JP2007525183A - Nucleic acids and corresponding proteins called 109P1D4 useful in the treatment and detection of cancer - Google Patents

Abstract

A novel gene 109P1D4 and its encoded protein, and variants thereof, have been described, wherein 109P1D4 exhibits tissue-specific expression in normal adult tissues and is abnormally expressed in the cancers listed in Table I The As a result, 109P1D4 provides a diagnostic, prognostic, prophylactic and / or therapeutic target for cancer. The 109P1D4 gene or a fragment thereof, or its encoded protein, or a variant thereof, or a fragment thereof can be used to elicit a humoral immune response or a cellular immune response; an antibody or T cell that is reactive with 109P1D4 is Can be used in active or passive immunity.

Description

(Statement of rights to inventions based on government-sponsored research)
N / A (Field of Invention)
The invention described herein relates to genes and proteins encoded thereby and variants thereof, referred to as 109P1D4 and expressed in certain cancers. The present invention also relates to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 109P1D4.

(Background of the Invention)
Cancer is the second leading cause of death in humans after coronary disease. Worldwide, millions of people die from cancer each year. In the United States alone, as reported by the American Cancer Society, cancer causes death of well over 500,000 people each year, and more than 1.2 million new cases are diagnosed each year. While deaths from heart disease have decreased significantly, deaths from cancer are generally on the rise. In the early part of the next century, cancer is predicted to be a major cause of death.

  Worldwide, several cancers are prominent as major killers. In particular, lung, prostate, breast, colon, pancreas, and ovarian carcinomas represent the leading cause of death from cancer. These and virtually all other carcinomas share common lethal characteristics. With very few exceptions, metastatic disease caused by carcinoma is fatal. Furthermore, even in the early stages of cancer patients who survived the primary cancer, a common experience has been shown that their lives have changed dramatically. Many cancer patients experience strong anxiety driven by recognizing the possibility of recurrence or treatment failure. Many cancer patients experience physical weakness after treatment. In addition, many cancer patients experience a recurrence.

  Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Scandinavia, prostate cancer is by far the most common cancer in men and the second leading cause of cancer death in men. In the United States alone, well after pulmonary cancer, more than 30,000 men die each year from this disease. Despite the magnitude of these numbers, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these procedures are ineffective for many and often have undesirable consequences.

  The lack of prostate tumor markers that can accurately detect early stage local tumors at the diagnostic site leaves significant limitations in the diagnosis and management of the disease. Serum prostate specific antigen (PSA) assay is a very useful tool, but its specificity and general utility are widely considered lacking some significant points.

  Advances in identifying additional specific markers for prostate cancer have been improved by the generation of prostate cancer xenografts that can reproduce different stages of the disease in mice. LAPC (Los Angels Prostate Cancer) xenografts have been shown to be capable of surviving passage in severe combined immunodeficiency (SCID) mice and mimicking the transition from androgen-dependent to androgen-independent. It is a graft (Klein et al., 1997, Nat. Med. 3: 402). More recently identified prostate cancer markers include: PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res, September 2, 1996 (9): 1445-51), STEAP (Hubert et al., Proc Natl Acad Sci USA. December 7, 1999; 96 (25): 14523-8) and prostate stem cell antigen ( (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

  Previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, but to further improve diagnosis and therapy, prostate cancer and related cancers There is a need to identify additional markers and therapeutic targets for.

  Renal cell carcinoma (RCC) accounts for about 3% of adult malignancies. Once the adenoma reaches a diameter of 2-3 cm, there is potential for malignancy. In adults, the two main malignant kidney tumors are adenomas of kidney cells and transitional cell carcinomas of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated to exceed 29,000 cases in the United States, and in 1998, more than 11,600 patients died from the disease. Transitional cell carcinoma is less frequent, with an incidence of about 500 cases per year in the United States.

  Surgery has been the main treatment for renal cell adenocarcinoma for decades. Until recently, metastatic disease was refractory to any systemic treatment. With the recent development of systemic treatment (especially immunotherapy), metastatic renal cell carcinoma can be aggressively approached in appropriate patients with the potential for an acceptable response. Nevertheless, there remains a need for effective treatments for these patients.

  Of all new cases of cancer in the United States, bladder cancer represents approximately 5% (5th of the most common neoplasms) in men and 3% (8th of the most common neoplasms) in women . The number of outbreaks increases slowly, consistent with an increase in the elderly population. In 1998, there were an estimated 54,500 cases, including 39,500 cases in men and 15,000 cases in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 for women. The 3: 1 historical male / female ratio may decrease in relation to smoking patterns in women. In 1998, an estimated 11,000 people died of bladder cancer (7,800 men and 3,900 women). Bladder cancer incidence and mortality increase greatly with age, and the problems increase as the population ages.

  Most bladder cancers recur in the bladder. Bladder cancer is managed using a combination of transurethral resection (TUR) of the bladder and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points to the limitations of TUR. Most muscle invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion are the most effective means to remove cancer, but possess undeniable effects on urine and sexual function. There continues to be a significant need for treatment modalities that are beneficial to patients with bladder cancer.

  In the United States in 2000, an estimated 130,200 colorectal cancers occurred, including 93,800 colon cancers and 36,400 rectal cancers. Colorectal cancer is the third most common cancer in men and women. Incidence decreased significantly between 1992 and 1996 (−2.1% per year). Research suggests that these reductions are due to increased screening and polyp removal, which prevents polyps from progressing to invasive cancers. In 2000, an estimated 56,300 people died (47,700 from colon cancer and 8,600 from rectal cancer), accounting for approximately 11% of all cancer deaths in the United States.

  Currently, surgery is the most common form of treatment for colorectal cancer and cancer that has not spread, which is often curative. Chemotherapy or radiation in addition to chemotherapy is provided to most patients whose cancer has penetrated deep into the intestinal wall or spread to lymph nodes before or after surgery. Permanent colostomy (formation of the abdominal opening to eliminate bodily excretion) is sometimes required for colon cancer and is often required for rectal cancer. There remains a need for effective diagnostic and treatment modalities for colorectal cancer.

  In 2000, there were an estimated 164,100 new cases of lung and bronchial cancer, accounting for 14% of all US cancer diagnoses. The incidence of lung and bronchial cancer has decreased significantly in men, from as high as 86.5 out of 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to decline. In 1996, the incidence in women was 42.3 out of 100,000.

  Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. Between 1992 and 1996, mortality from lung cancer decreased significantly among men (-1.7% per year), while mortality for women still increased significantly (1 0.9% per year). Since 1987, more women die each year from lung cancer than breast cancer (breast cancer has been the leading cause of cancer death in women for more than 40 years). The decrease in lung cancer incidence and mortality is most likely due to a decrease in smoking rates over the past 30 years; however, the decrease in smoking patterns among women is behind the decrease in men. While the decline in tobacco use in adults has slowed, it is significant that tobacco use in youth has increased again.

  Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy and chemotherapy. For many localized cancers, surgery is usually the preferred treatment. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often required in combination with surgery. Chemotherapy alone or in combination with radiation therapy is a selective treatment for small cell lung cancer; with this regimen, a large percentage of patients experience remission, which in some cases lasts long. However, there remains a need for effective treatment and diagnostic approaches for lung and bronchial cancer.

  An estimated 182,800 new invasive cases of breast cancer were predicted to occur among US women during 2000. In addition, approximately 1,400 new cases of breast cancer were predicted to be diagnosed in men in 2000. After an increase of about 4% per year in the 1980s, breast cancer incidence in women peaked at about 110.6 cases per 100,000 in the 1990s.

  In the United States alone, an estimated 41,200 people died in 2000 due to breast cancer (40,800 women and 400 men). Breast cancer is the second leading cause of cancer death among women. According to the closest data, mortality was significantly decreased between 1992 and 1996, with the greatest decrease among both white and black young women. These reductions were probably the result of earlier detection and treatment improvements.

  Taking into account the medical environment and patient preferences, breast cancer treatment may include: tumor removal (local removal of the tumor) and removal of armpit lymph nodes; mastectomy (surgical removal of the breast) And removal of armpit lymph nodes; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that for early-stage disease, long-term survival after radiation therapy in addition to tumor resection is similar to survival after modified radical mastectomy. Significant advances in reconstruction techniques offer several options for breast reconstruction after mastectomy. In recent years, such reconstruction has been performed simultaneously with mastectomy.

  Local resection of in situ ductal carcinoma (DCIS) with an appropriate amount of surrounding normal breast tissue may prevent local recurrence of DCIS. Chest radiation and / or tamoxifen can reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS can develop into invasive breast cancer if left untreated. Nevertheless, these treatments have serious side effects or sequelae. Therefore, there is a need for effective breast cancer treatment.

  There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. Ovarian cancer accounts for 4% of all cancers in women and is ranked second in gynecological cancers. Between 1992 and 1996, the incidence of ovarian cancer decreased significantly. As a result of ovarian cancer, an estimated 14,000 people died in 2000. Ovarian cancer causes more deaths than any other cancer in the female reproductive system.

  Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgical procedures typically include excision of one or both of the ovaries, Fallopian tube excision (fallopian ovariectomy), and uterine excision (hysterectomy). In some very early tumors, only the involved ovaries are removed, especially in young women who want to have children. In advanced disease, attempts are made to enhance the effectiveness of chemotherapy by removing all diseases in the abdominal cavity. There continues to be a significant need for effective treatment options for ovarian cancer.

  There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, the proportion of pancreatic cancer has decreased in men. The proportion among women remains roughly constant, but may begin to decrease. Pancreatic cancer caused an estimated 28,200 deaths in the United States in 2000. Over the past 20 years, mortality in men has been modestly decreased (significantly -0.9% per year), while that rate has increased slightly in women.

  Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can prolong survival and / or alleviate the symptoms in many patients, but do not appear to produce cure for most people. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

(Summary of the Invention)
The present invention relates to the gene designated 109P1D4, which has now been found to be overexpressed in the cancers listed in Table I. Northern blot expression analysis of 109P1D4 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide sequence (FIG. 2) and amino acid sequence (FIGS. 2 and 3) of 109P1D4 are provided. The tissue-related profile of 109P1D4 in normal adult tissues, combined with the overexpression observed in the tissues listed in Table 1, is that 109P1D4 is abnormally overexpressed in at least some cancers and is therefore listed in Table I It serves as a useful diagnostic, prophylactic, prognostic and / or therapeutic target for cancers of tissues such as the treated tissue.

  The present invention provides the following: polynucleotides corresponding to or complementary to all or part of the 109P1D4 gene, mRNA and / or coding sequence (preferably in isolated form), The following may be mentioned: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or Polynucleotides encoding 109P1D4-related proteins and 109P1D4-related fragments of more than 25 consecutive amino acids); at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 109P1D4-related protein of 100 or 100 consecutive amino acids, as well as the peptide / protein itself; 109P DNA, RNA, DNA / RNA hybrids, and related molecules, polynucleotides or oligonucleotides that are complementary or have at least 90% homology to the gene sequence or mRNA sequence of D4 or a portion thereof, and A polynucleotide or oligonucleotide that hybridizes to a 109P1D4 gene, mRNA or 109P1D4 encoding polynucleotide. Also provided are means for isolating cDNA and genes encoding 109P1D4. Also provided are recombinant DNA molecules comprising 109P1D4 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for expression of 109P1D4 gene products. The present invention further provides antibodies that bind to 109P1D4 proteins and polypeptide fragments thereof, which include polyclonal and monoclonal antibodies, mouse and other mammalian antibodies, chimeric antibodies, humanized antibodies and fully Human antibodies as well as antibodies labeled with a detectable marker or therapeutic agent are included. In certain embodiments, however, the entire nucleic acid sequence of FIG. 2 is not encoded and / or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and / or the entire amino acid sequence of FIG. 2 is prepared, any of which is a respective human unit dosage form.

  The invention further provides methods for detecting the presence and status of 109P1D4 polynucleotides and proteins in various biological samples, and methods for identifying cells that express 109P1D4. Exemplary embodiments of the present invention provide a method for monitoring 109P1D4 gene product in tissue or hematological samples that have or are suspected of having some form of growth dysregulation such as cancer To do.

  The present invention relates to various immunogenic or therapeutic compositions and strategies for treating 109P1D4 expressing cancers (eg, cancers of tissues listed in Table I) (109P1D4 transcription, translation, processing or Further provided are cancer vaccines, including treatments aimed at inhibiting function. In one aspect, the present invention provides compositions for treating cancers that express 109P1D4 in human subjects, as well as methods comprising them, wherein the composition comprises a suitable carrier for human use and A human unit dose of one or more agents that inhibit 109P1D4 production or function is included. Preferably, this carrier is a unique human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with the 109P1D4 protein. Non-limiting examples of such moieties include antibodies (eg, single chain, monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody or human antibody), those (naturally occurring or synthesized) Functional equivalents thereof, as well as combinations thereof, but are not limited thereto. These antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

  In another aspect, the agent binds to an HLA class I molecule in humans and induces a CTL response against 109P1D4. One or more peptides comprising a cytotoxic T lymphocyte (CTL) epitope and / or an HLA class in humans It includes one or more peptides comprising a helper T lymphocyte (HTL) epitope that binds to a II molecule and elicits an HTL response. The peptides of the invention can be the same or one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more nucleic acid molecules that express one or more of the peptides that stimulate a CTL or HTL response as described above. In yet another aspect of the invention, the one or more nucleic acid molecules can express a moiety that immunologically reacts with 109P1D4 as described above. The one or more nucleic acid molecules can also be or encode a molecule that inhibits the production of 109P1D4. Non-limiting examples of such molecules include molecules complementary to a nucleotide sequence essential for the production of 109P1D4 (eg, molecules that form a triple helix having an antisense sequence or a nucleotide double helix essential for 109P1D4 production). Or ribozymes effective to dissolve 109P1D4 mRNA.

  Determine the starting position of any peptide shown in Tables VIII-XXI and XXII-XLIX (collectively referred to as HLA Peptide Tables) with respect to the parent protein (eg, variant 1, variant 2, etc.) Note that for reference, the following three factors: specific variants, peptide lengths in the HLA peptide table, and search peptides in Table VII. In general, a unique search peptide is used to obtain a specific HLA peptide for a specific variant. The position of each search peptide relative to its respective parent molecule is listed in Table VII. Thus, if the search peptide starts at position “X”, the value “X-1” is assigned to each position in Tables VIII-XXI and XXII-XLIX to obtain the actual position of the HLA peptide in these parent molecules. Must be added. For example, if a particular search peptide starts at position 150 of its parent molecule, 150-1 or 149 must be added to each HLA peptide amino acid position to calculate the amino acid position in the parent molecule.

  One embodiment of the invention encompasses HLA peptides or oligonucleotides encoding HLA peptides that collectively appear at least twice in Table VIII to Table XXI and Table XXII to Table XLIX. Another embodiment of the invention encompasses an HLA peptide that appears at least once in Table VIII to Table XXI and at least once in Table XXII to Table XLIX, or an oligonucleotide encoding the HLA peptide.

Another embodiment of the invention is an antibody epitope comprising a peptide region having one, two, three, four, or five of the following features, or an oligonucleotide encoding the peptide region:
i) A peptide region of at least 5 amino acids of the particular peptide of FIG. 3 (in any integer increment up to the full length of the protein in FIG. 3) (in the hydrophilic profile of FIG. 5, 0.5, 0.6, 0. Including amino acid positions having a value greater than or equal to 7, 0.8, 0.9 or equal to 1.0);
ii) A peptide region of at least 5 amino acids of the particular peptide of FIG. 3 (in any integer increment up to the full length of the protein in FIG. 3) (in the hydropathic profile of FIG. 6, 0.5, 0.4, 0. Including amino acid positions having values less than or equal to 3, 0.2, 0.1 or equal to 0.0);
iii) a peptide region of at least 5 amino acids of the particular peptide of FIG. 3 (in any integer increment up to the full length of the protein in FIG. 3) (in the Percent Accessible Residues profile of FIG. Including amino acid positions having values greater than or equal to 5, 0.6, 0.7, 0.8, 0.9 or equal to 1.0);
iv) A peptide region of at least 5 amino acids of the particular peptide of FIG. 3 (in any integer increment up to the full length of the protein in FIG. 3) (in the Average Flexibility profile of FIG. 8, 0.5, Including amino acid positions having a value greater than or equal to 0.6, 0.7, 0.8, 0.9 or equal to 1.0); or
v) A peptide region of at least 5 amino acids of the particular peptide of FIG. 3 (in any integer increment up to the full length of the protein in FIG. 3) (in the β-turn profile of FIG. 9, 0.5, 0.6, 0. Including amino acid positions having a value greater than 7, 0.8, 0.9 or equal to 1.0).

(Detailed description of the invention)
(Summary of section)
I. ) Definition II. ) 109P1D4 polynucleotide II. A. ) Use of 109P1D4 polynucleotides II. A. 1. ) Monitoring genetic abnormalities II. A. 2. ) Antisense Embodiments II. A. 3. ) Primers and primer pairs II. A. 4). ) Isolation of 109P1D4-encoding nucleic acid molecule II. A. 5). ) Recombinant nucleic acid molecules and host-vector systems III. ) 109P1D4-related protein III. A. ) Embodiment of motif-bearing protein III. B. ) Expression of 109P1D4-related protein III. C. ) Modification of 109P1D4-related protein III. D. ) Use of 109P1D4-related proteins IV. ) 109P1D4 antibody ) 109P1D4 cell immune response VI. ) 109P1D4 transgenic animal VII. ) 109P1D4 detection method VIII. ) Methods for monitoring the status of 109P1D4-related genes and their products IX. ) Identification of molecules that interact with 109P1D4. ) Treatment methods and compositions X. A. ) Anti-cancer vaccine X. B. ) 109P1D4 as a target for antibody-based therapy
X. C. ) 109P1D4 as a target for cellular immune response
X. C. 1. Minigene vaccine X. C. 2. Combination of helper peptide and CTL peptide X. C. 3. Combination of T cell priming agent and CTL peptide X. C. 4). Vaccine composition comprising DC pulsed with CTL peptide and / or HTL peptide X. D. ) Adoptive immunotherapy X. E. ) Administration of vaccines for therapeutic or prophylactic purposes XI. ) 109P1D4 diagnostic and prognostic embodiments XII. ) Inhibition of 109P1D4 protein function XII. A. ) Inhibition of 109P1D4 using intracellular antibodies XII. B. ) Inhibition of 109P1D4 using recombinant protein XII. C. ) Inhibition of transcription or translation of 109P1D4 XII. D. ) General considerations for therapeutic strategies XIII. ) Identification, characterization and use of 109P1D4 modulators XIV. ) Kit / manufactured article.

(I.) Definition)
Unless defined otherwise, all technical terms, notations, and other scientific terms or terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Is intended. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or quick reference, and inclusion of such definitions herein. Should not be construed as representing substantial differences over the general understanding in the art. Many techniques and procedures described or referenced herein are generally described by conventional methodologies (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbour, Fully understood and commonly used by those skilled in the art using the widely used molecular cloning methodologies described in N.Y. Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer, unless otherwise noted.

  The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancer that has spread through the prostate capsule and the American Urological Association Including stage C disease under the (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N + disease under the TNM (tumor, node, metastasis) system Is meant. In general, surgery is not recommended for patients with locally advanced disease, and these patients are treated with patients with prostate cancer that are clinically localized (organ limited) In comparison, it has substantially less favorable results. Locally advanced disease is clinically identified by clear evidence of sclerosis beyond the lateral edges of the prostate, or asymmetry or sclerosis on the prostate base. Locally advanced prostate cancer is now physiological after a thorough prostatectomy if the tumor penetrates or penetrates the prostate capsule, extends to the surgical boundary, or enters the seminal vesicle. Diagnosed.

  “Modifying the native glycosylation pattern” is, for purposes herein, removing one or more carbohydrate moieties found in the native sequence 109P1D4 (removing an endogenous glycosylation site). Meaning or by removing glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites not present in the native sequence 109P1D4 Intended to be. In addition, this phrase involves qualitative changes in glycosylation of the native protein and relates to changes in the nature and proportion of the presence of various carbohydrate moieties.

  The term “analog” refers to a molecule that is structurally similar or that shares similar or consistent properties with another molecule (eg, 109P1D4-related protein). For example, an analog of 109P1D4 protein can be specifically bound by an antibody or T cell that specifically binds to 109P1D4.

  The term “antibody” is used in the broadest sense. Thus, an “antibody” can be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma technology. Anti-109P1D4 antibodies include monoclonal and polyclonal antibodies as well as fragments that contain an antigen binding domain and / or one or more complementary determining regions of these antibodies.

  An “antibody fragment” is defined as at least a portion of a variable region of an immunoglobulin molecule that binds to a target (ie, an antigen binding region). In one embodiment, it specifically comprises a single anti-109P1D4 antibody and clones thereof (including agonists, antagonists and neutralizing antibodies) and an anti-109P1D4 antibody composition having polyepitopic specificity. Is included.

  The term “codon optimized sequence” refers to a nucleotide sequence that is optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences optimized for expression in a given host species by codon optimization plus removal of pseudopolyadenylation sequences, exon / intron splicing signals, removal of transposon-like repeats and / or optimization of GC content , Referred to herein as “expression enhancing sequences”.

  A “combinatorial library” is a collection of diverse chemical compounds generated either by chemical synthesis or biosynthesis by combining a number of chemical “basic units” such as reagents. For example, linear combinatorial chemical libraries (eg, polypeptide (eg, mutein) libraries) are amino acids in all possible ways for a given compound length (ie, number of amino acids in a polypeptide compound). It is formed by combining chemical basic units called A number of chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37 (9): 1233-1251 (1994)).

  The preparation and screening of combinatorial libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (eg, US Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37: 487-493). (1991), Houghton et al., Nature, 354: 84-88 (1991)), peptoids (PCT publication number WO 91/19735), coding peptides (PCT publication WO 93/20242), random bio-oligomers (PCT). Published WO 92/00091), benzodiazepines (US Pat. No. 5,288,514), diversomers (eg, hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. S). ci.USA90: 6909-6913 (1993)), vinyllogous polypeptide (Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), non-peptidal peptide with β-D-glucose backbone Mimics (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-9218 (1992)), organic synthesis of similar small compound libraries (Chen et al., J. Amer. Chem. Soc. 116: 2661 (1994) )), Oligocarbonates (Cho et al., Science 261: 11303 (1993)) and / or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59: 658 (1994)), generally Gordon et al., J. Med. Chem. 37: 1385 (1994), nucleic acid libraries (see, eg, Stratagene, Corp.), peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083) Antibody libraries (see, eg, Vaughn et al., Nature Biotechnology 14 (3): 309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science 274: 1520-1522). (1996) and US Pat. No. 5,593,853) and small organic libraries (eg, benzodiazepines, Baum, C & EN, January 18, p. 33 (1993); isoprenoids, US patents). No. 5,56 No. 9,588; thiazolidinone and metathiazanone, US Pat. No. 5,549,974; pyrrolidine, US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506, 337; benzodiazepines, US Pat. No. 5,288,514;

  Devices for the preparation of combinatorial libraries are commercially available (eg, 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, Applied Biosystems 50; , Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for liquid phase chemistry. These systems include automated workstations (eg, automated synthesizers developed by Takeda Chemical Industries, LTD. (Osaka, Japan)) and many robotic systems that utilize robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass .; Orca, Hewlett-Packard, Palo Alto, Calif.) (Which mimics manual synthesis operations performed by chemists)). Any of the above devices are suitable for use with the present invention. Those skilled in the art will understand the nature and implementation modifications (if any) to these devices so that they can operate as described herein. In addition, numerous combinatorial libraries are commercially available per se (eg, ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).

The term “cytotoxic agent” refers to a substance that inhibits or prevents cell expression activity, cell function and / or causes destruction of cells. The term includes radioactive isotope chemotherapeutic agents and toxins (eg, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof). Is intended. Examples of cytotoxic agents include, but are not limited to: auristatin, auromycin, maytansinoid, yttrium, bismuth, lysine, ricin A chain, comble Statins (dubratin), duocarmycin, dorostatin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposid, vincristine, colblastine, colblastine, colblastine, colblastine Diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, Ab , Abrin A chain, modeccin A chain, α-sarcin, gelonin, mitellolin, retstrectocin, phenomycin, enomycin, curicin, crotin ), Calicheamicin, Saponiaria officinalis inhibitors, and glucocorticoids, as well as other chemotherapeutic agents, and radioisotopes (eg At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or Bi ²¹³ , P ³² and Lu (including Lu ¹⁷⁷ )). The antibody can also be conjugated to an anti-cancer prodrug activating enzyme that can convert the prodrug to an active form.

  A “gene product” is sometimes referred to herein as a protein or mRNA. For example, “gene products of the invention” are listed herein as “cancer amino acid sequences”, “cancer proteins”, “cancer proteins listed in Table I”, “cancer mRNA”, “Table I”. It may also be referred to as “cancer mRNA”. In one embodiment, the oncoprotein is encoded by the nucleic acid of FIG. The oncoprotein can be a fragment or can be a full-length protein for the fragment encoded by the nucleic acid of FIG. In one embodiment, cancer amino acid sequences are used to determine sequence identity or sequence similarity. In another embodiment, the sequence is a naturally occurring allelic variant of the protein encoded by the nucleic acid of FIG. In another embodiment, the sequence is a sequence variant as further described herein.

  “High-throughput screening” assays for the presence, absence, quantification, or other properties of a particular nucleic acid or protein product are well known to those skilled in the art. Similarly, binding assays and reporter gene assays are well known. Thus, US Pat. No. 5,559,410 discloses a high-throughput screening method for proteins; US Pat. No. 5,585,639 discloses a high-throughput screening method for nucleic acid binding (ie, in an array). While US Pat. Nos. 5,576,220 and 5,541,061 disclose high-throughput screening methods for ligand / antibody binding.

  In addition, high-throughput screening systems are commercially available (eg, Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Institute, Inc. Inc., Natick, MA; These systems typically include pipetting all samples and reagents, formulating liquids, scheduled incubations and finally reading the microplate with a detector appropriate for the assay. Automate the whole procedure. These configurable systems provide high throughput and rapid start-up as well as a high degree of adaptability and customization. The manufacture of such systems provides detailed protocols for various high throughput systems. Thus, for example, Zymark Corp. Provides a technical presentation describing screening systems for detecting modulation of gene transcription, ligand binding, and the like.

  The term “homologue” refers to a molecule that exhibits homology to another molecule (eg, a molecule having a sequence of chemical residues that are similar or similar at corresponding positions).

  “Human leukocyte antigen” or “HLA” is a human class I or class II major histocompatibility complex (MHC) protein (see, for example, Stites et al., IMMUNOLOGY, 8th edition, Lange Publishing, Los Altos, (See CA (1994)).

  As used in the context of polynucleotides, the terms “hybridize”, “hybridizing”, “hybridize” and the like refer to conventional hybridization conditions (preferably, for example, 50% formamide / 6 × SSC / 0.1% SDS / hybridization in 100 μg / ml ssDNA). Here, the temperature of hybridization is above 37 ° C. and the temperature for washing in 0.1 × SSC / 0.1% SDS is higher than 55 ° C.

  The phrase “isolated” or “biologically pure” refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Thus, an isolated peptide according to the present invention preferably does not contain substances normally associated with the peptide in an in situ environment. For example, if the polynucleotide corresponds to or is complementary to a gene other than the 109P1D4 gene, or is substantially separated from a contaminant polynucleotide encoding a polypeptide other than the 109P1D4 gene product or fragment thereof, Said to be “isolated”. One skilled in the art can readily utilize nucleic acid isolation procedures to obtain isolated 109P1D4 polynucleotides. A protein is said to be “isolated” if, for example, physical, mechanical or chemical methods are utilized to remove 109P1D4 protein from cellular components normally associated with the protein. One skilled in the art can readily utilize standard purification methods to obtain isolated 109P1D4 protein. Alternatively, isolated protein can be prepared by chemical means.

  The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

  The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancer that has spread to local lymph nodes or distant sites, and stage D disease under the AUA system, and TNM It is meant to include the stage T × N × M + under the system. In the case of locally advanced prostate cancer, surgery is generally not intended for patients with metastatic disease and hormonal (androgen excision) treatment is preferred Treatment style. Patients with metastatic prostate cancer eventually develop an androgen therapy refractory condition within 12-18 months of the start of treatment. Nearly half of these androgen refractory patients die within 6 months after the onset of the condition. The most common site of prostate cancer metastasis is the bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (ie, resulting in net bone formation). Bone metastases are most frequently found in the spine, followed by the femur, pelvis, rib cage, skull, and humerus. Other common sites of metastasis include lymph nodes, lungs, liver, and brain. Metastatic prostate cancer is typically diagnosed by open pelvic lymphadenectomy or laparoscopic pelvic lymphadenectomy, whole body radionuclide scan, skeletal radiography, and / or biopsy of bone lesions Is done.

  As used herein, the terms “modulator” or “test compound” or “drug candidate” or their synonyms are used to describe a cancer phenotype or expression of a cancer sequence (eg, a nucleic acid sequence or protein). Sequence), or any molecule (eg, protein, oligopeptide, small organic molecule) that is tested for the ability to directly or indirectly alter the effects of cancer sequences (eg, signal transduction, gene expression, protein interactions, etc.) , Polysaccharides, polynucleotides, etc.). In one aspect, the modulator neutralizes the effects of the oncoprotein of the present invention. “Neutralize” means that the activity of the protein is inhibited or blocked, with a lasting effect on the cell. In another aspect, the modulator neutralizes the effects of the gene of the invention and its corresponding protein by normalizing the protein level. In preferred embodiments, the modulator alters the expression profile, or the expression profile of a nucleic acid or protein provided herein, or a downstream effector pathway. In one embodiment, the modulator suppresses a cancer phenotype (eg, a phenotype for a normal tissue fingerprint). In another embodiment, the modulator induces a cancer phenotype. In general, multiple assay mixtures are run in parallel at different reagent concentrations, resulting in different responses to different concentrations. Typically, one of these concentrations serves as a negative control (ie, zero concentration or below detection level).

  Modulators, drug candidates, or test compounds comprise many chemical classes, but typically they are organic molecules, preferably small organic compounds having a molecular weight greater than 100 and less than about 2,500 daltons. is there. Preferred small molecules are less than 2000D, or less than 1500D or less than 1000D or less than 500D. Candidate factors include functional groups necessary for structural interaction (particularly hydrogen bonding) with proteins, and typically include at least one amine group, carbonyl group, hydroxyl group, or carboxyl group, preferably Contains at least two functional chemical groups. Candidate factors frequently include ring carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also include biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Peptides are particularly preferred. One class of modulator is a peptide, for example a peptide of about 5 to about 35 amino acids, preferably about 5 to about 20 amino acids, particularly preferably about 7 to about 15 amino acids. Preferably, the cancer modulator protein is soluble, includes a non-transmembrane region, and / or has an N-terminal Cys that aids solubility. In one embodiment, the C-terminus of the fragment is maintained as a free acid and the N-terminus is a free amine to aid in coupling (ie, coupling to cysteine). In one embodiment, the oncoproteins of the invention are conjugated to immunogenic factors as discussed herein. In one embodiment, the oncoprotein is bound to BSA. Polypeptides of the invention (eg, preferred length polypeptides) can be linked to each other or linked to other amino acids to produce longer peptides / proteins. The modulator peptide can be a digested product of a naturally occurring protein, a random peptide, or a “biased” random peptide, as outlined above. In a preferred embodiment, the peptide / protein based modulator is an antibody and fragments thereof as defined herein.

  The modulator of cancer can also be a nucleic acid. Nucleic acid modulators can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digested products of prokaryotic or eukaryotic genomes can be used in a similar approach as described above for proteins.

  The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the antibodies comprising this population are identical except for possible naturally occurring mutations (present in small amounts).

  A “motif” as a biological motif of a 109P1D4-related protein is any pattern of amino acids that form part of the primary sequence of a protein (specific function (eg, protein-protein interaction, protein-DNA interaction, etc.)). , Or modification (eg, phosphorylation, glycosylation, or amidation), or localization (eg, secretion sequence, nuclear localization sequence, etc.), or immunogenic (either humoral or cellular) )) And related sequences. Motifs can either be adjacent to or aligned with a particular location generally associated with a particular function or property. In the context of HLA motifs, a “motif” is a residue in a peptide of a defined length that is recognized by a particular HLA molecule (usually about 8 to about 13 amino acids for a class I HLA motif, and class II A pattern of about 6 to about 25 amino acid peptides for the HLA motif. The peptide motif for HLA binding is typically different for each protein encoded by each human HLA allele and is different in the pattern of primary and secondary anchor residues.

  “Pharmaceutical excipients” include substances such as adjuvants, carriers, pH adjusting and buffering agents, isotonic agents, wetting agents, preservatives and the like.

  “Pharmaceutically acceptable” refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

  The term “polynucleotide” refers to a polymorphic form of nucleotides (either ribonucleotides or deoxynucleotides, or a modified form of either nucleotide type) of at least 10 bases or base pairs in length. And is meant to include single and double stranded forms of DNA and / or RNA. In the art, this term is often used interchangeably with “oligonucleotide” if necessary. A polynucleotide can comprise the nucleotide sequence disclosed herein, wherein thymidine (T) (eg, as shown in FIG. 2) can also be uracil (U); And the difference between the chemical structures of RNA, particularly the finding that one of the four main bases in RNA is uracil (U) instead of thymidine (T).

  The term “polypeptide” means a polymer of at least about 4, 5, 6, 7 or 8 amino acids. Throughout this specification, standard three-letter or single-letter code for amino acids is used. In the art, this term is often used interchangeably with “peptide” or “protein”.

  The “primary anchor residue” of HLA is an amino acid at a specific position along the peptide sequence that is understood to provide a point of contact between the immunogenic peptide and the HLA molecule. The primary anchor residues within one to three, usually two, defined length peptides generally define a “motif” for an immunogenic peptide. It is understood that these residues are fitted in close contact with the peptide binding groove of the HLA molecule and their side chains embedded in the specific pocket of this binding glove. In one embodiment, for example, the primary anchor residue for an HLA class I molecule is in accordance with the present invention at position 2 (from the amino terminal position) and residue peptide epitopes 8, 9, 10 in the carboxy terminal position. , Localized at the 11th or 12th position. Alternatively, in another embodiment, the primary anchor residues of the peptide that bind to the HLA class II molecule are spaced apart relative to each other rather than at the end of the peptide. Here, the peptides are generally at least 9 amino acids long. The position of the primary anchor for each motif and supermotif is shown in Table IV. For example, analog peptides can be made by altering the presence or absence of certain residues at the primary anchor positions and / or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and / or population coverage of peptides containing specific HLA motifs or supermotifs.

