HK1132911B - Cancer vaccine - Google Patents

Description

Cancer vaccine

Technical Field

The present invention relates generally to the fields of cancer diagnosis, prognosis, treatment and prevention. More particularly, the present invention relates to methods for diagnosing, treating and preventing lung cancer. In particular, aspects of the invention are directed to methods of diagnosing, treating and preventing small cell lung cancer. The invention provides methods of treating, diagnosing and/or preventing such cancers using nucleic acids and/or proteins that are differentially expressed in tumor cells, and immunospecific antibodies directed against such proteins.

Background

Cancer is primarily characterized 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 or distant sites (metastasis). Clinical data and molecular biological studies indicate that cancer is a multistep process that starts with subtle precancerous changes that may progress to neoplasia under certain conditions.

Precancerous abnormal cell growth is exemplified by hyperplasia, tissue transformation, or, very particularly, dysplasia (for a review of these abnormal growth conditions, see robblins & Angell, 1976, Basic Pathology, 2d Ed., W.B.Saunders Co., Philadelphia, pp.68-79). Neoplastic lesions may progress clonally and develop increasing growth capacity, metastasis and heterogeneity, especially under conditions in which neoplastic cells escape host immune surveillance (Roitt, i., Brostoff, j.and Kale, d., 1993, Immunology, 3rd ed., Mosby, st.louis, pps.17.1-17.12). In the united states, Cancer epidemiology estimates over 1,300,000 new cases and over 550,000 deaths in 2003 (Jemal et al, 2003, CA Cancer j.clin., 53, 5-26). Lung Cancer is one of the most common cancers in the united states, with approximately 172,000 new cases and 157,000 deaths in 2003 (Jemal et al, 2003, CA Cancer j. clin., 53, 5-26). Lung cancer is generally classified as Small Cell Lung Cancer (SCLC) or non-small cell lung cancer (NSCLC). SCLC accounts for approximately 20% of all lung cancers while NSCLC accounts for the remaining approximately 80%. NSCLC is further divided into Adenocarcinoma (AC) (about 30-35% of all cases), Squamous Cell Carcinoma (SCC) (about 30% of all cases), and Large Cell Carcinoma (LCC) (10% of all cases). Other subtypes of NSCLC, not clearly defined in the literature, include adenosquamous carcinoma (ASCC) and bronchioloalveolar carcinoma (BAC).

Lung Cancer is the leading cause of Cancer death worldwide, and more precisely NSCLC accounts for approximately 80% of all disease cases (Cancer Facts and regulations, 2002, american Cancer Society, Atlanta, p.11.). NSCLC has 4 major types, including adenocarcinoma, squamous cell carcinoma, bronchioloalveolar carcinoma, and large cell carcinoma. Based on cellular morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC (Travis et al, 1996, Lung Cancer Principles and Practice, Lippincott-Raven, New York, pps.361-395). Adenocarcinomas are characterized by their more peripheral location in the lung and often have mutations in the K-ras oncogene (Gazdar et al, 1994, Anticancer Res.14: 261-267). Squamous cell carcinomas are generally more central and often carry a mutation in the p53 gene (Niklinska et al, 2001, Folia histochem. cytodiol.39: 147-148).

Lung cancer is the first killer among cancer patients for which there is no adequate treatment, corresponding to approximately one fifth of all cancer deaths (IARC) in europe. The growing burden of the population is perhaps best illustrated in recent studies in the united states, which showed that between 1960 and 1990, the number of deaths from lung cancer in women increased by over 400% over that of contemporary breast cancer.

There are currently no adequate treatment regimens for different types of lung cancer. By conventional treatment, median survival time for subtypes of SCLC is 15 months for localized disease and 9 months for widespread disease, while long-term survival is very low.

The major obstacles to successful treatment and eradication of lung cancer are late diagnosis, highly metastatic nature, resistance to chemotherapy and the impossibility of surgically removing all cancer cells. In principle, cancer vaccines are the most promising approach to the treatment of cancer in general and lung cancer in particular. The major hurdles in developing successful vaccines are the lack of targeted cancer specific antigens and the lack of tools to evaluate immunotherapies based on these targets. Another problem is the heterogeneity of lung cancer as described above.

Lung Cancer treatment has not resulted in a significant improvement in survival rates during the last decade compared to many other types of Cancer, showing an overall 5-year survival of only 14% (Haura EB.2001, Cancer Control; 8: 326-.

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To date, the decision of a treatment regimen is guided, inter alia, by the subdivision into SCLC and NSCLC. Unlike other types of lung cancer, SCLC is sensitive to chemotherapy. Initial response to chemotherapy can be noted in approximately 75% of SCLC cases, with a clinically complete response in approximately 35% of all cases (Johnson DH, et al, 1987; Am J Med Sci 293: 377 389). Unfortunately, however, in most cases a deterioration occurs, resulting in a 3-year survival rate of only 5-10%, and a 5-year survival rate of approximately 1% (Minna JD, et al 1985, Cancer of the long. in: Cancer. principles and practice of science 2 nd); within SCLC, corresponding divisions between classical and variant SCLCs can be made clinically. The variant types of SCLC appear to be even less sensitive to chemotherapy and radiation therapy. Therefore, the median survival time for patients with variant types of SCLC is significantly shorter than that for patients with classical types of SCLC (radius PA, et al, 1982, Cancer; 50: 2894-. It was also observed that patients with combined SCLC had a poorer prognosis than patients with classical SCLC (Hirsch FR et al, 1983, Cancer; 52: 2144-. Approximately 75% to 80% of cases have NSCLC histology, and most patients have locally advanced disease (stage III) or metastatic disease (stage IV). Importantly, patients undergoing curative surgical resection for a clearly localized disease have survival rates between 50% and 80%, suggesting a need for better systemic therapy to treat latent micrometastasis disease. In NSCLC, treatment with chemotherapy is generally unsuccessful (Minna JD, et al, 1985, Cancer of the lung in: Cancer. principles and dictionary of oncology; 2nd ed.). Thus, in addition to a high cure rate for surgical treatment of truly localized disease, the prognosis of patients with NSCLC is severe (Mulshine JL, et al 1986, J Clin Oncol; 4: 1704-1715). However, in a small fraction of patients, a response to chemotherapy can be observed. In part, these cases may represent NSCLC in which SCLC occurs, since such a heterogeneous component is common in lung cancer (see above). From these data, it may be apparent that alternative treatment modalities are critical for these patients.

The main objective of the present invention is to develop a new model for the biology and antigenicity of lung cancer and a new concept for lung cancer vaccine therapy. The method aims at the discovery of targets-antigens and immunization strategies specific for SCLC and NSCLC.

A large number of tumor-associated antigens have been identified in human lung cancer and used as targets for a common lung cancer vaccine. These antigens include carcinoembryonic antigen (CEA), human epithelial mucin MUC-1, cancer-testis antigen NY-ESO-1 and ganglioside Fuc-GM1(Haura EB.2001, cancer control, 8: 326-336; www.medscape.com/vacuroticle/409059). Several decades ago, vaccines for tumor patients with inactivated tumor cells have been tried without much success. The heat inactivated mycobacteria are taken as an adjuvant and are injected into SCLC lesion together with autologous tumor cells in a tumor manner to achieve certain effect.

NSCLC can elicit specific humoral and cellular anti-tumor immune responses in some patients (SalgiaR, et al 2003; J clin Oncol; 21: 624-. Serological-based cloning strategies have identified a variety of tumor-associated antigens, including eIF4G, aldolase, annexin XI, Rip-1, and NY-LU-12. Humoral responses to autologous lung cancer cells may be associated with prolonged survival. Similarly, T cell-based cloning strategies have revealed different targets in NSCLC, including Her2/neu, SART-1, SART-2, KIAA0156, ART-1, ART-4, cyclophilin B, mutated elongation factor 2, malic enzyme and alpha-actinin-4. The development of a cytotoxic T lymphocyte response to NSCLC may also be associated with prolonged survival.

In clinical practice, the definitive diagnosis of various subtypes of cancer is important, as therapy selection, prognosis, and the likelihood of response to therapy all vary widely depending on diagnosis. The accurate prognosis, or determination of distant metastasis-free survival, may allow oncologists to design the administration of adjuvant chemotherapy, giving more aggressive treatment to patients with poorer prognosis. In addition, accurate prediction of poor prognosis will greatly impact clinical trials for new lung cancer therapies, as potential study patients can be ranked according to prognosis. The test can be limited to patients with poor prognosis, making it easier to discern whether the experimental therapy is effective. To date, no satisfactory predictor has been identified for prognosis based solely on clinical information.

Therefore, it would be beneficial to provide specific methods and reagents for the diagnosis, staging, prognosis, monitoring and treatment of cancer, including lung cancer. It would also be beneficial to provide a method of identifying individuals with a predisposition to develop lung cancer and other types of cancer, which are thus suitable subjects for prophylactic therapy.

Summary of The Invention

The present inventors identified and characterized NCAM-180 and variants thereof using differential expression methods, the expression of which correlates with lung cancer and other types of cancer. The findings of the present inventors have made it possible to utilize the NCAM-180 molecule and variants thereof for the treatment, prevention and diagnosis of cancer (including but not limited to lung cancer), particularly in the treatment, prevention and diagnosis of neurological and neuroendocrine cancers.

Novel NCAM-180 has an up-regulated expression pattern in cancer tissues and cell lines, such as the lung cancer tissues and cell lines shown and described. Exon18_ MUM (a fragment of NCAM-180 that retains at least one functional characteristic of the full-length wild-type NCAM-180 gene) was also shown and described. Methods of using the gene, gene products, and antagonists or agonists of the gene or gene products (NCAM-180 and variants thereof, cDNA, RNA, and/or protein) as targets for diagnosis, drug screening, and therapy of cancer are also shown and described. Also disclosed is the use of the gene or gene product or derivative thereof as a vaccine against cancer. In one embodiment, methods of treating, preventing and diagnosing lung cancer using NCAM-180 protein (e.g., SEQ ID NO: 4), fragments thereof, particularly Exon18_ MUM (e.g., SEQ ID NO: 2 and 6) and variants thereof, or nucleic acids encoding the same are provided.

An isolated nucleotide sequence of NCAM-180 gene cDNA or a variant thereof is provided. In particular, isolated cDNA sequences of human NCAM-180 and its Exon18_ MUM fragment are provided herein (SEQ ID NOS: 1,3 and 5). Also provided is a polypeptide consisting of SEQ ID NO: 1,3 and 5 (which are designated SEQ ID NOs: 2, 4 and 6). The invention further relates to methods for the diagnostic evaluation and prognosis of cancer in a test animal. Preferably, the subject is a mammal, more preferably the subject is a human. In a preferred embodiment, the present invention relates to a method for the diagnostic evaluation and prognosis of lung cancer. For example, the nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis to detect abnormal expression of the NCAM-180 gene.

Antibodies or other binding partners to NCAM-180 or variants thereof can be used in diagnostic assays to detect the presence of NCAM-180 in a bodily fluid, cell or tissue biopsy. In particular embodiments, serum or cellular NCAM-180 and variants thereof can be measured to detect or classify lung cancer, such as adenocarcinoma, squamous cell carcinoma, bronchioloalveolar carcinoma or large cell carcinoma; in particular detecting or typing of SCLC.

In addition, methods for treating cancer, including lung cancer, are presented. These methods comprise administering a composition capable of modulating the level of NCAM-180 and variants thereof, including modulating the level of NCAM-180 gene expression and/or NCAM-180 gene product activity in a subject. The subject may be any animal, preferably a mammal, more preferably a human.

