WO2004090113A2

WO2004090113A2 - L53, a lung cancer associated antigen and uses thereof

Info

Publication number: WO2004090113A2
Application number: PCT/US2004/009951
Authority: WO
Inventors: Joseph M. Petroziello; Charlotte F. Mcdonagh
Original assignee: Seattle Genetics, Inc.
Priority date: 2003-03-31
Filing date: 2004-03-31
Publication date: 2004-10-21
Also published as: WO2004090113A3

Abstract

The present invention relates to L53 genes and gene products that are differentially expressed in cancer tissues and cell lines. In a particular aspect of the invention, L53 genes and gene products are differentially expressed in lung cancer tissues and cell lines. In accordance with the present invention, L53 nucleic acid sequences, amino acid sequences and antibodies thereto, and methods of use thereof are presented. The L53 molecules and methods of the invention may be used to monitor expression levels of L53, wherein the detection of aberrant levels of L53 molecules provides a positive diagnostic indicator of lung cancer and/or other L53 associated cancers and a useful prognostic indice of the state of the diseases. Also provided are compounds capable of modulating L53 activity, which are identified using the L53 molecules and methods of the invention. Such L53 modulating compounds may be used efficaciously to treat patients with lung cancer, or other L53 antigen positive cancers.

Description

L53, A LUNG CANCER ASSOCIATED ANTIGEN AND USES THEREOF

[0001] This application claims priority under 35 USC § 119(e) from U.S Provisional Application Serial No 60/459,221 filed 31 March 2003, which application is herein specifically incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] The invention relates generally to the field of cancer diagnosis, prognosis, treatment and prevention. More particularly, the present invention relates to methods of diagnosing, treating and preventing lung cancer. Methods of using a nucleic acid and a protein, differentially expressed in tumor cells, and antibodies against the protein, to treat, diagnose or prevent cancer, are provided for by the present invention. The instant invention provides compositions comprising, and methods of using products of a gene termed L53 and associated splice variants thereof. Such L53 gene products include L53 proteins and nucleic acids and variants thereof. Such gene products, as well as their binding partners and antagonists or agonists, can be used for the prevention, diagnosis, prognosis and treatment of lung cancer, and other cancers.

2. BACKGROUND OF THE INVENTION

[0003] Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood- borne spread of malignant cells to regional lymph nodes and to distant sites (metastases). Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.

[0004] Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for a review of such abnormal growth conditions, see Robbins & Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79). A neoplastic lesion may evolve clonally and develop an increasing capacity for growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance (Roitt, I., Brostoff, J. and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).

[0005] Lung cancer is the leading cause of cancer deaths worldwide, and more specifically non- small cell lung cancer (NSCLC) accounts for approximately 80% of all disease cases (Cancer Facts and Figures, 2002, American Cancer Society, Atlanta, p. 11.). In 2002, there were an estimated 169,500 cases of lung cancer resulting in 154,900 deaths (Cancer Facts and Figures, 2002, American Cancer Society, Atlanta, p. 11.). There are four major types of non-small cell lung cancer, including adenocarcinoma, squamous cell carcinoma, bronchioalveolar carcinoma, and large cell carcinoma. Adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC based on cellular morphology (Travis et al., 1996, Lung Cancer Principles and Practice, Lipprncott-Raven, New York, pps. 361-395). Adenocarc nomas are characterized by a more peripheral location in the lung and often have a mutation in tfre^'Λ-raron'cogene (Gazdar et at., 1994, Anticancer Res. 14:261-267). Squamous cell carcinomas are typically more centrally located and frequently carry p53 gene mutations (Niklinska et al, 2001, Folia Histochem. Cytobiol. 39:147-148). A comprehensive understanding of the disease genes associated with these two main NSCLC subtypes has not been described.

[0006] Several genes have been previously described as potential diagnostic markers or prognostic indicators for lung cancer, including: CYFRA 21-1, TPA, and CA125 (Hatzakis et al, 2002, Respiration. 69(l):25-29); CEA (Sawabata et al., 2002, Ann Thorac Surg. 74(1): 174-179); p53 and HER2-neu (Han et al, 2002, Hum Pathol. 33(1): 105-110); NSE (Kulpa et al, 2002, Clin Chem. 48(11):1931-1937); and JL-8 (Yuan et al, 2000, Am JRespir Crit Care Med. 162:1957-1963).

[0007] A marker-based approach to tumor identification and characterization promises improved diagnostic and prognostic reliability. Typically, the diagnosis of lung cancer and other types of cancer requires histopathological proof of the presence of die tumor. In addition to diagnosis, histopathological examinations also provide information about prognosis and selection of treatment regimens. Prognosis may also be established based upon clinical parameters such as tumor size, tumor grade, the age of the patient, and lymph node metastasis.

[0008] In clinical practice, accurate diagnosis of various subtypes of cancer is important because treatment options, prognosis, and the likelihood of therapeutic response all vary broadly depending on the diagnosis. Accurate prognosis, or determination of distant metastasis-free survival could allow the oncologist to tailor the administration of adjuvant chemotherapy, with patients having poorer prognoses being given the most aggressive treatment. Furthermore, accurate prediction of poor prognosis would greatly impact clinical trials for new lung cancer therapies, because potential study patients could then be stratified according to prognosis. Trials could then be limited to patients having poor prognosis, in turn making it easier to discern if an experimental therapy is efficacious. To date, no set of satisfactory predictors for prognosis based on the clinical information alone has been identified.

[0009] It would, therefore, be beneficial to provide specific methods and reagents for the diagnosis, staging, prognosis, monitoring and treatment of cancer, including lung cancer, and to provide methods that would identify individuals with a predisposition for the onset of lung cancer, and other types of cancer, and hence are appropriate subjects for preventive therapy.

3. SUMMARY OF THE INVENTION [0010] Intensive and systematic evaluation of gene expression patterns is essential for understanding the physiological mechanisms associated with cellular transformation and metastasis associated with cancer. Several techniques that permit comparison of gene expression in normal and cancerous cells are known in the art. Examples of these techniques include: Serial Analysis of Gene Expression (SAGE) (Velculescu et al., 1995, Science 270:484); Restriction Enzyme Analysis of Differentially Expressed Sequences (READS) (Prasher et al, 1999, Methods in Enzymology 303:258); Amplified Fragment Length Polymorphism (AFLP) (Bachem et al., 1996, Plant Journal 9:745); Representational Difference Analysis (RDA) (Hubank et al, 1994, Nucleic Acid Research 22:(25):5640); differential display ^"(Liang et al. ,^"1992, Cancer Research 52(24):6966); and suppression subtractive hybridization (SSH) (Diatchenko et al, 1996, Proc. Natl. Acad. Sci. USA 93:6025). Such differential expression methods have led the present inventors to the identification and characterization of the L53 gene and variants thereof, as genes whose expression is associated with lung cancer and other types of cancer. This discovery by the present inventors has made possible the use of L53 molecules and variants thereof for the treatment, prevention and diagnosis of cancers, including but not limited to lung cancer.

[0011] The present invention relates to the discovery of a novel gene designated L53 which has an expression pattern that is up-regulated in cancer tissues and cell lines, e.g., lung cancer tissues and cell lines. Also encompassed by the invention are L53 variants and fragments that retain at least one functional characteristic of the full length, wild type L53. The invention relates to the use of said gene, gene products, and antagonists or agonists of said gene or gene products (L53 and variants thereof, cDNA, RNA, and /or protein) as targets for diagnosis, drug screening and therapies for cancer. The present invention also relates to the use of said genes or gene products or derivatives thereof as vaccines against cancer. In a particular embodiment, the invention provides methods of using the L53 protein and variants thereof, or nucleic acids that encode said proteins for the treatment, prevention and/or diagnosis of lung cancer.

[0012] In particular, the methods of the present invention include using nucleic acid molecules that encode the L53 protein and variants thereof, and recombinant DNA molecules, cloned genes or degenerate variants thereof, and in particular naturally occurring variants that encode L53 related gene products. The methods of the present invention additionally include using cloning vectors, including expression vectors, containing the nucleic acid molecules encoding L53 and variants thereof, and hosts that contain such nucleic acid molecules. The methods of the present invention also encompass the use of L53 gene products and variants thereof, including fusion proteins, and antibodies directed against such L53 gene products or conserved variants or fragments thereof. In one embodiment, a fragment or other derivative of an L53 protein is at least 10 amino acids long. In another embodiment, a fragment of an L53 nucleic acid and variants thereof, including nucleic acid or derivative thereof is at least 10 nucleotides long.

[0013] The nucleotide sequence of the cDNA of a human L53 gene is provided. The nucleotide sequences of the L53 ORF in the L53 gene, as well as the amino acid sequences of the encoded gene products, are also provided. The full-length L53 gene of 3089 nucleic acids was cloned by polymerase chain reaction (PCR). The L53 transcript encodes a protein of 929 amino acids. An in-frame start and stop was observed by sequence analysis for L53. The L53 transcript was detected at elevated levels in both lung cancer cell lines and lung tumor isolates as compared to normal tissues. Elevated transcript levels for L53 were also detected in additional tumor types and cancer cells as described in Section 6.

[0014] The present invention further relates to methods for the diagnostic evaluation and prognosis of cancer in a subject animal. Preferably the subject is a mammal, more preferably the subject is a human. In a particular embodiment the invention relates to methods for diagnostic evaluation and prognosis of lung cancer. For example, nucleic acid molecules of the invention can be used as diagnostic hfbtidllittϊϋϊf'piδbesOτ s primers for diagnostic PCR analysis for detection of abnormal expression of the L53 gene.

[0015] Antibodies or other binding partners to L53 and variants thereof can be used in a diagnostic test to detect the presence of the L53 gene products in body fluids, cells or in tissue biopsy. In specific embodiments, measurement of serum or cellular L53 gene products and variants thereof can be made to detect or stage lung cancer, e.g., adenocarcinoma, squamous cell carcinoma, bronchioalveolar carcinoma, or large cell carcinoma.

[0016] The present invention also relates to methods for the identification of subjects having a predisposition to cancer, e.g., lung cancer. The subject can be any animal, but preferably the subject is a mammal, and most preferably the subject is a human. In a non-hmiting example nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for quantitative reverse transcriptase-PCR (RT- PCR) analysis to determine expression levels of the L53 gene products and variants thereof in a sample from a subject. In another example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of L53 and variants thereof, naturally occurring or non-naturally occurring gene mutations, allelic variations and regulatory defects in the L53 gene.

[0017] Imaging methods, for imaging the localization and/or amounts of L53 gene products in a patient, are also provided for diagnostic and prognostic use.

[0018] Use of nucleic acid molecules of the invention as diagnostic hybridization probes or as primers for diagnosing a patient with a cancer associated with elevated L53 gene product levels is also encompassed. Also included is the use of detection agents, such as antibodies immunologically specific for an L53 gene product, as means for determining the level of the L53 gene product in a sample of a subject, which in turn serves as an indicator of the predisposition of the subject to develop cancer. Use of the above nucleic acid molecules and detection agents in imaging methods for diagnostic and prognostic assessments is also envisioned. The subject or patient can be any animal, but preferably the subject is a mammal, and most preferably the subject is a human.

[0019] Further, methods are presented for the treatment of cancer, including lung cancer. Such methods comprise the administration of compositions that are capable of modulating the level of L53 and variants thereof, including L53 gene expression and/or the level of the L53 gene product activity in a subject. The subject can be any animal, preferably a mammal, more preferably a human.

[0020] Accordingly, the present invention encompasses using agents identified by the screening methods described herein and compositions comprising such agents to treat a subject in need thereof. Such subjects include subjects afflicted with cancer, particularly those subjects afflicted with a cancer associated with elevated levels of an L53 gene product or elevated levels of L53 gene product activity. Agents identified using the screening methods of the invention are capable of modulating the level of L53 and variants thereof, including L53 gene expression and/or the level of the L53 gene product activity. In a particular aspect, such agents modulate L53 expression and/or activity by reducing the level of an L53 gene product and or the level ot L53 gene product activity. The subject can be any animal, preferably a mammal, and more preferably a human.

[0021] Still further, the present invention relates to methods for the use of the L53 gene and variants thereof for the identification of compounds that modulate L53 gene expression and/or the activity of L53 gene products. Such compounds may be used as agents to prevent and/or treat lung cancer or any cancer wherein L53 and variants thereof are expressed at levels that are elevated with respect to the expression level in corresponding normal tissue. Such compounds can also be used to palliate the symptoms of the disease, and control the metastatic potential of lung cancer or any cancer wherein L53 and variants thereof are expressed at elevated levels relative to those of normal tissue.

[0022] The invention also provides methods for treating or preventing cancer wherein a product of the L53 gene or variants thereof are administered to a subject in an amount effective to elicit an immune response in a subject. The subject may be any animal, preferably a mammal, more preferably a human. The invention also provides methods for treating or preventing cancer by administering a nucleic acid sequence encoding an L53 protein or a variant thereof to a subject such that expression of the L53 protein or variant results in the production of these polypeptides in an amount effective to elicit an immune response. The invention further provides methods of treating or preventing cancer by administering an L53 protein or a peptide thereof, in an amount effective to elicit an immune response. The immune response may be humoral, cellular, or a combination of both. In a particular embodiment the invention provides a method of immunizing to confer protection against the onset of lung cancer.

[0023] The invention also entails use of a product of the L53 gene or variants thereof in the treatment or prevention of cancer in a subject, wherein the product of the L53 gene or variants thereof are administered to a subject in an amount effective to elicit an immune response in the subject. Such L53 gene products or variants thereof may be administered alone or in a composition. Alternatively, use of a nucleic acid sequence encoding an L53 protein or a variant thereof in the treatment or prevention of cancer in a subject is also envisioned. Administration and expression of L53 encoding nucleic acid sequences results in the production of L53 polypeptides in an amount effective to elicit an immune response in a subject. L53 nucleic acid sequences may be used alone or in a composition. The subject may be any animal, preferably a mammal, more preferably a human. The immune response elicited may be humoral, cellular, or a combination of both.

[0024] The invention relates to screening assays to identify antagonists or agonists of the L53 gene or gene product and variants thereof. Thus, the invention relates to methods of identifying agonists or antagonists of the L53 gene or gene product and variants thereof, and the use of said agonist or antagonist as a medicament for the treatment and/or prevention of lung cancer or other types of cancer.

[0025] The invention also provides methods of treating cancer by providing therapeutic amounts of an anti-sense nucleic acid molecule. An anti-sense nucleic molecule is a nucleic acid molecule that is the complement of all or a part of the L53 gene sequences or L53 ORFs and which therefore can hybridize to the L53 gene and variants thereof, or fragments thereof. Hybridization of the anti-sense molecule inhibits expression of the L53 gene. In a preferred embodiment the method is used to treat a subject with lung- cancer. Use of these L53 anu-sense nucleic molecules as medicaments for the treatment of a subject with lung cancer results in a reduction of tumor burden and/or of symptoms associated with the disease.

[0026] The invention also includes a kit for assessing whether a patient is afflicted with lung cancer or other types of cancer. This kit comprises reagents for assessing expression of an L53 gene product.

[0027] In another aspect, the invention relates to a kit for assessing the suitability of each of a plurality of compounds for inhibiting cancer including lung cancer in a patient. The kit comprises a reagent for assessing expression of an L53 gene product, and may also comprise a plurality of compounds.

[0028] In another aspect, the invention relates to a kit for assessing the presence of cancer cells. This kit comprises an antibody, wherein the antibody binds specifically with a protein corresponding to an L53 gene product and variants thereof. The kit may also comprise a plurality of antibodies, wherein the plurality binds specifically with different epitopes on an L53 gene product and variants thereof.

[0029] The invention also includes a kit for assessing the presence of cancer cells, wherein the kit comprises a nucleic acid (e.g., oligonucleotide) probe. The probe binds specifically to a transcribed polynucleotide corresponding to an L53 gene product and variants thereof. The kit may also comprise a plurality of probes, wherein each of the probes binds specifically to a transcribed polynucleotide corresponding to a different region of the mRNA sequence transcribed from the L53 gene and variants thereof.

[0030] Kits for diagnostic use, comprising in a container, primers for use in PCR that can amplify L53 cDNA and variants thereof, including the L53 cDNA and/or genes and, in a separate container, a standard amount of L53 cDNA are also provided.

[0031] The invention also provides transgenic non-human animals (e.g., mice) that express the L53 nucleic acids and proteins encoded by a transgene. Transgenic, non-human knockout animals (e.g., mice), and an L53 gene and variants thereof are also provided.

[0032] Accordingly, the present invention provides a method of diagnosing cancer in a subject comprising detecting or measuring an L53 gene product in a sample derived from said subject, wherein said L53 gene product is (a) an RNA corresponding to SEQ ID NO: 1, or SEQ ID NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ ID NO: 3 (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby; in which elevated levels of the L53 gene product and variants thereof, compared to a non-cancerous sample or a pre-determined standard value for a noncancerous sample, indicates the presence of cancer in the subject. In one embodiment of the foregoing diagnostic method, the subject is a human. In another embodiment, the cancer is lung cancer. In yet other embodiments, the sample is a tissue sample, a plurality of cells, or a bodily fluid.

[0033] The present invention further provides methods of staging cancer in a subject comprising detecting or measuring an L53 gene product and variants thereof, in a sample derived from said subject, \ ιefeιn s'iMUSB gene p oduct andNariants thereof, is (a) an RNA corresponding to SEQ ID NO: 1, or SEQ ID NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ ID NO: 3 (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (d) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ JJD NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (e) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as deterrnined using the NBLAST algorithm, or a protein encoded thereby; in which elevated levels of the L53 gene product and variants thereof, compared to a non-cancerous sample or a pre-determined standard value for a noncancerous sample, indicates an advanced stage of cancer in the subject.

[0034] The present invention further provides methods for treating cancer in a subject, comprising administering to the subject an amount of a compound which reduces the level and/or antagonizes the activity of an L53 gene product and variants thereof, wherein said L53 gene product is (a) an RNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ ID NO: 3, (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby. In one embodiment, the gene product whose expression is being decreased is a protein encoded by a nucleic acid comprising a nucleotide sequence with at least 90% sequence identity to SEQ JD NO: 1 or SEQ JD NO: 2. In another embodiment, the compound decreases expression of an RNA corresponding to SEQ JD NO: 1 or SEQ JD NO: 2. The antagonist can be (i) a protein; (ii) a peptide; (iii) an organic molecule with a molecular weight of less than 500 daltons; (iv) an inorganic molecule with a molecular weight of less than 500 daltons; (v) an antisense oligonucleotide molecule that binds to said RNA and inhibits translation of said RNA; (vi) a ribozyme molecule that targets said RNA and inhibits translation of said RNA; (vii) an antibody that specifically or selectively binds to an L53 gene product and variants thereof; (viii) a double stranded oligonucleotide that forms a triple helix with a promoter of an L53 gene and variants thereof, wherein said L53 gene is a nucleic acid at least 80% homologous to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement as determined using the NBLAST algorithm; or (ix) a double stranded oligonucleotide that forms a triple helix with a promoter of an L53 gene, wherein said L53 gene is a nucleic acid at least 80% homologous to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement as determined using the NBLAST algorithm. Wherein the compound is an L53 antagonist antibody, the antibody immunospecifically binds to a protein comprising the amino acid sequence of SEQ JD NO: 3 or fragments thereof, and thereby reduces or inhibits an activity of L53.

[0035] Use of a compound which reduces the level and/or antagonizes the activity of an L53 gene product and variants thereof, wherein said L53 gene product is as described herein above in (a)-(d), ^" as a medicament ror the treatment of a subject with cancer is also encompassed by the present invention. Suitable antagonists are described herein above in (i)-(ix) and can be used alone or in a composition.

[0036] The present invention further provides methods of vaccinating a subject against cancer comprising administering to the subject a molecule that elicits an immune response to an L53 gene product, wherein said L53 gene product is (a) an RNA corresponding to SEQ ID NO: 1, or SEQ ID NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3, (c) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby. In one embodiment, the immune response is a cellular immune response. In another embodiment, the immune response is a humoral immune response. In yet another embodiment, the immune response is both a cellular and a humoral immune response.

[0037] Use of L53 gene products as agents for vaccinating a subject against cancer are also described herein.

[0038] The present invention yet further provides methods of determining if a subject is at risk for developing cancer, said method comprising (I) measuring an amount of an L53 gene product in a sample derived from the subject, wherein said L53 gene product is: (a) an RNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1, or SEQ ID NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as deteπnined using the NBLAST algorithm; or a protein encoded thereby; and (II) comparing the amount of said L53 gene product in the subject with the amount of L53 gene product present in a non-cancerous sample or predetermined standard for a noncancerous sample, wherein an elevated amount of said L53 gene product in the subject compared to the amount in the non-cancerous sample or pre-determined standard for a noncancerous sample indicates a risk of developing cancer in the subject.

[0039] The present invention yet further provides methods of determining if a subject suffering from cancer is at risk for metastasis of said cancer, said method comprising measuring an amount of an L53 gene product in a sample derived from the subject, wherein said gene product is (a) an RNA corresponding to SEQ ID NO: 1, or SEQ ID NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ ID NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby, wherein an elevated amount of L53 gene products in the subject compared to the amount in the non-cancerous sample, or in the sample from the subject with the non-metastasizing cancer, or the amount in the predetermined^" standard tor a noncancerous or non-metastasizing sample, indicates a risk of developing metastasis of said cancer in the subject.

[0040] The present invention yet further provides methods of screening for a compound capable of binding to an L53 molecule, said method comprising (I) contacting the L53 molecule with a candidate agent, wherein said L53 molecule is (a) an RNA corresponding to SEQ ID NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ ID NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby and (II) determining whether or not the candidate agent binds the L53 molecule. The screening assay can be performed in vitro. In one embodiment, the L53 molecule, or variants thereof, is anchored to a solid phase. In another embodiment, the candidate agent is anchored to a solid phase. In other embodiments, the screening assay is performed in the liquid phase. In yet other embodiments, the L53 molecule and/or variants thereof, are expressed on the surface of a cell or in the cytosol of a cell in step (I). In these embodiments, the L53 molecule or variants thereof, are expressed naturally in the cell; alternatively, a cell can be engineered to express the L53 molecule or variants thereof. In the foregoing screening methods, the candidate agent is preferably labeled, for example radioactively or enzymatically.

[0041] The present invention provides methods of screening for a cellular protein capable of interacting with an L53 gene product, said method comprising (I) immunoprecipitating the L53 gene product from a cell lysate, wherein said L53 gene product is (a) an RNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1, or SEQ LO NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby; and (II) determining whether or not any cellular proteins bind to or form a complex with the L53 gene product in the immunoprecipitate.

[0042] The present invention yet further provides methods of screening for a candidate agent capable of modulating the expression level of an L53 gene, and variants thereof, said method comprising (I) contacting said L53 gene with a candidate agent, wherein said L53 gene is a nucleic acid at least 80% homologous to SEQ ID NO: 1, or SEQ JD NO: 2, as deteimined using the NBLAST algorithm; and (II) measuring the level of expression of an L53 gene product, said gene product selected from the group consisting of an mRNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a protein comprising SEQ JD NO: 3, wherein an increase or decrease in said level of expression relative to said level of expression in the absence of said candidate agent indicates that the candidate agent is capable of modulating expression of an L53 gene. 0&43] The present invention yet further provides a vaccine formulation for the prevention of cancer comprising (I) an immunogenic amount of an L53 gene product, wherein said L53 gene product is: (a) an RNA corresponding to SEQ ID NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ ID NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement as determined using the NBLAST algorithm; or a protein encoded thereby; and (II) a pharmaceutically acceptable excipient.

[0044] The present invention yet further provides an immunogenic composition comprising (I) a purified L53 gene product in an amount effective for eliciting an immune response, wherein said gene product is (a) an RNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; (b) a protein comprising SEQ JD NO: 3; (c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; or (d) a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement as deteranned using the NBLAST algorithm; or a protein encoded thereby; and (II) an excipient.

[0045] The present invention yet further provides a pharmaceutical composition comprising an antibody that specifically or selectively binds to a protein consisting essentially of SEQ ID NO: 3; and a pharmaceutically acceptable carrier.

[0046] The present invention yet further provides a pharmaceutical composition comprising an antibody that specifically or selectively binds to a protein comprising SEQ JD NO: 3; and a pharmaceutically acceptable carrier.

[0047] The present invention yet further provides pharmaceutical compositions comprising (I) an L53 gene product, and (II) a pharmaceutically acceptable carrier.

[0048] The present invention yet further provides a pharmaceutical composition comprising (I) a purified nucleic acid comprising SEQ ID NO: 1, or SEQ JD NO: 2, and (JJ) a pharmaceutically acceptable carrier.

[0049] Use of these pharmaceutical compositions as medicaments for the treatment of a subject with cancer are also envisioned.

[0050] The pharmaceutical compositions of the present invention can be formulated, inter alia, for delivery as an aerosol, for parenteral delivery, or for oral delivery.

[0051] The present invention yet further provides methods of diagnosing cancer in a subject comprising (I) administering to said subject a compound that specifically binds a protein comprising an amino acid sequence of SEQ ID NO: 3, wherein said compound is bound to an imaging agent; and (II) obtaining an internal image of said subject by use of said imaging agent; wherein the localization or amount of said image indicates whether or not cancer is present in said subject. In a particular embodiment, the compound is an antibody. In a particular mode of this embodiment, the antibody is conjugated to a radioactive metal and said obtaining step comprises recording a scintographic image obtained from the decay of the radioactive metal.

[0052] Use of the above-mentioned compounds as imaging agents for obtaining internal images of a subject are also contemplated.

[0053] The present invention yet further provides kits that are useful for practicing the present methods. In one embodiment, such a kit comprises, in one or more containers, a pair of oligonucleotide primers, each primer comprising a nucleotide sequence with at least 5 complementary nucleotides to a different strand of a double-stranded nucleic acid comprising SEQ JD NO: 1, or SEQ JD NO: 2, and, in a separate container, a purified double-stranded nucleic acid comprising SEQ ID NO: 1, or SEQ JD NO: 2. In specific modes of the embodiment, each primer comprises a nucleotide sequence with at least 8, more preferably at least 10, yet more preferably at least 12, and most preferably at least 15 complementary nucleotides to a different strand of a double-stranded nucleic acid comprising SEQ ID NO: 1, or SEQ ID NO: 2.

[0054] The present invention yet further provides transgenic non-human animals which express from a transgene an L53 gene product, for example, an RNA corresponding to SEQ ID NO: 1, or SEQ JD NO: 2, or a protein comprising SEQ JD NO: 3.

[0055] The present invention yet further provides a method of testing the effects of a candidate therapeutic compound comprising administering said compound to a transgenic non-human animal which expresses from a transgene an L53 gene product; and determining any effects of said compound upon said transgenic non-human animal.

[0056] The present invention further provides host cells comprising nucleic acids encoding the polypeptides of the invention operably linked to a promoter, and methods of expressing such polypeptides and variants thereto by culturing the host cells under conditions in which the nucleic acid molecule is expressed.

