WO2004015390A2

WO2004015390A2 - Lung cancer target proteins and use thereof

Info

Publication number: WO2004015390A2
Application number: PCT/US2003/024698
Authority: WO
Inventors: Stephen Hoffman; Ruobing Wang; Gangadharan Subramanian
Original assignee: Applera Corp
Current assignee: Applied Biosystems Inc
Priority date: 2002-08-09
Filing date: 2003-08-08
Publication date: 2004-02-19
Anticipated expiration: 2005-02-09
Also published as: AU2003258134A1; AU2003258134A8; WO2004015390A3

Abstract

The present invention provides five lung cancer target proteins that are differentially expressed in lung cancer cells or tissues. The invention provides a method for detecting lung cancer. The invention further provides a vaccine for preventing and treating lung cancer. The vaccine comprises immunogenic peptides derived from these five proteins and analogs thereof. The present invention further provides antibodies that specifically target the lung cancer proteins and the diagnostic and therapeutic use thereof.

Description

LUNG CANCERTARGETPROTEINSAND USE THEREOF

FIELD OF MENTION

BACKGROUND OF THE INVENTION

Lung cancer is the most common fatal cancer in the United States and most countires worldwide. There were an estimated 164,100 new cases of lung cancer and an estimated 156,900 deaths from lung cncer in the United States in 2000. The life time risk for diagnosing lung cancer is 8% in man and 6% in woman. There are two major types of lung cancer: non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer is much more common. It usually spreads to different parts of the body more slowly than small cell lung cancer. Squamous cell carcinoma, ademocarcinoma, and large cell carcinoma are three types of non-small cell lung cancer. Small cell lung cancer also called oat cell cancer, accounts for about 20% of all lung cancer.

Early detection is difficult since clinical symptoms are often not seen until the disease has reached an advanced stage. Currently, diagnosis is aided by the use of chest x-rays, analysis of the type of cells contained in sputum and fiberoptic examination of the bronchial passages. Treatment regimens are determined by the type and stage of the cancer, and include surgery, radiation therapy and/or chemotherapy. In spite of considerable research into therapies for the disease, lung cancer remains difficult to treat.

Screening efforts to date to identify predictive markers in lung cancer have been unsuccessful. Identifying a protein marker of disease would be a landmark finding in lung cancer and might improve basic understanding as well as reinvigorate screening and chemoprevention approaches, and be highly complementary with new imaging techniques. Currently, both screening and treatment for lung cancer are problematic. Identifying protein constituents that are associated with lung cancer would have broad implications to basic research and clinical practice.

The study of the genetics of complex disease in general and lung cancer in particular has been a major focus in cancer etiology, but to date, the genetic influence on most common cancers is poorly understood. Although much is known about the environmental causes of lung cancer (tobacco smoke, asbestos, radon, etc.) little is known about the effect of genetic background in the development of this complex disease.

The genetic contribution is recognized from segregation/association/mechanistic studies but no specific genes have been implicated. In spite of a decade of study the results remain unclear, and current approaches may have missed important candidates. Protein entities associated with cancer may provide important clues or specifically implicate particular genes or pathways that will lead o better understanding and treatment.

In the field of human cancer immunology, the last two decades has seen intensive efforts to characterize genuine cancer specific antigens. In particular, effort has been devoted to the analyses of antibodies to human tumour antigens. The prior art suggests that such antibodies could be used both for diagnostic and therapeutical purposes, for instance in connection with an anti-cancer agent. One problem is that antibodies can only bind to tumour antigens that are exposed on the surface of tumour cells. For this reason the efforts to produce a cancer treatment based on the immune system of the body has been less successful than expected.

Antibodies typically recognize free antigen in native conformation and can potentially recognize almost any site exposed on the antigen surface. In contrast to the antibodies produced by the B cells, T cells recognize antigens only in the context of major histocompatibility complex (MHC) molecules, designated human leucocyte antigen (HLA) in humans, and only after appropriate antigen processing, usually consisting of proteolytic fragmentation of the protein, resulting in peptides that fit into the groove of the MHC molecules. This enables T cells also to recognize peptides derived from intracellular proteins. T cells can thus recognize aberrant peptides derived from anywhere in the tumor cell, in the context of MHC molecules on the surface of the tumor cell, and subsequently can be activated to eliminate the tumor cell harboring the aberrant peptide. The HLA molecules are encoded by the HLA region on the human chromosome number 6. The class I molecules are encoded by the HLA A, B and C subloci, and the class JJ molecules are encoded by the DR, DP and DQ subloci. T-lymphocytes recognize antigen in association with Class I or Class II MHC molecules in the form of apeptide fragment bound to an MHC molecule. The degree of peptide binding to a given MHC allele is based on amino acids at particular positions within the peptide (Parker et al. (1992) Journal of Immunology 149:3580; Kubo, et al. (1994) Journal of Immunology 52:3913-3924; Ruppert J. et al. (1993) Cell 74:929-937; Falk et al. (1991) Nature 351:290-296).