The invention also provides a method for preventing cancer, wherein NCAM-180 or a fragment thereof, in particular Exon18_ MUM or a fragment thereof capable of inducing a humoral or cellular immune response in a mammal, is administered to a subject in an amount effective to elicit an immune response in the subject. Exon18_ MUM fragments were further characterized in that they comprise 5 to 30 amino acids, in particular 9 to 15 amino acids and comprise an epitope selected from the group consisting of PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

The subject may be any animal, preferably a mammal, more preferably a human. The invention also provides methods of treating or preventing cancer by administering to a subject a nucleic acid sequence encoding NCAM-180 or a variant thereof, particularly Exon18_ MUM or a fragment thereof capable of inducing a humoral or cellular immune response in a mammal, such that expression of NCAM-180 or the variant results in the production of an effective amount of these polypeptides to elicit an immune response. The immune response may be humoral, cellular, or a combination of both. In a preferred embodiment, the invention also provides methods of immunization to confer protection against carcinogenesis.

The invention also provides methods of treating cancer by providing a therapeutic amount of an antisense nucleic acid molecule. An antisense molecule is a nucleic acid molecule that is the complement of all or part of the sequence of the NCAM-180 gene, and therefore, can hybridize to the NCAM-180 gene and variants thereof, particularly to Exon18_ MUM (a fragment of NCAM-180). Thus, hybridization of the antisense molecule can reduce or inhibit the expression of NCAM-180. In a preferred embodiment, the method is for treating lung cancer.

The invention also includes kits for assessing whether a patient has lung cancer or other types of cancer. The kit comprises reagents for assessing the expression level of NCAM-180 or variants, including fragments thereof (e.g., Exon18_ MUM or fragments thereof capable of inducing a humoral or cellular immune response in a mammal). In another aspect, the kit comprises an antibody, wherein the antibody specifically binds to a protein corresponding to NCAM-180 and variants thereof, in particular Exon18_ MUM or fragments thereof capable of inducing a humoral or cellular immune response in a mammal. The kit may further comprise a plurality of antibodies, wherein the plurality specifically binds to different epitopes of NCAM-180 and variants thereof, in particular to an Exon18_ MUM fragment, said Exon18_ MUM fragment comprising 5 to 30 amino acids, in particular 9 to 15 amino acids, and comprising an epitope selected from PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

In an alternative embodiment, the kit comprises nucleic acid (e.g., oligonucleotide) probes. The probe specifically binds to the transcribed polynucleotide corresponding to the NCAM-180 gene product and variants thereof. The kit may further comprise a plurality of probes, wherein each probe specifically binds to a transcribed polynucleotide corresponding to a different region of an mRNA sequence transcribed from the NCAM-180 gene and variants thereof, in particular to a nucleic acid sequence encoding Exon18_ MUM (a fragment of NCAM-180). In a more specific embodiment, the probe consists essentially of a sequence of 15-50, 18-35, 15-24, 18-30, 18-21 or 21-24 nucleotides encoding a polypeptide of the invention or the complement thereof. In a still further embodiment, a kit for diagnostic use is provided comprising primers capable of amplifying NCAM-180cDNA and variants thereof used in PCR, further comprising the corresponding cDNA and/or gene and a standard amount of NCAM-180 cDNA.

Accordingly, the present invention provides a method of diagnosing cancer in a subject comprising detecting or measuring in a sample derived from said subject an NCAM-180 product or variant thereof (including fragments thereof, e.g., Exon18_ MUM), wherein said NCAM-180 product is (a) a nucleic acid sequence corresponding to SEQ id no: 1,3 or 5, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm, compared to SEQ ID NO: 1,3 or 5 or the complement thereof, or a protein encoded thereby, wherein the level provided by the NCAM-180 product or variant thereof, as compared to a non-cancerous sample or a predetermined standard value for a non-cancerous sample, is indicative of the presence of cancer in the subject.

In one embodiment of the foregoing diagnostic method, the subject is a human, and in another embodiment, the cancer is lung cancer. In another embodiment, the sample is a tissue sample, a plurality of cells or a body fluid.

The invention further provides a method of treating cancer in a subject comprising administering to the subject an amount of a compound that reduces the level of and/or antagonizes the activity of NCAM-180 product and variants thereof (including fragments such as Exon18_ MUM and other fragments as defined herein above), wherein the NCAM-180 product is (a) a polypeptide corresponding to SEQ id no: 1,3 or 5, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm, compared to SEQ ID NO: 1,3 or 5 or the complement thereof, or a protein encoded thereby. In one embodiment, the gene product whose expression is reduced is encoded by a polynucleotide comprising a nucleotide sequence identical to SEQ ID NO: 1,3 or 5, or a nucleic acid encoding a nucleotide sequence having at least 90% sequence identity thereto. In another embodiment, the compound reduces the expression of a polypeptide corresponding to SEQ ID NO: 1,3 or 5. The antagonist may be (i) a protein; (ii) a polypeptide; (iii) organic molecules having a molecular weight of less than 2000 daltons; (iv) inorganic molecules having a molecular weight of less than 2000 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 the NCAM-180 product and variants thereof as defined herein; (viii) a double-stranded oligonucleotide forming a triple helix with the promoter of the NCAM-180 gene and variants thereof, wherein the NCAM-180 gene is complementary to the sequence of seq id NO: 1,3 or 5 or the complement thereof is at least 80% homologous; or (ix) a double-stranded oligonucleotide forming a triple helix with the L gene promoter, wherein the NCAM-180 gene is a nucleotide sequence identical to SEQ ID NO: 1,3 or 5 or the complement thereof is at least 80% homologous. Wherein the compound is an NCAM-180 antagonist antibody that immunospecifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6, and thereby reducing or knowing the activity of the protein according to the invention.

The invention further provides a method of vaccinating a subject against cancer comprising administering to the subject a molecule that elicits an immune response against an NCAM-180 gene product, wherein the NCAM-180 gene product is (a) a polypeptide corresponding to SEQ ID NO: 1 or 3, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2 or 6; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1 or 3 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm to compare SEQ ID NO: 1 or 3 or the complement thereof, 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 a cellular and humoral immune response.

The present invention still further provides a vaccine formulation for preventing or delaying the onset of cancer comprising (I) an immunogenic amount of an NCAM-180 product, wherein the NCAM-180 product is: (a) corresponding to SEQ ID NO: 1,3 or 5, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6 or SEQ ID NO: 2, 4 or 6, wherein said fragment is capable of inducing a humoral or cellular response in a mammal, in particular a Cytotoxic T Lymphocyte (CTL) response in a mammal; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm to compare SEQ ID NO: 1,3 or 5 or the complement thereof, or a protein encoded thereby; and (II) a pharmaceutically acceptable excipient. In a specific embodiment, the fragment capable of inducing a humoral or cellular response in a mammal comprises SEQ ID NO: 2, 4 or 6, and comprises an epitope selected from the group consisting of PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

The present invention still further provides a pharmaceutical composition comprising a polypeptide that specifically or selectively binds to a polypeptide consisting essentially of SEQ ID NO: 2, 4 or 6; and a pharmaceutically acceptable carrier. In yet another embodiment, the pharmaceutical composition comprises a polypeptide that specifically or selectively binds SEQ ID NO: 2, 4 or 6, wherein said fragment comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6, more particularly from 9 to 15 amino acids.

The invention further provides host cells comprising a nucleic acid encoding a polypeptide of the invention operably linked to a promoter, and methods of expressing such polypeptides and variants thereof by culturing the host cells under conditions in which the nucleic acid molecule is expressed. It is therefore an object of the present invention to provide the use of said host cell in a method for treating or preventing cancer in a subject, comprising administering to the subject (I) an amount of a host cell expressing an immunogenic amount of an NCAM-180 product, wherein said NCAM-180 product is: (a) corresponding to SEQ ID NO: 1,3 or 5, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6 or SEQ ID NO: 2, 4 or 6, wherein said fragment is capable of inducing a humoral or cellular response, in particular a CTL response as defined above, in a mammal; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm to compare SEQ ID NO: 1,3 or 5 or the complement thereof, or a protein encoded thereby; and optionally (II) a pharmaceutically acceptable excipient.

It is therefore an object of the present invention to provide a method of vaccinating a subject against cancer, said method comprising administering to the subject an amount of host cells expressing an immunogenic amount of an NCAM-180 product, wherein said NCAM-180 product is as defined above, comprising an Exon18_ MUM fragment and a fragment capable of inducing a CTL response in a mammal; and optionally (II) a pharmaceutically acceptable excipient. In a particular embodiment, the NCAM-180 product is selected from: (a) corresponding to SEQ ID NO: 1,3 or 5, or a nucleic acid derived therefrom; (b) comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6; (c) comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; (d) determined using the NBLAST algorithm to compare SEQ ID NO: 1,3 or 5 or the complement thereof, or a protein encoded thereby. The subject may be any animal, preferably a mammal, more preferably a human.

These and other aspects of the invention are described in more detail herein.

Description of sequences

SEQ ID NO: 1 is the nucleotide sequence of human Exon18_ MUM.

SEQ ID NO: 2 is the amino acid sequence of human Exon18_ MUM.

SEQ ID NO: 3 is the nucleotide sequence of human NCAM-180.

SEQ ID NO: 4 is the amino acid sequence of human NCAM-180.

SEQ ID NO: 5 is the nucleotide sequence of a fragment of human Exon18_ MUM.

SEQ ID NO: 6 is a polypeptide consisting of SEQ ID NO: 5 amino acid sequence of a fragment of human Exon18_ MUM encoded thereby.

Brief Description of Drawings

FIG. 1: schematic representation of the principle of differential expression PCR. For primer set a, the forward primer was designed in exon 17 and the reverse primer in exon 19. For primer set B, both forward and reverse primers were designed in Exon18_ MUM. PCR amplification of NCAM-140 expressing cells produced a 180bp PCR product with primer set A and no amplicon with primer set B. NCAM-180 expressing cells were used with primer set B to generate a 600bp PCR amplicon. Cells expressing NCAM-140 and NCAM-180 produced PCR products using 2 primer sets.

FIG. 2: overexpression of Exon18_ MUM as part of NCAM-180 in cell cultures derived from neuroendocrine tumor-derived cell lines (SH-SYSY and CCI) and in cell lines derived from small cell lung cancer (H69, H82, GLC-1, GLC 1-M13). Cells expressing Exon18_ MUM produced a PCR product of 604bp in MUM-specific PCR. Cells expressing the NCAM-140 splice variant had 180bp product in the exon 17-19PCR amplification reaction. The high expression marker is (+ +), medium expression (+), low expression (- +), and undetectable expression (-).

FIG. 3: purified truncated (panel a) and full-length (panel B) His-NCAM-Exon18_ MUM proteins produced in e.coli and purified on a Ni-chelate column.

FIG. 4: CTL response measured after restimulation with Exon18_ MUM overlapping 9-peptide library in vitro. Mice were immunized with Exon18_ MUM protein and/or DNA. Spleens were isolated, harvested and plated in 96-well culture plates. Subsequently, ex on18_ MUM overlapping 9 peptide library was used for in vitro restimulation. Measuring each 10 by flow cytometry⁵I in CD8+ T cellsThe number of FN-gamma secreting cells. IPP-irrelevant peptide restimulation with a peptide unrelated to the NCAM Exon18_ MUM protein region.

Each group was immunized 2 times. The agent for priming is represented by before and the agent for boosting is represented by after. The adjuvant used was Abisco-100.

A：PBS/PBS

B: adjuvant/adjuvant

C: exon18_ MUM protein in adjuvant/Exon 18_ MUM protein in adjuvant

D: exon18_ MUM protein/Exon 18_ MUM protein

E: empty pCI vector/empty pCI vector

F：Exon18_MUM pCI/Exon18_MUM pCI

G: exon18_ MUM protein in Exon18_ MUM pCI/adjuvant

FIG. 5: cytotoxic T cell responses were measured in mice immunized with Exon18_ MUM protein. Mouse splenocytes were restimulated in vitro with 9 peptide alone. The number of CD8 positive T cells containing intracellular IFN- γ was measured using a FACS-based reader.

FIG. 6: a cytotoxic T cell responses were measured in mice immunized with Exon18_ MUM DNA. Mouse splenocytes were restimulated in vitro with 9 peptide alone. The number of CD8 positive T cells containing intracellular IFN- γ was measured using a FACS-based reader. B-predictive score H-2^dbA haplotype.