“Radioisotopes” include but are not limited to the following (non-limiting exemplary uses are also indicated):
Examples of medical isotopes:
Description of isotope use
Actinium-225
(AC-225)
See Thorium-229 (Th-229). Actinium-227
(AC-227)
The parent bismuth-212 of radium-223 (Ra-223), an alpha emitter used to treat cancer (ie, breast and prostate cancer) and intraskeletal metastases resulting from cancer radioimmunotherapy
(Bi-212)
See Thorium-228 (Th-228), Bismuth-213
(Bi-213)
See Thorium-229 (Th-229) Cadmium-109
(Cd-109)
Cancer detection cobalt-60
(Co-60)
Radiation source copper-64 for cancer radiotherapy, food irradiation, and pharmaceutical sterilization
(Cu-64)
Positron emitter copper-67 used in cancer therapy and SPECT imaging
(Cu-67)
Β / γ emitter dysprosium-166 used for cancer radioimmunotherapy and diagnostic tests (ie, breast cancer, colon cancer and lymphoma)
(Dy-166)
Cancer radioimmunotherapy erbium-169
(Er-169)
Rheumatoid arthritis treatment Europium-152, especially for small joints connected with fingers and toes
(Eu-152)
Radiation source Europium-154 for food irradiation and pharmaceutical sterilization
(Eu-154)
Radiation source gadolinium-153 for food irradiation and pharmaceutical sterilization
(Gd-153)
Osteoporosis Detection and Nuclear Medical Quality Assurance Device Gold-198
(Au-198)
Transplantation and intracavitary treatment of ovarian, prostate and brain cancer Holmium-166
(Ho-166)
Multiple myeloma treatment, cancer radioimmunotherapy, bone marrow dissection, and rheumatoid arthritis treatment iodine-125 in targeted bone treatment
(I-125)
Osteoporosis detection, diagnostic imaging, follow-up drugs, brain cancer treatment, radiolabeling, tumor imaging, receptor mapping in the brain, interstitial radiation therapy, brachytherapy for prostate cancer treatment, measurement of glomerular filtration rate (GFR) , Measurement of plasma volume, detection of deep vein thrombosis in the leg iodine-131
(I-131)
Thyroid function assessment, detection of thyroid disease, treatment of thyroid cancer and other non-malignant thyroid diseases (ie, Graves disease, goiter, and hyperthyroidism), leukemia using radioimmunotherapy, lymphoma, and another cancer Treatment of forms of iridium-192 (eg breast cancer)
(Ir-192)
Brachytherapy, treatment of brain tumors and myeloma, treatment of arterial occlusion (ie, arteriosclerosis and restenosis), and implant lutetium-177 for breast and prostate tumors
(Lu-177)
Cancer radioimmunotherapy and arterial occlusion treatment (ie, arteriosclerosis and restenosis)
Molybdenum-99
(Mo-99)
The parent of Technetium-99m (Tc-99m) used for imaging of the brain, liver, lungs, heart, and other organs. Currently, Tc-99m is used for imaging of various cancers and diseases including brain, heart, liver, lung, and is the most radioisotope used for detection of deep vein thrombosis in the legs Widely used.
Osmium-194
(Os-194)
Cancer Radioimmunotherapy Palladium-103
(Pd-103)
Prostate cancer treatment platinum-195m
(Pt-195m)
Test of cisplatin, biodistribution and metabolism of chemotherapeutic drugs Phosphorus-32
(P-32)
Treatment of polycythemia vera (blood cell disease) and leukemia, diagnosis / treatment of bone cancer; treatment of colon cancer, pancreatic cancer, and liver cancer; radiolabeling of nucleic acids for in vitro testing, diagnosis of surface tumors, arterial occlusion Treatment of (ie, arteriosclerosis and restenosis) and intraluminal therapy phospho-33
(P-33)
Treatment of leukemia, diagnosis / treatment of bone disease, radiolabeling, and arterial occlusion (ie, arteriosclerosis and restenosis) Radium-223
(Ra-223)
Actinium-227 (Ac-227) Reference Rhenium-186
(Re-186)
Reduction of bone cancer pain, rheumatoid arthritis treatment, and diagnosis of lymphoma, bone cancer, breast cancer, colon cancer, and liver cancer using radioimmunotherapy, and treatment rhenium-188
(Re-188)
Diagnosis and treatment of cancer using radioimmunotherapy, reduction of bone cancer pain, treatment of rheumatoid arthritis, and treatment of prostate cancer Rhodium-105
(Rh-105)
Cancer radioimmunotherapy samarium-145
(Sm-145)
Eye Cancer Treatment Samarium-153
(Sm-153)
Cancer radioimmunotherapy and bone cancer pain relief scandium-47
(Sc-47)
Cancer Radioimmunotherapy and Bone Cancer Pain Relief Selenium-75
(Se-75)
Radiotracer used for brain examination, imaging of the adrenal cortex using γ-scintigraphy, lateral location of steroid-secreting tumors, pancreatic scanning, detection of hyperparathyroidism, bile acids from the endogenous pool Decrease rate measurement Strontium-85
(Sr-85)
Bone cancer detection and brain scan strontium-89
(Sr-89)
Reduction of bone cancer pain, treatment of multiple myeloma, and osteoblast therapy Technetium-99m
(Tc-99m)
See Molybdenum-99 (Mo-99) Thorium-228
(Th-228)
Thorium-229, parent of bismuth-212 (Bi-212), an alpha emitter used in cancer radioimmunotherapy
(Th-229)
Parent of actinium-225 (Ac-225) and grandparents thulium-170 of bismuth-213 (Bi-213) which are alpha emitters used in cancer radioimmunotherapy
(Tm-170)
Gamma ray source for blood irradiator, energy source tin-117m for medical device for implantation
(Sn-117m)
Cancer radioimmunotherapy and bone cancer pain relief Tungsten-188
(W-188)
Parental xenon for rhenium-188 (Re-188) used in cancer diagnosis / treatment, bone cancer pain reduction, rheumatoid arthritis treatment, and arterial occlusion (ie, arteriosclerosis and restenosis) 127
(Xe-127)
Neuroimaging of brain disorders, high resolution SPECT examination, pulmonary function examination, and cerebral blood flow examination ytterbium-175
(Yb-175)
Cancer radioimmunotherapy yttrium-90
(Y-90)
Micro-species yttrium-91 obtained from irradiation of yttrium-89 (Y-89) for the treatment of liver cancer
(Y-91)
Yttrium-90 (Y-90) gamma radiation used for cancer radioimmunotherapy (ie, lymphoma, breast cancer, colon cancer, kidney cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, and inoperable liver cancer) “Randomized” as applied herein in nucleic acids and proteins, or grammatical equivalents thereof, each of the nucleic acids and peptides is essentially It is meant to consist of randomized nucleotides and amino acids. These randomized peptides (or nucleic acids as discussed herein) can incorporate any nucleotide or amino acid at any position. This synthetic process generates random proteins or nucleic acids, allowing the formation of all or most possible combinations over the length of the sequence, thereby rendering live candidate bioactive proteinaceous drugs randomized. Can be designed to form a rally.

  In one embodiment, the library is “fully randomized” and there is no sequence trend or sequence constancy at any position. In another embodiment, the library is a “biased randomized” library. That is, some locations within this array are either kept constant or selected with limited probability. For example, nucleotide or amino acid residues can be generated, for example, by the generation of nucleic acid binding domains, the generation of cysteines for cross-linking, the proline for SH3 domains, serine for phosphorylation sites, etc., threonine, tyrosine or histidine, etc. Random in a defined class that is a hydrophobic amino acid residue, a hydrophilic amino acid residue, or a spatially biased (either small or large) residue for purines, etc. It becomes.

  A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that is subjected to molecular manipulation in vitro.

  Non-limiting examples of small molecules include compounds that bind to or interact with 109P1D4 ligands (hormones, neuropeptides, chemokines, odorants, phospholipids, and preferably their function to inhibit 109P1D4 protein function. Equivalents). Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably less than about 9 kDa, about 8 kDa, about 7 kDa, about 6 kDa, about 5 kDa or about 4 kDa. In certain embodiments, the small molecule physically associates with or binds to 109P1D4 protein; is not found in naturally occurring metabolic pathways; and / or is more soluble in aqueous solution than non-aqueous solution .

  The “stringency” of a hybridization reaction is readily determinable by one of ordinary skill in the art, and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured nucleic acid sequence to anneal again when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures result in less. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

  “Stringent conditions” or “high stringent conditions”, as defined herein, may be identified by, but not limited to: (1) low ionic strength and Use high temperature (eg, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate, 50 ° C.); (2) Denaturing agents (eg, formamide) during hybridization (E.g. 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 75 mM 50 mM sodium phosphate buffer pH 42 with sodium acid, 42 ° C.); or (3) 50% pH Muamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated Salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate were used at 42 ° C., at 42 ° C. in 0.2 × SSC (sodium chloride / sodium citrate), It is then washed at 55 ° C. in 50% formamide, followed by a high stringency wash consisting of 0.1 × SSC containing EDTA at 55 ° C. “Moderate stringent conditions” are described by, but not limited to, Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and more than the hybridization conditions described above. Includes the use of low stringency wash solutions and hybridization conditions (eg, temperature, ionic strength and% SDS). Examples of moderate stringency conditions are 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% Of dextran sulfate and 20 mg / mL denatured sheared salmon sperm DNA overnight incubation at 37 ° C. followed by about 37 ° C.-50 ° C. in 1 × SSC Cleaning the filter with Those skilled in the art will recognize methods for adjusting temperature, ionic strength, etc., as needed to accommodate factors such as probe length.

  An “super motif” of HLA is a peptide binding specific shared by HLA molecules encoded by two or more HLA alleles. The frequency of all phenotypes of HLA supertypes in various racial populations is shown in Table IV (F). Non-limiting components of the various supertypes are as follows:

異なるＨＬＡスーパータイプの組み合わせによって得られる計算された集団の適用範囲は、表ＩＶ（Ｇ）に示される。

The calculated population coverage obtained by the combination of different HLA supertypes is shown in Table IV (G).

  As used herein, “treating” or “therapeutic” and grammatically related terms refer to any consequence of the disease (eg, prolonged survival, less morbidity, and / or Any improvement in side effects (reduction of side effects of alternative treatment modalities; complete eradication of the disease is not required).

  A “transgenic animal” (eg, a mouse or rat) is an animal having cells that contain the transgene, which has been introduced into the animal or is ancestor of the animal prior to birth (eg, at the embryonic stage). Was introduced. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

  As used herein, an HLA or cellular immune response “vaccine” refers to a composition comprising or encoding one or more peptides of the invention. Such vaccines (eg, one or more distinct peptides; one or more peptides of the invention encompassed by the polyepitope peptide; or such separate peptides or polyepitopes (eg, mini-encoding polyepitope peptides) There are numerous embodiments of the nucleic acid) encoding the gene). “One or more peptides” refers to 1 to 150 or more (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of the present invention). 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 , 130, 135, 140, 145, or 150 or more peptides). Peptides or polypeptides can be modified as necessary (eg, by lipidation, targeting sequences, or addition of other sequences). The HLA class I peptides of the present invention can be mixed or linked with HLA class II peptides to promote the activation of both cytotoxic and helper T lymphocytes. The HLA vaccine may also include peptide pulse antigen presenting cells (eg, dendritic cells).

  The term “variant” refers to one or more different amino acid residues in the corresponding portion of the type or criterion described (eg, the specifically described protein (eg, 109P1D4 protein shown in FIG. 2 or FIG. 3)). A molecule that exhibits a modification from An analog is an example of a modified protein. Splicing isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

  The “109P1D4-related protein” of the present invention includes the proteins specifically identified herein, as well as allelic variants (methods outlined herein or methods readily available in the art). Conservative substitution variants, analogs and homologues that can be isolated / generated and characterized without performing experiments according to Also included are fusion proteins that bind to portions of different 109P1D4 proteins or fragments thereof, as well as 109P1D4 protein and heterologous polypeptide fusion proteins. Such 109P1D4 protein is collectively referred to as 109P1D4-related protein, or 109P1D4, which is the protein of the present invention. The term “109P1D4-related protein” means 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. 25 or more amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, Refers to a polypeptide fragment or 109P1D4 protein sequence of 500, 525, 550, 575 or more than 576 amino acids.

(II.) 109P1D4 polynucleotide)
One aspect of the present invention provides a polynucleotide, preferably in isolated form, corresponding to or complementary to all or part of the 109P1D4 gene, mRNA and / or coding sequence, which is associated with 109P1D4. Polynucleotides encoding proteins and fragments thereof, DNA, RNA, DNA / RNA hybrids, and related molecules, polynucleotides or oligonucleotides or portions thereof complementary to 109P1D4 gene or mRNA sequence, and 109P1D4 genes, mRNA, Or a polynucleotide or oligonucleotide that hybridizes to a 109P1D4 encoding polynucleotide (collectively "109P1D4 polynucleotide"). As referenced in this section, in all examples, T can also be U in FIG.

Embodiments of 109P1D4 polynucleotides include: 109P1D4 polynucleotide having the sequence shown in FIG. 2, 109P1D4 nucleotide sequence as shown in FIG. 2 (where T is U); At least 10 contiguous nucleotides of a polynucleotide having the sequence as shown; or at least 10 contiguous nucleotides of a polynucleotide having the sequence shown in FIG. 2 (where T is U). For example, embodiments of 109P1D4 nucleotides include, but are not limited to:
(I) a polynucleotide comprising a sequence as shown in FIG. 2, consisting essentially of such a sequence, or consisting of such a sequence (where T can also be U);
(II) a sequence as shown in FIG. 2A comprising nucleotide residue number 876 to nucleotide residue number 3911 (including a stop codon), consisting essentially of such a sequence, or such a sequence A polynucleotide consisting of (where T can also be U);
(III) a sequence as shown in FIG. 2B comprising, consisting essentially of, or consisting of nucleotide residue number 503 to nucleotide residue number 3667 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(IV) a sequence as shown in FIG. 2C comprising, consisting essentially of, or consisting of nucleotide residue number 846 to nucleotide residue number 4889 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(V) a sequence as shown in FIG. 2D comprising, consisting essentially of, or consisting of nucleotide residue number 846 to nucleotide residue number 4859 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(VI) a sequence as shown in FIG. 2E comprising, consisting essentially of, or consisting of nucleotide residue number 846 to nucleotide residue number 4778 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(VII) a sequence as shown in FIG. 2F comprising, consisting essentially of, or consisting of nucleotide residue no. 614 to nucleotide residue no. 3727 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(VIII) a sequence as shown in FIG. 2G comprising, consisting essentially of, or consisting of nucleotide residue number 735-nucleotide residue number 3881 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(IX) a sequence as shown in FIG. 2H comprising, consisting essentially of, or consisting of nucleotide residues 735-nucleotide residues 4757 (including stop codon) A polynucleotide consisting of (where T can also be U);
(X) a sequence as shown in FIG. 2I comprising, consisting essentially of, or consisting of nucleotide residue number 514 to nucleotide residue number 3627 (including a stop codon) A polynucleotide consisting of (where T can also be U);
(XI) a polynucleotide encoding a 109P1D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to the full length amino acid sequence shown in FIGS.
(XII) a polynucleotide encoding a 109P1D4-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the full length amino acid sequences shown in FIGS.
(XIII) a polynucleotide encoding at least one peptide shown in Tables VIII-XXI and XXII-XLIX;
(XIV) At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 having a value greater than 0.5 in the hydrophilic profile of FIG. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions, including at least 5 of the peptide of FIG. 3A 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35 amino acids to a polynucleotide encoding any integer increment peptide region up to 1021;
(XV) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, having a value greater than 0.5 in the hydropathic profile of FIG. At least 5, of the peptide of FIG. 3A, comprising 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, A polynucleotide encoding a peptide region in any integer increment from 31, 32, 33, 34, 35 amino acids to 1021;
(XVI) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, having a value greater than 0.5 in the% profile of accessible residues in FIG. Peptide of FIG. 3A comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, A polynucleotide encoding a peptide region in any integer increment from 29, 30, 31, 32, 33, 34, 35 amino acids to 1021;
(XVII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, having a value greater than 0.5 in the average flexibility profile of FIG. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions, including at least the peptide of FIG. 3A 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, A polynucleotide encoding a peptide region in any integer increment from 30, 31, 32, 33, 34, 35 amino acids to 1021;
(XVIII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 having a value greater than 0.5 in the β-turn profile of FIG. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions, including at least 5 of the peptide of FIG. 3A 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35 amino acids to a polynucleotide encoding any integer increment peptide region up to 1021;
(XIX) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, having a value greater than 0.5 in the hydrophilic profile of FIG. 3B, 3C and / or 3D, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, A polynucleotide encoding a peptide region of any integer increment from 28, 29, 30, 31, 32, 33, 34, 35 amino acids to 1050, 1347 and / or 1337, respectively;
(XX) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, having a value greater than 0.5 in the hydropathic profile of FIG. 3B, 3C and / or 3D, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, A polynucleotide encoding a peptide region of any integer increment from 28, 29, 30, 31, 32, 33, 34, 35 amino acids to 1050, 1347 and / or 1337;
(XXI) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, having a value greater than 0.5 in the% profile of accessible residues in FIG. 3B, 3C, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions And / or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, of 3D peptides A polynucleotide encoding a peptide region in any integer increment from 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids to 1050, 1347 and / or 1337;
(XXII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, having a value greater than 0.5 in the average flexibility profile of FIG. 3B, 3C and / or 16, including amino acid positions 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, of a 3D peptide A polynucleotide encoding a peptide region of any integer increment from 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids to 1050, 1347 and / or 1337;
(XXIII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 having a value greater than 0.5 in the β-turn profile of FIG. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions, including FIGS. 3B, 3C and / or At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 of 3D peptides , 28, 29, 30, 31, 32, 33, 34, 35 amino acids to any integer increment peptide region from 1054, 1347 and / or 1337;
(XXIV) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, having a value greater than 0.5 in the hydrophilic profile of FIG. 3E, 3F, 3G, 3H, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions And / or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, of 3I peptides Poly, encoding any integer increment peptide region from 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids up to 1310, 1037, 1048, 1340 and / or 1037, respectively. Nucleoti ;
(XXV) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, having a value greater than 0.5 in the hydropathy profile of FIG. 3E, 3F, 3G, 3H, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions And / or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, of 3I peptides Polynucleotide encoding any integer increment peptide region from 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids to 1310, 1037, 1048, 1340 and / or 1037, respectively. Plastid;
(XXVI) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, having a value greater than 0.5 in the% profile of accessible residues in FIG. 3E, 3F, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, of 3G, 3H and / or 3I peptides 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids up to 1310, 1037, 1048, 1340 and / or 1037 in any integer increment peptide region, respectively. Code, poly Kureochido;
(XXVII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, having a value greater than 0.5 in the average flexibility profile of FIG. 3E, 3F, 3G, including 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions 3H and / or 3I peptides at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 encoding amino acid in any integer increment from 1310, 1037, 1048, 1340 and / or 1037, respectively , Polynuk Fault;
(XXVIII) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 having a value greater than 0.5 in the β-turn profile of FIG. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, including amino acid positions, 3E, 3F, 3G, At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 of 3H and / or 3I peptides , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids up to 1310, 1037, 1048, 1340 and / or 1037, respectively, in any increment increment peptide region, Polinu Reochido;
(XXIX) a polynucleotide that is completely complementary to a polynucleotide of any one of (I)-(XXVIII);
(XXX) a polynucleotide that is completely complementary to a polynucleotide of any one of (I) to (XXIX);
(XXXI) a peptide encoded by any of (I)-(XXX); and;
(XXXII) a composition comprising a polynucleotide of any of (I) to (XXX) or a peptide of (XXXI) with a pharmaceutical excipient and / or in human unit dosage form;
(XXXIII) A method of using a polynucleotide of any one of (I) to (XXX) or a peptide of (XXXI) or a composition of (XXXII) in a method of modulating cells expressing 109P1D;
(XXXIV) A polynucleotide of any one of (I) to (XXX) or a peptide of (XXXI) or a composition of (XXXII) in a method for diagnosing, preventing, prognosing or treating an individual having cells expressing 109P1D How to use;
(XXXV) any one of (I) to (XXX) in a method for diagnosing, preventing, prognosing or treating an individual having cells expressing 109P1D (cells derived from cancer of the tissues listed in Table I) A method of using a nucleotide or a peptide of (XXXI) or a composition of (XXXII);
(XXXVI) a method of using a polynucleotide of any one of (I) to (XXX) or a peptide of (XXXI) or a composition of (XXXII) in a method for diagnosing, preventing, prognosing or treating cancer;
(XXXVII) A polynucleotide of any one of (I) to (XXX) or a peptide of (XXXI) or a composition of (XXXII) in a method for diagnosing, preventing, prognosing or treating cancer of the tissues listed in Table I How to use the object; and;
(XXXVIII) A polynucleotide of any one of (I) to (XXX) or a peptide of (XXXI) or a composition of (XXXII) in a method for identifying or characterizing a modulator of cells expressing 109P1D4 Method.

  As used herein, a range is understood to specifically disclose all of its unit positions.

Exemplary embodiments of the invention disclosed herein include 109P1D4 polynucleotides (and such sequences that encode specific portions of 109P1D4 mRNA sequences such as those that encode proteins and / or fragments thereof). Complementary polynucleotides) (for example:
(A) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 of 109P1D4 variant 1 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10 0, 1020, and 1021 or more contiguous amino acids; the longest relevant length for other variants is: variant 2, 1054 amino acids: variant 3, 1347 amino acids: variant 4, 1337 amino acids: Variant 5, 1310 amino acids: Variant 6, 1047 amino acids: Variant 7, 1048 amino acids: Variant 8, 1340 amino acids and Variant 9, 1037 amino acids).

  For example, representative embodiments of the invention disclosed herein include the following: Figure 2 or Figure with about 10 amino acids added to the end of the carboxyl terminal amino acid shown in Figure 2 or Figure 3 A polynucleotide encoding from about amino acid 1 to about amino acid 10 of the 109P1D4 protein shown in FIG. 3, and the encoded peptide itself, a polynucleotide encoding from about amino acid 10 to about amino acid 20 of the 109P1D4 protein shown in FIG. 2 or FIG. 2, a polynucleotide encoding from about amino acid 20 to about amino acid 30 of the 109P1D4 protein shown in FIG. 2 or FIG. 3, a polynucleotide encoding from about amino acid 30 to about amino acid 40 of the 109P1D4 protein shown in FIG. 2 or FIG. 2 or 109P1D shown in FIG. A polynucleotide encoding from about amino acid 40 to about amino acid 50 of the protein, a polynucleotide encoding from about amino acid 50 to about amino acid 60 of the 109P1D4 protein shown in FIG. 2 or FIG. 3, and of a 109P1D4 protein shown in FIG. 2 or FIG. A polynucleotide encoding from about amino acid 60 to about amino acid 70, a polynucleotide encoding from about amino acid 70 to about amino acid 80 of the 109P1D4 protein shown in FIG. 2 or FIG. 3, and about amino acid of the 109P1D4 protein shown in FIG. 2 or FIG. A polynucleotide encoding a polynucleotide encoding from 80 to about amino acid 90, a polynucleotide encoding from about amino acid 90 to about amino acid 100 of the 109P1D4 protein shown in FIG. 2 or FIG. Thus, a polynucleotide encoding a portion of the amino acid sequence of amino acid 100 to the carboxyl terminal amino acid (approximately 10 amino acids) of 109P1D4 protein is an embodiment of the present invention. Here, it is understood that each specific amino acid position discloses a position of ± 5 amino acid residues.

  Polynucleotides that encode a relatively long portion of the 109P1D4 protein are also within the scope of the invention. For example, a polynucleotide encoding from about amino acid 1 (or 20 or 30 or 40 or the like) to about amino acid 20 (or 30 or 40 or 50 or the like) of the 109P1D4 protein shown in FIG. 2 or FIG. 3 or a “variant” thereof Can be generated by various techniques well known in the art. These polynucleotide fragments can comprise any portion of the 109P1D4 sequence as shown in FIG.

  Further exemplary embodiments of the invention disclosed herein include 109P1D4 polynucleotide fragments that encode one or more biological motifs contained within a 109P1D4 protein sequence or “variant” sequence. Includes one or more motif-bearing partial sequences of the 109P1D4 protein or “variant” shown in Tables VIII-XXI and XXII-XLIX. In another embodiment, a representative polynucleotide fragment of the invention encodes one or more regions of a 109P1D4 protein or variant that exhibits homology to a known molecule. In another embodiment of the invention, a representative polynucleotide fragment is an N-glycosylation site, cAMP-dependent and cGMP-dependent protein kinase phosphorylation site, casein kinase II phosphorylation site of a 109P1D4 protein or variant. Alternatively, it may encode one or more of N-myristoylation sites, as well as amidation sites.

  To determine the starting position of each of the peptides shown in Tables VIII-XXI and Tables XXII-XLIX (collectively, HLA peptide tables) relative to its parent protein (eg, variant 1, variant 2, etc.) Note that reference is made to the following three factors: specific variants, peptide lengths in the HLA peptide table, search peptides listed in Table VII. In general, unique search peptides are used to obtain HLA peptides for specific variants. The position of each search peptide for its respective parent molecule is listed in Table VII. Thus, when search peptides start at position “X”, to obtain the actual position of the HLA peptides in their parent molecule, at each position in Tables VIII-XXI and Tables XXII-IL, “X-1 Must be added. For example, if a particular search peptide starts at position 150 of its parent molecule, 150-1 (ie, 149) is added to the amino acid position of each HLA peptide to calculate the position of the amino acid in the parent molecule. Must.

(II.A.) Use of 109P1D4 polynucleotide)
(II.A.1. Monitoring of gene abnormality)
The above items of polynucleotides have a number of different specific uses. The human 109P1D4 gene is mapped to the chromosomal location shown in the example titled "109P1D4 chromosomal mapping". For example, since the 109P1D4 gene maps to this chromosome, polynucleotides encoding different regions of the 109P1D4 protein characterize cytogenic abnormalities at this chromosomal location (eg, abnormalities identified as being associated with various cancers). Used for. In certain genes, various chromosomal abnormalities, including rearrangements, have been identified as frequent cytogenetic abnormalities in a number of different cancers (eg Krajinovic et al., Mutat. Res. 382 (3-4): 81- 83 (1998); see Johansson et al., Blood 86 (10): 3905-3914 (1995) and Finger et al., PNA S. 85 (23): 9158-9162 (1988)). Thus, a polynucleotide encoding a specific region of 109P1D4 protein could previously be used to account for cytogenetic abnormalities in the chromosomal region encoding 109P1D4, which may contribute to a malignant phenotype Provide new tools that are more accurate than tools. In this context, these polynucleotides meet the need in the art for expanding the susceptibility of chromosomal screening to identify more subtle and less common chromosomal abnormalities (eg, Evans Et al., Am. J. Obstet. Gynecol 171 (4): 1055-1057 (1994)).

  Furthermore, since 109P1D4 has been shown to be highly expressed in prostate and other cancers, 109P1D4 polynucleotides are used in methods to assess 109P1D4 gene product status in normal versus cancerous tissues . Typically, a polynucleotide encoding a specific region of the 109P1D4 protein has a disorder in a specific region of the 109P1D4 gene (eg, a region containing one or more motifs) (eg, a deletion, insertion, Used to assess the presence of point mutations or modifications). Exemplary assays include RT-PCR assays and single strand conformation polymorphism (SSCP) analysis (see, eg, Marrogi et al., J. Cutan. Pathol. 26 (8): 369-378 (1999)). Both of which utilize a polynucleotide encoding a particular region within the protein to test these regions within the protein.

(II.A.2. Antisense Embodiment)
The embodiments of the invention disclosed herein relating to other specifically contemplated nucleic acids are genomic DNA, cDNA, ribozyme and antisense molecules, and whether they are derived from natural sources or synthetic Regardless, an alternative backbone-based nucleic acid molecule or nucleic acid molecule comprising an alternative base, and includes molecules capable of inhibiting 109P1D4 RNA or protein expression. For example, antisense molecules can be RNA or other molecules, including peptide nucleic acids (PNA) or non-nucleic acid molecules (eg, phosphorothioate derivatives) that specifically bind to DNA or RNA in a base-pair dependent manner. . One of skill in the art can readily obtain these classes of nucleic acid molecules using the 109P1D4 polynucleotides and polynucleotide sequences disclosed herein.

  Antisense technology involves the administration of exogenous oligonucleotides that bind to target polynucleotides located within the cell. The term “antisense” refers to the fact that such an oligonucleotide is complementary to its intracellular target (eg, 109P1D4). See, for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1: 1-5 (1988). 109P1D4 antisense oligonucleotides of the invention include derivatives (eg, S-oligonucleotides (see phosphorothioate derivatives or S-oligos, see Jack Cohen (supra)), which are enhanced cancer cells. S-oligo (nucleoside phosphorothioate) is an isoelectronic analog of an oligonucleotide (O-oligo) in which the non-bridging oxygen atom of the phosphate group is replaced by a sulfur atom. -Oligos can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent, eg, Iyer, RP et al., J. Org. Chem. 55: 4693-4698 (1990); r, R.P., et al., J. Am.Chem.Soc.112: 1253-1254 (1990) Additional 109P1D4 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art. (See, eg, Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

  The 109P1D4 antisense oligonucleotides of the invention are typically complementary to the first 100 5 ′ codons or the last 100 3 ′ codons of the 109P1D4 genomic sequence or corresponding mRNA, and It can be RNA or DNA that hybridizes stably. Although complete complementarity is not required, a high degree of complementarity is preferred. The use of an oligonucleotide complementary to this region allows selective hybridization to 109P1D4 mRNA, but does not allow selective hybridization to mRNAs that specify other regulatory subunits of protein kinases. . In one embodiment, the 109P1D4 antisense oligonucleotide of the invention is a 15-30 mer fragment of an antisense DNA molecule having a sequence that hybridizes to 109P1D4 mRNA. Optionally, the 109P1D4 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5 'codons or the last 10 3' codons of 109P1D4. Alternatively, the antisense molecule is modified to use a ribozyme in the inhibition of 109P1D4 expression (see, eg, LA Couture & DT Stinchcomb; Trends Genet 12: 510-515 (1996)). thing).

(II.A.3. Primers and primer pairs)
More specific embodiments of this nucleotide of the invention include primers and primer pairs (which allow specific amplification of the polynucleotide of the invention or any particular portion thereof) and probes (which Hybridize selectively or specifically to the nucleic acid molecule of the present invention or any part thereof. The probe can be labeled with a detectable marker (eg, radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator or enzyme). Such probes and primers are used as a means for detecting the presence of 109P1D4 polynucleotide in a sample and for detecting cells expressing 109P1D4 protein.

  Examples of such probes include a polynucleotide comprising all or part of the human 109P1D4 cDNA sequence shown in FIG. Examples of primer pairs that can specifically amplify 109P1D4 mRNA are also described in the Examples. As will be appreciated by those skilled in the art, a large number of different primers and probes can be prepared based on the sequences provided herein and effectively used to amplify and / or detect 109P1D4 mRNA. Can be done.

  The 109P1D4 polynucleotides of the present invention are useful for a variety of purposes, including but not limited to the following: Probes and primers for amplification and / or detection of 109P1D4 gene, mRNA or fragments thereof As a reagent for the diagnosis and / or prognosis of prostate cancer and other cancers; as a coding sequence capable of directing the expression of 109P1D4 polypeptide; regulating or inhibiting 109P1D4 gene expression and / or 109P1D4 transcript translation As a tool for; and as a therapeutic agent.

  The present invention provides any of the methods described herein for identifying and isolating 109P1D4 nucleic acid sequences or 109P1D4 related nucleic acid sequences from naturally occurring sources (eg, humans or other mammals). As well as the isolated nucleic acid sequence itself, which includes all or most of the sequences found in the probe used.

(II.A.4.) 109P1D4-encoding nucleic acid molecule isolation)
The 109P1D4 cDNA sequences described herein are used to isolate other polynucleotides that encode the 109P1D4 gene product, as well as 109P1D4 gene product homologs, alternative spliced isoforms, allelic variants and 109P1D4 gene products. Allows isolation of polynucleotides encoding mutant forms as well as polynucleotides encoding analogs of 109P1D4 related proteins. Various molecular cloning methods that can be used to isolate the full-length cDNA encoding the 109P1D4 gene are well known (eg, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al. (Eds.), Wiley and Sons, 1995). For example, the λ phage cloning methodology can be conveniently used using a commercially available cloning system (eg, Lambda ZAP Express, Stratagene). Phage clones containing 109P1D4 gene cDNA can be identified by probing with labeled 109P1D4 cDNA or fragments thereof. For example, in one embodiment, 109P1D4 cDNA (eg, FIG. 2) or a portion thereof can be synthesized and used as a probe to search for duplication to 109P1D4 gene and full-length cDNA corresponding to 109P1D4 gene. The 109P1D4 gene itself can be isolated by screening a genomic DNA library, a bacterial artificial chromosome library (BAC), a yeast artificial chromosome library (YAC) or the like using a 109P1D4 DNA probe or primer.

(II.A.5.) Recombinant nucleic acid molecules and host-vector systems)
The invention also includes recombinant DNA or RNA molecules comprising 109P1D4 polynucleotides, fragments, analogs, or homologs thereof (phages, plasmids, phagemids, cosmids, YACs, BACs, and various viral vectors and non-well known in the art. Cells, including but not limited to viral vectors), and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, eg, Sambrook et al., 1989, supra).

  The present invention further provides a host-vector system comprising a recombinant DNA molecule comprising a 109P1D4 polynucleotide, fragment, analog, or homolog thereof in a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include yeast cells, plant cells, or animal cells (eg, mammalian cells or insect cells (eg, baculovirus infectious cells such as Sf9 cells or High Five cells)). Examples of suitable mammalian cells include various prostate cancer cell lines (eg, DU145 and TsuPr1), other transfectable or transducable prostate cancer cell lines, primary cells (PrEC), and recombinant proteins A number of mammalian cells conventionally used for the expression of (eg, COS cells, CHO cells, 293 cells, 293T cells), or more particularly, a polynucleotide comprising the coding sequence of 109P1D4, or Fragments, analogs or homologs are routinely used in the field It is, and extensively known any number of host - with a vector system, may be used to generate 109P1D4 protein or fragment thereof.

  A wide range of host-vector systems suitable for the expression of 109P1D4 protein or fragments thereof are available (see, eg, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include, but are not limited to, pcDNA3.1 myc-His-tag (Invitrogen) and retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11: 1785). Using these expression vectors, 109P1D4 can be expressed in several prostate cancer cell lines and non-prostate cell lines, including, for example, 293, 293T, rat-1, NIH 3T3, and TsuPr1. The host-vector system of the present invention is useful for producing 109P1D4 protein or fragments thereof. Such host-vector systems can be used to study the functional properties of 109P1D4 and 109P1D4 variants or analogs.

  Recombinant human 109P1D4 protein or analog or homologue or fragment thereof can be produced by mammalian cells transfected with a construct encoding 109P1D4 related nucleotides. For example, 293T cells can be transfected with an expression plasmid encoding 109P1D4 or a fragment, analog, or homolog thereof, 109P1D4-related proteins are expressed in 293T cells, and recombinant 109P1D4 protein is expressed using standard purification methods. (Eg, affinity purification using anti-109P1D4 antibody). In another embodiment, the 109P1D4 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and various mammalian cell lines (eg, NIH 3T3, TSuPr1, 293, and rat− Used to infect 1). Various other expression systems well known in the art can also be used. An expression construct encoding a leader peptide linked in-frame to the 109P1D4 coding sequence can be used for the production of a secreted form of the recombinant 109P1D4 protein.

  As discussed herein, genetic code redundancy allows variation in the 109P1D4 gene sequence. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one skilled in the art can adapt the disclosed sequences to be preferred in the desired host. . For example, a preferred analog codon sequence typically has a rare codon (ie, a codon having a usage frequency of less than about 20% in the known sequence of the desired host) replaced with a more frequent codon. . The codon preference for a particular species is calculated, for example, by using a codon usage table available on the internet (eg, URL dna.affrc.go.jp/˜nakamura/codon.html).

  Further sequence modifications are known to enhance protein expression in cellular hosts. These include removal of sequences encoding pseudopolyadenylation signals, exon / intron splice site signals, transposon-like repeats, and / or other such well-characterized sequences that are detrimental to gene expression. . The GC content of the sequence is adjusted to an average level (calculated by reference to known genes expressed in that host cell) for a given cellular host. When possible, the sequence is modified to avoid the predicted hairpin secondary mRNA structure. Other useful modifications include Kozak, Mol. Cell. Biol. 9: 5073-5080 (1989), including the addition of a translation initiation consensus sequence at the start of the open reading frame. One skilled in the art understands that the general role that eukaryotic ribosomes initiate translation exclusively at the 5 ′ proximal AUG codon is eliminated only under rare conditions (eg, Kozak PNAS 92 ( 7): 2662-2666, (1995) and Kozak NAR 15 (20): 8125-8148 (1987)).

(III.) 109P1D4-related protein)
Another aspect of the invention provides 109P1D4-related proteins. Particular embodiments of the 109P1D4 protein include polypeptides having all or part of the amino acid sequence of human 109P1D4 as shown in FIG. 2 or FIG. Alternatively, 109P1D4 protein embodiments include variants, homologues, or analog polypeptides having changes in the 109P1D4 amino acid sequence shown in FIG. 2 or FIG.

