGGAGTTCAATGGCTGTCAT TTCAGAAAAGCTTGGTGACTTTACAAGCTGTGTCAAGGCCATTTATCAAGACCT ATACTGGGTATCGGTGCAGCAGGTCCATTCTCCACTGACCTGGGAGCAGCTCAC TCAGCTTGCCCGGTTCTGGCGGCCTCTATGTGCTCAGGTGCAGACCTTCTATTT TATAGCCACCCAGGTAGGTTATGTCTTCCCTGCTAAGAACTGGCTTGTTTCAGC CACATTGCCTGGCCCTGGGGATCCAGCCCTGGATAGAGAAGCCCATCCCT The protein-encoding open reading frame (ORF) from SEQ. ID. No.1 mRNA is the nucleotide sequence shown below (SEQ. ID. No.2): SEQ. ID. No.2 (ORFl) ATGGGAGGTTTTGGAAATGAGGGTCGGTGGGAACTGTGCTGGCGAGTATCTCAC TCACATGCTTCTTTTGCGGGGATCTGTCCAGGAGAGGAGCACAGCTTGATGTTG AAAAAGAAACTAAAGTCCTCCACTTTGCACAGCAATAAACTCAAGGAGACCTGT GATGCCCACCACAGAGGGCCTTTCATGACTCACACTGGGTGCACCAAGCAGCAC AGGTCTCAGACCCTAGGAGTTCAATGGCTGTCATTTCAGAAAAGCTTGGTGACT TTACAAGCTGTGTCAAGGCCATTTATCAAGACCTATACTGGGTATCGGTGCAGC AGGTCCATTCTCCACTGA

This ORF is one of several possible ORFs in SEQ. ID. No. 1, but based on the potential protein's high homology to another human protein, as described below, SEQ. ID. No. 2 was identified as the protein coding sequence. The protein encoded by SEQ. ID. No. 2 has the following amino acid sequence (SEQ. ID. No. 3): SEQ. ID. No. 3 (pSHML-ORFl) MGGFGNEGRWELCWRVSHSHASFAGICPGEEHSLMLKKKLKSSTLHSN KLKETCDAHHRGPFMTHTGCTKQHRSQTLGVQWLSFQKSLVTLQAVSR PFIKTYTGYRCSRSILH

Sequence comparison of SEO. ID. No. 3 with FRG2 and other sequences from LOC146481

SEQ. ID. No. 3 has significant homology to another known protein, called FRG2 (FSHD region gene 2), the sequence of which is given in Figure 2, along with other information on the gene encoding FRG2 protein. FRG2 is a gene implicated in facioscapulohumeral muscular dystrophy (T. Rijkers et al, FRG2, an FSHD candidate gene, is transcriptionally up-regulated in differentiating primary myoblast cultures of FSHD patients. J. MedGenet. 2004 ;41; 826-836).

Alignment of the SEQ. ID. No. 3 with FRG2 protein sequence is shown in Figure

3. In this alignment, 50 of 113 amino acids in SEQ. ID. No. 3 protein are identical to those in FRG2, for 44% overall homology. The most remarkable features of this alignment are that amino acids 24 and 90 of SEQ. ID. No. 3 protein align with FRG2 amino acids 110-177 with only a single gap. Across this 67 amino acid sequence of

SEQ. ID. No. 3 protein, there is identity with FRG2 amino acids at 39 sites (58% homology), with complete homology over a 9 amino acid sequence at sites 52-60 in

SEQ. ID. No. 3 and sites 139-147 of FRG2. These proteins are therefore highly likely to be related.

The protein sequence of FRG2 is annotated as having two sequences that confer nuclear localisation, at amino acids 96-99 (sequence RKRK) and 157-160 (sequence KRHR). The SEQ. ID. No. 3 has a homologous protein sequence for only the second sequence at amino acids 70-73 (sequence KQHR). SEQ. ID. No. 3 does have a potential nuclear localisation signal (sequence LKKKLKS) that is not present in FRG2 (using PredictNLS online http://cubic.bioc.columbia.edu/cgi/var/nair/resonline.pl'). Based on these findings it is therefore likely that the SEQ. ID. No. 3 protein will localise to the nucleus of the cell after translation, as does FRG2.

The sequence SNKL at amino acids 47-50 in the SEQ. ID. No. 3 protein is a potential protein kinase C (PKC) phosphorylation site, implicating PKC as a regulator of the protein's function. The SEQ. ID. No. 3 protein is thus potentially capable of post- translational modification by phosphorylation at this site. As a precedent, there are other nuclear factors involved in melanocyte differentiation such as MITF (microophthalmia-associated transcription factor) and CREB (cAMP-responsive element binding protein) that are regulated by phosphorylation. Sequence comparison of SEQ. ID. No. 1 and SEQ. ID. No. 3 with other sequences from LOC146481

The genomic locus LOC146481 had been predicted by NCBI to encode mRNAs (XR_017714.1, XR 017753.1, and XR_041323.1), different from that identified herein as SEQ. ID. No. 1. These predicted mRNAs (XR_017714.1, XR_017753.1, and XR 041323.1), had been predicted to encode a protein different from SEQ. ID. No. 3 described herein (predicted hypothetical protein XP J)01723191.1). One mRNA sequence previously filed in GenBank (National Institutes of Health Mammalian Gene Collection project (NIH MGC) also differs from SEQ. ID. No. 1, and has several reading frames. No protein sequence had ever been predicted from this single mRNA sequence, primarily because: a lack of any data regarding its potential function; doubts about its veracity given it was only partially homologous to the predicted mRNAs XR Ol 7714.1, XR 017753.1, and XR_041323.1; and uncertainty about which of several potential open-reading frames within BC023607 encoded protein.

In analysing SEQ. ID. No. 1 of the present invention, it was found that one of several open-reading frames encoded a protein with strong homology to another human protein, FRG2.

Based on analytical and comparative studies described above, particularly the recognition that the sequence was restricted to cells of the melanocytic lineage and, on the basis of strong homology to another protein, (that only one of the open-reading frames present in SEQ. ID. No. 1 was likely to encode protein) the present invention provides a novel nucleotide sequence, SEQ. ID. No.l, the mRNA sequence included therein, SEQ. ID. No. 2, and the corresponding protein sequence, SEQ. ID. No. 3. The restriction of the sequences to cells of the melanocytic lineage, indicates a function related to the specialised properties of such cells, as described below. Expression of SHML transcripts in human tissues and cells

Gene expression analysis by quantitative RT-PCR conducted on cDNA from normal human tissues showed that expression of "SHML" gene was restricted to tissues containing cells of melanocytic lineage, namely skin and neural tissue (Fig 4). Similar analysis on normal cell lines showed that SHML is expressed by cultured melanocytes, but by neither keratinocytes nor fibroblasts (Fig 4). These results identify the SHML as expressed specifically in cells of melanocytic lineage. A majority of melanoma cell lines were also found to express SHML (Fig 4). One well-characterised melanoma cell line MZ2 expresses SEQ ID No. 1 but not the nucleic acid encoding melanA, gplOO or tyrosinase, which are commonly used to identify cells as being of melanocyte lineage. This single experiment confirms the utility of SEQ ID No.l, the mRNA sequence, SEQ ID No. 2, and the corresponding protein sequence, SEQ ID No. 3, as markers in cancer diagnosis (Fig 4). The gene and its corresponding protein will therefore have value as a diagnostic marker for confirmation of diagnosis of cancers such as melanoma, a marker of a melanocyte cell lineage, as a therapeutic drug target, for example as a target for immunotherapy of melanoma or a drug target for melanoma or disorders of pigmentation. Its corresponding antibody will also have value as a diagnostic tool or as a treatment/therapy for melanoma in its various forms and for disorders associated with melanoma and/or disorders of pigmentation. Functional roles of SHML protein

To investigate the function of the SHML protein, the SHML mRNA was knocked down using siRNA, as shown in Fig 5. A melanoma cell line (DAGI) was exposed to siRNAs targeting SHML mRNA (both siRNAs shown in Fig 6), as well as control siRNAs targeting the melanocyte marker tyrosinase. Subsequent quantitative RT-PCR analysis confirmed knockdown of SHML mRNA by at least 60% even at 1OnM concentrations of siRNAs (Fig 5). This was equivalent to the knock down in tyrosinase mRNA concentrations in the same experiment, carried out with siRNAs that had been previously validated. DAGI cells with and without SHML mRNA knockdown were subjected to whole genome microarray analysis, to detect gene modulated by SHML.

Without wishing to be bound by theory or any particular mechanism of action, analysis of SHML siRNA knockdown using human whole genome microarrays, revealed that DKKl transcription is regulated by SHML. This finding was subsequently confirmed using quantitative RT-PCR (data not shown).