FIG. 7: body fluid response measured by NCAM Exon18_ MUM protein ELISA. Mice were immunized with truncated Exon18_ MUM protein and/or truncated Exon18_ MUM DNA. The mice were bled, and the sera were separated and diluted 1/200 with buffer. ELISA plates were coated with Exon18_ MUM protein and detection of bound antibody was performed using HRP-conjugated anti-mouse IgG. OD was measured at 450 nm.

FIG. 8: body fluid response measured by NCAM Exon18_ MUM protein ELISA. Mice were immunized with the truncated Exon18_ MUM protein. Mice (50 total, 5 per group) were bled, and sera were separated and diluted with buffer (range 1/50-1/135.000). ELISA plates were coated with full-length Exon18_ MUM protein a and truncated Exon18_ MUM protein B, respectively. Detection of bound antibody was performed using HRP-conjugated anti-mouse IgG. OD was measured at 450 nm.

FIG. 9: fluorescence microscopy of HCT-116 (NCAM-negative), H82(NCAM Exon18_ MUM positive) and H69(NCAM Exon18_ MUM positive) cells stained with monoclonal antibody (IgM) produced by the MUM hybridoma MUMI21B 2. An Alexa Fluor 488-labeled goat anti-mouse μ chain secondary antibody (Invitrogen) was used as the secondary antibody to detect bound antibody.

FIG. 10: confocal microscopy images of H69 cells expressing NCAM Exon18_ MUM stained with monoclonal antibody (IgM) produced by the MUM hybridoma MUMI21B2 (dilution 1/2000). An Alexa Fluor 488-labeled goat anti-mouse μ chain secondary antibody (Invitrogen) was used as the secondary antibody to detect bound antibody.

Detailed Description

The following definitions are set forth to illustrate aspects of the invention: specific or selective: a nucleic acid used in a reaction, such as a probe used in a hybridization reaction, a primer used in PCR, or a nucleic acid present in a pharmaceutical formulation, is said to be "selective" if it hybridizes or reacts with a specified target more frequently, more rapidly, or for a longer duration than it hybridizes or reacts with a selectable substance. Similarly, a polypeptide is said to be "selective" if it binds to a given target (e.g., a ligand, hapten, substrate, antibody or other polypeptide) more frequently, more rapidly, or for a longer duration than it binds to a selectable substance. An antibody is said to be "selective" if it binds to a given target more frequently, more rapidly, or for a longer duration through at least one antigen recognition site than it binds to a selectable substance. A marker that is selective for a particular cell or tissue type if it is predominantly expressed in or on the particular cell or tissue type (particularly for a biological sample of interest).

Variants: variants of a polynucleotide or polypeptide, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variant polynucleotides are generally limited such that the nucleotide sequences of the reference and variant are closely related as a whole and identical in many regions. The change in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acid sequence encoded by the polynucleotide. When the alteration is limited to this type of silent change, the variant will encode a polypeptide having the same amino acid sequence as the reference. Alternatively, a change in the nucleotide sequence of a variant may alter the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Variant polypeptides are generally limited, such that the sequences of the reference and variant are closely similar overall and, in many regions, identical. For example, a variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations (which may or may not be present in any combination).

Equivalent or corresponding: between nucleic acids, "corresponding" means homologous or complementary to a particular sequence or portion of a sequence of a nucleic acid. Between a nucleic acid and a polypeptide, "corresponding" refers to a sequence or portion of a sequence in which the amino acids of the peptide are sequentially derived from the nucleic acid or its complement. Between polypeptides (or peptides and polypeptides), "corresponding" refers to a sequence or portion of a sequence from which the amino acids of a first polypeptide (or peptide) are sequentially derived.

NCAM-180 gene: as used herein, unless otherwise stated, refers to novel splice variants of NCAM found herein that are upregulated in certain cancers (e.g., lung cancer) and subclasses thereof.

NCAM-180 product or NCAM-180 gene product: as used herein, unless otherwise stated, the NCAM-180 product is: corresponding to SEQ ID NO: 1,3 or 5, or a nucleic acid derived therefrom; comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6; comprising a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1,3 or 5 or a complement thereof, or a protein comprising a sequence encoded by said hybridizable sequence; determined using the NBLAST algorithm to compare SEQ ID NO: 1,3 or 5 or the complement thereof is at least 90% homologous; and SEQ ID NO: 1 or a fragment or derivative of any of the foregoing proteins or nucleic acids, in particular a nucleic acid that is at least 90% homologous to one of Exon18_ MUM (a fragment of NCAM-180, hereinafter also referred to as MUM protein or NCAM-MUM, encoded by any of SEQ ID NO: 3 or SEQ ID NO: 5); in a further embodiment, the fragment of NCAM-180 or Exon18_ MUM is characterized in that it comprises 5 to 30 amino acids, more particularly 9 to 15 amino acids, and comprises an epitope selected from the group consisting of PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

A control element: as used herein, all refer to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, all of which provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control elements are required so long as the selected coding sequence is capable of replication, transcription and translation in an appropriate host cell.

A promoter region: as used herein in its ordinary sense, means a nucleotide region that comprises a DNA regulatory sequence derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3' -direction) coding sequence.

And (3) effective connection: as used herein, means an arrangement of elements wherein the components are configured to perform their usual functions. Thus, a control element operably linked to a coding sequence is capable of effecting expression of the coding sequence. The control elements need not be linked to the coding sequence, so long as they function to direct the expression of the coding sequence.

Exogenous: as used herein, the term exogenous means those obtained or originating from outside. When used in the context of exogenous expression of a gene or protein, the term means a gene or protein that is expressed in a cell or tissue that does not normally express the gene or protein. When used in the context of nucleic acid sequences, the term can also be used to refer to the association of 2 or more nucleic acid sequences (which are not normally operably linked in nature) that are operably linked, for example.

Treatment of cancer or tumors: as used herein, the terms "treating cancer or tumor" or "treating cancer or tumor" in a mammal mean one or more of alleviating the symptoms of cancer or tumor, correcting the underlying molecular or physiological disorder of cancer or tumor, reducing the frequency or severity of the pathological or detrimental physiological consequences of cancer or tumor in the mammal. By way of example, and not by way of limitation, detrimental physiological consequences of a cancer or tumor may include uncontrolled proliferation, metastasis and invasion of other tissues, and suppression of immune responses.

Immunologically specific: for the antibodies of the present invention, the term "immunologically specific" means an antibody that binds to one or more epitopes of a protein of interest (e.g., the Exon18_ MUM protein), but which does not substantially recognize and bind other molecules in a sample comprising a mixed population of antigenic biological molecules.

Essentially consisting of. The term "consisting essentially of," when referring to a specific nucleotide or amino acid, refers to a polypeptide having the amino acid sequence of a given SEQ ID NO: the sequence of properties of (a). For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence. Such an amino acid sequence exhibits a sequence identical to a given SEQ id no: is 80% or more, i.e., 85%, 90%, 95%, 97%, 98%, 99% or more. In particular, such amino acid sequence exhibits a sequence identical to a given SEQ ID NO: is 80% or more, i.e., 85%, 90%, 95%, 97%, 98%, 99% or more.

Antagonists: as used herein, a compound capable of reducing the level and/or activity of NCAM-180 or a variant thereof may be referred to herein as an NCAM-180 antagonist.

Agonist(s): as used herein, a compound capable of increasing the level and/or activity of NCAM-180 or a variant thereof may be referred to herein as an NCAM-180 agonist.

NCAM-180 Activity: as used herein, the term "NCAM-180 activity", "NCAM-180 product activity", or "NCAM-180 mediated activity" means an activity associated with the expression of NCAM-180 and/or NCAM-180 product. Such activities include, but are not limited to, changes in cell proliferation, cell motility, cell differentiation, and/or cell adhesion associated with changes in the expression level of NCAM-180. As used herein, the terms "increased", "overexpressed", "upregulated", or "increased" mean that the expression of NCAM-180 transcripts and/or proteins is increased by about 2-fold or more compared to those of control tissues expressing baseline levels of NCAM-180.

Nucleic acids

Nucleic acids as used in the methods of the invention include DNA (including genomic and cDNA) and RNA. When the nucleic acid according to the invention comprises RNA, reference to the sequences shown in the appended list is to be understood as referring to RNA equivalents, replacing T with U.

The nucleic acids of the invention may be single-stranded or double-stranded. The single-stranded nucleic acid of the present invention includes antisense nucleic acids. Thus, it will be understood that reference to SEQ ID NO: 1 or a polypeptide comprising SEQ ID NO: 1 or a fragment thereof includes the complementary sequence unless the context clearly dictates otherwise. The above also applies to SEQ ID NO: 3 or 5.

Typically, the nucleic acid according to the invention is provided as an isolate, in isolated and/or purified form, or as free or substantially free material (to which it is naturally bound), e.g., free or substantially free nucleic acid flanking a human genome, except for one or more regulatory sequences that may be used for expression. The nucleic acid may be wholly or partially synthetic and may comprise genomic DNA, cDNA or RNA.

The invention also provides nucleic acids that are fragments of a nucleic acid encoding a polypeptide of the invention. In one aspect, the invention provides nucleic acid primers or probes consisting essentially of 15-50, e.g., 15-35, 18-35, 15-24, 18-30, 18-21 or 21-24 nucleotides of a sequence encoding a polypeptide of the invention or its complement.

The term "consisting essentially of means a nucleic acid that does not include any additional 5 'or 3' nucleic acid sequences. In yet another aspect of the invention, a nucleic acid of the invention consisting essentially of 15-30 nucleotides as defined above may be linked at the 3 'but preferably 5' end to a short (e.g. 4-15, e.g. 4-10 nucleotides) additional sequence (which is not naturally linked to the additional sequence). Such additional sequences are preferably linkers comprising a restriction enzyme recognition site to facilitate cloning when the nucleic acid of the invention is used as e.g.a PCR primer.

The primers and probes of the invention are desirably capable of selectively hybridizing to nucleic acids encoding the polypeptides of the invention. By "selective" it is meant selective with respect to sequences encoding other purine receptors, particularly receptors other than adenine receptors. The ability of a sequence to selectively hybridize can be determined experimentally or computationally.

For example, one method of calculating the Tm value of a primer is by reference to a formula for calculating the Tm value of a primer of a homologous target sequence. The formula is Tm (° C) 2(a + T) +4(G + C) -5. This provides the Tm values under the conditions of 3 XSSC and 0.1% SDS (where SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7). This formula is generally applicable to primers up to 50 nucleotides in length. In the present invention, this formula can be used as an algorithm for calculating a Tm value for a primer rated with respect to a specific sequence derived from a sequence encoding the polypeptide of the present invention. The Tm value can be compared to the Tm value calculated for human and rat GPCR sequences, based on the maximum number of matches to any portion of these other sequences.

Suitable conditions for hybridization of the primer to the target sequence can also be determined experimentally. Suitable experimental conditions include hybridizing candidate primers to nucleic acids encoding the polypeptides of the invention and to nucleic acids encoding other adenine receptors on a solid support under low stringency hybridization conditions (e.g., 6 x SSC, 55 ℃), washing at reduced SSC and/or higher temperatures, e.g., at 0.2 x SSC, 45 ℃, and gradually increasing the hybridization temperature to determine hybridization conditions that allow the primers to hybridize to nucleic acids encoding the polypeptides of the invention but not to nucleic acids encoding other purine receptors.

The nucleic acids of the invention, in particular the probes, may carry a revealing label. Suitable labels include radioisotopes such as³²P or³⁵S, fluorescent labels, enzyme labels, or other protein labels such as biotin. These labels may be added to the polynucleotides or primers of the invention and may be detected by using techniques known per se.

The primers of the present invention may be composed of synthetic nucleic acids, such as those having a modified backbone structure to increase the stability of the nucleic acids in cells. A large number of different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3 'and/or 5' ends of the molecule. For the purposes of the present invention, it is understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifetime of the polynucleotides of the invention.

Antisense sequences based on the nucleic acid sequences described herein are also encompassed by the present invention, preferably in the form of oligonucleotides, in particular stable oligonucleotides, or ribozymes.