Embodiments of 109P1D4 polypeptide include: 109P1D4 polypeptide having the sequence shown in FIG. 2, 109P1D4 peptide sequence shown in FIG. 2 (where T is U); having the sequence shown in FIG. At least 10 consecutive nucleotides of the polypeptide; or at least 10 consecutive peptides (where T is U) of the polypeptide having the sequence shown in FIG. For example, embodiments of the 109P1D4 peptide include, but are not limited to:
(I) a protein comprising, consisting essentially of, or consisting of the amino acid sequences shown in FIGS. 2A-I or 3A-I;
(II) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the entire amino acid sequence shown in FIG. 2A-I or FIG. 100% homologous 109P1D4-related protein;
(III) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the entire amino acid sequence shown in FIG. 2A-I or FIG. 3A-I or 109P1D4-related protein that is 100% identical;
(IV) a protein comprising at least one peptide described in Tables VIII-XLIX (although optionally, this protein is not the entire protein of FIG. 2);
(V) Collectively, a protein comprising at least one peptide described in Tables VIII-XXI, which is also collectively described in Tables XXII-XLIX, provided that the protein is Not the whole of the two proteins;
(VI) a protein comprising at least two peptides selected from the peptides listed in Tables VIII-XLIX (although optionally, this protein is not the entire protein of FIG. 2);
(VII) In summary, a protein comprising at least two peptides selected from the peptides listed in Tables VIII-XLIX (however, this protein is not a contiguous sequence derived from the amino acid sequence of FIG. 2);
(VIII) a protein comprising at least one peptide selected from the peptides listed in Tables VIII-XXI; and a protein comprising at least one peptide selected from the peptides listed in Tables XXII-XLIX, wherein The protein is not a continuous sequence derived from the amino acid sequence of FIG.
(IX) At least 5, 6, 7, 8, of the proteins of FIGS. 3A, 3B, 3C, 3D and / or 3E (any total increase up to 1021, 1050, 1347, 1337, and / or 1310, respectively) 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, A polypeptide comprising 35 amino acids, the peptide having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, having a value greater than 0.5 in the hydrophilic profile of FIG. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Amino acid position No;
(X) at least 5, 6, 7, 8, 9 of the proteins of FIGS. 3A, 3B, 3C, 3D and / or 3E (up to any total number of 1021, 1054, 1347, 1337 and / or 1310, respectively) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , A polypeptide comprising 35 amino acids, the polypeptide having a value of less than 0.5 in the hydropathy profile of FIG. 6, at least 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 34, 35 Including amino acid positions;
(XI) at least 5, 6, 7, 8, of the proteins of FIGS. 3A, 3B, 3C, 3D and / or 3E (any total increase up to 1021, 1050, 1347, 1337, and / or 1310, respectively) 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, A polypeptide comprising 34, 35 amino acids, the polypeptide having at least 1, 2, 3, 4, 5, 6, 7 having a value greater than 0.5 in the% profile of accessible residues of FIG. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 33, 34, Including the amino acid position of 5;
(XII) at least 5, 6, 7, 8, of the proteins of FIGS. 3A, 3B, 3C, 3D and / or 3E (up to any total number of 1021, 1050, 1347, 1337 and / or 1310, respectively) 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, A polypeptide comprising 34, 35 amino acids, wherein the polypeptide has at least 1, 2, 3, 4, 5, 6, 7, 8 having a value greater than 0.5 in the average flexibility profile of FIG. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35 Including the amino acid position;
(XIII) at least 5, 6, 7, 8, 9 of the proteins of FIGS. 3A, 3B, 3C, 3D and / or 3E (any total increase up to 1021, 1050, 1347, 1337, and / or 1310, respectively) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , A polypeptide comprising 35 amino acids, wherein the polypeptide has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, having a value greater than 0.5 in the β-turn profile of FIG. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Including amino acid positions;
(XIV) at least 5, 6, 7, 8, 9, 10, of the proteins of FIGS. 3F, 3G, 3H and / or 3I (up to any total increase of 1037, 1048, 1340, and / or 1037, respectively) 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids included A polypeptide having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, having a value greater than 0.5 in the hydrophilic profile of FIG. Includes amino acid positions 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 ;
(XV) at least 5, 6, 7, 8, 9, 10, 11 of the proteins of FIGS. 3F, 3G, 3H and / or 3I (up to any total number up to 1037, 1048, 1340, and / or 1037, respectively) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 A polypeptide comprising amino acids, wherein the polypeptide has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 having a value of less than 0.5 in the hydropathy profile of FIG. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 The amino acid position of No;
(XVI) at least 5, 6, 7, 8, 9, 10, of the proteins of FIGS. 3F, 3G, 3H and / or 3I (up to any total increase of 1037, 1048, 1340, and / or 1037, respectively) 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids Comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9 having a value greater than 0.5 in the% profile of accessible residues of FIG. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35 amino acids Including the location;
(XVII) at least 5, 6, 7, 8, 9, 10, of the proteins of FIGS. 3F, 3G, 3H and / or 3I (any total increase up to 1037, 1048, 1340 and / or 1037, respectively) 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids Wherein the polypeptide has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 having a value greater than 0.5 in the average flexibility profile of FIG. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Amino acid position Including;
(XVIII) at least 5, 6, 7, 8, 9, 10, 11 of the proteins of FIGS. 3F, 3G, 3H, and / or 3I (up to any total number of 1037, 1048, 1340, and / or 1037, respectively) , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids A polypeptide comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, having a value greater than 0.5 in the β-turn profile of FIG. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positions The No;
(XIX) Peptides that collectively appear at least twice in Tables VIII-XXI and Tables XXII-XLIX;
(XX) In summary, peptides that appear at least three times in Tables VIII-XXI and Tables XXII-XLIX;
(XXI) In summary, peptides that appear at least four times in Tables VIII-XXI and Tables XXII-XLIX;
(XXII) Peptides collectively appearing at least 5 times in Tables VIII-XXI and Tables XXII-XLIX;
(XXIII) Peptides that appear at least once in Tables VIII-XXI and at least once in Tables XXII-XLIX;
(XXIV) Peptides appearing at least once in Tables VIII-XXI and at least twice in Tables XXII-XLIX;
(XXV) a peptide that appears at least twice in Tables VIII-XXI and at least once in Tables XXII-XLIX;
(XXVI) a peptide that appears at least twice in Tables VIII-XXI and at least twice in Tables XXII-XLIX;
(XXVII) A peptide comprising one, two, three, four, or five of the following features or comprising an oligonucleotide encoding such a peptide:
i) A region of at least 5 amino acids of the specific peptide of FIG. 3 (in any total increase up to the full length of the protein in FIG. 3), which is 0.5 in the hydrophilic profile of FIG. Including amino acid positions having a value equal to or greater than 0.6, 0.7, 0.8, 0.9, or a value equal to 1.0;
ii) a region of at least 5 amino acids of the specific peptide of FIG. 3 (in any total increase up to the full length of the protein in FIG. 3), which is 0.5, in the hydropathy profile of FIG. Including amino acid positions having a value equal to or less than 0.4, 0.3, 0.2, 0.1 or a value equal to 0.0;
iii) a region of at least 5 amino acids of the particular peptide of FIG. 3 (in any total increase up to the full length of the protein in FIG. 3), which in the% profile of accessible residues in FIG. Including amino acid positions having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or a value equal to 1.0;
iv) A region of at least 5 amino acids of the specific peptide of FIG. 3 (in any total increase up to the full length of the protein in FIG. 3), which in the mean flexibility profile of FIG. Including amino acid positions having a value greater than or equal to 5, 0.6, 0.7, 0.8, 0.9, or a value equal to 1.0;
v) A region of at least 5 amino acids of the specific peptide of FIG. 3 (in any total increase up to the full length of the protein in FIG. 3), which is 0.5, in the β-turn profile of FIG. Including amino acid positions having a value equal to or greater than 0.6, 0.7, 0.8, 0.9, or a value equal to 1.0;
(XXVIII) a composition comprising a peptide of (I) to (XXVII), or an antibody or a binding region thereof, together with a pharmaceutical excipient and / or in human unit volume form;
(XXIX) a method for modulating a cell expressing 109P1D4, the method using a peptide of (I) to (XXVII), or an antibody or a binding region thereof, or a composition of (XXVIII);
(XXX) A peptide of (I) to (XXVII), or an antibody or a binding region thereof, or a composition of (XXVIII) in a method for diagnosing, preventing, predicting or treating an individual having cells expressing 109P1D4 Method used;
(XXXI) (I) to (XXVII) in a method for diagnosis, prevention, prognosis, or treatment of an individual having cells that express 109P1D4 (the cells described above derived from cancers of tissues listed in Table I) Or a binding region thereof, or a method of using a composition of (XXVIII);
(XXXII) A method using a peptide of (I) to (XXVII), or an antibody or a binding region thereof, or a composition of (XXVIII) in a method for diagnosing, preventing, prognosing, or treating cancer;
(XXXIII) A peptide of (I) to (XXVII), or an antibody or a binding region thereof, or a composition of (XXVIII) in a method for diagnosing, preventing, prognosing, or treating cancer of the tissues listed in Table I Using a method; and
(XXXIV) using a peptide of (I) to (XXVII), or an antibody or a binding region thereof, or a composition of (XXVIII) in a method for identifying or characterizing a modulator of a cell that expresses 109P1D4 Method.

  As used herein, a range is understood to specifically disclose all of its unit positions.

Exemplary embodiments of the invention disclosed herein include 109P1D4 polynucleotides (and polynucleotides complementary to such sequences) that encode specific portions of the 109P1D4 mRNA sequence (eg, polynucleotides complementary to such sequences). (A) 109P1D4 variant 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, A polynucleotide encoding a protein and / or fragment thereof of 1010, 1020, and 1021 or more contiguous amino acids; the maximum length for other variants is: variant 2, 1054 amino acids; variant 3, 1347 amino acids, (Variant 4, 1337 amino acids, variant 5, 1310 amino acids, variant 6; 1037 amino acids, variant 7; 1048 amino acids, variant 8; 1340 amino acids, and variant 9; 1037 amino acids)
In general, naturally occurring allelic variants of human 109P1D4 share a high degree of structural identity and homology (eg, greater than 90% homology). Typically, allelic variants of the 109P1D4 protein contain conservative amino acid substitutions in the 109P1D4 sequences described herein, or contain amino acid substitutions from corresponding positions in the 109P1D4 homolog. One class of 109P1D4 allelic variants shares a high degree of homology with at least a small portion of a particular 109P1D4 amino acid sequence, but fundamental deviations from that sequence (eg, non-conservative substitutions, truncations, insertions) Or a frame shift). In comparing protein sequences, the terms similarity, identity, and homology each have a distinct meaning as understood in the field of genetics. Furthermore, orthology and paralogy can be an important concept that describes the relationship of members of a given protein family in one organism to members of the same family in another organism.

  Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can often occur in a protein without altering either the protein's conformation or function. The proteins of the present invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such modifications include substitution of any of these hydrophobic amino acids for isoleucine (I), valine (V), and leucine (L); aspartic acid with glutamic acid (E) ( Substitution of D) and vice versa; substitution of glutamine (Q) with asparagine (N) and vice versa; and substitution of serine (S) with threonine (T) and vice versa. Other substitutions can also be considered conservative depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). The relatively hydrophobic methionine (M) can often be exchanged for leucine and isoleucine, and sometimes can be exchanged for valine. Lysine (K) and arginine (R) are often interchangeable at positions where an important feature of their amino acid residues is their charge and the different pKs of these two amino acid residues are not important. Still other changes may be considered “conservative” in certain circumstances (eg, Table III herein; pages 13-15 “Biochemistry”, 2nd edition, edited by Lubert Stryer (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270 (20): 11882-6).

  Embodiments of the invention disclosed herein include a wide variety of art-recognized variants or analogs of the 109P1D4 protein (eg, polypeptides having amino acid insertions, deletions, and substitutions). . 109P1D4 variants can be made using methods known in the art (eg, site-directed mutagenesis, alanine scanning, and PCR mutagenesis). Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34: 315 (1985)), restriction selective mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)), or other known techniques to produce 109P1D4 variant DNA Can be performed on the cloned DNA.

  Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence associated with a particular biological activity (eg, protein-protein interaction). Relatively small and neutral amino acids are included in preferred scanning amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically the preferred scanning amino acid in this group. This is because alanine is less likely to eliminate the side chain behind the beta carbon and alter the conformation of the variant backbone. Alanine is also typically preferred because it is the most common amino acid. In addition, alanine is often found in both buried and exposed positions (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150: 1 (1976)). If the alanine substitution does not yield a sufficient amount of modification, an equal volume of amino acids can be used.

  As defined herein, a 109P1D4 variant, analog, or homolog has the characteristic property of having at least one epitope that is “cross-reactive” with the 109P1D4 protein having the amino acid sequence of FIG. . As used in this sentence, “cross-reactivity” means that an antibody or T cell that specifically binds to a 109P1D4 variant also specifically binds to a 109P1D4 protein having the amino acid sequence shown in FIG. means. If the polypeptide does not contain an antibody that specifically binds to the starting 109P1D4 protein or any epitope that can be recognized by T cells, it will not be a variant of the protein shown in FIG. Those skilled in the art will recognize that antibodies recognizing proteins bind to epitopes of different sizes and a group of about 4 or 5 amino acids (whether contiguous or not) has a representative number of amino acids in the minimal epitope. Understand what is considered. For example, Nair et al. See Immunol 2000 165 (12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26 (9): 865-73; Schwartz et al., J Immunol (1985) 135 (4): 2598-608.

  Other classes of 109P1D4-related protein variants share more than 70%, 75%, 80%, 85%, or 90% similarity with the amino acid sequence of FIG. 3 or fragments thereof. Another specific class of 109P1D4 protein variants or analogs includes one or more of the 109P1D4 biological motifs described herein or currently known in the art. Thus, analogs of 109P1D4 fragments (nucleic acids or amino acids) with altered functional (eg, immunogenic) properties compared to the original fragment are encompassed by the present invention. It is understood that motifs that are currently part of the field or part of the field apply to the nucleic acid or amino acid sequences of FIG. 2 or FIG.

  As discussed herein, embodiments of the invention include a polypeptide comprising an amino acid sequence that is less than the full length of the 109P1D4 protein shown in FIG. 2 or FIG. For example, exemplary embodiments of the invention include any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 109P1D4 protein shown in FIG. 2 or FIG. Includes peptides / proteins with more consecutive amino acids.

  Further, exemplary embodiments of the invention disclosed herein include a polypeptide consisting of about 1 to about 10 amino acids of the 109P1D4 protein shown in FIG. 2 or FIG. 3 throughout the 109P1D4 amino acid sequence, 2 or a polypeptide consisting of about 10 to about 20 amino acids of 109P1D4 protein shown in FIG. 3, a polypeptide consisting of about 20 to about 30 amino acids of 109P1D4 protein shown in FIG. 2 or FIG. 3, FIG. 2 or FIG. A polypeptide consisting of about 30 to about 40 amino acids of 109P1D4 protein shown in FIG. 2, a polypeptide consisting of about 40 to about 50 amino acids of 109P1D4 protein shown in FIG. 2 or FIG. 3, 109P1D4 shown in FIG. 2 or FIG. About protein amino acids A polypeptide consisting of 0 to about 60 amino acids, a polypeptide consisting of about 60 to about 70 amino acids of the 109P1D4 protein shown in FIG. 2 or FIG. 3, and about 70 to about amino acids of the 109P1D4 protein shown in FIG. 2 or FIG. A polypeptide consisting of 80, a polypeptide consisting of about 80 to about 90 amino acids of the 109P1D4 protein shown in FIG. 2 or FIG. 3, and a polypeptide consisting of about 90 to about 100 amino acids of the 109P1D4 protein shown in FIG. 2 or FIG. Including peptides. Furthermore, a polypeptide consisting of about 1 amino acid (or 20 or 30 or 40 or the like) to about 20 amino acid (or 130 or 140 or 150 or the like) of the 109P1D4 protein shown in FIG. 2 or FIG. 3 is an embodiment of the present invention. is there. It should be understood that the start and stop positions in this paragraph refer to a particular position and its position plus 5 or residue minus 5 residue positions.

  109P1D4-related proteins are produced using standard peptide synthesis techniques or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode 109P1D4-related proteins. In one embodiment, the nucleic acid molecule provides a means of generating a defined fragment of 109P1D4 protein (or a variant, homolog or analog thereof).

(III. A.) Embodiment of motif-bearing protein)
Further exemplary embodiments of the invention disclosed herein are 109P1D4 polys containing amino acid residues of one or more biological motifs contained in the 109P1D4 polypeptide sequence shown in FIG. 2 or FIG. Includes peptides. Various motifs are known in the art, and proteins are available from many publicly available internet sites (eg, URL address: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc -predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; Epimatrix TM and Epimer ^™ , Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMA S, bimas.dcrt.nih.gov/) can be evaluated for the presence of such motifs.

  Motifs carrying the sequences of all 109P1D4 modified proteins are shown and identified in Tables VIII-XXI and XXII-XLIX.

  Table V shows some frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns in Table V are: (1) Motif name abbreviation, (2) Identity found between different members of this motif family (%), (3) Motif name or details, and (4) Most common The location information is included when the motif is related to location.

  Polypeptides containing one or more 109P1D4 motifs discussed above are in view of the observation that the 109P1D4 motif discussed earlier is associated with growth dysregulation and why 109P1D4 is overexpressed in certain cancers Are useful for elucidating specific features of malignant phenotypes (see, eg, Table I). Casein kinase II, cAMP and camp dependent protein kinase, and protein kinase C are, for example, enzymes known to be associated with the expression of malignant phenotypes (eg, Chen et al., Lab Invest., 78 (2 ): 165-174 (1998); Gaiddon et al., Endocrinology 136 (10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24 (6): 1119-1126 (1996); Peterziel et al., Oncogene 1846 ): 6322-6329 (1999), and O'Brian, Oncol. Rep. 5 (2): 305-309 (1998)). Furthermore, both glycosylation and myristoylation are protein modifications that are also associated with cancer and cancer progression (eg, Dennis et al., Biochem. Biophys. Acta 1473 (1): 21-34 (1999); Raju et al., Exp. Cell Res. 235 (1): 145-154 (1997)). Amidation is another protein modification that is also associated with cancer and cancer progression (see, eg, Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, the protein of the invention comprises one or more immunoreactive epitopes identified according to art recognized methods (eg, peptides shown in Tables VIII-XXI and XXII-XLIX). CTL epitopes can be determined using specific algorithms to identify peptides in the 109P1D4 protein that can optimally bind to specific HLA alleles (eg, Table IV: Epimatrix ^™ and Epimer ^™ , Brown University, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/). Furthermore, processes for identifying peptides with sufficient binding affinity for HLA molecules and associated with being immunogenic epitopes are well known in the art and without undue experimentation To be implemented. In addition, processes for identifying peptides that are immunogenic epitopes are well known in the art and are performed without undue experimentation, either in vitro or in vivo.

  The principles for making analogs of such epitopes to modulate immunogenicity are also known in the art. For example, one skilled in the art begins with an epitope having a CTL or HTL motif (see, eg, HLA class I and HLA class II motif / supermotif in Table IV). This epitope is made analog by replacing one amino acid at a particular position and replacing it with another amino acid specified for that position. For example, based on the residues defined in Table IV, one of ordinary skill in the art can substitute harmful residues in favor of any other residue (eg, a preferred residue); Can be replaced with a priority residue; or a pre-existing priority residue can be replaced with another priority residue. Substitutions can occur at major anchor positions or other positions in the peptide; see, for example, Table IV.

  Various references reflect techniques relating to the identification and generation of epitopes in proteins of interest and analogs thereof. See, eg, WO 97/33602 to Chesnut et al; Sette, Immunogenetics 1999 50 (3-4): 201-212; Sette et al. Immunol. 2001 166 (2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58 (1): 12-20; Kondo et al., Immunogenetics 1997 45 (4): 249-258; Sidney et al., J. Biol. Immunol. 1996 157 (8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255: 1261-3 (1992); Parker et al., J. Biol. Immunol. 149: 3580-7 (1992); Parker et al., J. Biol. Immunol. 152: 163-75 (1994); Kast et al., 1994 152 (8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61 (3): 266-278; Alexander et al., J. Biol. Immunol. 2000 164 (3); 164 (3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Biol. Immunol. 1991 147 (8): 2663-2669; Alexander et al., Immunity 1994 1 (9): 751-761, and Alexander et al., Immunol. Res. 1998 18 (2): 79-92.

  Related embodiments of the invention include different motifs shown in Table VI, and / or one or more putative CTL epitopes of Tables VIII-XXI and XXII-XLIX, and / or one or more putatives of Tables XLVI-XLIX Polypeptides comprising HTL epitopes and / or combinations of one or more T cell binding motifs known in the art. Preferred embodiments do not include insertions, deletions or substitutions, either within the polypeptide motif or intervening sequences. Furthermore, embodiments that include any of a number of N-terminal and / or C-terminal amino acid residues on either side of these motifs may be desirable (eg, a larger portion of the polypeptide structure in which the motif is located). To include). Typically, the number of N-terminal and / or C-terminal amino acid residues on either side of the motif is between about 1 and about 100 amino acid residues, preferably between 5 and about 50 amino acid residues. .

  109P1D4-related proteins are embodied in many forms, preferably in isolated forms. A purified 109P1D4 protein molecule is substantially free of other proteins or molecules that impair 109P1D4 binding to antibodies, T cells or other ligands. The nature and degree of isolation and purification will depend on the intended use. Embodiments of 109P1D4-related proteins include purified 109P1D4-related proteins and functional soluble 109P1D4-related proteins. In one embodiment, the functional soluble 109P1D4 protein or fragment thereof maintains the ability to be bound by antibodies, T cells or other ligands.

  The present invention also provides a 109P1D4 protein comprising a biologically active fragment of the 109P1D4 amino acid sequence shown in FIG. 2 or FIG. Such proteins exhibit the properties of the starting 109P1D4 protein as follows: the ability to induce the production of antibodies that specifically bind to epitopes associated with the starting 109P1D4 protein; the ability to bind to such antibodies; HTL Or the ability to induce activation of CTL; and / or the ability to be recognized by HTL or CTL that also specifically binds to this starting protein.

  109P1D4-related polypeptides containing specific interesting structures include various analytical techniques well known in the art (eg, Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis methods). And can be predicted and / or identified based on immunogenicity. Fragments containing such structures are particularly useful in the production of subunit-specific anti-109P1D4 antibodies or T cells or in the identification of cellular factors that bind to 109P1D4. For example, a hydrophilic profile can be generated and immunogenic peptide fragments can be obtained from Hopp, T .; P. And Woods, K .; R. , 1981, Proc. Natl. Acad. Sci. U. S. A. 78: 3824-3828 can be identified. Hydropathic profiles can be generated and immunogenic peptide fragments are described in Kyte, J. et al. And Doolittle, R .; F. , 1982, J. et al. Mol. Biol. 157: 105-132. A profile of% accessible residues can be generated and immunogenic peptide fragments can be obtained from Janin J. et al. 1979, Nature 277: 491-492. An average flexibility profile can be generated and Bhaskaran R. Ponuswamy P. K. , 1988, Int. J. et al. Pept. Protein Res. The method of 32: 242-255 can be used to identify immunogenic peptide fragments. A β-turn profile can be generated and immunogenic peptide fragments can be obtained as described in Release, G. et al. , Roux B. , 1987, Protein Engineering 1: 289-294.

CTL epitopes can be identified using a specific algorithm (eg, World Wide Web URL symphpeithi.bmi-heidelberg.) To identify polypeptides within the 109P1D4 protein that can optionally bind to designated HLA alleles. com / SYFPEITHI site; Tables IV (A)-(E); Epimatrix ^™ and Epimer ^™ , Brown University, URL (brown. edu / Research / TB-HIV_Lab / epimatr. URL bimas.dcrt.nih.gov/). To illustrate this, 109P1D4-derived peptide epitopes (eg, HLA-A1, A2, A3, A11, A24, B7 and B35) shown in the context of human MHC class I molecules were predicted (eg, Table VIII- See XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 109P1D4 protein and related portions of other variants (ie, for the prediction of HLA class I, located on either side of the point mutation or exon junction corresponding to this variant) 9 neighboring residues, and for HLA class II prediction, 14 neighboring residues located on either side of the point mutation or exon junction) can be found on the Bioinformatics and Molecular Analysis Section (BIMAS) website (URL The HLA Peptide Motif Search algorithm found at (in addition to the site SYFPEITHI at syffpeithi.bmi-heidelberg.com/).

  This HLA peptide motif search algorithm is based on the binding of specific peptide sequences in HLA class I molecules, particularly HLA-A2 grooves. Developed by Ken Parker (eg, Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255: 1261-3 (1992)); Parker et al., J. Immunol. 149: 3580-7 (1992) Parker et al., J. Immunol. 152: 163-75 (1994)). This algorithm locates and ranks 8-mer, 9-mer, and 10-mer peptides from the complete protein sequence for predicted binding to HLA-A2 and many other HLA class I molecules. enable. A number of HLA class I binding peptides are 8-mer, 9-mer, 10-mer or 11-mer. For example, for class I HLA-A2, the epitope preferably includes leucine (L) or methionine (M) at position 2 and valine (V) or leucine (L) at the C-terminus (eg, Parker et al. J. Immunol. 149: 3580-7 (1992)). The results of selecting 109P1D4 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, the 9-mer and 10-mer that are candidates for selection for each family member are shown along with their position, the amino acid sequence of each particular peptide, and the putative binding score. In Tables XLVI-XLIX, the 15-mers that are candidates for selection for each family member are shown along with their position, the amino acid sequence of each particular peptide, and a putative binding score. This binding score corresponds to the estimated half-life of dissociation of the complex containing the peptide at 37 ° C. and pH 6.5. The peptide with the highest binding score is predicted to be bound most closely to HLA class I on the cell surface over the best period and represent the best immunogenic target for T cell recognition.

  The actual binding of the peptide to the HLA allele can be assessed by stabilizing HLA expression on the antigen processing deficient cell line T2 (eg, Xue et al., Prostate 30: 73-8 (1997) and Peshwa et al. Prostate 36: 129-38 (1998)). The immunogenicity of a particular peptide can be assessed in vitro by stimulating CD8 + cytotoxic T lymphocytes (CTLs) in the presence of antigen presenting cells (eg, dendritic cells).

By HLA class I motifs or HLA class II motifs predicted by BIMAS sites, Epimer ^TM sites and Epimatrix ^TM sites or available in the field or part of the fields as shown in Table IV All epitopes specified according to the invention (or determined using the World Wide Web site URL symphpeithi.bmi-heidelberg.com/ or BIMAS, bimas.dcrt.nih.gov /) are in accordance with the present invention 109P1D4 protein It is understood that “applies” to: As used in this context, “applied” means that the 109P1D4 protein is evaluated by computer-based pattern recognition methods, eg, visually or as understood by one skilled in the art. All partial sequences of 109P1D4 protein of 8, 9, 10 or 11 amino acid residues carrying the HLA class I motif, or partial sequences of 9 amino acid residues or more carrying the HLA class II motif are within the scope of the present invention. Is within.

(III.B.) Expression of 109P1D4-related protein)
In the embodiments described in the Examples below, 109P1D4 is expressed using commercially available expression vectors (eg, C-terminal 6 × His and MYC tag (pcDNA3.1 / mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). It can be conveniently expressed in cells (eg, 293T cells) transfected with a CMV driven expression vector encoding 109P1D4. The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of secreted 109P1D4 protein in transfected cells. Secreted HIS-tagged 109P1D4 in the culture medium can be purified using, for example, a nickel column using standard techniques.

(III.C.) 109P1D4-related protein modification)
Modifications of 109P1D4-related proteins (eg, covalent modifications) are included within the scope of the present invention. One type of covalent modification involves reacting a targeted amino acid residue of a 109P1D4 polypeptide with an organic derivatization factor (a selected side chain residue or N-terminal or C-terminal residue of 109P1D4 protein). And reacting with). Another type of covalent modification of the 109P1D4 polypeptide included within the scope of the invention involves altering the native glycosylation pattern of the protein of the invention. Another type of covalent modification of 109P1D4 is the modification of 109P1D4 polypeptide to one of a variety of non-proteinaceous polymers (eg, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene). No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; No. 4,791,192; or No. 4,179 , 337, and the like.

  The 109P1D4-related proteins of the present invention can also be modified to form a chimeric molecule that comprises 109P1D4 fused to another heterologous polypeptide or amino acid sequence. Such chimeric molecules can be synthesized chemically or recombinantly. A chimeric molecule may have a protein of the invention fused to another tumor associated antigen or fragment thereof. Alternatively, a protein according to the invention may comprise a fusion (amino acid or nucleic acid) of a fragment of the 109P1D4 sequence, so that over its length, it is directly homologous to the amino acid or nucleic acid sequence shown in FIG. 2 or FIG. Not a molecule is created. Such a chimeric molecule can comprise multiple identical partial sequences of 109P1D4. The chimeric molecule can comprise a fusion of a 109P1D4-related protein with a polyhistidine epitope tag, which provides a cytokine or growth factor for the epitope to which immobilized nickel can selectively bind. The epitope tag is generally placed at the amino terminus or carboxyl terminus of the 109P1D4 protein. In an alternative embodiment, the chimeric molecule may comprise a fusion of a 109P1D4-related protein with an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of the chimeric molecule (also referred to as "immunoadhesin"), such a fusion may be to the Fc region of an IgG molecule. This Ig fusion preferably comprises a substitution with a soluble (deleted or inactivated transmembrane domain) form of 109P1D4 polypeptide in place of at least one variable region within the Ig molecule. In a preferred embodiment, the immunoglobulin fusion comprises the hinge region, CH2 region and CH3 region, or hinge region, CH1 region, CH2 region and CH3 region of an IgGI molecule. For the production of immunoglobulin fusions, see, for example, US Pat. No. 5,428,130 (issued June 27, 1995).

(III.D.) Use of 109P1D4-related protein)
The proteins of the present invention have many different specific uses. When 109P1D4 is highly expressed in prostate cancer and other cancers, 109P1D4-related proteins are used in methods of assessing the status of normal 109P1D4 gene products against cancer tissue, thereby revealing the malignant phenotype Become. Typically, polypeptides derived from specific regions of the 109P1D4 protein assess the presence of perturbations (eg, deletions, insertions, point mutations, etc.) in those regions (eg, regions containing one or more motifs) Used to do. An exemplary assay is one or more biologicals included in an antibody, or 109P1D4 polypeptide sequence, to assess normal region characteristics against cancerous tissue or to elicit an immune response against an epitope. A T cell targeted 109P1D4-related protein containing the amino acid residues of the motif is used. Alternatively, 109P1D4 related proteins containing one or more biological motif amino acid residues in 109P1D4 protein are used to screen for factors that interact with the 109P1D4 region.

  109P1D4 protein fragments / subsequences are agents that bind to 109P1D4 or a particular structural domain thereof in creating and characterizing domain specific antibodies (eg, antibodies that recognize an extracellular or intracellular epitope of 109P1D4 protein) It is particularly useful for identifying cellular factors and in a variety of therapeutic and diagnostic situations, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

  Proteins encoded by the 109P1D4 gene, or proteins encoded by analogs, homologues or fragments thereof, have a variety of uses, including the production of antibodies and ligands that bind to the 109P1D4 gene product. Methods include but are not limited to methods for identifying other drugs and cell constructs. Antibodies raised against 109P1D4 protein or fragments thereof are imaging methods in diagnostic and prognostic assays, and management of human cancers characterized by expression of 109P1D4 protein (eg, cancers as listed in Table I) Useful for. Such antibodies can be expressed intracellularly and used in methods for treating patients with such cancers. 109P1D4-related nucleic acids or 109P1D4-related proteins are also used in generating HTL or CTL responses.

  Various immunological assays useful for detecting 109P1D4 protein are used, including various types of radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay ( ELIF), immunocytochemical methods and the like, but are not limited thereto. The antibody can be labeled and used as an immunological imaging reagent capable of detecting 109P1D4-expressing cells (eg, in radioscintigraphic imaging methods). The 109P1D4 protein is also particularly useful in making cancer vaccines, as further described herein.

(IV.) 109P1D4 antibody)
Another aspect of the invention provides an antibody that binds to a 109P1D4-related protein. Preferred antibodies specifically bind to 109P1D4-related proteins and do not bind (or bind weakly) to peptides or proteins that are not 109P1D4-related proteins. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline ( 0.9% NaCl); 4) animal serum (eg, human serum); or 5) any combination of 1) -4); preferably these reactions have a pH of 7.5, or a pH of 7.0. To be carried out in the range of ~ 8.0, or in the range of pH 6.5 to 8.5; and these reactions are carried out at a temperature between 4C and 37C. For example, an antibody that binds to 109P1D4 can bind to a 109P1D4 related protein (eg, a homolog or analog thereof).

  The 109P1D4 antibodies of the invention are particularly useful in diagnostic and prognostic assays for cancer (see, eg, Table I), and imaging methodologies. Similarly, such antibodies are useful in treating, diagnosing and / or prognosing other cancers to the extent that 109P1D4 is also expressed or overexpressed in these other cancers. . In addition, antibodies that are expressed intracellularly (eg, single chain antibodies) are therapeutically useful in the treatment of cancers that involve 109P1D4 expression (eg, advanced or metastatic prostate cancer).

  The present invention also provides various immunological assays useful for the detection and quantification of 109P1D4 protein and mutant 109P1D4 related proteins. Such assays can include one or more 109P1D4 antibodies, where appropriate, that can recognize and bind 109P1D4-related proteins. These assays can be performed within a variety of immunological assay formats well known in the art, including various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISAs), enzymes Examples include, but are not limited to, a binding immunofluorescence assay (ELIFA).

  The immunological non-antibody assays of the present invention also include T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

  In addition, immunological imaging methods that can detect prostate and other cancers that express 109P1D4 are also provided by the present invention, including radionuclide imaging methods that use labeled 109P1D4 antibodies. Although it is mentioned, it is not limited to these. Such assays are clinically useful in the detection, monitoring, and prognosis of cancers that express 109P1D4 (eg, prostate cancer).

  The 109P1D4 antibody can also be used in methods for purifying 109P1D4-related proteins, as well as methods for isolating 109P1D4 homologs and 109P1D4-related molecules. For example, a method for purifying 109P1D4-related protein includes the following steps: 109P1D4 antibody bound to a solid matrix, together with lysate or other solution containing 109P1D4-related protein, 109P1D4 antibody becomes 109P1D4-related protein. Incubating under conditions capable of binding; washing the solid matrix to remove impurities; and eluting 109P1D4-related protein from the bound antibody. Other uses of the 109P1D4 antibody according to the present invention include making anti-idiotype antibodies that mimic the 109P1D4 protein.

  Various methods for preparing antibodies are well known in the art. For example, antibodies can be prepared in isolated or immunoconjugate form by immunizing a suitable mammalian host using 109P1D4-related proteins, peptides, or fragments (Antibodies: A Laboratory Manual, CSH Press, Hen, Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, 109P1D4 fusion proteins such as 109P1D4 GST fusion proteins can also be used. In certain embodiments, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is generated and then used as an immunogen to generate a suitable antibody. In another embodiment, 109P1D4-related protein is synthesized and used as an immunogen.

  In addition, naked DNA immunization techniques known in the art are used (with or without purified 109P1D4-related protein or 109P1D4-expressing cells) to generate an immune response against the encoded immunogen (as a review). (See Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

  A particular region of the 109P1D4 protein can be selected for generating antibodies by analyzing the amino acid sequence of the 109P1D4 protein shown in FIG. 2 or FIG. For example, hydrophobicity analysis and hydrophilicity analysis of the 109P1D4 amino acid sequence is used to identify hydrophilic regions in the 109P1D4 structure. Regions of the 109P1D4 protein that exhibit immunogenic structure, as well as other regions and domains, can be obtained from various other methods known in the art (eg, Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson). -Wolf analysis) can be easily identified. The hydrophilic profile is described in Hopp, T .; P. And Woods, K .; R. , 1981, Proc. Natl. Acad. Sci. U. S. A. 78: 3824-3828 can be used. Hydropathic profiles are described in Kyte, J. et al. And Doolittle, R .; F. , 1982, J. et al. Mol. Biol. 157: 105-132. The accessible percent (%) residue profile is given by Janin J. et al. 1979, Nature 277: 491-492. The average flexibility profile was determined by Bhaskaran R.C. Ponuswamy P. K. , 1988, Int. J. et al. Pept. Protein Res. 32: 242-255. The β-turn profile is described in Release, G. et al. , Roux B. , 1987, Protein Engineering 1: 289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for making 109P1D4 antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of a carrier (eg, BSK, KLH or other carrier protein) and protein are also well known in the art. In some situations, for example, direct conjugation using a carbodiimide reagent is used; in other examples, Pierce Chemical Co. Binding reagents supplied by Rockford, IL are effective. Administration of the 109P1D4 immunogen is often performed by injection using an appropriate adjuvant over an appropriate period of time, as is understood in the art. During the immunization schedule, antibody titers can be collected to determine the validity of antibody formation.

  The 109P1D4 monoclonal antibody can be produced by various means well known in the art. For example, an immortalized cell line that secretes the desired monoclonal antibody is prepared using standard hybridoma technology from Kohler and Milstein, or a modification that immortalizes antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibody are screened by immunoassay (in this assay the antigen is a 109P1D4-related protein). If a suitable immortalized cell culture is identified, the cells can be expanded and antibodies can be produced from either in vitro culture or ascites.

  The antibodies or fragments of the invention can also be produced by recombinant means. Regions that specifically bind to the desired region of the 109P1D4 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies from multiple species. Humanized 109P1D4 antibodies or human 109P1D4 antibodies can also be produced and are preferred for use in therapeutic situations. Methods for humanizing mouse antibodies and other non-human antibodies by replacing one or more non-human antibody CDRs with the corresponding human antibody sequences are well known (eg, Jones et al., 1986, Nature). 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285, and Sims et al., 1993, J. MoI. Immunol. See also 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display methods and transgenic methods (for a review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 109P1D4 monoclonal antibodies can be generated using cloning techniques using large human Ig gene combinatorial libraries (ie, phage display) (Protein Engineering of Antibiotic for Proliferative and Therapeutic Applications, Therapeutic Applications, Therapeutic Applications, Therapeutic Applications, Ed.), Nottingham Academic, pages 45-64 (1993), Griffiths and Hoogenboom, Building an in vitro immune.
system: human antigens from phage display libraries; Burton and Barbas, Human Antibodies from combinatorial libraries (ibid.) pp. 65-82). The fully human 109P1D4 monoclonal antibody is also a transgenic engineered to contain human immunoglobulin loci as described in PCT patent application WO 98/24893 (Kucherlapati and Jakobovits et al.) Published December 3, 1997. Can be produced using mice (Jakobovits, 1998, Exp. Opin. Invest. Drugs 7 (4): 607-614; US Pat. No. 6,162,963 (issued December 19, 2000); See also 6,150,584 (issued 12 November 2000); and US Pat. No. 6,114,598 (issued 5 September 2000)). This method avoids the in vitro manipulation required for phage display technology and efficiently produces high affinity authentic human antibodies.

  The reactivity of the 109P1D4 antibody with 109P1D4 related proteins can be established by a number of well-known methods including, as appropriate, using 109P1D4 related proteins, 109P1D4 expressing cells or extracts thereof, Immunoprecipitation, ELISA, and FACS analysis are included. The 109P1D4 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators or enzymes. In addition, bi-specific antibodies specific for two or more 109P1D4 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be made by cross-linking techniques known in the art (eg, Wolff et al., Cancer Res. 53: 2560-2565).