DKKl is a secreted protein involved in regulation of the Wnt signalling pathway. Wnt proteins interact with a cell surface receptor complex consisting of Frizzled and LRP5/6, resulting in stabilisation of cytoplasmic β-catenin. β-catenin accumulates in the cytoplasm and moves to the nucleus. Once in the nucleus β-catenin interacts with the LEF/TCF family of transcription factors to activate transcription of TCF/LEF target genes. DKKl is able to inhibit Wnt signalling at the cell surface by binding to LRP5/6 component of the Wnt receptor complex. The complex is endocytosed upon DKKl binding, thereby blocking the ability of Frizzled to send the Wnt signal upon binding of Wnt. (1)

In melanocytes Wnt signalling is crucial in development, differentiation, and pigmentation. Wnt signalling is required to maintain melanocyte precursor cells in a stem cell like state during development. Wnt signalling has been shown to control pigmentation by activation of Mitf-M transcription via the TCF/LEF1 binding site in the Mitf-M promoter. Wnt signalling in melanocytes results in the β-catenin/TCF/LEF complex increasing transcription of Mitf-M. Downstream of Mitf-M the pigmentation pathway is activated and melanosomes are produced. (2-4) SHML protein therefore appears to be a negative regulator of DKKl expression.

Reduction in SHML mRNA levels resulted in increased DKKl mRNA expression levels, as demonstrated herein. SHML protein is a putative transcription factor and may regulate DKKl mRNA levels via interaction with the DKKl promoter. Thus, SHML protein is a melanocyte-specific regulator of the Wnt signalling pathway, which has been shown to be required for melanocyte development, differentiation and pigmentation. The tissue specificity of SHML protein expression enables systemic drug treatment with a tissue specific effect on Wnt signalling. Successful inhibition of SHML gene expression with siRNA

As shown in Fig 56, siRNAs were used to successfully knock down expression of SHML mRNA in melanoma cells by at least 60%, with subsequent effects on the transcription of other genes, notably DKKl. The location of the sequences successfully targeted by siRNAs are shown in Fig 6, along with the siRNA sequences. These sequences are located at residues 552-570, and 637-655 of the SEQ. ID. No. 1, and were targeted by siRNAs 293438 and 293439 (provided by Applied Biosystems/Ambion, Austin, TX, USA). The siRNAs have similar efficacy when used individually as when combined. These experiments demonstrated that expression of SHML mRNA could be successfully knocked down using an RNA inhibition approach, with subsequent effects on the expression of other genes. Alternate transcripts from SHML Analysis of genomic data available from public databases (including human genome sequences and expressed sequences, including Expressed Sequence Tags and other mRNAs) allows the full length sequences of possible alternate transcripts from SHML to be deduced. Most of these alternate transcripts encode the protein SEQ. ID. No. 3, and these transcripts are shown in Fig 7, including the full length mRNA that incorporates SEQ. ID. No. 1 (labelled "transcript 1", SEQ. ID. No. 4). As shown in Fig 8, the transcripts differ in their inclusion of a 12bp sequence at the end of the first exon, and the inclusion of an additional lOObp exon before the coding sequence. Experimental evidence shows transcripts including the 1 OObp additional sequence shown in transcripts 2 and 4 (SEQ. ID. No. 5 and SEQ. ID. No. 7) because RT-PCR of cDNA from cells of melanocytic origin using primers that span this region of cDNA occasionally produce two bands that differ in size by lOObp (data not shown).

There are also Single Nucleotide Polymorphisms (SNPs) in the genomic DNA that encodes these transcripts. Therefore allelic variants of all 4 alternate transcripts are possible, though none of these affect the encoded protein, because they are located in the untranslated regions of each mRNA. The positions of the 3 possible polymorphic residues in each transcript are noted in Fig 7 with bolded, capitalised letters. AU 4 alternate transcripts have a possible AJC polymorphism at residue 376 (SNP_ID=3751731). All 4 alternate transcripts also have a possible G/A polymorphism at residue 416 (SNP_ID=238004). A G/T polymorphism (SNPJD=I 1541272) exists at the other site in bold capitals in each transcript (the precise location changes due to the alternate splicing upstream). It is therefore possible that any of the 4 alternate transcripts may carry any one of the 3 nucleotide changes designated at these SNP sites, so up to 32 different mRNA sequences are possible that may all encode the protein sequence SEQ. ID. No. 3 in different individuals. Only the 4 most likely alternative transcripts are shown in Fig 7, although all variants due to SNPs, as described above are included in the scope of the present invention. Promoter analysis Analysis of over 4kb upstream of the transcription initiation site (the beginning of the transcripts shown in Fig 7) revealed clusters of numerous sequences that are likely to be involved in regulating gene expression, especially by binding transcription factors and regulatory proteins. As shown in Fig 8, these regulatory elements include eight TATA boxes, five CCAAT boxes, and one SPl binding sites, likely to be involved in binding general transcription factors and related proteins. There are 6 putative binding sites for MITF (microophthalmia-associated transcription factor) indicating this crucial transcription factor for melanocyte differentiation is involved in regulating "SHML" expression. There are also two cAMP-response elements (CREs) implicating cAMP signalling in the regulation of SHML transcription.

A SNP in this promoter region that might conceivably affect transcription or transcript stability, SNP # 11647911, is a C/T polymorphism transcript start site, as shown at position 4018. This is not predicted to be a site involved in transcription factor binding or transcription initiation, but it is still possible that it will affect the transcription of the gene in different individuals, potentially affecting melanocyte structure or function.

Novel protein encoded by ORF2

A second open reading frame was also identified in the SHML gene described above. This nucleotide sequence is designated herein ORF2.

ORF2 sequence (SEQ ID NO. 10 ) is as shown below:

ATGGCTGTCATTTCAGAAAAGCTTGGTGACTTTACAAGCTGTGTCAAG GCCATTTATCAAGACCTATACTGGGTATCGGTGCAGCAGGTCCATTCTCCAC TGACCTGGGAGCAGCTCACTCAGCTTGCCCGGTTCTGGCGGCCTCTATGTGC TCAGGTGCAGACCTTCTATTTTATAGCCACCCAGGTAGGTTATGTCTTCCCTG CTAAGAACTGGCTTGTTTCAGCCACATTGCCTGGCCCTGGGGATCCAGCCCT GGATAGAGAAGCCCATCCCTTGCCTGGGCAGGAGATAACTGAGCCTGTCAG TGGGTCAGATGAGGCTTGA pSHML-ORF2 amino acid sequence (SEQ ID NO. 11) is as shown below:

MAVISEKLGDFTSCVKAIYQDLYWVSVQQVHSPLTWEQLTQLARFWRP LCAQVQTFYFIATQVGYVFPAKNWLVSATLPGPGDPALDREAHPLPGQEITEPV SGSDEA pSHML-ORF2 has different properties and cellular localisation when compared to pSHML-ORFl. Both pSHML-ORFl and pSHML-ORF2 have been expressed in prokaryotic and eukaryotic cells, and the isolated proteins characterised further as described herein. Further, the isolated recombinant proteins have been used to prepare antibodies that may be useful for analytical, diagnostic and/or therapeutic applications.

The novel proteins described herein, or parts thereof, represent new and useful targets for diagnosis and treatment of skin and other conditions that are caused or mediated by abnormal melanocyte function, growth, proliferation and/or localisation. The active agents described herein, including anti-sense and sense nucleic acid approaches, and antibodies, for manipulating expression of the proteins of the present invention can be applied to both diagnostic and therapeutic uses in the treatment of melanocyte-mediated disorders, the most important of which is skin cancer such as melanoma. However, cosmetic applications are also contemplated such as alteration of skin and hair pigmentation, masking or removal of skin blemishes and the- like. Manipulation of regulatory processes within melanocytes has been clearly demonstrated herein using siRNAs that modulate expression of the proteins of the present invention. The structure of the gene and its regulatory regions, and the protein sequences provided herein enable other known therapeutic and diagnostic approaches to be adopted for manipulating the expression and/or function of the proteins of the invention and hence melanocytes and melanocyte-mediated disorders.

A preferred embodiment of the invention will now be described in more detail by reference to the following non-limiting examples.

EXAMPLES EXAMPLE 1 : General Methods General laboratory procedures not specifically described herein can be found in the general molecular biology or immunology texts including, for example, Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory: Co Id Spring Harbor, NY, and Harlow et al. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory: Co Id Spring Harbor, NY. (i) Melanocyte Cell Culture

Seed Cascade melanocytes (catalogue # C-024-5C, Cascade Biologies Inc. 1341 SW Custer Drive, Portland OR 97219, USA, ph 503 292 9521) at a density of 5xlO³ cells/cm² in a 25 cm² flask with Cascade medium 254 (cat # M254-500) with PMA free melanocyte supplement (cat # S-015-5C).