T cells may control the development and growth of cancer by a variety of mechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLA Class II restricted CD4+, may directly kill tumor cells carrying the appropriate tumor antigens. CD4+ helper T cells are needed for cytotoxic T cell responses as well as for antibody responses, and for inducing macrophage and LAK cell killing. One approach to an immunological cancer therapy is through administration of interleukin-2 combined with specific lymphocytes, so called lymphokine-activated killer cells (LAK cells), or tumor infiltrating lymphocytes (TIL cells). The beneficial effects achieved for some patients having specific cancers are not of a general nature. Further, the side effects experienced by some of the patients are quite unpleasant and sometimes severe. (Steven A. Rosenberg, Scientific American, May 1990, Adoptive Immunotherapy for Cancer.) Attempts have been made to develop cancer vaccines based on injection of cancer cells from patient's own cancer or insoluble fragments of said cells or such cells mixed with other nonspecific stimulators of the immune system, such as BCG, interferons or interleukins.

As a result of the poor prognosis for lung cancer patient, much attention and interest has focused on adjuvant therapy for lung cancer. Immunotherapy has been considered as an adjuvant or even sole therapy for lung cancer. Tumor immunotherapy is based on the hypothesis that tumors have unique signatures in the form of tumor antigens, and that the immune system can be redirected to recognize and eliminate cells bearing these signatures. While tumor antigens have been identified in many types of cancer. It is evident that in most cases, the patient's immune system responds to them weakly or not at all. This unresponsiveness can be attributed to both passive (through immune evasion) (Elgert, K.D., et al. 1998. J. Leukoc. Biol. 64, 275-290) and active (through generation of immunosuppressive environments) (Alizadeh, A.A., et al. 2000. Nature 403:503-511) anti-immune strategies adopted by the tumor.

Identification of tumor antigens is likely to impact significantly on other areas of lung cancer treatment. The identification of more and more tumor antigens may provide the ability to classify tumors more accurately, resulting in more effective therapeutic regimens. For example, using genomic signatures, diffuse large B cell lymphomas can be divided into two distinct categories, with widely different outcomes (Schultze, J.L, et al. 2001, Trends in Immunology 22:517-523). Furthermore, identification of these antigens may have valuable prognostic implications as well, particularly in areas such as early detection of relapse.

A key characteristic of a tumor antigen is its ability to induce an immune response in vivo sufficient to eliminate the tumor. However, not all tumor antigens function as tumor rejection antigens (Gilboa, E., et al.1999. Immunity 11 :263-270). While ultimate fitness as a tumor rejection antigen can only be evaluated in vivo, large amounts of information can be gleaned from in vitro studies. Hence, there is a need for the identification and evaluation of optimal T cell epitopes on candidate tumor antigens.

SUMMARY OF THE INVENTION

The present invention provides immunogenic peptides having the amino acid sequences depicted in Figures 1- 4 for use in lung cancer vaccines.

The invention also provides immunogenic peptide analogs having the amino acid sequences listed in Figure 4 for use in lung cancer vaccines, wherein analogs of these peptides in which anchor residues are modified to enhance the immunogenicity of the peptides are also included in the invention.

The invention also provides peptide sequences for MHC or HLA class I- restricted epitopes (or restricting allele) from HLA-A*0101, HLA-A*0201, HLA- A*0301, HLA-A*2401, and HLA B*0701 that serve as specific targets of T lymphocytes.

In addition, the present invention also provides peptide sequences for MHC or HLA class LT-restricted epitopes (restricting allele) from HLA-DR*0101 and HLA- DR*0401 that may serve as specific targets of T lymphocytes. The peptides of the present invention also are utilized as therapeutic T cell vaccines themselves or as components of a polyepitope therapeutic T cell vaccine.

The protein of the present invention provides for polyepitope contractions comprising of identified overlapping epitopes including T-cell epitopes restrictied by both HLA-A*0201 and HLA-DRB*0101, orboth HLA-A*0201 and HLA- DRB*0401, as well as gene domains encoding epitope-rich regions.

The invention provides cancer vaccines comprising all or part of the nucleic acid sequence encoding peptides for preventing or treating lung cancer.

The invention provides cancer vaccines comprising peptides depicted in Figures 1 - 4 in combination with at least one pharmaceutically acceptable carrier. The peptides are preferred to be derived from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5.

The invention also provides multivalent vaccines comprising all or part of the LCT nucleic acid sequence or its corresponding protein or peptide and at least one other immunogenic molecule capable of eliciting the production of antibodies in a mammal to lung cancer antigens.

The invention also provides methods for prophylactic or therapeutic uses involving all or part of the nucleic acid sequence encoding LCT proteins and LCT corresponding protein or peptide derived from the amino acid sequence. ₍ The invention also provides methods for prophylactic or therapeutic uses involving LCT proteins or peptides derived from the amino acid sequences.

The invention also provides a method for preventing or treating lung cancer utilizing all or part of the LCT nucleic acid sequence or its corresponding protein in gene therapy protocols.

The invention further provides methods of identifying an agent or a small molecule against LCT and the said small molecule can be used to treat lung cancer.

The present invention provides B-cell epitopes/peptides for preparing monoclonal and polyclonal antibodies, wherein the preferred peptides are listed in Figure 6.

The invention also provides monoclonal or polyclonal antibodies reactive with LCT protein, peptides in a form for use in treating lung cancer.