(V.) 109P1D4 cell immune response)
The mechanism by which T cells recognize antigens has been elucidated. An effective peptide epitope vaccine composition of the present invention induces a therapeutic or prophylactic immune response in a very broad segment of the worldwide population. In order to understand the value and efficacy of the compositions of the present invention that induce cellular immune responses, a brief review of immunology related techniques is provided.

  Complexes of HLA molecules and peptide antigens act as ligands recognized by HLA-restricted T cells (Bus, S et al., Cell 47: 1071, 1986; Babbitt, BP et al., Nature 317: 359, 1985). Townsend, A. and Bodmer, H., Annu. Rev. Immunol.7: 601, 1989; German, RN, Annu.Rev. Immunol. Through sequencing studies of antigenic analogs with single amino acid substitutions and endogenously processed naturally processed peptides, key residues corresponding to motifs required for specific binding to HLA antigen molecules are identified And described in Table IV (see, eg, Southwood et al., J. Immunol. 160: 3363, 1998; Ramsensee et al., Immunogenetics 41: 178, 1995; Ramsensee et al., SYFPEITHI (URL 134. via the World Wide Web). 2.96.221 / scripts.hlaserber.dll / home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10: 478, 1998; gelhard, V.H., Curr.Opin.Immunol.6: 13,1994; Sette, A. and Grey, HM, Curr.Opin.Immunol.4: 79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol.6: 52, 1994; Ruppert et al., Cell 74: 929-937, 1993; Kondo et al., J. Immunol.155: 4307-4312, 1995; Sidney et al., J. Immunol. 3490, 1996; Sidney et al., Human Immunol. 45: 79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 Nov; 50 (3-4): 201-12, total That of the reference).

  Furthermore, X-ray crystallographic analysis of the HLA-peptide complex reveals a pocket within the peptide binding gap / groove of the HLA molecule that accommodates residues carried by the peptide ligand in an allele-specific manner; These residues determine the HLA binding ability of the peptide in which this residue is present (eg, Madden, DR Annu. Rev. Immunol. 13: 587, 1995; Smith et al., Immunity 4: 203, 1996). Fremont et al., Immunity 8: 305, 1998; Stern et al., Structure 2: 245, 1994; Jones, EY Curr. Opin.Immunol. 33, 1993; Guo, H .; USA 90: 8053, 1993; Guo, HC et al., Nature 360: 364, 1992; Silver, ML, et al., Nature 360: 367, 1992; M. et al., Science 257: 927, 1992; Madden et al., Cell 70: 1035, 1992; Fremont, DH et al. , DC, J. Mol. Biol. 219: 277, 1991).

  Thus, the definition of class I and class I allele-specific HLA binding motifs, or class I or class II supermotifs, allows the identification of regions within proteins that are associated with binding to specific HLA antigens.

  Thus, the identification process of HLA motifs has identified candidate epitope-based vaccines; such candidates determine the binding affinity and / or duration of the association of the epitope with its corresponding HLA molecule. In addition, it can be further evaluated by an HLA-peptide binding assay. From these vaccine candidates, further validation experiments can be performed to select epitopes with favorable characteristics in terms of population coverage and / or immunogenicity.

A variety of strategies can be used to assess cellular immunogenicity, including the following:
1) Evaluation of primary T cell cultures from normal individuals (eg, Wentworth, PA et al., Mol, Immunol. 32: 603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA). 91: 2105, 1994; Tsai, V. et al., J. Immunol. 158: 1796, 1997; Kawashima, I. et al., Human Immunol. 59: 1, 1998). This procedure involves stimulating peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells for a period of several weeks in vitro. T cells specific for this peptide are activated during this time and detected using, for example, a lymphokine release assay or ⁵¹ Cr release assay involving target cells sensitized with the peptide.

2) Immunization of HLA transgenic mice (eg Wentworth, PA et al., J. Immunol. 26:97, 1996; Wentworth, PA et al., Int. Immunol. 8: 651, 1996; Alexander, J Et al., J. Immunol. 159: 4753, 1997). For example, in such a method, peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks after immunization, splenocytes are removed and cultured in vitro in the presence of the test peptide for about a week. Peptide-specific T cells are detected using, for example, a ⁵¹ Cr release assay that includes target cells sensitized with peptides and target cells that express endogenously generated antigens.

3) Demonstration of recall T cell responses from either effectively vaccinated immunized individuals and / or chronically ill patients (eg, Rehermann, B. et al., J. Exp. Med. 181: 1047, 1995; Doolan, DL, et al. Immunity.
7:97, 1997; Bertoni, R .; Et al. Clin. Invest. 100: 503, 1997; Threlkeld, S .; C. Et al. Immunol. 159: 1648, 1997; M.M. Et al. Virol. 71: 6011, 1997). Thus, a recall response is obtained by culturing PBL from a subject that has been exposed to an antigen due to disease and thus produced a “naturally” immune response, or from a patient vaccinated against the antigen. Detected. Subject-derived PBLs are cultured in vitro for 1-2 weeks in the presence of test peptide + antigen presenting cells (APCs) and can activate "memory" T cells compared to "unstimulated" T cells To. At the end of this culture period, T cell activity is detected using assays involving ⁵¹ Cr release, T cell proliferation or lymphokine release including peptides sensitized targets.

(VI.) 109P1D4 transgenic animal)
Nucleic acids encoding 109P1D4-related proteins can also be used to create either transgenic or “knock-out” animals, which are then useful in the development and screening of therapeutically useful reagents. . By established techniques, cDNA encoding 109P1D4 can be used to clone genomic DNA encoding 109P1D4. This cloned genomic sequence can then be used to create transgenic animals containing cells that express DNA encoding 109P1D4. Methods for producing transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in US Pat. No. 4,736,866 (April 12, 1988). And No. 4,870,009 (issued on Sep. 26, 1989). Typically, specific cells are targeted for 109P1D4 transgene integration using a tissue-specific enhancer.

  Transgenic animals containing one copy of the transgene encoding 109P1D4 can be used to test the effect of increasing the expression of DNA encoding 109P1D4. Such animals can be used, for example, as tester animals for reagents believed to confer protection from pathological conditions associated with its overexpression. According to this aspect of the invention, the animal is treated with a reagent and the reduced incidence of pathological condition compared to an untreated animal with the transgene is a potential for this pathological condition. Indicates therapeutic intervention.

  Alternatively, a non-human homologue of 109P1D4 can be used to construct a 109P1D4 “knockout” animal, which 109P1D4 “knockout” animal has an endogenous gene encoding 109P1D4 and 109P1D4 introduced into the embryonic cells of the animal. As a result of homologous recombination with the altered genomic DNA encoding, have a defective or altered gene encoding 109P1D4. For example, cDNA encoding 109P1D4 can be used to clone genomic DNA encoding 109P1D4 according to established techniques. A portion of the genomic DNA encoding 109P1D4 can be deleted or replaced with another gene (eg, a gene encoding a selectable marker that can be used to monitor integration). Typically, several kilobases of unaltered flanking DNA (both 5 'and 3' ends) are included in the vector (see, eg, Thomas and Capecchi, for a description of homologous recombination vectors). Cell, 51: 503 (1987)). This vector is introduced into an embryonic stem cell line (eg, by electroporation), and cells in which the introduced DNA is homologously recombined with endogenous DNA are selected (eg, Li et al., Cell, 69: 915 (1992)). The selected cells are then injected into an animal (eg, mouse or rat) blastocyst to form an aggregate chimera (eg, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Edited by Robertson (IRL, Oxford, 1987), pp. 113-152). The chimeric embryo can then be transferred to a suitable pseudopregnant female surrogate mother and the embryo is born to create a “knockout” animal. Offspring with DNA homologously recombined in germ cells can be identified by standard techniques and used to breed animals that contain DNA in which all cells are homologous recombination. Knockout animals can be characterized, for example, for the ability to protect against a particular pathological condition or for the development of a pathological condition resulting from the absence of 109P1D4 polypeptide.

(VII.) 109P1D4 Detection Method)
Another aspect of the invention relates to methods for detecting 109P1D4 polynucleotides and 109P1D4-related proteins, and methods for identifying cells that express 109P1D4. The expression profile of 109P1D4 makes 109P1D4 a diagnostic marker for metastatic disease. Thus, the status of the 109P1D4 gene product provides useful information for estimating a variety of factors including susceptibility to advanced stage disease, rate of progression, and / or tumor aggressiveness. As discussed in detail herein, the status of 109P1D4 gene product in a patient sample can be analyzed by various protocols well known in the art including immunohistochemical analysis, various Northern Blot techniques (including in situ hybridization), RT-PCR analysis (eg, for laser captured microdissection samples), Western blot analysis and tissue array analysis.

  More particularly, the present invention provides assays for the detection of 109P1D4 polynucleotides in biological samples (eg, serum, bone, prostate and other tissues, urine, semen, cell preparations, etc.). Detectable 109P1D4 polynucleotides include, for example, recombinant DNA or RNA molecules comprising 109P1D4 gene or fragments thereof, 109P1D4 mRNA, alternative splice variants 109P1D4 mRNA, and 109P1D4 polynucleotides. Numerous methods for amplifying 109P1D4 polynucleotide and / or detecting the presence of 109P1D4 polynucleotide are well known in the art and can be used in the practice of this aspect of the invention.

  In one embodiment, a method for detecting 109P1D4 mRNA in a biological sample uses at least one primer to generate cDNA from this sample by reverse transcription; 109P1D4 as a sense primer and an antisense primer. Using the polynucleotide to amplify the cDNA thus generated to amplify 109P1D4 cDNA therein; and detecting the presence of the amplified 109P1D4 cDNA. If necessary, the sequence of this amplified 109P1D4 cDNA can be determined.

  In another embodiment, a method of detecting 109P1D4 gene in a biological sample first comprises isolating genomic DNA from the sample; using 109P1D4 polynucleotides as sense and antisense primers, Amplifying the isolated genomic DNA; and detecting the presence of the amplified 109P1D4 gene. Any number of suitable sense and antisense probe combinations can be designed from the 109P1D4 nucleotide sequence (see, eg, FIG. 2) and used for this purpose.

  The present invention also provides an assay for detecting the presence of 109P1D4 protein in a tissue or other biological sample (eg, serum, semen, bone, prostate, urine, cell preparation, etc.). Methods for detecting 109P1D4-related proteins are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA, and the like. For example, a method for detecting the presence of a 109P1D4-related protein in a biological sample first comprises: Contacting; then detecting the binding of 109P1D4-related protein in the sample.

  Methods for identifying cells that express 109P1D4 are also within the scope of the present invention. In one embodiment, the assay for identifying a cell that expresses a 109P1D4 gene comprises detecting the presence of 109P1D4 mRNA in the cell. Methods for detecting specific mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (eg, in situ hybridization using labeled 109P1D4 riboprobe, Northern blot and Related technology), and various nucleic acid amplification assays (eg, RT-PCR using complementary primers specific for 109P1D4, and other amplified detection methods (eg, branched DNA, SISBA, TMA, etc.) )). Alternatively, an assay for identifying a cell that expresses a 109P1D4 gene includes detecting the presence of a 109P1D4-related protein that is present in or secreted by the cell. Various methods for the detection of proteins are well known in the art and are used for the detection of 109P1D4-related proteins and for the detection of cells expressing 109P1D4-related proteins.

  109P1D4 expression analysis is also useful as a tool for identifying and evaluating factors that modulate 109P1D4 gene expression. For example, 109P1D4 expression is significantly upregulated in prostate cancer and is expressed in cancers of tissues listed in Table I. Identification of molecules or biological factors that inhibit 109P1D4 expression or 109P1D4 overexpression in cancer cells has therapeutic value. For example, such factors can be identified by using screens that quantify 109P1D4 expression by RT-PCR, nucleic acid hybridization, or antibody binding.

(VIII.) 109P1D4-related genes and methods for monitoring the status of their products)
Tumor formation is known to be a multi-step process in which cell growth becomes progressively dysregulated and cells progress from a normal physiological state to a pre-cancerous state and then to a cancerous state (eg, Allers Et al., Lab Invest. 77 (5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this situation, testing biological samples for evidence of dysregulated cell growth (eg, abnormal 109P1D4 expression in cancer) makes pathological conditions such as cancer more restrictive for treatment options It is possible to detect such abnormal physiology early before proceeding to a stage and / or before prognosis worsens. In such a test, the status of 109P1D4 in the biological sample of interest is, for example, in the corresponding normal sample (eg, a sample from this individual or another alternative individual not affected by pathology). It can be compared with the state of 109P1D4. Changes in the status of 109P1D4 in biological samples (as compared to normal samples) provide evidence of dysregulated cell growth. In addition to using a biological sample unaffected by pathology as a normal sample, a predetermined normative value (eg, a predetermined normal level of mRNA expression) (eg, Grever et al., J. Comp. Neurol). Dec. 9, 1996; 376 (2): 306-14 and US Pat. No. 5,837,501) can also be used to compare the status of 109P1D4 in samples.

  In this context, the term “state” is used according to the art recognized meaning and refers to the condition or state of a gene and its products. Typically, one of skill in the art uses a number of parameters to assess the condition or state of a gene and its product. This includes the location of the expressed gene product (including the location of 109P1D4 expressing cells), as well as the level and biological activity of the expressed gene product (eg, 109P1D4 mRNA, polynucleotides and polypeptides). However, it is not limited to these. Typically, the change in 109P1D4 status comprises a change in the location of 109P1D4 and / or 109P1D4 expressing cells and / or increased expression of 109P1D4 mRNA and / or 109P1D4 protein.

  The status of 109P1D4 in the sample can be analyzed by a number of means well known in the art, including immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser-captured microdissection samples, Western blot analysis and Examples include, but are not limited to, tissue array analysis. Representative protocols for assessing the status of the 109P1D4 gene and gene products are described, for example, in Ausubel et al., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunton). 18 (PCR Analysis). Thus, the status of 109P1D4 in a biological sample can be assessed by various methods used by those skilled in the art, including genomic Southern analysis (eg, to test perturbation in the 109P1D4 gene). ), Northern analysis and / or PCR analysis of 109P1D4 mRNA (eg, to test changes in polynucleotide sequence or expression level of 109P1D4 mRNA), and Western analysis and / or immunohistochemical analysis (eg, changes in polypeptide sequence) To test for changes in the localization of the polypeptide in the sample, changes in the expression level of 109P1D4 protein, and / or association of 109P1D4 protein with the polypeptide binding partner) Although not mentioned, it is not limited to these. Detectable 109P1D4 polynucleotides include, for example, 109P1D4 gene or fragments thereof, 109P1D4 mRNA, alternative splice variants, 109P1D4 mRNA, and recombinant DNA molecules or recombinant RNA molecules comprising 109P1D4 polynucleotides.

  The expression profile of 109P1D4 makes 109P1D4 a diagnostic marker for local and / or metastatic disease and provides information about the potential for growth or tumor formation of biological samples. In particular, 109P1D4 status provides information useful for estimating susceptibility to a particular disease stage, progression and / or tumor aggressiveness. The present invention provides methods and assays for determining the status of 109P1D4 and diagnosing cancers that express 109P1D4 (eg, cancers of tissues listed in Table I). For example, since 109P1D4 mRNA is highly expressed in prostate and other cancers compared to normal prostate tissue, an assay that evaluates the level of 109P1D4 mRNA transcript or 109P1D4 protein in a biological sample is 109P1D4. Can be used to diagnose diseases associated with dysregulation of cancer and provide prognostic information useful in defining appropriate treatment options.

  109P1D4 expression status assesses information to predict susceptibility to various stages of disease, including the presence, stage and location of dysplastic cells, precancerous cells and cancer cells, and / or tumor aggressiveness Provide information for Furthermore, the expression profile makes 109P1D4 useful as an imaging reagent for metastatic disease. Consequently, an aspect of the invention is a sample from an individual suffering from a biological sample (eg, a pathology characterized by dysregulated cell growth (eg, cancer), or affected by such a pathology. The present invention relates to various molecular prognostic methods and molecular diagnostic methods for testing the status of 109P1D4 in samples from individuals suspected of being

  As described above, the status of 109P1D4 in a biological sample can be tested by a number of procedures well known in the art. For example, the status of 109P1D4 in a biological sample taken from a particular location in the body evaluates this sample for the presence or absence of 109P1D4 expressing cells (eg, cells expressing 109P1D4 mRNA or 109P1D4 protein). Can be tested. This test can provide evidence of dysregulated cell growth, for example, if 109P1D4 expressing cells are found in biological samples that normally do not contain such cells (eg, lymph nodes) . This is because such changes in the state of 109P1D4 in biological samples are often associated with dysregulated cell growth. In particular, one indication of dysregulated cell growth is metastasis of cancer cells from an organ of origin (eg, prostate) to a different region of the body (eg, lymph nodes). In this situation, evidence of dysregulated cell growth is important. This is because, for example, in a significant proportion of patients with prostate cancer, latent lymph node metastases can be detected and such metastases are associated with known predictors of disease progression (eg, Murphy et al., Prostate 42 (4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18 (1): 17-28 (2000) and Freeman et al., J Urol 1995 Aug 154 (2 Pt 1). ): See 474-8).

  In one aspect, the present invention determines the status of a 109P1D4 gene product expressed by cells from an individual suspected of having a disease (eg, hyperplasia or cancer) associated with dysregulated cell proliferation, A method for monitoring 109P1D4 gene product is provided by comparing the status determined in this way to the status of 109P1D4 gene product in a corresponding normal sample. The presence of an abnormal 109P1D4 gene product in the test sample compared to a normal sample provides an indication of the presence of dysregulated cell proliferation in the individual's cells.

  In another aspect, the present invention provides an assay useful in determining the presence of cancer in an individual, wherein the assay is a test cell sample compared to the expression level in corresponding normal cells or normal tissues. Or detecting a significant increase in the expression of 109P1D4 mRNA or protein in the test tissue sample. For example, the presence of 109P1D4 mRNA can be assessed in tissues including but not limited to the tissue samples listed in Table I. The presence of significant 109P1D4 expression in any of these tissues is useful to indicate the development, presence and / or severity of cancer. This is because the corresponding normal tissue does not express 109P1D4 mRNA or only expresses it at a low level.

  In related embodiments, 109P1D4 status is determined at the protein level rather than at the nucleic acid level. For example, such methods determine the level of 109P1D4 protein expressed by cells in a test tissue sample and compare the level thus determined to the level of 109P1D4 expressed in the corresponding normal sample. Process. In one embodiment, the presence of 109P1D4 protein is assessed using, for example, immunohistochemical methods. A 109P1D4 antibody or 109P1D4 binding partner capable of detecting 109P1D4 protein expression is used for this purpose in various assay formats well known in the art.

  In further embodiments, the status of 109P1D4 nucleotide and 109P1D4 amino acid sequences in a biological sample can be evaluated to identify variations in the structure of these molecules. These variations can include insertions, deletions, substitutions, and the like. Such an evaluation is useful. Because variations in nucleotide and amino acid sequences are observed in a number of proteins associated with a growth dysregulated phenotype (eg, Marrogi et al., 1999, J. Cutan. Pathol. 26 (8): 369-378). For example, mutations in the 109P1D4 sequence may indicate the presence of a tumor or an oncogenic aid. Thus, such assays have diagnostic and predictive value when mutations in 109P1D4 indicate potential loss of function or increased tumor growth.

  A wide variety of assays for observing variations in nucleotide and amino acid sequences are well known in the art. For example, the nucleic acid or amino acid sequence size and structure of the 109P1D4 gene product is observed by the Northern, Southern, Western, PCR, and DNA sequencing protocols discussed herein. In addition, other methods for observing variations in nucleotide and amino acid sequences (eg, single chain conformational polymorphism analysis) are well known in the art (eg, US Pat. No. 5,382,510 ( (See September 7, 1999) and 5,952,170 (issued January 17, 1995).

  In addition, the methylation status of the 109P1D4 gene in a biological sample can be tested. Aberrant demethylation and / or hypermethylation of CpG islands in the 5 'regulatory region of genes often occurs in immortalized and transformed cells and can result in altered expression of various genes. For example, promoter hypermethylation of the π class glutathione S-transferase (a protein that is expressed in normal prostate and not expressed in more than 90% of prostate cancers) seems to permanently stop transcription of this gene. And is the most frequently detected genomic alteration in prostate cancer (De Marzo et al., Am. J. Pathol. 155 (6): 1985-1992 (1999)). Furthermore, this change is present in at least 70% of cases of advanced prostate intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7: 531-536). In another example, the expression of a LAGE-I tumor-specific gene (which is not expressed in normal prostate but is expressed in 20-50% of prostate cancer) is caused by deoxy-azacytidine in lymphoblastoid cells Induced, suggesting that tumor expression is due to demethylation (Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998)). Various assays for testing the methylation status of a gene are well known in the art. For example, in a Southern hybridization approach, methylation sensitive restriction enzymes that cannot cleave sequences containing methylated CpG sites can be used to assess the methylation status of CpG islands. Furthermore, MSP (methylation specific PCR) can rapidly profile the methylation status of all CpG sites present in the CpG island of a given gene. This procedure is specific for DNA that is first methylated with sodium bisulfite, which converts all unmethylated cytosine to uracil, and then methylated against unmethylated DNA. Amplification using simple primers. Protocols involving methylation interference are also described in, for example, Current Protocols In Molecular Biology, Unit 12, Frederick M. et al. As edited by Ausubel et al., 1995.

  Gene amplification is an additional method for assessing 109P1D4 status. Gene amplification is based on the sequences provided herein, eg, conventional Southern blotting or Northern blotting (Thomas, 1980, for quantifying mRNA transcription, using appropriately labeled probes). Proc. Natl. Acad. Sci. USA, 77: 5201-5205), dot blotting (DNA analysis), or in situ hybridization. Alternatively, antibodies that recognize specific duplexes (including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes) are used. The antibody is then labeled and an assay is performed in which the duplex is bound to a surface such that when a duplex is formed on the surface, the duplex is The presence of antibody bound to can be detected.

  Biopsy tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using, for example, Northern analysis, dot blot analysis or RT-PCR analysis to detect 109P1D4 expression. The presence of 109P1D4 mRNA capable of RT-PCR amplification provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of many human solid tumors. In the field of prostate cancer, this assay includes RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995). J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem. 41: 1687-1688).

  A further aspect of the invention is an assessment of the susceptibility an individual has for developing cancer. In one embodiment, the method for predicting susceptibility to cancer comprises detecting 109P1D4 mRNA or 109P1D4 protein (the presence of which indicates susceptibility to cancer) in a tissue sample, wherein 109P1D4 mRNA expression. The degree of correlates with the degree of sensitivity. In certain embodiments, the presence of 109P1D4 in the prostate or other tissue is tested, wherein the presence of 109P1D4 in this sample provides an indication of prostate cancer susceptibility (or the development or presence of prostate tumor). . Similarly, the integrity of 109P1D4 nucleotide and 109P1D4 amino acid sequences in biological samples can be assessed to identify variations in the structure of these molecules (eg, insertions, deletions, substitutions, etc.). The presence of one or more variations of the 109P1D4 gene product in the sample is an indication of cancer susceptibility (or tumor onset or presence).

  The invention also includes a method for assessing tumor aggressiveness. In one embodiment, the method for assessing tumor aggressiveness comprises the step of determining the level of 109P1D4 mRNA or 109P1D4 protein expressed by tumor cells, the level thus determined can be referred to the same individual or normal tissue. Comparing the level of 109P1D4 mRNA or 109P1D4 protein expressed in the corresponding normal tissue taken from the sample, wherein the degree of expression of 109P1D4 mRNA or 109P1D4 protein in the tumor sample relative to the normal sample is aggressive Indicates the degree. In certain embodiments, tumor aggressiveness is assessed by determining the extent to which 109P1D4 is expressed in tumor cells, where a higher expression level indicates a more aggressive tumor. Another embodiment is an assessment of the integrity of this 109P1D4 nucleotide and amino acid sequence in a biological sample to identify variations in the structure of 109P1D4 nucleotides and amino acids (eg, insertions, deletions, substitutions, etc.) . The presence of one or more fluctuations indicates a more aggressive tumor.

  Another embodiment of the invention relates to a method for observing the progression of malignancy in an individual over time. In one embodiment, a method for observing the progression of malignant tumors in an individual over time is determined by determining the level of 109P1D4 mRNA or 109P1D4 protein expressed by cells in a sample of tumors, thus. Comparing the levels of 109P1D4 mRNA or 109P1D4 protein expressed in equivalent tissue samples taken from the same individual at different times, wherein 109P1D4 mRNA or 109P1D4 protein in tumor samples over time The degree of expression provides information about the progression of the cancer. In certain embodiments, cancer progression is assessed by determining 109P1D4 expression in tumor cells over time, wherein increased expression over time indicates cancer progression. Also, the integrity of 109P1D4 nucleotide and 109P1D4 amino acid sequences in biological samples can be assessed to identify variations in the structure of these molecules (eg, insertions, deletions, substitutions, etc.), where 1 The presence of one or more fluctuations indicates cancer progression.

  The above diagnostic approach can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention may be used as a means for diagnosing and predicting 109P1D4 gene expression and 109P1D4 gene product expression (or 109P1D4 gene variation and 109P1D4 gene product variation) and tissue sample status. The present invention relates to a method for observing co-occurrence between factors associated with malignant tumors. A wide variety of factors associated with malignant tumors can be used, such as genes associated with malignant tumors (eg, expression of PSA, PSCA and PSM for prostate cancer, and macroscopic cytological findings) (For example, Bocking et al., 1984, Anal. Quant. Cytol. 6 (2): 74-88; Epstein, 1995, Hum. Pathol. 26 (2): 223-9; Thorson et al., 1998, Mod. Pathol. 11). (6): 543-51; see Baisden et al., 1999, Am. J. Surg. Pathol. 23 (8); Methods for observing co-occurrence between expression of 109P1D4 gene and expression of 109P1D4 gene product (or variation in 109P1D4 gene and 109P1D4 gene product) and another factor associated with malignancy are useful. This is because, for example, the presence of a particular set of factors that coincide with the disease provides important information for diagnosing and predicting the condition of a tissue sample.

  In one embodiment, the method for observing co-occurrence between expression of 109P1D4 gene and expression of 109P1D4 gene product (or variation in 109P1D4 gene and 109P1D4 gene product) and another factor associated with malignancy is: Detecting overexpression of 109P1D4 mRNA or 109P1D4 protein in a tissue sample, detecting overexpression of PSA mRNA or PSA protein (or PSCA expression or PSM expression) in a tissue sample, and of 109P1D4 mRNA or 109P1D4 protein It involves observing the coincidence of overexpression and overexpression of PSA mRNA or PSA protein (or PSCA expression or PSM expression). In certain embodiments, the expression of 109P1D4 mRNA and PSA mRNA in prostate tissue is tested, wherein the co-occurrence of 109P1D4 mRNA overexpression and PSA mRNA overexpression in this sample is indicative of the presence of prostate cancer, prostate cancer Indicates the onset or status of susceptibility or prostate tumor.

  Methods for detecting and quantifying 109P1D4 mRNA or protein expression are described herein, and standard nucleic acid and protein detection and quantification techniques are well known in the art. Standard methods for detection and quantification of 109P1D4 mRNA include in situ hybridization using labeled 109P1D4 riboprobe, Northern blot using 109P1D4 polynucleotide probe and related techniques, using primers specific for 109P1D4 RT-PCR analysis, as well as other amplification type detection methods (eg, branched DNA, SISBA, TMA, etc.) can be mentioned. In certain embodiments, semi-quantitative RT-PCR is used to detect and quantify 109P1D4 mRNA expression. Any number of primers capable of amplifying 109P1D4 can be used for this purpose, including but not limited to the various primer sets specifically described herein. . In certain embodiments, polyclonal or monoclonal antibodies that specifically react with wild-type 109P1D4 protein may be used in immunohistochemical assays of biopsy tissues.

(Identification of molecules that interact with IX.109P1D4)
The 109P1D4 protein and nucleic acid sequences disclosed herein allow one of ordinary skill in the art to use any one of proteins, small molecules and other agents that interact with 109P1D4, and protocols accepted in various fields. Through which makes it possible to identify the pathway activated by 109P1D4. For example, one of the so-called interaction trap systems (also referred to as “two hybrid assays”) may be utilized. In such a system, the molecule interacts with and reconstitutes a transcription factor that directs expression of the reporter gene, while assaying for expression of this reporter gene. Other systems identify protein-protein interactions in vivo via reconstruction of eukaryotic transcriptional activators (eg, US Pat. No. 5,955,280 issued Sep. 21, 1999). No. 5,925,523 issued July 20, 1999, No. 5,846,722 issued December 8, 1998, and issued December 21, 1999 No. 6,004,746). Algorithms are also available in the art for genome-based prediction of protein function (see, eg, Marcotte et al., Nature 402: November 4, 1999, 83-86).

  Alternatively, peptide libraries can be screened to identify molecules that interact with the 109P1D4 protein sequence. In such methods, peptides that bind to 109P1D4 are identified by screening a library that encodes a random or controlled collection of amino acids. Peptides encoded by this library are expressed as bacteriophage coat protein fusion proteins, and then the bacteriophage particles are screened against the 109P1D4 protein.

  Thus, peptides having a wide variety of uses (eg, therapeutic, prognostic or diagnostic agents) are identified without previous information regarding the structure of the expected ligand or receptor molecule. Exemplary peptide libraries and screening methods that can be used to identify molecules that interact with 109P1D4 protein sequences are described, for example, in US Pat. No. 5,723,286, issued Mar. 3, 1998, and This is disclosed in US Pat. No. 5,733,731 issued on March 31, 1998.

Alternatively, cell lines expressing 109P1D4 are used to identify protein-protein interactions mediated by 109P1D4. Such interactions can be tested using immunoprecipitation techniques (see, eg, Hamilton BJ et al., Biochem. Biophys. Res. Commun, 1999, 261: 646-51). 109P1D4 protein can be immunoprecipitated from a 109P1D4 expressing cell line using an anti-109P1D4 antibody. Alternatively, antibodies against His-tag can be used in cell lines engineered to express a fusion of 109P1D4 and His-tag (the above vector). This immunoprecipitation complex can be tested for proteins along procedures such as Western blot, ³⁵ S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

  Small molecules and ligands that interact with 109P1D4 can be identified through related embodiments of such screening assays. For example, small molecules (including molecules that interfere with 109P1D4's ability to mediate phosphorylation and dephosphorylation) may cause interference with protein function to be an indication of modulation of the cell cycle, second messenger signaling, or tumorigenesis. As an interaction with a DNA or RNA molecule. Similarly, small molecules that modulate 109P1D4-related ion channels, protein pumps, or cell communication functions are identified and used to treat patients with cancer that express 109P1D4 (eg, Hille, B., Ionic Channels). of Excitable Membranes 2nd edition, Sinauer Assoc., Sunderland, MA, 1992). In addition, ligands that modulate 109P1D4 function can be identified based on their ability to bind 109P1D4 and activate the reporter construct. Exemplary methods are discussed, for example, in US Pat. No. 5,928,868, issued July 27, 1999, and to form hybrid ligands in which at least one ligand is a small molecule. Including methods. In an exemplary embodiment, cells engineered to express 109P1D4 fusion protein and DNA binding protein are used to co-express the hybrid ligand / small molecule fusion protein and the cDNA library transcription activation protein. The cell further comprises a reporter gene (the expression of which is regulated adjacent to each other of the first and second fusion proteins), and this event causes the hybrid ligand to bind to the target sites of both hybrid proteins. Only occurs when Those cells that express the reporter gene are selected and the unknown small molecule or unknown ligand is identified. This method provides a means to identify modulators that activate or inhibit 109P1D4.

  Embodiments of the invention include a method of screening for molecules that interact with the 109P1D4 amino acid sequence shown in FIG. 2 or FIG. 3, which comprises contacting a group of molecules with a 109P1D4 amino acid sequence, Interacting the molecule with the 109P1D4 amino acid sequence under conditions that facilitate interaction, determining the presence of a molecule that interacts with the 109P1D4 amino acid sequence, and then interfacing with the 109P1D4 amino acid sequence, Separating from molecules that interact with the 109P1D4 amino acid sequence. In certain embodiments, the method further comprises purifying, characterizing and identifying molecules that interact with the 109P1D4 amino acid sequence. This identified molecule can be used to modulate the function performed by 109P1D4. In a preferred embodiment, the 109P1D4 amino acid sequence is contacted with a peptide library.

X. ) Therapeutic Methods and Compositions Identifying 109P1D4 as a protein that is normally expressed in a limited set of tissues but is also expressed in cancers as listed in Table I is the treatment of such cancers. Open the door to many therapeutic approaches.

  It should be noted that targeted anti-tumor therapies are useful even when the targeted protein is expressed in normal tissues (even organ tissues necessary for life support). Organs necessary for life support are organs necessary for life support, such as the heart or colon. Organs that are not necessary for life support are organs that can be removed, in which case the individual can still survive. Examples of organs that are not necessary for life support are the ovary, breast and prostate.

  For example, Herceptin® is an FDA-approved drug that has as its active ingredient antibodies immunoreactive with proteins known variously as HER2, HER2 / neu and erb-b-2. Herceptin is marketed by Genentech and is a commercially successful anti-tumor agent. Herceptin sales reached approximately $ 4 billion in 2002. Herceptin is a treatment for HER2-positive metastatic breast cancer. However, HER2 expression is not limited to such tumors. The same protein is expressed in many normal tissues. In particular, HER2 / neu is known to be present in normal kidney and heart, and thus these tissues are present in all human recipients of Herceptin. The presence of HER2 / neu in normal kidneys is also reported by Latif, Z. et al. Et al. J. et al. U. International (2002) 89: 5-9. Both protein and mRNA are generated in benign renal tissue as shown in this document (evaluating whether renal cell carcinoma is a preferred indicator for anti-HER2 antibodies (eg, Herceptin)) . Of note, the HER2 / neu protein was very overexpressed in benign kidney tissue. Despite the fact that HER2 / neu is expressed in such life-sustaining tissues (eg heart and kidney), Herceptin is very useful, approved by the FDA, and commercially It is a successful drug. Only the effect of Herceptin on heart tissue (ie, “cardiotoxicity”) is a side effect of treatment. If the patient is treated with Herceptin alone, significant cardiotoxicity occurs in a very low percentage of patients.

  Of particular note, although kidney tissue has been shown to exhibit normal expression (probably higher expression than heart tissue), the kidney does not have any measurable Herceptin side effects. Furthermore, according to the various arrays of normal tissues in which HER2 is expressed, few side effects occur. Only heart tissue has any measurable side effects. In tissues such as the kidney, HER2 / neu expression is particularly prominent and is not the basis for any side effects.

  Furthermore, favorable therapeutic effects have been found for anti-tumor therapeutic agents that target epidermal growth factor receptor (EGFR). EGFR is also expressed in many normal tissues. There have been very limited side effects in normal tissues after the use of anti-EGFR therapeutics.

  Thus, the expression of a target protein in normal tissues (even normal tissues that are necessary for life support) indicates that the targeting agent for that protein as a therapeutic agent for a particular tumor in which the protein is also overexpressed It does not invalidate usefulness.

  Accordingly, therapeutic approaches that inhibit the activity of 109P1D4 protein are useful for patients suffering from cancers that express 109P1D4. These therapeutic approaches are generally divided into two classes. One class includes various methods for inhibiting the binding or association of 109P1D4 protein with its binding partner or other proteins. The other class includes various methods for inhibiting 109P1D4 gene transcription or 109P1D4 mRNA translation.

X. A. ) Anti-cancer vaccine The present invention provides a cancer vaccine containing 109P1D4-related protein or 109P1D4-related nucleic acid. Due to the expression of 109P1D4, cancer vaccines prevent and / or treat cancers expressing 109P1D4 with minimal or no effect on non-target tissues. The use of tumor antigens in vaccines that produce humoral and / or cell-mediated immune responses as anti-cancer treatments is well known in the art and in human prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J, Cancer 63: 231-237; Fong et al., 1997, J, Immunol. 159: 3113-3117).

  Such methods employ 109P1D4-encoding nucleic acid molecules and recombinant vectors capable of expressing and displaying 109P1D4-related proteins or 109P1D4 immunogens, which typically contain numerous antibodies or T cell epitopes. Can be easily implemented. One skilled in the art understands that a wide variety of vaccine systems for the delivery of immunoreactive epitopes are known in the art (eg, Herln et al., Ann Med 1999 Feb 31 (1): 66-78; Maruyama). Et al., Cancer Immunol Immunoother 2000 Jun 49 (3): 123-32.). Briefly, methods for generating an immune response (eg, a humoral immune response and / or a cell-mediated immune response) in such a mammal can cause the mammalian immune system to undergo immunoreactive epitopes (eg, FIG. To the 109P1D4 protein present in 109P1D4 protein, or analogs or homologues thereof, whereby the mammal produces an immune response specific for that epitope (eg, specifically recognizes that epitope) To produce an antibody that produces In a preferred method, the 109P1D4 immunogen is a biological motif (see, eg, Tables VIII-XXI and XXII-XLIX) or the size from 109P1D4 shown in FIGS. 5, 6, 7, 8 and 9. Including a range of peptides.