Thaw vial of 0.5 million cells in a 37⁰C water bath. Open vial in a laminar flow hood and resuspend cell pellet using a ImL pipette. Remove lOμL and determine cell number using a haemocytometer. Seed cells at density specified above. Incubate at ^•

37°C/5%CO2 for 24 hours then replace the media and remove dead cells. Change media every 2-3 days and passage 1:2 whenever flask becomes confluent. Passage using 2x trypsin. fin DAGI Cell Culture Thaw vial of 0.5 million cells in a 37°C water bath. Open vial in a laminar flow hood and resuspend cell pellet using a ImL pipette. Transfer cells to a 15mL tube then add

1OmL RPMI1640/10% FBS/lxPSG(penicillin/streptomycin/glutamate).

Mix cells then centrifuge for 5min at 18Og. Remove supernatant, add 5mL

RPMI1640/10% FBS/lxPSG, resuspend cells and transfer to a 25 cm² flask. Incubate at 37°C/5%CO2 for 24 hours then replace the media and remove dead cells.

Change media every 2-3 days and passage 1:2 whenever flask becomes confluent.

Passage using 2x trypsin.

(iii) Extraction of RNA and cDNA synthesis for microarray and qPCR analysis

Materials: TrizoL Invitrogen cat # 15596-026

Mini RNA extraction kit, Qiagen cat # 74104

1^st strand cDNA synthesis kit, Invitrogen cat #18080-093

RNA synthesis:

Remove media and add 750μL trizol, using a pipette ensure that cell pellet is fully resuspended. Shear genomic DNA by passing sample through a 21 gauge needle 5 times. Rock the tube at room temperature (RT) for 5 min. Add 150μL chloroform shake vigorously for 15s then incubate at RT for 3min. Spin at 1200Og for 5min at 4°C.

Transfer upper aqueous phase to a fresh non-stick RNAse free tube. Add an equal volume of 70% ethanol to the tube and add mix to spin column in 2mL collection tube, centrifuge at 800Og for 15s, discard flow through. Using the Qiagen RNA extraction kit complete the remaining steps following manufacturers instructions. Add 700μL buffer

RWl to the column, centrifuge at 800Og for 15s, discard flow through. Add lOμL of

DNAse I stock to 70μL buffer RDD, add to column and incubate at RT for 15min. Add 700μL buffer RWl to the column, centrifuge at 800Og for 15 s, discard flow through and collection tube.

Transfer column to a clean 2mL collection tube and add 500μL buffer RPE to column, centrifuge at 800Og for 15s, discard flow through. Add 500μL buffer RPE to column, centrifuge at 800Og for 2min, discard flow through & collection tube. Transfer spin column to 1.5mL non-stick RNAse free Eppendorf tube, add minimum 15μL water to centre of the column, centrifuge at 8000g for lmin.

Transfer spin column to 1.5mL non-stick RNAse free Eppendorf tube, add minimum

45 μL water to centre of the column, centrifuge at 8000g for lmin. Assess quality and quantity of RNA using the nano-drop of both elutes.

Continue only with enough sample for qPCR only, the rest is stored at -80⁰C until ready for microarray analysis.

1^st strand cDNA synthesis:

Use cDNA synthesis kit from Invitrogen and follow manufacturers instructions. Make same mix for NoRT control, half volume and omit the SSRTIII:

200ng RNA (to 4μL for a lOμL rxn)

0.5μL 50μM oligodT primer

0.5 μL 50ng/μL random hexamers

0.5μL 1OmM dNTPs Incubated @ 65°C for 5 min then kept on ice for at least 1 min.

Combine following components together before adding to RNAmix: lμL 10x RT buffer

2μL 25mM MgCl₂ lμL O.lM DTT 0.5μL RNAseOUT

0.5 μL SSRTIII

Add to RNA mix

Incubated @ 25⁰C for 10 min, then incubated @ 50⁰C for 50 min.

Terminate reaction @ 85⁰C for 5 min, then chilled on ice. Spin to collect reaction, add 0.5μL RNAseH & incubated @ 37°C for 20 min.

Stored @ -20⁰C until used for qRTPCR.

(iv) qPCR for measuring "Target 1" ( SHML) siRNA knockdown in DAGI:

Source of nucleic acid: cDNA of siRNA knockdown in DAGI Materials:

Applied Biosystems FAST Taqman PCR mastermix, cat # 4352042

Hypothetical Protein Hs01390151_ml Taqman primer/probe mix, Applied Biosystems cat # 4331182 Methods:

Add 2μL with IOng of sample cDNA all required wells

Add 2μL with corresponding amount to the actual sample for NoRT sample to each required well.

5μL of 2x PCR mastermix 0.5 μL 2Ox primer/pro be mix

2.5 μL water

Primer/probe sets

HPRTl

SHML

PCR cycling:

20s @ 95°C

40 cycles of — ls @ 95°C

20s @ 60⁰C

(v) siRNA knockdown of SHML expression in the melanoma cell line DAGI for microarray analysis:

Cell Sources: DAGI Materials:

Ambion siRNA # 293438, 293439, sequences as shown in Figure 6.

Lipofectamine 2000 ("LKOOO"). Invitrogen (USA) cat #11668-027

BlockiT Fluorescent oligo, Invitrogen cat #2013

Methods: Seed 5x10⁵ cells/well in a 6 well plate with 2mL of RPMI 1640 (Invitrogen) with 10% foetal bovine serum.

Make up LF2000/siRNA mixes -

Add 5μL LF2000 per lOOμL. Incubate at room temperature for 5min

Add siRNA to lOOμL OM. Mix siRNA & LF2000 mixes & incubate at room temperature for 20min. Add siRNA mixes to wells

Change media after 24 hours.

Harvested and analyse after 48 hours.

All microarray/qPCR samples were harvested into non-stick RNAse free 1.5mL tubes.

Analyse by microarray, qPCR and flow cytometry for BlockiT transfection

(vi) Microarray Information:

Chips used - Affymetrix Human Genome U133 Plus 2.0 Array

Genechip Users protocol - httpV/www.affymetrix.com/support/technical/manual/expression manual.affx Used as protocol for Genechip use from RNA to producing expression data EXAMPLE 2: Determination of tissue expression specificity of the nucleic acid encoding SHML in humans using quantitative real time PCR.

Using Clontech Human Tissue libraries (Clontech Laboratories Inc. 1290 Terra Bella Ave, Mountain View, CA 94043, USA, ph 800.662.2566, catalogue # 636643) SHML mRNA levels were measured by quantitative real time PCR (qPCR) using the Taqman specific assay ID # Hs01390151_ml. Since SHML was suspected to be a melanocyte specific gene, expression of two known melanocyte specific genes, tyrosinase and melanA, were also measured using Applied Biosystems Taqman assays, ID # HsOOl 65976_ml & Hs00924233_ml respectively. All qPCR reactions were 45 cycles, triplicates with IOng of each tissue cDNA. Referring to Figure 5, using the Clontech tissue library, SHML mRNA and the two melanocyte specific genes were all detected in brain, spinal cord & the testes only.

SHML mRNA was also tested in normal human melanocytes (Cascade Biologies), samples of normal human skin and melanoma tumor-infiltrated lymph nodes (TILNs) (obtained from consenting patients), and a number of melanoma cell lines, obtained from collaborating research groups.

Referring to Figure 3, SHML mRNA was shown to be expressed in skin and at a higher level in normal human melanocytes. SHML mRNA was also expressed in most melanoma cell lines including MZ2 where no other markers of melanocytic origin were expressed.

EXAMPLE 3: Sequencing of the nucleic acid encoding SHML in normal human melanocytes

PCR fragments of the nucleic acid sequence encoding SHML were generated from cDNA synthesized using RNA extracted from normal human melanocytes (Cascade Biologies catalogue # C-024-5C) using the primers 5 'TGGTCAGGATGGAGGGGAAG & 3'-ATGAGGCTTGAGCTGGGTGC.

The resulting PCR product was gel purified using the Qiagen Qiaex II Gel

Extraction kit (Qiagen catalogue # 20021). The PCR product was sequenced using the

PCR primers, at 5μM, with approximately 50ng of PCR product per reaction, on a 3130XL capillary sequencer from Applied Biosystems using Bigdye version 3.1 terminator chemistry.