The invention also provides monoclonal or polyclonal antibodies reactive with LCT protein, peptides in a form for use in diagnosing lung cancer. The invention also provides methods for detecting the LCT genes or mRNA in a biological sample for use in diagnosing the presence, absence or progression of lung cancer.

The invention also provides methods for detecting the LCT proteins or peptides in a biological sample for use in diagnosing the presence, absence or progression, metastatic stage and thrapeutical potential of lung cancer.

The invention also provides methods for detecting the LCT proteins, preferably, the sequence from SEQ ID NO: 1-5 or combination thereof in a biological sample for use in diagnosing the presence, absence or progression, metastatic stage and thrapeutical potential of lung cancer.

DESCRIPTION OF THE FIGURE SHEETS

FIGURE 1. 249 Class I-restricted T-cell epitopes, 9-10 amino acid in length: T-cell epitopes restricted by 5 alleles; HLA-A*0201, *0101, *0301, *2401, and B*0701 Epitope sequences were based on Celera-generated epitope prediction algorithm, selection based on the high, intermediate, low or no binding capacity prediction. The epitope sequences with high, intermediate binding affinity were selected for testing.

FIGURE 2. 38 Class JJ-restricted T-cell epitopes, 15 amino acid in length: T-cell epitopes restricted by 2 alleles; HLA-DRB*0101, HLA-DRB*0401. Epitope prediction was based on SYFPEITHI algorithm in the public domain, the selected epitopes with the top 2% high binding score.

FIGURE 3. 49 Class I/II overlapping T-cell epitopes, 15 amino acid in length: T-cell epitopes restricted by both HLA- A*0201 and HLA-DRB*0101, or HLA-A*0201 and HLA-DRB*0401. High or intermediate binding peptides (9-10 amino acid residues) restricted by HLA-A*0201 are nested in the HLA-DRB*0101 or HLA-DRB*0401 epitopes (15 amino acid residues).

FIGURE 4.216 Analogs: HLA-A*0201-restricted T-cell epitopes at intermediate and low binding capacity with fixed anchor residues [Position (P2, AP9 or P10)] that may serve as specific targets of T lymphocytes with increased peptide class I MHC- binding affinity, compared to that with wide type sequences, for improvement of tumor-specific CTL induction.

FIGURE 5. Differentially expressed proteins: Lul (SEQ ID NO: 1); Lu2 (SEQ JD NO: 2); Lu3 (SEQ ID NO: 3); Lu4 (SEQ ID NO: 4); Lu5 (SEQ JD NO: 5).

FIGURE 6. B-cell epitopes/peptides for making immunogenic antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:

General Description

The present invention is based on the discovery from SAGE™ database of protein(s) that are differentially expressed in lung cancer samples versus normal lung samples. These proteins, and the encoding nucleic acid sequences are associated with lung cancer, as lung cancer target proteins (LCT), wherein the proteins are referred as Lul, Lu2, Lu3, Lu4 and Lu 5 and are listed in Figure 5 as SEQ ID NO: 1, SEQ ID NO: 2, SEQ LD NO: 3, SEQ ID NO: 4, SEQ JD NO: 5.

The protein of the present invention demonstrated more than 17 fold of differential expression in lung cancer cell lines compared to expression data from a panel of vital tissues.

Based on the discoveries, the present invention provides T-cell epitopes that stimulate T-cell responses to the LCT, antibodies that specifically bind to the LCTs, and small molecule assays. The present invention also provides methods for early detection, diagnosis, prognosis, prevention and treatment of lung cancers. The present invention also provides markers of lung cancer at different stage, in the form of LCT peptide/proteins or nucleic acid sequences isolated from human lung tumors, sera, or lung cancer cell lines.

Definitions:

The terms "vaccine", "immunogen", or "immunogenic composition" are used herein to refer to a compound or composition, as appropriate, that is capable of conferring a degree of specific immunity when administered to a human or animal subject. The vaccines, immunogens, and immunogenic compositions of this invention are active vaccines, which mean that they are capable of stimulating a specific immunological response (such as an anti-tumor antigen or anti-cancer cell response) mediated at least in part by the immune system of the host. The immunological response may comprise antibodies, immunoreactive cells (such as helper/inducer or cytotoxic cells), or any combination thereof, and is preferably directed towards an antigen that is present on a tumor towards which the treatment is directed. The response may be elicited or restimulated in a subject by administration of either single or multiple doses. Nothing further is required of a composition in order for it to qualify as a vaccine, unless otherwise specified.

A compound or composition is "immunogenic" if it is capable of either: a) generating an immune response against an antigen (such as a tumor antigen) in a naive individual; or b) reconstituting, boosting, or maintaining an immune response in an individual beyond what would occur if the compound or composition was not administered. A composition is immunogenic if it is capable of attaining either of these criteria when administered in single or multiple doses.

The term "analog" are peptides in which anchor residues are modified to enhance th eimmunogenicity of the peptide, for example the peptides shown in Figure 4.

"Eliciting" an immune or immunological response refers to administration of a compound or composition that initiates, boosts, or maintains the capacity for the host's immune system to react to a target substance, such as a foreign molecule, an allogeneic cell, or a tumor cell, at a level higher than would otherwise occur.