3.1 DEFINITIONS

[0057] SPECIFIC OR SELECTIVE: a nucleic acid used in a reaction, such as a probe used in a hybridization reaction, a primer used in a PCR, or a nucleic acid present in a pharmaceutical preparation, is referred to as "selective" or "specific' f it hybridizes or reacts with the intended target more frequently, more rapidly, or with greater duration than it does with alternative substances. Similarly, a polypeptide is referred to as "selective" or "specific'lf it binds an intended target, such as a ligand, hapten, substrate, antibody, or other polypeptide more frequently, more rapidly, or with greater duration than it does to alternative substances. An antibody is referred to as "selective" or "specific" if it binds via at least one antigen recognition site to the intended target more frequently, more rapidly, or with greater duration than it does to alternative substances. A marker is selective to or specific for a particular cell or tissue type if it is expressed predominantly in or on that cell or tissue type, particularly with respect to a biological sample of interest. [0058] VARIANT (S): A variant (v) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that are different from a reference polynucleotide or polypeptide, respectively.

[0059] Variant polynucleotides are generally limited so that the nucleotide sequence of the reference and the variant are closely related overall and, in many regions, identical. Changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acid sequence encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Alternatively, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence.

[0060] Variant polypeptides are generally limited so that the sequences of the reference and the variant are closely similar overall and, in many regions, identical. For example, a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions, and truncations, which may be present or absent in any combination.

[0061] CORRESPOND OR CORRESPONDING: Between nucleic acids, "corresponding" means homologous to or complementary to a particular sequence or portion of the sequence of a nucleic acid. As between nucleic acids and polypeptides, "corresponding" refers to amino acids of a peptide in an order derived from the sequence or portion of the sequence of a nucleic acid or its complement. As between polypeptides (or peptides and polypeptides), "corresponding" refers to amino acids of a first polypeptide (or peptide) in an order derived from the sequence or portion of the sequence of a second polypeptide.

[0062] L53 GENE PRODUCT: AS used herein, unless otherwise indicated, an L53 gene product is: an RNA corresponding to SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleic acid derived therefrom; a protein comprising SEQ JD NO: 3; a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or SEQ JD NO: 2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; a nucleic acid at least 90% homologous to SEQ JD NO: 1 or SEQ JD NO: 2 or its complement as determined using the NBLAST algorithm; or a fragment or derivative of any of the foregoing proteins or nucleic acids.

[0063] CONTROL ELEMENTS: As used herein refers collectively to promoter regions, polyadenylation signals, transcription teπriination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0064] PROMOTER REGION: Is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. [0065]' OPERABLY LINKED: As used herein refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.

[0066] TO TREAT A CANCER OR A TUMOR: As used herein, the phrase "to treat a cancer or a tumor" or "treating a cancer or a tumor" in a mammal means one or more of alleviating a symptom of, con-ecting an underlying molecular or physiological disorder of, or reducing the frequency or severity of a pathological or deleterious physiological consequence of a cancer or a tumor in a mammal. By way of example, and not by limitation, the deleterious physiological consequences of a cancer or a tumor can include uncontrolled proliferation, metastasis and invasion of other tissues, and suppression of an immune response.

[0067] TO STAGE A TUMOR: As used herein, to "stage a tumor" or to "determine the stage of progression of a tumor" means to ascertain the stage of progression of a tumor along the continuum from non-invasive to invasive, or from non-metastatic to metastatic. Typically tumors are staged from grades I- IV with IV being the most malignant or metastatic.

[0068] IMMJJNOLOGICALLY SPECIFIC: With respect to antibodies of the invention, the term "inrmunologically specific" refers to antibodies diat bind to one or more epitopes of a protein of interest (e.g., L53 protein), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0069] CONSISTING ESSENTIALLY OF: The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ JD NO:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

[0070] MODULATE: As used herein, a compound which is capable of increasing or decreasing the level and/or activity of an L53 molecule may be referred to herein as an L53 modulator.

[0071] ANTAGONIST: As used herein, a compound capable of reducing the level and/or activity of an L53 molecule or a variant thereof may be refeπed to herein as an L53 antagonist.

[0072] AGONIST: As used herein, a compound capable of increasing the level and/or activity of an L53 molecule or a variant thereof may be referred to herein as an L53 agonist.

[0073] L53 ACTIVITY: As used herein, the term "L53 activity" refers to any L53-mediated function, such as those activities that contribute to or affect onset, progression, and metastatic spread of lung cancer and/or other cancers. L53 activity is also used in the conext of methods for identifying compounds that may interact with L53 and modulate or alter such L53-mediated activity.

[0074] ELEVATED L53 LEVELS: As used herein the terms "elevated", "over-expressed", "up- regulated", or "increased" L53 levels refer to an approximately three-fold or greater increase in the expression of L53 transcript and/or protein as compared to that of a control tissue, which expresses a baseline level of L53.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1. L53 transcript of 3089bp (SEQ JD NO: 1) with a coding sequence (CDS) ^' spanning 168-2954bp.

[0076] FIG. 2. Semi-quantitative RT-PCR results using various NSCLC cell lines. RT-PCR products were visualized by ethidium bromide staining. Samples are loaded as follows: (1) A549, (2) NCI-H920, (3) NCI-H969, (4) NCI-H647, (5) NCI-H226, (6) NCI-H1869, (7) NCI-H1385, (8) NCI- HI 155, (9) NCI-H1155, (10) NCI-H358, and (11) NCI-H650. The control gene EF-1 was included for comparison.

[0077] FIG. 3. Semi-quantitative RT-PCR results using various ATCC tumor cell lines. RT- PCR products were visualized by ethidium bromide staining. Samples are loaded as follows: (1) MIAPaCa-2, (2) CAPAN-2, (3) HCT116, (4) HCT15, (5) HT29, (6) RCA, (7) MCF-7, (8) T47D, (9) SKBR-3, (10) MDA-MB231, (11) MDA-MB435s, (12) MDA-MB453, (13) BT-549, (14) Hs578T, and (15) JJVTR-32. The control gene EF-1 was included for comparison.

[0078] FIG. 4. Evaluation of normal tissue expression levels for L53 and EF-1 using the Multiple Tissue Expression (MTE™) Array. L53 cDNA was amplified and used as a probe (B). EF-1 was included as a control gene for comparison of sample loading equivalence (A).

[0079] FIG. 5. The L53 protein, including 929 amino acids (SEQ ID NO: 3), encoded by the L53 nucleic acid sequence of SEQ ID NO: 1 (FIG. 1).

5. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention relates to the discovery that the L53 gene is over-expressed in cancer cells and tissues such as lung cancer cells. The invention relates to methods of using the L53 gene and variants thereof, to diagnose, treat, monitor treatment efficacy and prevent cancer, e.g., lung cancer. The invention further relates to methods of using the L53 gene to evaluate the prognosis of a patient diagnosed with cancer. The invention also relates to the discovery that the L53 gene is over-expressed in metastatic cancer cells. Thus, the invention contemplates the use of the L53 gene and variants thereof, to evaluate a cancer patient's risk of metastasis of said cancer, e.g., lung cancer.

[0081] In the development of lung neoplasia and other cancers, subsets of genes are specifically and differentially expressed at various stages of disease progression. Some of these genes/gene subsets are critical for progression of the cancer, and are associated with a particular stage of the disease, for example, metastasis. As described by way of example herein, a profile of gene expression patterns which correlates with the neoplastic development of lung carcinoma was identified using Suppression Subtractive Hybridization (SSH). See An et al, 2003, hit J Cancer. 103(2): 194-204; Shen et al., 2002, Lung Cancer. 38(3):235-241; and Zhang et al, 2000, J Surg Res. 93(1):108-119. SSH generated cDNA libraries derived from lung cancer cell lines were screened using microanays for genes that were expressed at elevated levels in the cancerous cells as compared to those of various normal tissues and cultured cells. A total of >2000 clones were spotted onto nylon membranes for subsequent expression anay screening.^' Several previously identified lung cancer associated genes, as well as the novel L53 gene of the present invention were identified using this analysis. The details concerning the isolation and characterization of the L53 cDNA, its expression level in various cancer cell lines and tissues, and the significance of its expression in carcinogenic processes are described in detail in the examples provided.

[0082] The present invention encompasses methods for the diagnosis, prognosis and staging of lung cancer and other cancers, as well as methods for treating a patient with cancer and/or monitoring of the effect of a therapeutic treatment. Further provided are methods for the use of the L53 gene products in the identification of compounds that are capable of modulating the expression of L53 or the activity of an L53 gene product. Expression of the L53 gene and variants thereof, is upregulated in various types of cancer cells including lung cancer cell lines and tissues. As such, L53 gene products can be involved in the mechanisms underlying the onset and development of lung cancer and other types of cancer as well as the regional infiltration and metastatic spread of cancer. Thus, the present invention also provides methods for the prevention and/or treatment of lung cancer and other types of cancer, and for the control of metastatic spread of lung cancer and other types of cancer, wherein such regimens are based on modulating the expression and/or activity of an L53 molecule. In a specific embodiment, the invention is directed to methods wherein antagonists or agonists of L53 activity are used to efficaciously treat a cancer patient.

[0083] The invention further provides for screening assays and methods of identifying agonists and antagonists of an L53 gene or gene product. The invention also provides methods of vaccinating an individual against cancer (e.g., lung cancer), by administering an L53 gene, gene product, or fragment thereof, in an amount that effectively elicits an immune response in a subject who has cancer or is at risk of developing cancer.

5.1. THE L53 GENE

[0084] A nucleotide sequence comprising the open reading frame of the L53 gene is described herein. The L53 cDNA (3089bp) SEQ ID NO: 1 was cloned by PCR using gene-specific primers. The L53 sequence contains an open reading frame (SEQ JD NO: 2) spanning 168-2954bp within SEQ JD NO: 1 that encodes a protein of 929 amino acids (SEQ JD NO: 3).

[0085] The L53 nucleic acids and derivatives used in the present invention include but are not limited to RNA corresponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived tlierefrom, including but not limited to RNAs comprising SEQ JD NO: 1, or SEQ ID NO: 2; a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ ID NO: 2, or a complement of any of the foregoing nucleic acids; a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ JD NO: 2, or at least 90% homologous to the complement of any of the foregoing nucleic acids (e.g., as determined using the NBLAST algorithm under default parameters). As used herein an "RNA conesponding to SEQ JD NO: 1, or SEQ ID NO: 2, means an RNA comprising a sequence that is the same or the (inverse) complement of SEQ JD NO: 1, or SEQ ID NO: 2, except that thymidines (T's) can be replaced with uridines (U's). Such RNAs corresponding to SEQ JD NO: 1, or SEQ ID NO: 2, include, for example, RNA encoded by a gene that gives rise to a cDNA of SEQ LD NO: 1, or SEQ JD NO: 2, as well as RNA of which the cDNA of SEQ JD NO: 1, or SEQ ID NO: 2, is a copy. A nucleic acid derived from such RNA includes but is not limited to cDNA of said RNA, and cRNA (e.g., RNA that is derived from said cDNA; see, e.g., U.S. Patent Nos. 5,545,522; 5,891,636; 5,716,785). In the present invention, the ability to hybridize may be detennined under low, moderate, or high stringency conditions and preferably is under conditions of high stringency.

[0086] The L53 protein and derivatives used in the present invention include, but are not limited to proteins (and other molecules) comprising SEQ JD NO: 3, proteins comprising a sequence encoded by the hybridizable (complementary) portion of a nucleic acid hybridizable to SEQ ID NO: 1, or SEQ JD NO: 2, or their complements, and proteins encoded by a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ ID NO: 2, or their complement, e.g., as detennined using the NBLAST algorithm.

[0087] The L53 nucleic acids used in the present invention include but are not limited to (a) a nucleic acid sequence comprising the DNA sequence shown in FIG. 1 (SEQ ID NO: 1 and SEQ LD NO: 2), or a complement thereof; (b) any DNA sequence that hybridizes to the DNA sequences or their complements that encode the amino acid sequences shown in FIG.5, under low, moderate or highly stringent conditions, as disclosed infra in Section 5.1.1; as well as proteins encoded by such nucleic acids. In a specific embodiment, nucleic acids used in the invention encode a gene product that has at least one conservative or silent substitution. The encoded proteins are also provided for use. Additional molecules that can be used in the invention include, but are not limited to, protein derivatives that can be made by altering their sequences by substitutions, additions or deletions, and their encoding nucleic acids. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as a component gene or cDNA can be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the component protein gene that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a component protein, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a functionally silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (a "conservative amino acid substitution") that acts as a functional equivalent, resulting in a conservative alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycfne, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. fθ088]" The invention includes the use of the L53 gene coding sequences which preferably hybridize under highly stringent or moderately stringent conditions as described infra in Section 5.1.1 to at least about 6, preferably about 12, more preferably about 18, consecutive nucleotides of the L53 gene sequences described above as being useful for the detection of an L53 gene product for the diagnosis and prognosis of cancer, e.g., an RNA conesponding to SEQ JD NO: 1, or SEQ JD NO: 2, or a nucleic acid derived therefrom; a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency; a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1, or SEQ JD NO: 2, or its complement under conditions of high stringency; a nucleic acid at least 90% homologous to SEQ ID NO: 1, or SEQ JD NO: 2, or its complement as detennined using the NBLAST algorithm.

[0089] The invention also includes the use of nucleic acid molecules, preferably DNA molecules, that preferably hybridize under highly stringent or moderately stringent conditions as described infra in Section 5.1.1 to, and are therefore the inverse complements of, the nucleic acid sequences (a) and (d)-(i), described, inter alia, in Section 3 above. These nucleic acid molecules may encode or act as L53 gene coding sequence antisense molecules useful, for example, in L53 gene regulation. With respect to L53 gene regulation, such techniques can be used to modulate, for example, the phenotype and metastatic potential of lung cancer or other cancer cells. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for L53 gene regulation and thus may be used for the treatment and/or prevention of cancer.

[0090] In one embodiment, the invention encompasses methods of using the L53 gene coding sequence or fragments and degenerate variants of DNA sequences which encode the L53 gene or gene product, including naturally occurring and non-naturally occurring variants thereof. A non-naturally occurring variant is one that is engineered by man. A naturally occurring L53 gene, gene product, or variant thereof is one that is not engineered by man. In the methods of the invention wherein an L53 gene product in a sample derived from a subject is detected or measured, naturally occuning L53 gene products are detected, including, but not hmited to wild-type L53 gene products as well as mutants, allelic variants, splice variants, polymorphic variants, etc. In general, such mutants and variants are believed to be highly homologous to SEQ JD NO: 1 or SEQ JD NO: 2 or at least 90% homologous and/or hybridizable under high stringency conditions. In specific embodiments, the mutants and variants being detected or measured may comprise (or, if nucleic acids, encode) not more than 1, 2, 3, 4, or 5 point mutations (substitutions) relative to SEQ JD NO: 1, or SEQ JD NO: 2 and/or comprise or encode only conservative amino acid substitutions.

[0091] In other methods of the invention, wild-type, or naturally occuning variant, or non- naturally occurring variant L53 sequences may be used in the methods of the invention (e.g., in vaccination, immunization, antisense, or ribozyme procedures).

[0092] An L53 gene fragment may be a complementary DNA (cDNA) molecule or a genomic DNA molecule that may comprise one or more intervening sequences or introns, as well as regulatory regions located beyond the 5' and 3' ends of the coding region or within an intron. [0093] The present invention provides for methods of using isolated nucleic acid molecules encoding an L53 protein, polypeptide, or fragments, derivatives, and variants thereof that include, both naturally occuning and non-naturally occuning variants or mutants. The invention also contemplates, for use in the methods of the invention, the use of 1) any nucleic acid that encodes an L53 polypeptide of the invention; 2) any nucleic acid that hybridizes to the complement of the sequences disclosed herein, preferably under highly stringent conditions as disclosed infra in Section 5.1.1, and encodes a functionally equivalent gene product; and/or 3) any nucleic acid sequence that hybridizes to the complement of the sequences disclosed herein, preferably under moderately stringent conditions, as disclosed infra in Section 5.1.1 yet which still encodes a gene product that displays a functional activity of L53.

[0094] As discussed above, the invention also contemplates the use of isolated nucleic acid molecules that encode a variant protein or polypeptide. The variant protein or polypeptide can occur naturally or non-naturally. It can be engineered by introducing nucleotide substitutions, e.g., point mutations, or additions or deletions into the nucleotide sequence of SEQ ED NO: 1, or SEQ ID NO: 2. In a specific embodiment, one or more, but not more than 5, 10, or 25 amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0095] In a specific embodiment, the invention provides for the use of L53 derivatives and analogs of the invention which are functionally active, i.e. , they are capable of displaying one or more known functional activities associated with a (wild-type) L53 encoded protein. Such functional activities include but are not limited to antigenicity/immunogenicity (e.g., ability to bind or compete with L53 for binding to an anti-L53 antibody, ability to generate antibody which binds to L53), ability to bind or compete with L53 for binding to other proteins or fragments thereof, ability to bind or compete with L53 for binding to a receptor for L53.

[0096] Using all or a portion of the nucleic acid sequences of SEQ ID NO: 1, or SEQ JD NO: 2, for example SEQ ID NO: 1, or SEQ ID NO: 2, or portions thereof, as a hybridization probe, nucleic acid molecules encoding an L53 gene product can be isolated using standard hybridization and cloning techniques (See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) for use in the methods of the invention.

[0097] In addition, gene products encoded by L53, including L53 peptide fragments, as well as specific or selective antibodies thereto, can be used for construction of fusion proteins to facilitate recovery, detection, or localization of another protein of interest. In addition, genes and gene products encoded by L53 (e.g., L53) can be used as research reagents, e.g., for genetic mapping.

[0098] Additionally, the present invention contemplates use of the nucleic acid molecules, polypeptides, and/or antagonists or agonists of gene products encoded by the L53 gene to screen, diagnose, prevent and/or treat disorders characterized by abenant expression and/or activity of the L53 polypeptides, which include, cancers, such as but not limited to cancer of the lung, pancreas, colon, breast, and brain.

[0099] The present invention encompasses the use of L53 nucleic acid molecules comprising cDNA, genomic DNA, introns, exons, promoter regions, 5' and 3' regulatory regions of the gene, RNA, hnRNA, mRNA, regulatory regions within RNAs, and degenerate variants thereof in the methods of the invention. Promoter sequences for L53 can be determined by promoter-reporter gene assays and in vitro binding assays.

[0100] In one embodiment, the invention comprises the use of a variant L53 nucleic acid sequence that hybridizes to a naturally-occuning or non-naturally occurring variant L53 nucleic acid molecule under stringent conditions as described infra in Section 5.1.1. In another embodiment, the invention contemplates the use of an L53 variant nucleic acid sequence that hybridizes to a naturally- occurring or non-naturally occuning variant L53 nucleic acid molecule under moderately stringent conditions as described infra in Section 5.1.1.

[0101] A nucleic acid molecule is intended to include DNA molecules (e.g., cDNA, genomic DNA), RNA molecules (e.g., hnRNA, pre-mRNA, mRNA), and DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded.

[0102] The L53 sequences used in the methods of the invention are of human origin, however, homologs of L53 isolated from other mammals may also be used in the methods of the invention. Thus, the invention also includes the use of L53 homologs isolated from non-human animals such as: non- human primates; rats; mice; farm animals including, but not limited to: cattle; horses; goats; sheep; pigs; etc.; household pets including, but not limited to: cats; dogs; etc. in the methods of the invention.

[0103] Still further, such molecules may be used as components of diagnostic and/or prognostic methods whereby, for example, the presence of a particular L53 allele or alternatively spliced L53 transcript responsible for causing or predisposing one to lung cancer or other cancers may be detected.

[0104] The invention also includes the use of transcriptional regulators that control the level of expression of an L53 gene product. A transcriptional regulator can include, e.g., a protein which binds a DNA sequence and which up-regulates or down-regulates the transcription of the L53 gene. A transcriptional regulator can also include a nucleic acid sequence that can be either upstream or downstream from the L53 gene and which binds an effector molecule that enhances or suppresses L53 gene transcription.

[0105] Still further, the invention encompasses the use of L53 gene coding sequences or fragments thereof as a screen in an engineered yeast system, including, but not limited to, the yeast two hybrid system as a method to identify proteins, peptides or nucleic acids related to the onset and/or metastatic spread of cancer, including lung cancer.

[0106] The invention also encompasses the use of (a) DNA vectors that contain any of the foregoing L53 coding sequences and/or their complements (e.g., antisense); (b) DNA expression vectors that contain any of the foregoing L53 coding sequences operatively associated with a regulatory element that directs the expression ot the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing L53 coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. Cell lines and/or vectors which contain and/or express L53 can be used to produce the L53 gene product for use in the methods of the invention, e.g., vaccination against lung cancer or other cancers in which expression of L53 is found to be elevated and screening assays for antagonists and agonists that bind, or interact with L53 or suppress or enhance expression of L53.

[0107] As used herein, regulatory elements include, but are not limited to inducible and non- inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus (hCMV) immediate early promoter, the early or late promoters of S V40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors.

[0108] The invention includes the use of fragments or derivatives of any of the nucleic acids disclosed herein in any of the methods of the invention. In various embodiments, a fragment or derivative comprises 10, 20, 50, 100, or 200 nucleotides of SEQ JD NO: 1, or SEQ JD NO: 2 or encodes all or a fragment of SEQ JD NO: 1 or SEQ JD NO: 2. In the same or alternative embodiments, a nucleic acid is not more than 500, 1000, 2000, 5000, 7500, or 10,000 nucleotides in size.

[0109] In addition to the use of the L53 gene sequences described above, homologs of such sequences, exhibiting extensive homology to the L53 gene product present in other species can be identified and readily isolated, and used in the methods of the invention without undue experimentation by molecular biological teclmiques well known in the art. Further, there can exist homologous genes at other genetic loci within the genome that encode proteins that have extensive homology to L53. Alternatively, such homologous genes can encode a single protein with homology to L53. These genes can also be identified via similar techniques and used in the methods of the invention. Still further, there can exist alternatively spliced variants of the L53 gene. The invention thus includes the use of any of these homologs in the methods of the invention.

[0110] As an example, in order to clone a mammalian L53 gene homolog or variants using isolated human L53 gene sequences as disclosed herein, such human L53 gene sequences are labeled and used to screen a cDNA library constructed from rnRNA obtained from appropriate cells or tissues (e.g., bronchial epithelial cells) derived from the organism of interest. With respect to the cloning of such a mammalian L53 homolog, a mammalian lung cancer cell cDNA library may, for example, be used for screening. In one embodiment, such a screen would employ a probe conesponding to all or a portion of the L53 open reading frame SEQ JD NO: 2. hi yet another embodiment, such a screen would employ one or more probes conesponding to all or a portion of the coding sequence for L53 (SEQ JD NO: 2), for example, a probe conesponding to the L53 cDNA (SEQ JD NO: 2). [ l^'ll] The hybndrzation and wash conditions used should be of a low stringency, as described infra in Section 5.1.1 when the cDNA library is derived from a different type of organism than the one from which the labeled sequence was derived.

[0112] Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions well known to those of skill in the art.

[0113] Further, an L53 gene homolog may be isolated from nucleic acid of the organism of interest by perfoπning PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within an L53 encoded gene product. The template for the reaction may be cDNA obtained by reverse transcription of either total RNA or mRNA prepared from, for example, mammalian cell lines or tissue known or suspected to express an L53 gene homology or allele.

[0114] The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an L53 related nucleic acid sequence. The PCR fragment may then be used to isolate an L53 cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

[0115] PCR technology may be utilized to isolate cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (e.g., one known, or suspected, to express an L53 gene, such as, for example, lung cancer cell lines). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific or selective for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of PCR technology and cloning strategies which may be used, see, e.g., PCR Primer, 1995, Dieffenbach et al, ed., Cold Spring Harbor Laboratory Press; Sambrook et al, 1989, supra.

[0116] L53 gene coding sequences may additionally be used to isolate L53 gene alleles and mutant L53 gene alleles. Such mutant alleles may be isolated from individuals either known or susceptible to or predisposed to have a genotype that contributes to the development of cancer, e.g., lung cancer, including metastasis. Such mutant alleles may also be isolated from individuals either known or susceptible to or predisposed to have a genotype that contributes to resistance to the development of cancer, e.g., lung cancer, including metastasis. Mutant alleles and mutant allele products may then be utilized in the screening, therapeutic and diagnostic methods and systems described herein. Additionally, such L53 gene sequences can be used to detect L53 gene regulatory (e.g., promoter) defects that can affect the development and outcome of cancer. Mutants can be isolated by any technique known in the art, e.g., PCR, screening genomic libraries, screening expression libraries.

[0117] As described below, the invention also relates to the use of an L53 gene coding sequence or gene product in the methods of the invention. An L53 gene coding sequence or gene product includes, but'is^'not limited to an RNA conesponding to SEQ JD NO: 1, or SEQ JD NO: 2, a nucleic acid derived therefrom, a protein comprising SEQ JD NO: 3, or a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, or SEQ JD NO: 2, under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence or a nucleic acid at least 90% homologous to SEQ JD NO: 1, or SEQ ID NO: 2, as determined using the NBLAST algorithm or a protein encoded thereby.

5.1.1 HYBRIDIZATION CONDITIONS

[0118] A nucleic acid which is hybridizable to an L53 nucleic acid (e.g., having a sequence as set forth in SEQ ID NO: 1, or SEQ JD NO: 2, or to its reverse complement, or to a nucleic acid encoding an L53 derivative, or to its reverse complement under conditions of low stringency) can be used in the methods of the invention to detect the presence of an L53 gene and/or presence or expression level of an L53 gene product. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% FicoU, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% FicoU, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10^δ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and re-exposed to film. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).

[0119] A nucleic acid which is hybridizable to an L53 nucleic acid (e.g., having a sequence as set forth in SEQ JD NO: 1, or SEQ JD NO: 2, or to its reverse complement, or to a nucleic acid encoding an L53 derivative, or to its reverse complement under conditions of high stringency) is also provided for use in the methods of the invention. By way of example and not limitation, procedures using such conditions of high stringency are as follows, Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% FicoU, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. Other conditions of high stringency that may be used are well known in the art.

[0120] A nucleic acid which is hybridizable to an L53 nucleic acid (e.g., having a sequence as set forth in SEQ ID NO: 1, or SEQ JD NO: 2, or to its reverse complement, or to a nucleic acid encoding an L53^' derivative, or to its reverse complement under conditions of moderate stringency) is also provided for use in the methods of the invention. For example, but not limited to, procedures using such conditions of moderate stringency are as follows: filters comprising immobUized DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with 5-20 x 10⁶ cpm ³²P-labeled probe. Filters are incubated in hybridization mixture for 18-20 hours at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing IX SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.1% SDS. Other conditions of moderate stringency that may be used are well known in the art. (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see also, Ausubel et al., eds., in the Cunent Protocols in Molecular Biology series of laboratory technique manuals, 1987-1997 Cunent Protocols,© 1994-1997 John Wiley and Sons, Inc.).

5.2. PROTEINPRODUCTS OF THE L53 GENE

[0121] In another embodiment, the present invention provides for the use of L53 gene products, including L53, or peptide fragments thereof which can be used for the generation of antibodies, in diagnostic assays, or for the identification of other cellular gene products involved in the development of cancer, such as, for example, lung cancer.

[0122] The amino acid sequences depicted in FIG. 5 represent an example of an L53 gene product, i.e., L53 (SEQ ID NO: 3). The L53 gene products, sometimes refened to herein as an "L53 protein" or "L53 polypeptides," may additionally include those gene products encoded by the L53 gene sequences (SEQ ID NOs: 1 and 2) described hereinabove.