Antisense oligonucleotides can be designed to hybridize to a complementary sequence of a nucleic acid, a pre-mRNA or mature mRNA, interfering with the production of the polypeptide encoded by a given target DNA sequence, thereby reducing or completely preventing its expression. Ribozymes can be designed to cleave mRNAs encoded by the nucleic acid sequence encoding NCAM-180 of the invention, ideally on a target sequence (including a sequence according to SEQ ID NO: 1,3 or 5) specific for the NCAM-180 gene as defined above. Construction of antisense sequences and their use are described in Peyman and Ulman, Chemical ReviewS, 90: 543-; 32: 329-, (1992) and Zamecnik and Stephenson, P.N.A.S., 75: 280-284, (1974). Construction of ribozymes and their use are described, for example, in Gibsonand Shillitoe, Molecular Biotechnology7 (2): 125, 137, (1997).

The cDNA sequence of NCAM-180 of the present invention can be cloned by standard PCR (polymerase chain reaction) cloning techniques. This involves making a sequence for SEQ ID NO: 1 by contacting the primers with mRNA or cDNA obtained from mammalian cells under conditions that cause amplification of the desired region, performing a polymerase chain reaction, isolating the amplified fragments (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. The above also applies to seq id NO: 3 or 5.

The amino acid sequence not identical to SEQ ID NO: 1,3 or 5 but encodes SEQ ID NO: 2, 4 or 6 or other polypeptides of the invention.

For example, SEQ ID NO: 1,3 or 5. This is useful, for example, when the sequence requires silent codon changes to optimize codon preference for the particular host cell in which the polynucleotide sequence is expressed. Additional sequence changes may be required to introduce restriction enzyme recognition sites, or to alter the properties or function of the polypeptide encoded by the polynucleotide. Additional changes may be required to represent particular coding changes (such changes are required to provide, for example, conservative substitutions).

The nucleic acids of the invention may comprise additional sequences at the 5 'or 3' end. For example, a synthetic or natural 5' leader sequence may be attached to a nucleic acid encoding a polypeptide of the invention. Additional sequences may also include 5 'or 3' untranslated regions required for transcription of a nucleic acid of the present invention in a particular host cell.

In addition, other animals, particularly mammals (e.g., monkeys or rabbits), more particularly primates (including monkeys), can obtain homologs of NCAM-180 and be used in the methods of the invention. By making or obtaining a cDNA library from a segmented cell or tissue or a genomic DNA library from other animal species and isolating the cDNA from the segmented cell or tissue and using a DNA molecule comprising SEQ ID NO: 1,3 or 5 under moderate or high stringency conditions (e.g., 0.03M sodium chloride and 0.03M sodium citrate at from about 50 ℃ to about 60 ℃) to search these libraries.

The invention further extends to an isolated DNA sequence comprising a sequence encoding a polypeptide of the invention and wherein the coding sequence is divided into 2 or more (preferably no more than 5, for example 4 or 3) exons. Such exon sequences may be natural and obtained from genomic clones or synthetic. Exon sequences may be used in the construction of minigene sequences comprising nucleic acids encoding polypeptides of the invention whose sequence is interrupted by one or more exon sequences.

Heterologous exons from any eukaryotic source can be used to construct minigenes.

Polypeptides

The isolated polypeptides used in the methods of the invention are those as defined above, which are free or substantially free, in isolated form, from material with which they are naturally associated, e.g., other polypeptides with which they are associated found in a cell. Of course, the polypeptide may be formulated with diluents or adjuvants and isolated for practical purposes-for example if used to coat microtiter plates for use in immunoassays, the polypeptide may typically be mixed with gelatin or other carriers. The polypeptides may be glycosylated, either naturally or by heterologous eukaryotic cell systems, or they may be (e.g., if produced by expression in a prokaryotic cell) unglycosylated. The polypeptide may be phosphorylated and/or acetylated.

The polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, for example 95%, 98% or 99% of the polypeptide in the preparation is the polypeptide of the invention.

The polypeptides of the invention may be modified, for example, by the addition of histidine residues to aid their purification or by the addition of signal sequences to facilitate their secretion from cells.

In one embodiment, the invention relates to a fusion protein comprising an NCAM-180 polypeptide, fragment or derivative of the invention and a heterologous protein or part of a protein as a fusion partner. The proteins and fusion proteins of the invention may be chemically conjugated but are preferably expressed as recombinant fusion proteins in heterologous expression systems. The fusion partner may be an immunological fusion partner (which may assist in providing T helper epitopes, or as an expression enhancer). Thus the immunological fusion protein can act by a bystander helper effect linked to the secretion of an activation signal (by a large number of T cells specific for foreign proteins or peptides), thereby enhancing the induction of immunity to the NCAM-180 protein. Preferably, the heterologous partner is selected to be recognized by T cells in most humans. The expression enhancer will allow an increase in the level of NCAM-180 protein to be produced (compared to the native recombinant protein) and includes partners that assist in initial protein folding, particularly the HSP70 or HSP60 family and the C-terminal domain of the heat shock protein (which is known to interact with substrate proteins and immunogenic peptides). Preferably, the fusion partner is an immunological fusion protein and an expression enhancer partner. Accordingly, the present invention provides a fusion protein, fragment or derivative of NCAM-180 according to the invention linked to a fusion partner. In a further embodiment, the fusion partner is selected from the group consisting of the nonstructural proteins from influenza viruses, NS1 (hemagglutinin) or the C-terminal domain of the HSP70 or HSP60 family. Further examples of immunological fusion partners are provided in EP 804156.

Has a sequence similar to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is a polypeptide having at least 50%, e.g., 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, an allele, derivative or mutant, and provided by the invention. For example, such polypeptides may have amino acid sequences corresponding to those shown in SEQ ID NOs: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a pharmaceutically acceptable salt thereof.

The percent identity of a polypeptide sequence can be calculated using commercially available algorithms that compare a reference sequence (e.g., SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 of the invention) to a query sequence. Further details of assessing identity are described below.

When the query sequence is determined to have a sequence identical to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and which sequence is a sequence of a polypeptide that retains the activity of the NCAM-180 protein, such a polypeptide forms part of the present invention.

The polypeptides according to the invention can be isolated and/or purified (e.g. using antibodies), for example after production by expression from the encoding nucleic acid. The isolated and/or purified polypeptide may be used in the formulation of a composition, which may comprise at least one additional component, e.g., a pharmaceutical composition comprising a pharmaceutically acceptable excipient, vehicle or carrier.

The polypeptides according to the invention can be used as immunogens or to obtain specific antibodies. Antibodies are useful in purification and other manipulations of polypeptides, diagnostic screening and therapeutic contexts.

The polypeptides of the invention may be labelled with a display tag. The indicative label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, for example¹²⁵I, fluorescent dyes, enzymes, antibodies, polynucleotides and linkers such as biotin. The labeled polypeptides of the invention can be used in diagnostic procedures, such as immunoassays, to determine the amount of the polypeptide of the invention in a sample. The polypeptides or labeled polypeptides of the invention may also be used in serological or cell-mediated immunoassays using standard methods to detect immunoreactivity for the polypeptides in animals and humans.

The polypeptide or labelled polypeptide of the invention or fragments thereof may also be immobilised to a solid phase, for example the surface of an immunoassay well or dipstick.

Such labeled and/or immobilized polypeptides may be packaged in kits in suitable containers with appropriate reagents, controls, instructions, and the like.

Such polypeptides and kits may be used in methods of antibody detection (by immunoassay) of the presence of these polypeptides or active portions or fragments thereof in a sample.

Immunoassays are well known in the art and typically include:

(a) providing a polypeptide comprising an epitope that can be bound by an antibody against said protein;

(b) incubating a biological sample with the polypeptide under conditions that allow formation of an antibody-antigen complex; and

(c) determining whether an antibody-antigen complex comprising the polypeptide is formed.

Sequence identity

The percent identity of nucleotide and polypeptide sequences can be calculated using commercially available algorithms that compare reference and query sequences. Homology/identity can be determined using the following program (provided by national center for Biotechnology Information): BLAST, gapped BLAST, BLASTN and PSI-BLAST, which may use default parameters.

The GAP algorithm (Genetics Computer Group, Madison, Wis.) uses the Needleman and Wunsch algorithms to align two complete sequences, which maximizes the number of matches and minimizes the number of GAPs. Typically, the default parameters used are a gap creation penalty of 12 and a gap extension penalty of 4.

Another method for determining the best overall match between a nucleic acid sequence or portion thereof and a query sequence uses the FASTDB computer program based on the Brutlag et al algorithm (Comp.App.biosci., 6; 237-. The program provides a global sequence alignment. The overall sequence alignment results in percent identity. Suitable parameters used in FASTDB studies of DNA sequences to calculate percent identity are: the matrix is unitary, k-tuple 4, mismatch penalty 1, binding penalty 30, random group length 0, cutoff score 1, gap penalty 5, gap size penalty 0.05, and window size 500 or the nucleotide base length of the query sequence, whichever is shorter. Suitable parameters for calculating the percent identity and similarity of an amino acid alignment are: the matrix is PAM150, k-tuple 2, mismatch penalty 1, binding penalty 20, random group length 0, cutoff score 1, gap penalty 5, gap size penalty 0.05, and window size 500 or the nucleotide base length of the query sequence, whichever is shorter.

Carrier

The nucleotide sequences of the present invention may be incorporated into vectors, particularly expression vectors. The nucleic acid may be replicated in a compatible host cell using a vector. Thus in a further embodiment, the invention provides for making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which cause replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in conjunction with expression vectors.

Preferably, the polynucleotide in the vector of the invention is operably linked to a control sequence capable of providing for expression of the coding sequence in a host cell, i.e., the vector is an expression vector.

The term "operably linked" means a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Suitable vectors may be selected or constructed which contain appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. The vector may be an appropriate plasmid, viral, e.g.phage, phagemid or baculovirus, cosmid, YACs, BACs, or PACs. Vectors include gene therapy vectors, such as those based on adenoviral, adeno-associated viral, retroviral (e.g., HIV or MLV) or alphaviral vectors.

The vector may be equipped with an origin of replication, optionally a promoter for expression of the polynucleotide and optionally a regulator of the promoter. The vector may comprise one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. The vectors may be used in vitro, for example for the production of RNA or for the transfection or transformation of host cells. The vector may also be suitable for in vivo use, for example in methods of gene therapy. Systems for the cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammals and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells, and many others.

Promoters and other expression regulatory signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, yeast promoters include the Saccharomyces cerevisiae (S.cerevisiae) GAL4 and ADH promoters, the Schizosaccharomyces (S.pombe) nmtl and ADH promoters. Mammalian promoters include metallothionein promoters (which can be induced in response to heavy metals such as cadmium). Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may be used. All of these promoters are readily available in the art.

The vector may contain other sequences, such as promoters or enhancers, to drive expression of the inserted nucleic acid, nucleic acid sequence, such that the polypeptide is produced as a fusion protein and/or a secretion signal encoded by the nucleic acid, such that the polypeptide produced in the host cell is secreted from the cell.

Vectors for producing the polypeptide of the present invention used in gene therapy include vectors carrying the minigene sequence of the present invention.

For further details see, for example, Molecular Cloning: a laboratory manua 1: 2nd edition, Sambrook et al, 1989, Cold Spring harbor laboratory Press. Many known techniques and methods for manipulating nucleic acids, such as preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Current Protocols in Molecular Biology, Ausubel et al, John Wiley & Sons, 1992.

The vector may be transformed into a suitable host cell as described above to provide for expression of the polypeptide of the invention. Thus, in another aspect, the invention provides a method for producing a polypeptide according to the invention, comprising culturing a host cell transformed or transfected with an expression vector as described above under conditions which provide for expression of the vector comprising a coding sequence encoding the polypeptide, and recovering the expressed polypeptide. The polypeptide may be expressed in an in vitro system, such as in reticulocyte lysate.