The siRNAs specific for SHML (Ambion siRNA #293438 & # 293439) can be used to knockdown SHML mRNA levels as in the melanoma cell line DAGI. RNA from the siRNA knockdown can be used for microarray experiments. RNA is extracted using the Qiagen Rneasy Mini kit (Qiagen catalogue #74104). Microarray analysis of the RNA generated from these knockdowns can be done using the Affymetrix U 133 plus 2.0 human gene expression quantitation arrays. (Affymetrix "Expression Analysis Technical Manual" Affymetrix, 3420 Central Expressway, Santa Clara, CA 95051, USA. TeL 408-731-5000. Fax. 408-731-5380.) Results:

SHML mRNA was found to be expressed in many melanoma cell lines and tumour samples, but was absent from nearly all normal human tissues. Commercial cDNA libraries were sourced from volunteers by the respective companies and information on these donors and tissue processing is available on the company websites; normal human skin and melanoma tumor-infiltrated lymph nodes were sourced from consenting volunteers undergoing plastic surgery and lymph node resection respectively, under appropriate ethics committee oversight, and part of each sample was snap frozen in liquid nitrogen as soon as practicable and stored at -80°C until RNA extraction. However, it was discovered that SHML mRNA was also switched on in skin and some neural samples.

Subsequently, SHML mRNA was found to be expressed in melanocytes, the normal cells that melanoma develops from, and which are found in skin and some neural tissue. Indeed, the expression pattern of SHML mRNA in human cell lines and tissues mirrors other genes that are only switched on in melanocytes. This finding made it highly likely that the gene is specific to cells of the melanocyte lineage, and may even play a role in some of the unique functions of these cells, such as pigmentation. EXAMPLE 4: Expression of SHML protein The nucleic acid sequence encoding SHML is amplified from a cDNA template using PCR and cloned into expression vectors for protein expression in E. coli, yeast and mammalian cells. The confirmed plasmid is then lransfected into the appropriate cell line/cells and expression of the protein can be induced. (Invitrogen Life Technologies Instruction Manual for E.coli Expression System with Gateway Technology, from Invitrogen Life Technologies Ltd, 18-24 Botha Road, Penrose, Auckland 1006, New Zealand.) Recombinant SHML protein is purified from these cellular sources by the use of tags attached to the protein, that are encoded in the expression vector. For expression in mammalian cells the Invitrogen V5 tag system is appropriate for this purpose, since it allows affinity purification using anti-V5 antibodies. (Invitrogen Life Technologies Ltd, 18-24 Botha Road, Penrose, Auckland 1006, New Zealand.) Purified SHML protein is then used as an immunogen for the production of antibodies to SHML EXAMPLE 5: Antigen Synthesis and Conjugation

Production of antibodies is according to standard techniques know, for example those described in Harlow et al. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory:Cold Spring Harbor, NY. Other suitable techniques would be known to those skilled in the art. Immunization and Sera Collection:

Using rabbits as hosts, the purified Shml protein is injected subcutaneously at multiple sites. The initial immunization is given in Complete Freund's Adjuvant with all subsequent immunizations given in Incomplete Freund's Adjuvant. The animals are bled at intervals to ascertain the titer of the antiserum. Antisera are used unpurified. Immunization/Bleed Protocol: A detailed immunization and bleeding schedule is provided below:

EXAMPLE 6: SHML Protein Experiments

DNA Amplification and Cloning

ORFl and ORF2 were cloned using the Gateway^® cloning system, which uses a nested PCR method. Nested PCR involves two rounds of amplification in which the second round uses the product of the first round as template. Gene-specific primers are used in the first round PCR to amplify the gene of interest and generic primers are used in the second round amplification to incorporate the required recombination sites for subsequent cloning. The gene-specific primers are designed to have a 9 or 12 base pair overlap with the generic primers. Both generic primers have attB recombination sites

(bold) while generic forward primer includes TEV recognition site (underlined bold) for cleavage of the recombinant protein from tag (Table 1). Table 1. Primers used in SHML amplification

The open reading frames ORFl and 0RF2 were amplified from a cDNA clone using PrimeStar HS DNA polymerase (Takara Bio Inc., Japan). A temperature gradient for the annealing step (50-70⁰C) was applied during the first round of Gateway^® amplification using gene-specific primers. The second Gateway^® amplification was carried out at the constant annealing temperature of 45°C (10 cycles) and then 55⁰C (20 cycles) using the Gateway^® generic primers. All reactions with different annealing temperatures showed bands of correct size on an agarose gel (Figure 9).

The Gateway^® cloning system uses the site-specific recombination system of bacteriophage lambda which involves two sequential, BP and LR, reactions. The BP reaction creates entry clones by incorporating PCR products (flanked by atiQ recombination sites) into an entry vector (pDONR221). The subsequent LR reaction recombines the entry clones with a destination vector (pDEST15, pDEST17 or pDEST566) to produce the expression vector. The BP reaction was performed on both ORFs after successful amplification. 25 femtomoles of the PCR product and the donor vector were used for each BP reaction. The equation below was used to convert between femtomoles and nanograms of PCR products: ng = 660N/10⁶¹ final where N is the length of PCR products in bases and fmol is the amount of the required DΝA in femtomoles.

A typical recipe for the BP reaction is shown in Table 2. Reactions were incubated at ambient temperature for 5 hours. 0.5 μL proteinase K was added to the reaction and incubation continued at 37⁰C for 15 minutes. 1-2 μL of each BP reaction was then transformed into E. colt ToplO cells and positive clones were selected on LB agar plates containing 25 μg/μL kanamycin.

Table 2. BP reaction set up

A single colony was picked up from the selection plates and resuspended in 5 mL of sterile LB medium for overnight cultures. After overnight growth, the cells were centrifuged and plasmid was purified using the QIAprep Miniprep kit (Qiagen). Restriction digestion with BsrGI (Table 3) was then used to confirm insert incorporation (Figure 10). Digestion reactions were performed at 37 ⁰C for 1 hour.

Table 3. BsrGI digestion reaction

Positive clones were then sequenced using Ml 3 primers. 5 μL of plasmid DNA were sent for sequencing (150-200 ng/μL) to the DNA Sequencing Facility, School of Biological Sciences, Auckland University. Resulting sequences were compared with those of the appropriate genes by BLAST alignment using T-Coffee.

Following successful sequencing results, LR reactions were performed (Table 4), which transferred both ORFs to three destination vectors, pDEST15, pDEST17 and pDEST566, producing N-terminal GST-, His- and MBP-tag proteins, respectively.

Table 4. LR reaction set up

After completion of LR reactions at ambient temperature, 1-2 μL of each reaction was used to transform E. coli ToplO cells. Positive clones were selected on LB agar plates containing 100 μg/μL ampicillin. BsrGI digestion was again used for further characterisation of positive clones (Figure 11).

Protein Expression

The main E. coli expression strain used during this project is BL21(DE3)pRP. E. coli BL21(DE3) cells expressing high concentrations of DnaK, DnaJ, GrpE, CIpB and

GroESL chaperones were also used for co-expression with His-tagged ORFl and ORF2 constructs. These strains have a copy of T7 RNA polymerase integrated into their genome under control of the lacUV5 promoter. This promoter enables expression of RNA polymerase by inducers like lactose or IPTG. T7 RNA polymerase, in turn, can induce expression of genes cloned under the T7 promoter.

LB medium was used for IPTG induction of expression cultures, although auto induction media was used more often for expression. PA-0.5G medium (Table 5) was used for non-inducing cultures and ZYP-5052 medium (Table 6) for autoinduction.

Table 5. PA-0.5G medium

Table 6. ZYP-5052 medium

ZY, 2Ox NPS and 50^χ 5052 solutions for preparation of the above-mentioned media were prepared using the protocols shown in Table 7. Table 7. Buffers and solutions required for expression media

The 17 amino acid mix contains no Cys, Tyr or Met. AU other amino acids are present at 10 mg/mL each. After dissolving all 17 amino acids in water, they were filter-sterilized and kept at 4°C.

The 100Ox metal mix was prepared by mixing the listed metal salts in water (Table 8). The mixture was filtered through 0.2 μm membrane filters and kept at ambient temperature.

Table 8. 1000 x metal mix

Protein Purification

(i) Small-Scale Protein Expression and Solubility Testing

Three expression constructs for each ORF were transformed into E. coli BL21pRP cells and plated on LB agar medium containing lOO μg/mL ampicillin and 34 μg/mL chloramphenicol. Expression experiments were performed in autoinduction media supplemented with the appropriate antibiotics. A single colony was used to inoculate a seeder culture, grown overnight at 37 ⁰C in PA-0.5G non-inducing medium. The PA-

05. G culture was used at a dilution of 1:1000 to seed the autoinduction medium ZYP-

5052 for expression. Expression cultures were routinely grown overnight at 37 ⁰C or for 4 h at 37 °C followed by 20 h at 18 ⁰C for low temperature experiments.