[0123] In addition, L53 derivatives may include proteins that have conservative amino acid substitution(s) and/or display a functional activity of an L53 gene product. Such a derivative may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the L53 gene sequences described, above, in Section 5.1, but which result in a silent change, thus producing a functionally equivalent L53 gene product.

[0124] In a specific embodiment, the invention provides a functionally equivalent protein that exhibits a substantially similar in vivo activity as that of an endogenous L53 gene product encoded by an L53 gene sequence described in Section 5.1, above. An in vivo activity of the L53 gene product can be exhibited by, for example, preneoplastic and/or neoplastic transformation of a ceU upon overexpression of the gene product, such as for example, may occur in the onset, progression, and/or metastasis of lung cancer.

[0125] An L53 gene product sequence preferably comprises an amino acid sequence that exhibits at least about 65% sequence similarity to L53, more preferably exhibits at least 70% sequence similarity to L53, yet more preferably exhibits at least about 75% sequence similarity to L53. In other embodiments, the L53 gene product sequence preferably comprises an amino acid sequence that exhibits ^"at least 85% sequence sixnilarity to L53, yet more preferably exhibits at least 90% sequence similarity to to L53, and most preferably exhibits at least about 95% sequence similarity to L53.

[0126] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A prefened, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc Natl Acad Sci. 87:2264- 2268, modified as in Karlin and Altschul (1993) Proc Natl Acad Sci. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403- 410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0127] Another particular, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=l , single aligned amino acids are examined, ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. For a further description of FASTA parameters, see http:/ bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2.

[0128] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps, hi calculating percent identity, only exact matches are counted. However, conservative substitutions should be considered in evaluating sequences that have a low percent identity with the L52 sequences disclosed herein.

[0129] In a specific embodiment, molecules or protein comprising at least 10, 20, 30, 40 or 50 amino acids of SEQ JD NO: 3, or at least 10, 20, 30, 40, 50, 75, 100, or 200 a ino acids of SEQ JD NO: 3 are used in the present invention. 5.2.1 FUSION PROTEINS

[0130] L53 gene products can also include fusion proteins comprising an L53 gene product sequence as described above operatively associated to a heterologous, component, e.g., peptide for use in the methods of the invention. Heterologous components can include, but are not limited to sequences that facilitate isolation and purification of fusion protein, or label components. Heterologous components can also include sequences that confer stability to the L53 gene product. Such isolation and label components are well known to those of skill in the art.

[0131] The present invention encompasses the use of fusion proteins comprising the protein or fragment thereof encoded by the L53 gene open reading frame of SEQ ID NO: 2 and a heterologous polypeptide (i.e., an unrelated polypeptide or fragment thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide). The fusion can be direct, but may occur through linker sequences. The heterologous polypeptide may be fused to the N-terminus or C-terminus of an L53 gene product.

[0132] A fusion protein can comprise an L53 gene product fused to a signal sequence at its N- teirminus. Various signal sequences are commercially available. Eukaryotic heterologous signal sequences include, but are not limited to, the secretory sequences of honeybee melittin (Invitrogen Corporation; Carlsbad, Calif.) and human placental alkaline phosphatase (Stratagene; La Jolla, California). Prokaryotic heterologous signal sequences useful in the methods of the invention include, but are not limited to, the phoA secretory signal (Sambrook et al, eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

[0133] The L53 protein or fragment thereof encoded by the L53 open reading frame of SEQ ID NO: 2 or a fragment thereof can be fused to nucleic acid sequences encoding a tag sequence, e.g., a hexa- histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA, 91311). Additional tag moieties are commercially available and may be used to advantage to in the methods of the invention. As described in Gentz et αl, 1989, Proc. Nαtl. Acαd. Sci. USA, 86:821-824, for instance, hexa- histidine provides for convenient purification of the fusion protein. Other examples of peptide tags are the hemagglutinin "HA" tag, which conesponds to an epitope derived from the influenza hemagglutinin protein (Wilson et cd., 1984, Cell, 37:767) and the "flag" tag (KnappUc et αl., 1994, Biotechniques, 17(4):754-761). These tags are especially useful for purification and detection of recombinantly produced polypeptides of the invention.

[0134] Any fusion protein may be readily purified by utilizing an antibody specific or selective for the fusion protein being expressed. For example, a system described by Janknecht et αl. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et αl, 1991, Proc. Nαtl. Acαd. Sci. USA 88:8972). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an arnino- teπninal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Nr'-mtnloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

[0135] An affinity label can also be fused at its amino terminal to the carboxyl terminal of the protein or fragment thereof encoded by an L53 open reading frame SEQ JD NO: 2 for use in the methods of the invention. The precise site at which the fusion is made in the carboxyl terminal is not critical. The optimal site can be detennined by routine experimentation. An affinity label can also be fused at its carboxyl terminal to the amino terminal of the L53 gene product for use in the methods of the invention.

[0136] A variety of affinity labels known in the art may be used, such as, but not limited to, the immunoglobulin constant regions, (Petty, 1996, Metal-chelate affinity chromatography, in Cunent Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell Bio. 4:220-229), the E. coli maltose binding protein (Guan et al, 1987, Gene 67:21-30), and various cellulose binding domains (U.S. Patent Nos. 5,496,934; 5,202,247; 5,137,819; Tomme et al, 1994, Protein Eng. 7:117-123), etc. Other affinity labels may impart fluorescent properties to an L53 gene product, e.g., green fluorescent protein and the like. Other affinity labels are recognized by specific binding partners and thus facilitate isolation by affinity binding to the binding partner that can be immobUized onto a solid support. Some affinity labels may afford the L53 gene product novel structural properties, such as the ability to form multimers. These affinity labels are usually derived from proteins that normally exist as homopolymers. Affinity labels such as the extracellular domains of CD8 (Shiue et al, 1988, /. Exp. Med. 168:1993-2005), or CD28 (Lee et al, 1990, J. Immunol 145:344-352), or fragments of the immunoglobulin molecule containing sites for interchain disulfide bonds, could lead to the formation of multimers.

[0137] As will be appreciated by those skilled in the art, many methods can be used to obtain the coding region of the above-mentioned affinity labels, including but not limited to, DNA cloning, DNA amplification, and synthetic methods. Some of the affinity labels and reagents for their detection and isolation are available commercially.

[0138] A prefened affinity label is a non-variable portion of the immunoglobulin molecule. Typically, such portions comprise at least a functionally operative CH2 and CH3 domain of the constant region of an immunoglobulin heavy chain. Fusions are also made using the carboxyl terminus of the Fc portion of a constant domain, or a region immediately arrjdno-terminal to the CHI of the heavy or light chain. Suitable immunoglobulin-based affinity label may be obtained from IgG-1, -2, -3, or -4 subtypes, IgA, IgE, IgD, or IgM, but preferably IgGl. Preferably, a human immunoglobulin is used when the L53 gene product is intended for in vivo use for humans. Many DNA encoding immunoglobulin light or heavy chain constant regions are known or readily available from cDNA libraries. See, for example, Adams et al, Biochemistry, 1980, 19:2711-2719; Gough et αZ., 1980, Biochemistry, 19:2702-2710; Dolby et al, 1980, Proc. Natl. Acad. Sci. U.S.A., 77:6027-6031; Rice et al, 1982, Proc. Natl. Acad. Sci. U.S.A., 79:7862-7865; Falkner et al, 1982, Nature, 298:286-288; and Monison et al, 1984, Ann. Rev. Immunol, 2:239-256. Because many immunological reagents and labeling systems are available for the detection of immunoglobulins, the L53 gene product-Ig fusion protein can readily be detected and quantified by a variety of immunological techniques known in the art, such as the use of enzyme-linked immunosorbent assay (ELISA), i munoprecipitation, fluorescence activated cell sorting (FACS), etc. Similarly, if the affinity label is an epitope with readily avaUable antibodies, such reagents can be used with the techniques mentioned above to detect, quantitate, and isolate the L53 gene product containing the affinity label. In many instances, there is no need to develop specific or selective antibodies to the L53 gene product.

[0139] A fusion protein can comprise an L53 gene product fused to the Fc domain of an immunoglobulin molecule or a fragment thereof for use in the methods of the invention. A fusion protein can also comprise an L53 gene product fused to the CH2 and/or CH3 region of the Fc domain of an immunoglobulin molecule. Furthermore, a fusion protein can comprise an L53 gene product fused to the CH2, CH3, and hinge regions of the Fc domain of an immunoglobulin molecule (see Bowen et al, 1996, J. Immunol. 156:442-49). This hinge region contains three cysteine residues that are normally involved in disulfide bonding with other cysteines in the Ig molecule. Since none of the cysteines are required for the peptide to function as a tag, one or more of these cysteine residues may optionally be substituted by another amino acid residue, such as for example, serine.

[0140] Various leader sequences known in the art can be used for the efficient secretion of the L53 gene product from bacterial and mammalian cells (von Heijne, 1985, J. Mol. Biol. 184:99-105). Leader peptides are selected based on the intended host cell, and may include bacterial, yeast, viral, animal, and mammalian sequences. For example, the herpes virus glycoprotefn D leader peptide is suitable for use in a variety of mammalian cells. A prefened leader peptide for use in mammalian cells can be obtained from the V-J2-C region of the mouse immunoglobulin kappa chain (Bernard et al, 1981, Proc. Natl. Acad. Sci. 78:5812-5816). Prefened leader sequences for targeting L53 gene product expression in bacterial cells including, but not limited to, the leader sequences of the E.coli proteins OmpA (Hobom et al, 1995, Dev. Biol. Stand. 84:255-262), Pho A (Oka et al, 1985, Proc. Natl. Acad. Sci 82:7212-16), OmpT (Johnson et al, 1996, Protein Expression 7:104-113), La B and OmpF (Hoffman & Wright, 1985, Proc. Natl. Acad. Sci. USA 82:5107-5111), β-lactamase (Kadonaga et al, 1984, J. Biol Chem. 259:2149-54), enterotoxins (Morioka-Fujimoto et al, 1991, J. Biol. Chem. 266:1728- 32), Staphylococcus aureus protein A (Abrahmsen et al, 1986, Nucleic Acids Res. 14:7487-7500), and the B. subtϊlis endoglucanase (Lo et al, Appl Environ. Microbial 54:2287-2292), as well as artificial and synthetic signal sequences (Maclntyre et al, 1990, Mol. Gen. Genet. 221:466-74; Kaiser et al, 1987, Science, 235:312-317).

[0141] A fusion protein can comprise an L53 gene product and a cell permeable peptide, which facilitates the transport of a protein or polypeptide across the plasma membrane for use in the methods of the invention. Examples of cell permeable peptides include, but are not limited to, peptides derived from hepatitis B virus surface antigens (e.g., the PreS2- domain of hepatitis B virus surface antigens), herpes simplex virus VP22, antennapaedia, 6H, 6K, and 6R. See, e.g., Oess et al, 2000, Gene Ther. 7:750-758, DeRossi et al, 1998, Trends Cell Biol 8(2):84-7, and Hawiger, 1997, J. Curr Opin Immunol 9(2):189-94.

[0142] Fusion proteins can be produced by standard recombinant DNA teclmiques or by protein synthetic techniques, e.g., by use of a peptide synthesizer. For example, a nucleic acid molecule encoding rfusionprotein can oe synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be canied out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, 1992).

[0143] The nucleotide sequence coding for a fusion protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The expression of a fusion protein may be regulated by a constitutive, inducible or tissue-specific, or selective promoter. It will be understood by the skilled artisan that fusion proteins, which can facilitate solubility and/or expression, and can increase the in vivo half-life of an L53 protein (SEQ ID NO: 3) or fragment thereof and thus are useful in the methods of the invention. The L53 gene products or peptide fragments thereof, or fusion proteins can be used in any assay that detects or measures L53 gene products or in the calibration and standardization of such assays.

[0144] The methods of the invention encompass the use of L53 gene products or peptide fragments thereof, which may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the L53 gene polypeptides and peptides of the invention by expressing nucleic acid containing L53 gene sequences are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing L53 gene product coding sequences including but not limited to SEQ JD NO: 2 and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al, 1989, supra, and Ausubel et al, 1989, supra. Alternatively, RNA capable of encoding L53 gene product sequences may be chemically synthesized using, for example, synthesizers (see e.g., the techniques described in Oligonucleotide Synthesis, 1984, Gait, M.J. ed., IRL Press, Oxford).

5.2.2 EXPRESSION SYSTEMS

[0145] A variety of host-expression vector systems may be utilized to express the L53 gene coding sequences for use in the methods of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the L53 gene product of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtϊlis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing L53 gene product coding sequences; yeast (e.g., Saccharomyces, Picliia) transformed with recombinant yeast expression vectors containing the L53 gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) conta iing the L53 gene product coding sequences; plant ceU systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing L53 gene product codmg sequences; or mammalian ceU systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metaUofhionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0146] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the L53 gene product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of L53 protein or for raising antibodies to L53 protein, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO J. 2:1791), in which the L53 gene product coding sequence may be ligated into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose bead followed by elution in the presence of free glutathione. The pGEX vectors are designed to include, e.g., thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0147] In an insect system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The L53 gene coding sequence may be cloned into a non-essential region (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of L53 gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., see Smith et al, 1983, J. Virol. 46:584; Smith, U.S. Patent No. 4,215,051).

[0148] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the L53 gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may be inserted in the adenovirus genome by in vitro or in vivo recombination, insertion in a non-essential region of the viral genome (e.g. , region El or E3) results in a recombinant virus that is viable and capable of expressing an L53 gene product in infected hosts. (See, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655). Specific initiation signals may also be required for efficient translation of inserted L53 gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire L53 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional ranslational control signals may be needed. However, in cases where only a portion of the L53 gene coding sequence is mserted, exogenous translational control signals, including, perhaps, an ATG initiation codon, may be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al, 1987, Methods in Enzymol 153:516).

[0149] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the conect modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, lung cancer cell lines such as, for example, A549, NCI-H920, NCI-H969, NCI- H23, NCI-H226, NCI-H647, NCI-H1869, NCI-HH1385, NCI-H460, NCI-H1155, NCI-H358, and NCI- H650.

[0150] For long-term, high-yield production of recombinant proteins, stable expression is prefened. For example, cell lines that stably express the L53 gene product may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter and/or enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the L53 gene product. Such engineered cell lines may be particularly useful in the screening and evaluation of compounds that are capable of altering endogenous activity of the L53 gene product.

[0151] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidme kinase (Wigler et al, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to mefhotrexate (Wigler et al, 1980, Proc Natl. Acad. Sci. USA 77:3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (MuUigan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers ^"resistance to the aminoglycoside G418 (Colbene-Garapin et al, 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santene et al, 1984, Gene 30:147).

5.2.3 L53 TRANSGENIC ANIMALS

[0152] L53 gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, sheep, pigs, micro-pigs, goats, and non- human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate L53 transgenic animals.

[0153] Transgenic animals that over- or mis-express an L53 gene product may be used in any of the methods of the invention. For example transgenic animals may be used to study the in vivo effects of enhanced expression levels of L53 and the onset, diagnosis and/or prognosis of cancer. Transgenic animals are useful for screening compounds to identify antagonists or agonists of L53 activity. Transgenic animals could be used to screen the in vivo effects of anti-sense or ribozyme therapeutic molecules in the treatment of cancer. Transgenic animals could be used to screen for methods of vaccinating against cancer using an L53 gene product or a portion thereof.

[0154] Further, L53 knock out animals are also useful in the methods of the invention. For example, animals with disruptions in only L53 are useful in assessing the relative contribution of L53 gene products to the cancer state, as well as assessing the positive effect of a cancer therapeutic candidate.

[0155] For over- or mis-expression of an L53 gene product, any technique known in the art may be used to introduce the L53 gene product into animals to produce founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into genn lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci. USA 82:6148); gene targeting in embryonic stem cells (Thompson et al, 1989, Cell 56:313); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803); and sperm-mediated gene transfer (Lavitrano et al, 1989, Cell 57:717); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115: 171.

[0156] The methods of the invention provide for the use of transgenic animals that carry the L53 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e. , mosaic animals.

[0157] The transgene may be integrated as a single transgene or in concatamers, e.g., head-to- head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al, 1992, Proc. Natl. Acad. Sci. USA 89:6232). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and are known to skilled artisans.

[0158] When it is desired that the L53 transgene be integrated into the chromosomal site of the endogenous L53 gene to disrupt the expression of the endogenous L53 gene, for example, targeting is prefened. Briefly, when such a technique is utilized, vectors containing some nucleotide sequences homologous to the endogenous L53 gene are designed for the purpose of promoting integration into the endogenous gene via homologous recombination. Such chromosomal integration may partially or wholly disrupt the function of the nucleotide sequence of the endogenous L53 gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous L53 gene in only that ceU type, by following, for example, the teaching of Gu et al. (Gu et al, 1994, Science 265:103). The regulatory sequences required for such a cell-type specific inactivation wiU depend upon the particular cell type of interest, and are apparent to those of skill in the art.

[0159] Methods for the production of single-copy transgenic animals with chosen sites of integration are also well known to skilled practitioners. See, for example, Bronson et al. (Bronson, S.K. et al, 1996, Proc. Natl Acad. Sci. USA 93:9067).

[0160] Once transgenic animals have been generated, the expression of the recombinant L53 gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot or PCR analysis of tissue derived from experimental animals to determine which animals possess an integrated transgene. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using teclmiques which include, but are not limited to: Northern blot analysis, in situ hybridization analysis, and RT-PCR. L53 gene-expressing samples may also be evaluated immunocytochemically using antibodies specific or selective for an L53 gene product.

5.3. ANTIBODIES TO L53 GENE PRODUCTS [0161] The methods of the present invention encompass the use of antibodies or fragments thereof capable of specifically or selectively recognizing one or more L53 gene product epitopes or epitopes of conserved variants or peptide fragments of the L53 gene products. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, Fv fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0162] Such antibodies may be used, for example, in the detection of an L53 gene product in a biological sample and may, therefore, be utilized as part of a diagnostic and/or prognostic technique whereby patients may be tested for abnormal or elevated levels of L53 gene products, and/or for the presence of abnormal forms of the such gene products. Such antibodies may also be included as a reagent in a kit for use in a diagnostic and/or prognostic technique. Such antibodies may also be utilized in conjunction with, for example, compound screening methods, as described, below, in Section 5.5, for the evaluation of the effect of test compounds on L53 gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with gene therapy techniques described below in Section 5.6.4, to, for example, evaluate the normal and/or engineered L53 expressing cells prior to their introduction into a patient.

[0163] Antibodies to the L53 gene product may additionally be used in a method for the inhibition of L53 gene product activity. Thus, such antibodies may, therefore, be utilized as part of cancer treatment methods. [0164] Described herein are methods for the production of antibodies or fragments thereof. Any of such antibodies or fragments thereof may be produced by standard irnmunological methods or by recombinant expression of nucleic acid molecules encoding the antibody or fragments thereof in an appropriate host organism.

[0165] For the production of antibodies against an L53 gene product, various host animals may be immunized by injection with an L53 gene product, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, for example. Various adjuvants may be used to increase the irnmunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium paiyum.

[0166] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as an L53 gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with L53 gene product(s) supplemented with adjuvants as described above.

[0167] Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen or epitope thereof, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495; and U.S. Patent No. 4,376,110), the human B- cell hybridoma technique (Kosbor et al, 1983, Immunology Today 4:72; Cole et al, 1983, Proc. Natl. Acad. Sci. USA 80:2026), and the EBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo renders this method a particularly prefened method of production of L53 antibodies.

[0168] Teclmiques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81, 6851-6855; Neuberger et al., 1984, Nature 312, 604-608; Takeda et al., 1985, Nature 314, 452-454), whereby the genes from a mouse antibody molecule of appropriate antigen specificity are spliced to genes from a human antibody molecule of appropriate biological activity, are also encompassed by the present invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., CabUly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 5,816,397). The invention thus contemplates chimeric antibodies that are specific or selective for an L53 gene product.

[0169] Examples of techniques that have been developed for the production of humanized antibodies are known in the art. (See, e.g., Queen, U.S. Patent No. 5,585,089 and Winter, U.S. Patent No. 5,225,539) An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, refened to as complementarity-deteπriining regions (CDRs). The extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Irnmunological Interest", Kabat, E. et al., U.S. Department of Health and Human Services (1983). Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and framework regions from a human immunoglobulin molecule. The invention includes the use of humanized antibodies that are specific or selective for an L53 gene product in the methods of the invention.

[0170] The methods of the invention encompass the use of an antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three complementarity-determining regions (CDRs), in which said set of CDRs are from a monoclonal antibody to a gene product encoded by an L53 nucleic acid sequence (e.g., SEQ ID NO: 1 or SEQ JD NO: 2), and (b) a set of four framework regions, in which said set of framework regions differs from the set of framework regions in the L53 monoclonal antibody, and in which said antibody or derivative thereof immunospecifically binds to the gene product encoded by an L53 gene sequence. Preferably, the set of framework regions is from a human monoclonal antibody, e.g., a human monoclonal antibody that does not bind the gene product encoded for by the L53 gene sequence.

[0171] Phage display technology can be used to increase the affinity of an antibody to an L53 gene product. This technique is useful i obtaining high affinity antibodies to an L53 gene product that are useful for the diagnosis and/or prognosis of a subject with cancer. The technology, refened to as affinity maturation, employs mutagenesis or CDR walking and re-selection using an L53 gene product antigen to identify antibodies that bind with higher affinity to the antigen when compared with the initial or parental antibody (see, e.g., Glaser et al, 1992, J. Immunology 149:3903). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be screened by contact with the immobilized mutants containing labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity to the antigen (e.g., ELISA) (See Wu et al, 1998, Proc Natl Acad Sci. USA 95:6037; Yelton et al, 1995, J. Immunology 155:1994). CDR walking which randomizes the light chain is also possible (See Schier et al, 1996, /. Mol. Bio. 263:551).

[0172] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242:423; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879; and Ward et al, 1989, Nature 334:544) can be adapted to produce single chain antibodies against L53 gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skena et al. , 1988, Science 242: 1038). [0173] The methods of the mvention include using an antibody to an L53 polypeptide, peptide or other derivative, or analog thereof that is a bispecific antibody (see generally, e.g. , Fanger and Drakeman, 1995, Drug News and Perspectives 8:133-137). Bispecific antibodies can be used for example to treat or prevent cancer in a subject that expresses elevated levels of an L53 gene product. Such a bispecific antibody is genetically engineered to recognize both (1) an epitope and (2) one of a variety of "trigger" molecules, e.g., Fc receptors on myeloid cells, and CD3 and CD2 on T-cells, that have been identified as being able to cause a cytotoxic T-cell to destroy a particular target. Such bispecific antibodies can be prepared either by chemical conjugation, hybridoma, or recombinant molecular biology techniques known to the skilled artisan.

[0174] Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al, 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

5.4. USES OF THE L53 GENE, GENE PRODUCTS, AND ANTD3QDIES

[0175] In various embodiments, the present invention provides various uses of the L53 gene, L53 polypeptides and peptide fragments thereof, and of antibodies directed against the L53 polypeptides and peptide fragments. Such uses include, for example, prognostic and diagnostic evaluation of cancer, and the identification of subjects with a predisposition to a cancer, as described, below. The invention also includes methods of treating and/or preventing cancer. The invention includes methods for vaccinating against cancer. The methods of the invention can be used for the treatment, prevention, vaccination, diagnosis, staging and/or prognosis of any cancer or tumor, including those listed below in Table 1, which is provided by way of non-limiting example.

[0176] Malignancies and related disorders that may be treated according to the methods of the present invention, include but are not limited to those listed in Table 1 (for a review of such disorders, see Fishman et al, 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):

TABLE 1

MALIGNANCIES AND RELATED DISORDERS

Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic monocytic erytliroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia

Polycythemia vera TABLE 1 Continued

Lymphoma

Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrόm's macroglobulmemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma

Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma

Wilms' tumor cervical cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma nemangioblastoma acoustic neuroma oligodendroglioma menangioma melanoma neuroblastoma retinoblastoma [0177] in a prefened embodiment, the methods of the invention are directed to diagnosis, prognosis, treatment and/or prevention of lung cancer. In other embodiments, the cancer is ovarian cancer, skin cancer, or a cancer of the lymphoid system.

[0178] The invention further provides for screening assays to identify antagonists or agonists of the L53 gene or gene product. Thus, the invention relates to methods to identify molecules that are capable of modulating (e.g., increase or decrease) the expression and/or activity of L53 molecules.

[0179] The nucleic acid molecules, proteins, protein homologs, and antibodies described herein may be used in one or more of the following methods, including but not limited to: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring efficacy of clinical trials, and pharmacogenomics); and d) methods of treatment (e.g., therapeutic and prophylactic). For example, an L53 gene product can be used to modulate (i) cellular proliferation; (ii) ceUular differentiation; and/or (iii) cellular adhesion. Isolated nucleic acid molecules that encode the L53 gene or a fragment thereof can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and/or to modulate expression/activity of an L53 polypeptide. In addition, an L53 gene product may be used to screen drugs or compounds to identify drugs or compounds capable of modulating the expression and/or activity of an L53 gene product. Such drugs or compounds may be used to treat disorders characterized by insufficient or excessive production of the L53 gene product or production of a form the L53 gene product which has decreased or abeπant activity as compared to that of the wild type protein. In addition, antibodies that specifically or selectively bind to an L53 gene product may be used to detect, isolate, and/or modulate activity of an L53 gene product.

[0180] In one embodiment, the present invention provides a variety of methods for the diagnostic and prognostic evaluation of cancer, including lung cancer. Such methods may, for example, utilize reagents such as the L53 gene nucleotide sequences described in Sections 5.1, and antibodies directed against L53 gene products, including peptide fragments thereof, as described above in Section 5.2. Specifically, such reagents may be used, for example, for: (1) the detection of the presence of L53 gene mutations, or the detection of abeπant expression of L53 gene mRNA relative to that of normal cells, or the qualitative or quantitative detection of other allelic forms of L53 transcripts which may conelate with lung cancer or susceptibihty toward neoplastic changes, and (2) the detection of an over-abundance of an L53 gene product relative to the non-disease state or relative to a predetermined non-cancerous standard or the presence of a modified (e.g., less than full-length) L53 gene product which coπelates with a neoplastic state or a progression toward neoplasia or metastasis.

[0181] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic test kits comprising at least one specific or selective L53 gene nucleic acid or anti-L53 antibody reagent described herein, which may be conveniently used, e.g., in clinical settings or in home settings, to diagnose patients exhibiting preneoplastic or neoplastic abnormalities, and to screen and identify those individuals exhibiting a predisposition to such neoplastic changes. [0182] Nucleic acid-based detection techniques are described, below, in Section 5.4.1. Peptide detection techniques are described, below, in Section 5.4.2.