  The entire 109P1D4 protein, its immunogenic regions or epitopes can be combined and delivered by various means. Such vaccine compositions include, for example, lipopeptides (eg, Vitiello, A. et al., J. Clin. Invest. 95: 341, 1995), poly (DL-lactide-co-glycolide) (“PLG”) Peptide compositions encapsulated in microspheres (eg Eldridge, et al., Molec. Immunol. 28: 287-294, 1991: Alonso et al., Vaccine 12: 299-306, 1994; Jones et al., Vaccine 13: 675-681, 1995), peptide compositions contained in immunostimulatory complexes (ISCOMS) (eg Takahashi et al., Nature 344: 873-875, 1990; Hu et al., Clin Exp Immunol. 113: 235-24). 3, 1998), multiple antigen peptide systems (MAP) (eg, Tam, JP, Proc. Natl. Acad. Sci. USA 85: 5409-5413, 1988; Tam, JP, J. Immunol. Methods 196: 17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems (typically crystallized peptides , Viral delivery vectors) (Percepts in vaccine development, Kaufmann, SH, Ed., Perkus, ME et al., P. 379, 1996; Chakrabarti, S. et al., Nature 320: 535, 1986; Hu S.L., et al., Nature 320: 537. Kieny, MP et al., AIDS Bio / Technology 4: 790, 1986; Top, F. H. et al., J. Infect. Dis. 124: 148, 1971; : 535, 1990), particles of viral or synthetic origin (eg, Kofler, N., et al., J. Immunol. Methods. 192: 25, 1996; Eldridge, J. H., et al., Sem. 1993; Falo, LD, Jr., et al., Nature Med. 7: 649, 1995), adjuvant (Warren, HS, Vogel, FR, and Chedid, LA Annu. Rev.). Immunol. 4: 369, 1986; Gupta, R .; K. Et al., Vaccine 11: 293, 1993), liposomes (Reddy et al., J. Immunol. 148: 1585, 1992; Rock, KL, Immunol. Today 17: 131, 1996) or naked cDNA or incorporated into particles. CDNA (Ulmer, J.B. et al., Science 259: 1745, 1993; Robinson, HL, Hunt, LA and Webster, RG, Vaccine 11: 957, 1993; Concepts in vaccine) Shiver, JW, et al., development, Kaufmann, SHE, ed., p.423, 1996, Cease, KB and Berzofsky, JA, Annu.Rev.Immuno. .12:. 923,1994 and Eldridge, J. H. et al., Sem.Hematol.30: 16,1993) may be mentioned. Toxin targeted delivery technology, also known as receptor-mediated targeting (also known as, for example, Avant Immunotherapeutics, Inc. (Needham, Massachusetts)), can also be used.

  In patients with 109P1D4-related cancer, the vaccine compositions of the invention are also used in combination with other treatments used for cancer (eg, surgery, chemotherapy, drug therapy, radiation therapy, etc.). And combined use of an immune adjuvant (for example, IL-2, IL-12, GM-CSF, etc.).

(Cell vaccine)
CTL epitopes can be determined using specific algorithms to identify peptides within the 109P1D4 protein that bind to the corresponding HLA allele: (eg, Table IV; Epimer ^™ and Epimatrix ^™ , Brown University (URL brown. edu / Research / TB-HIV_Lab / epimatrix / epimatrix.html); In a preferred embodiment, the 109P1D4 immunogen comprises one or more amino acid sequences identified using techniques well known in the art, which are the sequences shown in Table VIII-Table XXI and Table XXII-Table XLIX. Or a peptide of 8, 9, 10 or 11 amino acids identified by HLA class I motif / supermotif (eg, Table IV (A), Table IV (D), or Table IV (E)) And / or a peptide of at least 9 amino acids comprising an HLA class II motif / supermotif (eg, Table IV (B), or Table IV (C)). As understood in the art, the HLA class I binding groove is essentially closed, so that only peptides of a certain size range can fit into the groove and be bound, In general, HLA class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA class II binding groove is essentially openly terminated, so that peptides of about 9 or more amino acids can be bound by HLA class II molecules. Due to the difference in the binding groove between HLA class I and HLA class II, the HLA class I motif is length specific. That is, position 2 of the class I motif is the second amino acid from the amino direction to the carboxyl direction of this peptide. The amino acid positions of class II motifs only correlate with each other and not with the whole peptide. That is, additional amino acids can be attached to the amino terminus and / or carboxyl terminus of the motif bearing sequence. HLA class II epitopes are often 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long Long, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids, or longer than 25 amino acids.

(Antibody-based vaccine)
A wide variety of methods for generating an immune response in a mammal are known in the art (eg, as a first step in the generation of hybridomas). A method of generating an immune response in a mammal includes exposing the mammal's immune system to an immunogenic epitope on a protein (eg, 109P1D4 protein), thereby generating an immune response. Exemplary embodiments contact a sufficient amount of at least one 109P1D4B cell or cytotoxic T cell epitope or analog thereof with the host; and after at least one periodic interval, the 109P1D4B cell or cytotoxicity It consists of a method for generating an immune response against 109P1D4 in a host by re-contacting the sex T cell epitope or analog thereof with the host. Certain embodiments consist of a method of generating an immune response against a 109P1D4-related protein or an artificial multi-epitope peptide comprising a 109P1D4 immunogen (eg, 109P1D4 protein or peptide fragment thereof, 109P1D4 fusion protein or analog, etc.) In a vaccine preparation to a human or another mammal. Typically, such vaccine preparations are prepared using suitable adjuvants (see, eg, US Pat. No. 6,146,635) or universal helper epitopes such as PADRE ^™ peptides (Epimmune Inc., San Diego, For example, Alexander et al., J. Immunol. 2000 164 (3); 164 (3): 1625-1633; Alexander et al., Immunity 1994 1 (9): 751-761 and Alexander et al., Immunol. Res. 2): 79-92). An alternative method involves in vivo administration of a DNA molecule comprising a DNA sequence encoding a 109P1D4 immunogen, a DNA sequence operably linked to a regulatory sequence that controls expression of the DNA sequence, to an individual body muscle or skin. Generating an individual immune response against the 109P1D4 immunogen; wherein the DNA molecule is taken up by the cell, the DNA sequence is expressed in the cell, and the immune response is (See, eg, US Pat. No. 5,962,428). Optionally, genetic vaccine facilitators (eg, anionic lipids; saponins; lectins; estrogen compounds; hydroxylated lower alkyls; dimethyl sulfoxides; and ureas) are also administered. In addition, anti-idiotypic antibodies that mimic 109P1D4 can be administered to generate a response to the target antigen.

(Nucleic acid vaccine)
The vaccine composition of the present invention comprises a nucleic acid mediated mode. DNA or RNA encoding the protein of the invention can be administered to a patient. Genetic immunization methods can be utilized to generate prophylactic or therapeutic humoral and cellular immune responses against cancer cells expressing 109P1D4. A construct comprising DNA encoding a 109P1D4-related protein / immunogen and appropriate regulatory sequences can be injected directly into the muscle or skin of an individual so that the muscle or skin cells take up the construct and code Expressed 109P1D4 protein / immunogen. Alternatively, the vaccine comprises 109P1D4-related protein. Expression of the 109P1D4-related protein immunogen results in the development of prophylactic or therapeutic humoral and cellular immunity against cells carrying the 109P1D4 protein. Various prophylactic and therapeutic gene immunization techniques known in the art can be used (for review, see information and references published at the internet address genweb.com). Nucleic acid-based delivery is described, for example, by Wolff et al., Science 247: 1465 (1990) and US Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery techniques include “naked DNA”, enhanced (bupivacaine, polymer, peptide mediated) delivery, cationic lipid complexes, and particle mediated delivery (“gene gun”) or pressure Mediated delivery (see, eg, US Pat. No. 5,922,687).

  For therapeutic or prophylactic immunization purposes, the proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the present invention include vaccinia virus, fowlpox virus, canarypox virus, adenovirus, influenza virus, poliovirus, adeno-associated virus, lentivirus, and Sindbis virus (eg, Ristifo, 1996, Curr. Opin. Immunol. 8: 658-663; see Tsang et al., J. Natl. Cancer Inst. 87: 982-990 (1995)). Non-viral delivery systems can also be utilized by introducing naked DNA encoding a 109P1D4-related protein into the patient (eg, intramuscularly or intradermally) to induce an anti-tumor response.

  The vaccine virus is used, for example, as a vector for expressing a nucleotide sequence encoding the peptide of the present invention. Upon introduction into the host, the recombinant vaccine virus expresses a protein immunogenic peptide and thereby elicits a host immune response. Vaccine vectors and methods useful in immunization protocols are described, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacile Calmette Guerin). BCG vectors are described in Stover et al., Nature 351: 456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the present invention (eg, adenoviral vectors and adeno-associated viral vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.) Will be apparent to those skilled in the art from the description herein.

  Thus, gene delivery systems are used to deliver 109P1D4-related nucleic acid molecules. In one embodiment, full length human 109P1D4 cDNA is used. In another embodiment, 109P1D4 nucleic acid molecules that encode specific cytotoxic T lymphocytes (CTLs) and / or antibody epitopes are used.

(Ex vivo vaccine)
A variety of ex vivo strategies can also be used to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DCs) that present the 109P1D4 antigen to the patient's immune system. Dendritic cells express MHC class I and MHC class II molecules, B7 costimulators, and IL-12 and are therefore highly specific antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with a peptide of prostate specific membrane antigen (PSMA) are used in phase I clinical trials to stimulate the immune system of prostate cancer patients (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate 29: 371-380). Thus, dendritic cells can be used to present 109P1D4 peptides to T cells in the contents of MHC class I or MHC class II molecules. In one embodiment, autologous dendritic cells are pulsed with a 109P1D4 peptide that can bind to MHC class I and / or MHC class II molecules. In another embodiment, dendritic cells are pulsed with the complete 109P1D4 protein. Yet another embodiment relates to manipulating overexpression of the 109P1D4 gene in dendritic cells using various execution vectors known in the art (see, eg, adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869). Or tumor-induced RNA transfection (Ashley et al., 1997, J. Exp. Med. 186: 1177-1182)). Cells expressing 109P1D4 can also be engineered to express immunomodulators (eg, GM-CSF) and used as immunizing agents.

(XB 109P1D4 as a target for antibody-based therapy)
109P1D4 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art to target both extracellular and intracellular molecules (see, eg, complement and ADCC mediated killing and use in the body). 109P1D4 is expressed by various lines of cancer cells against the corresponding normal cells, so that toxic effects, non-specific effects and / or non-targets produced by binding immunoactive compositions to non-target organs and tissues A systemic administration of 109P1D4 immunoactive composition is prepared that exhibits excellent sensitivity without effect. Antibodies that specifically react with the domain of 109P1D4 treat cancers that develop 109P1D4 systemically, either in combination with toxins or therapeutic agents, or as naked antibodies that can inhibit cell proliferation or function Useful for.

  The 109P1D4 antibody allows the antibody to bind to 109P1D4 and to modulate function (eg, interaction with a binding partner) and consequently mediate tumor cell destruction and / or inhibit tumor cell growth. Can be introduced to the patient. The mechanisms by which such antibodies exert their therapeutic effects include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulating 109P1D4 physiological functions, inhibiting ligand binding or signaling pathways, Modulating tumor cell differentiation, altering tumor angiogenic profile, and / or apoptosis may be involved.

  One skilled in the art understands that antibodies can be used to specifically target and bind immunogenic molecules (eg, the immunogenic region of the 109P1D4 sequence shown in FIG. 2 or FIG. 3). Furthermore, those skilled in the art understand that it is conventional to conjugate antibodies to cytotoxic agents (see, eg, Slivers et al., Blood 93:11 3678-3684 (June 1, 1999)). ) Cytotoxic agents and / or therapeutic agents are delivered directly to the cells, for example, by conjugating the agents to antibodies specific for molecules expressed by the cells (eg, 109P1D4). In some cases, the cytotoxic agent exerts a known biological effect (eg, cytotoxicity) on those cells.

  A wide variety of compositions and methods for killing cells using antibody-cytotoxic drug conjugates are known in the art. In the context of cancer, an exemplary method involves administering to a mammal having a tumor a biologically effective amount of a conjugate, wherein the conjugate is expressed or accessible for binding. Or a selected cytotoxic agent and / or therapeutic agent linked to a target reagent (eg, anti-109P1D4 antibody) that binds to a marker (eg, 109P1D4) that is localized on the cell surface. An exemplary embodiment is a method of delivering a cytotoxic agent and / or therapeutic agent to a cell that expresses 109P1D4, wherein the method includes applying a cytotoxic agent to an antibody that immunospecifically binds to a 109P1D4 epitope. Conjugating and exposing the cell to the antibody drug conjugate. Another exemplary embodiment is a method of treating an individual suspected of having metastatic cancer, wherein the method comprises treating a therapeutically effective amount of an antibody conjugated to a cytotoxic agent and / or therapeutic agent. Administering the pharmaceutical composition comprising it to an individual parenterally.

Cancer immunotherapy using anti-109P1D4 antibodies is made according to various approaches that have been successfully used to treat other types of cancer, including but not limited to: colon cancer (Arlen). 1998, Crit. Rev. Immunol. 1992, Cancer Res. 52: 2771-2777), B cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19: 93-101), leukemia (Zhong et al., 1996, Le k.Res.20: 581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54: 6160-6166; Velders et al., 1995, Cancer Res. 55: 4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11: 117-127). Some therapeutic approaches include the binding of naked antibodies to toxins or radioisotopes (eg, binding of Y ⁹¹ or I ¹³¹ to anti-CD20 antibodies (eg, Zevalin ^™ , IDEC Pharmaceuticals Corp., or Bexar ^™ , Coulter). Other approaches include co-administration of antibodies and other therapeutic agents (eg, Herceptin ^™ (with trastuzumab and paclitaxel (Genentech, Inc.)). For the treatment of prostate cancer, for example, 109P1D4 antibody may be administered in combination with irradiation, chemotherapy or hormonal resection, and the antibody may be calibamitin (c Licheamicin) ^{(e.g., Mylotarg TM, Wyeth-Ayerst,} Madison, NJ, a recombinant humanized IgG ₄ kappa antibody conjugated to antitumor antibodies Karikiamichin), or maytansinoid (maytansinoid) (e.g., taxane-based Tumor Can be conjugated to toxins such as activated prodrugs, TAPs, platforms, immunogens, Cambridge, MA, also see, eg, US Pat. No. 5,416,064).

  109P1D4 antibody therapy is useful for all stages of cancer, but may be particularly appropriate in advanced or metastatic cancer. Treatment with the antibody therapy of the present invention is indicated for patients undergoing one or more series of chemotherapy. Alternatively, the antibody therapy of the invention is combined with a chemotherapeutic or irradiation regimen for patients who have not received chemotherapy treatment. In addition, antibody therapy may allow the use of reduced dosage co-chemotherapy, particularly for patients who are less tolerant of the toxicity of the chemotherapeutic agent. Fan et al. (Cancer Res. 53: 4637-4642, 1993), Prewett et al. (International J. of Onco. 9: 217-224, 1996), and Hancock et al. (Cancer Res. 51: 4575-4580, 1991) are: The use of various antibodies in conjunction with chemotherapeutic agents is described.

  109P1D4 antibody therapy is useful for all stages of cancer, but may be particularly appropriate in advanced or metastatic cancer. Treatment with the antibody therapy of the present invention is indicated for patients undergoing one or more series of chemotherapy. Alternatively, the antibody therapy of the invention is combined with a chemotherapeutic or irradiation regimen for patients who have not received chemotherapy treatment. In addition, antibody therapy may allow the use of reduced dosage co-chemotherapy, particularly for patients who are less tolerant of the toxicity of the chemotherapeutic agent.

  Cancer patients are preferably assessed for the presence and level of 109P1D4 expression using immunohistochemical assessment of tumor tissue, quantitative 109P1D4 imaging, or other techniques that can reliably indicate the presence and extent of 109P1D4 expression. Can be done. Tumor biopsies or immunohistochemical analysis of surgical specimens are preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

  Anti-109P1D4 monoclonal antibodies that treat prostate cancer and other cancers include antibodies that can initiate a strong immune response against the tumor, or antibodies that can be directly cytotoxic. In this regard, anti-109P1D4 monoclonal antibody (mAb) can trigger tumor cell lysis by either complement-mediated cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC) mechanisms, both of which are Interaction with the effector cell Fc receptor site on the body protein requires the intact Fc portion of the immunoglobulin molecule. Furthermore, anti-109P1D4 mAbs that exert a direct biological effect on tumor growth are useful for treating cancers that express 109P1D4. The mechanisms by which cytotoxic mAbs act directly include inhibition of cell proliferation, regulation of cell differentiation, regulation of tumor angiogenic factor profiles, and induction of apoptosis. The mechanism by which a particular anti-109P1D4 mAb exerts an anti-tumor effect can be any number of cells that assess cell death such as ADCC, ADMMC, complement-mediated cell lysis, as is generally known in the art. Assessed using in vitro assays.

  In some patients, the use of murine or other non-human monoclonal antibodies, or the use of human / mouse chimeric mAbs can induce a moderate to strong immune response against non-human antibodies. This can lead to clearance of the antibody from the circulation and reduced efficacy. In the most severe cases, such an immune response can result in extensive formation of immune complexes that can potentially cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention specifically bind to the target 109P1D4 antigen with high affinity, but exhibit low or no antigenicity in the patient. An antibody that is either human or humanized.

  The therapeutic methods of the present invention contemplate administration of a single anti-109P1D4 mAb, as well as administration of different mAb combinations or cocktails. Such mAb cocktails combine mAbs that contain mAbs that target different epitopes, use different effector mechanisms, or depend on immune effector functionality directly with cytotoxic mAbs. May have advantages. Such mAbs in combination may show a synergistic therapeutic effect. In addition, the anti-109P1D4 mAb is administered concurrently with other therapeutic agents including, but not limited to, various chemotherapeutic agents, androgen blockers, immunomodulators (eg, IL-2, GM-CSF), surgery or radiation. Can be done. Anti-109P1D4 mAbs can be administered in their “naked” or unbound form, or can have therapeutic agents attached to them.

  The anti-109P1D4 antibody formulation is administered via any route that can deliver the antibody to the tumor cells. Administration routes include, but are not limited to, intravenous routes, intraperitoneal routes, intramuscular routes, intratumoral routes, intradermal routes, and the like. Treatment is typically typically about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, Via an acceptable route of administration (eg, intravenous injection (IV)) at doses ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg; Includes repeated administration of anti-109P1D4 antibody preparations. In general, doses ranging from 10 to 1000 mg mAb per week can be effective and well tolerated.

Based on clinical experiments using Herceptin ^™ mAb in the treatment of metastatic breast cancer, an initial loading dose of about 4 mg / kg patient weight (IV) of anti-109P1D4 mAb preparation was followed by a weekly dose of about 2 mg / kg (IV). The dose indicates an acceptable dosing regimen. Preferably, this initial loading dose is administered as an infusion over 90 minutes. If the initial dose is well tolerated, a periodic maintenance dose is administered as an infusion over 30 minutes. As those skilled in the art will appreciate, a variety of factors can affect the ideal dosage regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the Ab or mAb used, the extent of 109P1D4 expression in the patient, the extent of circulating 109P1D4 antigen, the desired steady state antibody Concentration levels, frequency of treatment, and the effects of chemotherapeutic drugs or other factors used in combination with the treatment methods of the present invention as well as the health status of a particular patient.

  If necessary, patients are evaluated for levels of 109P1D4 in a given sample (eg, levels of circulating 109P1D4 antigen and / or 109P1D4 expressing cells) to help determine the most effective dosing regimen, etc. Should. Such assessments are also used for monitoring purposes throughout the treatment, and other parameters such as urinary cytology and / or ImmunoCyt levels in bladder cancer treatment or, according to analogy, prostate cancer Useful for assessing therapeutic success in combination with assessing serum PSA levels in therapy.

  Anti-idiotype anti-109P1D4 antibodies can also be used as vaccines to elicit immune responses against cells expressing 109P1D4-related proteins in anti-cancer therapy. In particular, the production of anti-idiotype antibodies is well known in the art; this methodology can be readily adapted to generate anti-idiotype anti-109P1D4 antibodies that mimic epitopes on 109P1D4-related proteins (eg, Wagner et al., 1997, Hybridoma 16: 33-40; see Foon et al., 1995, J. Clin. Invest. 96: 334-342; Herlyn et al., 1996, Cancer Immunol.Immunother. 43: 65-76). . Such anti-idiotype antibodies can be used in cancer vaccine strategies.

(XC 109P1D4 as a target for cellular immune response)
Methods for preparing vaccines and vaccines comprising an immunologically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, the vaccine according to the invention comprises a composition of one or more claimed peptides. The peptides can be present individually in the vaccine. Alternatively, the peptides can exist as homopolymers containing multiple copies of the same peptide or as heteropolymers of various peptides. The polymer has the advantage of increased immunological response, and when different peptide epitopes are used to make up the polymer, different antigenic determinants of pathogenic organisms or tumor-related peptides that target the immune response It has the additional ability to induce reactive antibodies and / or CTLs. The composition can be a naturally occurring region of the antigen or can be prepared, for example, by recombinant or chemical synthesis.

Carriers that can be used with the vaccines of the invention are well known to those skilled in the art and include, for example, thyroglobulin, albumin (eg, human serum albumin), tetanus toxoid, polyamino acids (eg, poly L-lysine, poly L-glutamic acid). , Influenza, hepatitis B virus core protein, and the like. The vaccine may include a physiologically acceptable (ie, acceptable) diluent (eg, water or saline, preferably phosphate-releasing saline). A vaccine may also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Further, as disclosed herein, a CTL response conjugated a peptide of the invention to a lipid (eg, tripalmitoyl-S-glycerylcysteinyl seryl-serine (P ₃ CSS)). Can be primed. Further, adjuvants (eg, synthetic cytokine phosphorothiolated guanine-containing (CpG) oligonucleotides) were found to increase CTL responses by 10-100 fold (eg, Davila and Celis, J. Immunol. 165). : 539-547 (2000)).

  Upon immunization with a peptide composition according to the present invention by injection, aerosol, oral, transdermal, transmucosal, intrathoracic, intrathecal, or other suitable route, the host immune system is specific for the desired antigen. It responds to the vaccine by producing large amounts of CTL and / or HTL. As a result, the host is at least partially immunized for the late development of cells expressing or overexpressing 109P1D4 antigen, or provides at least some therapeutic benefit if the antigen was tumor associated.

  In some embodiments, it may be desirable to combine a class I peptide component with a neutralizing antibody to the target antigen and / or a component that induces or promotes a helper T cell response. Preferred embodiments of this composition comprise class I and class II epitopes according to the invention. Alternative embodiments of such compositions include class I and / or class II epitopes according to the invention, and cross-reactive HTL epitope (eg, PADRETM (Epimmune, San Diego, CA) molecules (eg, U.S. Pat. No. 5,736,142).

  The vaccines of the present invention can also include antigen presenting cells (APC) (eg, dendritic cells (DC)) as a vehicle for presenting the peptides of the present invention. After the dendritic cells are mobilized and collected, thereby creating a load of dendritic cells in vitro, a vaccine composition can be made in vitro. For example, dendritic cells are, for example, transfected with a minigene according to the invention or pulsed with a peptide. The dendritic cells can then be administered to the patient to elicit an immune response in vivo. Vaccine compositions (either DNA-based or peptide-based) can also be administered in vivo with dendritic cell mobilization, which causes dendritic cell loading in vivo.

  Preferably, the following principles are separate epitopes for inclusion in a polyepitope composition for use in a vaccine, or to be included in a vaccine and / or encoded by a nucleic acid (eg, a minigene) To select an array of epitopes. Each of the following principles is preferably balanced to make a selection: The multi-epitope incorporated into a given vaccine composition can be a contiguous sequence in the native antigen from which the epitope is derived, but need not be.

  1. When administered, epitopes are selected that mimic the observed immune response in correlation with tumor clearance. For HLA class I, this includes 3-4 epitopes derived from at least one tumor associated antigen (TAA). Similar principles are used for HLA class II; again, 3-4 epitopes are selected from at least one TAA (see, eg, Rosenberg et al., Science 278: 1447-1450). Epitopes from one TAA can be used in combination with epitopes from one or more additional TAAs to produce vaccines that target tumors with various expression patterns of frequently expressed TAAs.

2. ) Epitopes with the essential binding affinity established to be associated with immunogenicity are selected: IC _{50 of} 500 nM or less (often 200 nM or less) for HLA class I; and 1000 nM or less for class II IC ₅₀ .

  3. ) Sufficient supermotif-bearing peptides, or sufficient arrays of allele-specific motif-bearing peptides are selected to give a broad population range. For example, it is preferable to have a population range of at least 80%. Monte Carlo analysis (statistical assessments known in the art) can be used to assess the breadth or overlap of population ranges.

  4). ) When epitopes from cancer-associated antigens are selected, it is often useful to select an analog. This is because the patient may have developed resistance to the native epitope.

  5. ) Of particular relevance are epitopes called “nested epitopes”. A nested epitope occurs when at least two epitopes overlap with a given peptide sequence. The nested peptide sequence can comprise a B cell epitope, an HLA class I epitope and / or an HLA class II epitope. When providing nested epitopes, the general goal is to provide the highest number of epitopes per sequence. Thus, one aspect is to avoid providing peptides that are somewhat longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing multi-epitope sequences (eg, sequences containing nested epitopes), it is common to screen sequences to ensure that they do not have pathological or other deleterious biological properties Is important.

  6). ) When making a polyepitope protein, or when making a minigene, the goal is to make the smallest peptide that contains the epitope of interest. This principle is similar if it is not the same as that used when selecting peptides containing nested epitopes. However, when using artificial polyepitope peptides, the objective of size minimization is balanced against the need to incorporate any spacer sequence between the epitopes in the polyepitope protein. For example, spacer amino acid residues may be used to avoid junctional epitopes (epitopes that are recognized by the immune system, are not present in the target antigen, and are created only by artificial juxtaposition of epitopes) or between epitopes It can be introduced to facilitate cleavage and thereby enhance epitope presentation. Junction epitopes should generally be avoided. This is because the recipient can generate an immune response against that non-native epitope. Of particular interest is a junctional epitope which is a “dominant epitope”. Dominant epitopes can lead to a strong response such that the immune response to other epitopes is reduced or suppressed.

  7). ) If there are multiple variants of the same target protein, potential peptide epitopes can also be selected based on their conservation. For example, a criterion for conservation is that the entire sequence of HLA class I binding peptides or the entire 9-mer core of class II binding peptides is conserved at a specified percentage of the sequence evaluated for a particular protein antigen. Can be defined.

(X.C.1. Minigene vaccine)
Many different approaches are available that allow for co-delivery of multiple epitopes. Nucleic acids encoding the peptides of the present invention are particularly useful embodiments of the present invention. The epitope for inclusion in the minigene is preferably selected according to the guidelines described in the previous section. A preferred means of administering a nucleic acid encoding a peptide of the invention uses a minigene construct encoding a peptide comprising one epitope or multiple epitopes of the invention.

  The use of multi-epitope minigenes is described below and by Ishioka et al. Immunol. 162: 3915-3925, 1999; An, L .; And Whitton, J .; L. , J .; Virol. 71: 2292, 1997; Thomson, S .; A. Et al. Immunol. 157: 822, 1996; Whitton, J. et al. L. Et al. Virol. 67: 348, 1993; Hanke, R .; Et al., Vaccine 16: 426, 1998. For example, 109P1D4 derived supermotif-bearing epitopes and / or multi-epitope DNA plasmids encoding motif-bearing epitopes, PADRE® universal helper T cell epitopes or multiple HTL epitopes derived from 109P1D4 (eg, Tables VIII-XXI and XXII) ~ XLIX), as well as the endoplasmic reticulum translocation signal sequence. The vaccine may also include other TAA-derived epitopes.

  The immunogenicity of the multi-epitope minigene can be confirmed in transgenic mice to assess the magnitude of the CTL-induced response to the epitope being tested. Furthermore, the immunogenicity of DNA-encoded epitopes in vivo can correlate with the in vitro response of certain CTL lines to target cells transfected with a DNA plasmid. Thus, these experiments may show that the minigene serves both 1) producing a CTL response and 2) recognizing cells that express the encoded epitope.

  For example, the amino acid sequence of an epitope can be back-translated to create a DNA sequence that encodes an epitope (minigene) selected for expression in human cells. The human codon usage table can be used to guide codon selection for each amino acid. These epitope-encoding DNA sequences can be directly adjacent, resulting in a continuous polypeptide sequence when translated. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be back-translated into and included in a minigene sequence include: HLA class I epitope, HLA class II epitope, antibody epitope, ubiquitination signal sequence, and / or endoplasmic reticulum targeting signal . Furthermore, HLA presentation of CTL and HTL epitopes can be improved by including synthetic (eg, poly-alanine) or naturally-occurring flanking sequences flanking the CTL or HTL epitope; these larger peptides containing the epitope Is within the scope of the present invention.

  Minigene sequences can be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) can be synthesized, phosphorylated, purified, and annealed using well-known techniques and under appropriate conditions. The ends of the oligonucleotides can be joined using, for example, T4 DNA ligase. This synthetic minigene (encoding the epitope polypeptide) can then be cloned into the desired expression vector.

  Standard regulatory sequences well known to those skilled in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; E. coli origin of replication; E. coli selectable markers (eg ampicillin resistance or kanamycin resistance). A number of promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter. See, eg, US Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

  Further vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcription region of the minigene. Inclusion of mRNA stabilizing sequences and sequences for replication in mammalian cells can also be considered to increase minigene expression.

  Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is suitable E. coli. E. coli strains are transformed and DNA is prepared using standard techniques. The minigene orientation and DNA sequence, as well as all other elements contained in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells with the correct plasmid can be stored as a master cell bank and a working cell bank.

  Furthermore, immunostimulatory sequences (ISS or CpG) appear to play a role in the immunogenicity of DNA vaccines. These sequences can be included in the vector outside the minigene coding sequence if it is desired to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector is used that allows for the production of both a minigene-encoding epitope and a second protein (included to enhance or reduce immunogenicity). obtain. Examples of proteins or polypeptides that can advantageously enhance the immune response when co-expressed include cytokines (eg, IL-2, IL-12, GM-CSF), cytokine-inducing molecules (eg, LeIF), co- For stimulatory molecules or HTL responses, pan-DR binding proteins (PADRE ^™ , Epimmune, San Diego, CA) are included. Helper (HTL) epitopes can be bound to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows the orientation of HTL epitopes to a different cellular compartment than the CTL epitope To do. If required, this can facilitate more efficient entry of HTL epitopes into the class II pathway, thereby improving HTL induction. In contrast to HTL induction or CTL induction, specifically reducing the immune response by co-expression of an immunosuppressive molecule (eg, TGF-β) may be beneficial in certain diseases.

  A therapeutic amount of plasmid DNA is described, for example, in E. coli. It can be produced by fermentation in E. coli followed by purification. An aliquot from the cell bank is used to inoculate growth medium and grown to saturation in a shaker flask or bioreactor according to well known techniques. Plasmid DNA is available from QIAGEN, Inc. It can be purified using standard bioseparation techniques such as solid phase anion exchange resins supplied by (Valencia, California). If necessary, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other methods.

  Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate buffered saline (PBS). This approach (known as “naked DNA”) is currently used for intramuscular (IM) administration in clinical trials. In order to maximize the immunotherapeutic effect of the minigene DNA vaccine, alternative methods for formulating purified plasmid DNA may be desirable. Various methods have been described and new techniques may be available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (eg, WO 93/24640; Mannino & Gold-Fogate, BioTechniques 6 (7): 682 (1988); US Patents). No. 5,279,833; WO 91/06309; and Felgner et al., Proc. Nat'l Acad. Sci. USA 84: 7413 (1987)). In addition, peptides and compounds collectively referred to as protective interactive non-condensing compounds (PINCs) can also be complicated to purify plasmid DNA and can be variable (eg, stable, intramuscularly dispersed, or specific Affects transport to other organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, plasmid DNA is introduced into a mammalian cell line suitable as a target for a standard CTL chromium release assay. The transfection method used will depend on the final formulation. Electroporation can be used for “naked” DNA, but cationic lipids allow direct in vitro transfection. Plasmids expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescent cell activated cell sorting (FACS). These cells can then chrome ^{-51 (51} Cr) labeled and used as target cells for epitope-specific CTL lines; ^(detected by a 51 Cr release) cell lysis, the minigene encoding CTL epitope Both production and HLA presentation are shown. Expression of the HTL epitope can be assessed in a similar manner using an assay to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing the appropriate human HLA protein are immunized with the DNA product. The dose and route of administration will depend on the formulation (eg, IM for DNA in PBS, intraperitoneal (ip) for lipid complexed DNA). After 21 days of immunization, splenocytes are collected and restimulated for 1 week in the presence of peptides encoding each epitope to be tested. The CTL effector cells are then assayed for cytolysis of peptide-loaded ⁵¹ Cr labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loaded with a peptide epitope corresponding to the minigene-encoded epitope demonstrates DNA vaccine function for in vivo introduction of CTL. The immunogenicity of the HTL epitope is confirmed in transgenic mice in a similar manner.

  Alternatively, the nucleic acid can be administered using ballistic delivery, eg, as described in US Pat. No. 5,204,253. Using this technique, particles composed solely of DNA are administered. In a further alternative embodiment, the DNA can be attached to particles (eg, gold particles).

  Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art (eg, an expression construct encoding an epitope of the invention can be incorporated into a viral vector such as vaccinia). .

(XC.2.) Combination of CTL peptide and helper peptide)
Vaccine compositions comprising CTL peptides of the present invention are modified (eg, analogs) to provide the desired properties (eg, improved serum half-life, extended population coverage, or enhanced immunogenicity). ).

  For example, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence comprising at least one epitope that can induce a T helper cell response. CTL peptides can be linked directly to T helper peptides, but often CTL epitope / HTL epitope conjugates are linked by a spacer molecule. Spacers are typically composed of relatively small neutral molecules (eg, amino acids or amino acid mimetics) that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be appreciated that the optional spacers need not be composed of the same residues, and thus can be hetero-oligomers or homo-oligomers. When present, the spacer is usually at least one or two residues, more usually 3 to 6 residues, sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide, either directly or through a spacer, at either the amino terminus or the carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide can be acylated.

  In certain embodiments, T helper peptides are peptides that are recognized by T helper cells that are present in the majority of genetically diverse populations. This can be accomplished by selecting peptides that bind to many, most, or all HLA class II molecules. Examples of such amino acids that bind to many HLA class II molecules are the tetanus toxoid positions 830-843 QYIKANSKFIGITE (SEQ ID NO: 40), Plasmodium falciparum circumsporozoite (CS) protein positions 378-398 DIEKVSNK No. 41), and sequences from antigens such as Streptococcus 18 kD protein at positions 116-131 GAVDSILGGVATYGAA (SEQ ID NO: 42). Other examples include peptides having either the DR 1-4-7 supermotif or the DR3 motif.

Alternatively, amino acid sequences not found in nature can be used to prepare synthetic peptides that can stimulate T helper lymphocytes in a loose HLA-restricted fashion (eg, PCT publication). (See WO95 / 07707). These synthetic compounds, called Pan-DR binding epitopes (eg, PADRE ^™ , Epimmune, Inc., San Diego, Calif.) Are designed to bind most favorably to most HLA-DR (human HLA class II) molecules. Is done. For example, the formula: xKXVAAWTLKAAx (SEQ ID NO: 43) where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine. It has been found that the pan-DR binding epitope peptides that bind to most HLA-DR alleles and stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. It was. Alternatives to pan-DR binding epitopes all include the “L” natural amino acid and can be provided in the form of nucleic acids encoding the epitope.

  HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus increase their serum half-life, or they increase their biological activity. Can be conjugated to other molecules such as lipids, proteins, carbohydrates and the like. For example, a T helper peptide can be conjugated to one or more palmitic acid chains, either at the amino terminus or the carboxyl terminus.

(XC.3.) Combination of CTL peptide and T cell priming agent)
In some embodiments, it may be desirable to include in the pharmaceutical composition of the invention at least one component that primes B lymphocytes or T lymphocytes. Lipids have been identified as agents that can prime CTL in vivo. For example, palmitic acid residues can be linked to the ε-amino and α-amino groups of lysine residues, and then one or more linkages such as, for example, Gly, Gly-Gly, Ser, Ser-Ser, etc. It can be linked to an immunogenic peptide via a residue. The lipidated peptide can then be administered either directly in micelles or particles, can be incorporated into liposomes, or can be emulsified in an adjuvant (eg, incomplete Freund's adjuvant). In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to the ε-amino and α-amino groups of Lys (which is linked to the amino terminus of the immunogenic peptide (eg, Ser- Ser)).

As another example of lipid priming of a CTL response, E. coli lipoproteins (eg, tripalmitoyl-S-glycerylcysteinyl seryl-serine (P ₃ CSS)) can be used to prime virus-specific CTLs when covalently attached to the appropriate peptide (eg, Deres et al., Nature 342: 561, 1989). The peptides of the invention can be conjugated, for example, to P ₃ CSS, and the lipopeptide can be administered to an individual to specifically prime an immune response to a target antigen. In addition, since the introduction of neutralizing antibodies can also be primed with P ₃ CSS binding epitopes, two such compositions can be combined to elicit both humoral and cell-mediated responses more efficiently. Can be.