A further embodiment of the invention provides a host cell transformed or transfected with a vector for replication and expression of a polynucleotide of the invention. The cells are chosen to be compatible with the vector, and may be, for example, bacteria, yeast, insects, or mammals. The host cell can be cultured under conditions for gene expression so that the encoded polypeptide is produced. If the polypeptide is expressed with a suitable signal leader, it can be secreted from the cell into the culture medium. Following expression production, the polypeptide may be isolated and/or purified from the host cell and/or culture medium, as appropriate, and then used as desired, e.g., in the formulation of a composition, which may comprise one or more additional ingredients, e.g., a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, vehicles or carriers.

The polynucleotide according to the invention may be inserted into a vector as described above in an antisense orientation to provide for the production of antisense RNA or ribozymes.

Diagnostic method

In a preferred embodiment, the invention relates to a method for assessing the quantitative and qualitative aspects of NCAM-180 gene expression. In one embodiment, the increase in the expression of the NCAM-180 gene or gene product provided by the present invention is predictive of the presence of cancer in a subject or the risk of cancer metastasis in said subject. Techniques well known in the art, such as quantitative or semi-quantitative RT-PCR or northern blot analysis, can be used to measure the expression level of NCAM-180.

Measurement of the expression level of the NCAM-180 gene may include measurement of naturally occurring NCAM-180 transcripts and variants thereof as well as non-naturally occurring variants thereof. However, the diagnosis and/or prognosis of cancer in a subject is preferably direct detection of the naturally occurring NCAM-180 gene product and variants thereof. Accordingly, the present invention relates to a method of diagnosing and/or prognosing cancer in a subject by measuring the expression of the NCAM-180 gene in the subject. For example, an increase in the level of mRNA encoded by an NCAM-180 nucleic acid sequence (e.g., SEQ ID NO: 1,3 or 5).

Diagnostic methods for detecting NCAM-180 nucleic acid molecules in patient samples or other suitable cell sources may involve amplification of specific gene sequences, for example, by PCR (see Mullis, k.b., 1987, U.S. Pat. No. 4,683,202), followed by analysis of the amplified molecules using techniques well known to those skilled in the art (such as, for example, those listed above). By using analytical techniques such as these, the amplified sequence can be compared to levels in a control sample.

The diagnosis and/or prognosis of cancer is suitable for detecting a naturally occurring NCAM-180 polypeptide in a subject. The NCAM-180 polypeptide can be detected by any method known in the art.

The tissue or cell types to be analyzed generally include those known, or suspected, to express the NCAM-180 gene, such as, for example, cancer cells including lung cancer cells, ovarian cancer cells, skin cancer cells, lymphoid cancer cells, and metastatic forms thereof. Protein isolation procedures used herein may, for example, be those as 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 may be derived from a cell culture or a patient.

Preferred diagnostic methods for detecting the NCAM-180 gene product or a conservative variant thereof or a peptide fragment thereof (particularly Exon18_ MUM) may involve, for example, immunoassays in which the NCAM-180 gene product or a variant thereof, including peptide fragments such as Exon18_ MUM, is detected by its interaction with a selective antibody. For example, an antibody, or fragment of an antibody, can be used to quantitatively or qualitatively detect the presence of a polypeptide encoded by NCAM-180, or a naturally occurring variant thereof, or a peptide fragment thereof. The antibodies (or fragments thereof) useful in the present invention may additionally be used histologically, such as in immunofluorescence or immunoelectron microscopy, for in situ detection of the NCAM-180 gene product or a conservative variant thereof or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen, e.g., a paraffin-embedded tissue (e.g., lung tissue) section, from the subject and applying the labeled antibody of the invention thereto. The antibody (or fragment) is preferably applied by placing the labeled antibody (or fragment) on a biological sample. Since the NCAM-180 fragment Exon18_ MUM appears to be expressed primarily as an intracellular protein, it may be desirable to introduce the antibody into the cell, for example, by making the cell membrane permeable. Certain NCAM-180 immunogenic polypeptides may also be expressed on the cell surface, and thus cells may be directly labeled by the application of antibodies specific or selective for extracellular NCAM-180 polypeptides or fragments thereof.

By using these methods, it is possible to determine not only the presence of the NCAM-180 gene product or naturally occurring variants or peptide fragments thereof, but also its distribution in the tissue under examination. By using the methods of the present invention, those of ordinary skill will readily recognize that any of a number of histological methods (e.g., staining methods) can be modified to achieve such in situ detection.

Immunoassays for the NCAM-180 encoded polypeptide or a conservative variant thereof or a peptide fragment thereof, particularly Exon18_ MUM, will generally involve contacting a sample, such as a biological fluid, a tissue or tissue extract, freshly harvested cells, or a lysate of cells that have been incubated in cell culture, in the presence of an antibody that specifically or selectively binds to the NCAM-180 gene product (e.g., a detectable labeled antibody capable of identifying the NCAM-180 polypeptide or a conservative variant or peptide fragment thereof), and detecting the bound antibody by any of a number of techniques well known in the art (e.g., western blot analysis, ELISA, FACS). The biological sample may be contacted with and immobilized on a solid support or carrier, such as nitrocellulose, or other solid support capable of immobilizing cells, cell particles, or soluble proteins. The support is washed with a suitable buffer and subsequently treated with a blocking reagent and an antibody that selectively or specifically binds to a polypeptide encoded by NCAM-180. The solid support was washed twice with buffer to remove unbound antibody. The amount of label bound to the solid support can be detected by conventional methods. Alternatively, the antibody that selectively or specifically binds to the polypeptide encoded by NCAM-180 is immobilized and the biological sample comprises

By "solid phase support or carrier" is meant any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, nitrocellulose, natural or modified cellulose, polyacrylamide, and magnetite. For the purposes of the present invention, the nature of the carrier may be somewhat soluble or insoluble. The support material may have virtually any possible structural configuration, so long as the conjugated molecule is capable of binding an antigen or antibody. Thus, the support morphology may be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, or the like. Preferred supports include polystyrene beads. Those skilled in the art will know of many other suitable carriers for binding antibodies or antigens, or will be able to determine the same by routine experimentation.

anti-NCAM-180 antibodies can be labeled with antibodies detectable by linking them to enzymes and using The antibodies labeled in Enzyme Immunoassays (EIA) (Voller, A., "The Enzyme Linked immunological Assay (ELISA)," 1978, Diagnostic Horizons 2: 1, Microbiological associates Quaternary publication, Walkersville, MD; Voller, A.et. 1978, J.Clin.Pathol.31: 507-520; Butler, J.E., 1981, meth.E4Zy7.73: Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, FL; Ishiwa, E.et. edition., (1981, Enzyme), Shork, Enzyme Kyoya). The enzyme bound to the antibody will react with a suitable substrate, preferably fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Antibodies can also be produced by using fluorescent emitting metals such as¹⁵²Eu, or other lanthanide metals. These metals are prepared by using metal chelating groupsA moiety such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) is attached to the antibody. Commonly used fluorescent pigments are fluorescein, Texas Red (Texas Red) or other fluorescent pigments such as the Alexa Fluor series.

Antibodies can also be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescent-labeled antibody is detected by luminescence emitted during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, bioluminescent compounds may be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins is determined by detecting luminescence. Important bioluminescent compounds for labeling are luciferin, luciferase and aequorin.

In various embodiments, the invention provides methods for measuring NCAM-180 polypeptides using NCAM-180-specific or NCAM-180-selective antibodies and the use of these measurements in clinical applications.

Measurement of the NCAM-180 polypeptides of the invention is valuable for detecting and/or staging lung cancer or other cancers in a subject, for screening of lung cancer and other cancers in a population, for differential diagnosis of a physiological condition in a subject, and for monitoring the effect of a therapeutic therapy on a subject.

The invention also provides for detecting, diagnosing, or staging lung cancer and other cancers, or monitoring treatment of lung cancer and other cancers by measuring the expression level of the NCAM-180 polypeptide. In addition to the NCAM-180 polypeptide, at least one other marker, such as a receptor or differentiation antigen, 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 conjunction with NCAM-180 polypeptides to detect, diagnose, stage, or monitor the treatment of lung cancer and other cancers. In another embodiment, the prognostic indicator is the change observed in the levels of different markers that are correlated with one another, rather than the absolute level of the marker that occurs at any one time. These measurements can help predict treatment outcome as well as evaluate and monitor the overall disease state of the subject.

Method of vaccination

The invention also relates to the use of vaccines based on NCAM-180 as a tumor-associated antigen (TAA), its epitopes, mimotopes, specific or anti-idiotypic antibodies, for the preparation of a medicament, and kits for prophylactic and/or therapeutic automated vaccination against cancer. Tumor Associated Antigens (TAAs) are often the basis for the development of immunotherapeutic agents for the prevention and/or treatment of cancer. TAAs are structures that allow differentiation associated with benign tissues and are therefore considered targets for diagnostic and therapeutic applications of specific antibodies. TAAs are normally expressed on the plasma membrane of tumor cells, but may also be intracellular proteins, including nuclear, cytoplasmic and transmembrane proteins.

The direct therapeutic use of antibodies against TAA is based on passive immunotherapy, i.e. the specific antibody is administered systemically (or locally, e.g. into a vein flowing to the tissue) in a suitable amount to a cancer patient and has a therapeutic effect only if its concentration in the organism is sufficiently high for this. The biological half-life of these agents depends on their structure, ranging from a few hours to several days. Therefore, repetitive applications must be provided. However, when using xenogeneic antibodies (e.g. murine monoclonal antibodies, MAB), this leads to undesired immune reactions which may neutralize possible therapeutic activity and may lead to dangerous side effects (allergic reactions). Thus, such immunotherapeutic agents can only be administered for a limited time.

Different approaches for cancer immunotherapy are based on the selective activation of the immune system of cancer patients against malignant cells. This was attempted by a very diverse cancer vaccine. Among them are vaccines with irradiated or otherwise inactivated (non-proliferating) autologous or allogeneic tumor cells or autologous or allogeneic tumor cells modified chemically or molecularly, isolated TAAs or TAAs derived therefrom prepared by means of chemical or molecular biological methods, more recently also vaccines with DNA encoding TAAs or structures derived therefrom, and so on. An alternative approach is based on the use of anti-idiotype antibodies for immunization against cancer. Suitable anti-idiotype antibodies can immunologically mimic TAA. As foreign proteins (e.g. murine antibodies, goat antibodies, etc.) they induce a strong immune response in humans after immunization-unlike suitable human tumor antigens (which have self-structures and are usually only poorly immunogenic). Thus, anti-idiotype antibodies can be used as immunogenic substitutes for tumor antigens for immunization.

By using anti-tumor antibodies, in contrast to passive immunotherapy, in principle very small amounts of suitable vaccines can meet the needs of active immunotherapy for specific cancers so that immunity is induced for months or even years, which can be enhanced by boosting immunity when it becomes weak. In addition, in active immunization, humoral and cellular immunity can be induced, and their interaction can lead to effective protection.

In summary, the use of antibodies or derivatives thereof in cancer immunotherapy has so far been based essentially on 2 principles:

passive therapy with antibodies or derivatives thereof directed against TAA. In this case, the tumor cells are destroyed relatively specifically. (Immunology Today (2000), 21: 403-.

Active immunization (vaccination) with cells, TAAs, or antibodies, or derivatives thereof, respectively, directed against the idiotype of the antibody (with specificity for TAAs). Active vaccination triggers an immune response against TAA. This immune response is therefore also directed against the corresponding tumor cells (Ann. Med. (1999), 31: 66; Immunobiol. (1999), 201: 1).

The present object of the invention is now to provide a vaccine based on NCAM-180 as Tumor Associated Antigen (TAA), its epitopes, mimotopes, specific or anti-idiotypic antibodies, for the preparation of a medicament for prophylactic and/or therapeutic active immunization against cancer, optionally in combination with chemotherapy.