5.4.1. DETECTION OF L53 GENE NUCLEIC ACID MOLECULES

[0183] i a prefened embodiment, the invention involves methods to assess quantitative and qualitative aspects of L53 gene expression, i one example the increased expression of an L53 gene or gene product indicates a predisposition for the development of cancer. Alternatively, enhanced expression levels of an L53 gene or gene product are indicative of the presence of cancer in a subject or the risk of metastasis of a cancer in said subject. Techniques well known in the art, e.g. , quantitative or semi-quantitative RT PCR or Northern blot, can be used to measure expression levels of L53. Methods that describe both qualitative and quantitative aspects of L53 gene or gene product expression are described in detail in the examples infra. The measurement of L53 gene expression levels may include measuring naturally occurring L53 transcripts and variants thereof as well as non-naturally occurring variants thereof. The diagnosis and/or prognosis of cancer in a subject, however, is preferably directed to detecting a naturally occurring L53 gene product or variant thereof. Thus, the invention relates to methods of diagnosing and/or predicting cancer in a subject by measuring the expression of an L53 gene in a subject. For example an increased level of mRNA encoded by an L53 nucleic acid sequence (e.g., SEQ JD NO: 1 or SEQ JD NO: 2), or other gene product, as compared to a non-cancerous sample or a noncancerous predetermined standard is indicative of the presence of cancer in said subject or an increased risk of developing cancer in said subject.

[0184] i another aspect of the invention, an increased level of mRNA encoded by an L53 nucleic acid sequencd (e.g., SEQ JD NO: 1 or SEQ ID NO: 2), or other related gene product, as compared to that of a non-cancerous sample or a non-cancerous predetermined standard is indicative of the stage of disease or the risk of metastasis of the cancer in a subject or the likelihood of a poor prognosis for said subject.

[0185] In another example, RNA from a cell type or tissue known, or suspected, to express an L53 gene, such as lung cancer cells, or other types of cancer cells, may be isolated and tested utilizing hybridization or PCR techniques as described above. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of an L53 gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of an L53 gene, including activation or suppression of L53 gene expression and presence of alternatively spliced L53 transcripts.

[0186] In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest by reverse transcription. AU or part of the resulting cDNA is then used as a template for a nucleic acid amplification reaction, such as a PCR or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the L53 gene nucleic acid reagents described in Section 5.1. The prefened lengths of such nucleic acid reagents are at least 9-30 nucleotides.

[0187] For detection of the amplified product, a nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid visualizing method.

[0188] RT-PCR techniques can be utilized to detect differences in L53 transcript size that may be due to normal or abnonnal alternative splicing. Additionally, such techniques can be performed using standard techniques to detect quantitative differences between levels of L53 transcripts detected in normal individuals relative to those individuals having cancer or exhibiting a predisposition toward neoplastic changes.

[0189] In an application wherein detection of particular alternatively spliced species is desired, appropriate primers and/or hybridization probes can be used, such that, in the absence of such a sequence, for example, no amplification products are produced. Alternatively, primer pahs may be chosen utilizing the sequence data depicted in FIG. 1 which will yield fragments of differing size depending on whether a particular exon is present or absent from the transcript of L53 being utilized.

[0190] As an alternative to amplification techniques, standard Northern analyses can be performed if a sufficient quantity of the appropriate cells can be obtained. The prefened length of a probe used in a Northern analysis is 9-50 nucleotides. Utilizing such techniques, quantitative as weU as size related differences between L53 transcripts are also detectable.

[0191] Additionally, it is possible to perform such L53 gene expression assays in situ, i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (see, e.g., Nuovo, G.J., 1992, PCR In Situ Hybridization: Protocols And Applications, Raven Press, NY).

[0192] Mutations or polymorphisms within an L53 gene can be detected utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as a starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to skilled artisans. For detection of L53 mutations, any nucleated cell can be used as a source of genomic nucleic acid. For detection of L53 transcripts or L53 gene products, any cell type or tissue in which the L53 gene is expressed, such as, for example, lung cancer ceUs may be utilized.

[0193] Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving L53 gene structure, including point mutations, insertions, deletions and chromosomal reaπangements. Such assays may include, but are not limited to, direct sequencing (Wong, C. et al, 1987, Nature 330:384), single stranded conformational polymorphism analyses (SSCP; Orita, M. et al, 1989, Proc. Natl. Acad. Sci. USA 86:2766), heteroduplex analysis (Keen, TJ. et al, 1991, Genomics 11:199; Perry, D.J. & Caπell, R.W., 1992), denaturing gradient gel electrophoresis (DGGE; Myers, R.M. et al, 1985, Nucl Acids Res. 13:3131), chemical mismatch rleavage (Cotton, R.G. etal, 1^'98^'8, Proc. Natl. Acad. Sci. USA 85:4397) and oligonucleotide hybridization (Wallace, R.B. et al, 1981, Nucl Acids Res. 9:879; Lipshutz, R.J. et al, 1995, Biotechniques 19:442).

[0194] Diagnostic methods for the detection of L53 nucleic acid molecules, in patient samples or other appropriate cell sources, may involve the amplification of specific gene sequences, e.g., by PCR (See Mullis, K.B., 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skUl in the art, such as, for example, those listed above. Utilizing analysis techniques such as these, amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of the L53 gene in order to determine whether an L53 gene mutation exists.

[0195] Well-known genotyping techniques can be performed to type polymorphisms that are in close proximity to mutations in the L53 gene. Such polymorphisms can be used to identify individuals in families likely to cany mutations. If a polymorphism exhibits linkage disequilibrium with mutations hi the L53 gene, it can also be used to identify individuals in the general population likely to carry mutations. Polymorphisms detectable by such methods include restriction fragment length polymorphisms (RFLPs), which involve sequence variations in restriction enzyme target sequences, single-nucleotide polymorphisms (SNPs) and simple sequence repeat polymorphisms (SSLPs).

[0196] For example, Weber (U.S. Pat. No. 5,075,217) describes a DNA marker based on length polymorphisms in blocks of (dC-dA)n(dG-dT)n short tandem repeats. The average separation of (dC- dA)n-(dG-dT)n blocks is estimated to be 30,000-60,000 bp. Markers that are so closely spaced exhibit a high frequency of co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the L53 gene, and the diagnosis of diseases and disorders related to L53 mutations.

[0197] Also, Caskey et al. (U.S. Pat.No. 5,364,759), describe a DNA profiling assay for detecting short tri- and tetra-nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the L53 gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map of an individual's DNA.

[0198] An L53 probe can be used to identify RFLPs directly. Additionally, an L53 probe or primers derived from the L53 sequence can be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids. The DNA contained in these clones can be screened for single-base polymorphisms or simple sequence length polymorphisms (SSLPs) using standard hybridization or sequencing procedures.

[0199] Alternative diagnostic methods for the detection of L53 gene expression, L53 gene mutations or polymorphisms include hybridization techniques which involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the specific or selective annealing of these reagents to their complementary sequences within the L53 gene. Preferably, the lengths of these nucleic acid reagents are at least 9 to 50 nucleotides. After incubation, all non- annealed nucleic acids are removed from the L53 hybrid molecule. The presence of nucleic acids that have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from a cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads or to a glass surface such as a microscope slide. For applications involving immobilization, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled L53 nucleic acid reagents is achieved using standard techniques well known to those in the art. The L53 gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal L53 gene sequence in order to determine whether an L53 gene mutation is present.

5.4.2. DETECTION OF L53 ENCODED PROTEINS

[0200] Detection of the L53 gene product includes the detection of the proteins encoded by SEQ ID NOs: 1 or 2. L53 proteins of the invention include SEQ ID NO: 3 and functional fragments thereof. Detection of elevated levels of L53 protein or polypeptides thereof, as compared to a non-cancerous sample or a non-cancerous predetermined standard, is indicative of the presence of, or predisposition to developing cancer in a subject. Detection of elevated levels of L53 protein or polypeptides thereof, in a subject as compared to a non-cancerous sample or a non-cancerous predetermined standard is also indicative of the likelihood of metastasis of a cancer in the subject, and/or poor prognosis for the subject. The diagnosis and/or prognosis of cancer pertain to the detection of naturally occuning L53 polypeptides in a subject. Detection of an L53 polypeptide may be by any method known in the art.

[0201] Antibodies directed against naturally occurring L53, or naturally occuning variants thereof or peptide fragments thereof, which are discussed in Section 5.2, may be used as diagnostics and prognostics, as described herein. Such diagnostic methods may be used to detect abnormalities in the level of L53 gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of the L53 encoded polypeptide. Antibodies, or fragments of antibodies, such as those described herein, may be used to screen therapeutic compounds in vitro to identify compounds capable of modulating L53 gene expression, L53 encoded polypeptide production and activity thereto. Compounds capable of modulating L53 activity and identified using the methods of the invention may be tested to determine their utUity as therapeutic compounds for the treatment of cancer patients (e.g., lung cancer patients). Accordingly, a skilled practitioner could determine a therapeuticaUy effective dose range for a cancer patient based on a number of parameters, including but not limited to the age, weight, and condition of the patient, the type and severity of the disease, and the treatment history of the patient.

[0202] The tissue or cell type to be analyzed generally includes those which are known, or suspected, to express the L53 gene, such as, for example, cancer cells including lung cancer cells, ovarian cancer ceUs, skin cancer cells, lymphoid cancer cells, and metastatic forms thereof. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step to test the effect of compounds on the expression of the L53 gene.

[0203] Prefened diagnostic methods for the detection of L53 gene products or conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the L53 gene products or conserved variants, including gene products which are the result of alternatively spliced transcripts, or peptide fragments are detected by their interaction with an anti-L53 gene product-specific or -selective antibody.

[0204] For example, antibodies, or fragments of antibodies, such as those described above in Section 5.3, may be used to quantitatively or qualitatively detect the presence of L53 encoded polypeptides or naturally occuning variants or peptide fragments thereof. The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in inrmunofluorescence or immunoelectron microscopy, for in situ detection of L53 gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a subject, such as paraffin embedded sections of tissue, e.g., lung tissue, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Since the L53 gene product appears to be expressed predominantly as an intracellular protein, it may be desirable to introduce the antibody inside the cell, for example, by making the cell membrane permeable. The L53 polypeptides may also be expressed on the cell surface, thus cells can be directly labeled by applying antibodies that are specific or selective for the L53 polypeptides or fragment thereof to the cell surface.

[0205] Through the use of such a procedure, it is possible to determine not only the presence of the L53 gene product, or naturally occuning variants thereof or peptide fragments, but also its distribution in the examined tissue. Using the methods of the present invention, those of ordinary skill wiU readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[0206] Immunoassays for L53 encoded polypeptides or conserved variants or peptide fragments thereof wiU typically comprise contacting a sample, such as a biological fluid, tissue or a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of an antibody that specifically or selectively binds to an L53 gene product, e.g., a detectably labeled antibody capable of identifying L53 polypeptides or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art (e.g. , Western blot, ELISA, FACS).

[0207] The biological sample may be brought in contact with and immobilized onto a solid phase support or canier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support is washed with suitable buffers followed by treatment with the uetectably labeled antibody that selectively or specificaUy binds to an L53 encoded polypeptide. The solid phase support is washed with buffer a second time to remove unbound antibody. The amount of bound label on a solid support may be detected by conventional means.

[0208] By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or caπiers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the canier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Prefened supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

[0209] The anti-L53 antibody can be detectably labeled by linking the same to an enzyme and using the labeled antibody in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Lrrmunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A. et al, 1978, J. Clin. Pathol 31: 507-520; Butler, J.E., 1981, Meth. Enzymol. 73:482; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al, (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not lir ited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

[0210] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect L53 encoded polyepeptides using radioi nmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

[0211] It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be detected due to fluorescence emission. Among the most commonly used fluorescent labeling compounds are fluorescein lsothiocyanate, rhodamme, phycoeiythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0212] The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals are attached to an antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or βthylenediaminetetraacetic acid (EDTA).

[0213] The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is detected by luniinescence that arises during the course of a chemical reaction. Examples of particularly useful cher luminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0214] Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of a chenriluminescent reaction. The presence of a bioluminescent protein is determined by detecting luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

[0215] In various embodiments, the present invention provides methods for the measurement of L53 polyepeptides, and the uses of such measurements in clinical applications using L53-specific or L53- selective antibodies.

[0216] The measurement of L53 polypeptides of the invention is valuable for detecting and/or staging lung cancer and other cancers in a subject, for screening of lung cancer and other cancers in a population, for differential diagnosis of the physiological condition of a subject, and for monitoring the effect of a therapeutic treatment on a subject.

[0217] The present invention also provides for detecting, diagnosing, or staging lung cancer and other cancers, or for monitoring the treatment of lung cancer and other cancers by measuring the level of expression of an L53 polypeptide. In addition to L53 polypeptides, at least one other marker, such as receptors or differentiation antigens can also be measured. For example, serum markers selected from, for example but not limited to, carcinoembryonic antigen (CEA), CA15-3, CA549, CAM26, M29, CA27.29 and MCA can be measured in combination with an L53 polypeptide to detect, diagnose, stage, or monitor treatment of lung cancer and other cancers. In another embodiment, the prognostic indicator is the observed change in different marker levels relative to one another, rather than the absolute levels of the markers present at any one time. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the overall disease status of a subject.

[0218] In a specific embodiment of the invention, soluble L53 polypeptide alone or in combination with other markers can be measured in any body fluid of the subject including but not limited to blood, serum, plasma, lymphatic fluid, milk, urine, saliva, pleural effusions, synovial fluid, spinal fluid, tissue infiltrations and tumor infiltrates. In another embodiment an L53 polypeptide is measured in tissue samples or cells directly. The present invention also contemplates a kit for measuring the level of L53 expression in a biological sample and the use of said kit to diagnose a subject with cancer. Alternatively said kit could be used to determine the prognosis of a cancer patient or the risk of metastasis of said cancer.

[0219] Any of numerous immunoassays can be used in the practice of the methods of the instant invention, such as those described in Section 5.4.2. Antibodies, or antibody fragments containing the binding domain, which can be employed include, but are not limited to, suitable antibodies among those in Section 5.3 and other antibodies known in the art or those which can be obtained by procedures standard in the art such as those described in Section 5.3.

5.4.2.1 IN VIVO IMAGING USING ANTIBODIES TO AN L53 POLYPEPTIDE [0220] Cunent diagnostic and therapeutic methods make use of antibodies to target imaging agents or therapeutic substances, e.g., to tumors. Thus, labeled antibodies specific or selective for an L53 polypepeptide are used in the methods of the invention for in vivo imaging, detection, and treatment of cancer in a subject.

[0221] Antibodies may be linked to chelators such as those described in U.S. Patent No. 4,741,900 or U.S. Patent No. 5,326,856. The antibody-chelator complex may then be radiolabeled to provide an imaging agent for diagnosis or treatment of disease. Antibodies of the invention may also be used in the methods disclosed in U.S. Patent No. 5,449,761 for creating a radiolabeled antibody for use in imaging or radiotherapy.

[0222] In in vivo diagnostic applications, specific tissues or even specific ceUular disorders, e.g., cancer, may be imaged by administration of a sufficient amount of a labeled antibody using the methods of the instant invention.

[0223] A wide variety of metal ions suitable for in vivo tissue imaging have been tested and utilized clinicaUy. For imaging with radioisotopes, the following characteristics are generally desirable: (a) low radiation dose to the patient; (b) high photon yield which permits a nuclear medicine procedure to be performed in a short time period; (c) ability to be produced in sufficient quantities; (d) acceptable cost; (e) simple preparation for administration; and (f) no requirement that the patient be sequestered subsequently. These characteristics generally translate into the following: (a) the radiation exposure to the most critical organ is less than 5 rad; (b) a single image can be obtained within several hours after infusion; (c) the radioisotope does not decay by emission of a particle; (d) the isotope can be readily detected; and (e) the half-life is less than four days (Lamb and Kramer, "Commercial Production of Radioisotopes for Nuclear Medicine", In Radiotracers For Medical Applications, Vol. 1, Rayudu (Ed.), CRC Press, Inc., Boca Raton, pp. 17-62). Preferably, the metal is technetium-99m.

[0224] By way of iUustration, the targets that one may image include any solid neoplasm, certain organs such a lymph nodes, parathyroids, spleen and kidney, sites of inflammation or infection (e.g., macrophages at such sites), myocardial infarction or thromboses (neoantigenic determinants on fibrin or platelets), and the like evident to one of ordinary skill in the art. Furthermore, the neoplastic tissue may be present in bone, internal organs, connective tissue, or skin. [0225J As is also apparent to one of ordinary skill in the art, one may use the methods of the present invention for in vivo therapeutics (e.g., using radiotherapeutic metal complexes), especially after having diagnosed a diseased condition via the in vivo diagnostic method described above, or in in vitro diagnostic application (e.g., using a radiometal or a fluorescent metal complex).

[0226] Accordingly, a method for diagnosing cancer by obtaining an image of an internal region of a subject is contemplated in the instant invention which comprises adimnistering to a subject an effective amount of an antibody composition specific or selective for an L53 polypeptide conjugated with a metal which is radioactively labeled, and recording the scintigraphic image obtained from the decay of the radioactive metal. Likewise, a method is contemplated for enhancing a magnetic resonance image (MRI) of an internal region of a subject which comprises administering to a subject an effective amount of an antibody composition containing a paramagnetic metal, and recording the MRI of an internal region of the subject.

[0227] Other methods include a method of enhancing a sonographic image of an internal region of a subject comprising administering to a subject an effective amount of an antibody composition containing a metal and recording the sonographic image of an internal region of the subject. In this latter application, the metal is preferably any non-toxic heavy metal ion. A method of enhancing an X-ray image of an internal region of a subject is also provided which comprises administering to a subject an antibody composition containing a metal, and recording the X-ray image of an internal region of the subject. A radioactive, non-toxic heavy metal ion is prefened.

5.4.3. DETECTINGAND STAGING CANCERINA SUBJECT

[0228] The methods of the present invention include measurement of naturally occurring L53 polypeptides, or naturally occuning variants thereof, or fragments thereof, soluble L53 polypeptides or mtracellular L53 polypeptides to detect lung cancer or other cancers in a subject or to stage lung cancer or other cancers in a subject.

[0229] Staging refers to the grouping of patients according to the extent of their disease. Staging is useful in choosing treatment for individual patients, estimating prognosis, and comparing the results of different treatment programs. Staging of lung cancer for example is performed initially on a clinical basis, according to a physical examination and laboratory radiologic evaluation. The most widely used clinical staging system is the one adopted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) Staging and End Results Reporting. It is based on the tumor-nodes- metastases (TNM) system as detailed in the 1988 Manual for Staging of Cancer. The revised International System for Staging Lung Cancer was completed in 1997 by the American Joint Committee on Cancer and the Union Internationale Contre le Cancer (Mountain et al., 1997, Chest. 111(6): 1710- 1717). Lung cancer diseases or conditions that may be detected and/or staged in a subject according to the present invention include but are not limited to those listed in Table 2. TABLE 2 TNM Classification for Lung Cancer

Stage Classification Definition

T TX Primary tumor not visual by imaging or bronchoscopy T TO No evidence of primary tumor T Tis Carcinoma in situ T Tl Tumor is < or = 3 cm T T2 Tumor is > 3 cm T T3 Tumor of any size that invades the chest wall or the structures of the chest's center

T4 Tumor of any size that invades vital structures, such as soft tissues of the mediastinum and the vertebral body

N NX Regional lymph nodes can't be assessed N NO No regional lymph node metastasis N NI Metastasis to ipsilateral peribronchial and/or ipsilateral nodes, and intrapulmonary nodes including involvement by extension of the primary tumor

N N2 Metastasis to ipsilateral mediastinal and/or subcarinal lymph nodes N N3 Metastasis to contralateral mediastinal, contralateral Hilar, ipsilateral or contralateral scalene, or supraclavicular lymph nodes

M MX Distant metastasis can't be assessed M MO No distant metastasis M Ml Presence of distant metastasis

[0230] Any immunoassay, such as those described in Section 5.4.2 can be used to measure the amount of L53 polypeptide or soluble L53 polypeptide and compare the measured level to that of a baseline level. This baseline level is the amount or range therof that is established to be present in a noncancerous tissue or body fluid (e.g., unaffected tissue) of subjects with various degrees of the disease or disorder. An amount present in the tissue or body fluid of the subject that is similar to a standard amount, established to be normally present in the tissue or body fluid of the subject during a specific stage of cancer or lung cancer, is indicative of the stage of the disease in the subject. The baseline level may also be the level present in the subject prior to the onset of disease or the amount present during remission of the disease.

[0231] In specific embodiments of this aspect of the invention, measurements of levels of an L53 polypeptide or soluble L53 polypeptide can be used in the detection of infiltrative ductal carcinoma (IDC) or the presence of metastases or both. Increased levels of L53 polypeptides or soluble L53 polypeptide may be associated with metastases.

[0232] In another embodiment of the invention, the measurement of soluble L53 polypeptide, intra-cellular L53 polypeptide, fragments thereof or immunologically related molecules can be used to differentially diagnose in a subject a particular disease phenotype or physiological condition from other phenotypes or physiological conditions. For example, measurements of L53 polypeptide or soluble L53 polypeptide levels may be used in the differential diagnosis of infiltrative ductal carcinoma, as distinguished from ductal carcinoma in situ or benign fibroadenomas. To this end, for example, the measured amount of the L53 polypeptide is compared with the amount of the molecule normally present in the tissue, cells or body fluid of a subject with one of the suspected physiological conditions. A measured amount of the L53 polypeptide similar to the amount normally present in a subject with one of the physiological conditions, and not normaUy present in a subject with one or more of the other physiological conditions, is indicative of the physiological condition of the subject.

[0233] As an alternative to measuring levels of L53 polypeptides in the foregoing staging methods, levels of L53 transcript can be measured, for example by the methods described in Section 5.4.1, supra.

5.4.4. MONITORING THE EFFECT OF A THERAPEUTIC TREATMENT

[0234] The present invention provides a method for monitoring the effect of a therapeutic treatment on the disease state of a subject.

[0235] The need for a clinical procedure(s) that can be used to monitor the efficacy of a cancer treatment is well recognized. As described herein, the detection of L53 transcripts and encoded polypeptides in lung cancer and other cancers associated with abenant L53 regulation provides a sensitive assay system with which to monitor therapeutic regimens. Therapeutic treatments that may be evaluated according to the present invention include, but are not limited to, radiotherapy, surgery, chemotherapy, vaccine administration, endocrine therapy, immunotherapy, and gene therapy, etc. The chemotherapeutic regimens include, but are not limited to administration of drugs such as, for example, methotrexate, fluorouracil, cyclophosphamide, doxorubicin, and taxol. The endocrine therapeutic regimens include, but are not limited to administration of tamoxifen and progestins.

[0236] The method of the invention comprises measuring at suitable time intervals before, during, or after tlierapy, the amount of an L53 transcript or polypeptide (including soluble polypeptide), or any combination of the foregoing. Any change or absence of change in the absolute or relative amounts of the L53 gene products can be identified and conelated with the effect of the treatment on the subject.

[0237] In particular, serum- or cell-associated levels of an L53 polypeptide may bear a direct relationship with the severity of lung cancer, or other cancer, the risk of metastasis of said cancer and poor prognosis. Since serum- or cell-associated L53 polypeptide levels are generally undetectable or negligible in normal individuals and up-regulated in cancer patients (e.g., lung cancer patients), generally, a decrease in the level of detectable L53 polypeptide after a therapeutic treatment is associated with efficacious treatment.

[0238] In a particular aspect, levels of soluble or cell-associated L53 polypeptide are measured at different time points and compared to baseline levels. The baseline level(s) may be established as the level present prior to treatment, during remission of disease, or during periods of stability. For some applications, the baseline level may conelate with the level of the L53 polypeptide present in normal, disease free individuals. Comparisons to baseline levels may be used to establish ratios of change (or relative comparisons), which may be conelated with the disease course or treatment outcome. 5.4.5. PROGNOSTIC ASSAYS

[0239] The methods described herein can furtliermore be utilized as prognostic assays to identify subjects having or at risk of developing cancer or another disease or disorder associated with abenant expression or activity of an L53 polypeptide. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing cancer, e.g., lung cancer, or another disorder associated with abenant expression or activity of an L53 polypeptide. Thus, the present invention provides a method in which a test sample is obtained from a subject and an L53 polypeptide or nucleic acid (e.g., mRNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant expression or activity of the L53 polypeptide, e.g., cancer. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

[0240] The prognostic assays described herein, for example, can be used to identify a subject having or at risk of developing disorders such as cancers, for example, hormone-sensitive cancer such as lung cancer.

[0241] In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing related disorders associated with expression of polypeptides or nucleic acids of the invention.

[0242] Furtliermore, the prognostic assays described herein can be used to detemiine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat cancer or another disease or disorder associated with abenant expression or activity of an L53 polypeptide. For example, such methods can be used to determine whether a subject can be treated effectively with a specific agent or class of agents (e.g., agents of a type which decrease activity or expression level of an L53 transcript or polypeptide). Thus, the present invention provides methods for deteπnining whether a subject can be effectively treated with an agent for a disorder associated with abenant expression or activity of the L53 transcript or polypeptide. Such methods may involve steps whereby a test sample is obtained and the L53 polypeptide or nucleic acid encoding the L53 polypeptide is detected. The presence of the polypeptide or nucleic acid in the sample indicates that the subject is a candidate for treatment with agents of the present invention.

[0243] The methods of the invention can also be used to detect genetic lesions or mutations in an L53 gene, thereby determining if a subject with the lesioned gene is at increased or reduced risk for a disorder characterized by abenant expression or activity of a polypeptide of the invention, e.g., cancer. In one embodiment, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding an L53 polypeptide, or the mis-expression of the gene encoding an L53 polypeptide. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from an L53 gene; 2) an addition of one or more nucleotides to an L53 gene; 3) a substitution of one or more nucleotides of an L53 gene i.e. a point mutation; 4) a chromosomal reanangement of an L53 gene; 5) an alteration in the level of a messenger RNA transcript of an L53 gene; 6) an abenant modification of an L53 gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an L53 gene; 8) a non-wUd type level of the protein encoded by an L53 gene; 9) an allelic loss of an L53 gene; and 10) an inappropriate post-translational modification of a protein encoded by an L53 gene. As described herein, there are a large number of assay techniques known in the art that can be used for detecting lesions in a gene.

[0244] In certain embodiments, methods for the detection of the lesion involve the use of a probe/primer in a polymerase chain reaction (PCR) (See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077; and Nakazawa et al (1994) Proc Natl Acad Sci. USA 91:360), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675). These methods are useful in the diagnosis and prognosis of cancer in a subject. This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, nα-RNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene or gene product (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be used as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0245] Mutations in a selected gene from a sample ceU or tissue can also be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA are isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are detennined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicate mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0246] In other embodiments, methods are provided whereby genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density aπays comprising hundreds or thousands of oligonucleotides probes (Cronin et al.1996, Human Mutation 2:244; Kozal et al. 1996, Nature Medicine 2:753). For example, genetic mutations can be identified in two-dimensional aπays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization aπay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear aπays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization aπay that allows the characterization of specific mutations by using smaUer, specialized probe aπays complementary to all variants or mutations detected. Each mutation aπay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0247] Sequencing reactions known in the art can be used to sequence the selected gene and detect mutations in the L53 gene by comparing the sequence of the sample nucleic acids with the conesponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (Maxim and Gilbert, 1977, Proc Natl Acad Sci. USA 74:560) or Sanger (Sanger et al. 1977, Proc Natl Acad Sci. USA 74:5463). Such methods are useful in the diagnosis and prognosis of a subject with cancer. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve et al, 1995, BioTechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al. 1996, Adv. Chromatogr. 36: 127; and Griffin et al, 1993, Appl Biochem. Biotechnol. 38147).