A "cell line" or "cell culture" denotes higher eukaryotic cells grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell.

The terms "tumor cell" or "cancer cell", used either in the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non- cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.

The term "tumor-associated antigen" is used herein to refer to a molecule or complex which is expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. Tumor-associated antigens maybe antigens not normally expressed by the host; they may be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they may be identical to molecules normally expressed but expressed at abnormally high levels; or they may be expressed in a context or milieu that is abnormal. Tumor- associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules. Knowledge of the existence or characteristics of a particular tumor-associated antigen is not necessary for the practice of the invention.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

The "pathology" associated with a disease condition is anything that compromises the well-being, normal physiology, or quality of life of the affected individual. This may involve (but is not limited to) destructive invasion of affected tissues into previously unaffected areas, growth at the expense of normal tissue function, irregular or suppressed biological activity, aggravation or suppression of an inflammatory or immunological response, increased susceptibility to other pathogenic organisms or agents, and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood alterations, and such other features as may be determined by an attending physician.

An "effective amount" is an amount sufficient to affect a beneficial or desired clinical result, particularly the generation of an immune response, or noticeable improvement in clinical condition. An immunogenic amount is an amount sufficient in the subject group being treated (either diseased or not) sufficient to elicit an immunological response, which may comprise either a humoral response, a cellular response, or both. In terms of clinical response for subjects bearing a neoplastic disease, an effective amount is amount sufficient to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. An effective amount maybe given in single or divided doses. Preferred quantities and cell ratios for use in an effective amount are given elsewhere in this disclosure.

An "individual" or "subject" is a vertebrate, preferably a mammal, more preferably a human. Non-human mammals include, but are not limited to, farm animals, sport animals, and pets.

Peptides for T-cell mediated immunotherapy

The peptides derived from LCT proteins of the present invention are useful as immunogenic peptides or epitopes for immunogenic response. The preferred peptides used for specific alleles are depicted in Figures 1-4.

An immunogenic peptide is a peptide, which comprises an allele-specific motif such that the peptide will bind the MHC allele (HLA in human) and be capable of inducing a CTL response. Thus, immunogenic peptides are capable of binding to an appropriate class I or II of MHC molecule and inducing a cytotoxic T cell or T helper cell or both response against the antigen from which the immunogenic peptide is derived.

Alternatively, amino acid sequence or analog of a peptide can be prepared by in the DNA, which encodes the peptide, or by peptide synthesis.

The peptides of present invention can be modified to increase immunogenicity by enhancing binding of a peptide to the MHC molecule with which the peptide is associated. By way of example, modification may include substitution, deletion or addition of an amino acid in the given immunogenic peptide sequence or mutation of existing amino acids within the given immunogenic peptide sequence, or derivatization of existing amino acids within the given immunogenic peptide sequence. Any amino acid comprising the immunogenic peptide sequence may be modified in accordance with this invention. In one aspect, at least one amino acid is substituted or replaced within the given immunogenic peptide sequence. Any amino acid may be used to substitute or replace a given amino acid within the immunogenic peptide sequence. Modified peptides are intended to include any immunogenic peptide obtained from differentially expressed proteins, which has been modified and exhibits enhanced binding to the MHC molecule with which it associates when presented to the T-cell.

This invention further includes analogs of these immunogenic modified peptides derived from the peptides in Figures 1-4, which have been modified. The term analog is intended to include any peptide, which displays the functional aspects of these modified peptides. The term analog also includes conservative substitutions or chemical derivatives of these modified peptides as described above which enhance the immunogenicity. These modified peptides may be synthetically or recombinantly produced by conventional methods.

In another embodiment, the peptides of the present invention comprise, or comprising essentially, or consisiting sequences of about 5-8, 8-10, 10-15 orl5-30 amino acids, preferably 9 to 10 amino acids or 15 amino acids, which are immunogemc, that is, capable of inducing an immune response when injected into a subject.

In one embodiment, a list of 9 or 10 amino aicd sequence derived from SEQ ID NO: 1-5 are immunogenic epitopes of Lul recognized by HLA- A or HLA-B family. The peptide is capable of activating CTLs in the absence of cancer antigen, and thus is useful for augmenting the immune system of normal and cancer patients, as well as in the study of the Class I antigen processing pathway for LCT proteins. For example, in Figure 1, the 56 peptides ranges from 9 to 10 amino acids are from various regions for interaction with the HLA A*0201; Other peptides from Figures 1 and 2 were also identified from LCT proteins for intereacting with HLA- A *1010, HLA-A *0301, HLA-A *2401, HLA-B *0701, HLA-DRB1 *0101, HLA-DRB1 *0401.

In yet another embodiement, 49 peptides having overlapping regions among LCT proteins are used for interacting HLA-A *0201 and DRBl *0101, or HLA-A *0201 and DRBl *0401 (see Figure 3)

In yet another emodiment, 216 peptide analogs from LCT proteins are used for interacting with HLA A*0201, HLA-A *1010, HLA-A *0301, HLA-A *2401, HLA- B *0701, HLA-DRB1 *0101, HLA-DRB1 *0401 (see Figure 4).