(XC.4.) Vaccine composition comprising DC pulsed with CTL peptide and / or HTL peptide)
Embodiments of vaccine compositions according to the present invention include ex vivo administration of a cocktail of epitope-bearing peptides to PBMCs derived from patient blood, or isolated DCs derived therefrom. Drugs that facilitate DC collection, such as Progenipoietin ^™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF / IL-4, can be used. After DC is pulsed with peptide and before reinfusion into the patient, the DC is washed to remove unbound peptide. In this embodiment, the vaccine comprises peptide-pulsed DC presenting a pulsed peptide epitope complexed with HLA molecules on their surface.

  DCs can be pulsed ex vivo with a cocktail of peptides, some of which stimulate a CTL response to 109P1D4. Optionally, helper T cell (HTL) peptides (eg, natural or artificial loosely restricted HLA class II peptides) can be included to promote CTL responses. Therefore, the vaccine according to the invention is used to treat cancers that express or overexpress 109P1D4.

(X.D.) Adoptive immunotherapy)
Antigenic 109P1D4-related peptides are used to elicit CTL responses and / or HTL responses ex vivo. The resulting CTL cells or HTL cells can be used to treat tumors in patients who do not respond to other conventional forms of therapy or do not respond to therapeutic vaccine peptides or nucleic acids according to the present invention. An ex vivo CTL response or HTL response to a particular antigen is expressed in a tissue culture by replacing the patient's or genetically compatible CTL or HTL progenitor cells with antigen presenting cells (APCs) (eg, dendritic cells). Induced by incubating with the source and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), where the progenitor cells are activated and expand into effector cells, the cells are injected back into the patient, where Thus, they destroy (CTL) or promote destruction (HTL) of specific target cells (eg, tumor cells). Transfected dendritic cells can also be used as antigen presenting cells.

(X.E.) Administration of vaccines for therapeutic or prophylactic purposes)
The pharmaceutical and vaccine compositions of the invention are typically used to treat and / or prevent cancers that express or overexpress 109P1D4. In therapeutic applications, the peptide composition and / or nucleic acid composition elicits an effective B cell response, CTL response and / or HTL response to the antigen and cures symptoms and / or complications or Administered to the patient in an amount sufficient to at least partially stop or delay. An amount adequate to accomplish this is defined as a “therapeutically effective dose”. Effective amounts for this use will depend, for example, on the particular composition being administered, the method of administration, the stage and severity of the disease being treated, the weight and general condition of the patient, and the judgment of the prescribing physician. .

  For pharmaceutical compositions, the immunogenic peptides of the invention, or the DNA encoding them, are generally administered to an individual who already has a tumor that expresses 109P1D4. The peptides or the DNA encoding them can be administered individually or as a fusion of one or more peptide sequences. Patients can be treated with immunogenic peptides separately or in conjunction with other treatments (eg, surgery) as appropriate.

  For therapeutic use, administration should generally begin in the first diagnosis of 109P1D4-related cancer. This is continued by boosting the dose at least until symptoms are substantially stopped and for a period thereafter. Embodiments of vaccine compositions delivered to a patient (ie, including but not limited to embodiments such as peptide cocktails, polyepitope polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) May vary according to the stage of the disease or the health status of the patient. For example, in patients with tumors that express 109P1D4, vaccines containing 109P1D4-specific CTLs may be more effective at killing tumor cells in patients with advanced disease than alternative embodiments.

  It is generally important to provide an amount of peptide epitope delivered by a mode of administration sufficient to efficiently stimulate a cytotoxic T cell response; a composition that stimulates a helper T cell response is also Can be provided according to this embodiment of the present invention.

  Dosages for initial therapeutic immunization generally have a lower limit of about 1 μg, 5 μg, 50 μg, 500 μg, or 1,000 μg and an upper limit of about 10,000 μg; 20,000 μg; 30 Present in a unit dose range that is 50,000 μg; or 50,000 μg. Dosage values for humans typically range from about 500 μg to about 50,000 μg per 70 kg patient. According to a boost regimen ranging from weeks to months, a boost dosage of between about 1.0 μg to about 50,000 μg of peptide is determined by measuring the specific activity of CTL and HTL obtained from the patient's blood Depending on the patient's response and condition. Administration should continue until at least clinical symptoms or laboratory tests indicate that neoplasia is eliminated or diminished and thereafter for a period of time. Dosages, routes of administration, and dosage schedules are adjusted according to methodologies known in the art.

  In certain embodiments, the peptides and compositions of the invention are used in severe disease states (ie, life-threatening or potentially life-threatening conditions). In such cases, the preferred composition of the present invention has a substantial excess compared to these stated dosages as a result of the minimal amount of extraneous material and the relatively non-toxic nature of the peptide. Of these peptide compositions can be administered and may be felt as desired by the treating physician.

  The vaccine composition of the present invention can also be used purely as a prophylactic agent. In general, the dosage for initial prophylactic immunization is generally about 1 μg, 5 μg, 50 μg, 500 μg or 1000 μg for the lower value and about 10,000 μg for the higher value; 20,000 μg; 30 Present in a unit dosage range that is 50,000 μg; or 50,000 μg. Dosage values for humans typically range from about 500 μg to about 50,000 μg per 70 kg patient. This is followed by a boost dosage of between about 1.0 μg and about 50,000 μg of peptide at defined intervals about 4 weeks to 6 months after the initial vaccine administration. The immunogenicity of a vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from patient blood samples.

  Pharmaceutical compositions for therapeutic treatment are parenteral, topical, oral, nasal, intrathecal, or local (e.g., Intended as a cream or topical ointment). Preferably, the pharmaceutical composition is administered parenterally (eg intravenously, subcutaneously, intradermally or intramuscularly). Accordingly, the present invention provides a composition for parenteral administration comprising a solution of an immunogenic peptide dissolved or suspended in an acceptable carrier (preferably an aqueous carrier).

  A variety of aqueous carriers can be used (eg, water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc.). These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solution can be packaged as is for use or lyophilized (the lyophilized preparation is combined with a sterile solution prior to administration).

  This composition may be pharmaceutically acceptable auxiliary substances (eg, pH and buffering agents, tonicity adjusting agents, wetting agents, preservatives, etc.) as necessary to approximate physiological conditions (eg, , Sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like)).

  The concentration of the peptide of the invention in the pharmaceutical formulation can vary widely (ie, from less than about 0.1%, usually from about 2% or at least from about 2% to 20% to 50%). Can vary up to as much as% by weight or more) and is selected primarily by fluid volume, viscosity, etc., according to the particular mode of administration selected.

The human unit dose form of the composition is typically included in a pharmaceutical composition comprising a human unit dose of an acceptable carrier (in one embodiment, an aqueous carrier), and such composition to humans. Volumes / amounts known to those skilled in the art to be used for administration of products (see, eg, Remington's Pharmaceutical Sciences, 17th Edition, Editor A. Gennaro, Mack Publishing Co., Easton, Pennsylvania, 1985). ). For example, the peptide dose for the first immunization can be about 1 μg to about 50,000 μg, generally 100 μg to 5,000 μg for a 70 kg patient. For example, for nucleic acids, the initial immunization can be performed using IM (or SC or ID) administered at multiple sites in amounts of 0.5 mg to 5 mg and using the expression vector in the form of naked nucleic acids. Nucleic acids (0.1-1000 μg) can also be administered using a gene gun. After a 3-4 week incubation period, a booster dose is then administered. The booster can be a recombinant fowlpox virus that is administered at a dose of 5 × 10 ⁷ pfu to 5 × 10 ⁹ pfu.

For antibodies, treatment is generally administered via an acceptable route of administration, such as intravenous injection (IV), typically at doses ranging from about 0.1 mg / kg body weight to about 10 mg / kg body weight. Including repeated administration of 109P1D4 antibody preparations. In general, doses in the range of 10 mg to 500 mg mAb per week are effective and well tolerated. In addition, an anti-109P1D4 mAb preparation given at an initial loading dose of about 4 mg / kg patient body weight IV, followed by weekly IV doses of about 2 mg / kg represents an acceptable dosing regimen. As will be appreciated by those skilled in the art, various factors can affect the ideal dosage in a particular case. Such factors include, for example, the half-life of the composition, the binding affinity of the Ab, the immunogenicity of the substance, the degree of 109P1D4 expression in the patient, the degree of circulating shed 109P1D4 antigen, as desired Examples include steady state concentration levels, frequency of treatment, and the effects of chemotherapeutic or other agents used in combination with the treatment methods of the present invention, and the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μl to 1 mg, 1 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 400 mg to 500 mg, 500 mg to 600 mg, 600 mg to 700 mg, 700 mg to 800 mg, 800 mg. -900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose ranges from: 2 mg / kg body weight to 5 mg / kg body weight (eg, a weekly dose of 1 mg / kg to 3 mg / kg); 0.5 mg / kg body weight, 1 mg / kg Body weight, 2 mg / kg body weight, 3 mg / kg body weight, 4 mg / kg body weight, 5 mg / kg body weight, 6 mg / kg body weight, 7 mg / kg body weight, 8 mg / kg body weight, 9 mg / kg body weight, 10 mg / kg body weight (for example, weekly 2 weeks, 3 weeks or 4 weeks); 0.5 mg / kg body weight to 10 mg / kg body weight (eg, weekly dose for 2 weeks, 3 weeks or 4 weeks); weekly 225 mg / m ² body surface area, 250 mg / ^{m 2} body surface area, 275 mg / ^{m 2} body surface area, 300 mg / ^{m 2} body surface area, 325 mg / ^{m 2} body surface area, 350 mg / ^{m 2} body surface area, 375 mg / ^{m 2} body Surface area, 400 mg / ^{m 2} body surface area; weekly 1 to 600 mg / ^{m 2} body surface area; weekly 225~400mg / ^{m 2} body surface area product; these, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, Can be followed by weekly doses for 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more.

  In one embodiment, the human unit dosage form of the polynucleotide comprises a suitable dosage range or effective amount that provides any therapeutic effect. As will be appreciated by those skilled in the art, the therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, the molecular weight of the polynucleotide, and the route of administration. The dosage is generally selected by a physician or other medical professional according to various parameters known in the art, such as the severity of symptoms, the patient's medical history, and the like. In general, for polynucleotides of about 20 bases, the dosage ranges are, for example, about 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg. 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg From an independently selected lower limit such as / kg or 500 mg / kg, greater than the lower limit by about 60 mg / kg, 80 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg kg, 750 mg / kg, 1000 mg / kg, 1500 mg / kg, 2000 mg / kg, 000mg / kg, 4000mg / kg, 5000mg / kg, 6000mg / kg, 7000mg / kg, 8000mg / kg, may be selected from up to the limit independently selected of 9000 mg / kg or 10,000mg / kg. For example, the dose can be almost any of the following: 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 10 mg / kg, 1 mg / kg to 500 mg / kg, 100 mg / kg to 400 mg / kg, 200 mg / kg to 300 mg / kg, 1 mg / kg to 100 mg / kg, 100 mg / kg to 200 mg / kg, 300 mg / kg to 400 mg / kg kg, 400 mg / kg to 500 mg / kg, 500 mg / kg to 1000 mg / kg, 500 mg / kg to 5000 mg / kg, or 500 mg / kg to 10,000 mg / kg. In general, parenteral routes of administration may require higher doses of polynucleotides compared to more direct application of polynucleotides to diseased tissue. Increasing lengths of polynucleotides also require higher doses of polynucleotides.

In one embodiment, the human unit dosage form of T cells comprises an appropriate dosage range or effective amount that provides any therapeutic effect. As will be appreciated by those skilled in the art, the therapeutic effect depends on a number of factors. The dosage is generally selected by a physician or other medical professional according to various parameters known in the art, such as the severity of the symptoms, the patient's medical history, and the like. The dose can be from about 10 ⁴ cells to about 10 ⁶ cells, from about 10 ⁶ cells to about 10 ⁸ cells, from about 10 ⁸ cells to about 10 ¹¹ cells, or from about 10 ⁸ cells to about 5 × 10 ¹⁰ cells. The dose can also be about 10 ⁶ cells / m ² to about 10 ¹⁰ cells / m ² , or about 10 ⁶ cells / m ² to about 10 ⁸ cells / m ² .

  The proteins of the invention and / or nucleic acids encoding the proteins can also be administered via liposomes, which can also serve for the following: 1) Targeting proteins to specific tissues such as lymphoid tissues 2) selective targeting to diseased cells; or 3) increased half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, layered layers, and the like. In these preparations, the peptide to be delivered is either alone or in combination with a molecule that binds to a receptor that is prevalent in lymphoid cells (eg, a monoclonal antibody that binds to the CD45 antigen) or other therapeutic composition. In combination with a product or immunogenic composition. Thus, liposomes that are either filled or decorated with the desired peptide of the present invention can be directed to the site of lymphoid cells, and then to the lymphoid cells, the liposomes have a peptide composition. Deliver. Liposomes for use in accordance with the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids, and sterols (eg, cholesterol). The choice of lipid is generally guided by considerations of, for example, the size of the liposome, acid instability, and the stability of the liposome in the bloodstream. For preparing liposomes, see, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. Various methods can be used.

  In order to target cells of the immune system, the ligand to be incorporated into the liposome can include, for example, an antibody or fragment thereof specific for the cell surface determinant of the desired immune system cell. Liposomal suspensions containing peptides can be administered intravenously, locally, topically, in doses that vary according to the mode of administration, the peptide being delivered, and the stage of the disease being treated, among others. It can be administered by administration or the like.

  For solid compositions, conventional non-toxic solid carriers may be used including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Can be mentioned. For oral administration, a pharmaceutically acceptable non-toxic composition is generally 10% to 95% with any of the commonly used excipients such as the carriers listed above. Of active ingredient (ie, one or more peptides of the present invention) and more preferably by incorporating active ingredient at a concentration of 25% to 75%.

  For aerosol administration, the immunogenic peptide is preferably supplied in finely divided form together with a surfactant and a propellant. A typical percentage of peptides is about 0.01% to 20% by weight, preferably about 1% to 10% by weight. Of course, the surfactant must be non-toxic and preferably soluble in the propellant. Representative examples of such agents include fatty acids containing about 6 to 22 carbon atoms (eg, caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid). ), And oleic acid) with aliphatic polyhydric alcohols or cyclic anhydrides thereof and partial esters. Mixed esters such as mixed glycerides or natural glycerides can be used. The surfactant may constitute from about 0.1% to 20%, preferably from about 0.25% to 5% by weight of the composition. The rest of the composition is usually a propellant. A carrier may also be included if desired, such as in the case of lecithin for intranasal delivery.

(XI) 109P1D4 diagnostic and prognostic embodiments)
As disclosed herein, 109P1D4 polynucleotides, 109P1D4 polypeptides, 109P1D4 reactive cytotoxic T cells (CTL), 109P1D4 reactive helper T cells (HTL) and anti-109P1D4 polypeptide antibodies are dysregulated. Conditions associated with sex cell proliferation (eg, cancers, in particular those listed in Table I (eg, specific patterns of their tissue expression, and, for example, the expression analysis of 109P1D4 in normal tissues and patient specimens) (See both its overexpression in specific cancers as described in the appended examples))) used in well-known diagnostic, prognostic, and therapeutic assays.

  109P1D4 can be similar to PSA, a prostate-related antigen. PSA is a prototype marker that has been used by healthcare professionals for several years to identify and monitor the presence of prostate cancer (eg, Merrill et al., J. Urol. 163 (2): 503-5120 (2000). Polasik et al., J. Urol. Aug; 162 (2): 293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91 (19): 1635-1640 (1999)). A variety of other diagnostic markers are also used under similar circumstances and include p53 and K-ras (eg, Tulcinsky et al., Int J Mol Med 1999 Jul 4 (1): 99-102). And Minimoto et al., Cancer Detect Prev 2000; 24 (1): 1-12). Thus, with this disclosure regarding 109P1D4 polynucleotides and 109P1D4 polypeptides (and 109P1D4 polynucleotide probes and anti-109P1D4 antibodies used to identify the presence of these molecules) and their properties, one skilled in the art was used. These molecules can be utilized in a manner similar to that (eg, in various diagnostic assays relating to testing conditions associated with cancer).

  Representative embodiments of 109P1D4 polynucleotides, 109P1D4 polypeptides, 109P1D4 reactive T cells, and diagnostic methods utilizing anti-109P1D4 antibodies include, for example, PSA polynucleotides, PSA polypeptides and PSA reactive T cells. And similar to methods from well-established diagnostic assays using anti-PSA antibodies. For example, to observe the presence and / or level of PSA mRNA in methods of monitoring PSA overexpression or prostate cancer metastasis, PSA polynucleotides can be probed (eg, in Northern analysis (see, eg, Sharief et al., Biochem. Mol. Biol. Int. 33 (3): 567-74 (1994))) and primers (eg, in PCR analysis (eg, Okagawa et al., J. Urol. 163 (4): 1189-1190)). 2000)) 109P1D4 polynucleotides described herein are expressed in the same way as over-expression of 109P1D4 or metastasis of prostate cancer and other cancers that express this gene. Used in the same manner to detect Get. Alternatively, a PSA polypeptide is used to generate a PSA-specific antibody, which is then expressed by overexpression of the PSA protein (see, eg, Stephan et al., Urology 55 (4): 560-3 (2000)). ) Or the presence and / or level of PSA protein in a method that monitors metastasis of prostate cells (see, eg, Alanen et al., Pathol. Res. Pract. 192 (3): 233-7 (1996)) Just as it is used, the 109P1D4 polypeptides described herein are used in the detection of 109P1D4 overexpression or metastasis of prostate and other cancer cells expressing this gene. Can be used to generate antibodies for

  Specifically, since metastasis involves the migration of cancer cells from the original organ (eg, lung or prostate) to a different region of the body (eg, lymph nodes), 109P1D4 polynucleotide and / or 109P1D4 polypeptide Assays that test biological samples for the presence of expressing cells can be used to provide evidence of metastasis. For example, biological samples from tissues that normally do not contain 109P1D4-expressing cells (lymph nodes), such as 109P1D4 expression found in LAPC4 and LAPC9 (xenografts isolated from lymph nodes and bone metastases, respectively) If found to contain 109P1D4-expressing cells, this finding indicates metastasis.

  Alternatively, the 109P1D4 polynucleotide and / or 109P1D4 polypeptide may be expressed in a biological sample that does not normally express 109P1D4 or express 109P1D4 at different levels, for example, 109P1D4 or increased expression of 109P1D4. To provide evidence of cancer when found to have (eg, 109P1D4 expression in the cancers listed in Table I and patient samples shown in the accompanying figures) Can be used. In such an assay, one of ordinary skill in the art can supplement the evidence for metastasis by testing the biological sample (in addition to 109P1D4) for the presence of a second tissue restriction marker (eg, PSA, PSCA, etc.). May be further desired (see, eg, Alanen et al., Pathol. Res. Pract. 192 (3): 233-237 (1996)).

  The use of immunohistochemistry to identify the presence of 109P1D4 polypeptide in a tissue section can indicate an altered state of a particular cell in that tissue. It is well known in the art that the ability of an antibody to localize against a polypeptide expressed in cancer cells is a method of diagnosing the presence, disease state, progression, and / or tumor invasiveness of a disease. To be understood. Such antibodies can also detect altered distribution of the polypeptide in the cancer cells when compared to the corresponding non-malignant tumor tissue.

  The 109P1D4 polypeptides and immunogenic compositions are also useful in view of the phenomenon of altered subcellular protein localization in disease states. A change in cells from a normal state to a disease state causes a change in cell morphology. This is often associated with changes in subcellular protein localization / distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in the distribution of that protein in a non-polar manner across the cell surface.

  The phenomenon of altered subcellular protein localization in disease states has been demonstrated with MUC1 and Her2 proteins by using immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1 and some subcellular localization of its glycoprotein, while malignant lesions often show a nonpolar staining pattern (Diaz et al., The Breast). Journal, 7: 40-45 (2001); Zhang et al., Clinical Cancer Research, 4: 2669-2676 (1998); Furthermore, normal breast epithelium is Her2 protein negative or shows only a basolateral distribution. On the other hand, malignant cells can express this protein throughout the cell surface (De Potter et al., International Journal of Cancer 44: 969-974 (1989); McCormick et al., 117: 935-943 (2002)). Alternatively, the protein distribution can vary from a surface-only localization to include diffuse cytoplasmic expression in the disease state. Such an example can be observed in the case of MUC1 (Diaz et al., The Breast Journal 7: 40-45 (2001)).

  Changes in protein localization / distribution in the cells as detected by immunohistochemistry may also provide valuable information regarding the convenience of a particular treatment modality. This latter point is indicated by the situation where the protein can be in the cell in normal tissues but on the cell surface in malignant cells. The cell surface location allows the cells to be advantageously subjected to antibody-based diagnostic and treatment regimens. If such changes in protein localization occur with 109P1D4, this 109P1D4 protein and the immune response associated therewith are very useful. Thus, the ability to determine whether changes in subcellular protein localization have occurred for 109P1D4 makes the 109P1D4 protein and its associated immune response very useful. The use of the 109P1D4 composition allows one skilled in the art to make important diagnostic and therapeutic decisions. Immunohistochemical reagents specific for 109P1D4 are also useful for detecting metastasis of tumors that express 109P1D4 when the 109P1D4 polypeptide is present in tissues where 109P1D4 is not normally produced.

  Accordingly, 109P1D4 polypeptides and 109P1D4 antibodies resulting from immune responses thereto are useful in a variety of important settings known to those skilled in the art (eg, diagnostic, prognostic, prophylactic and / or therapeutic purposes).

  Just as PSA polynucleotide fragments and PSA polynucleotide variants are utilized by those skilled in the art for use in methods of monitoring PSA, 109P1D4 polynucleotide fragments and 109P1D4 polynucleotide variants are expressed in a similar manner. used. In particular, typical PSA polynucleotides used in methods for monitoring PSA are probes or primers composed of fragments of PSA cDNA sequences. Taking this as an example, the primer used to PCR amplify a PSA polynucleotide must contain a sequence that is shorter than the entire PSA sequence in order to function in the polymerase chain reaction. In the context of such a PCR reaction, one of ordinary skill in the art generally creates a variety of different polynucleotide fragments that can be used as primers to amplify different portions of the polynucleotide of interest or to optimize the amplification reaction. (See, eg, Caetano-Anoles, G. Biotechniques 25 (3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98: 121-154 (1998)). A further illustration of the use of such a fragment is provided in the Example entitled “109P1D4 Expression Analysis in Normal Tissue and Patient Samples”, where the 109P1D4 polynucleotide fragment is of 109P1D4 RNA in cancer cells. Used as a probe to show expression. Furthermore, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNA in PCR and Northern analyses (eg, Sawai et al., Fetal Diagnostic. Ther. 1996 Nov-Dec; 11 ( 6): 407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al., 1995). Polynucleotide fragments and variants are useful in situations where they can bind to a target polynucleotide sequence (eg, 109P1D4 polynucleotide shown in FIG. 2 or variants thereof) under high stringency conditions.

  In addition, PSA polypeptides that contain an epitope that can be recognized by an antibody or T cells that specifically bind to that epitope are used in methods of monitoring PSA. 109P1D4 polypeptide fragments, and 109P1D4 polypeptide analogs or 109P1D4 variants can also be used in a similar manner. This practice of generating antibodies (eg, anti-PSA antibodies or T cells) using polypeptide fragments or polypeptide variants is well known in the art with a wide variety of systems such as fusion proteins used by practitioners. (See, eg, Current Protocols In Molecular Biology, Volume 2, Unit 16, edited by Frederick M. Ausubel et al., 1995). In this situation, each epitope functions to provide a structure to which the antibody or T cell is reactive. Typically, those skilled in the art generate a variety of different polypeptide fragments that can be used to generate an immune response specific for different portions of the polypeptide of interest (see, eg, US Pat. No. 5,840). , 501 and U.S. Pat. No. 5,939,533). For example, utilizing a polypeptide comprising one of the 109P1D4 biological motifs discussed herein or one of the motif-bearing subsequences readily identified by one of ordinary skill in the art based on motifs available in the art May be preferred. Polypeptide fragments, variants or analogs typically include epitopes that are capable of producing antibodies or T cells specific for the target polypeptide sequence (eg, 109P1D4 polypeptide shown in FIG. 3). Insofar as useful in this situation.

  As shown herein, 109P1D4 and 109P1D4 polypeptides (and 109P1D4 polynucleotide probes and anti-109P1D4 antibodies or T cells used to identify the presence of these molecules) are listed in Table I. It shows the specific properties that make them useful in diagnosing cancers such as those listed. Diagnostic assays that measure the presence of the 109P1D4 gene product to assess the presence or development of the disease states described herein (eg, prostate cancer) appear to have been performed very successfully using PSA. In addition, it is used to identify patients for prophylactic measurement or further monitoring. In addition, these substances cannot be made based on tests for PSA alone, for example, a clear diagnosis of metastasis of prostate origin (eg Alanen et al., Pathol. Res. Pract. 192 (3): 233-237). (See 1996)), and as a result, substances such as 109P1D4 and 109P1D4 polypeptides (and 109P1D4 polynucleotide probes and anti-109P1D4 antibodies used to identify the presence of these molecules) In situations where it is necessary to be used to confirm metastases of prostate origin, the need in the art for molecules with similar or complementary characteristics to PSA is met.

  Finally, in addition to their use in diagnostic assays, the 109P1D4 polynucleotides disclosed herein are chromosomal regions to which the 109P1D4 gene is mapped (see the example below, titled “Chromosome Mapping of 109P1D4”). Have many other uses, such as their use in the identification of oncogene-related chromosomal abnormalities. Furthermore, in addition to their use in diagnostic assays, the 109P1D4-related proteins and 109P1D4-related polynucleotides disclosed herein are useful in other applications such as their use in forensic analysis of tissues of unknown origin. (See, for example, Takahama K, Forensic Sci Int 1996 Jun 28; 80 (1-2): 63-9).

  Furthermore, the 109P1D4-related proteins or 109P1D4-related polynucleotides of the present invention may be used to treat pathological conditions characterized by 109P1D4 overexpression. For example, the amino acid sequence or nucleic acid sequence of FIG. 2 or FIG. 3, or any fragment can be used to generate an immune response against the 109P1D4 antigen. An antibody or other molecule reactive with 109P1D4 can be used to modulate the function of this molecule, thereby providing a therapeutic benefit.

(XII) Inhibition of 109P1D4 protein function)
The present invention includes various methods and compositions for inhibiting 109P1D4 from binding to its binding partner or inhibiting its binding to other proteins, and methods for inhibiting the function of 109P1D4. .

(XII.A.) Inhibition of 109P1D4 using intracellular antibodies)
In one approach, a recombinant vector encoding a single chain antibody that specifically binds 109P1D4 is introduced into cells expressing 109P1D4 via gene transfer techniques. Thereby, the encoded single chain anti-109P1D4 antibody is expressed intracellularly, binds to 109P1D4 protein and thereby inhibits its function. Methods for manipulating such intracellular single chain antibodies are well known. Such intracellular antibodies (also known as “intrabodies”) are specifically targeted to specific compartments within the cell and provide control over where the inhibitory activity of the treatment is focused . This technique has been successfully applied in the field (for review, see Richardson and Marasco, 1995, TIBTECH, Volume 13). Intrabodies have been shown to substantially eliminate the expression of other abundant cell surface receptors (eg, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al. 1994, J. Biol. Chem. 289: 23931-23936; Desane et al., 1994, Gene Ther. 1: 332-337).

  A single chain antibody comprises a heavy chain variable domain and a light chain variable domain linked by a flexible polypeptide linker and is expressed as a single polypeptide. Optionally, single chain antibodies are expressed as single chain variable region fragments linked to their light chain constant regions. Well-known intracellular transport signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies to accurately target the internal antibody to the desired intracellular compartment. For example, an internal antibody targeted to the endoplasmic reticulum (ER) is engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal (eg, KDEL amino acid motif). An internal antibody that is intended to exert activity in the nucleus is engineered to contain a nuclear localization signal. A lipid moiety is linked to the internal antibody to connect it to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic internal antibodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural destination in the cell.

  In one embodiment, an internal antibody is used to capture 109P1D4 in the nucleus, thereby preventing its activity in the nucleus. A nuclear targeting signal is engineered into such 109P1D4 internal antibody to achieve the desired targeting. Such 109P1D4 internal antibodies are designed to specifically bind to a specific 109P1D4 domain. In another embodiment, a cytosolic internal antibody that specifically binds to 109P1D4 protein is used to prevent 109P1D4 from approaching the nucleus, thereby causing 109P1D4 to exert any biological activity in the nucleus. (Eg, prevent 109P1D4 from forming transcription complexes with other factors).

  In order to specifically direct the expression of such an internal antibody to a particular cell, the transcription of the internal antibody is placed under the regulatory control of an appropriate tumor-specific promoter and / or tumor-specific enhancer. For example, PSA promoters and / or PSA promoter / PSA enhancers can be utilized to specifically target expression of internal antibodies to the prostate (eg, US patents issued on July 6, 1999). No. 5,919,652).

(XII.B.) Inhibition of 109P1D4 using recombinant protein)
In another approach, the recombinant molecule binds to 109P1D4, thereby preventing 109P1D4 function. For example, such recombinant molecules prevent or inhibit 109P1D4 from accessing / binding to its binding partner and binding to other proteins. Such recombinant molecules can include, for example, the reactive portion of a 109P1D4-specific antibody molecule. In certain embodiments, the 109P1D4 binding domain of the 109P1D4 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein is linked to two 109P1D4 linked to the Fc portion of human IgG (eg, human IgG1). Contains a ligand binding domain. Such an IgG portion can include, for example, a C _H 2 domain and a C _H 3 domain, and a hinge region, but does not include a C _H 1 domain. Such a dimeric fusion protein is administered in a soluble form to a patient suffering from cancer associated with expression of 109P1D4, whereby the dimeric fusion protein specifically binds to 109P1D4. And block the interaction of 109P1D4 with its binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking techniques.

(XII.C.) 109P1D4 transcription or translation inhibition)
The present invention also includes various methods and compositions for inhibiting transcription of the 109P1D4 gene. Similarly, the present invention also provides methods and compositions for inhibiting 109P1D4 mRNA to protein translation.

  In one approach, the method of inhibiting 109P1D4 gene transcription comprises contacting the 109P1D4 gene with a 109P1D4 antisense polynucleotide. In another approach, a method of inhibiting the translation of 109P1D4 mRNA comprises contacting 109P1D4 mRNA with an antisense polynucleotide. In another approach, 109P1D4 specific ribozymes are used to inhibit translation by cleaving the 109P1D4 message. Such antisense-based and ribozyme-based methods can also be directed to the regulatory regions of the 109P1D4 gene (eg, 109P1D4 promoter element and / or 109P1D4 enhancer element). Similarly, a protein capable of inhibiting 109P1D4 gene transcription factor is used to inhibit the transcription of 109P1D4 mRNA. Various polynucleotides and compositions useful in the above methods are described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

  Other factors that inhibit 109P1D4 transcription by interfering with 109P1D4 transcriptional activation are also useful for treating 109P1D4 expressing cancers. Similarly, factors that interfere with the processing of 109P1D4 are useful for treating cancers that express 109P1D4. Cancer treatment methods utilizing such factors are also within the scope of the present invention.

(XII.D.) General considerations for therapeutic strategies)
In order for gene transfer and gene therapy techniques to deliver therapeutic polynucleotide molecules (ie, antisense, ribozyme, polynucleotide encoding internal antibodies, and other 109P1D4 inhibitory molecules) to tumor cells that synthesize 109P1D4. Can be used. A number of gene therapy approaches are known in the art. Recombinant vectors encoding 109P1D4 antisense polynucleotides, 109P1D4 ribozymes, factors that can interfere with transcription of 109P1D4, and the like can be delivered to target tumor cells using such gene therapy approaches.

  The therapeutic approaches described above can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approach of the present invention uses reduced dosages of chemotherapy (or other therapies) and is advantageous for all patients and in particular for patients who do not tolerate chemotherapeutic toxicity. The use of less frequent administration may be possible.

  The anti-tumor activity of a particular composition (eg, antisense, ribozyme, internal antibody), or a combination of such compositions can be assessed using a variety of in vitro and in vivo assay systems. In vitro assays to assess therapeutic activity include cell proliferation assays, soft agar assays, and other assays that exhibit tumor promoting activity, binding that can determine the extent to which the therapeutic composition inhibits binding of 109P1D4 to a binding partner Examples include assays.

  In vivo, the efficacy of a 109P1D4 therapeutic composition can be evaluated in an appropriate animal model. For example, a xenoprostate cancer model (where human prostate cancer explants or passaged xenograft tissue is introduced into immunocompromised animals (eg, nude mice or SCID mice)) is used (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT patent application WO 98/16628 and US Pat. No. 6,107,540 may repeat the formation of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of later stages of the disease, Various xenograft models of human prostate cancer are described. Efficacy can be predicted using assays that measure inhibition such as tumor formation, tumor regression or metastasis.

  In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor-bearing mice treated with the therapeutic composition can be tested for the presence of apoptotic lesions and compared to untreated control xenograft-bearing mice. The degree to which apoptotic lesions are found in the tumors of treated mice provides an indication of the therapeutic efficacy of this composition.

  The therapeutic composition used in the practice of the foregoing method can be formulated into a pharmaceutical composition comprising a carrier suitable for the desired delivery method. Suitable carriers include any substance that, when combined with a therapeutic composition, retains the antitumor function of the therapeutic composition and is generally unresponsive to the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers (eg, sterile phosphate buffered saline solution, bacteriostatic water, etc., generally Remington's Pharmaceutical Sciences). (See 16th edition), A. Osal., 1980).

  The therapeutic formulation can be solubilized, and it can be administered via any route that can deliver the therapeutic composition to the tumor site. Potentially effective routes of administration include intravenous routes, parenteral routes, intraperitoneal routes, intramuscular routes, intratumoral routes, intradermal routes, intraorgan routes, orthotopic routes and the like. However, it is not limited to these. Preferred formulations for intravenous injection include the therapeutic composition in a solution of preserved bacteriostatic water, the therapeutic composition in a solution of sterile non-preserved water, and / or The therapeutic composition diluted in a polyvinyl chloride or polyethylene bag (USP) containing 9% sterile sodium chloride. The therapeutic protein preparation can be lyophilized and stored as a sterile powder, preferably under reduced pressure, and then reconstituted in bacteriostatic water (eg, containing benzyl alcohol preservatives) or sterile water prior to injection. Can be built.

  Dosages and administration protocols for the treatment of cancer using the methods described above will vary with the method and target cancer and will generally depend on a number of other factors understood in the art.

(XIII.) Identification, characterization and use of 109P1D4 modulators)
(Identification methods and use of regulators)
In one embodiment, screening is performed to identify modulators that induce or suppress specific expression profiles, induce or suppress specific pathways, and preferably thereby generate related phenotypes. In another embodiment, when identifying differentially expressed genes that are important in a particular state; screening to identify modulators that alter (increase or decrease) the expression of individual genes Execute. In another embodiment, screening is performed to identify modulators that alter the biological function of the expression product of a differentially expressed gene. Again, in identifying the importance of a gene in a particular state, screening is performed to identify factors that bind to and / or modulate the biological activity of the gene product.

  In addition, screening is performed on genes that are induced in response to the candidate agent. After identifying a regulator (a regulator that suppresses the cancer expression pattern that causes the normal expression pattern, or a regulator of the oncogene that causes the gene expression as in normal tissues), a screening is performed to Identify genes that are specifically regulated in response. Comparison of expression profiles between normal and drug-treated cancer tissues reveals genes that are not expressed in normal or cancerous tissues but are expressed in drug-treated tissues or vice versa . These drug specific sequences are identified and used by the methods described herein for oncogenes or oncoproteins. In particular, these sequences and the proteins they encode are used in the production or identification of cells treated with drugs. In addition, antibodies are raised against drug-inducible proteins and used to target new therapeutic agents to treated cancer tissue samples.

(Identification and screening assays related to modulators)
(Assays related to gene expression)
The proteins, nucleic acids and antibodies of the invention are used in screening assays. Cancer-related proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences can be used in screening assays (eg, “gene expression profiles”, polypeptide expression profiles or effects of drug candidates on altered biological function). Used in evaluation). In one embodiment, the expression profile is preferably used in combination with high-throughput screening techniques to monitor genes after treatment with a candidate agent for the expression profile (eg, Davis, GF et al., J Biol Screen 7:69 (2002); Zlokarnik et al., Science 279: 84-8 (1998); Heid, Genome Res 6: 986-94, 1996).

  Cancer proteins, antibodies, nucleic acids, modified proteins, and cells containing native or modified cancer proteins or genes are used in screening assays. That is, the present invention includes a method for screening for a composition that modulates the cancer phenotype or physiological function of the cancer protein of the present invention. This can be done on the gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, the expression profile is preferably used in combination with a high throughput screening technique that allows monitoring after treatment with a candidate agent. See Zlokamik (supra).