[0248] Furthermore, the presence of an L53 nucleic acid molecule or polypeptide of the invention can be conelated with the presence or expression level of other cancer-related proteins, such as for example, the androgen receptor, the estrogen receptor, adhesion molecules (e.g., E-cadherin), proliferation markers (e.g., MJB-l), tumor-suppressor genes (e.g., TP53, retinoblastoma gene product), vascular endothelial growth factor (Lissoni et al, 2000, hit J Biol Markers. 15(4):308), Rad51 (Maacke et al, 2000, IntJ Cancer. 88(6):907), cyclin Dl, BRCA1, BRCA2, or carcinoembryonic antigen.

[0249] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one nucleic acid probe or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or a famUy history of a disease or illness involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue, e.g., preferably cancerous lung cells or tissue, in which the L53 gene is expressed may be utilized in the prognostic assays described herein.

5.5. SCREENING FOR L53 ACTIVITY

[0250] The present invention further provides methods for the identification of compounds that, through their interaction with the L53 gene or L53 gene product, affect the onset, progression and/or metastatic spread of lung cancer and/or other cancers.

[0251] The following assays are designed to identify: (i) compounds that bind to L53 gene products; (ii) compounds that bind to other proteins that interact with an L53 gene product; (iii) compounds that interfere with the interaction of the L53 gene product with other proteins; and (iv) compounds that modulate the activity of an L53 gene (i.e., modulate the level of L53 gene expression, including transcription of the L53 gene and/or translation of its encoded transcript), and/or modulate the level of L53-encoded polyepeptide activity).

[0252] Assays may additionally be utilized which identify compounds that bind to L53 gene regulatory sequences (e.g., promoter sequences), which may modulate the level of L53 gene expression (see e.g., Platt, K.A., 1994, J. Biol. Chem. 269:28558). [0253] Such proteins that interact with L53 may be involved in the onset, development and/or metastatic spread of lung cancer or other cancers.

[0254] The present invention also provides methods of using isolated L53 nucleic acid molecules, or derivatives thereof, as probes that can be used to screen for DNA-binding proteins, including but not limited to proteins that affect DNA conformation or modulate transcriptional activity (e.g., enhancers, transcription factors). In another embodiment, such probes can be used to screen for RNA-binding factors, including but not limited to proteins, steroid hormones, or other small molecules. In yet another embodiment, such probes can be used to detect and identify molecules that bind or affect the pharmacokinetics or activity (e.g. , enzymatic activity) of the L53 gene or gene product. The proteins or nucleic acid binding factors or transcriptional modulators identified by a screening assay provide an appropriate target for anti-cancer therapeutics.

[0255] In one embodiment, a screening assay of the invention can identify a test compound that is useful for increasing or decreasing the translation of an L53 ORF, for example, by binding to one or more regulatory elements in the 5' untranslated region, the 3' untranslated region, or the coding regions of the mRNA. Compounds that bind to mRNA can, inter alia, increase or decrease the rate of mRNA processing, alter its transport in a cell, prevent or enhance binding of the mRNA to ribosomes, suppressor proteins or enhancer proteins, or alter mRNA stability. Accordingly, compounds that increase or decrease mRNA translation can be used to treat or prevent disease. For example, diseases such as cancer, associated with overproduction of proteins, such as L53, can be treated or prevented by decreasing translation of the mRNA that codes for the overproduced protein, thus inhibiting production of the protein.

[0256] Accordingly, in one embodiment, a compound identified by a screening assay of the invention inhibits the production of an L53 protein, h a further embodiment, the compound inhibits the translation of an L53 mRNA. In yet another embodiment, the compound inhibits transcription of the L53 gene.

[0257] The invention provides a method for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which hind to the L53 gene product or fragments thereof or have a stimulatory or inhibitory effect on, for example, expression and/or activity of the L53 gene product or fragments thereof. Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the L53 gene product, and for ameliorating symptoms of lung cancer or other types of cancer. Assays for testing the effectiveness of compounds, identified by, for example, techniques such as those described in Section 5.5.1, are discussed in Section 5.5.3. It is to be noted that the compositions of the invention include pharmaceutical compositions comprising one or more of the compounds identified via such methods. Such pharmaceutical compositions can be formulated, for example, as discussed in Section 5.7.

5.5.1. IN VITRO SCREENING FOR COMPOUNDS THAT BIND TO THE L53 GENE

[0258] In vitro systems may be designed to identify compounds capable of interacting with, e.g., binding to, an L53 gene product of the invention. Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant L53 gene products, may be useful in elaborating the biological function of the L53 gene product, may be utilized in screens for identifying compounds that disrupt normal L53 gene product interactions, or may disrupt such interactions directly. Thus, said compounds are useful for treating, preventing and/or diagnosing cancer. In a particular embodiment, said compounds are used for the treatment, prevention and/or diagnosis of lung cancer.

[0259] The principle of the assays used to identify compounds that interact with the L53 gene product involves preparing a reaction mixture of an L53 gene product and the test compound under conditions and for a time sufficient to allow the two components to interact, e.g., bind, thus forming a transient or stable complex that can be removed from and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay involves anchoring an L53 gene product or the test substance onto a solid phase and detecting L53 gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the L53 gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

[0260] In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific or selective for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

[0261] In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, umeacted components are removed (e.g., by washing) under conditions such that any complexes formed remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific or selective for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

[0262] Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific or selective for L53 gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific or selective for the other component of the possible complex to detect anchored complexes.

5.5.2 ASSAYS FOR PROTEINS THAT INTERACT WITH THE L53 GENE [0263] Any method suitable for detecting protein-protein interactions may be employed for identifying L53 protein-protein interactions. Proteins that interact with L53 are potential therapeutics for the treatment of cancer. Thus, the assays described below are useful for identifying proteins that can be used in methods to treat cancer. Proteins that interact with L53 can also be used for the diagnosis of cancer. Thus, the assays described below are also useful in methods to diagnose cancer.

[0264] Traditional methods for the detection of protein-protein interactions include, without limitation, co-immunoprecipitation, crosslinking, and co-purification through gradients or chromatographic columns (e.g., size exclusion chromatography). Utilizing procedures such as these allows for the isolation of cellular proteins that interact with L53 gene products. Once isolated, such a cellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify additional proteins with which it interacts. For example, at least a portion of the amino acid sequence of a cellular protein that interacts with an L53 gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide rriixtures that can be used to screen for gene sequences encoding such cellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and screening are well known in the art. (See, e.g. , Ausubel, supra, and PCR Protocols: A Guide to Methods and Applications, 1990, mis, M. et al, eds. Academic Press, Inc., New York).

[0265] Additionally, methods may be employed which result in the simultaneous identification of genes which encode a protein interacting with the L53 protein. These methods include, for example, probing expression libraries with labeled L53 protein, using L53 protein in a manner similar to the technique of antibody probing of λgtl 1 libraries.

[0266] One method that detects protein interactions in vivo, the two-hybrid system, may also be used to advantage. Many versions of this system have been described (See e.g., Chien et al, 1991, supra). The system described by Chien etal. (1991, supra) is commercially available from Clontech (Palo Alto, CA).

5.5.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH L53 INTERACTION

[0267] The L53 gene product may, in vivo, interact with one or more macromolecules, such as proteins or nucleic acids. With regard to the present invention, such macromolecules are refened to herein as "interacting partners". Compounds that disrupt L53 interactions with interacting partners are useful in regulating the activity of the L53 gene product, including mutant L53 gene products. Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in Section 5.5.1. Thus, the assays described below are useful for identifying proteins and/or nucleic acids that can be used in methods to treat cancer. Proteins and nucleic acids that interact with L53 can also be used in the diagnosis of cancer, e.g., lung cancer. Thus, the assays described below are also useful for methods to diagnose cancer, e.g., lung cancer.

[0268] The basic principle of the assay systems used to identify compounds that interfere with the interaction between an L53 gene product and its interacting partner or partners involves preparing a reaction mixture containing the L53 gene product and the interacting partner under conditions and for a time sufficient to allow uie two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initiaUy included in the reaction mixture, or may be added at a time subsequent to the addition of L53 gene product and its cellular interacting partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between an L53 protein and an interacting partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the L53 protein and the interacting partner. Additionally, complex formation within reaction mixtures containing the test compound and normal L53 protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant L53 protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal L53 gene proteins.

[0269] The assay for compounds that interfere with the interaction of the L53 gene product or protein and interacting partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the L53 gene product or the interacting partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction, hi homogeneous assays, the entire reaction is caπied out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the L53 gene products and the interacting partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; le., by adding the test substance to the reaction mixture prior to or simultaneously with the L53 gene protein and cellular interacting partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

[0270] hi a heterogeneous assay system, either the L53 gene product or the interacting partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non- covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the L53 gene product or interacting partner and drying. Alternatively, an immobilized antibody specific or selective for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

[0271] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-inrmobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific or selective for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

[0272] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific or selective for one of the interacting components to anchor any complexes formed in solution, and a labeled antibody specific or selective for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex formation or which disrupt preformed complexes can be identified.

[0273] In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the L53 gene protein and the interacting partner is prepared in which either the L53 gene product or its interacting partner is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein). The addition of a test substance that competes with and displaces one of the species from the preformed complex results in the generation of a signal above background, hi this way, test substances that disrupt L53 gene protein/cellular interacting partner interaction can be identified.

[0274] In a particular embodiment, an L53 gene product can be prepared for immobilization using recombinant DNA techniques such as those described in Section 5.1. For example, the L53 coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its interacting activity is maintained in the resulting fusion protein. The cellular interacting partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described in Section 5.2. This antibody can be labeled with the radioactive isotope

I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-L53 or GST-L53 fusion protein can be anchored to glutathione-agarose beads. The cellular interacting partner can then be added in the presence or absence of the test compound in a manner that allows interaction, e.g., binding, to occur. At the end of the reaction period, unbound material is washed away, and the labeled monoclonal antibody is added to the system and allowed to bind to the complexed components. The interaction between the L53 gene protein and the cellular interacting partner is detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by a test compound results in a decrease in measured radioactivity.

[0275] Alternatively, the GST-L53 or GST-L53 gene fusion protein and the cellular interacting partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after complex formation. This mixture is then added to the glutathione-agarose beads and unbound material is washed away. The extent of inhibition of L53 gene product/interacting partner interaction can be detected by the addition of a labeled antibody and measuring the radioactivity associated with the beads. 5.5.4. CELL-BASED ASSAYS FOR L53 ACTIVITY

[0276] Cell-based methods are presented herein which identify compounds capable of treating lung cancer and other cancers by modulating L53 activity and/or expression levels. Specifically, such assays identify compounds that affect L53 dependent processes, such as but not limited to changes in cell morphology, cell division, differentiation, adhesion, motility, phosphorylation, or dephosphorylation of cellular proteins. Such assays can also identify compounds that affect L53 expression levels and/or gene activity directly. Compounds identified via such methods can, for example, be utilized in methods for treating lung cancer and other cancers and metastasis thereof.

[0277] In one embodiment, an assay is a cell-based assay in which a cell that expresses a membrane-bound form of the L53 gene product, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined. In another embodiment the L53 gene product is cytosolic. The cell, for example, may be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125^ 35§₎ 14 _or 3JJ^ either directly or indirectly, and the radioisotope detected by direct counting of radio-emission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In a prefened embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide reveals the ability of the test compound to bind preferentially to the polypeptide or a biologically active portion thereof as compared to the known compound.

[0278] In another embodiment, the cell-based assays are based on expression of the L53 gene product in a mammalian cell and measuring L53-dependent processes. Any mammalian cells that can express the L53 gene and allow the functioning of the L53 gene product can be used, in particular, cancer cells derived from the lung, such as A549, NCI-H920, NCI-H969, NCI-H23, NCI-H226, NCI-H647, NCI- H1869, NCI-HH1385, NCI-H460, NCI-H1155, NCI-H358, and NCI-H650. Normal bronchial cell lines such as, for example, HBECs and SAECs, may also be used provided that an L53 gene product is produced. Other mammalian cell lines that can be used include, but are not limited to CHO, HeLa, NTH3T3, and Vero cells. Recombinant expression of the L53 gene in these cells can be achieved by methods described in Section 5.2. In these assays, cells producing functional L53 gene products are exposed to a test compound for an interval sufficient for the compound to modulate the activity of the L53 gene product. The activity of an L53 gene product can be measured directly or indkectly through the detection or measurement of L53-dependent cellular processes. As a control, a cell not producing the L53 gene product may be used for comparisons. Depending on the cellular process, any techniques known in the art may be applied to detect or measure it.

[0279] In another embodiment a cell or cell line that is capable of expressing L53 is contacted with a test compound that is believed to modulate expression of the L53 gene. Expression levels of the L53 gene can be monitored in the presence or absence of the test compound. Alternatively, expression levels can be monitored in the presence of a test compound as compared to expression levels of the L53 gene in the presence of a control compound or a placebo. Any method known in the art can be used to monitor L53 gene expression. As an example, but not as a limitation, such methods can include Western blot, Northern Blot, and real-time quantitative RT-PCR.

[0280] In yet another embodiment, cells which express the L53 gene product, e.g., NCI-H460 cells are made permeable, e.g., by treatment with a mild detergent and exposed to a test compound. Binding of the test compound can be detected directly (e.g. , radioactively labeling the test compound) or indirectly (antibody detection) or by any means known in the art.

[0281] Any compound can be used in a cell-based assay to test if it affects L53 activity or expression levels. The compound can be a protein, a peptide, a nucleic acid, an antibody or fragment thereof, a small molecule, an organic molecule or an inorganic molecule, (e.g., steroid, pharmaceutical drug). A small molecule is considered a non-peptide compound with a molecular weight of less than 500 daltons.

5.6. METHODS FOR TREATMENT OF CANCER

[0282] Described below are methods and compositions for treating cancer, e.g., lung cancer, using the L53 gene or gene product as a therapeutic target. The outcome of a treatment is to at least produce in a treated subject a healthful benefit, which hi the case of cancer, including lung cancer, includes but is not limited to remission of the cancer, palliation of the symptoms of the cancer, and/or control of metastatic spread of the cancer.

[0283] AU such methods comprise methods that modulate L53 gene activity and/or expression, that in turn, modulate the phenotype of the treated cell.

[0284] As discussed, above, successful treatment of lung cancer or other cancers can be brought about by techniques that serve to decrease L53 activity. Activity can be decreased by, for example, directly decreasing L53 gene product activity and/or by decreasing the level of L53 gene expression. Thus, the invention provides methods for treating a subject with cancer by administering to said subject an effective amount of a compound that antagonizes an L53 gene product.

[0285] For example, compounds that decrease L53 activity (using assays described in Section 5.5) can be used in accordance with the invention to treat lung cancer or other cancers. As discussed in Section 5.5, such molecules can include, but are not limited to proteins, nucleic acids, peptides, including soluble peptides, and small organic or inorganic molecules, and can be refened to as L53 antagonists or agonists. 'Techniques for the deteπmnation of effective doses and administration of such compounds are described in Section 5.7.

[0286] Further, antisense and ribozyme molecules which inhibit L53 gene expression can also be used in accordance with the invention to reduce the level of L53 gene expression, thus effectively reducing the level of L53 gene product present, thereby decreasing the level of L53 activity. The invention therefore relates to a pharmaceutical composition comprising an L53 gene product. Still further, triple helix molecules can be utilized for reducing the level of L53 gene activity. Such molecules can be designed to reduce or inhibit either wild type, or if appropriate, mutant target gene activity. SmaU organic or inorganic molecules can also be used to inhibit L53 gene expression and/or inhibit production or activity of an L53 gene product. Techniques for the production and use of such molecules are well known to those of skill in the art.

5.6.1. ANTISENSE MOLECULES

[0287] Anti-sense nucleic acid molecules which are complementary to nucleic acid sequences contained within the L53 gene as shown in FIG. 1 (SEQ ID NO: 1 and SEQ JD NO: 2), including but not limited to anti-sense nucleic acid molecules complementary to (SEQ ID NO: 1 and SEQ JD NO: 2), can be used to treat any cancer, in which the expression level of the L53 gene is elevated in cancerous cells or tissue as compared to that of normal cells or tissue or a predetermined non-cancerous standard. Thus, in one embodiment of the invention a method for treating lung cancer is provided whereby a patient suffering from lung cancer is treated with an effective amount of an L53 anti-sense nucleic acid molecule.

[0288] Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to L53 gene mRNA. The antisense oligonucleotides will bind to the complementary L53 gene mRNA transcripts and prevent translation. Absolute complementarity, although prefened, is not required. A sequence "complementary" to a portion of an RNA, as refened to herein, means a sequence having sufficient complementarity to be able to hybridize with the non-poly A portion of the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and stUl form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0289] Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, are considered prefened for antisense applications because, in general, they efficiently inhibit translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have also been shown to be effective at inhibiting translation of mRNAs as weU. (See generally, Wagner, R., 1994, Nature 372:333). Thus, oligonucleotides complementary to the 5 '-non-translated region, the 3 '-non-translated region, or the non- translated, non-coding region between the L53 open reading frame of the L53 gene (refened to herein after as the mtervenmg region , as shown, for example, hi FIG. 1) could be used in an antisense approacn to inhibit translation of endogenous L53 gene mRNA.

[0290] Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient mhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'-, intervening, or coding region of L53 gene mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

[0291] Regardless of the choice of target sequence, it is prefened that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is prefened that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also prefened that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared to those obtained using a control oligonucleotide. It is prefened that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

[0292] The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The ohgonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host ceU receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA 86:6553; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. USA 84:648; PCT Publication No. WO88/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published April 25, 1988), hybridization-triggered cleavage agents, (see, e.g., Krol et al, 1988, BioTechniques 6:958) or intercalating agents, (see, e.g., Zon, 1988, Pharm. Res. 5:539). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[0293] The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- lsopentenyiadenrne, uracil-i-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thιocytosιne, 5-methyl-2-thiouracil, 2-thiouracU, 4-thiouracU, 5-methyluracU, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0294] The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0295] In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphorarnidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

[0296] In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al, 1987, Nucl Acids Res. 15:6625). The oligonucleotide is a 2 -0-methylribonucleotide (Inoue et al, 1987, Nucl. Acids Res. 15:6131), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327).

[0297] The L53 antisense nucleic acid sequence can comprise the complement of any contiguous segment within the sequence of the L53 gene (SEQ ID NO: 1).

[0298] In one embodiment of the present invention, the L53 antisense nucleic acid sequence is about 50 bp in length. In certain specific embodiments, the L53 antisense nucleic acid sequence comprises the sequence complementary to nucleotides 1-50, 51-100, 101-150, 151-200, 201-250, 251- 300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801- 850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251- 1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701- 1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101- 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701- 2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951- 3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801- 3850, 3851-3900 (SEQ JD NO: 1).

[0299] In another embodiment the L53 antisense nucleic acid sequence is about 100 bp in length. In certain specific embodiments, the L53 antisense nucleic acid sequence comprises the sequence from nucleotides 1-100, 51-150, 101-200, 151-250, 201-300, 251-350, 301-400, 351-450, 401-500, 451-550, 501-600, 551-650, 601-700, 651-750, 701-800, 75-850, 801-900, 851-950, 901-1000, 951-1050, 1001- 1100, 1051-1150, 1101-1200, 1151-1250, 1201-1300, 1251-1350, 1301-1400, 1351-1450, 1401-1500, 1451-1550, 1501-1600, 1551-1650, 1601-1700, 1651-1750, 1701-1800, 1751-1850, 1801-1900, 1951- 2050, 2001-2100, 2051-2150, 2101-2200, 2151-2250, 2201-2300, 2251-2350, 2301-2400, 2351-2450, 2401-2500, 2451-2550, 2501-2600, 2551-2650, 2601-2700, 2651-2750, 2701-2800, 2751-2850, 2801- ^•29U0, 2951-3050, 3001-3100, 3151-3250, 3201-3300, 3251-3350, 3301-3400, 3351-3450, 3401-3500, 3451-3550, 3501-3600, 3551-3650, 3601-3700, 3651-3750, 3701-3800, 3751-3850, 3801-3900, and 3851-3950 (SEQ JD NO: 1)

[0300] In another embodiment the L53 antisense nucleic acid sequence is about 200 bp in length. In a particular embodiment, the L53 antisense nucleic acid sequence comprises the sequence from nucleotides 1-200, 101-300, 201-400, 301-500, 401-600, 501-700, 601-800, 701-900, 801-1000, 901- 1100, 1001-1200, 1101-1300, 1201-1400, 1301-1500, 1401-1600, 1501-1700, 1601-1800, 1701-1900, 1801-2000, 1901-2100, 2001-2200, 2101-2300, 2201-2400, 2301-2500, 2401-2600, 2501-2700, 2601- 2800, 2701-2900, 2801-3000, 2901-3100, and 3001-3200 (SEQ ID NO: 1).

[0301] In another embodiment the L53 antisense nucleic acid sequence is about 400 bp in length, hi a particular embodiment, the L53 antisense nucleic acid sequence comprises the sequence from nucleotides 1-400, 101-500, 201-600, 301-700, 401-800, 501-900, 601-1000, 701-1100, 801-1200, 901- 1300, 1001-1400, 1101-1500, 1201-1600, 1301-1700, 1401-1800, or 1501-1900, 1601-2000, 1701-2100, 1801-2200, 1901-2300, 2001-2400, 2101-2500, 2201-2600, 2301-2700, 2401-2800, 2501-2900, 2601- 3000, 2701-3100, 2801-3200, 2901-3300, and 3001-3400 (SEQ ID NO: 1).

[0302] Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448), etc.

[0303] While antisense nucleotides complementary to the L53 coding region could be used, those complementary to the transcribed untranslated region are most prefened.

[0304] The antisense molecules should be delivered to cells that express the L53 gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the deshed cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

[0305] It is often difficult, however, to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore, a prefened approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol flT or pol IT promoter. The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that form complementary base pahs with the endogenous L53 gene transcripts and thereby prevent translation of the L53 gene mRNA. For example, a vector can be introduced in vivo such that it can be taken up by a cell and direct the transcription of an antisense RNA. Such a vector may remain episomal or become chrornosomally integrated, as long as it can be transcribed to produce the deshed antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known m the art, used ior replication and expression in mammalian ceUs. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304), the promoter contained in the 3' long teπninal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22:787), the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. USA 7_8:1441), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the deshed tissue.

[0306] The effective dose of L53 antisense oligonucleotide to be administered during a treatment cycle ranges from about 0.01 to 0.1, 0.1 to 1, or 1 to 10 mg/kg/day. The dose of L53 antisense oligonucleotide to be administered can be dependent on the mode of administration. For example, intravenous administration of an L53 antisense oligonucleotide would likely result in a significantly higher full body dose than a full body dose resulting from a local implant containing a pharmaceutical composition comprising L53 antisense oligonucleotide. In one embodiment, an L53 antisense oligonucleotide is administered subcutaneously at a dose of 0.01 to 10 mg/kg/day. In another embodiment, an L53 antisense oligonucleotide is administered intravenously at a dose of 0.01 to 10 mg/kg/day. In yet another embodiment, an L53 antisense oligonucleotide is administered locally at a dose of 0.01 to 10 mg/kg/day. It will be evident to one skilled in the art that local administrations may result in lower total body doses. For example, local administration methods such as intratumor administration, intraocular injection, or implantation, can produce locally high concentrations of L53 antisense oligonucleotide, but represent a relatively low dose with respect to total body weight. Thus, in such cases, local administration of an L53 antisense oligonucleotide is contemplated to result in a total body dose of about 0.01 to 5 mg/kg/day.

[0307] In another embodiment, a particularly high dose of L53 antisense oligonucleotide, which ranges from about 10 to 50 mg/kg/day, is administered during a treatment cycle.

[0308] Moreover, the effective dose of a particular L53 antisense oligonucleotide may depend on additional factors, including the type of disease, the disease state or stage of disease, the oligonucleotide' s toxicity, the oligonucleotide' s rate of uptake by cancer cells, as well as the weight, age, and health of the individual to whom the antisense ohgonucleotide is to be administered. Because of the many factors present in vivo that may interfere with the action or biological activity of an L53 antisense oligonucleotide, one of ordinary skill in the art can appreciate that an effective amount of an L53 antisense oligonucleotide may vary for each individual.

[0309] In another embodiment, an L53 antisense oligonucleotide is administered at a dose which results in circulating plasma concentrations of an L53 antisense oligonucleotide that are at least 50 nM (nanomolar). As wiU be apparent to the skilled artisan, lower or higher plasma concentrations of an L53 antisense ohgonucleotide may be prefened depending on the mode of administration. For example, plasma concentrations of an L53 antisense oligonucleotide of at least 50 nM can be appropriate in connection witn, e.g., intravenous, subcutaneous, intramuscular, controlled release, and oral administration methods. In another example, relatively low circulating plasma levels of an L53 antisense ohgonucleotide can be desirable, however, when using local administration methods such as, for example, intratumor administration, intraocular administration, or implantation, which nevertheless can produce locally high, clinically effective concentrations of L53 antisense oligonucleotide.

[0310] The high dose may be achieved by several administrations per cycle. Alternatively, the high dose may be administered in a single bolus adnxinistration. A single administration of a high dose may result in circulating plasma levels of L53 antisense oligonucleotide that are transiently much higher than 50 nM.

[0311] Additionally, the dose of an L53 antisense oligonucleotide may vary according to the particular L53 antisense oligonucleotide used. The dose employed is likely to reflect a balancing of considerations, among which are stability, localization, cellular uptake, and toxicity of the particular L53 antisense oligonucleotide. For example, a particular chemically modified L53 antisense oligonucleotide may exhibit greater resistance to degradation, or may exhibit higher affinity for the target nucleic acid, or may exhibit increased uptake by the cell or cell nucleus; all of which may permit the use of low doses. In yet another example, a particular chemically modified L53 antisense oligonucleotide may exhibit lower toxicity than other antisense oligonucleotides, and therefore can be used at high doses. Thus, for a given L53 antisense oligonucleotide, an appropriate dose to administer can be relatively high or low. The invention contemplates the continued assessment of optimal treatment schedules for particular species of L53 antisense oligonucleotides. The daily dose can be administered in one or more treatments.

[0312] A "low dose" or "reduced dose" refers to a dose that is below the normally administered range, i.e., below the standard dose as suggested by the Physicians' Desk Reference. 54^th Edition (2000) or a similar reference. Such a dose can be sufficient to inhibit cell proliferation, or demonstrates ameliorative effects in a human, or demonstrates efficacy with fewer side effects as compared to standard cancer treatments. Normal dose ranges used for particular therapeutic agents and standard cancer treatments employed for specific diseases can be found in the Physicians' Desk Reference, 54^th Edition (2000) or in Cancer: Principles & Practice of Oncology, DeVita, Jr., Hellman, and Rosenberg (eds.) 2nd edition, Philadelphia, PA: J.B. Lippincott Co., 1985.