The recombinant, synthetic, or natural protein, peptides, or analogs of LCT, or modified peptides, or analogs thereof may be used as a vaccine either prophylactically or therapeutically. When provided prophylactically the vaccine is provided in advance of any evidence of lung cancer. The prophylactic administration of the lung cancer vaccine should serve to prevent or attenuate lung cancer in a mammal.

In a preferred embodiment, mammals, preferably human, at high risk for lung cancer are prophylactically treated with the vaccines of this invention. Examples of such mammals include, but are not limited to, humans with a family history of lung cancer, humans with a history of lung disease, or humans afflicted with lung cancer previously resected and therefore at risk for reoccurrence. When provided therapeutically, the vaccine is provided to enhance the patient's own immune response to the tumor antigen present on the lung cancer or metastatic lung cancer. The vaccine, which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector, cell lysates from cells transfected with a recombinant expression vector, or a culture supernatant containing the expressed protein. Alternatively, the immunogen is a partially or substantially purified recombinant or synthetic protein, peptide or analog thereof or modified peptides or analogs thereof. The proteins or peptides may be conjugated with lipoprotein or administered in liposomal form or with adjuvant.

While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.

The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.

All methods include the step of bringing into association the active ingredient with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions, which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1- 3.0 osmoles, preferably in the range of 0.8-1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, anti-adsorption agent maybe used.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the lung cancer specific protein, peptides and analogs thereof into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly (methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.

Preparation of vaccine is using recombinant protein or peptide expression vectors comprising all or part of nucleic acid sequence of LCT proteins encoding peptides in Figures 1-4. Examples of vectors that may be used in the aforementioned vaccines include, but are not limited to, defective retroviral vectors, adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, or other viral vectors (Mulligan, R. C, (1993) Science 260:926-932). The viral vectors carrying all or part of nucleic sequence of SEQ JD Nos: 1-5 can be introduced into a mammal either prior to any evidence of lung cancer or to mediate regression of the disease in a mammal afflicted with lung cancer. Examples of methods for administering the viral vector into the mammals include, but are not limited to, exposure of cells to the viras ex vivo, or injection of the retro viras or a producer cell line of the viras into the affected tissue or intravenous administration of the viras. Alternatively the viral vector carrying all or part of the LCT nucleic acid sequence that encode peptides in Figures 1-4 may be administered locally by direct injection into the lung cancer lesion or topical application in a pharmaceutically acceptable carrier. The quantity of viral vector, carrying all or part of the LCT nucleic acid sequence, to be administered is based on the titer of virus particles. A preferred range of the immunogen to be administered may be about 10 to about 10 viras particles per mammal, preferably a human. After immunization the efficacy of the vaccine can be assessed by production of antibodies or immune cells that recognize the antigen, as assessed by specific lytic activity or specific cytokine production or by tumor regression. One skilled in the art would know the conventional methods to assess the aforementioned parameters. If the mammal to be immunized is already afflicted with lung cancer, the vaccine can be administered in conjunction with other therapeutic treatments. Examples of other therapeutic treatments includes, but are not limited to, adoptive T cell immunotherapy, coadministration of cytokines or other therapeutic drugs for lung cancer. Alternatively all or parts thereof of a substantially or partially purified the LCT or their peptides may be administered as a vaccine in a pharmaceutically acceptable carrier. Ranges of the protein that may be administered are about 0.001 to about 100 mg per patient, preferred doses are about 0.01 to about 100 mg per patient. In a preferred embodiment, the peptides or analogs thereof is administered therapeutically or prophylactically to a mammal in need of such treatment. The peptide may be synthetically or recombinantly produced. Immunization is repeated as necessary, until a sufficient titer of anti-immunogen antibody or immune cells has been obtained.

In yet another alternative embodiment a viral vector, such as a retroviral vector, can be introduced into mammalian cells. Examples of mammalian cells into which the retroviral vector can be introduced include, but are not limited to, primary mammalian cultures or continuous mammalian cultures, COS cells, NTH3T3, or 293 cells (ATTC #CRL 1573), dendritic cells. The means by which the vector carrying the gene maybe introduced into a cell includes, but is not limited to, microinj ection, electroporation, transfection or transfection using DEAE dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art (Sambrook et al. (EDS) (1989) in "Molecular Cloning. A laboratory manual", Cold Spring Harbor Press Plainview, N.Y.).

The vaccine formulation of the present invention comprises an immunogen that induces an immune response directed against the lung cancer associated antigens such as the protein LCT antigen. The vaccine formulations may be evaluated first in animal models, initially rodents, and in nonhuman primates and finally in humans. The safety of the immunization procedures is determined by looking for the effect of immumzation on the general health of the immunized animal (weight change, fever, appetite behavior etc.) and looking for pathological changes on autopsies. After initial testing in animals, lung cancer patients can be tested. Conventional methods would be used to evaluate the immune response of the patient to determine the efficiency of the vaccine.