  Various assays are performed on the genes and proteins of the invention. Assays are performed for individual nucleic acid or protein levels. That is, once a particular gene is identified as being upregulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the oncoproteins of the invention. In this context, “modulation” includes an increase or decrease in gene expression. The preferred amount of modulation is based on the natural change in gene expression in normal versus cancer-affected tissue, and this change is at least 10%, preferably 50%, more preferably 100-300%, and some In embodiments, it is 300-1000% or more. Thus, if the gene shows a 4-fold increase in cancer tissue compared to normal tissue, a reduction of about 4-fold is often desirable; similarly, the gene is cancerous compared to normal tissue. A target value of a 10-fold increase in expression by a test compound is often desirable if it exhibits a 10-fold decrease in tissue. Regulators that exacerbate the type of gene expression observed in cancer are also useful as upregulated targets, for example, in further analysis.

  The amount of gene expression is monitored using nucleic acid probes and quantification of gene expression levels, or the gene product itself is monitored by using, for example, antibodies against cancer proteins and standard immunoassays. Is done. Proteomics and separation techniques also allow quantification of expression.

(Expression monitoring to identify compounds that alter gene expression)
In one embodiment, gene expression monitoring (ie, expression profile) is monitored for multiple entities simultaneously. Such a profile typically includes one or more of the genes of FIG. In this embodiment, for example, a cancer nucleic acid probe is attached to the biochip to detect and quantify cancer sequences in specific cells. Alternatively, PCR can be used. Thus, for example, a series of microtiter plate wells can be used with primers distributed to the desired wells. A PCR reaction can then be performed and analyzed for each well.

  Expression monitoring is performed to identify compounds that alter the expression of one or more cancer-related sequences (eg, the polynucleotide sequences set forth in FIG. 2). In general, test modulators are added to the cells prior to analysis. In addition, the screen also modulates cancer, modulates a cancer protein of the invention, binds to a cancer protein of the invention, or prevents binding of a cancer protein of the invention and an antibody or other binding partner. Provided to identify drugs.

  In one embodiment, the high-throughput screening method includes providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify library members (specific chemical species or subclasses) that exhibit the desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds”, as compounds for screening, or as therapeutic agents.

  In certain embodiments, a combinatorial library of potential modulators is screened for the ability to bind to a cancer polypeptide or to modulate activity. Traditionally, a new chemical entity with useful properties identifies a chemical compound (referred to as a “lead compound”) that has any desired property or activity (eg, inhibitory activity) and creates a variant of this lead compound. And by evaluating the properties and activities of these variant compounds. Often, high throughput screening (HTS) methods are used for such analyses.

  As noted above, gene expression monitoring is conveniently used to test candidate modulators (eg, proteins, nucleic acids or small molecules). After adding the candidate compound and incubating the cells for a period of time, a sample containing the target sequence to be analyzed is added to, for example, a biochip.

  If desired, the target sequence is prepared using known techniques. For example, the sample is processed to lyse cells using known lysis buffers, electroporation, etc., and purification and / or amplification (eg, PCR) is performed as needed. For example, in vitro transcription using a label fed to a nucleotide is performed. In general, these nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.

  The target sequence can be labeled with, for example, a fluorescent signal, a chemiluminescent signal, a chemical signal, or a radioactive signal to provide a means of detecting specific binding of the target sequence to the probe. The label can also be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which, when provided with an appropriate substrate, produces a product that is detected. Alternatively, the label is a labeled compound or small molecule (eg, an enzyme inhibitor that binds to the enzyme but is not catalyzed or altered by the enzyme). The label can also be a moiety or compound (eg, biotin that specifically binds to an epitope tag or streptavidin). For the biotin example, streptavidin is labeled as described above, thereby providing a detectable signal to the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.

  As will be appreciated by those skilled in the art, these assays may be direct hybridization assays or may include “sandwich assays”, which include the use of multiple probes and generally include: U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591 No. 5,571,670; No. 5,580,731; No. 5,571,670; No. 5,591,584; No. 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, generally the target nucleic acid is prepared as outlined above and then added to a biochip containing a plurality of nucleic acid probes under conditions that allow the formation of a hybridization complex. The

  A variety of hybridization conditions as outlined above (including high stringency conditions, moderate stringency conditions and low stringency conditions) are used in the present invention. These assays are generally performed under stringency conditions that allow the formation of labeled probe hybridization complexes only in the presence of the target. Stringency is by changing process parameters that are thermodynamic variables, including but not limited to temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc. Can be controlled. These parameters can also be used to control non-specific binding, as generally outlined in US Pat. No. 5,681,697. Thus, it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

  The reactions outlined herein can be accomplished in a variety of ways. The components of these reactions can be added simultaneously or sequentially in a different order and preferred embodiments are outlined below. In addition, the reaction can include a variety of other reagents. This includes salts, buffers, neutral proteins (eg, albumin), detergents, etc., to facilitate optimal hybridization and detection and / or non-specific interactions or Can be used to reduce background interactions. In other cases, reagents that improve the efficiency of the assay (eg, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.) may also be used as needed, depending on the sample preparation method and target purity. The assay data is analyzed to determine the expression levels of individual genes and the changes in expression levels between states to create a gene expression profile.

(Assays related to biological activity)
The present invention provides methods for identifying or screening for compounds that modulate the activity of the cancer-related genes or proteins of the present invention. This method includes the step of adding a test compound as described above to a cell containing the cancer protein of the present invention. These cells contain a recombinant nucleic acid encoding the oncoprotein of the present invention. In another embodiment, the library of candidate agents is tested on a plurality of cells.

  In one aspect, the assay may include physiological signals (eg, hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutic agents, radiation, carcinogenic or other cells ( That is, it is evaluated in the presence or absence of exposure before or after cell-cell contact)) or before or after exposure. In another example, this determination is made at different stages of the cell cycle process. In this manner, compounds that modulate the gene or protein of the invention are identified. A compound having pharmacological activity can increase the activity of the cancer protein of the present invention or can interfere with the activity of the cancer protein of the present invention. Once identified, similar structures are evaluated to identify important structural properties of the compound.

  In one embodiment, a method of modulating (eg, inhibiting) cancer cell division is provided; the method includes administration of a cancer modulator. In another embodiment, a method of modulating (eg, inhibiting) cancer is provided; the method includes administration of a cancer modulator. In a further embodiment, a method of treating a cell or individual having cancer is provided; the method includes administration of a cancer modulator.

  In one embodiment, a method is provided for modulating the state of a cell that expresses a gene of the invention. As used herein, conditions include parameters accepted in the art such as cellular growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction and the like. In one embodiment, the cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. Various cell growth, proliferation, and metastasis assays are known to those skilled in the art, as described herein.

(High-throughput screening to identify regulators)
Assays to identify appropriate modulators are acceptable for high throughput screening. Accordingly, preferred assays detect oncogene transcription increase or inhibition, polypeptide expression inhibition or increase, and polypeptide activity inhibition or increase.

  In one embodiment, the modulator evaluated in a high-throughput screening method is a protein, often a naturally occurring protein, or a fragment of a naturally occurring protein. Thus, for example, cell extracts containing proteins, or random or directed digests of proteinaceous cell extracts are used. In this manner, a library of proteins is created for screening in the methods of the invention. Bacterial, fungal, viral and mammalian protein libraries are particularly preferred in this embodiment, the latter is preferred, and human proteins are particularly preferred. Particularly useful test compounds are directed to the class of protein to which the target belongs (eg, substrates for enzymes, or ligands and receptors).

(Use of soft agar growth and colonization to identify and characterize modulators)
Normal cells require a solid support to bind and grow. When cells are transformed, they lose their phenotype and grow and detach from the support. For example, transformed cells can be grown in stirred suspension cultures or suspended in semi-solid media (eg, semi-solid agar or soft agar). This transformed cell, when transfected with a tumor suppressor gene, can regenerate a normal phenotype and again requires a solid support to bind and grow. Soft agar growth or colony formation in the assay is used to identify regulators of cancer sequences that, when expressed in host cells, inhibit abnormal cell growth and transformation. Regulators reduce or eliminate the ability of host cells suspended in solid or semi-solid media (eg, agar) to grow.

  Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells of Manual of Basic Technique (3rd edition, 1994). See also the method section of Garkavtsev et al. (1996) (supra).

(Evaluation of contact inhibition and growth density limitation to identify and characterize modulators)
Normal cells typically grow in a flat organized pattern in cell culture until they come into contact with other cells. When the cells come into contact with another cell, they are contact-inhibited and stop growing. However, transformed cells are not contact-inhibited and continue to grow to high density in unorganized lesions. Thus, the transformed cells grow to a higher saturation density than the corresponding normal cells. This is morphologically detected by the formation of undirected monolayers of cells or cells in the lesion. Alternatively, the labeling index with ( ³ H) -thymidine at saturation density is used to measure the density limit of growth, and similarly, the MTT assay or Alamar blue assay is used to determine whether the cell's growth capacity and regulators are To clarify the ability to influence the growth ability of See Freshney (1994) (supra). Transformed cells can regenerate a normal phenotype when transfected with a tumor suppressor, and are contact-inhibited and grow to low density.

In this assay, the saturation density ^{(3 H)} - labeling index of thymidine is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with cancer-associated sequences and grown at saturating density for 24 hours in non-limiting medium conditions. The percentage of cell labeling with ( ³ H) -thymidine is determined by the incorporated cpm.

  Contact-independent growth is used to identify regulators of cancer sequences that have resulted in abnormal cell growth and transformation. Regulators reduce or eliminate contact-independent growth and return cells to a normal phenotype.

(Evaluation of growth factors or serum dependence to identify and characterize modulators)
Transformed cells have lower serum dependence than their normal counterparts (eg, Temin, J. Natl. Cancer Inst. 37: 167-175 (1966); Eagle et al., J. Exp. Med 131: 836). -879 (1970); Freshney, (supra)). This is partly due to the release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of the transformed host cell can be compared to that under control. For example, cellular growth factor or serum dependence is monitored in a method for identifying and characterizing compounds that modulate cancer-associated sequences of the invention.

(Use of tumor-specific marker levels to identify and characterize modulators)
Tumor cells release an increased amount of certain factors (hereinafter “tumor-specific markers”) over their normal counterparts. For example, plasminogen activator (PA) is released from human gliomas at a higher level than from normal brain cells (eg, Gullino, Angiogenesis, Tumor Vascularization, and Potential interference with Tumor Growth, in Biologon Cancer, pages 178-184 (see Mich (ed.) 1985). Similarly, tumor angiogenic factor (TAF) is released in tumor cells at a higher level than its normal counterpart. See, for example, Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)). On the other hand, bFGF is released from endothelial cell tumors (Ensoli, B et al.).

  Various techniques for measuring the release of these factors are described in Freshney (1994) (supra). Unkless et al. Biol. Chem. 249: 4295-4305 (1974); Strickland & Beers, J. Am. Biol. Chem. 251: 5694-5702 (1976); Whur et al., Br. J. et al. Cancer 42: 305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, Biological Responses in Cancer, pp. 178h 184ch (A). 5: 111-130 (1985). For example, tumor-specific marker levels are monitored with a method for identifying and characterizing compounds that modulate the cancer-associated sequences of the invention.

(Invasion to matrigel to identify and characterize modulators)
The degree of invasiveness to Matrigel or extracellular matrix components can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and the invasiveness of cells into Matrigel or some other extracellular matrix component. In this assay, tumorigenic cells are typically used as host cells. The expression of tumor suppressors in these host cells reduces the invasiveness of the host cells. Cancer Res. 1999; 59: 6010; Freshney (1994) (supra) can be used. Briefly, the level of host cell invasion is measured by using a filter coated with Matrigel or some other extracellular matrix component. Penetration into the gel or through the distal side of the filter is rated as invasive and is pre-labeled by the number of cells and the distance traveled or by ¹²⁵ I and the distal side of the filter Or histologically rated by counting the activity at the bottom of the dish. See, for example, Freshney (1984) (supra).

(Evaluation of tumor growth in vivo to identify and characterize modulators)
The effect of cancer associated sequences on cell proliferation is tested in transgenic or immunosuppressed organisms. Transgenic organisms are prepared in a variety of ways accepted in the art. For example, a knockout transgenic organism (eg, a mammal such as a mouse) is produced in which the oncogene is disrupted or the oncogene is inserted. Knockout transgenic mice are created by inserting a marker gene or other heterologous gene into the endogenous oncogene site in the mouse genome via homologous recombination. Such mice can also be made by replacing an endogenous oncogene with a mutated version of an oncogene or by mutating the endogenous oncogene, for example by exposure to a carcinogen.

  In order to prepare a transgenic chimeric animal (eg, a mouse), a DNA construct is introduced into the nucleus of an embryonic stem cell. Cells containing the newly engineered genetic lesion are injected into the host mouse embryo, which is reimplanted into the recipient female. Some of these embryos develop into chimeric mice where some germ cells are derived from mutant cell lines. Thus, by breeding this chimeric mouse, it is possible to obtain a new strain of mouse containing the introduced genetic lesion (see, eg, Capecchi et al., Science 244: 1288 (1989)). Chimeric mice are described in US Pat. No. 6,365,797 issued April 2, 2002; US Pat. No. 6,107,540 issued August 22, 2000; Hogan et al., Manipulating the Mouse. Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approch, Robert. C. , (1987).

Alternatively, various immunosuppressed or immunodeficient host animals can be used. For example, genetically athymic “nude” mice (see, eg, Giovanella et al., J. Natl. Cancer Inst. 52: 921 (1974)), SCID mice, thymectomized mice, or irradiated Mice (see, for example, Bradley et al., Br. J. Cancer 38: 263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as the host. Transplantable tumor cells (typically about 10 ⁶ cells) injected into a syngeneic host produce invasive tumors in high proportions, while normal cells of similar origin Does not produce invasive tumors. In a host that has developed an invasive tumor, cells expressing cancer-associated sequences are injected subcutaneously or in a normal position. The mice are then separated into groups comprising a control group and a treatment experimental group (eg, treated with a modulator). After an appropriate length of time (preferably 4-8 weeks), tumor growth is measured (eg, by volume or its two largest dimensions, or weight) and compared to a control. Tumors with statistically significant reduction (eg, using Student's T test) are said to have inhibited growth.

(In vitro assay to identify and characterize modulators)
Assays to identify compounds with modulatory activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for an appropriate amount of time (eg, 0.5-48 hours). In one embodiment, the level of cancer polypeptide is determined in vitro by measuring the level of protein or mRNA. Protein levels are measured using immunoassays (eg, Western blotting, ELISA, etc.) using antibodies that selectively bind to cancer polypeptides or fragments thereof. For measurement of mRNA, amplification (eg, PCR, LCR, or hybridization assay (eg, Northern hybridization, RNAse protection, dot blotting)) is preferred. Protein or mRNA levels can be detected directly or indirectly labeled detection reagents (eg, fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically as described herein). For example, using a labeled antibody).

  Alternatively, a reporter gene system can be invented using an oncoprotein promoter operably linked to a reporter gene (eg, luciferase, green fluorescent protein, CAT, or P-gal). The reporter construct is typically transfected into the cell. After treatment with potential regulators, the amount of transcription, translation, or activity of the reporter gene is measured according to standard techniques known to those skilled in the art (Davis GF, supra; Gonzalez, J. & Neglescu , P. Curr. Opin. Biotechnol. 1998: 9: 624).

  As outlined above, in vitro screening is done on individual genes and gene products. That is, if a particular differentially expressed gene is identified as being important in a particular state, expression of this gene or screening for regulators of the gene product itself is performed.

  In one embodiment, screening for regulators of expression of a particular gene is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, the screen is designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for their ability to modulate differentially expressed activity. Furthermore, once the first candidate compound is identified, variants can be further screened to better evaluate the structure-activity relationship.

(Binding assay to identify and characterize modulators)
In a binding assay according to the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated against the protein of the invention and an immunoassay is performed to determine the amount and / or location of the protein. Alternatively, cells containing oncoproteins are used in the assay.

  Accordingly, these methods include the steps of mixing a cancer protein of the invention with a candidate compound, such as a ligand, and determining binding of the compound to the cancer protein of the invention. Preferred embodiments utilize human oncoproteins; animal models of human disease can also be developed and used. Other similar mammalian proteins may also be used as understood by those skilled in the art. Furthermore, in some embodiments, variants or derived oncoproteins are used.

  In general, the oncoproteins or ligands of the invention are non-diffusibly bound to an insoluble support. The support can be, for example, one having a region for receiving an isolated sample (microtiter plate, array, etc.). An insoluble support can be made from any composition to which the composition can be bound, is easily separated from soluble material, and is otherwise compatible with the overall screening method. The surface of such a support can be solid or porous and can be of any convenient shape.

  Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (eg, polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon®. Microtiter plates and arrays are particularly advantageous. This is because multiple assays can be performed simultaneously using small amounts of reagents and samples. The particular manner in which the composition is bound to the support is not critical so long as it is compatible with the reagents and overall method of the invention, maintains the activity of the composition and is non-diffusible. Preferred methods of conjugation include ligand binding sites when attaching the protein to a support (direct attachment to a “sticky” or ionic support, chemical cross-linking, protein or drug synthesis on the surface, etc.) And the use of antibodies that neither sterically block the activation sequence. Following binding of the protein or ligand / binding agent to the support, excess unbound material is removed by washing. The sample receiving area can then be blocked by incubating with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.

  Once the oncoprotein of the invention is bound to the support, a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and then the oncoprotein of the invention is added. Binding agents include specific antibodies, non-natural binding agents identified by screening chemical libraries, peptide analogs, and the like.

  Of particular interest are assays for identifying agents that have low toxicity to human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding. Functional assays (such as phosphorylation assays).

  Determination of the binding of test compounds (ligands, binding agents, modulators, etc.) to the oncoproteins of the invention can be made in a number of ways. The test compound can be labeled and binding can be accomplished, for example, by attaching all or part of the oncoprotein of the invention to a solid support, adding a labeled candidate compound (eg, a fluorescent label), and removing excess reagents. It can be determined directly by washing away and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled. For example, the protein or ligand of the invention is labeled. Alternatively, more than one component is labeled with a different label (eg, I ¹²⁵ for proteins and fluorophores for compounds). Proximity reagents (eg, quenching agents or energy transfer agents) are also useful.

(Competitive binding to identify and characterize modulators)
In one embodiment, binding of a “test compound” is determined by a competitive binding assay using a “competitor”. A competitor is a binding moiety that binds to a target molecule (eg, a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands and the like. Under certain circumstances, the competitive binding between the test compound and the competitor replaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both are added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity (typically between 4 ° C. and 40 ° C.). Incubation times are typically optimized, for example, to facilitate rapid high-throughput screening; typically between 0 and 1 hour is sufficient. Excess reagent is generally removed or washed away. A second component is then added and the presence or absence of the labeled component is followed to indicate binding.

  In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound binds to the oncoprotein, and thus can bind to the oncoprotein and potentially modulate the activity of the oncoprotein. In this embodiment, any component can be labeled. Thus, for example, if the competitor is labeled, the presence of the label in the post-test compound wash solution indicates replacement by the test compound. Alternatively, when the test compound is labeled, the presence of the label on the support is an indication of displacement.

  In an alternative embodiment, the test compound is added first, incubated and washed, and then the competitor is added. The absence of binding by the competitor indicates that the test compound binds to the oncoprotein with a higher affinity than the competitor. Thus, when the test compound is labeled, the presence of the label on the support (combined with the lack of binding of the competitor) causes the test compound to bind to the cancer protein of the invention and thus potentially bind the protein of the invention. It is an index of adjusting to.

  Thus, competitive binding methods include differential screening to identify agents that can modulate the activity of the oncoproteins of the present invention. In this embodiment, the method includes mixing the oncoprotein and the competitor in the first sample. The second sample contains the test compound, oncoprotein, and competitor. The binding of the competitor is determined for both samples, and the change or difference in binding between these two samples is indicative of the presence of an agent that can bind to the cancer protein and potentially modulate its activity. Show. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent can bind to the oncoprotein.

  Alternatively, differential screening is used to identify drug candidates that bind to the native oncoprotein but cannot bind to the modified oncoprotein. For example, the structure of oncoproteins can be modeled and used in rational drug design to synthesize factors that interact with the site, i.e., factors that do not generally bind to site-modified proteins. . In addition, drug candidates that affect the activity of native oncoproteins are also identified by screening drugs for the ability to either enhance or decrease the activity of such proteins.

  Positive and negative controls can be used in this assay. Preferably, control and test samples are performed at least in triplicate to obtain statistically significant results. Incubation of all samples is performed for a time sufficient to allow binding of this factor to the protein. Following incubation, the sample is washed free of non-specific binding material and the amount of genetically labeled factor bound is determined. For example, if a radiolabel is used, these samples can be counted using a scintillation counter to determine the amount of compound bound.

  A variety of other reagents may be involved in this screening assay. These reagents include salts, neutral protein (eg, albumin) surfactants, etc. that are used to facilitate optimal protein-protein binding and / or reduce non-specific or background interactions. And the like. Reagents that improve the efficiency of the assay in other ways (eg, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.) can also be used. The mixture of ingredients is added in an order that provides the requisite binding.

(Use of polynucleotides to down-regulate or inhibit proteins of the invention)
Polynucleotide modulators of cancer can be introduced into cells containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to: cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugate of the ligand binding molecule does not interfere sufficiently with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or a sense or antisense oligonucleotide or conjugate thereof to the cell. Do not block the entry of versions sufficiently. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence by formation of a polynucleotide-lipid complex, eg, as described in WO 90/10448. It will be appreciated that the use of antisense molecules or knockout models and knock-in models can also be used in screening assays as discussed above, in addition to processing methods.

(Inhibition and antisense nucleotides)
In certain embodiments, the activity of a cancer-associated protein is associated with an antisense polynucleotide or inhibitory micronuclear RNA (snRNA) (ie, a coding mRNA nucleic acid sequence (eg, a cancer protein, mRNA of the invention, or a subsequence thereof). Complementary and preferably down-regulated or completely inhibited by the use of nucleic acids) that are preferably capable of hybridizing to them. Binding of the antisense polynucleotide to the mRNA reduces the translation and / or stability of the mRNA.

  In the context of the present invention, antisense polynucleotides can include naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or related homologues thereof. Antisense polynucleotides can also have altered sugar moieties or linkages between sugars. Phosphorothioates and other sulfur-containing species known to be used in the art are illustratively included among these. Analogs are included in the invention as long as they function effectively so that they hybridize to the nucleotides of the invention. See, for example, Isis Pharmaceuticals, Carlsbad, CA; See, Natick, MA.

  Such antisense polynucleotides can be readily synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is commercially available from a variety of vendors including Applied Biosystems. The preparation of other oligonucleotides (eg, phosphorothioates and alkylated derivatives) is also well known to those skilled in the art.

  As used herein, an antisense molecule includes an antisense oligonucleotide or a sense oligonucleotide. Sense oligonucleotides can be used, for example, to block transcription by binding to the antisense strand. The antisense and sense oligonucleotides comprise a single stranded nucleic acid sequence (either RNA or DNA) that can bind to a target mRNA (sense) sequence or target DNA (antisense) sequence for the cancer molecule. An antisense oligonucleotide or sense oligonucleotide according to the invention generally comprises a fragment of at least about 12 nucleotides, preferably from about 12 nucleotides to about 30 nucleotides. The ability to derive an antisense or sense oligonucleotide based on a cDNA sequence encoding a given protein is described, for example, by Stein & Cohen (Cancer Res. 48: 2659 (1988) and van der Krol et al. (BioTechniques 6: 958 (1988)). )It is described in.

(Ribozyme)
In addition to antisense polynucleotides, ribozymes can be used to target or inhibit transcription of cancer-associated nucleotide sequences. Ribozymes are RNA molecules that catalytically cleave other RNA molecules. Various types of ribozymes have been described. These ribozymes include group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, eg, Castanotto et al., Adv. In Pharmacology 25 for a general review of the properties of various ribozymes). : 289-317 (1994)).

  The genetic features of hairpin ribozymes are described, for example, in Hampel et al., Nucl. Acids Res. 18: 299-304 (1990); European Patent Application No. 0360257; U.S. Pat. No. 5,254,678. Preparation methods are well known to those skilled in the art (eg, WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90: 6340-6344 (1993); Yamada et al., Human Gene Therapy 1: 39-45). (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92: 699-703 (1995); 1994)).

(Use of regulators in phenotypic screening)
In one embodiment, the test compound is administered to a population of cancer cells having an associated cancer expression profile. As used herein, “administering” or “contacting” refers to this modulation in such a manner that the modulator acts on the cell either by uptake and intracellular action or by action on the cell surface. It means adding a factor to the cell. In some embodiments, a nucleic acid encoding a proteinaceous factor (ie, a peptide) is added to a viral construct (eg, an adenoviral construct or a retroviral construct) and added to a cell to express the peptide factor. Is achieved (eg PCT US97 / 01019). Regulatable gene therapy systems can also be used. Once the modulator is administered to the cells, the cells are washed if desired and incubated for some period of time under favorable physiological conditions. These cells are then collected and a new gene expression profile is created. Thus, for example, cancerous tissue is screened for factors that modulate (eg, induce or suppress) the cancer phenotype. A change in at least one gene, preferably many genes, in the expression profile indicates that this factor has an effect on the activity of the cancer. Similarly, altering a biological function or signal transduction pathway indicates the activity of a modulator. By defining such a signature for the cancer phenotype, screening for new drugs that alter the phenotype is devised. In this approach, the drug target need not be known, need not be represented in the original gene / protein expression screening platform, and the level of transcript for the target protein need not change. Regulators that inhibit function serve as surrogate markers.

  Screening is performed to evaluate the gene or gene product as outlined above. That is, a step of identifying a specific differentially expressed gene as important in a specific state and a step of screening for any regulator of the expression of the gene or gene product itself are performed.

(Use of modulators affecting the peptides of the invention)
Measurement of cancer polypeptide activity, or cancer phenotype, is performed using various assays. For example, the effect of a modulator on the function of a cancer polypeptide is measured by testing the above parameters. Physiological changes that affect activity are used to assess the effect of a test compound on the polypeptides of the invention. When functional results are determined using intact cells or animals, various effects are known (eg, by solid tumor, tumor growth, tumor metastasis, neovascularization, hormone release, eg, by Northern plot) In the case of cancers associated with transcriptional changes to both genetic and uncharacterized genetic markers, changes in cellular metabolism (eg, cell growth or pH changes) and changes in intracellular secondary messengers (eg, cGNIP) Can be evaluated).

(Method for identifying and characterizing cancer-related sequences)
The expression of various gene sequences is associated with cancer. Accordingly, a disorder based on a mutant oncogene or a modified oncogene is determined. In one embodiment, the present invention provides a method for identifying a cell comprising a variant oncogene (eg, determining the presence of all or part of a sequence of at least one endogenous oncogene in the cell). provide. This is accomplished using any sequencing technique. The invention encompasses a method of identifying an individual's cancer genotype (eg, determining all or part of the sequence of at least one gene of the invention in an individual). This is generally done in at least one tissue of the individual (eg, the tissue shown in Table I) and may include evaluation of multiple tissues or different samples of the same tissue. The method includes the step of comparing the sequence of the sequenced gene with the sequence of a known oncogene (ie, wild type gene) to determine the presence of a family member, homolog, variant, or variant. obtain. The sequence of all or part of this gene can then be compared to the sequence of known oncogenes to determine if any differences exist. This is done using any known homology program (eg BLAST, Bestfit, etc.). The presence of sequence differences between a patient oncogene and a known oncogene is associated with a disease state or trend for a disease state, as outlined herein.

  In a preferred embodiment, these oncogenes are used as probes to determine the copy number of oncogenes in the genome. These oncogenes are used as probes for determining the chromosomal location of the oncogene. Information such as chromosomal location finds use in providing diagnosis or prognosis, particularly when chromosomal abnormalities (eg, translocations, etc.) are identified at the oncogene locus.

(XIV.) Kit / article of manufacture)
Kits are also within the scope of the present invention for use in the laboratory, prognostic, preventive, diagnostic and therapeutic applications described herein. Such a kit is for use with a carrier, package or a compartmented container to receive one or more containers (eg, vials, tubes, etc.) (eg, the uses described herein). For example, the container may contain a probe that is detectably labeled or can be labeled, each such probe being a gene of the present invention. Or a message-specific antibody or polynucleotide If the method uses nucleic acid hybridization to detect the target nucleic acid, the kit also contains a nucleotide for amplifying the target nucleic acid sequence. The kit may comprise a reporter (eg, a biotin binding protein (eg, an enzyme label, a fluorescent label or Avidin or streptavidin coupled to a reporter molecule such as a radioactive label)) can be provided, and such a reporter can be used with, for example, a nucleic acid or an antibody. All or part of the amino acid sequence or analogs thereof, or a nucleic acid molecule encoding such an amino acid sequence.

  The kits of the present invention typically contain the desired materials (buffers, diluents, filter papers, needles, syringes; carriers, packages, containers, vials, and / or the above containers and commercial and user aspects). Or one or more other containers in connection with the present invention, including tubes, labels listing the contents and / or instructions for use, and package inserts with instructions for use) including.

  The label may be present on or with the container to indicate that the composition is to be used for a particular therapeutic or non-therapeutic application (eg prognostic application, prophylactic application, diagnostic application or research application) and in vivo or in vitro Instructions for use in (eg, use described herein) may be given. Instructions and / or other information may also be included with the kit or on an insert or label provided on the kit.

  The label can be on the container or attached to the container. If letters, numbers or other features forming this label are formed on or etched into the container itself, the label can be on the container; it is also a receptacle or carrier that holds this container If present (eg, package insert), a label can be attached to the container. The label may indicate that the composition is used for diagnosing, treating, preventing or prognosing certain conditions (eg, tissue neoplasia as shown in Table I).

  The terms “kit” and “article of manufacture” may be used as synonyms.

  In another embodiment of the invention, the composition (eg, amino acid sequence, small molecule, nucleic acid sequence, and / or antibody (eg, diagnosis of neoplasia of tissue, prognosis, prevention and / or as shown in Table I) Or a substance useful for treatment) is provided, which typically comprises at least one container and at least one label, suitable containers include, for example, bottles, These include vials, syringes, and test tubes, which can be formed from a variety of materials, such as glass, metal, or plastic, which contain amino acid sequences, small molecules, nucleic acid sequences, cell populations, and / or In one embodiment, the container is a polynucleotide for use in testing cellular mRNA expression profiles. In another embodiment, the container may be used to assess protein expression of P1091D4 in cells, tissues, or related laboratory purposes, prognosis. Comprising antibodies, binding fragments of antibodies, or specific binding proteins for purposes, diagnostic purposes, prophylactic purposes and therapeutic purposes; indications and / or instructions for such use on such containers, Or may be provided with a container and provided with reagents and other compositions or tools for use for these purposes, hi another embodiment, the container is a substance for eliciting a cellular or humoral immune response In another embodiment, the container is a substance for adoptive immunotherapy (e.g., an example). For example, cytotoxic T cells (CTL) or helper T cells (HTL)) with associated indications and / or instructions; reagents and other compositions or tools used for such purposes are also included. May be included.

  The container may alternatively comprise a composition effective for treating, diagnosing, prognosing or preventing the condition and may comprise a sterile access port (eg, the container may be an intravenous solution bag or subcutaneous injection) It can be a vial with a stopper that can be penetrated by a needle). The active agent in the composition can be an antibody that can specifically bind 109P1D4 and modulate the function of 109P1D4.

  The article of manufacture may further comprise a second container, the second container being a pharmaceutically acceptable buffer (eg, phosphate buffered saline, Ringer's solution, and / or dextrose solution). including. The container may contain other materials desired from a commercial and user standpoint (other buffers, diluents, filter paper, stir bars, needles, syringes, and / or package inserts and / or uses with instructions. For example).

  Various aspects of the invention are further described and illustrated by the following examples. None of these examples are intended to limit the scope of the invention.

(Example 1: Isolation obtained by SSH of 109P1D4 gene cDNA fragment)
To isolate genes overexpressed in prostate cancer, suppressive subtractive hybridization (SSH) means using cDNA from prostate cancer tissue was used. This 109P1D4 SSH cDNA sequence is derived from an experiment in which androgen-inducible LNCaP cell-derived cDNA was subtracted from LNCaP cell-derived cDNA stimulated with mibolerone for 9 hours.

(Materials and methods)
(Human tissue)
Patient cancer and normal tissues were purchased from different sources (eg, NDRI (Philadelphia, PA)). MRNA for some normal tissues was purchased from different companies (eg, Clontech, Palo Alto, CA).

(Isolation of RNA)
Tissue was homogenized in Trizol reagent (Life Technologies, Gibco BRL) with 10 ml / g of tissue to isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total RNA and mRNA were quantified by spectrophotometric analysis (OD 260/280 nm) and analyzed by gel electrophoresis.

(Oligonucleotide)
The following HPLC purified oligonucleotides were used.

(DPNCDN (cDNA synthesis primer))
5 ′ TTTTGATCAAGCTT ₃₀ 3 ′ (SEQ ID NO: 44)
(Adapter 1)
5 ′ CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGGAG 3 ′ (SEQ ID NO: 45)
3 ′ GGCCCGTCCTAG5 ′ (SEQ ID NO: 46)
(Adapter 2)
5 ′ GTAATACGACTCACTATAGGGCAGCGGTGGTCCGCGCCGAG3 ′ (SEQ ID NO: 47)
3′CGGCTCCTAG5 ′ (SEQ ID NO: 48)
(PCR primer 1)
5 ′ CTAATACGACTCACTATAGGC3 ′ (SEQ ID NO: 49)
(Nested primer (NP) 1)
5′TCGAGCGGCCCGCCCGGGCAGGA3 ′ (SEQ ID NO: 50)
(Nested primer (NP) 2)
5′AGCGTGGTCGCGCGCGAGGA3 ′ (SEQ ID NO: 51)
(Suppression subtractive hybridization)
Suppressive subtractive hybridization (SSH) was used to identify cDNAs corresponding to genes that could be differentially expressed in prostate cancer. This SSH reaction utilized cDNA derived from LNCaP prostate cancer cells.

  The 109P1D4 sequence was derived from the cDNA subtraction of LNCaP in the absence of LNCaP-androgen stimulated with mibololeone. The SSH DNA sequence (FIG. 1) was identified.

CDNA derived from androgen-deficient LNCaP cells was used as the source of “driver” cDNA, while cDNA derived from androgen-stimulated LNCaP cells was used as the source of “tester” cDNA. Double stranded cDNA corresponding to the tester cDNA and double stranded cDNA corresponding to the driver cDNA were isolated from the relevant xenograft tissue as described above using the CLONTECH PCR-Select cDNA Subtraction Kit. Synthesized from 2 μg poly (A) ⁺ RNA and 1 μg oligonucleotide DPNCD as primer. First strand synthesis and second strand synthesis were performed as described in the kit user manual protocol (CLONTECH protocol number PT1117-1, catalog number K1804-1). The resulting cDNA was digested with DpnII for 3 hours at 37 ° C. Digested cDNA was extracted with phenol / chloroform (1: 1) and precipitated with ethanol.

  Tester cDNA was made by diluting DpnII digested cDNA (1 μl) from the relevant tissue source (see above) (400 ng) in 5 μl of water. This diluted cDNA (2 μl, 160 ng) was then used in a separate ligation reaction using 400 u of T4 DNA ligase (CLONTECH) in a total volume of 10 μl, overnight at 16 ° C., 2 μl of Adapter 1 and Adapter 1 overnight. 2 (10 μM). Ligation was terminated using 1 μl of 0.2 M EDTA heated at 72 ° C. for 5 minutes.

  The first hybridization was performed by adding driver cDNA (1.5 μl, 600 ng) to each of the two tubes containing 1.5 μl (20 ng) of Adapter 1 ligated tester cDNA and Adapter 2 ligated tester cDNA. . In a final volume of 4 μl, these samples were covered with mineral oil, denatured with an MJ Research thermal cycler for 1.5 minutes at 98 ° C., and then hybridized at 68 ° C. for 8 hours. The two hybridization products were then mixed with additional fresh denatured driver cDNA (1 μl) and hybridized at 68 ° C. overnight. This second hybridization product was then diluted in 20 mM Hepes (pH 8.3), 50 mM NaCl, 0.2 mM EDTA (200 μl), heated at 70 ° C. for 7 minutes, and stored at −20 ° C.

(PCR amplification, cloning and sequencing of gene fragments generated from SSH)
Two PCR amplifications were performed to amplify the gene fragment obtained from the SSH reaction. In the first PCR reaction, the diluted final hybridization mixture (1 μl) is mixed with 1 μl PCR primer 1 (10 μM), 0.5 μl dNTP mixture (10 μM), 2.5 μl 10 × reaction buffer (CLONTECH) and To 0.5 μl of 50 × Advantage cDNA polymerase mixture (CLONTECH) was added in a final volume of 25 μl. PCR1 was performed using the following conditions: 75 ° C for 5 minutes, 94 ° C for 25 seconds, then 27 cycles of 94 ° C for 10 seconds, 66 ° C for 30 seconds, 72 ° C for 1.5 minutes. Five separate first PCR reactions were performed for each experiment. The product was pooled and diluted 1:10 with water. For the second PCR reaction, 1 μl from the pooled and diluted first PCR reaction was used for PCR1, except that primers NP1 and NP2 (10 μM) were used instead of PCR1 primer. Added to the same reaction mixture. PCR2 was performed using 10-12 cycles of 94 ° C. for 10 seconds, 68 ° C. for 30 seconds and 72 ° C. for 1.5 minutes. The PCR product was analyzed using 2% agarose gel electrophoresis.