[0313] Reduced doses of an L53 nucleic acid molecule, an L53 polypeptide, an L53 antagonist, and/or a combination therapeutic may demonstrate reduced toxicity, such that fewer side effects and toxicities are observed in connection with administering an L53 antagonist and one or more cancer therapeutics for shorter duration and/or at lower doses when compared to other treatment protocols and dosage formulations, including the standard treatment protocols and dosage formulations as described in the Physicians' Desk Reference, 54^th Edition (2000) or in Cancer: Principles & Practice of Oncology, DeVita, Jr., Hellman, and Rosenberg (eds.) 2nd edition, Philadelphia, PA: J.B. Lippincott Co., 1985.

[0314] A "treatment cycle" or "cycle" refers to a period during which a single therapeutic or sequence of therapeutics is administered. In some instances, one treatment cycle may be deshed, such as, for example, in the case where a significant therapeutic effect is obtained after one treatment cycle. The present invention coniemplates at least one treatment cycle, generaUy preferably more than one treatment cycle.

[0315] Other factors to be considered in determining an effective dose of an L53 antisense oligonucleotide include whether the oligonucleotide will be administered in combination with other therapeutics. In such cases, the relative toxicity of the other therapeutics may indicate the use of an L53 antisense oligonucleotide at low doses. Alternatively, treatment with a high dose of L53 antisense oligonucleotide can result in combination therapies with reduced doses of therapeutics. In a specific embodiment, treatment with a particularly high dose of L53 antisense oligonucleotide can result in combination therapies with greatly reduced doses of cancer therapeutics. For example, treatment of a patient with 10, 20, 30, 40, or 50 mg/kg/day of an L53 antisense oligonucleotide can further increase the sensitivity of a subject to cancer therapeutics. In such cases, the particularly high dose of L53 antisense oligonucleotide is combined with, for example, a greatly shortened radiation therapy schedule. In another example, the particularly high dose of an L53 antisense oligonucleotide produces significant enhancement of the potency of cancer therapeutic agents.

[0316] Additionally, the particularly high doses of L53 antisense oligonucleotide may further shorten the period of administration of a therapeuticaUy effective amount of L53 antisense oligonucleotide and/or additional therapeutic, such that the length of a treatment cycle is much shorter than that of the standard treatment.

[0317] The invention contemplates other treatment regimens depending on the particular L53 antisense oligonucleotide to be used, or depending on the particular mode of administration, or depending on whether an L53 antisense oligonucleotide is adr nistered as part of a combination therapy, e.g., in combination with a cancer therapeutic agent. The daily dose can be administered in one or more treatments.

5.6.2. RIBOZYME MOLECULES [0318] Ribozyme molecules that are complementary to RNA sequences transcribed from the L53 gene (shown in FIG. 1) may be used to treat any cancer, including lung cancer. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (For a review see, for example Rossi, J., 1994, Current Biology 4:469). The mechanism of ribozyme action involves sequence specific or selective hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage (See U.S. Pat. No. 5,093,246). As such, within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins. Ribozyme molecules designed to catalyticaUy cleave L53 mRNA transcripts can also be used to prevent translation of L53 mRNA to protein. (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al, 1990, Science 247:1222). While ribozymes that cleave mRNA at site-specific recognition sequences-can be useα ro destroy L53 mRNAs, the use of hammerhead ribozymes is prefened. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pahs with the target mRNA. The sole requirement is that the target mRNA have the foUowing sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fuUy in Haseloff and Gerlach, 1988, Nature 334:585. Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the L53 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

[0319] The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetra ymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Cech and collaborators (Zaug et al, 1984, Science 224:574; Zaug and Cech, 1986, Science 231:470; Zaug et al, 1986, Nature 324:429; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell 47:207). The Cech-type ribozymes have an eight base pah active site that hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses Cech-type ribozymes that target eight base-pah active site sequences that are incorporated into an L53 transcript.

[0320] As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the L53 gene in vivo. A prefened method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol JJJ or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous L53 gene messages and inhibit translation. Ribozymes, unlike antisense molecules, are catalytic and require a lower intracellular concentration for their overall efficiency.

[0321] Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include teclmiques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

[0322] Various well-known modifications to the DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. 5.6.3. THERAPEUTIC ANTIBODIES

[0323] Antibodies exhibiting the ability to downregulate L53 gene product activity can be utilized to treat lung cancer and other cancers wherein the L53 expression levels are elevated. Such antibodies can be generated against wild type or mutant L53 proteins, or against peptides conesponding to portions of the proteins using standard techniques as described in Section 5.3. The antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, and the like.

[0324] Antibodies that recognize any epitope on the L53 protein can be used as therapeutic reagents for the treatment of a patient with a cancer associated with abenant L53 activity.

[0325] Because L53 is generally expressed as an intracellular protein, it is prefened that internalizing antibodies be used. However, lipofectin or liposomes can be used to deliver the L53 antibody or an L53 binding fragment of the Fab region into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the L53 is prefened. For example, peptides having an amino acid sequence conesponding to the domain of the variable region of the antibody that binds to L53 can be used. Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g., see Creighton, 1983, supra; and Sambrook et al, 1989, supra). Alternatively, single chain antibodies, such as neutralizing antibodies, which bind to intracellular epitopes can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993, Proc. Natl. Acad. Sci. USA 90:7889).

[0326] Also contemplated by the methods of the invention are antibodies that are conjugated to a cytostatic and/or a cytotoxic agent. Such conjugated antibodies are useful for treating a patient with cancer because they target cancer cells expressing the antigen for which the antibody is specific, thereby inhibiting the proliferation of these cells and/or killing these cells. A useful class of such cytotoxic or cytostatic agents includes, but is not limited to, the following non-mutually exclusive classes of agents: alkylating agents, anthracyclines, antibiotics, antifolates, antimetabolites, antitubulin agents, auristatins, chemotherapy sensitizers, DNA minor groove binders, DNA replication inhibitors, duocarmycins, etoposides, fluorinated pyrimidines, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, and vinca alkaloids.

[0327] Individual cytotoxic or cytostatic agents encompassed by the invention include but are not limited to an androgen, anthrarnycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorabicin, decarbazine, docetaxel, doxorubicin, estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicrn, ifosfamide, irinotecan, lomustine (CCNU), mechloremamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitrohnidazole, pachtaxel, plicamycin, procarbizrne, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.

[0328] In a prefened embodiment, the cytotoxic or cytostatic agent is an antimetabolite. The anthnetabolite can be a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dUiydrofolate reductase inhibitor (e.g., methotrexate), acyclovh, gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, and trifluridine.

[0329] Techniques for conjugating such therapeutic moieties to proteins, and in particular to antibodies, are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, 1982, Immunol. Rev. 62:119-58.

5.6.4. TARGETED DISRUPTION OF L53 EXPRESSION

[0330] As briefly described in Section 5.2.4, endogenous L53 gene expression can also be reduced by inactivating or "knocking out" the gene or its promoter using targeted homologous recombination, (e.g., see Smithies et al, 1985, Nature 317:230; Thomas & Capecchi, 1987, Cell 51:503; Thompson et al, 1989 Cell 5:313). For example, a mutant, non-functional L53 gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous L53 gene (either the coding regions or regulatory regions of the L53 gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express L53 gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results hi inactivation of the L53 gene. Such approaches are particularly useful for modifications to ES (embryonic stem) cells that can be used to generate animal offspring with an inactive L53 gene homolog (e.g., see Thomas & Capecchi 1987 supra and Thompson 1989, supra). Such techniques can also be utilized to generate animal models of lung cancer and other types of cancer. It should be noted that this approach can be adapted for use in humans provided the recombinant DNA constructs are dhectly administered or targeted to the requhed site in vivo using appropriate vectors, e.g., herpes virus vectors, retrovirus vectors, adenovirus vectors, or adeno associated virus vectors.

[0331] Alternatively, endogenous L53 gene expression can be reduced by targeting deoxyribonucleoti.de sequences complementary to the regulatory region of the L53 gene (le., the L53 gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the L53 gene m target cells in the body, ώee generally, Helene, 1991, Anticancer Drug Des. θ(o :Doy; neiene eτ al, 1992, Ann, NY. Acad. Sci. 660:27; and Maher, 1992, Bioassays 14(12):807).

5.6.5. COMBINATION THERAPIES

[0332] The administration of an L53 antagonist can potentiate the effect of anti-cancer agents. In a prefened embodiment, the invention further encompasses the use of combination therapy to prevent or treat cancer. In one embodiment, the L53 antagonist selectively or specifically antagonizes L53 expression and/or activity.

[0333] In one embodiment, lung cancer and other cancers (e.g., pancreatic, colon, breast, and brain cancer) can be treated with a pharmaceutical composition comprising an L53 antagonist in combination with 5-fluorouracU, cisplatin, docetaxel, doxorubicin, Herceptin®, gemcitabine (Seidman, 2001, Oncology 15:11-14), JL-2, paclitaxel, and/or VP-16 (etoposide).

[0334] These combination therapies can also be used to prevent cancer, prevent the recunence of cancer, or prevent the spread or metastasis or cancer.

[0335] Combination therapy also includes, in addition to administration of an L53 antagonist, the use of one or more molecules, compounds or treatments that aid in the prevention or treatment of cancer (i.e., cancer therapeutics), which molecules, compounds or treatments include, but are not limited to, chemoagents, immunotherapeuti.es, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapies, gene therapies, and radiotherapies.

[0336] In one embodiment, one or more chemoagents, in addition to an L53 antagonist, is administered to treat a cancer patient. A chemoagent (or "anti-cancer agent" or "anti-tumor agent" or "cancer therapeutic") refers to any molecule or compound that assists in the treatment of tumors or cancer. Examples of chemoagents contemplated by the present hivention include, but are not limited to, cytosine arabinoside, taxoids (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel, docetaxel, epothilone B, or its analogues), macrolides (e.g., rhizoxin ) cisplatin, carboplatin, adriamycin, tenoposide, mitozantron, discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin), antimetabolites (e.g., mefhotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, flavopiridol, 5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolamide), asparaginase, Bacillus Calmette and Guerin, diphtheria toxin, hexamethylmelamine, hydroxyurea, LYSODREN®, nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan (CAMPTOSAR, CPT-11), vincristine, vinca aUcyloids such as vinblastine), podophyllotoxin (including derivatives such as epipodophyllotoxin, VP-16 (etoposide), VM-26 (teniposide)), cytochalasin B, colchine, gramicidin D, ethidium bromide, emetine, mitomycin, procarbazine, mechlorethamine, anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin, doxorubicin hposomal), dmydroxyanfhracindione, mitoxantrone, mithramycin, actinomycin u, procarne, tetracame, lidocaine, propranolol, puromycin, anti-mitotic agents, abnn, ncm A, pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, aldesleukin, allutamine, anastrozle, bicalutamide, biaomycin, busulfan, capecitabine, carboplain, chlorabusil, cladribine, cylarabine, daclinomycin, estramusine, floxuridhe, gamcitabine, gosereine, idarubicin, itosfamide, lauprolide acetate, levamisole, lomusline, mechlorethamine, magestrol, acetate, mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxhnab, campath-1, straplozocin, thioguanine, tretinohi, vinorelbrne, or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof. Compositions comprising one or more chemoagents (e.g., FLAG, CHOP) are also contemplated by the present invention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.

[0337] In one embodiment, said chemoagent is gemcitabine at a dose ranging from 100 to 1000 mg/m²/cycle. In one embodiment, said chemoagent is dacarbazine at a dose ranging from 200 to 4000 mg/m²/cycle. In a prefened embodiment, said dose ranges from 700 to 1000 mg/m²/cycle. In another embodiment, said chemoagent is fludarabine at a dose ranging from 25 to 50 mg/m²/cycle. In another embodiment, said chemoagent is cytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000 mg/m²/cycle. In another embodiment, said chemoagent is docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle. In another embodiment, said chemoagent is paclitaxel at a dose ranging from 5 to 15 mg/kg/cycle. In yet another embodiment, said chemoagent is cisplatin at a dose ranging from 5 to 20 mg/kg/cycle. In yet another embodiment, said chemoagent is 5-fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In yet another embodiment, said chemoagent is doxorubicin at a dose ranging from 2 to 8 mg kg/cycle. In yet another embodiment, said chemoagent is epipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle. In yet another embodiment, said chemoagent is cyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle. hi yet another embodiment, said chemoagent is irinotecan at a dose ranging from 50 to 75, 75 to 100, 100 to 125, or 125 to 150 mg/m²/cycle. In yet another embodiment, said chemoagent is vinblastine at a dose ranging from 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11, or 11 to 18.5 mg/m²/cycle. In yet another embodiment, said chemoagent is vincristine at a dose ranging from 0.7 to 1.4, or 1.5 to 2 mg/m²/cycle. In yet another embodiment, said chemoagent is methotrexate at a dose ranging from 3.3 to 5, 5 to 10, 10 to 100, or 100 to 1000 mg/m²/cycle.

[0338] In a prefened embodiment, the invention further encompasses the use of low doses of chemoagents when administered as part of an L53 antagonist treatment regimen. For example, initial treatment with an L53 antagonist increases the sensitivity of a tumor to subsequent challenge with a dose of chemoagent, which dose is near or below the lower range of dosages when the chemoagent is administered without an L53 antagonist. In one embodiment, an L53 antagonist and a low dose (e.g., 6 to 60 mg/m day or less) of docetaxel are administered to a cancer patient. In another embodiment, an L53 antagonist and a low dose (e.g., 10 to 135 mg/m²/day or less) of paclitaxel are adrninistered to a cancer patient. In yet another embodiment, an L53 antagonist and a low dose (e.g., 2.5 to 25 mg/m /day or less) of fludarabine are administered to a cancer patient. In yet another embodiment, an L53 antagonist and a low dose (e.g., 0.5 ^"to 1.5 g/m /day or less) of cytosine arabinoside (Ara-C) are administered to a cancer patient.

[0339] The invention, therefore, contemplates the use of one or more L53 antagonists or agonists, which is administered prior to, subsequently, or concuπently with low doses of chemoagents, for the prevention or treatment of cancer.

[0340] In one embodiment, said chemoagent is gemcitabine at a dose ranging from 10 to lOOmg/mVcycle.

[0341] In one embodiment, said chemoagent is cisplatin, e.g., PLATINOL™ or PLATJNOL- AQ™(Bristol Myers), at a dose ranging from 5 to 10, 10 to 20, 20 to 40, or 40 to 75 mg/m²/cycle. In another embodiment, a dose of cisplatin ranging from 7.5 to 75 mg/m²/cycle is administered to a patient with ovarian cancer or other cancer. In another embodiment, a dose of cisplatin ranging from 5 to 50 mg/m²/cycle is administered to a patient with bladder cancer or other cnacer.

[0342] In another embodiment, said chemoagent is carboplatin, e.g., PARAPLATIN™(Bristol Myers), at a dose ranging from 2 to 4, 4 to 8, 8 to 16, 16 to 35, or 35 to 75 mg/m²/cycle. In another embodiment, a dose of carboplatin ranging from 7.5 to 75 mg/m²/cycle is administered to a patient with ovarian cancer or other cancer. In another embodiment, a dose of carboplatin ranging from 5 to 50 mg/m²/cycle is administered to a patient with bladder cancer or other cancer. In another embodiment, a dose of carboplatin ranging from 2 to 20 mg/m /cycle is administered to a patient with testicular cancer or other cnacer.

[0343] In another embodiment, said chemoagent is docetaxel, e.g., TAXOTERE™ (Rhone Poulenc Rorer) at a dose ranging from 6 to 10, 10 to 30, or 30 to 60 mg/m²/cycle.

[0344] In another embodiment, said chemoagent is paclitaxel, e.g., TAXOL™ (Bristol Myers Squibb), at a dose ranging from 10 to 20, 20 to 40, 40 to 70, or 70 to 135 mg kg/cycle.

[0345] In another embodiment, said chemoagent is 5-fluorouracil at a dose ranging from 0.5 to 5 mg kg/cycle.

[0346] hi another embodiment, said chemoagent is doxorubicin, e.g., ADRIAMYCIN^TM (Pharmacia & Upjohn), DOXJL (Alza), RUBEX™ (Bristol Myers Squibb), at a dose ranging from 2 to 4, 4 to 8, 8 to 15, 15 to 30, or 30 to 60 mg/kg/cycle.

[0347] hi another embodiment, an L53 antagonist is administered in combination with one or more immunotherapeutic agents, such as antibodies and irrrmunomodulators, which include, but are not limited to, Herceptin®, Retuxan®, OvaRex, Panorex, BEC2, JMC-C225, Vitaxin, Campath I H, Smart MI95, LymphoCide, Smart I D10, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.

[0348] In another embodiment, an L53 antagonist is administered in combination with one or more anti-angiogenic agents, which include, but are not limited to, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor) anti-tlirombin, 29 kDa N-termhial and a 40 kDa C-teiτninal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13-amino acid peptide conesponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer Res. 51:2077), a 14-amino acid peptide conesponding to a fragment ot collagen I (Tolma et al., 1993, J. Cell Biol. 122:497), a 19 amino acid peptide conesponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J. Cell Biol 122:497), a 20-amino acid peptide conesponding to a fragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-), or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof.

[0349] Other peptides that inhibit angiogenesis and conespond to fragments of laminin, fibronectin, procoUagen, and EGF have also been described (See the review by Cao, 1998, Prog. Mol. Subcell. Biol. 20:161). Monoclonal antibodies and cyclic pentapeptides, which block certain integrins that bind RGD proteins (le., possess the peptide motif Arg-Gly-Asp), have been demonstrated to have anti- vascularization activities (Brooks et al, 1994, Science 264:569; Hammes et al, 1996, Nature Medicine 2:529). Moreover, inhibition of the urokinase plasminogen activator receptor by antagonists or agonists inhibits angiogenesis, tumor growth and metastasis (Min et al, 1996, Cancer Res. 56:2428-33; Crowley et al, 1993, Proc Natl Acad Sci. USA 90:5021). Use of such anti-angiogenic agents in combination with the L53 modulators is also contemplated by the present invention.

[0350] In another embodiment, an L53 antagonist is administered in combination with a regimen of radiation.

[0351] In another embodiment, an L53 antagonist is administered in combination with one or more cytokines, which include, but are not limited to, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokines, lymphotoxin-α, lymphotoxhi-β, interferon-α, interferon-β, macrophage inflammatory proteins, granulocyte monocyte colony stimulating factor, interleukins (including, but not limited to, interleukin-1, interleukin-2, mterleukin-6, interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD137 ligands, Fas-Fas ligand, 4-1BBL, endothelial monocyte activating protein or any fragments, family members, or derivatives thereof, including phannaceutically acceptable salts thereof.

[0352] In yet another embodiment, an L53 antagonist is administered in combination with a cancer vaccine. Examples of cancer vaccines include, but are not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins (e.g., gplOO, MART-1/MelanA, TRP-1 (gp75), tyrosinase, widely shared tumor-associated, including tumor-specific, antigens (e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3, N-acetylglucosaminyltransferase-V, pl5), mutated antigens that are tumor-associated (β-catenin, MUM-1, CDK4), nonmelanoma antigens (e.g., HER-2/neu (breast and ovarian carcinoma), human papillomavirus-E6, E7 (cervical carcinoma), MUC-1 (breast, ovarian and pancreatic carcinoma). For human tumor antigens recognized by T-cells, see generally Robbins and Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may or may not be purified preparations.

[0353] In yet another embodiment, an L53 antagonist is used in association with a hormonal treatment. Hormonal therapeutic treatments comprise hormonal agonists, hormonal antagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, betamethasone, cortisol, cortisone, prednisone, denydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), and antiandrogens (e.g., cyproterone acetate).

[0354] In yet another embodiment, an L53 antagonist is used in association with a gene therapy program in the treatment of cancer. In one embodiment, gene therapy with recombinant cells secreting interleukin-2 is administered in combination with an L53 antagonist to prevent or treat cancer, particularly lung cancer (See, e.g., Deshmukh et al, 2001, /. Neurosurg. 94:287).

[0355] In one embodiment, an L53 antagonist is administered, in combination with at least one cancer therapeutic agent, for a short treatment cycle to a cancer patient. The duration of treatment with the cancer therapeutic agent may vary according to the particular cancer therapeutic agent used. The invention also contemplates discontinuous administration or daily doses divided into several partial administrations. Appropriate treatment time-lines for cancer therapeutic agents wiU be appreciated by those skilled in the art, and the invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent.

[0356] The present invention contemplates at least one cycle, preferably more than one cycle during which a single therapeutic or sequence of therapeutics is administered. An appropriate period of time for one cycle will be appreciated by the skilled artisan, as will the total number of cycles, and the interval between cycles. The invention contemplates the continued assessment of optimal treatment schedules for each L53 antagonist and cancer therapeutic agent.

5.7. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

[0357] The compounds, proteins, peptides, nucleic acid sequences and fragments mereof, described herein can be administered to a patient at therapeuticaUy effective doses to treat cancer, e.g., lung cancer wherein the expression level of the L53 gene is elevated compared to a non-cancerous sample or a predeteπmned non-cancerous standard. A therapeuticaUy effective dose refers to that amount of a compound sufficient to result in a healthful benefit in the treated subject.

5.7.1. EFFECTIVE DOSE

[0358] Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experhnental animals, e.g., for deteπnining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅o (the dose therapeuticaUy effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅0 ED₅₀. Compounds that exhibit large therapeutic indices are prefened. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.

[0359] The data obtained from the cell culture assays and animal studies can be used in formulating a dose range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. ±<or any compound used in the method of the invention, the therapeuticaUy effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (le., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately deteπnine efficacious doses for administration to humans. Levels in plasma can be measured by any technique known in the art, for example, by high performance hquid chromatography.

5.7.2. FORMULATIONS AND USE

[0360] The invention relates to pharmaceutical compositions, including, but not limited to pharmaceutical compositions comprising an L53 gene product, or antagonists or agonists thereof, for the treatment or prevention of cancer.

[0361] Pharmaceutical compositions for use in accordance with the present invention, e.g., methods to treat or prevent cancer, can be formulated in a conventional manner using one or more physiologically acceptable caπiers or excipients.

[0362] Thus, the compounds and their physiologically acceptable salts and solvents can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

[0363] For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceuticaUy acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyπohdone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystaUine cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g_, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0364] Preparations for oral administration can be suitably formulated to provide controlled release of the active compound.

[0365] For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0366] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use ot a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be deteirnined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0367] The compounds can be formulated for parenteral administration (i.e., intravenous or intramuscular) by injection, via, for example, bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0368] The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0369] In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be adrriinistered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

5.8. VACCINE THERAPY

[0370] L53 nucleic acids and L53 polypeptides and peptides encoded therefrom and fragments thereof, may be used as vaccines by administering to an individual at risk for developing cancer an amount of said protein, peptide, or nucleic acid that effectively stimulates an immune response against an L53-encoded polypeptide and protects that individual from cancer. The invention thus contemplates a method of vaccinating a subject against cancer wherein said subject is at risk for developing cancer.

[0371] Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous route. Various adjuvants may be used to increase the irnmunological response, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

[0372] The nucleotides of the invention, including variants and derivatives, can be used as vaccines, e.g., by genetic immunization. Genetic immunization is particularly advantageous as it stimulates a cytotoxic T-cell response but does not utilize live attenuated vaccines, which can revert to a virulent form and infect the host causing complications from infection. As used herein, genetic immunization comprises inserting the nucleotides of the invention into a host cell, wherein the encoded proteins are expressed. These translated proteins are then either secreted or processed by the host cell for presentation to immune cells and an immune reaction is stimulated. Preferably, the immune reaction is a cytotoxic T cell response, however, a humoral response or macrophage stimulation is also useful in preventing initial or additional tumor growth and metastasis or spread of the cancer. The skilled artisan will appreciate that there are various methods for introducing foreign nucleotides into a host animal and subsequently into cells for genetic immunization, for example, by intramuscular injection of about 50 mg of plasmid DNA encoding the proteins of the invention solubilized in 50 ml of sterile saline solution, with a suitable adjuvant (See, e.g., Weiner and Kennedy, 1999, Scientific American 7:50-57; Lowrie et al., 1999, Nature 400:269-271).

[0373] The invention thus provides a vaccine formulation for the prevention of cancer comprising an immunogenic amount of an L53 gene product. The invention further provides for an immunogenic composition comprising a purified L53 gene product.

5.9. KITS

[0374] The invention includes a kit for assessing the presence of cancer cells including lung cancer cells (e.g., in a sample such as a patient sample). The kit comprises a plurality of reagents, each of which is capable of binding specifically with a nucleic acid or polypeptide conesponding to a marker of the invention, e.g., the L53 gene or gene product or fragment thereof. Suitable reagents for binding with a polypeptide conesponding to a marker of the invention include antibodies, antibody derivatives, labeled antibodies, antibody fragments, and the like. Suitable reagents for binding to a nucleic acid (e.g., a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pahs of PCR primers, molecular beacon probes, and the like.

[0375] The kit of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit may comprise fluids (e.g. , SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody to a protein for which it is immunologically specific, one or more sample compartments, an instructional material which describes performance of a method of the invention, a sample of normal cells, a sample of cancer cells, and the like.

6. EXAMPLES [0376] Methodology utilizing Suppression Subtractive Hybridization (SSH) and high-throughput cDNA microaπays were combined to analyze gene expression patterns in lung cancer cells, hi an aspect of the invention, genes over-expressed in lung cancer were identified. In a particular aspect of the invention, a novel lung cancer-associated antigen L53 was identified using these methods. In accordance with the present invention, L53 nucleic acid sequences, a ino acid sequences and antibodies thereto, and methods of use thereof are provided herein.

[0377] The results provided herein underscore the utility of combining SSH and cDNA microaπays in providing lung cancer-specific gene expression profiles. Sequence analysis confirmed the presence of several previously identified cancer-specific genes and identified additional novel molecules, including L53. The L53 cDNA (FIG. 1) was cloned by PCR and sequenced by automated fluorescent sequencing (Applied Biosystems, Foster City, CA). The expression pattern of the tumor selective antigen L53 demonstrated the utility of the L53 molecules of the invention as diagnostic/prognostic markers for lung cancer and other L53 positive cancers and/or as potential therapeutic targets for regimens dhected to treating patients with lung cancer or other L53 positive cancers.

6.1. INTRODUCTION

[0378] Lung cancer is the leading cause of cancer deaths worldwide, and more specifically non- small cell lung cancer (NSCLC) accounts for approximately 80% of all disease cases (Cancer Facts and Figures, 2002, American Cancer Society, Atlanta, p. 11.). In 2002, there were an estimated 169,500 cases of lung cancer resulting in 154,900 deaths (Cancer Facts and Figures, 2002, American Cancer Society, Atlanta, p. 11.). Adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC based on cellular morphology (Travis et al., 1996, Lung Cancer Principles and Practice, Lippincott- Raven, New York, pps. 361-395). Adenocarcinomas are characterized by a more peripheral location in the lung and often have a mutation in the K-ras oncogene (Gazdar et al, 1994, Anticancer Res. 14:261- 267). Squamous cell carcinomas are typicaUy more centrally located and frequently cany p53 gene mutations (Niklinska et al., 2001, Folia Histochem. Cytobiol. 39:147-148). A systematic evaluation of gene expression profiling data for each of the conesponding NSCLC subtypes using a combination of SSH and DNA aπays may be useful for the identification of additional novel targets of utility in disease detection and as therapeutic targets for lung cancer treatment modalities.