Measurement of candidate tumor antigen or vaccine expression in lung cancer patients is the first step of the present invention. Subsequent steps will focus on measuring immune responses to these candidate antigens or vaccine. Sera from cancer patients and healthy donors will be screened for antibodies to the candidate antigens as well as for levels of circulating tumor derived antigens. Peptides corresponding to epitopes in Figures 1-4 are synthesized and their ability to elicit an immune response from a patient and a control subject is measured. Peptides inducing strong T cell responses are considered as tumor rejection antigens.

In one embodiment of this invention all, part, or parts of the LCT protein or peptides or analogs thereof, or modified peptides or analogs thereof, may be exposed to dendritic cells cultured in vitro. The cultured dendritic cells provide a means of producing T-cell dependent antigens comprised of dendritic cell modified antigen or dendritic cells pulsed with antigen, in which the antigen is processed and expressed on the antigen activated dendritic cell. The LCT antigen activated dendritic cells or processed dendritic cell antigens may be used as immunogens for vaccines or for the treatment of lung cancer. The dendritic cells should be exposed to antigen for sufficient time to allow the antigens to be internalized and presented on the dendritic cells surface. The resulting dendritic cells or the dendritic cell process antigens can than be administered to an individual in need of therapy. Such methods are described in Steinman et al. (WO93/208185) and in Banchereau et al. (EPO Application 0563485A1).

Examples of where T-lymphocytes can be isolated from include but are not limited to, peripheral blood lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes (TJL). Such lymphocytes can be isolated from the individual to be treated or from a donor by methods known in the art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol. 142: 2453-3461). Lymphocytes are cultured in media such as RPMI or RPMI 1640 or ATM V for 1-10 weeks. Viability is assessed by trypan blue dye exclusion assay. The lymphocytes are exposed to all peptides in Figure 1-4 for part or all of the culture duration. Examples of how these sensitized T- cells can be administered to the mammal include but are not limited to, intravenously, intraperitoneally or intralesionally. Parameters that may be assessed to determine the efficacy of these sensitized T-lymphocytes include, but are not limited to, production of immune cells in the mammal being treated or tumor regression. Conventional methods are used to assess these parameters. Such treatment can be given in conjunction with cytokines or gene modified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).

Diagnostic and therapeutic use:

The LCT proteins and their peptides are used in substantial and specific assays related to the functional information; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for lung cancer or tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state).

Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a lung cancer specific protein-effector protein interaction or lung cancer specific protein-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products for detecting and treating lung cancer.

In one embodiment, LCT proteins isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drag, particularly in modulating a biological or pathological response in a cell or tissue that expresses the LCT protein. A large percentage of pharmaceutical agents are being developed that modulate the activity of LCT. The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The LCT protein is useful for biological assays related to lung cancer. Such assays involve any of the known LCT functions or activities or properties useful for diagnosis and treatment of lung cancer.

There are a variety of assay formats known to those of ordinary skill in the art for using a binding partner to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of binding partner immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a second binding partner that contains a reporter group. Suitable second binding partners include antibodies that bind to the binding partner/polypeptide complex. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding partner after incubation of the binding partner with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding partner is indicative of the reactivity of the sample with the immobilized binding partner.

The solid support may be any material known to those of ordinary skill in the art to which the antigen may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Irnmunotechnology Catalog and Handbook, 1991, at A12- A13).

In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a second antibody (containing a reporter group) capable of binding to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is that period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with breast cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of antibody to reporter group maybe achieved using standard methods known to those of ordinary skill in the art. i

The second antibody is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound second antibody is then removed and bound second antibody is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

In one embodiment, LCT proteins are targets for diagnosing a lung cancer or predisposition to lung cancer mediated by the peptides. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the LCT proteins or peptides such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array. To determine the presence or absence of lung cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. Jn one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without lung cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for lung cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%—specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for lung cancer.

In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized antibody as the sample passes through the membrane. A second, labeled antibody then binds to the antibody-polypeptide complex as a solution containing the second antibody flows through the membrane. The detection of bound second antibody may then be performed as described above. In the strip test format, one end of the membrane to which antibody is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second antibody and to the area of immobilized antibody. Concentration of second antibody at the area of immobilized antibody indicates the presence of lung cancer. Typically, the concentration of second antibody at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

Of course, numerous other assay protocols exist that are suitable for use with the antigens or antibodies of the present invention. The above descriptions are intended to be exemplary only.

In another embodiment, the above polypeptides may be used as markers for the progression of lung cancer, or diagnosis of cancer in symptomatic or high risk subject, stage thereof. In this embodiment, assays as described above for the diagnosis of lung cancer may be performed over time, and the change in the level of reactive polypeptide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, lung cancer is progressing in those patients in whom the level of polypeptide detected by the binding agent increases over time. In contrast, lung cancer is not progressing when the level of reactive polypeptide either remains constant or decreases with time.

Antibodies for use in the above methods may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the antigenic polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats), hi this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, e.g. antigenic peptides depicted in Figure 6, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques maybe employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention maybe used in the purification process in, for example, an affinity chromatography step.

In general, the antibodies are also useful for inhibiting protein function, for example, blocking the binding of the LCT peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane.

In one specific embodiment, the antibody binds specifically to Lul, which express on the cell surface.