Preferably, the TAA is selected from a peptide or protein as described herein, in particular NCAM-180 or Exon18_ MUM as described above. In a further experimental protocol, the TAA is selected from:

essentially consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6; or

A fragment of said isolated protein, wherein said fragment is capable of inducing a humoral or cellular immune response in a mammal and comprises an epitope selected from the group consisting of:

PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

The TAAs used in the vaccine according to the invention preferably induce a functional immune response against tumor cells. In doing so, not only the tumor cells during their cell division, but also those in the quiescent state (e.g., the recently demonstrated tumor stem cells) are attacked to effectively treat "minimal residual disease," or to reduce metastatic potential.

Preferably, according to the present invention, epithelial cancers, such as breast cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, ovarian cancer and lung cancer, for example SCLC ("small cell lung cancer") and NSCLC ("non-small cell lung cancer") will be treated or can be treated.

In a specific embodiment, at least 2 identical or different epitopes of NCAM-180TAA are provided or mimicked, respectively, in a vaccine according to the invention. Thus, by active immunization, a number of antibodies can be generated that have specificity for the same molecule but for different NCAM-180 binding sites.

The vaccine may also comprise glycosylated antibodies, in particular epitopes which can mimic carbohydrate epitopes of TAAs if the glycosylation itself is also capable. Such an antibody may mimic a cellular tumor antigen particularly well and, therefore, it results in the desired immune response inhibiting epithelial tumor cells.

According to the present invention, the vaccine can be used for active immunization, and therefore, it can be administered in only a small amount. No particular side effects, in particular no fever, no rise in glucose levels are therefore to be expected, even if the immunogenic active substances used according to the invention originate from non-human species, such as murine antibodies. However, it is assumed that recombinant, chimeric and humanized or human active substances which bind to murine and human components are particularly suitable for administration to humans. On the other hand, the murine part of the active substance, because it is foreign, can additionally stimulate an immune response in humans.

Although the vaccines used according to the invention may naturally comprise active substances derived from natural antibodies, which may be isolated from an organism or a patient, antibody derivatives preferably selected from antibody fragments, conjugates or homologues, and complexes and adsorbates are generally used. In any case, it is preferred that the antibody derivative comprises at least part of a Fab fragment, preferably together with at least part of a F (ab') 2-fragment, and/or part of a hinge region and/or an Fc-portion of a lambda or kappa antibody. In addition, as defined in the present invention, single chain antibody derivatives, such as so-called single chain antibodies, may also be used in the vaccine. Preferably, an immunoglobulin type antibody, such as IgG, IgM, or IgA, is used. It is particularly preferred to use IgG2 a-antibodies, since IgG2a antibodies exhibit particularly good complement activation, leading to an increase in vaccine immunogenicity. In addition, this has the advantage of further reducing the content of antibodies in the vaccine.

According to the present invention, it is preferred to also use a vaccine comprising an antibody or antibody derivative (i.e. abl) against a tumor-associated antigen. The specificity of the antibody is preferably selected from the group of TAAs mentioned above, in particular from the group NCAM-180 or Exon18_ MUM.

It is particularly preferred that the vaccine used comprises an antibody that specifically binds to the antibody. Thus, the tumor vaccine comprises anti-idiotype antibodies (i.e. ab2), in particular for active immunization. According to the invention, the anti-idiotype antibody used preferably again recognizes the idiotype of the antibody directed against TAA. Thus, TAA epitopes have been formed in the paratope of anti-idiotypic antibodies as TAA mimetics. Here, the preferred choices are also from the group of TAAs indicated above.

The vaccines used according to the invention are advantageously provided in suitable formulations. Preferably these formulations have a pharmaceutically acceptable carrier. This includes, for example, auxiliary substances, buffers, salts, preservatives. The vaccine may, for example, be used for the prevention and treatment of cancer-related conditions such as metastasis formation and minimal residual disease in cancer patients. In this case, the antigen presenting cells are specifically regulated in vivo or ex vivo to specifically generate an immune response against TAAs and tumor cells.

It is therefore also an object of the present invention, in the case of active immunization, to provide vaccine formulations comprising an immunogenically active substance, in most cases only in low concentrations, for example in an immunogenic amount ranging from 0.01[ mu ] g to 10 mg. Depending on the TAA, its epitope, mimotope, nature of the specific or anti-idiotypic antibody, on the use of the sequence or derivative of the foreign species, and also on the respective adjuvant or adjuvant used, a suitable immunogenic dose can be selected, for example ranging from 0.01[ mu ] g to 750[ mu ] g, preferably from 100[ mu ] g to 500[ mu ] g. However, the storage vaccine (which will be delivered to the organism over an extended period of time) may also contain much higher amounts of antibodies, for example from at least 1mg up to over 10 mg. The concentration depends on the amount of liquid or suspension vaccine administered. The vaccine is typically provided as a concentrated solution or suspension, respectively, in an easy to use syringe having a volume of 0.01 to 1ml, preferably 0.1 to 0.75 ml.

For passive immunization as described above, the invention further provides a vaccine formulation comprising TAA-specific antibodies (ranging from 1mg to 10gr, preferably from 10mg to 1 gr).

According to the present invention, the vaccine used is preferably presenting the immunogen or the antibody in a pharmaceutically acceptable carrier suitable for subcutaneous, intramuscular or intradermal or transdermal administration. An alternative mode of administration is vaccination by a mucosal route, for example by nasal or oral administration. If solids are used as adjuvants for vaccine formulations, for example, adsorbates or suspension mixtures of the antibody and adjuvant are administered. In particular embodiments, the vaccine is administered as a solution or liquid vaccine, respectively, in an aqueous solvent.

Preferably, one or more vaccine units of the tumor vaccine are provided in a suitable easy-to-use syringe. Since antibodies are relatively stable compared to TAAs, vaccines based on antibodies or derivatives thereof have the fundamental advantage that they can be marketed in an easy-to-use form as a stably stored solution or suspension. However, ingredients of preservatives (e.g., thimerosal or other preservatives having improved resistance) are not necessary, but may be provided in the formulation to maintain a longer shelf life at storage temperatures (from refrigerator temperatures up to room temperature). However, the vaccines used according to the invention may also be provided in frozen or lyophilized form, which may be thawed, or reconstituted, respectively, if desired.

In any case, it has proven suitable to increase the immunogenicity of the active substances used according to the invention by using adjuvants in vaccine formulations. Preferably, vaccine adjuvants suitable for this purpose are aluminium hydroxide (aluminium gel) or aluminium phosphate, and also growth factors, lymphokines, cytokines, for example IL-2, IL-12, GM-CSF, lambda interferon or complement factors, for example C3d, in addition to liposomal formulations, and formulations with additional antigens against which the immune system has developed a strong immune response, such as tetanus toxoid, bacterial toxins, such as pseudomonas exotoxin and derivatives of lipid a.

Adjuvants that allow the drug to be administered without any side effects are preferred. The term side effects includes, for example, increased glucose levels or fever; localized reddening or slight swelling at the site of application is not considered a specific side effect, but indicates that an immune response is proceeding. For the formulation of the vaccine, it is also possible to further use known methods for conjugating or denaturing vaccine components in order to further increase the immunogenicity of the active substance. Particular embodiments of the vaccine for use according to the invention further comprise vaccination antigens, in particular additional anti-idiotype antibodies, as well as mixtures of immunogenic antibodies with simultaneously administered antibodies of various kinds.

When combined with chemotherapy, the vaccine according to the invention is preferably used early in chemotherapy. Preferably, chemotherapy is initiated within 1 to 2 weeks. For practical reasons, vaccination simultaneously with or during chemotherapy is also preferred. The patient is already undergoing clinical treatment and other treatments are readily available. If immunotherapy is active on the first day of chemotherapy or on the first 2-3 days, the immune system may be activated at an early point in time, even before the body is adversely affected by chemotherapy. Indeed, chemotherapy does have side effects, such as weakened immune system, which makes patients increasingly susceptible to infection. For this reason, it is surprising that immunotherapy can be successfully used immediately before or during chemotherapy. Thus, it can be observed that the immune response after vaccination with tumor vaccine can be induced on the first day, several hours before the initial phase of chemotherapy, to the same extent as without chemotherapy treatment. In any case, the serum levels of immunoglobulins and inoculated antigen-specific antibodies continue to increase with and without chemotherapy. There is even evidence that the specific immune response is enhanced by chemotherapy.

The mode of administration of the vaccine according to the invention preferably comprises not only primary vaccinations within the scope of chemotherapy but also several booster vaccinations at specific time intervals (which may for practical reasons be equal to the intervals of chemotherapy). After chemotherapy, long-term immunotherapy of months and years is also a suitable approach. Preferably, both the initial vaccination and the subsequent booster vaccination are effected with the same vaccine. Combination with adjuvants or palliative chemotherapy is preferred. Combinations with monotherapy or combination therapy (polytherapy) are possible. Due to the different mechanisms of action, it is preferred that the vaccine is combined with a combination chemotherapy.

Preferred agents for chemotherapy are alkylating pharmaceutical agents. Thus, for example, agents comprising taxanes, anthracyclines or platinum are preferred. All conventional formulations for the treatment of various cancers can be combined according to the present invention. Chemotherapy agents are typically administered intravenously or orally. Administration of an oral form of a chemotherapeutic agent may also be administered together with an oral form of a vaccine according to the invention (as a combined preparation).

Vaccination as defined by the present invention may be performed for both therapeutic and prophylactic treatment in principle. Such prophylactic vaccination is particularly-although not exclusively-directed to patients diagnosed with cancer and patients with small (single resting cell, small metastases) metastases but who currently have no evidence of disease. In such cases, the vaccine is a prophylactic vaccine because it induces an immune response that may be beneficial against future outbreaks of cancer.

In a preferred embodiment, the vaccine used according to the invention comprises the NCAM-180 gene product or an antibody directed against this gene product, or a corresponding anti-idiotypic antibody, respectively. Methods of formulating vaccines suitable for the inventive method are known:

NCAM-180TAA, its derivatives, epitopes or mimetics can be obtained from natural and synthetic sources. The antibody component may also be chemically synthesized and then bound to the epitope structure, or it may be synthesized routinely. In the chemical synthesis of antibody carrier molecules, reactive groups may be introduced at specific sites to enable control over the extent of conjugation to the epitope and the type and location of binding.

-immunogenic NCAM-180TAA, epitopes, mimetics or antibodies thereof can also be prepared as recombinant molecules by genetic engineering methods. Suitable derivatives can be produced, for example, by altering the genetically engineered nucleic acid encoding the native molecule. Glycosylation of recombinant gene products and the corresponding tumor-associated polysaccharide structures can also be influenced by production in cells that have been genetically modified so that they will thus glycosylate proteins. Such cells may be natural isolates (cell clones) or may be found by appropriate screening for the desired glycosylation. However, the cells may also be modified so that they express the corresponding enzymes required for the desired glycosylation, in order to find the desired glycosylation, in particular on recombinant polypeptides or proteins (Glycoconj. J. (1999), 16: 81). However, it is also possible to enzymatically generate or alter, respectively, the glycosylation pattern of a protein (Clin. chem. Lab. Med. (1998), 36: 373).

According to a particular embodiment of the invention, the vaccine used according to the invention comprises a nucleic acid molecule which is a precursor of NCAM-180TAA, wherein the nucleic acid molecule encodes an NCAM-180 gene product as defined in the invention. The obtained DNA vaccine is administered as a protein-based tumor vaccine.

The invention also relates to kits suitable for prophylactic and/or therapeutic treatment of cancer-related conditions. The kit comprises a) a vaccine based on NCAM-180 tumor-associated antigen, epitopes, mimotopes, specific or anti-idiotypic antibodies, and optionally

b) An agent for chemotherapy.

The selection of the components of the kit according to the invention and their combination is carried out as described above. Preferably, the kit further comprises suitable application means, such as, for example, a syringe, an infusion device or the like. If the vaccine is provided in lyophilized form, the kit will further comprise a suitable reconstitution solution, optionally comprising a specific stabilizer or reconstitution accelerator. Further preferred kits comprise several units of the vaccine for use according to the invention, which will be used for the initial vaccination as well as one or more, preferably up to 3, booster vaccinations.