[0379] As described herein, tumor-enriched NSCLC SSH libraries were constructed and aπayed to selectively screen for tumor-specific genes. SSH is a technique well known in the art for its effectiveness in characterizing and prioritizing differentially expressed genes: (Chu et al, 1997, Proc. Natl. Acad. Sci. 94(19): 10057; Gurskaya et al, 1996, Anal. Biochem. 240: 90; Kuang et at., 1998, Nuc. Acid Res. 26: 1116; von Stein et al, 1997, Nuc. Acid Res. 25: 2598; Wong et al, 1997, /. Biol. Chem. 272(40): 25190; and Yokomizo et al, 1997, Nature 387: 620). As described herein, the novel lung cancer-associated molecule L53 was discovered utilizing these techniques.

[0380] Intensive and systematic evaluation of gene expression patterns is crucial for understanding the physiological mechanisms associated with cellular transformation and metastasis. Cuπently, several technical platforms are being used to accomplish this goal. They include: Serial Analysis of Gene Expression (SAGE) (Velculescu et al, 1995, Science 270: 484); Restriction Enzyme Analysis of Differentially Expressed Sequences (READS) (Prasher et al, 1999, Methods Enzymol. 303: 258); Amplified Fragment Length Polymorphism (AFLP) (Bachem et al, 1996, Plant J. 9: 745); Representational Difference Analysis (RDA) (Hubank et al. , 1994, Nucleic Acid Kes. _u/^)' 0O Ui; Differential Display (Liang et al, 1992, Cancer Res. 52(24): 6966); and SSH (Diatchenko et al, 1996, Proc. Natl. Acad. Sci. 93: 6025) as detailed in this text. SSH is very similar to RDA with the exception of an additional normalization step that is included to increase the relative abundance of rare transcripts. The combination of SSH and cDNA microaπays offers several advantages over the aforementioned technologies for the discovery of novel tumor-associated proteins and antigens (TAA's). The use of SSH for identifying novel cancer targets is an attractive approach because it does not rely on previously characterized cDNA sets. SSH efficiently normalizes both frequent and rare transcripts at equivalent levels and preferentiaUy amplifies only those which are differentially expressed. The use of expression anays further increases the chances of identifying lead targets by examining thousands of genes in a single experiment.

6.2. MATERIALS AND METHODS

6.2.1. CELL CULTURE

[0381] NSCLC cell lines including: A549, NCI-H23, NCI-H920, NCI-H969, NCI-H647, NCI- H226, NCI-H1869, NCI-H1385, NCI-H460, NCI-H1155, NCI-H358, and NCI-H650 (ATCC, Manassas, VA) were grown in SAGM medium® (Clonetics, San Diego, CA) supplemented with 0.5% fetal bovine serum (Sigma, St. Louis, MO). AU tumor cell lines were passaged once per week by trypsinization and replated at 2500-3000 cells/cm² (Clonetics, San Diego, CA). Normal human bronchial epithelial cells (NHBEs) (Clonetics, San Diego, CA) were grown in SAGM medium® supplemented with 0.5% fetal bovine serum.

6.2.2. RNA ISOLATION

[0382] Total RNA was isolated from cultured cells using RNA-Bee™ (Tel-Test, Inc., Friendswood, TX). Poly A+ RNA was extracted using the Oligotex mRNA Midi kit ® (Qiagen, Inc., Valencia, CA).

6.2.3. GENERATION OF SSH cDNA LD3RARIES

[0383] Two NSCLC-specific SSH cDNA libraries were constructed as described by Diatchenko et al, 1996, Proc. Natl. Acad. Sci. 93:6025. Library one was constructed using a pool of NSCLC cell lines (tester RNA) including: A549, NCI-H23, NCI-H226, and NCI-H460 vs. a pool of normal patient tissue RNAs (driver RNA) including colon, kidney, lung, and liver (Origene, Inc., Rockville, MD), pancreas (Clontech, Palo Alto, CA), and cultured NHBEs. Library two was constructed using a pool of NSCLC cell lines (tester RNA) including: A549, NCI-H23, NCI-H920, NCI-H969, NCI-H358, and NCI- H650 vs. a pool of normal patient tissue RNAs (driver RNA) including: colon, kidney, lung, and liver (Origene, Inc., Rockville, MD), and pancreas and spleen (Clontech, Palo Alto, CA).

[0384] Driver cDNA was synthesized from poly A+ RNA using 1 ul of 10 uM cDNA synthesis primer 5'-TTTTGTACAAGCTT₃₀NιN-3' (SEQ ID NO: 4) and 1 ul of 200 u/ul Superscript B Reverse ^"Transcπptase® (Invitrogen, Carlsbad, CA). The resulting cDNA peUets were pooled and digested witn 1.5 ul of lOu/ul of Rsa I restriction enzyme. Driver cDNA's were then precipitated with 100 ul of 10M Ammonium Acetate (Sigma, St. Louis, MO), 3 ul of 20mg/ml glycogen (Roche Molecular Biochemicals, Indianapolis, IN) and 1 ml of ethanol (Sigma, St. Louis, MO). The cDNA preparations were then resuspended in 5 ul of diethyl pyrocarbonate (DEPC) treated water.

[0385] Tester cDNA was synthesized from poly A+ RNA as described above for the driver. The resulting cDNA peUets were pooled and digested with 1.5 ul of lOu/ul of Rsa I restriction enzyme. Rsa I digested tester cDNA was diluted in 5 ul of DEPC treated water prior to adaptor ligation. Diluted tester cDNA (2 ul) was ligated to 2 ul of 10 uM adaptor 1 (5'-

CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3') (SEQ ID NO: 5) and 2 ul of 10 uM adaptor 2R (5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3') (SEQ JD NO: 6) in separate reactions ushig 0.5 units of T4 DNA ligase (Invitrogen, Carlsbad, CA).

[0386] Driver cDNA (600 ng) was added separately to each of the two tubes containing adaptor- 1 ligated tester (20 ng) and adaptor 2R ligated tester (20 ng). The samples were mixed, ethanol precipitated as described above, and resuspended in 1.5 ul of hybridization buffer (50 mM Hepes pH 8.3, 0.5 M NaCl/0.0.2 mM EDTA pH 8.0). The reaction mixture was placed in hot start PCR tubes, (Molecular BioProducts, San Diego, CA), denatured at 95°C for 1.5 min. and then incubated at 68°C for 8 hrs. After this initial hybridization, the samples were combined and excess heat denatured driver cDNA (150 ng) was added. This secondary reaction mixture was incubated overnight at 68°C. The final hybridization mixture was diluted in 200 ul of dilution buffer (20 mM Hepes pH 8.3, 50mM NaCl, 0.2 mM EDTA) and stored at -20°C.

[0387] Two rounds of PCR amplification were performed for each SSH library. The primary PCR was performed in 25 ul. The reaction mixture contained 1 ul of diluted subtracted cDNA, 1 ul of 10 uM PCR primer 1 (5'-CTAATACGACTCACTATAGGGC-3') (SEQ JD NO: 7), lOx PCR buffer consisting of (166 mM NH C₂H₃0₂, 670 mM Tris pH 8.8, 67 mM MgCl₂, and lOOmM 2- mercaptoethanol), 1.5 ul of lO M dNTP's, 1.5 ul dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO), and 0.25 ul of 5 u/ul of Taq polymerase (Brinkmann, Westbury, NY). PCR was performed with the following cycling conditions: 75°C for 7 min.; 94°C for 2 min.; 94°C for 30 sec, 66°C for 30 sec, and 72°C for 1.5 min.; and a final extension at 72°C for 5 min. A secondary PCR was performed using 1 ul of the primary PCR as template with the same reaction components as above. Nested PCR primers NP1 (5'- TCGAGCGGCCGCCCGGGCAGGT-3') (SEQ JD NO: 8) and NP2R (5'- AGCGTGGTCGCGGCCGAGGT-3') (SEQ JD NO: 9) were used in place of PCR primer 1. The secondary PCR was performed with the following cycling conditions: 94°C for 2 min.; 94°C for 30 se , 68°C for 30 sec, and 72°Cfor 1.5 min.; and a final extension at 72°C for 5 min. The PCR products were analyzed on 1.5% ultrapure agarose gels (Invitrogen, Carlsbad, CA) and visualized by ethidium bromide (Fisher Chemical, Fair Lawn, NJ). L0388J Subtraction efficiency was confirmed by PCR depletion of EF-1 and Tubulin. EF-1 primers were EF-1 (5'-CTGTTCCTGTTGGCCGAGTC-3') (SEQ ID NO: 10) and EF-2

(5' -CGATGCATTGTTATCATTAAC-3') (SEQ ID NO: 11). Tubulin primers were Tu-1 (5'-

CACCCTGAGCAGCTCATCAC-3') (SEQ ID NO: 12) and Tu2 (5'-GGCCAGGGTCACATTTCACC-3')

(SEQ ID NO: 13).

6.2.4. CLONING OF SSH POOLS INTO pCR4-TOPO [0389] The SSH-cDNA pools were cloned into the pCR4-TOPO® vector (Invitrogen, Carlsbad, CA) and transformed into chemically competent TOP 10 cells® (Invitrogen, Carlsbad, CA). The library was plated on LB agar plates (Becton Dickinson, Sparks, MD) containing 50μg/ul kanamycin (Sigma, St. Louis, MO). Cloning efficiency and size distribution for each library was determined by amplification using M13 (-20) (5'-GTAAAACGACGGCCAGT-3') (SEQ JD NO: 14) and Ml 3R (5' - CAGGAAACAGCTATGACC-3') (SEQ JD NO: 15) universal primers.

6.2.5. CUSTOM ARRAY GENERATION [0390] SSH clones containing cDNA sequences of interest were amplified using Ml 3 (-20) and M13R universal primers. PCR products were purified using 96-weU MultiScreen PCR Purification Plates (Millipore, Bedford, MA). Microaπays were prepared by spotting targets in duplicate on positively charged nylon membranes (Hybond-XL®, Amersham Pharmacia Biotech, Piscataway, NJ) at concentrations of 2 ng DNA/spot using a Biomek 2000 Robot® (Beckman Coulter Inc., FuUerton, CA). For probe construction, mRNA was isolated from cell lines as described above. Poly A+ RNA (1 ug) was converted to cDNA and labeled with (α-P32) dCTP (Amersham Pharmacia Biotech, Piscataway, NJ) by reverse transcription using Superscript II RT® (Invitrogen, Carlsbad, CA). Hybridizations were performed overnight at 42°C in 6X Saline Sodium Citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), 50% deionized formamide, and 5X Denhardf s solution (1% FicoU Type 400, 1% polyvinylpyπolidone, and 1% bovine serum albumin) (Research Genetics, Huntsville, AL). Wash conditions were 4 times in 2X SSC/0.1% SDS for 10 min. each at room temperature, followed by 4 high stringency washes in 0.1X SSC/0.1%SDS at 65°C for 30 min. each.

6.2.6. ARRAY DATA ANALYSIS [0391] Hybridization Intensities were quantitated on the Phosphorlmager SI® (Molecular Dynamics, Sunnyvale, CA) using AnayVision 6.0 Software® (Imaging Research, St. Catharines, ON, CA). Average signal intensities were determined for each set of duplicate spots. For each membrane analyzed, relative quantitative values were detennined based on normalization to multiple housekeeping genes spotted at various locations on each membrane. 6.2.7. SEMI-QUANTITATIVE RT-PCR [0392] DNA was synthesized from 5 ug total RNA using the Superscript First-Strand cDNA Synthesis System for RT-PCR® (Invitrogen, Carlsbad, CA). Gene specific primers were selected for L53 and EF-1 to obtain semi-quantitative mRNA levels. They were as follows: L53-RT1 (5'- TCTGGGTGGAAGTATTCAGC -3') (SEQ JD NO: 16), andL53-RT2 (5 -

TTCTTGATGATCTGCTGCAC-3') (SEQ ID NO: 17). Primers for EF-1 were as follows: EF-1 (5'- CTGTTCCTGTTGGCCGAGTC-3') (SEQ JD NO: 10) and EF-2 (5' CGATGCATTGTTATCATTAAC- 3') (SEQ JD NO: 11).

6.2.8. MULTIPLE TISSUE EXPRESSION ARRAY (MTE™) [0393] The MTE™ (Clontech, Palo Alto, CA) array was used to determhie relative expression of L53 in various normal tissues. Gene specific primers were selected for L53 and EF-1 to amplify a probe for use in this experiment. They were as follows: L53-RT1 (5'-TCTGGGTGGAAGTATTCAGC -3') (SEQ ID NO: 16), and L53-RT2 (5'-TTCTTGATGATCTGCTGCAC-3') (SEQ JD NO: 17). Primers for EF-1 were as follows: EF-1 (5'-CTGTTCCTGTTGGCCGAGTC-3') (SEQ JD NO: 10) and EF-2 (5' CGATGCATTGTTATCATTAAC-3') (SEQ JD NO: 11). Fifty ng of PCR product was labeled using Ready-to-go Beads® (Amersham Biosciences Corporation, Piscataway, NJ) and α-P32 dCTP at 3000 Ci mmol (Amersham Biosciences Corporation, Piscataway, NJ). The housekeeping control, EF-1, was used to evaluate the spot-to-spot variability within the experiment. Images were analyzed using the PhosPhor Imager SI (Amersham Biosciences, Piscataway, NJ).

6.2.9 QUANTITATIVE REAL-TIME PCR USING SYBR GREEN I CHEMISTRY [0394] The ABI PRISM® 7000 Real-Time PCR Sequence Detection System (Applied Biosystems, Foster City, CA) was used to determine the cancer-selectivity for L53. Primers and probes for L53 were as follows: L53-FP (5'-CAAGTTTGGCTGCCATGCTTA-3') (SEQ JD NO: 18), L53-RP (5'-AATACTTCCACCCAGAAAATGATGAT-3') (SEQ JD NO: 19), and L53-Probe (5'- CGGGATACTGCACATGA-3') (SEQ JD NO: 20). Primers and probes for EFl were as follows: EFl-FP (5'-ATGACCCACCAATGGAAGCA-3') (SEQ ID NO: 21), EF1-RP (5'-

GCCTGGATGGTTCAGGATAATC-3') (SEQ JD NO: 22), and EFl-Probe (5'-CTGGCTTCACTGCTC- 3') (SEQ ID NO: 23). EF-1 was used as the normalization gene for all ABI PRISM® 7000 experiments.

[0395] The Comparative Ct Method (Applied Biosystems, Foster City, CA) was used in calculating tumor vs. normal ratios for L53. The amount of target (L53), normalized to an endogenous reference gene (EF-1) and relative to a calibrator, is given by the arithmetic formula: 2^"MCt where ΔΔCt is the change in threshold cycle between target and reference.

6.2.10. BIOINFORMATICS ANALYSIS [0396] After completion of the aπay data analysis sorting process, interesting novel targets were retained and analyzed further using several computational programs. The derived L53 cDNA was analyzed using Vector NTI Suite 6.0® (InforMax, Inc., Bethesda, MD). Transmembrane (TM) domain and protein localization analysis was performed using the ExPASy Proteomics Tools Programs® (Swiss Institute of Bioinformatics, Geneve, Switzerland). The PSORT algorithm (Nakai et al, 1999, Trends Biochem. Sci. 24(1):34) and ScanProsite (Gattiker et al, 2002, Applied Bioinformatics. 1:107-108) were also used for bioinformatic analysis of L53.

6.2.11 SUBCELLULAR LOCALIZATION FOR L53 [0397] The subceUular localization patterns for L53 were determined using green fluorescent protein (GFP) reporter constructs. L53 cDNA clones were amplified by PCR using gene-specific primers: L53 (GFP1) 5'- CACCATGACTAAAAAAAGAAAACGC-3' (SEQ ID NO: 24) and L53 (GFP2) 5- ATACACTGTAGCCAGTGCACTGGGC-3' (SEQ JD NO: 25) and cloned into pENTR/D-TOPO® vector (Invitrogen, Carlsbad, CA). These constructs were transiently transfected into human kidney 293 cells by electroporation. The subceUular localization patterns for L53 green fluorescence signals were monitored by fluorescence microscopy.

6.3 RESULTS 6.3.1. ISOLATION OF THE L53 cDNA

[0398] The L53 nucleic acid sequence (FIG. 1) was amplified from lung carcinoma cell line RNA using gene-specific primers and cloned into the pCR 4.0® TOPO TA vector (Invitrogen, Carlsbad, CA). The L53 nucleic acid sequence (FIG. 1) (SEQ ID NO: 1) was sequence verified using custom prhners (Sigma-Genosys, Woodlands, TX) and automated fluorescent sequencing (PE Applied Biosystems, Foster City, CA).

6.3.2. L53 CANCER-SELECTIVITY BY SEMI-QUANTITATIVE PCR [0399] L53 displayed cancer-selectivity on various lung carcinoma cell lines (FIG. 2). A cDNA region specific for L53 was amplified in this experiment. L53 mRNA was positive in various NSCLC cell lines including: NCI-H920 (adenocarcinoma, and lymph node metastases), NCI-H647 (adenosquamous carcinoma, pleural effusion, and metastases), NCI-H1869 (squamous cell carcinoma, pleural effusion, and metastases), NCI-H1385 (squamous cell carcinoma, and lymph node metastases), NCI-H460 (large cell carcinoma, pleural effusion, and tumorigenic in mice), NCI-H1155 (large cell carcinoma, neuroendocrine origin, and lymph node metastases), NCI-H358 (bronchioalveolar carcinoma, and tumorigenic in mice), and NCI-H650 (bronchioalveolar carcinoma, and lymph node metastases).

[0400] L53 also displayed positive mRNA expression in other tumor cell lines (FIG. 3). Positive tumor cell lines for L53 included: CAPAN-2 (pancreas adenocarcinoma), HCT 116 (colon carcinoma), HCT 15 (colon adenocarcinoma), RCA (colon adenocarcinoma) (Sjogren et al, 1997, Cancer Res. 57(20):4530-4536), T-47D (breast ductal carcinoma, pleural effusion, and metastases), and IMR-32 (neuroblastoma). 6.3.3. L53 EVALUATION OF NORMAL EXPRESSION BY MTE™ ARRAY

[0401] 53 mRNA expression levels in normal tissues were evaluated using the Multiple Tissue

Expression (MTE™) Anay. A cDNA region specific for L53 was amplified and used as a probe for this experiment. The MTE™ Anay contains 76 tissue-specific polyA+ RNA isolates (Table 3). Overall, L53 displayed minimal expression in normal tissues (FIG. 4B) using a 6-day exposure. A cDNA region specific for the control gene EF-1 was amplified and used to examine loading equivalence (FIG. 4A) using a 2-day exposure. L53, based on its highly restrictive normal tissue expression and its observed NSCLC selectivity, is an interesting target for therapeutic consideration.

TABLE 3: Table of RNA used in the MTE™ Array

6.3.4. L53 QUANTITATIVE REAL-TIME PCR USING SYBR GREEN I CHEMISTRY

[0402] L53 displayed lung cancer-selectivity using tumor and conesponding normal tissue RNAs (BioChain Institute, Hayward, CA, and Ambion, Austin, TX) and the ABI PRISM® 7000 Real-Time PCR Sequence Detection System. Ten lung tumors and conesponding normal tissues were used for comparative L53 mRNA expression analysis (Table 4). The comparative C method was used in calculating quantitative T: N ratios while ushig the endogenous control gene EF-1 as a reference. In total, L53 was overexpressed in 5 of 10 lung cancer patients (50%) at T: N levels > 3-fold (Table 5). Interleukin 8 (IL-8), a previously reported NSCLC marker (Yuan et al, 2000, Am J Respir Crit Care Med. 162:1957-1963), and Mucin (MUC-1) were used in assessing overall patient tissue RNA integrity (Table 5). JL-8 data was consistent with previously published reports (Yuan et al, 2000, Am J Respir Crit Care Med. 162:1957-1963) (Table 6). hi a direct comparison with IL-8 and MUC-1, L53 exhibited similar levels of over-expression using NSCLC and adjacent normal tissue RNAs (Table 6). Based on its elevated tumor selective expression and minimal normal tissue cross reactivity, L53 can be useful as a potential indicator for disease progression. TABLE 4: Summarv of NSCLC patient tissue RNAs

Tissue Tumor Type Sex Age

Lung Tumor 1 Adenocarcinoma M 44 Lung Tumor 2 Adenocarcinoma M 62 Lung Tumor 3 Adenocarcinoma F 58 Lung Tumor 4 Adenocarcinoma M 60 Lung Tumor 5 Bronchioalveo i!lar Carcinoma F 73 Lung Tumor 6 Squamous Ce] 11 Carcinoma M 78 Lung Tumor 7 Squamous Ce] .1 Carcinoma M 62 Lung Tumor 8 Squamous Ce] 1 Carcinoma F 63 Lung Tumor 9 Squamous Ce .1 Carcinoma M 64 Lung Tumor 10 Squamous Ce .1 Carcinoma M 52

TABLE 5: L53 quantitative expression in individual NSCLC RNAs using SYBR Green I Assays

Tissue L53Ct EF-lCt ΔCt ΔACt T: N

Lung Tumor 1 27.61 18.84 8.77 0.06 0.9 Lung Normal 1 28.78 20.08 8.71 Lung Tumor 2 28.61 21.11 7.50 -2.56 5.9 Lung Normal 2 31.53 21.47 10.06 Lung Tumor 3 28.05 19.02 9.04 -0.27 1.2 Lung Normal 3 28.60 19.29 9.31 Lung Tumor 4 29.22 19.94 9.28 0.25 0.8 Lung Normal 4 28.84 19.81 9.03 Lung Tumor 5 33.51 24.18 9.33 0.54 0.7 Lung Normal 5 33.29 24.49 8.79 Lung Tumor 6 29.07 20.94 8.13 -1.55 3.0 Lung Normal 6 31.55 21.88 9.68 Lung Tumor 7 31.88 25.02 6.87 -2.98 7.9 Lung Normal 7 34.25 24.40 9.85 Lung Tumor 8 29.15 22.00 7.15 -0.43 1.4 Lung Normal 8 29.15 21.57 7.58 Lung Tumor 9 30.38 22.09 8.30 -3.73 13.3 Lung Normal 9 35.37 23.35 12.03 Lung Tumor 10 26.31 19.03 7.28 -2.84 7.2 Lung Normal 10 32.46 22.34 10.12

TABLE 6: L53. IL-8. and MUC-1 mRNA expression levels using SYBR Green I Assays Tissue L53 T: N IL-8 T: N MUC-1 T: N

Lung Tumor 1 0.9 1.1 2.7 Lung Normal 1 Lung Tumor 2 5.9 7.1 9.4 Lung Normal 2 Lung Tumor 3 1.2 0.5 1.1 Lung Normal 3 Lung Tumor 4 0.8 1.7 2.0 Lung Normal 4 Lung Tumor 5 0.7 2.8 3.2 Lung Normal 5 Lung Tumor 6 3.0 0.2 1.0 Lung Normal 6 Lung Tumor 7 7.9 6.7 3.2 Lung Normal 7 Lung Tumor 8 1.4 2.1 0.7 Lung Normal 8 Lung Tumor 9 13.3 13.9 >20 Lung Normal 9 Lung Tumor 10 7.2 5.7 0.60 Lung Normal 10 6.3 5 L53 AMINO ACID SEQUENCE, PUTATIVE TM PREDICTIONS, AND SEQUENCE

MOTIFS

[0403] The L53 protein (SEQ ID NO: 3) (FIG. 5) was analyzed using the Bioinformatics

Algorithms associated with the ExPASy Proteomics Tools Programs® (Swiss Institute of Bioinformatics, Geneve, Switzerland). The TMpred algorithm (Hofinann et al, 1993, J. Biol. Chem. 347:166) was used in identifying putative transmembrane (TM) regions (Table 7) for the L53 protein (SEQ ID NO: 3) (FIG.

5). According to the PSORT algorithm (Nakai et al, 1999, Trends Bi Ochem. Sci. 24(1):34), results of the k- Nearest Neighbors Method for classifying cellular localization predi cted a 52.2 % likelihood of L53 being associated with the plasma membrane (Nakai et al, 1997, Intelli g_t ent Systems for Molecular Biology. 5:147-152).

[0404] The L53 protein (SEQ JD NO: 3) (FIG. 5) was also analyzed using ScanProsite (Gattiker et al, 2002, Applied Bioinformatics. 1:107-108) for interesting functional motifs (Table 8). L53 has a cAMP phosphorylation site at position 216-219 (RRLT) (Table 8). Numerous studies have been completed in assessing the specificity of cAMP vs. cGMP dependent protein kinases (Glass et al, 1986, J Biol Chem. 261:2987-2993). Divergence in cAMP phosphorylation sites between normal and transformed lung tissues have been previously reported (Gasa et al, 1987, J Biol Chem. 262(3): 1230-1238).

[0405] L53 has numerous protein kinase C (PKC) phosphorylation sites (Table 8) that often exhibit a preference for C-terminal serine or threonine residues (Woodget et al, 1986, Eur J Biochem. 161:177-184). Alterations in PKC isoforms have been reported to promote cellular survival and chemotherapeutic resistance in non-small lung cancer cells (Clark et al, 2003, Cancer Res. 63(4):780- 786). Various NSCLC ceU lines exhibited altered expression of specific PKC isoforms in comparison with normal lung epithelial cells (Clark et al, 2003, Cancer Res. 63(4):780-786).

[0406] L53 contains several casein kinase π phosphorylation sites (Table 8). Casein kinase JJ (CK2) is a protein serine/threonine kinase that functions in a cyclic nucleotide and calcium independent fashion. CK2 phosphorylates a variety of different proteins via numerous substrate specificities, including: Ser residues are more efficiently phosphorylated acceptor sites than Thr residues; Asp or Glu must be present three residues from the C-teπninal of the phosphate acceptor site; additional acidic residues in positions +1, +2, +4, and +5 increase the rate of phosphorylation; Asp is prefened to Glu as an acidic provider; and a basic residue at the N-tenriinal of the acceptor site decreases the rate of phosphorylation (Pinna et al, 1990, Biochem Biophys Acta. 1054:267-284). CK2, reportedly over- expressed in lung tumors vs. conesponding non-neoplastic tissue, has been previously reported to play a significant role in cellular proliferation (Yaylim et al, 2002, Anticancer Res. 1A:215-218).

[0407] N-teιτninal N-myristoylation is a lipid anchor modification of eukaryotic and viral proteins that facilitates membrane targeting and alters cellular functioning (Maurer-Stroh et al, 2002, J Mol Biol 317(4):523-540). L53 contains multiple N-myristoylation sites (Table 8) that may assist in defining its putative biological role. N-myristoyl proteins have been described in association with carcinogenesis, including colon cancer (Schultz et al, 1985, Science. 227:427-429). Protein myristoylation has been described in relation to several different biological pathways, including: signal transduction, apoptosis, and alternative extracellular protein export (Maurer-Stroh et al, 2002, J Mol Biol. 317(4):523-540). An additional tumor-associated gene hMMTAG2, with similar molecular characteristics as L53, including N-myristoylation sites, phosphorylation sites, and nuclear localization signals has been previously described with significant transformation capabilities (Tian et al, 2003, Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 35(2): 143-148). L53, based on its similarity with such tumor-associated genes, may possess the same unique transformation capabihties.