In yet another embodiment, the LCT antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at a range of about hng to about 100 mg per patient. Thus, antibodies can be passively administered alone or in conjunction with other anti-cancer therapies to a mammal afflicted-with lung cancer. Examples of anti- cancer therapies include, but are not limited to, chemotherapy, radiation therapy, adoptive immunotherapy therapy with TIL.

In yet another embodiment, LCT antibodies, prefereably monoclonal antibodies of the present invention may also be used as therapeutic reagents, to diminish or eliminate breast tumors. The antibodies may be used on their own (for instance, to inhibit metastases) or coupled to one or more therapeutic agents. Suitable agents in this regard include radionuchdes, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuchdes include ⁹⁰ Y, ^m 1, ¹²⁵1, ¹³¹ 1, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, and ^2U Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.

In addtion, LCT proteins can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant protein. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Screening Use

In one embodiment, LCT proteins isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drag, particularly in modulating a biological or pathological response in a cell or tissue that expresses the LCT protein. For example, the LCT proteins are also used to identify compounds that modulate lung activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the lung cancer. A large percentage of pharmaceutical agents are being developed that modulate the activity of LCT. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

In one embodiment, LCT proteins are used as a drug target to screen compounds such as small molecules for the ability to stimulate or inhibit interaction between the LCT and a molecule that normally interacts with these proteins. Such assays typically include the steps of combining the LCT proteins with a candidate compound under conditions that allow the these proteins, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the lung cancer specific protein and the target, such as any of the associated effects of signal transduction such as cell communication, receptor linked signaling, protein phosphorylation, cAMP turnover, and adenylate cyclase activation. Small molecules include small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

In addition to small molecules, candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al, Nature 354:82-84 (1991); Houghten et al, Nature 354:84-86 (1991)) and 'combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, Cell 72:161-11% (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') , Fab expression library fragments, and epitope-binding fragments of antibodies).

One candidate compound is a soluble fragment of the LCT that competes for substrate or ligand binding. Other candidate compounds include mutant LCT or appropriate fragments containing mutations that affect the protein function and thus compete for substrate or hgand. Accordingly, a fragment that competes for substrate or ligand, for example with a higher affinity, or a fragment that binds substrate or ligand but does not allow release, is encompassed by the invention.

Binding and/or activating compounds can also be screened by using chimeric LCT in which the amino terminal domain, or parts thereof, the entire subregions, and the carboxy terminal domain, or parts thereof, can be replaced by heterologous domains or subregions.

Protein Array

The present invention provides the sequences (SEQ JD NOS: 1-5) of Lul-Lu5 proteins. These proteins are directly useful in protein analysis devices (PADs), particularly protein arrays, in the form of peptide probes and, alternatively, may readily be used to design protein capture agents (PCAs) which may be incorporated into PADs for detecting these proteins. The amino acid sequences of these proteins are provided in SEQ ID NOS: 1-5 in the Sequence Listing and in Figure 5. The present invention thus provides proteins, detection elements such as peptide probes and protein capture agent (PCAs such as antibodies, aptamers) based on these proteins, and PADs such as protein arrays comprising one or more of these detection elements.

Given the protein sequences provided by the present invention, or fragments thereof, readily implementable compositions of matter, such as detection elements (e.g., peptide probes, PCAs , and protein analysis devices (e.g., protein arrays, etc.), can be made using methods well known in the art. Such detection elements and devices can be used to track the presence, abundance, expression, interactions (e.g., with other proteins, small molecules, ligands, substrates, etc.), etc. of all of the proteins disclosed herein, or rationally selected subsets thereof, defined by a user. For example, the devices can be used to detect and/or quantify the presence or expression of many proteins, even all of the proteins, or rationally selected subsets thereof, provided herein.

Another embodiment of the present invention is directed to collections of these proteins and proteins fragments, as well as antibodies that are specific for these proteins. The proteins of the present invention include the proteins provided in the Sequence Listing (SEQ JD NOS: 1-5), fragments thereof, proteins comprising these sequences, and collections/sets/subsets of these proteins.

The following Examples are offered by way of illustration and not by way of limitation. EXAMPLES: Synthetic peptides:

MHC class I-restricted peptides, 9-10 amino acids in length, and > 80% purity, were synthesized and purified by Jerini AG (Berlin, Germany). MHC-DR-restricted peptides, 15 amino acid in length and >90% purity, were synthesizedand purified by SynPep Corporation (Dublin, CA). The homogeneity was confirmed by analytical HPLC. Purity was determined on an analytical reverse-phase column and their composition ascertained by amino acid analysis and/or mass spectrometry analysis. Lyophilized peptides were reconstituted at 20 mg/ml with 100% dimethyl sulfoxide (Sigma) and stored at -80°C until used.

Peptide Binding Assay: The synthetic peptides including the T epitopes in Figures are tested for their capacity to bind purified HLA-A*0201, *0101, *0301, 2401, -B*0701, and DRBl molecules in vitro. Binding of test peptides to MHC molecules is measured by determining the level of competition induced by the test peptide for binding of a rodiolabeled standard peptide to purified HLA molecules. The percentage of MHC- bound radioactivity is determined by gel filtration and the concentration of test peptide that inhibites 50% of the binding of the labeled standard peptide (IC50%) is calculated. The peptide is considered a high binding affinity if IC 50 is equal or less than 50nM, moderate binding affinity with IC50 at 50-500 nM, and low binding affinity with IC50 at >500nM. The peptides bound to MHC molecules with high or moderate binding affinity are selected for in vitro immunogenecity testing.