However, the number of booster inoculations may also be higher, for which a kit comprising several vaccine units alone, without being combined with a chemotherapeutic agent, is provided. Optionally, the frequency ranges from 1 to 12 times per year, particularly preferably, from 4 to 8 times per year. The dose may remain equal or may be reduced. The booster inoculation can be carried out at regular intervals, in principle for life. Suitable intervals range from about 6 to 24 months and can be determined by examining the titer of the induced CTL response (which should be boosted immediately once the titer of the induced CTL response has significantly decreased).

The present invention may be better understood by reference to the following experimental details, but those skilled in the art will readily appreciate that these are only exemplary illustrations of the invention described more fully in the claims that follow. In addition, throughout this application, a number of publications are referenced. These publications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention pertains.

Examples

The following examples illustrate the invention. Other embodiments will occur to those skilled in the art from consideration of the specification.

Experiment to study the differential expression of NCAM-180(Exon18_ MUM) in different cell lines

Differential expression of NCAM-180 was assessed in different cancer cell lines and healthy controls using methods known in the art. The method comprises the following steps:

RNA extraction and cDNA synthesis according to standard methods; and

PCR amplification to evaluate expression of Exon19_ MUM according to the principle described in figure 1.

Expression of NCAM Exon18_ MUM as part of NCAM-180 was found in cell cultures derived from neuroendocrine tumors (SH-SYSY and CCI), more particularly over-expression in Small Cell Lung Carcinoma (SCLC) cell lines (FIG. 2). The results for the other cell lines are summarized in table 1.

Table 1: differential expression of Exon18_ MUM (NCAM-180) in cancer cell lines and healthy controls. Symbol: high expression (+ +), normal expression (+/-), low expression (+/-), no expression (-/-).

Cloning and purification of NCAM Exon18_ MUM

NCAM Exon18_ MUM clone

Designing PCR primers. The complete Exon18_ MUM nucleotide sequence (816nt) (SEQ ID NO.1) and the nucleotide sequence of a truncated form of Exon18_ MUM (369nt) (SEQ ID NO.5) were obtained from cDNA of H69 cells. These cells showed overexpression of Exon18_ MUM. Both the forward and reverse primers contain restriction sites for subcloning into pRSET (protein production) and pCI (DNA immunization) vectors.

NCAM Exon18_ MUM encoding DNA, full-length and truncated Exon18_ MUM, were successfully cloned into:

●pRSET(Invitrogen), a construct for protein production, resulting in a His-tagged recombinant protein with the following protein sequence (SEQ ID No.2) in e.coli; (truncated version shown in italics-SEQ ID NO.6)

1 lpadttatve dmlpsvttvt tnsdtitetf ataqnsptse tttltssiap

51 patatpdsns vpagqatpsk gpsasapspa pasapkvapl vdlsdtptst

101 paasnlsssv lanqgavlsp sapagvgeas kappaskptp apvptptgaa

151 splaaaaapa teapqakqea pstkgpdpep tqpgaakspa eaatalaspk

201 seaasvsttn psggedfkmd egnfktpdid lakdvfaalg spapaagasg

251 qapelapsta dssvspapak t

●pCI(Promega), a construct for DNA immunization.

NCAM Exon18_ MUM protein production and purification

Bacteria were transformed with expression constructs encoding Exon18_ MUM (pRSET, Invitrogen). We used a pRSET construct encoding a truncated NCAM Exon18_ MUM protein (marked in italics in the NCAM Exon18_ MUM protein sequence described above) and a pRSET construct encoding the full-length NCAM Exon18_ MUM protein sequence. Transformation was performed according to the manufacturer's instructions. The 2 forms of protein were all expressed as His-tagged proteins and purified on Ni-chelated columns according to standard methods. His-tagged proteins were eluted from the column with 300mM imidazole, and the eluted protein solution was then renatured with standard dialysis against PBS. The purity of the NCAM Exon18_ MUM protein product is shown in fig. 3A and B.

Experiments to validate the immunogenic potential of NCAM-180 and the NCAM-180 gene product as candidate antigens

C. Immunization and immune response assays

NCAM Exon18_ MUM immunization induces an intracellular immune response

Based on differential expression PCR, NCAM Exon18_ MUM splice variants were identified as targets for lung cancer immunotherapy. As described above, for this novel antigen, DNA and recombinant proteins were prepared. Using the methods described above, truncated MUM proteins were produced in e.coli, purified and subsequently used for immunization of Balb/c mice.

C1.1 immunization with Exon18_ MUM protein and/or Exon18_ MUM DNA and

in vitro restimulation with overlapping 9-peptide libraries

In the first type of experiment, some mice were pre-sensitized, also referred to hereinafter as immunized, with a truncated Exon18_ MUM protein in PBS, with or without adjuvant (e.g., Abisco-100), and some were immunized with Exon18_ MUM encoding DNA. In addition, immunization with Exon18_ MUM protein was combined with Exon18_ MUM DNA immunization. On day 21, these different groups were boosted as follows:

description of the drawings:

the _Exon18_ MUM protein is referred to in the tables as the MUM protein.

Exon18_ MUM DNA is referred to in the tables as MUM DNA.

The adjuvant used was Abisco-100.

Immunization t-0 boost t-21 days

14 BALB/c female PBS

24 BALB/c female PCI empty vector

Abisco adjuvant 34 BALB/c female Abisco adjuvant

44 BALB/c female MUM protein in Abisco while MUM protein in Abisco

54 BALB/c female MUM protein w/o adjuvant

64 BALB/c female MUM DNA

74 BALB/c female MUM DNA MUM protein in Abisco

For protein immunization, we used:

● 10 μ g recombinant Exon18_ MUM protein in PBS, optionally formulated with 12 μ g Abisco-100.

For DNA immunization, we used:

● 2X 50. mu.g Exon18_ MUM DNA (in pCI vector) (50. mu.g injected into the tibialis anterior of each of 2 hind limbs)

Balb/c mice (n-4 per group) were immunized on day 0 and boosted on day 21. 30 days after the first immunization, the spleen of the mouse was isolated, and a mouse spleen cell suspension was prepared, and 1.510⁶Density seeding of cells/well. In vitro restimulation was performed at 37 ℃ for 4 hours. (5% CO)₂). Restimulation was performed with an overlapping 9-peptide library covering the full-length amino acid sequence of the truncated MUM protein. A total of 45 overlapping peptides were used. For all immunized mice, we are at 10⁵The number of cells containing intracellular IFN- γ was calculated from CD8 positive cells as determined by flow cytometry (FIG. 4 panels A-G). Our data show that there are a small number of CD8 positive T cells containing intracellular IFN- γ in animals immunized and boosted with Exon18_ MUM protein, representing a limited CTL response (fig. 4 panel D). Mice immunized with Exon18_ MUM protein dissolved in Abisco-100 adjuvant apparently did not induce CTL responses at all (fig. 4, panel C). For mice immunized with Exon18_ MUM DNA, we found that intramuscular injection of truncated Exon18_ MUM encoding DNA significantly induced a MUM-specific cytotoxic T cell response when compared to mice immunized with pCI vector alone (performed as a control immunization) (fig. 4 groups F and G). We count up to 10⁵727 cells containing intracellular IFN- γ were found in CD8 positive T cells (0.7%). Our data even suggested that there was a slight increase in the number of CD8 positive T cells containing intracellular IFN- γ (887 intracellular IFN- γ containing cells) in mice immunized with Exon18_ MUM DNA and boosted with Exon18_ MUM protein and Abisco-100Cell/10⁵CD8 positive T cells, 0.9%). In vitro restimulation with an Irrelevant Peptide Pool (IPP) was performed for all spleens as a negative control. Here, a peptide library covering a region of NCAM other than the Exon18_ MUM sequence (such as, for example, the NCAM Exon 7pi8 region of the NCAM protein) served as a control. In the present example, IPP consists of 17 overlapping peptides (9 peptides, 5 amino acids overlap) covering 76 amino acid sequences (NCAM exon 7pi8 region of the NCAM protein).

For animals immunized with recombinant Exon18_ MUM protein, and animals immunized with Exon18_ MUM DNA, no CD8 positive T cells containing intracellular IFN- γ were found upon restimulation with this IPP. Since T cells from immunized animals cannot be restimulated with IPP, we conclude that MUM immunization induces MUM-specific T cells.

Based on this experiment, we concluded that immunization with recombinant Exon18_ MUM protein and Exon18_ MUM encoding DNA induced a MUM-specific T cell response. Immunization with Exon18_ MUM protein resulted in only a limited number of CD8 positive T cells containing intracellular IFN- γ, whereas immunization with Exon18_ MUM encoding DNA induced a significant intracellular MUM-specific immune response.

Based on the results of these experiments, we concluded that:

● immunization with Exon18_ MUM DNA induced a significant MUM-specific cytotoxic T cell response.

● the truncated Exon18_ MUM protein alone was immunogenic and induced a MUM-specific CTL response as determined by restimulation with a MUM-related overlapping 9 peptide library.

C1.2 immunization with Exon18_ MUM protein and/or Exon18_ MUM DNA restimulation in vitro with overlapping 9-peptide library

In the next type of experiment, we used a single 9-peptide (9-peptide, 5 amino acid overlap) covering the truncated Exon18_ MUM protein sequence instead of the 9-peptide library as described above for in vitro restimulation. In vitro restimulation was performed with the 9 peptide alone to map the CTL epitope on the Exon18_ MUM protein sequence in detail. 11 animals, 1 negative control mouse and 2 groups of mice (5 animals per group) were used in each experiment, the 2 groups of mice were immunized on day 0 as shown below and boosted on day 21 as shown below:

group 1: 1 mouse, immunized and boosted with 100. mu.g of pCI empty vector

Group 2: 5 mice, immunized and boosted with 100. mu.g MUM DNA

Group 3: 5 mice, with 210 u g NCAM18MUM protein for immunization and enhancement.

We performed 9 experiments, 11 animals per experiment. 45 animals were immunized with truncated Exon18_ MUM protein, 45 animals were immunized with truncated Exon18_ MUM DNA, and 9 animals were immunized with pCI empty vector.

In each experiment, mice were sacrificed 12-14 days after the boost. Spleens were excised, spleen cell suspensions were prepared, seeded onto multi-well 96 plates, and restimulated in vitro with NCAM Exon18_ MUM9 peptide alone. In each experiment, 5 peptides were tested for in vitro restimulation, and the experiment was repeated 9 times for a total of 45 peptides tested. The establishment of this epitope map allowed us to locate the best CTL epitope in the truncated Exon18_ MUM protein.

In vitro restimulation was performed with a total of 45 MUM overlapping 9 peptides covering the AA sequence of the truncated Exon18_ MUM protein (table 2).

Table 2: NCAM Exon18_ MUM protein overlap 9 peptide sequence

23 PLVDLSDTP 46 TQPGAAKSP

24 LSDTPTSTP 47 AAKSPAEAA

25 PTSTPAASN 48 PAEAATALA

26 PAASNLSSS 49 ATALASPKS

27 NLSSSVLAN 50 ASPKSEAAS

28 SVLANQGAV 51 SEAASVSTT

29 NQGAVLSPS 52 SVSTTNPSQ

30 VLSPSAPAG 53 TNPSQGEDF

31 SAPAGVGEA 54 QGEDFKMDE

32 GVGEASKAP 55 FKMDEGNFK

33 ASKAPPASK 56 EGNFKTPDI

34 PPASKPTPA 57 KTPDIDLAK

35 KPTPAPVPT 58 IDLAKDVFA

36 APVPTPTGA 59 KDVFAALGS

37 TPTGAASPL 60 AALGSPAPA

38 AASPLAAAA 61 SPAPAAGAS

39 LAAAAAPAT 62 AAGASGQAP

40 AAPATEAPQ 63 SGQAPELAP

41 TEAPQAKQE 64 PELAPSTAD

42 QAKQEAPST 65 PSTADSSVS

43 EAPSTKGPD 66 DSSVSPAPA

44 TKGPDPEPT

45 DPEPTQPGA

For each polypeptide, restimulation was performed 5-fold. Cells were permeabilized, fixed and stained for intracellular IFN-. gamma.and surface expressed CD8 using fluorescently labeled antibodies. Each 10 was determined by FACS analysis⁵The number of cells containing intracellular IFN- γ among the individual CD8 positive cells.