Cell pellets from 30 mL of culture were resuspended in 1 mL of lysis buffer containing 20 mM buffer, 150 mM NaCl and 1 mM β-mercaptoethanol. The pH of the lysis buffer varied depending on the target to ensure at least one unit difference between the buffer pH and theoretical pi of the protein (calculated using the Prot Param tool). Resuspended cells were lysed using sonication. Following lysis and centrifugation (13000 rpm, lO min, 4 ⁰C) the soluble and insoluble fractions were used for SDS-PAGE analysis (Figures 12 and 13).

Small-scale expression experiments showed that GST- and MBP-tagged pSHML- ORFl were partially soluble. In addition, all three pSHML-ORF2 fusion proteins were partially soluble. In order to reach large-scale expression, a 19.5-litre fermentor (New Brunswick Scientific, USA) or 2-litre baffled flasks in a shaking incubator were used.

(ii) Large-Scale Purification

MBPTrap HP column (Amersham Bio sciences) was used for purification of MBP- fusion pSHML-ORFl. The column was washed in 5 CV of water, followed by 5 CV of lysis buffer. After loading the sample using peristaltic pump, it was washed in 5 CV of lysis buffer to remove all unbound and non-specifϊcally bound protein. The bound protein was eluted using lysis buffer containing 10 mM maltose (Figure 14).

All cloned proteins had a TEV protease cleavage site, allowing removal of the tag using rTEV protease prepared in the lab. After affinity purification, rTEV protease was added to cut the MBP tag from pSHML-ORFl (Figure 15). Further purification of the protein using Ni-NTA and gel filtration steps proved that the protein is aggregated. In addition, the protein was precipitating after cutting the tag off the protein. Analysing samples of the protein fractions on DNA agarose gel indicated presence of high concentrations of DNA (Figure 16) which may explain a reason for very high molecular weight in gel filtration and dynamic light scattering experiments.

In order to prepare MBP-pSHML-ORFl samples for injection into rabbits, three consecutive purification steps were carried out to minimize the presence of DNA. High salt concentration (1 M) helps to dissociate DNA from proteins and using positively charged resins (Ni-NTA and QFF columns) traps free and protein-bound DNA. A small fraction of MBP-ORFl protein was pure enough for antibody production.

The fermentor-grown E. coli cells expressing MBP-pSHML-ORFl were resuspended in 20 mM MES pH 6.0, 1 M NaCl, 10 mM EDTA and 1 mM β-ME. Cell lysis was performed using sonication for 40 min in the presence of 50 μg/mL lysozyme and protease inhibitor tablets (Roche). After centrifugation of the cell suspension at 15000 rpm for 45 min, the supernatant was loaded onto an MBPTrap HP column using a peristaltic pump. The column was washed using the above-mentioned buffer with lowered EDTA concentration to 1 mM to wash all unbound and non-specifically bound proteins. The bound MBP-pSHML-ORFl was eluted using the buffer containing 10 mM maltose. The eluted protein was loaded onto a Ni-NTA column and washed using the buffer without EDTA. The column was then run on a gradient of imidazole from 0-500 mM over 100 mL. The appropriate fractions were pooled and dialyzed against the buffer with 50 mM NaCl and passed through QFF anion exchange column. The flow through contained the MBP-pSHML-ORFl which was then dialyzed against PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄ and 1.47 mM KH₂PO₄, pH 7.4).

Mass Spectrum Analysis

Following SDS-PAGE analysis of the expressed proteins, the band corresponding to the target protein was excised and submitted to our in-house facility for mass spectra analyses. The samples were treated in the facility by dehydration in acetonitrile, followed by reduction in 10 mM DTT at 56°C for 1 hour. Samples were then alkylated in 55 mM iodoacetamide before again being dehydrated in acetonitrile. An overnight digestion with 12.5 mM sequencing grade trypsin (Promega) was performed at 37°C to release peptides. The digests were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

LC-MS/MS was performed on a QSTAR XL mass spectrometer (Applied Bio systems). Peptides were separated on a Zorbax Cl 8 0.3x150 mm column (Agilent) using a 10-

35% gradient of acetonitrile in 0.1% formic acid. A 300-1600 m/z scan was performed, followed by fragmentation of the two most abundant multiply-charged peptide ions from each scan. Peptide masses were submitted to the database search program Mascot

(Matrix Science, London, UK) for peptide mass mapping against the NCBI protein database.

The mass spectrometry identified 12 different peptides matching to the pSHML-ORFl sequence outlined in bold text:

MGGFGNEGRWELCWRVSHSHASFAGICPGEEHSLMLKKKLKSSTLHSNKLK ETCDAHHRGPFMTHTGCTKQHRSQTLGVQWLSFQKSLVTLQAVSRPFIKTY TGYRCSRSILH

EXAMPLE 7: Mammalian Expression Plasmid Construction

ORFl and ORF2 were cloned into the Sigma-Aldritch pFLAG-CMV-2 vector (Catalogue number E7398) for over expression of the protein in mammalian cells under the CMV promoter. When the proteins are expressed in the pFLAG-CMV-2 vector a FLAG tag (MDYKDDDDK) is added to the N terminus of the expressed protein. Having the FLAG tag on present on the expressed protein enables subcellular localisation analysis of the expressed protein using immunocytochemistry techniques with an anti-FLAG monoclonal antibody (Sigm-Aldritch catalogue number Fl 804- IMG) . ORFl nucleotide sequence (SEQ ID NO. 2)

ATGGGAGGTTTTGGAAATGAGGGTCGGTGGGAACTGTGCTGGCGAGTATCTC ACTCACATGCTTCTTTTGCGGGGATCTGTCCAGGAGAGGAGCACAGCTTGAT GTTGAAAAAGAAACTAAAGTCCTCCACTTTGCACAGCAATAAACTCAAGGA GACCTGTGATGCCCACCACAGAGGGCCTTTCATGACTCACACTGGGTGCACC AAGCAGCACAGGTCTCAGACCCTAGGAGTTCAATGGCTGTCATTTCAGAAAA GCTTGGTGACTTTACAAGCTGTGTCAAGGCCATTTATCAAGACCTATACTGG GTATCGGTGCAGCAGGTCCATTCTCCACTGA pSHML-ORFl amino acid sequence with N terminal FLAG tag (bold text) (SEQ ID NO. 12) MDYKDDDDKLAAAGGFGNEGRWELCWRVSHSHASFAGICPGEEHSLMLKKK LKSSTLHSNKLKETCDAHHRGPFMTHTGCTKQHRSQTLGVQWLSFQKSLVTLQ AVSRPFIKTYTGYRCSRSILH*

ORF2 nucleotide sequence (SEQ ID NO. 10)

ATGGCTGTCATTTCAGAAAAGCTTGGTGACTTTACAAGCTGTGTCAAGGCCA TTTATCAAGACCTATACTGGGTATCGGTGCAGCAGGTCCATTCTCCACTGAC CTGGGAGCAGCTCACTCAGCTTGCCCGGTTCTGGCGGCCTCTATGTGCTCAG GTGCAGACCTTCTATTTTATAGCCACCCAGGTAGGTTATGTCTTCCCTGCTAA GAACTGGCTTGTTTCAGCCACATTGCCTGGCCCTGGGGATCCAGCCCTGGAT AGAGAAGCCCATCCCTTGCCTGGGCAGGAGATAACTGAGCCTGTCAGTGGG TCAGATGAGGCTTGA pSHML-ORF2 amino acid sequence with N terminal FLAG tag (bold text) (SEQ ID NO. 13)

MDYKDDDDKLAAAAVISEKLGDFTSCVKAIYQDLYWVSVQQVHSPLTWEQLT QLARFWRPLCAQVQTFYFIATQVGYVFPAKNWLVSATLPGPGDPALDREAHPL PGQEITEPVSGSDEA*

PCR products for ORFl and ORF2 cloning into pFLAG-CMV-2 were generated with Notl and Xbal ends using the primers below with IMAGE clone # 3916231 in the pCMVSPORTβ plasmid as template following the "PCR" protocol.