Methods for identification and characterization of tumor-specific T cell epitopes or antigens:

The tumor-specific T cell activation is assayed by cytokine ELISPOT and cytotoxicity assays to assess their capability to induce cytotoxic T lymphocytes (CTLs) that kill tumor cells or homologous target cells sensitized with depicted epitopes in vitro with periperal blood mononuclear cells (PBMCs) from healthy donors, or recognized by PBMCs or TILs from cancer patients as compared to PBMCs from HLA-matched normal donors. Sources of T cells are mononuclear cells (PBMCs) separated from peripheral blood or tumor infiltrating lymphocytes (TILs) purified from lung specimens from NSLC patients. Recognition is assessed based on response of T cells incubated with synthetic peptides, recombinant proteins including the T epitopes, antigen presenting cells transfected with plasmids expressing each of the individual proteins, or recombinant poxviruses expressing each of the individual proteins or expanded tumor cell populations and cell lines shown to express the appropriate HLA molecules and antigens.

ELISPOT assay:

T cell activation is evaluated as the number of cells secreting IFN-γ after stimulation with a tumor antigen-specific fashion. The number of spots corresponding to cytokine producing cells in wells (spot forming cells; SFCs) are enumerated with an automated spot counting system (Zeiss ELISPOT system). Responses are expressed as the mean number of SFCs/10⁶ PBMCs. A response is considered positive if the mean of interferon gamma spot forming cells in triplicate or quadruplicate experimental wells is significantly greater than the mean of interferon gamma spot forming cells (SFCs) in control wells (p<0.05), the ratio of SFCs in experimental vs control wells is greater than 2, and the difference between SFCs in experimental vs control wells is greater than 5 SFCs. Cytotoxic T cell assay:

The target cells (expanded tumor cell populations, or homologous target cells pulsed with the predicted T epitopes) are labelled with ⁵¹Cr prior to mixing with lymphocytes from normal donors or cancer patients, and release of ⁵¹Cr from killed targets is measured. Standard in vitro effector restimulation and chromium release assays are performed. Percent lysis is defined as (experimental release - medium control release)/(maximum release - medium control release) x 100. Percent specific lysis is determined by subtracting the % lysis of targets sensitized with the control or unrelated A2-restricted peptide from the % lysis of targets incubated with the experimental peptide. CTL responses are considered positive only if % specific lysis post-immunization is >10% for at least two effector: target (E:T) ratios in the same assay and if % specific lysis pre-immunization was < 10%. Spontaneous release values are always < 25%.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention, which are obvious to those skilled in the field of molecular biology or related fields, are intended to be within the scope of the following claims.

Claims

ClaimsThat which is claimed is:

1. A method of eliciting an immune response in a subj ect, comprising introducing into the subject a composition comprising purified peptides in Figures 1-4 from SEQ JD NO: 1, 2, 3, 4 or 5 that binds to a specific human leukocyte antigen (HLA) allele.

2. The method of claim 1, wherein the peptide is selected from an amino acid sequences of SEQ JD NO: 1 (Lul).

3. The method of claim 1 , wherein the peptide is selected from an amino acid sequence of SEQ JD NO: 2 (Lu2).

4. The method of claim 1 , wherein the peptide is selected from an amino acid . sequence of SEQ ID NO: 3 (Lu3).

5. The method of claim 1, wherein the peptide is selected from an amino acid . sequence of SEQ ID NO: 4 (Lu4).

6> The method of claim 1 , wherein the peptide is selected from an amino acid sequence of SEQ ID NO: 5 (Lu5).

7. A method of eliciting a T cell response in a human, comprising vaccinating said human with a therapeutical effective amount of composition comprising peptide amino acid sequence selected from a group consisting of Figures 1, 2, 3 or 4.

8. A composition comprising purified antigen selected from a peptide from Figures 1, 2, 3 or 4 and a carrier.

9. A composition comprising purified antigen of claim 8 and an adjuvant.

10. A composition comprising purified antigen of claim 8 conjugate to immunotoxin agent.

11. A vaccine comprising at least one peptide from Figures 1-4 in combination with at least one pharmaceutically acceptable carrier.

12. An antiboby specifically bind to a protein of SEQ ID NOS: 1-5 or peptides in Figure 6.

13. A method for diagnosing lung cancer in a subject using combination of LCT proteins contained in a sample of the subject, comprising:

(a) determining the amount of a Lu 1 contained in the sample;

(b) determining the amount of a Lu 2 contained in the sample;

(c ) determining the amount of a Lu 3 contained in the sample;

(d) determining the amount of a Lu 4 contained in the sample;

(e) determining the amount of a Lu 4 contained in the sample;

(f) determining the level of the Lul, Lu2, Lu3, Lu4 and/or Lu5 wherein higher level expression of one or all proteins compare to a healthy subject indicating the likelihood of having lung cancer.