TABLE 7 L53 putative TM regions using the TMpred algorithm

TM # TM Position TM Sequence

1 431-451 LLLNLTLSDLMVSLANASTLQ (SEQ ID NO: 26)

2 641-663 IMGRLSSSLAAMLIGILHMRSSF (SEQ ID NO: 27) 3 673-692 WLMSDVDYFSFLFSTLTGFS (SEQ ID NO: 28) 4 761-783 LVGLTVIPDSTAGCVFGVICKLL (SEQ ID NO: 29) 5 786-808 TCVVSETLLPFLASCCYSLLYFL (SEQ ID NO: 30) 6 825-844 LWGGVCVSILALLPRVLRLML (SEQ ID NO: 31)

TABLE 8 L53 functional motifs using the ScanProsite algorithm

Motif Position Sequence

Glycosylation 290-293 NVSS (SEQ ID NO: 32)

Glycosylation 415-418 NKSI (SEQ ID NO: 33)

Glycosylation 434-437 NLTL (SEQ ID NO: 34)

Glycosylation 446-449 NAST (SEQ ID NO: 35)

Glycosylation 890-893 NITT (SEQ ID NO: 36) cAMP phospho site 216-219 RRLT (SEQ ID NO: 37)

PKC phospho site 2-4 TKK

PKC phospho site 108-110 TDK

PKC phospho site 160- 162 SLK

PKC phospho site 176-178 TGR

PKC phospho site 242-244 SSR

PKC phospho site 376-378 THK

PKC phospho site 417-419 SIK

PKC phospho site 486-488 SWR

PKC phospho site 538-540 SCR

PKC phospho site 669-671 SAK

PKC phospho site 701-703 SLR

PKC phospho site 846-848 SLR

PKC phospho site 893-895 TLK

CK2 phospho site 108-111 TDKD (SEQ ID NO: 38)

CK2 phospho site 305-308 SGVD (SEQ ID NO: 39)

CK2 phospho site 312-315 SSTE (SEQ ID NO: 40)

CK2 phospho site 436-439 TLSD (SEQ ID NO: 41)

CK2 phospho site 455-458 SWIE (SEQ ID NO: 42)

CK2 phospho site 467-470 TLED (SEQ ID NO: 43)

CK2 phospho site 669-672 SARD (SEQ ID NO: 44)

CK2 phospho site 676-679 SDVD (SEQ ID NO: 45)

CK2 phospho site 692-695 SKEE (SEQ ID NO: 46)

CK2 phospho site 721-724 TDLD (SEQ ID NO: 47)

CK2 phospho site 898-901 SVQE (SEQ ID NO: 48)

N-myristoylation site 48-53 GTLPTN (SEQ ID NO: 49)

N-myristoylation site 72-77 GVKQSA (SEQ ID NO: 50)

N-myristoylation site 286-291 GSQPNV (SEQ ID NO: 51)

N-myristoylation site 302-307 GGLSGV (SEQ ID NO: 52)

N-myristoylation site 306-311 GVDEGL (SEQ ID NO: 53)

N-myristoylation site 310-315 GLSSTE (SEQ ID NO: 54)

N-myristoylation site 471-476 GSRLNS (SEQ ID NO: 55)

N-myristoylation site 773-778 GCVFGV (SEQ ID NO: 56)

N-myristoylation site 827-832 GVCVSI (SEQ ID NO: 57)

Amidation site 18-21 VGKK (SEQ ID NO: 58)

Nuclear targeting site 5-21 RKRQHDFQKVKLKVGKK (SEQ ID NO: 59)

Nuclear targeting site 6-22 KRQHDFQKVKLKVGKKK (SEQ ID NO: 60)

Leucine zipper site 548-569 LSRWLAGLPLQLAHLGSRNPEL (SEQ ID NO: 61) TABLE 8Continued

Heat repeat site 99-137 ILSEVTAWTDKDANVRLAAVQLLQFLAPKIRAEQISPFL

(SEQ ID NO: 62)

6.3.6 L53 SUBCELLULAR LOCALIZATION

[0408] SubceUular localization patterns for L53 were analyzed using confocal microscopy. Specifically, transient expression and subceUular localization pattern recognition of L53/GFP constructs were analyzed using 293 human kidney cells. SubceUular localization patterns for L53 were consistent with previously reported data demonstrating the nuclear localization of unrelated molecules (Simpson et al, 2000, EMBO reports, 3 : 287-292). The biological function of many important proteins and transcription factors are regulated via nuclear localization. Small molecular weight inhibitors which facilitate nuclear or cytoplasmic sequestering are cunently under therapeutic investigation (Kau et al, 2003, Drug Discov Today. 8(2):78-85). The identification of additional molecules with properties similar to those of L53, including an involvement in cellular transformation, may also assist in defining the therapeutic potential of agents that disrupt nuclear locahzation of molecules implicated in the onset and/or progression of cancer (Tian et al, 2003, Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 35(2)143- 148). Future studies to investigate a proposed targeted therapy utilizing the L53 antigen will be helpful in defining a functional role for this class of targets.

6.4. DISCUSSION [0409] Gene expression profiling provides a systematic approach for studying the mechanisms associated with progression from normal to metastatic disease. As described herein, SSH and cDNA microaπay techiniques were utilized to identify the novel lung cancer-associated antigen, L53. Combining SSH and cDNA microaπay methodology provides a rapid and effective approach for high- throughput screening and identification of novel tumor associated antigens (TAAs). The principle of SSH allows for the preferential amplification of differentially expressed sequences while suppressing those present at equal abundance within the initial mRNA (Diatchenko et al, supra). The high level of enrichment, low level of background, and efficient normalization of sequences makes this an attractive approach for the rapid identification of novel targets. L53 molecules of the present invention and variants thereof may be used to advantage as diagnostic, prognostic, and/or therapeutic targets for lung cancer and other cancers in which L53 is abeπantly regulated or expressed. Notably, L53 displayed tumor-selective expression in lung cancer, and other cancers, while displaying minimal expression in normal tissues. Based on its elevated level of tumor-selective expression and overall hydrophobicity, L53 nucleic acid sequences, encoded polypeptides and antibodies thereto, and methods of use thereof may be utilized effectively to detect upregulated expression of L53, the detection of which serves as a diagnostic and/or prognostic indicator of cancer. Moreover, the L53 molecules of the invention and compounds identified using the methods of the invention which are capable of modulating L53 expression levels and/or activity also provide novel reagents with which to treat cancer patients. Such treatment modalities may be administered to a patient to amehorate the symptoms of the disease, inhibit the disease by, for example, reducing tumor burden, and/or inhibit the progression of the disease by, for example, preventing metastasis.

[0410] The identification of L53 nucleic and amino acid sequences, L53 antibodies, methods of use of these L53 reagents, and L53 modulating compounds provides valuable reagents with which to investigate the mechanism(s) involved in lung cancer development and progression. As demonstrated herein, gene expression profiling studies using SSH and aπays are useful for identifying novel cancer- selective genes, such as L53, whose role(s) in lung cancer onset/progression have been heretofore previously unrecognized. Additional studies, based on the novel findings set forth herein, may elucidate the functional role of tumor associated antigens in biochemical pathways and mechanisms involved in carcinogenesis and disease progression. Such information may then be applied to the design of improved and novel therapeutic regimens for the treatment of lung cancer and other L53 associated cancers.

7. REFERENCES CITED

[0411] AU references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0412] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

What is claimed is:

1. A method of diagnosing cancer in a subject comprising detecting or measuring an L53 gene product in a sample derived from said subject, wherein the L53 gene product is:

(a) an RNA conesponding to SEQ JD NO: 1 , or a nucleic acid derived thereof;

(b) an RNA conesponding to SEQ JD NO:2, or a nucleic acid derived thereof;

(c) a protein comprising SEQ JD NO: 3 ;

(d) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(e) a nucleic acid comprising a sequence hybridizable to SEQ ID NO:2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(f) a nucleic acid at least 90% homologous to SEQ ID NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or

(g) a nucleic acid at least 90% homologous to SEQ JD NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; in which elevated levels of the L53 gene product compared to a non-cancerous sample or a pre- detenriined standard value for a noncancerous sample, indicates a presence of cancer in the subject.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 1 or 2, wherein the cancer is lung cancer or any L53 positive cancer.

4. The method of claim 1 or 2, wherein the sample is a tissue sample.

5. The method of claim 1 or 2, wherein the sample is a plurality of cells.

6. The method of claim 1 or 2, wherein the sample is a bodily fluid.

7. The method of claim 1, wherein the L53 gene product is a protein comprising SEQ JD NO:3.

8. The method of claim 1, wherein the L53 gene product is an mRNA conesponding to SEQ JD NO:l.

9. The method of claim 1, wherein the L53 gene product is an mRNA conesponding to SEQ ID NO:2.

10. The method of claim 1, wherein an antibody that is specific for the L53 gene product is used for detecting or measuring the L53 gene product.

11. The method of claim 10, wherein the antibody immunospecifically binds to SEQ ID NO: 1 or SEQ

JD NO:2.

12. The method of claim 10, wherein the antibody immunospecifically binds to SEQ ID NO:3.

13. The method of claim 1, wherein an oligonucleotide that is specific for the L53 gene product is used for detecting or measuring the L53 gene product.

14. The method of claim 13, wherehi the oligonucleotide is a DNA oligonucleotide.

15. A method of staging cancer in a subject comprising detecting or measuring an L53 gene product in a sample derived from the subject, wherein the L53 gene product is:

(a) an RNA conesponding to SEQ JD NO: 1 , or a nucleic acid derived thereof; (a) an RNA conesponding to SEQ ID NO:2, or a nucleic acid derived thereof;

(c) a protein comprising SEQ JD NO:3 ;

(d) a nucleic acid comprising a sequence hybridizable to SEQ ID NO:l or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(e) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(g) a nucleic acid at least 90% homologous to SEQ ID NO:2 or its complement as detennined using an NBLAST algorithm, or a protein encoded thereby; in which elevated levels of the L53 gene product compared to a non-cancerous sample or a predetermined standard value for a noncancerous sample, indicates an advanced stage of cancer in the subject.

16. The method of claim 15, wherein the subject is a human.

17. The method of claim 15, wherein the cancer is lung cancer or any L53 positive cancer.

18. The method of claim 15, wherein the cancer involves regional lymph nodes.

19. The method of claim 15, wherein the cancer involves distant metastases.

20. The method of claim 15, wherein the sample is a tissue sample.

21. The method of claim 15, wherein the sample is a plurality of cells.

22. The method of claim 15, wherein the sample is a bodily fluid.

23. The method of claim 15, wherein the L53 gene product is a protein comprising SEQ ID NO:3.

24. The method of claim 15, wherein the L53 gene product is an mRNA conesponding to SEQ ID NO:l.

25. The method of claim 15, wherein the L53 gene product is an mRNA conesponding to SEQ JD NO:2.

26. The method of claim 15, wherein an antibody that is specific for the L53 gene product is used for detecting or measuring the L53 gene product.

27. The method of claim 26, wherein the antibody immunospecifically binds a protein comprising an amino acid sequence of SEQ JD NO:3.

28. The method of claim 15, wherein an oligonucleotide that is specific for the L53 gene product is used for detecting or measuring the L53 gene product.

29. The method of claim 28, wherein the oligonucleotide is a DNA oligonucleotide.

30. A method for treating cancer in a subject, comprising administering to the subject an amount effective for treating cancer of a compound that modulates expression levels of an L53 gene product or an activity of an L53 gene product, wherein said L53 gene product is:

(a) an RNA conesponding to SEQ ID NO: 1, or a nucleic acid derived thereof;

(b) an RNA conesponding to SEQ ID NO: 2, or a nucleic acid derived thereof;

(c) a protein comprising SEQ JD NO:3;

(d) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (e) a nucleic acid comprising a sequence hybridizable to SEQ ID NO:2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(f) a nucleic acid at least 90% homologous to SEQ JD NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or

(g) a nucleic acid at least 90% homologous to SEQ ID NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby.

31. The method of claim 30, wherein the compound decreases expression of the L53 gene product and the L53 gene product is a protein comprising SEQ JD NO:3.

32. The method of claim 30, wherein the compound decreases expression of the L53 gene product and the L53 gene product is an RNA conesponding to SEQ JD NO:l.

33. The method of claim 30, wherein the compound decreases expression of the L53 gene product and the L53 gene product is an RNA conesponding to SEQ ID NO:2.

34. The method of claim 30, wherein the cancer is lung cancer or any L53 positive cancer.

35. The method of claim 30, wherein the compound is a protein, a peptide, an organic molecule with a molecular weight of less than 500 daltons, an inorganic molecule with a molecular weight of less than 500 daltons, an antisense ohgonucleotide molecule that binds to said RNA and inhibits translation of said RNA, a ribozyme molecule that targets said RNA and inhibits translation of said RNA, or an antibody immunologically specific for an L53 gene product.

36. The method of claim 35, wherein the antibody immunologically specific for an L53 gene product binds to a protein comprising an amino acid sequence of SEQ ID NO:3.

37. The method of claim 30, wherein the compound is a double stranded oligonucleotide that forms a triple helix with a promoter of an L53 gene, wherein the L53 gene is a nucleic acid at least 80% homologous to SEQ JD NO:l or its complement as detennined using an NBLAST algorithm.

38. The method of claim 30, wherein the compound is a double stranded oligonucleotide that forms a triple helix with a promoter of an L53 gene, wherein the L53 gene is a nucleic acid at least 80% homologous to SEQ JD NO:2 or its complement as determined using an NBLAST algorithm.

39. A method of vaccinating a subject against cancer comprising administering to me subject a molecule that elicits an immune response to an L53 gene product, wherein the L53 gene product is: (a) an RNA conesponding to SEQ ID NO: 1, or a nucleic acid derived thereof;

(b) an RNA conesponding to SEQ ID NO:2, or a nucleic acid derived thereof;

(c) a protein comprising SEQ ID NO:3;

(d) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(f) a nucleic acid at least 90% homologous to SEQ ID NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby;

(g) a nucleic acid at least 90% homologous to SEQ ID NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby;

(h) a DNA molecule comprising SEQ JD NO: 1 ; or (i) a DNA molecule comprising SEQ ID NO:2.

40. The method of claim 39, wherein the subject is a human.

41. The method of claim 39, wherein the molecule is an isolated DNA molecule comprising SEQ JD NO:l, an isolated DNA molecule comprising SEQ ID NO: 2, or an isolated protein comprising SEQ JD NO:3.

42. The method of claim 39, wherein the cancer is lung cancer or any L53 positive cancer.

43. The method of claim 39, wherein the immune response comprises a cellular immune response, a humoral immune response, or both a cellular and a humoral immune response.

44. A method of determining if a subject is at risk for developing cancer, said method comprising: (a) measuring an amount of an L53 gene product in a sample derived from the subject, wherein said L53 gene product is:

(i) an RNA conesponding to SEQ ID NO: 1 , or a nucleic acid derived thereof;

(ii) an RNA conesponding to SEQ ID NO:2, or a nucleic acid derived thereof;

(hi) a protein comprising SEQ JD NO:3;

(iv) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (v) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (vi) a nucleic acid at least 90% homologous to SEQ ID NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or (vii) a nucleic acid at least 90% homologous to SEQ JD NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; and

(b) comparing the amount of the L53 gene product in the subject with the amount of L53 gene product present in a non-cancerous sample or predetermined standard for a noncancerous sample, wherein an elevated amount of the L53 gene product in the subject compared to the amount in the noncancerous sample or predetermined standard for a noncancerous sample indicates a risk of develophig cancer in the subject.

45. The method of claim 44, wherein the subject is a human.

46. The method of claim 44, wherein the cancer is lung cancer or any L53 positive cancer.

47. The method of claim 44, wherein the L53 gene product is a protein comprising SEQ ID NO:3, an mRNA conesponding to SEQ JD NO:l, or an mRNA conesponding to SEQ ID NO:2.

48. The method of claim 44, wherein an antibody hnmunologicaUy specific for the L53 gene product is used for measuring the amount of the L53 gene product.

49. The method of claim 48, wherein the antibody antibody hnmunologicaUy specific for the L53 gene product binds to a protein comprising an amino acid sequence of SEQ JD NO: 3.

50. The method of claim 44, wherein an oligonucleotide specific for the L53 gene product is used for measuring the amount of the L53 gene product.

51. The method of claim 50, wherein the oligonucleotide is a DNA oligonucleotide.

52. A method of determining if a subject suffering from cancer is at risk for metastasis of said cancer, the method comprising measuring an amount of an L53 gene product in a sample derived from the subject, wherein the gene product is:

(a) an RNA conesponding to SEQ ID NO: 1 , or a nucleic acid derived thereof; (a) an RNA conesponding to SEQ JD NO:2, or a nucleic acid derived thereof;

(c) a protein comprising SEQ JD NO : 3 ; (d) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence;

(f) a nucleic acid at least 90% homologous to SEQ JD NO:l or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or

(g) a nucleic acid at least 90% homologous to SEQ JD NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; wherein an elevated amount of L53 gene product in the subject compared to the amount in the noncancerous sample, or in the sample from the subject with the non-metastasizing cancer, or the amount in the predetermined standard for a noncancerous or non-metastasizing sample, indicates a risk of developing metastasis of said cancer in the subject.

53. The method of claim 52, wherein the subject is a human.

54. The method of claim 52, wherein the cancer is lung cancer or any L53 positive cancer.

55. The method of claim 52, wherein the sample is a tissue sample, a plurality of cells, or a bodily fluid.

56. The method of claim 52, wherein the L53 gene product is a protein comprising SEQ ID NO:3, an mRNA conesponding to SEQ ID NO. , or an mRNA conesponding to SEQ JD NO:2.

57. The method of claim 52, wherein an antibody immunospecific for the L53 gene product is used for detecting or measuring the L53 gene product.

58. The method of claim 57, wherein the antibody immunospecific for the L53 gene product binds to SEQ JD NO:l, SEQ JD NO:2, or SEQ JD NO:3.

59. The metliod of claim 52, wherein an ohgonucleotide specific for the L53 gene product is used for detecting or measuring the L53 gene product.

60. The method of claim 59, wherein the oligonucleotide is a DNA ohgonucleotide.

61. A method of screening for a compound capable of binding to an L53 molecule, said method comprising: (a) contacting the L53 molecule with a candidate agent, wherein said L53 molecule is: (i) an RNA conesponding to SEQ JD NO: 1 , or a nucleic acid derived thereof; (ii) an RNA conesponding to SEQ JD N0:2, or a nucleic acid derived thereof;

(iii) a protein comprising SEQ JD NO : 3 ;

(iv) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprismg a sequence encoded by said hybridizable sequence; (v) a nucleic acid comprising a sequence hybridizable to SEQ JD NO:2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (vi) a nucleic acid at least 90% homologous to SEQ JD NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or (vii) a nucleic acid at least 90% homologous to SEQ JD NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; and

(b) determining whether or not the candidate agent binds the L53 molecule.

62. The method of claim 61, wherein said L53 molecule is an RNA molecule conesponding to SEQ ID NO:l, a DNA molecule that is at least 90% homologous to SEQ ID NO:l or its complement as determined using an NBLAST algorithm, or a protein comprising SEQ ID NO:3.

63. The method of claim 61, wherein the screening assay is performed in vitro.

64. The method of claim 61, wherein the L53 molecule is anchored to a solid phase.

65. The method of claim 61, wherein the candidate agent is anchored to a solid phase.

66. The method of claim 61, wherein the screening assay is performed in liquid phase.

67. The method of claim 61, wherein said L53 molecule is expressed on a surface of a cell or inside a cell in step (a).

68. The method of claim 67, wherein the cell is engineered to express the L53 molecule.

69. The method of claim 68, wherein the L53 molecule is a protein comprising SEQ ID NO:3.

70. The method of claim 69, wherein the protein is expressed on a surface of the ceU or inside the cell.

71. The method of claim 61 , wherein the candidate agent is labeled radioactively or enzymatically.

72. The method of claim 68, wherein the L53 molecule is at least 80% homologous to SEQ ID NO: 1 as determined using the NBLAST algorithm.

73. The method of clahn 72, wherein the screening assay is performed in vitro.

74. The method of claim 72, wherein the L53 molecule is isolated from said cell and anchored to a solid phase.

75. The method of claim 72, wherein the candidate agent is anchored to a solid phase.

76. The method of claim 72, wherein the L53 molecule is isolated from said cell and the screening assay is performed in a liquid phase.

77. The method of claim 72, wherein the L53 molecule is expressed on a surface of a cell or inside a cell hi step (a).

78. The method of clahn 77, wherein the cell is engineered to express the L53 molecule.

79. The method of claim 72, wherein the candidate agent is labeled radioactively or enzymatically.

80. A method of screening for a cellular protein capable of interacting with an L53 gene product, the method comprising:

(a) immunoprecipitating the L53 gene product from a cell lysate, wherein the L53 gene product is:

(i) an RNA conesponding to SEQ JD NO:l, or a nucleic acid derived thereof;

(ii) an RNA conesponding to SEQ JD NO:2, or a nucleic acid derived thereof;

(iii) a protein comprising SEQ JD NO:3 ;

(iv) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (v) a nucleic acid comprising a sequence hybridizable to SEQ JD NO:2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (vi) a nucleic acid at least 90% homologous to SEQ ID NO: 1 or its complement as detemrined using an NBLAST algorithm, or a protein encoded thereby; or (vii) a nucleic acid at least 90% homologous to SEQ JD NO: 2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; and (b) deteiTnining whether or not any cellular proteins bind to or form a complex with the L53 gene product in the immunoprecipitate.

81. A method of screening for a candidate agent capable of modulating an expression level of an L53 gene, the method comprising:

(a) contacting the L53 gene with a candidate agent, wherein the L53 gene is a nucleic acid at least 80% homologous to SEQ ID NO:l as determined using an NBLAST algorithm; and

(b) measuring the expression level of an L53 gene product, wherein the L53 gene product is an mRNA conesponding to SEQ ID NO:l or 2, or a protein comprising SEQ JD NO:3, wherein an increase or decrease in the expression level in a presence of the candidate agent relative to the expression level in an absence of the candidate agent indicates that the candidate agent is capable of modulating expression of an L53 gene.

82. The method of claim 81, wherein the L53 gene product is a protein comprising SEQ ID NO:3, or an mRNA conesponding to SEQ JD NO: 1 or SEQ ID NO:2.

83. An immunogenic composition comprising:

(a) a purified L53 gene product in an amount effective for eliciting an immune response, wherein said gene product is:

(i) an RNA conesponding to SEQ ID NO: 1, or a nucleic acid derived thereof; (ii) an RNA conesponding to SEQ ID NO:2, or a nucleic acid derived thereof; (iii) a protein comprising SEQ ID NO:3; (iv) a nucleic acid comprising a sequence hybridizable to SEQ JD NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (v) a nucleic acid comprising a sequence hybridizable to SEQ ID NO:2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (vi) a nucleic acid at least 90% homologous to SEQ ID NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or (Vii) a nucleic acid at least 90% homologous to SEQ ID NO:2 or its complement as detennined using an NBLAST algorithm, or a protein encoded thereby; and (b) a pharmaceutically acceptable excipient.

84. The immunogenic composition of claim 83, wherein the L53 gene product is a nucleic acid at least 90% homologous to SEQ JD NO. as determined using an NBLAST algorithm.

85. The immunogenic composition of claim 83, wherein the L53 gene product is a protein comprising SEQ J NO.-3.

86. A pharmaceutical composition comprising:

(a) an antibody immunologically specific for a protein comprising SEQ ID NO:3; and

(b) a pharmaceutically acceptable carrier.

87. The pharmaceutical composition of claim 86, wherein the composition is formulated for delivery as an aerosol, parenterally, or orally.

88. A pharmaceutical composition comprising:

(a) an L53 gene product, wherein said gene product is:

(i) an RNA conesponding to SEQ ID NO: 1 , or a nucleic acid derived thereof;

(ii) an RNA conesponding to SEQ JD NO:2, or a nucleic acid derived thereof; (iii) a protein comprising SEQ JD NO:3 ; (iv) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (v) a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 2 or its complement under conditions of high stringency, or a protein comprising a sequence encoded by said hybridizable sequence; (vi) a nucleic acid at least 90% homologous to SEQ JD NO: 1 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; or (vii) a nucleic acid at least 90% homologous to SEQ ID NO:2 or its complement as determined using an NBLAST algorithm, or a protein encoded thereby; and (b) a pharmaceutically acceptable carrier,

89. The pharmaceutical composition of claim 88, wherein the L53 gene product is an mRNA conesponding to SEQ JD NO.l or SEQ ID NO:2, or a protein comprising SEQ ID NO:3.

90. The pharmaceutical composition of claim 88, wherein the gene product is purified.

91. The pharmaceutical composition of claim 88, wherein the composition is formulated for delivery as an aerosol, parenterally, or orally.

92. A pharmaceutical composition comprising:

(a) a purified nucleic acid comprising SEQ JD NO: 1 or 2; and

(b) a pharmaceutically acceptable carrier.

93. The pharmaceutical composition of claim 92, wherein the composition is formulated for delivery as an aerosol, parenterally, or orally.

94. A method of diagnosing cancer in a subject comprising:

(a) administering to the subject a compound that specifically binds a protein comprising an amino acid sequence of SEQ JD NO:3, wherein the compound is bound to an imaging agent; and

(b) obtaining an internal image of the subject by use of the imaging agent; wherein localization or amount of the imaging agent detected indicates whether or not cancer is present in the subject.

95. The method of claim 94, wherein the compound is an antibody.

96. The method of claim 95, wherein the antibody is conjugated to a radioactive metal and said obtaining step comprises recording a scintographic image obtained from decay of the radioactive metal.

97. A kit comprising:

(a) in one or more containers, a pah of oligonucleotide primers, each primer comprising an at least 5 nucleotide sequence complementary to a different strand of a double-stranded nucleic acid comprising SEQ ID NO:l; and

(b) in a separate container, a purified double-stranded nucleic acid comprising SEQ ID NO: 1.

98. A transgenic non-human animal, which expresses from a transgene an RNA conesponding to SEQ JD NO.l, an RNA conesponding to SEQ ID NO:2, or a protein comprising SEQ ID NO:3.

99. A method of testing effects of a candidate therapeutic compound, said method comprising administering said compound to the transgenic non-human animal of claim 98, and determhiing any effects of the compound upon the transgenic non-human animal.

100. An isolated polypeptide comprising at least 8-50 amino acids of SEQ ID NO:3.

101. An isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1.

102. An isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid comprising the nucleotide sequence of SEQ JD NO:2.

103. An antibody immunologically specific for a polypeptide of any one of claims 100, 101, or 102.

104. A host cell comprising a nucleic acid encoding the polypeptide of claim 100 operably linked to a promoter.

105. A host cell comprising a nucleic acid encoding the polypeptide of claim 101 operably linked to a promoter.

106. A host cell comprising a nucleic acid encoding the polypeptide of claim 102 operably linked to a promoter.

107. A method of producing an isolated polypeptide comprising at least 8-50 amino acids of SEQ JD NO: 3, said method comprising culturing the host cell of claim 104 under conditions in which the nucleic acid molecule is expressed.

108. A method of producing an isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l, said method comprising culturing the host cell of claim 105 under conditions in which the nucleic acid molecule is expressed.

109. A method of producing an isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2, said method comprising culturing the host cell of claim 106 under conditions in which the nucleic acid molecule is expressed.