ORFl 5' Primer with Noll end GCGCGCGGCCGCGGGAGGTTTTGGAAATGAGGG ORFl 3' Primer with Xbal end (reverse complemented) GCGCTCTAGATCAGTGGAGAATGGACCTGC ORF2 5' Primer with Notl end GCGCGCGGCCGCGGCTGTCATTTCAGAAAAGCTTG ORF2 3' Primer with Xbal end (reverse complemented) GCGCTCTAGATCAAGCCTCATCTGACCCA

The PCR products for both ORFs were digested with Notl and Xbal according to the "Restriction Enzyme Digestion of PCR Products" protocol. The digested PCR products were gel excised according to the "PCR Product Gel Extraction" protocol in order to remove the PCR template and stop it from creating a high background in the transformations of the PCR product ligations into pFLAG-CMV-2. The pFLAG-CMV- 2 vector was digested with Notl and Xbal according to the "Restriction Enzyme Digest of a DNA Vector" protocol. The digested PCR products were ligated into the digested pFLAG-CMV-2 according to the "Ligation" protocol, transformed following the "Transformation" protocol and then the colonies were screened for positives using the "PCR Screen for Positive Colonies" protocol. Any positive colonies were miniprepped and the plasmid DNA extracted according to the "DNA Miniprep" protocol and sequence confirmed with the CMV30 sequencing primer (AATGTCGTAATAACCCCGCCCCGTTGACGC).

DNA Cloning Protocols

(i) PCR

All reagents used are from Invitrogen's Platinum Taq DNA Polymerase Kit (Catalogue number 10966-018) unless otherwise specified. The following PCR reaction components were combined in 0.2mL PCR tubes (Eppendorf catalogue number 0030 124.332) on ice. 5μL of 10x PCR buffer, lμL of 1OmM dNTPs, 1.5μL of 5OmM MgCl₂ ,0.5μL of 10μM 5' primer, 0.5μL of 10μM 3' primer, 100-300ng cDNA template/50- lOOng plasmid template, 0.2μL of Platinum Taq DNA Polymerase, and water up to 50μL. The PCR reaction was performed on an Applied Biosystems 96 well Gene Amp 9700 PCR (Catalogue number N8050200) with the following cycling parameters. 94°C for 3min, 35 cycles of denaturing at 94⁰C for 45 seconds, annealing at the appropriate temperature for the primer pair for 30 seconds, extension at 72°C for 1 minute per kilo base (kb) of PCR product, and a final extension at 72°C for lOmin. PCR reactions were analysed by gel electrophoresis on a 1% agarose gel (Invitrogen catalogue number 16500500) (ii) PCR Product Purification and Gel Extraction

PCR products were purified using the Qiagen QIAquick PCR Purification Kit (Catalogue number 28104) and eluted in 30μL of water.

PCR products were gel excised by running up to 50μL of PCR reaction on a 1% agarose gel and excising the band of interest using a clean scalpel. The PCR products were then purified from the gel slice using the Qiagen QIAEX II Gel Extraction Kit (Catalogue number 20021).

(iii) Restriction Enzyme Digestion of PCR Products To 44μL of purified PCR product lμL of the restriction enzyme 1 and 5μL of the appropriate 10x restriction enzyme buffer was added and incubated at 37°C for 2-3 hours. All traces of enzyme and buffer were removed using the Qiagen QIAquick PCR Purification Kit and the PCR product was eluted in 44μL of water. lμL of restriction enzyme 2 and 5μL of the appropriate 10x restriction enzyme buffer was added and incubated at 37°C for 2-3 hours. AU traces of enzyme and buffer was removed using the Qiagen QIAquick PCR Purification Kit and eluted in 30μL of water. lμL of the PCR product was run on a 1% agarose gel to check the PCR product was present. The PCR product was quantified using a Thermo Scientific Nanodrop 1000. (iv) Restriction Enzyme Digest of a DNA Vector To lOμg of the DNA vector 2μL of restriction enzyme 1, 5μL of the appropriate restriction enzyme buffer, and water up to 50μL was added and incubated at 37°C for 3- 4 hours. 1 μL of the digested vector was run on a 1 % agarose gel to ensure that all of the vector had been cut. The vector was purified using the Qiagen QIAquick PCR Purification Kit and eluted in 43μL of water. 2μL of restriction enzyme 2 and 5μL of the appropriate restriction enzyme buffer was added and incubated at 37°C for 3-4 hours. lμL of the digested vector was run on a 1% agarose gel to ensure that all of the vector had been cut. 1 unit of CIAP (Invitrogen catalogue number 18009019) was added to the vector and incubated at 50°C for 5 minutes to dephosphorylate the 5' termini to prevent self ligation. The enzyme was deactivated by adding EDTA to the equivalent concentration of magnesium present in the reaction and incubated at 65°C for 5 minutes. The vector was purified using the Qiagen QIAquick PCR Purification Kit and eluted in 30μL of water. lμL of the PCR product was run on a 1% agarose gel to check the vector was present. The vector was quantified using a Thermo Scientific Nanodrop 1000. (v) Ligation

Digested PCR products were ligated into DNA vectors using Invitrogen's T4 DNA Ligase (Catalogue number 15224090). 2μL of 5x T4 DNA Ligase buffer, lμL of T4 DNA Ligase, lOOng of digested DNA vector, 300ng of digested PCR product, and water up to 1 OμL were incubated at 16°C overnight, (vi) Transformation

DH5α cells from Invitrogen (Catalogue number 18258012) were used for all transformations. 5μL of a ligation or lμL of miniprepped DNA were added to 25 μL of DH5α cells and incubated on ice for 30 minutes. The cells were incubated at 42°C for 45 seconds and then transferred to ice for 2 minutes. 450μL of SOC medium was added to the cells which were then incubated at 37°C for 1 hour with shaking. 50μL of the cells was spread onto an LB agar plate containing the appropriate antibiotic (lOOμg/mL Kanamycin or lOOμg/mL Ampicillin) and labelled 10% sample. The remaining cells were pelleted and resuspended in lOOμL of the media before plating onto an LB agar plate containing the appropriate antibiotic and labelled 90% sample. LB agar plates were incubated overnight, or until colonies of the required size were seen, at 37°C. (vii) PCR Screen for Positive Colonies

Twelve colonies were labelled and half of each colony was picked, using a sterile pipette tip, into a 96 well PCR plate (Axygen catalogue number PCR-96-C) containing 9μL of Platinum PCR Supermix (Invitrogen catalogue number 12532-016), 0.5μL of the 5' primer of the insert at lOμM, and 0.5μL of the 3' primer of the insert at lOμM. The tips were removed, the PCR plate sealed and then the PCR reaction was performed on an Applied Biosystems 96 well Gene Amp 9700 PCR with the following PCR parameters, 94⁰C for 2 minutes, 35 cycles of denaturing at 94⁰C for 30 seconds, annealing at the appropriate temperature for the primer pair for 30 seconds, extension at 68°C for 1 minute per kb of PCR product length. 5μL of the PCR was run on a 1% agarose gel to identify positive colonies, (viii) DNA Miniprep A colony was picked, using a sterile pipette tip, into a test tube containing 3mL of LB broth with the appropriate antibiotic (lOOμg/mL Kanamycin or lOOμg/mL Ampicillin) and incubated at 37⁰C overnight with shaking. The resulting cells were pelleted and the plasmid DNA extracted. The DNA was quantified using a Thermo Scientific Nanodrop 1000. PlasmidDNA Transfection

All cell transfection experiments were set up in a sterile class 2 laminar flow hood. 5xlO⁴ HEK293 cells or ME275 melanoma cells were seeded in 250μL of RFlO media in each required well of a chamber slide (Becton-Dickinson catalogue number 354108) and incubated at 37°C/5%CO₂ for 24 hours. The media was removed from all wells and replaced with RFlO without PSG. lμL of Lipofectamine 2000 (Invitrogen catalogue number 11668-027) was added to 24μL of Opti-MEM reduced serum medium (Invitrogen catalogue number 22600) and incubated at room temperature for 5 minutes. 0.5μg of plasmid DNA was added to Opti-MEM, to make a total volume of 25μL. The lipofectamine in Opti-MEM was then added to each plasmid DNA sample and incubated at room temperature for 20 minutes to allow the plasmid DNA to complex with the lipofectamine. The plasmid DNA/lipofectamine complex was added to the cell sample and incubated at 37°C/5%CO₂ for 24 hours before immunocytochemistry analysis.