The results are shown in FIG. 5.

We found that immunization with the truncated Exon18_ MUM protein induced MUM-specific cytotoxic T cells as shown by in vitro restimulation with peptides 26-27, 30-32, 40-42, 45-47 and 55-58 (fig. 5). Interestingly, these data show that some specific regions of the truncated MUM recombinant protein are potential CTL epitopes. Furthermore, these data demonstrate that even without Abisco-100, the truncated MUM protein is highly immunogenic as found in the first immunization experiment. In addition, mice were also immunized with NCAMExon18_ MUM plasmid DNA. As shown in fig. 6A, DNA immunization induced a high MUM-specific CTL response. In vitro restimulation with peptides 25-28, 30-32, 40-44, 45-47, 52-54, 62 and 65 clearly showed that MUM-specific cytotoxic T cells were induced upon immunization of animals with Exon18_ MUM DNA. Peptides 26-27, 30-32, 40-42, 45-47 and 65 were also found in DNA-immunized animals when compared to CTL epitopes from protein-immunized mice. These data indicate that cytotoxic T cells are induced in both DNA and protein immunized animals against some epitopes.

Next, CTL epitopes found in the immunization with Exon18_ MUM DNA and Exon18_ MUM protein were compared with those predicted by SYPHEITI software. H2-D against Balb/c mice using this software^d/dHaplotype, MHC haplotype, score prediction was performed for all 9 peptides. Scores were predicted for all 9 peptides used in vitro restimulation, and these scores were calculatedShown in fig. 6B. Some peptides predicted to be potential CTL epitopes (which means that they have the potential to re-stimulate in vitro cytotoxic T cells corresponding to in vivo induction) were confirmed in the mice of our experiment. Our data indicate that in vitro restimulation of lymphocytes isolated from mice immunized with Exon18_ MUM with any of peptides 31, 53 and 56 effectively resulted in restimulation of pre-sensitized CD8 positive T cells in these mice.

D.NCAM Exon18_MUM ELISA

NCAM Exon18_ MUM ELISA was performed as follows. ELISA plates were coated with His-tagged NCAM Exon18_ MUM protein (full length or truncated) and blocked with skim milk powder (2%). Serial dilutions of sera from mice immunized with NCAM Exon18_ MUM protein were added. MUM-specific antibodies induced on the basis of immunization specifically bound to Exon18_ MUM protein on the coated plates. The specificity of the antibody was confirmed by detecting the binding of the antibody to plates coated with His-tagged negative control protein (which was also produced in e. Bound antibodies were detected with horseradish peroxidase conjugated anti-mouse Ig antibody. The conjugated enzyme oxidizes 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) substrates. By H₂SO₄The reaction was terminated and the OD of the oxidation product was measured at 450nm according to standard methods.

NCAMExon18_ MUM immunization induces a humoral immune response

We measured the titer of antibodies in the sera of Balb/c mice immunized with NCAM Exon18_ MUM protein and/or Exon18_ MUM plasmid DNA. Animals immunized with the truncated Exon18_ MUM protein in PBS or Abisco-100 adjuvant all showed serum antibody titers specific for anti-MUM as shown in fig. 7 panels C and D (dark bars). No binding of serum antibodies was shown when ELISA was performed with plates coated with His-tagged negative control protein (which was also produced in e.coli) (fig. 7 panels C and D (grey bars)). These data indicate that no antibodies against histidine or antibodies against e. Immunization with the plasmid encoding NCAM Exon18_ MUM DNA did not induce serum antibody titers. Significant induction of anti-MUM antibodies was only found when DNA-immunized mice were boosted with formulated NCAMExon18_ MUM protein (fig. 7 groups F and G). Our data clearly show that immunization of mice with the truncated NCAM Exon18_ MUM protein induces a significant MUM-specific humoral immune response.

D2. Full-length Exon18_ MUM protein vs truncated Exon18_ MUM protein based on ELISA

Serum antibody titers were determined for mice immunized with the truncated Exon18_ MUM protein. Sera were obtained from a total of 45 animals immunized with the truncated Exon18_ MUM protein and 5 mice immunized with the empty pCI vector. The humoral immune response detected on ELISA plates (coated with full-length Exon18_ MUM and truncated Exon18_ MUM recombinant protein) was compared (fig. 8).

As shown in figure 8, no significant difference was found in the detection of MUM-specific antibodies in the serum of immunized animals if coated with full-length or truncated Exon18_ MUM protein. These data indicate that all epitopes exposed in truncated Exon18_ MUM are still available when coated with the intact Exon18_ MUM protein.

E. Production of anti-MUM monoclonal antibodies

Mice were immunized/boosted with truncated NCAM Exon18_ MUM protein according to standard methods. Spleen cells were fused with NS0 myeloma cells. The fused cells were inoculated into HAT selection medium according to standard methods. Viable clones were selected and subsequently screened for secreted antibodies on cells expressing NCAM Exon18_ MUM according to standard immunocytochemistry methods.

The obtained monoclonal antibodies were screened on NCAM Exon18_ MUM positive H69 and H82 cells. Finally, one clone was selected, which hybridoma clone produced a monoclonal antibody that displayed a specific NCAM-like staining in immunocytochemical staining on cell lines H69 and H82 expressing NCAM Exon18_ MUM. We used Alexa fluor-labeled goat antibody against mouse IgG as a secondary antibody, visualized by fluorescence microscopy (fig. 9). Further selection of clone (MUMI 21B2) was based on the absence of staining on NCAM Exon18_ MUM negative HCT-116 cells. In addition, the MUMI21B2 staining demonstrated proper localization at the cell-cell contact area, as shown by confocal imaging (FIG. 10). For these experiments, we used antibodies enriched by production in the MiniPerm bioreactor.

In summary, the anti-MUM monoclonal antibody (MUMI 21B2) displayed similar staining of NCAM on cells expressing NCAM Exon18_ MUM, but not on NCAM Exon18_ MUM negative HCT-116 cells.

Experiments to evaluate the immunogenic potential of NCAM180 and Exon18_ MUM as candidate antigens

Performing an animal experiment in which

1) We evaluated the immunogenic potential of cells expressing NCAM-180 protein or Exon18_ MUM protein as a whole cell vaccine for the treatment of NCAM-180 expressing tumors.

RVIK (H-2k non-MHC matched) cells expressing full-length NCAM-180 or Exon18_ MUM irradiated with 200Gy were vaccinated as whole cell vaccines to mice.

NCAM-180 expressing tumors were induced in mice by injection of stably transfected, antigen expressing Renca (H-2b MHC matched) cells. The protection and immune efficiency of cells expressing NCAM-180 and Exon18_ MUM as whole cell vaccines was evaluated based on tumor volume and mouse survival. CTL and antibody responses were determined in splenocytes (isolated at week 9) and blood samples (sampled at the beginning of the study and after weeks 3, 5 and 7), respectively.

2) We evaluated the immunogenic potential of a DNA construct encoding Exon18_ MUM (as a DNA vaccine for the treatment of NCAM-180 expressing tumors).

Mice were vaccinated with a DNA construct encoding Exon18_ MUM as a DNA vaccine. NCAM-180 expressing tumors were induced in mice by using stably transfected, antigen expressing Renca (H-2b MHC matched) cells. The protection/immunization efficiency of the DNA vaccine construct encoding Exon18_ MUM was evaluated based on tumor volume and mouse survival. CTL and antibody responses were determined in splenocytes (isolated at week 9) and blood samples (sampled at the beginning of the study and after weeks 3, 5 and 7), respectively.

3) We evaluated the immunogenic potential of Exon18_ MUM protein (as a protein vaccine for the treatment of NCAM-180 expressing tumors).

Mice were vaccinated with Exon18_ MUM protein as a protein vaccine. NCAM-180 expressing tumors were induced in mice by injection of stably transfected, antigen expressing Renca (H-2b MHC matched) cells. The protective/immune efficiency of Exon18_ MUM protein was evaluated based on tumor volume and mouse survival. CTL and antibody responses were determined in splenocytes (isolated at week 9) and blood samples (sampled at the beginning of the study and after weeks 3, 5 and 7), respectively.

Vaccines were summarized to evaluate:

● cells expressing NCAM180 and Exon18_ MUM are used as in vivo effects of whole cell vaccines.

● Exon18_ MUM DNA in vivo Effect as DNA vaccine

● Exon18_ MUM protein as in vivo effect of protein vaccine

This model emphasizes that NCAM180 protein or Exon18_ MUM protein are potential antigens for vaccine development. In addition, the immunogenic potential of Exon18_ MUM as a whole cell vaccine, DNA vaccine or protein-based vaccine was compared using this in vivo model.

For each group:

using 100. mu.l (10)⁶Individual cells (RVIK); 200Gy irradiated or 50. mu.g of DNA) or 100. mu.g of protein dissolved in 30. mu.l. Vaccination was performed 3 times at weekly intervals.

100. mu.l (10) expressing NCAM-180⁵Cells (Renca)) induced tumors. The immunization was performed after the last vaccination and only once.

-measuring tumor growth in horizontal and vertical direction.

Claims (14)

1. An isolated polypeptide selected from the group consisting of: consisting of SEQ ID NO: 2, SEQ ID NO: 6; or SEQ ID NO: 2 or SEQ ID NO: 6, wherein said fragment is capable of inducing a humoral or cellular immune response in a mammal and consists of an epitope selected from the group consisting of: PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

2. An isolated nucleic acid molecule encoding the isolated protein of claim 1.

3. A vector comprising a nucleic acid molecule according to claim 2.

4. Comprising a nucleic acid molecule according to claim 2; or the vector of claim 3.

5. An antibody immunospecific for the polypeptide of claim 1.

6. Comprising the protein of claim 1; or a nucleic acid molecule as claimed in claim 2.

7. A vaccine according to claim 6 for use as a medicament.

8. A vaccine according to claim 6 or 7 for use in the treatment or prevention of lung cancer, including SCLC and NSCLC.

Use of an NCAM-180 gene product for the preparation of a diagnostic agent for the in vitro diagnosis of cancer, wherein said NCAM-180 gene product is:

(a) corresponding to SEQ ID NO: 1, or a nucleic acid derived therefrom;

(b) corresponding to SEQ ID NO: 3, or a nucleic acid derived therefrom;

(c) comprises the amino acid sequence of SEQ ID NO: 2 or 4.

10. The use of claim 9, wherein the NCAM-180 gene product is a polypeptide having the sequence of seq id NO: 2. SEQ ID NO: 4 or SEQ ID NO: 6.

11. Immunologically specific binding to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: 6 for the preparation of a composition for the diagnosis of cancer in a subject, wherein said fragment is capable of inducing a humoral or cellular immune response in a mammal and consists of an epitope selected from the group consisting of: PAASNLSSSVLAN (AA 101-113 of SEQ ID NO: 2), VLSPSAPAGVG (AA 117-127 of SEQ ID NO: 2), LAAAAAPATEAPQ (AA 153-165 of SEQ ID NO: 2), KGPDPEPTQPGA (AA 174-185 of SEQ ID NO: 2) and DFKMDEGNFK (AA 216-225 of SEQ ID NO: 2).

12. A pharmaceutical composition comprising: (a) a purified NCAM-180 gene product in an amount effective to elicit an immune response, wherein the gene product is:

(i) a polypeptide as defined in claim 1 or (i i) a nucleic acid molecule as defined in claim 2;

and a pharmaceutically acceptable excipient.

13. A pharmaceutical composition comprising: (a) an antibody immunologically specific for the protein of claim 1; and (b) a pharmaceutically acceptable carrier.

14. Use of a pharmaceutical composition according to any one of claims 12 to 13 in the manufacture of a medicament for treating lung cancer in a subject.