Immunocytochemistry The cells cultured on a slide were fixed by covering with 100% acetone for 5 minutes. The section was washed with Tris Buffered Saline (TBS) and blocked with DAKO serum free protein block (Catalogue number X0909) for 10 minutes. The protein block was removed and washed with TBS before adding lOOμL of 1:1000 anti FLAG monoclonal antibody (Sigma-Aldritch catalogue number F1804-1MG) in TBS with 1% FBS. After a 1 hour incubation in a humidity chamber the section was washed once with TBS followed by three 5 minute washes with TBS, with rocking. lOOμL of the secondary antibody, 1:200 goat anti mouse monoclonal antibody Alexa 488 (Invitrogen catalogue number A-21121), in TBS with 1% FBS was added to the section and incubated in a humidity chamber for 1 hour. The section was washed once with TBS then twice for 15 minutes with TBS with rocking. One drop of Invitrogen Prolong Gold Anti-Fade Reagent (Catalogue number P36930) was added to each section before covering with a glass coverslip, ensuring no air bubbles were trapped beneath. The coverslip was then sealed to the slide using a clear adhesive and was ready for fluorescent microscopy analysis. The results of these studies are shown in Figures 17, 18 and the table below. FIow

The cellular localisation of pSHML-ORFl and pSHML-ORF2 were quite different. Whereas pSHML-ORFl appears to be associated with the endoplasmic reticulum (ER), since it is only seen next to the nucleus, pSHML-ORF2 is cytoplasmic and vesicular. HEK293 cells and ME275 melanoma cells extended out cell structures reminiscent of melanocytes, which were particularly long in the ME275 melanoma cells, which have pSHML-ORF2 vesicular expression in these cellular extensions. EXAMPLE 8: T-cell responses to ORF-I and ORF-2 peptides

A convenient method was employed to measure whether melanoma patients had expanded populations of memory T cells capable of recognising proteins encoded by either ORF-I or ORF-2. These experiments allowed not only confirmation of the potential of either protein as a target for immune therapy of melanoma, but also a possible confirmation that the protein may expressed in vivo by melanoma cells. Methods: Part of a lymph node infiltrated with melanoma cells was obtained from a patient with melanoma at surgery. Expression levels of SHML were assessed by real-time PCR and found to be present (see Fig. 4). The cells from this lymph node were separated by mechanical disruption in standard tissue culture media (RMPI 1640 medium plus 5% human serum) and T cell lines established by stimulating the cells with Dynabeads T cell expander beads (Invitrogen, USA, cat. # 111.31D) at a 1:1 ratio with cells. After approximately 2 weeks in cell culture these T cell lines were then tested for their responses to antigenic peptides. The 4 peptide treatments were either (single letter amino acid coding): 1. Negative control: no peptide 2. Positive control:

MART-1/Melan-A 26-35 analogue - ELAGIGLTV

3. SHML-ORFl peptide pool (expressed first as residue number range and then amino acid sequence):

01-29 MGGFGNEGRWELCWRVSHSHASFAGICPG 22-50 SFAGICPGEEHSLMLKKKLKSSTLHSNKL 43 - 71 STLHSNKLKETCDAHHRGPFMTHTGCTKQ 64-92 THTGCTKQHRSQTLGVQWLSFQKSLVTLQ 85-113 QKSLVTLQAVSRPFIKTYTGYRCSRSILH

4. SHML-ORF2 peptide pool (expressed first as residue number range and then amino acid sequence):

01-28 MAVISEKLGDFTSCVKAIYQDLYWVSVQ 21-48 DLYWVSVQQVHSPLTWEQLTQLARFWRP 41-68 QLARFWRPLCAQVQTFYFIATQVGYVFP 61-88 TQVGYVFPAKNWLVSATLPGPGDPALDR 81-108 PGDPALDREAHPLPGQEITEPVSGSDEA

The peptides were all made into stock solutions by dissolving in dimethyl sulfoxide (DMSO) at a concentration of 1OmM. The pooled peptides (treatments 3. and 4.) were mixtures of 5 peptides made from 1OmM stock, so the final concentration of each peptide in the mixed stocks was 2mM.

For each peptide stimulation plate 1 x 10⁶ cells of the patient T cell lines were plated in 1 well of a round bottom 96 well plate in lOOμl RPMI1640 medium plus 5% human serum. lμl of the 4 peptide treatments were added to the wells - either lμl of DMSO as a negative control, lμl of the positive control at 1OmM (for lOOμM final concentration), or 1 μl of stock for either peptide pool (for 20μM final concentration of any one peptide within the pool).

The cells were mixed with the peptides by pipetting, and the cells were then pelleted by centrifugation for 5 mins at 18Og. The cells were incubated for 1 hour at 37°C in a humidified atmosphere with 5%

CO_2. Each well was then treated with 70μl GolgiStop (BD Biosciences, USA, cat #

554724), at 1 in 1000 dilution in RMPI 1640 plus 5% human serum), and the cells mixed with the GolgiStop solution by pipetting. The cells were pelleted by centrifugation for 5 mins at 18Og, and incubated for 5 hours at 37⁰C in a humidified atmosphere with 5% CO₂

75μl of the medium was then removed from each well, and the tissue culture plate placed on ice.

AU samples were stained with 1.25 μl anti-CD8 antibody conjugated to Peridinin Chlorophyll Protein Complex (PerCP-anti-CD8, Biolegend, USA, cat #301030), and incubated on ice for 20 minutes. All wells were then washed twice with 200μl phosphate-buffered saline containing 1% fetal bovine serum.

Cells were then fixed with lOOμl per sample of Cytofix/Cytoperm solution (BD

Biosciences cat # 554722), and incubated on ice for 20 minutes. After 2 washes in Perm Wash (BD Biosciences cat# 554723) all samples were stained with 0.4 μl anti-interferon gamma antibody conjugated to fluorescein (BD

Biosciences cat # 554551)

Cells were incubated on ice for 30 minutes then washed twice with 200 μl phosphate-buffered saline containing 1% fetal bovine serum. All cells were then analysed by flow cytometry using a FACSCalibur instrument

(Becton-Dickinson, USA).

Results:

Figure 19 summarises the results of these studies.

T cells derived from a human lymph node that had become infiltrated with melanoma tumour cells were exposed to either a negative control solution (Fig. 19, upper left panel), a positive control peptide (Fig 19, upper right panel), a pool of 5 peptides covering the entire SHML-ORFl sequence (Fig 19, lower left panel) or a pool of 5 peptides covering the entire SHML-ORF2 sequence (Fig 19, lower right panel). In the absence of any peptide, approximately 0.61% of the CD8+ T cells were synthesising interferon-gamma (IFNγ). In the presence of the positive control peptide, the proportion of CD8+ T cells synthesising IFNγ rose to 1.67%, approximately 1% above the negative control, indicating the presence of T cells specific for the positive control peptide comprising approximately 1% of the T cell population. In the presence of the SHML- ORFl peptides, the proportion of CD8+ T cells synthesising IFNγ rose to 1.32%, approximately 0.7% above the negative control, indicating the presence of T cells specific for SHML-ORFl comprising approximately 0.7% of the T cell population. In contrast, stimulation with the SHML-ORF2 peptides did not detect the presence of any T cells specific for SHML-ORF2, because the number of T cells synthesising IFNγ was lower than the in the negative control.

What these studies demonstrate is that at least ORF-I protein may be expressed by melanoma cells in situ, in a way that it can activate cytotoxic T cells in vivo that are specific for that protein. These observations form the basis for development of therapeutic strategies for treatment of cancers, such as melanoma and related skin cancers, using adoptive cell transfer approaches, targeted vaccine therapies, and the like

Although the invention has been described with reference to certain embodiments and examples it will be understood that variants in keeping with the disclosure and the spirit of the invention are also within its scope.

REFERENCES:

1. Niehrs, C. 2006. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25:7469-7481.

2. Yamaguchi, Y., S. Itami, H. Watabe, K. Yasumoto, Z. A. Abdel-Malek, T. Kubo, F. Rouzaud, A. Tanemura, K. Yoshikawa, and V. J. Hearing. 2004.

Mesenchymal-epithelial interactions in the skin: increased expression of dickkopfl by palmoplantar fibroblasts inhibits melanocyte growth and differentiation. J Cell Biol 165:275-285.

3. Yamaguchi, Y., T. Passeron, T. Hoashi, H. Watabe, F. Rouzaud, IC Yasumoto, T. Hara, C. Tohyama, I. Katayama, T. Miki, and V. J. Hearing. 2008. Dickkopf 1

(DKKl) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes. Faseb J 22: 1009-1020.

4. Yamaguchi, Y., T. Passeron, H. Watabe, K. Yasumoto, F. Rouzaud, T. Hoashi, and V. J. Hearing. 2007. The effects of dickkopf 1 on gene expression and Wnt signaling by melanocytes: mechanisms underlying its suppression of melanocyte function and proliferation. J Invest Dermatol 127:1217-1225.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

I. An isolated nucleic acid molecule that encodes a melanocyte-specific protein, comprising a nucleic acid sequence set out in SEQ. ID. No. 10 or functional fragments thereof.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule encodes a protein having SEQ ID No.11 or a functional fragment thereof.

3. An isolated nucleic acid molecule that encodes a melanocyte-specific protein, wherein the nucleic acid molecule has at least 75% sequence identity with SEQ. ID. No. 10.

4. The nucleic acid molecule according to claim 3, wherein the nucleic acid molecule has from 80% to 95% sequence identity with SEQ. ID. No. 10.

5. A nucleic acid molecule that is complementary to a nucleic acid sequence of SEQ. ID. No. 10 or fragments thereof.

6. A recombinant protein comprising a polypeptide sequence encoded by an isolated nucleic acid molecule comprising SEQ ID NO 2 or SEQ ID NO 10.

7. A cell marker comprising a peptide or polypeptide sequence encoded by an isolated nucleic acid molecule of any one of claims 1 to 4, or part thereof.

8. A cell marker comprising a peptide or polypeptide sequence comprising SEQ ID No. 11.

9. The cell marker according to claim 7 or claim 8, wherein the cell marker is a skin cell marker.

10. The cell marker according to claim 9, wherein the skin cell is a melanocyte.

I I. The cell marker according to any one of claims 7 to 10, wherein the cell marker is a marker for a skin-related neoplastic condition.

12. The cell marker according to claim 11, wherein the neoplastic condition is melanoma.

13. An antibody that binds to the peptide or polypeptide sequence encoded by a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or to a protein comprising SEQ ID No. 11, or part thereof.

14. The antibody according to claim 13, wherein the antibody is a monoclonal antibody or a functional fragment thereof.

15. A pharmaceutical composition comprising an antibody according to claim 13 or claim 14, optionally in combination with a pharmaceutically acceptable carrier.

16. A vector comprising a nucleic acid sequence set forth in any one of claims 1 to 5, or a part thereof, operatively linked to a promoter.

17. A prokaryotic or eukaryotic cell comprising the expression vector according to claim 16.

18. A prokaryotic or eukaryotic cell according to claim 17, wherein the prokaryotic cell is a bacterial cell or a fungal cell.

19. A prokaryotic or eukaryotic cell according to claim 17, wherein the eukaryotic cell is selected from a mammalian cell, an avian cell, an amphibian cell, a reptilian cell or an insect cell.

20. Use of a nucleic acid molecule of any one of claims 1 to 4, a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or a protein comprising said peptide or polypeptide, as a marker of a melanocyte cell lineage.

21. Use of a protein having SEQ ID No.11, or part thereof, as a marker of a melanocyte cell lineage.

22. Use of an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or a protein comprising said peptide or polypeptide, for identifying melanocyte cell lineage.

23. Use of a nucleic acid molecule comprising a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or a protein comprising said peptide or polypeptide, or a protein having SEQ ID No.11, or part thereof, or an antibody that specifically binds to said peptide or polypeptide and/or said protein, in the diagnosis and/or detection of a skin cancer.

24. Method of diagnosing and/or treating a skin cancer comprising the step of administering to a subject in need of said treatment an effective amount of an inhibitory or interfering RNA or DNA sense or antisense sequence capable of blocking or inhibiting expression of a nucleic acid molecule of any one of claims

1 to 4, or part thereof, or an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or part thereof, or a protein comprising said peptide or polypeptide, or a protein having SEQ ID No.i l.

25. A method of screening for the presence of a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, in a sample, comprising: a) contacting a biological sample with a nucleic acid having a sequence that is complementary to nucleic acid sequence of SEQ. ID. No. 10 or part thereof; and b) determining whether the nucleic acid binds to a nucleic acid molecule in the sample.

26. A method of screening for the presence of a peptide or polypeptide or part thereof, encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, in a sample, comprising: a) contacting a biological sample with an antibody that specifically binds to the peptide or polypeptide, or part thereof; and b) determining whether the antibody binds to the peptide or polypeptide, or part thereof, in the sample.

27. A method of screening for the presence of a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, and at least one other nucleic acid molecule which codes for a cell marker commonly used to identify cells of melanocyte lineage in a sample, comprising: a) contacting a biological sample with i) a nucleic acid molecule having a sequence that is complementary to nucleic acid sequence of SEQ. ID. No. 10 or part thereof, and ii) a nucleic acid molecule having a sequence that is complementary to the at least one other nucleic acid molecule or part thereof; and b) determining whether the nucleic acid molecules bind to the nucleic acid molecules in the sample.

28. A method of screening for the presence .of a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, and at least one other polypeptide which is a cell marker commonly used to identify cells of melanocyte lineage in a sample, comprising: a) contacting a biological sample with i) an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, and ii) an antibody that specifically binds to the at least one other polypeptide; and b) determining whether the antibodies bind to their corresponding polypeptides in the sample.

29. A method of diagnosing and/or detecting skin cancer or skin condition comprising: a) contacting a biological sample with a nucleic acid having a complementary sequence to nucleic acid sequence of SEQ. ID. No. 10 or part thereof; and b) detecting localisation of the nucleic acid as an indicator of the presence (and/or type) of skin cancer or skin condition.

30. A method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof; and b) detecting localisation of the antibody as an indicator of the presence (and/or type) of skin cancer or skin condition.

31. A method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a nucleic acid having a complementary sequence to nucleic acid sequence of SEQ. ID. No. or part thereof, and one or more other nucleic acids complementary to a nucleic acid molecule encoding a marker commonly used to identify cells of melanocyte lineage, or part thereof; and b) determining localisation of the nucleic acids as an indicator of the presence (and/or type) of skin cancer or skin condition.

32. A method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, and at least one other antibody that binds to a polypeptide that is a marker commonly used to identify cells of melanocyte lineage, or part thereof and b) determining localisation of the antibodies as indicator of the presence (and/or type) of skin cancer or skin condition.

33. A method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a panel of nucleic acid molecules, or parts thereof, encoding a marker commonly used to identify cells of melanocyte lineage, and a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof; and b) determining localisation of the nucleic acid molecules as an indicator of the presence (and/or type) of skin cancer or skin condition.

34. A method of detecting and/or diagnosing skin cancer or skin condition comprising: a) contacting a biological sample with a panel of antibodies that bind specifically to a marker commonly used to identify cells of melanocyte lineage, or part thereof, and a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof; and b) determining localisation of the antibodies as indicator of the presence (and/or type) of skin cancer or skin condition.

35. The method of any one of claims 26 to 34, wherein the biological sample is selected from the group consisting of a tissue biopsy, a biological fluid and a surgical specimen.

36. The method of any one of claims 26 to 35, wherein the detection or diagnosis is performed in vivo or ex vivo.

37. The method of any one of claims 30 to 36, wherein the skin cancer is melanoma.

38. The method of any one of claims 30 to 37, wherein the skin condition is a disorder of pigmentation, and/or abnormal function, localisation or growth of melanocytes.

39. A method of modulating expression of genes associated with skin cancer or skin condition comprising contacting a cell with a short interfering nucleic acid molecule directed against a nucleic acid sequence of SEQ. ID. No. 10 or part thereof.

40. The method of claim 39, wherein the short interfering nucleic acid molecule is selected from a group consisting of short interfering RNA, double stranded RNA, micro RNA and short hairpin DNA.

41. A method of modulating expression of genes associated with skin cancer or skin condition comprising contacting the cells with an antisense nucleic acid molecule directed against a nucleic acid sequence of SEQ. ID. No. 10 or part thereof.

42. Use of a nucleic acid molecule comprising a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, or an inhibitory or interfering RNA or DNA sense or antisense sequence, or an antibody that specifically binds to a peptide or polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof, or a protein comprising said peptide or polypeptide, in the treatment of disorders associated with melanoma and/or disorders of pigmentation, and/or abnormal function, localisation or growth of melanocytes. ^•

43. Use according to claim 42, wherein the disorders of pigmentation is selected from hypo -pigmentation and/or hyper-pigmentation.

44. A method of modulating expression of genes associated with changes in skin pigmentation or cosmetic lightening or darkening of skin tone, comprising contacting a cell with an inhibitory or interfering RNA or DNA, sense or antisense, sequence directed against a nucleic acid molecule of any one of claims 1 to 4, or a part or variant thereof

45. A method according to any one of claims 24, 42 or 44 wherein the inhibitory or interfering RNA is an siRNA.

46. A nucleic acid molecule encoding a protein having SEQ. ID. No. 11, comprising

3' and 5' regulatory regions.

47. A method according to any one of claims 27, 28, 31, 32 or 34, wherein the cell marker is selected from melanA, gplOO, tyrosinase, trp-1 and trp-2 48. A recombinant protein expressed in a prokaryotic or eukaryotic cell and having an amino acid sequence selected from SEQ ID NO 3 or SEQ ID NO 11.