  This PCR product was inserted into pCR2.1 using the T / A vector cloning kit (Invitrogen). Transformed E. coli The E. coli was subjected to blue / white sorting and ampicillin sorting. White colonies were selected and placed in 96-well plates and grown overnight in liquid culture. To identify the insert, PCR amplification was performed on 1 μl of bacterial culture using PCR1 conditions and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

  Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced and subjected to nucleic acid homology searches of the GenBank database, the dbest database, and the NCI-CGAP database.

(RT-PCR expression analysis)
First strand cDNA can be generated from 1 μg of mRNA using oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used, which included incubation with reverse transcriptase at 42 ° C for 50 minutes, followed by RNAse H treatment at 37 ° C for 20 minutes. After the reaction was complete, the volume could be increased to 200 μl with water prior to normalization. First strand cDNA from 16 different normal human tissues can be obtained from Clontech.

Normalization of first strand cDNA from multiple tissues was performed by amplifying β-actin using primers 5′ATATCCGCCGCCGCTCGTCCGTCGACAA3 ′ (SEQ ID NO: 52) and 5′AGCCACACGCAGCTCATTGTAGAAGG3 ′ (SEQ ID NO: 53) did. First strand cDNA (5 μl) was added in a total volume of 50 μl (0.4 μM primer, 0.2 μM dNTP, 1 × PCR buffer (Clontech, 10 mM Tris-HCl, 1.5 mM MgCl ₂ , 50 mM KCl, Amplified with pH 8.3) and 1 × Klentaq DNA polymerase (Clontech). 5 μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using the MJ Research thermal cycler under the following conditions: The first denaturation step was 94 ° C for 15 seconds, followed by 18 cycles, 20 cycles and 22 cycles of 94 ° C for 15 minutes. It may be 2 minutes at 65 ° C. and 5 seconds at 72 ° C. The final extension step at 72 ° C. was performed for 2 minutes. After agarose gel electrophoresis, the band intensities of 283 base pair β-actin bands from multiple tissues were compared by visual inspection. The dilution factor of the first strand cDNA was calculated to obtain equal β-actin band intensity in all tissues after 22 cycles of PCR. After 22 cycles of PCR, 3 normalizations may be required to achieve equal band intensity in all tissues.

To determine the expression level of the 109P1D4 gene, 5 μl of normalized first strand cDNA was analyzed by PCR using 26 and 30 cycles of amplification. Semi-quantitative expression analysis can be accomplished by comparing PCR products at a cycle number that gives light band intensity. Primers used for RT-PCR were designed using the 109P1D4 SSH sequence. These are listed below:
(109P1D4.1)
5′-TGGTCTTTCAGGTAATTGCTGTTG-3 ′ (SEQ ID NO: 54)
(109P1D4.2)
5′-CTCCATCAATGTTATGTTGCCTGT-3 ′ (SEQ ID NO: 55)
A representative RT-PCR expression analysis is shown in FIG.

(Example 2: Isolation of cDNA encoding full-length 109P1D4)
The 192 bp 109P1D4SSH sequence (FIG. 1) showed homology to protocadherin 11 (PCDH11), a cell adhesion molecule associated with calcium-dependent cadherin. The human cDNA sequence encodes a 1021 amino acid protein with an N-terminal leader sequence and a transmembrane domain. 4603 bp of 109P1D4v. 1 was cloned from a human prostate cancer xenograft LAPC-9AD cDNA library, thereby revealing a 1021 amino acid ORF (FIGS. 2 and 3). Other variants of 109P1D4 (transcripts and SNPs) were also identified and are listed sequentially in FIG. 2 and FIG.

(Example 3: Chromosome mapping of 109P1D4)
Chromosomal localization can relate genes to disease pathogenesis. Several chromosome mapping approaches are available, including fluorescence in situ hybridization (FISH), human / hamster radiation hybrid (RH) panel (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville AI ), Human-rodent somatic cell hybrid panels (e.g., as available from Coriell Institute (Camden, New Jersey)), and genomic viewers using BLAST homology to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).

  109P1D4 uses the 109P1D4 sequence and the NCBI BLAST tool (on the World Wide Web (.ncbi.nlm.nih.gov / genome / seq / page.cgi? F = HsBlast.html && ORG = Hs)). 3 is mapped. 109P1D4 was also identified in a region that is 99% identical to Xq21 on chromosome Yp11.2.

Example 4: Expression analysis of 109P1D4 in normal tissues and patient specimens
Expression analysis by RT-PCR and Northern analysis showed that normal tissue expression of the gene of FIG. 2 was predominantly restricted to the tissues shown in Table 1.

  The therapeutic application of the gene of FIG. 2 includes use as a small molecule therapy and / or vaccine (T cell or antibody) target. The diagnostic application of the gene of FIG. 2 includes its use as a diagnostic marker for local disease and / or metastatic relaxation. Limited expression of the gene of FIG. 2 in normal tissue makes it useful as a tumor target for diagnosis and therapy. The gene expression analysis of FIG. 2 provides useful information to predict disease in advanced state, rate of progression and / or susceptibility to tumor pathogenicity. The expression status of the gene of FIG. 2 in patient samples, tissue arrays and / or cell lines is as follows: (i) immunohistochemical analysis; (ii) in situ hybridization; (iii) on the microablated sample with laser capture Can be analyzed by RT-PCR analysis; (iv) Western blot analysis; and (v) Northern analysis.

  RT-PCR analysis and Northern blotting were used to assess gene expression in the selection of normal and cancerous urinary tissues. The results are summarized in FIGS.

  FIG. 14 shows the expression of 109P1D4 in a sample of a lymphoma cancer patient. RNA was extracted from peripheral blood lymphocytes, umbilical cord blood isolated from normal individuals, and lymphoma patient cancer samples. Northern blots with 10 μg total RNA were probed with the 109P1D4 sequence. Place a size standard in kilobase units next to it. The results showed 109P1D4 expression in lymphoma patient samples but not in normal blood cells tested.

  FIG. 15 shows the expression of 109P1D4 by RT-PCR. The first strand cDNA is derived from biological pool 1 (liver, lung and kidney), biological pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer. Ovarian cancer pool, breast cancer pool, cancer metastasis pool, and pancreatic cancer pool. Normalization was performed by PCR using primers for actin and GAPDH. Semi-quantitative PCR using primers for 109P1D4 was performed with 30 cycles of amplification. The results show strong expression of 109P1D4 in all cancer pools tested. Very low expression was detected in the biological pool.

  FIG. 16 shows 109P1D4 expression in normal tissues. Two multiple tissue Northern blots (Clontech), both using 2 μg of mRNA per lane, were probed with 109P1D4 SSH fragment. A kilobase (kb) size standard is shown next to it. The results show the expression of about 10 kb 109P1D4 transcript in the ovary. Weak expression was also detected in the placenta and brain, but not in other normal tissues tested.

  FIG. 17 shows the expression of 109P1D4 in human cancer cell lines. RNA was extracted from a number of human prostate and bone cancer cell lines. Northern blots with 10 μg total RNA per lane were probed with 109P1D4 SSh fragment. A size standard in kilobases (kb) is shown next to it. The results show the expression of 109P1D4 in the LAPC-9AD prostate cell line, LAPC-9AI prostate cell line, LNCaP prostate cell line, and bone cancer cell lines SK-ES-1 and RD-ES.

  Extensive expression of 109P1D4 in normal tissues is shown in FIG. 18A. A cDNA dot blot containing 76 different samples from human tissue was analyzed using a 109P1D4 SSH probe. Expression was detected only in multiple regions of the brain, placenta, ovary and fetal brain among all tissues examined.

  FIG. 18B shows 109P1D4 expression in patient cancer samples. The expression of 109P1D4 was assayed on a RNA dot blot in a panel of human cancers (T) and a panel of normal tissues that matched them (N). The expression of 109P1D4 in tumors that were up-regulated compared to normal tissue was observed in the uterus, lungs and stomach. Expression that was detected in normal adjacent tissues (isolated from diseased tissue) but not detected in normal tissues (isolated from healthy donors) is sufficient for these tissues to be sufficiently normal. Rather, it may suggest that 109P1D4 can be expressed in early stage tumors.

  FIG. 19 shows 109P1D4 expression in lung cancer patient samples. RNA was extracted from normal lung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES and lung cancer patient tumors. Northern blots with 10 μg total RNA were probed with 109P1D4. A size standard in kilobases (kb) is shown next to it. The results showed strong expression of 109P1D4 in lung tumor tissue and RD-ES cell line, but no expression in normal lung.

  The restricted expression of 109P1D4 in normal tissues and the expression detected in cancer patient samples suggests that 109P1D4 is a potential therapeutic target and a diagnostic marker for human cancer.

(Example 5: Splice variant of 109P1D4)
A transcriptional variant is a variant of the mature mRNA from the same gene that results from alternative transcription or alternative splicing. Selective transcripts are transcripts from the same gene but initiate transcription at different points. Splicing variants are mRNA variants that are differentially spliced from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the first RNA is spliced to have only the exon and be used for translation into an amino acid sequence Produces functional mRNA. Thus, a given gene can have 0 to many alternative transcripts, and each transcript can have 0 to many splicing variants. Each transcriptional variant has a unique exon makeup and may have different coding and / or non-coding portions (5 ′ or 3 ′ end) from the original transcript. A transcriptional variant may encode a similar or different protein having the same or similar function, may encode a protein having a different function, and may be expressed in the same tissue at the same time or at the same time It may be expressed in different tissues, expressed in the same tissue at different time points, or expressed in different tissues at different time points. Proteins encoded by transcriptional variants can have similar or different intracellular or extracellular localization (eg, secreted versus intracellular).

  Transcriptional variants are identified by various methods accepted in the art. For example, alternative transcripts and splicing variants are identified by full length cloning experiments or by use of full length transcripts and EST sequences. First, all human ESTs were grouped into clusters that showed direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to a consensus sequence or other full-length sequence. Each consensus sequence is a potential splicing variant for that gene. Even if a variant that is not a full-length clone is identified, that portion of the variant is very useful for antigen generation using techniques known in the art and for further cloning of full-length splicing variants.

  In addition, computer programs for identifying transcriptional variants based on genomic sequences are available in the art. As a genome-based transcriptional variant identification program, FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 22 (4): 51 (4): 51; URL compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of splicing variant identification protocols see, for example, Southan, C .; , A genomic perspective on human protocols, FEBS Lett. 2001 Jun 8; 498 (2-3): 214-8; de Souza, S .; J. et al. Identification of human chromosome 22 translated sequences with ORF expressed sequence tags, Proc. Natl Acad Sci USA. 2000 Nov 7; 97 (23): 12690-3.

  Various techniques are available in the art (eg, full length cloning, proteomic validation, PCR-based validation, and 5′RACE confirmation, etc.) to further confirm the parameters of the transcript variant (eg, Proteome confirmation: Brennan, S.O. et al., Albuminbanks peninsula: a new termination variant characterized by electrospray mass spectrometry 1-D2; splicing of pre-messenger RNA products multiple forms of maturity capsule alpha (s1) -casein, Eur J Biochem. 1997 Oct 1; 249 (1): 1-7, for PCR-based confirmation: Wellmann S etr. Vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 Apr .; 47 (4): 654, et al. Sing a genomics-based approach, Gene.2001 Jan 24; 263 (1-2): 211-8, for PCR-based confirmation and 5'RACE confirmation: Brigle, KE et al., Organization of the reduced for example, carrier carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353 (2): 191-8).

  It is known in the art that genomic regions are regulated in cancer. When the genomic region to which a gene is mapped is regulated in a particular cancer, alternative transcripts or splicing variants of that gene are also regulated. It is disclosed herein that 109P1D4 has a specific expression profile associated with cancer. Alternative transcripts and splicing variants of 109P1D4 may also be involved in cancer in the same or different tissues and thus serve as tumor associated markers / antigens.

  Using the full-length gene and EST sequence, eight transcriptional variants were identified and 109P14D v. 2, v. 3, v. 4, v. 5, v. 6, v. 7, v. 8 and v. It was expressed as 9. 109P1D4 which is the boundary of exons in the original transcript v. 1 is shown in Table LI. 1091D4 v. Compared to 1, transcription variant 109P1D4 v. 3 was modified 109P1D4 v.3 as shown in FIG. 1 to 2069-2395 are spliced. 109P1D4 v. 5, one exon is a variant 109P1D4 v. 1 to 5 'and 109P1D4 v. 2 bp extension to the 5 'end of 1 and 109P1D4 v. 288 bp extended to the 3 'end of 1. In theory, each different combination of exons in spatial rules (eg, exon 1 of v.5 and exon 1 and exon 2 of v.3 or v.4) is a potential splice variant.

  Tables LII to LV are based on individual variants. Tables LII (a)-(h) show the nucleotide sequences of the above transcriptional variants. Tables LIII (a)-(h) show the above transcript variants and 109P1D4 v. Alignment with one nucleic acid sequence is shown. Tables LIV (a)-(h) show amino acid translations for the identified reading frame directions of the transcriptional variants. Tables LV (a)-(h) show the amino acid sequence encoded by the splice variant and 109P1D4 v. The alignment with the amino acid sequence of 1 is shown.

Example 6: Single nucleotide polymorphism of 109P1D4
A single nucleotide polymorphism (SNP) is a single base pair modification at any given position in a nucleotide sequence. There are four possible nucleotide base pairs (A / T, C / G, G / C and T / A) at a particular point in the genome. A genotype refers to a specific base pair sequence at one or more positions in an individual's genome. A haplotype often refers to a base pair sequence of more than one position on the same DNA molecule (or the same chromosome in a higher organism) in the context of one gene or in the context of several closely linked genes. . SNPs that occur on cDNA are called cSNPs. These cSNPs can change the amino acids of the protein encoded by the gene and thus can change the function of the protein. Some SNPs cause genetic disorders, others contribute to quantitative variation in phenotype and response to environmental factors (including diet and drugs) between individuals. Thus, combinations of SNPs and / or alleles (called haplotypes) have many applications (diagnosis of genetic diseases, determination of drug response and dosage, identification of genes responsible for the disease, and between individuals (Including analysis of genetic relationships) (P. Nowotny, JM Kwon and AM Goate, “SNP analysis to detect human traits” Curr. Opin. Neurobiol. 2001 Oct; 11 (5): 637 M. Pirmohamed and B. K. Park, “Genetic Susceptibility to Adverse Drug Reactions”, Trends Pharmacol. Sci. 2001 Jun; 22 (6): 298-305; Riley, C. J. Allan, E. Lai, and A. Roses, “The use of single nucleotides in the isolation of common diseases genes,” J. 2000; 1. Pharmacogenics.1; C. Stephens and A. Windemuth, “The preferential power of haplotypes in clinical response” Pharmacogenomics. 2000 feb; 1 (1): 15-26).

  SNPs are identified by a variety of methods available in the art (P. Bean, “The proposing voice of SNP target discovery”, Am. Clin. Lab. 2001-10-11; 20 (9): 18. -20; KM Weiss, “In search of human variation”, Genome Res., July 1998; 8 (7): 691-697; MM She, “Enabling large-scale pharmacogenetics— throughputation detection and genotyping technologies ", Clin. Chem. February 2001; 47 (2): 16. -172). For example, SNPs can be identified by sequencing polymorphic DNA fragments (eg, restriction enzyme fragment length polymorphisms) by gel-based methods and denaturing gradient gel electrophoresis (DGGE). SNPs can also be discovered by direct sequencing of pooled DNA samples from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, SNPs can be discovered by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace”, Hum. Mutat. 1998; 12 (4): 221-225). By various methods, including direct sequencing and high-throughput microarrays, SNPs can be confirmed and the genotype and haplotype of an individual can be determined (PY Kwok, “Methods for genotyping single nucleotide polymorphisms”, Annu. Rev. Genomics Hum. Genet. 2001; 2: 235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Dusterhoeft, "High. -Throughput SNP genetyping with the Masscode system ", Mol. 5 (4): 329-340).

  Using the method described above, SNP is the original transcript 109P1D4 v. 1 and its variants (see FIGS. 2J and 2K). The SNP alleles are shown separately here, but can occur in any one of various combinations (haplotypes) and transcriptional variants containing SNP sites (eg, 109P1D4 v.4 or v.5). Transcriptional variant v. 4 and v. 5 modified these SNPs v. 3 in an exon shared with 3 and a transcriptional variant v. 9 is a variant v. All SNPs that occurred in 6 were included (see FIG. 10).

(Example 7: Production of recombinant 109P1D4 in prokaryotic systems)
To express recombinant 109P1D4 and 109P1D4 variants in prokaryotic cells, the full-length or partial-length 109P1D4 cDNA sequence and 109P1D4 variant cDNA sequence are cloned into any one of various expression vectors known in the art. . One or more of the following regions for 109P1D4 variants are expressed: the full-length sequence shown in FIGS. 2 and 3, or any 8, 9, 10, 11 from 109P1D4, variants or analogs thereof, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30 or more consecutive amino acids.

(A. In vitro transcription constructs and in vitro translation constructs)
pCRII: To create a 109P1D4 sense RNA probe and a 109P1D4 antisense RNA probe for RNA in situ studies, a pCRII construct (Invitrogen, Carlsbad CA) is generated, which encodes either the entire 109P1D4 cDNA or a fragment To do). The pCRII vector has an Sp6 promoter and a T7 promoter adjacent to the insert to drive transcription of 109P1D4 RNA for use as a probe in RNA in situ hybridization experiments. These probes are used to analyze 109P1D4 cell and tissue expression at the RNA level. Transcribed 109P1D4 RNA, which represents the cDNA amino acid coding region of the 109P1D4 gene, is used in an in vitro translation system, such as the TnT ^™ Coupled Reticulylate System (Promega, Corp., Madison, WI), to synthesize 109P1D4 protein.

(B. Bacterial construct)
pGEX construct: All or part of the 109P1D4 cDNA or variant is transformed into a GST-fusion of the pGEX family to produce a recombinant 109P1D4 protein (which is fused to a glutathione S-transferase (GST) protein) in bacteria. Cloning into a vector (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow for controlled expression of recombinant 109P1D4 protein sequences with GST fused at the amino terminus and six histidine epitopes at the carboxyl terminus (6 × His). This GST and 6 × His tag allow purification of the recombinant fusion protein from induced bacteria using an appropriate affinity matrix and recognition of the fusion protein using anti-GST and anti-His antibodies To. The 6 × His tag is created, for example, by adding six histidine codons to the cloning primer at the 3 ′ end of the open reading frame (ORF). A protein cleavage site (eg, the PreScience ^™ recognition site in pGEX-6P-1) can be used to allow cleavage of the GST tag from 109P1D4-related proteins. The ampicillin resistance gene and the pBR322 origin are E. coli. Allows selection and maintenance of the pGEX plasmid in E. coli.

  pMAL construct: In order to produce a recombinant 109P1D4 protein fused to maltose binding protein (MBP) in bacteria, all or part of the 109P1D4 cDNA protein coding sequence is expressed in pMAL-c2X and pMAL-p2X vectors (New England). Fusion to the MBP gene by cloning into Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant 109P1D4 protein sequences with MBP fused at the amino terminus and a 6 × His epitope tag at the carboxyl terminus. This MBP and 6 × His tag allow purification of the recombinant fusion protein from induced bacteria using an appropriate affinity matrix and recognition of the fusion protein using anti-MBP and anti-His antibodies To. The 6 × His epitope tag is created by the addition of 6 histidine codons to the 3 ′ cloning primer. The factor Xa recognition site allows cleavage of the pMAL tag from 109P1D4. The pMAL-c2X and pMAL-p2X vectors are each optimized to express the recombinant protein in the cytoplasm or periplasm. Periplasmic expression increases the folding of proteins containing disulfide bonds. In one embodiment, amino acids 24-419 of 109P1D4 variant 1 were cloned into the pMAL-c2X vector and used to express the fusion protein.

pET construct: In order to express 109P1D4 in bacterial cells, all or part of the 109P1D4 cDNA protein coding sequence is cloned into the pET family of vectors (Novagen, Madison, WI). These vectors provide fusion to proteins that increase solubility (eg, NusA and thioredoxin (Trx)), and epitope tags (eg, 6 × His and S-Tag ^™ ) to aid in purification and detection of recombinant proteins. Allows tightly controlled expression of recombinant 109P1D4 protein in bacteria with and without. For example, constructs are made utilizing the pET NusA fusion system 43.1 so that the region of 109P1D4 protein is expressed as an amino-terminal fusion to NusA. In two embodiments, amino acids 24-419 and 24-815 were cloned into the pET43.1 vector and used to express the fusion protein.

(C. Yeast construct)
pESC construct: In order to express 109P1D4 in the yeast species Saccharomyces cerevisiae, for the study of the production and function of recombinant proteins, all or part of the 109P1D4 cDNA protein coding sequence was transformed into a vector of the pESC family (each of which (Including one of four selectable markers, HIS3, TRP1, LEU2, and URA3) (Stratagene, La Jolla, Calif.). These vectors allow for controlled expression of up to two different genes or cloned sequences containing either Flag ^™ or Myc epitope tags in the same yeast cell from the same plasmid. This system is useful for confirming the protein-protein interaction of 109P1D4. Furthermore, expression in yeast results in similar post-translational modifications (eg, glycosylation and phosphorylation) that are found when expressed in eukaryotic cells.

pESP construct: In order to express 109P1D4 in the yeast species Saccharomyces pombe, all or part of the 109P1D4 cDNA protein coding sequence is cloned into a vector of the pESP family. These vectors allow for controlled high level expression of 109P1D4 protein sequences fused to GST, which aids the purification of the recombinant protein, either at the amino terminus or the carboxyl terminus. The Flag ^™ epitope tag allows the detection of recombinant proteins using anti-Flag ^™ antibodies.

(Example 8: Production of recombinant 109P1D4 in higher eukaryotic systems)
(A. Mammalian construct)
To express recombinant 109P1D4 in eukaryotic cells, the full length or partial length of the 109P1D4 cDNA sequence can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 109P1D4 are expressed in these constructs: 109P1D4 v. Amino acids 1 to 1021 from 1; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids; 109P1D4 v. Amino acids 1-1054 from 109, 109P1D4 v. Amino acids 1 to 1347 from 109, 109P1D4 v. Amino acids 1-1337 from 109, 109P1D4 v. Amino acids 1-1310 derived from 5, 109P1D4 v. Amino acids 1-1037 from 109, 109P1D4 v. Amino acids 1-1048 derived from 7 and 109P1D4 v. Amino acids 1-1340 from 8; or any 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 derived from 109P1D4 variants or analogs thereof 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids.

  These constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-109P1D4 polyclonal sera described herein.

pcDNA4 / HisMax construct: To express 109P1D4 in mammalian cells, 109P1D4, 109P1D4 ORF, or a portion thereof, is cloned into pcDNA4 / HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and SP16 translational enhancer. This recombinant protein has Xpress ^™ and 6 × His epitope fused to its amino terminus. The pcDNA4 / HisMax vector is also a bovine growth hormone (BGH) polyadenylation signal and transcription to increase mRNA stability, along with an SV40 origin for episomal replication and simple vector rescue in cell lines expressing large T antigen. Contains termination sequence. The zeocin resistance gene allows selection of mammalian cells that express the protein, and the ampicillin resistance gene and the ColE1 origin are E. coli. Allows selection and maintenance of the plasmid in E. coli.

  pcDNA3.1 / MycHis construct: To express 109P1D4 in mammalian cells, 109P1D4 ORF of 109P1D4 with a consensus Kozak translation initiation site or a part thereof, pcDNA3.1 / MycHis Version A (Invitrogen, Carlsbad, CA) Cloned in. Protein expression is driven from the cytomegalovirus (CMV) promoter. This recombinant protein has a myc epitope and a 6 × His epitope fused to its carboxyl terminus. The pcDNA3.1 / MycHis vector is also a bovine growth hormone (BGH) polyadenylation signal to increase mRNA stability, along with an SV40 origin for episomal replication and simple vector rescue in cell lines expressing large T antigen. And a transcription termination sequence. A neomycin resistance gene may be used. This is because the neomycin resistance gene allows selection of mammalian cells that express the protein, and the ampicillin resistance gene and the ColE1 origin are E. coli. This allows for the selection and maintenance of the plasmid in E. coli.

  109P1D4 v. 1 complete ORF was cloned into pcDNA3.1 / MycHis construct and 109P1D4. pcDNA3.1 / MycHis was generated.

  pcDNA3.1 / CT-GFP-TOPO construct: 109P1D4 ORF with a consensus Kozak translation initiation site to express 109P1D4 in mammalian cells and to allow detection of its recombinant protein using fluorescence, Alternatively, a portion thereof is cloned into pcDNA3.1 / CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant protein has a green fluorescent protein (GFP) fused to the carboxyl terminus, facilitating non-invasive in vivo detection and cell biology studies. The pcDNA3.1 / CT-GFP-TOPO vector is also a bovine growth hormone (BGH) to increase mRNA stability, along with an SV40 origin for episomal replication and simple vector rescue in cell lines expressing large T antigen. ) Contains a polyadenylation signal and a transcription termination sequence. The neomycin resistance gene allows selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin are derived from E. coli. Allows selection and maintenance of the plasmid in E. coli. Additional constructs with amino terminal GFP fusions are made in pcDNA3.1 / NT-GFP-TOPO over the entire length of 109P1D4 protein.

  PAPtag: 109P1D4 ORF or part thereof is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct produces an alkaline phosphatase fusion at the carboxyl terminus of the 109P1D4 protein, which, on the other hand, fuses an IgG kappa signal sequence to the amino terminus. A construct is also made in which alkaline phosphatase with an amino terminal IgG kappa signal sequence is fused to the amino terminus of the 109P1D4 protein. The resulting recombinant 109P1D4 protein can be optimized for secretion into the medium of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 109P1D4 protein . Protein expression is driven from the CMV promoter and the recombinant protein also contains a myc and 6 × His epitope fused at the carboxyl terminus to facilitate detection and purification. The zeocin resistance gene present in the vector allows selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene is E. coli. Allows selection of the plasmid in E. coli.

  pTag5: 109P1D4 ORF or a portion thereof was cloned into pTag-5. This vector is similar to pAPtag but does not contain an alkaline phosphatase fusion. This construct produces a 109P1D4 protein with an amino-terminal IgGκ signal sequence and a carboxyl-terminal myc and 6 × His epitope tag that facilitates detection and affinity purification. The resulting recombinant 109P1D4 protein was optimized for secretion into the medium of transfected mammalian cells. It is then used as an immunogen or ligand to identify proteins such as ligands or receptors that interact with 109P1D4 protein. Protein expression is driven from the CMV promoter. The zeocin resistance gene present in the vector allows selection of mammalian cells that express the protein, and the ampicillin resistance gene is E. coli. Allows selection of the plasmid in E. coli.

  PsecFc: 109P1D4 ORF or part thereof is also cloned into psecFc. This psecFc vector was assembled by cloning human immunoglobulin G1 (IgG) Fc (hinge region, CH2 region, CH3 region) into pSecTag2 (Invitrogen, California). This construct generates an IgGl Fc fusion at the carboxyl terminus of the 109P1D4 protein and at the same time fuses an IgGK signal sequence to the N-terminus. A 109P1D4 fusion utilizing the mouse IgG1 Fc region is also used. To optimize the resulting recombinant 109P1D4 protein for secretion into the medium of transfected mammalian cells and to identify a protein such as a ligand or receptor as an immunogen or that interacts with the 109P1D4 protein Can be used. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows selection of mammalian cells expressing the recombinant protein, and the ampicillin resistance gene is E. coli. Allows selection of plasmids in E. coli.

  pSRα construct: To create a mammalian cell line constitutively expressing 109P1D4, the 109P1D4 ORF of 109P1D4 or a portion thereof was cloned into the pSRα construct. Amphitrophic and ecotrophic retroviruses are transfected into pSRα constructs into 293T-10A1 packaging strain, respectively, or into 293 cells of pSRα and helper plasmids (including missing packaging sequences). This was prepared by co-transfection. This retrovirus is used to infect various mammalian cell lines, resulting in integration of the cloned gene 109P1D4 into the host cell line. Protein expression is driven from a long terminal repeat (LTR). The neomycin resistance gene present in the vector allows selection of mammalian cells expressing the protein, and the ampicillin resistance gene and the ColE1 origin are E. coli. Allows selection and maintenance of the plasmid in E. coli. This retroviral vector was then used for infection and production of various cell lines such as PC3 cells, NIH 3T3 cells, TsuPr1 cells, 293 cells or rat-1 cells.

Additional pSRα constructs were made. These have an epitope tag such as a FLAG ^™ tag fused to the carboxyl terminus of the 109P1D4 sequence, allowing detection using anti-Flag antibodies. For example, the FLAG ^™ sequence 5 ′ ga tac aag gat gac gac gat aag 3 ′ (SEQ ID NO: 45) is added to the cloning primer at the 3 ′ end of the ORF. Additional pSRα constructs are made to produce both the amino terminal GFP fusion protein of full length 109P1D4 and the carboxyl terminal GFP and myc / 6 × His fusion proteins.

  Additional viral vectors: Additional constructs are made for virus-mediated delivery and expression of 109P1D4. High viral titers leading to high level expression of 109P1D4 are achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The 109P1D4 coding sequence or fragment thereof is amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and viral packaging are performed according to the manufacturer's instructions for making adenoviral vectors. Alternatively, the 109P1D4 coding sequence or fragment thereof is cloned into an HSV-I vector (Imgenex) to create a herpesvirus vector. This viral vector is then used for infection of various cell lines such as PC3 cells, NIH 3T3 cells, 293 cells or rat-1 cells.

  Regulated expression system: To control the expression of 109P1D4 in mammalian cells, the 109P1D4 coding sequence, or part thereof, is converted to T-Rex System (Invitrogen), GeneSwitch System (Invitrogen), and tightly-regulated Ecdysystem (Ecdysyston). Cloning into a regulated mammalian expression system such as Stratagene). These systems allow the study of time-dependent and concentration-dependent effects of recombinant 109P1D4. These vectors are then used to control the expression of 109P1D4 in various cell lines such as PC3 cells, NIH 3T3 cells, 293 cells, or rat-1 cells.

(B. Baculovirus expression system)
To produce recombinant 109P1D4 protein in a baculovirus expression system, 109P1D4 ORF, or part thereof, is transferred to a baculovirus transfer vector, pBlueBac 4.5 (Invitrogen), which provides a His tag at the N-terminus Cloning in. Specifically, pBlueBac-109P1D4 is co-transfected into SF9 (Spodoptera frugiperda) insect cells with the helper plasmid pBac-N-Blue (Invitrogen) to produce recombinant baculovirus (for details see Invitrogen See the instruction manual). The baculovirus is then collected from the cell supernatant and purified by plaque assay.

  Recombinant 109P1D4 protein is then generated by infection of High Five insect cells (Invitrogen) with purified baculovirus. Recombinant 109P1D4 protein can be detected using anti-109P1D4 antibody or anti-His tag antibody. The 109P1D4 protein can be purified and used in various cell-based assays or as an immunogen to generate polyclonal and monoclonal antibodies specific for 109P1D4.

Example 9 Antigenic profile and secondary structure
FIGS. 5A-I, 6A-I, 7A-I, 8A-I, and 9A-I show the five amino acid profiles of 109P1D4 variants 1-9 (each evaluation is ProtScale of the ExParsy molecular biology server). Figure 2 illustrates an evaluation available by accessing the website (located on the world wide web at .expasy.ch / cgi-bin / protscale.pl).

  These profiles: FIG. 5, Hydrophilicity, (Hopp TP, Woods KR, 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828); (Kyte J., Doolittle RF, 1982. J. Mol. Biol. 157: 105-132); FIG. 7,% of accessible residues, (Janin J., 1979 Nature 277: 491-492). FIG. 8, average flexibility (Bhaskaran R., and Ponwaswamy P.K. 1988. Int. J. Pept. Protein Res. 32: 242-255); FIG. 9, β-turn (Deleage, G., Roux) B. 1987 Protein Engineering 1: 289-294); Beauty, if necessary, for example, as in ProtScale website, using other profiles available in the art to identify antigenic regions of the 109P1D4 protein. Each of the above amino acid profiles of 109P1D4 were generated for analysis using the following ProtScale parameters: 1) window size 9; 2) 100% weight of window edge compared to window center; and 3) 0 and 1 Amino acid profile values normalized to be between.

  Using the hydrophilic profile (FIG. 5), hydropathy profile (FIG. 6) and% accessible residue profile (FIG. 7), the hydrophobic amino acids (ie, hydropathic profile and% accessible residues) Stretch with a value greater than 0.5 in the profile and less than 0.5 in the hydropathy profile was determined. Such regions can be exposed to an aqueous environment, can be present on the protein surface, and are therefore available for immune recognition (eg, by antibodies).

  The average flexibility profile (FIG. 8) and β-turn profile (FIG. 9) are amino acids that are not constrained to secondary structures (eg, β-sheet and α-helix) (ie, β-turn profile and average flexibility profile). To determine a stretch with a value greater than 0.5). Such regions are also likely to be exposed portions on the protein and are therefore available for immune recognition (eg, by antibodies).

  For example, using the antigenic sequence of the 109P1D4 variant protein as shown by the profiles shown in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and / or FIG. To prepare therapeutic and diagnostic anti-109P1D4 antibodies. The immunogen is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 109, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, or 109P1D4 listed in FIG. It can be any amino acid derived from a protein variant, or a corresponding nucleic acid encoding it. In particular, the peptide immunogens of the present invention may comprise: at least five of FIGS. 2 and 3 in any integer increment, including amino acid positions having a value greater than 0.5 in the hydrophilic profile of FIG. A peptide region of at least 5 amino acids of FIG. 2 and FIG. 3 in any integer increments, including amino acid positions having a value of less than 0.5 in the hydropathy profile of FIG. 6; The peptide region of at least 5 amino acids of FIGS. 2 and 3 in any integer increments, including amino acid positions having a value greater than 0.5 in the% accessible residue profile; At least five of FIGS. 2 and 3 in any integer increments, including amino acid positions having a value greater than 0.5 in the profile Peptide region of amino acids; and an amino acid position having a value greater than 0.5 in the β- turn profile of Figure 9, at least 5 amino acids of a peptide region of Figures 2 and 3 in any whole number increment. The peptide immunogens of the invention can also include a nucleic acid encoding any of the above.

  All of the immunogens, peptides or nucleic acids of the invention can be implemented in human unit dosage form or can be contained by a composition comprising pharmaceutical excipients compatible with human physiology.

  The secondary structure of variant 1 of 109P1D4 (ie, the presumed presence and position of the α helix, extended strand, and random coil) was determined by the HNN-Hierarchical Neural Network method (Guermeur, 1997, http: //pbil.ibcp. fr / cgi-bin / npsa_automat.pl? page = npsa_nn.html) (accessed from the ExPassy molecular biology server (http://www.expasy.ch/tools/)) Deduced from the sequence. This analysis is shown in 13A-13I for protein variants 1-9, respectively. The percent structure for each variant consisting of an α helix, extended strand, and random coil is also shown.

Analysis of the possible presence of a transmembrane domain in 109P1D4 variants was performed using various transmembrane estimation algorithms accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/) Executed. The analysis results of 109P1D4 protein variants 1-9 using the TMpred program (upper panel) and TMHMM program (lower panel) are shown graphically in FIGS. 13J-R, respectively.
Analysis of variants using other structure prediction programs is summarized in Table VI and Table L.

(Example 10: Production of 109P1D4 polyclonal antibody)
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immune factor and (if desired) an adjuvant. Typically, the immune factor and / or adjuvant is injected into the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full-length 109P1D4 protein, computer algorithms are used in the design of immunogens with characteristics that are antigenic and available for recognition by the immune system of the immunized host, based on amino acid sequence analysis (See the example entitled “Antigenic Profile and Secondary Structure”). Such a region is presumed to be hydrophilic, flexible, in a β-turn conformation and exposed on the surface of the protein (eg 109P1D4 and variants thereof) (See FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles showing such regions).

  For example, a 109P1D4 bacterial recombinant fusion protein or 109P1D4 bacterial recombinant fusion peptide comprising the hydrophilic, flexible, β-turn region of 109P1D4 variant protein can be expressed as a polyclonal antibody in New Zealand white rabbits or “109P1D4 monoclonal in the examples. Used as an antigen to generate monoclonal antibodies as described under the heading "Antibodies (mAbs)". For example, such regions include amino acids 22-39, amino acids 67-108, amino acids 200-232, amino acids 454-499, amino acids 525-537, amino acids 640-660, amino acids 834-880 and amino acids of 109P1D4 variant 1. 929-942, but is not limited to these. It is useful to conjugate the immune factor to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In two embodiments, peptides encoding amino acids 77-90 and amino acids 929-942 of 109P1D4 variant 1 were synthesized, conjugated to KLH, and individual rabbits were immunized. Alternatively, the immune factor may comprise all or part of a 109P1D4 variant protein, analog thereof or fusion protein. For example, the amino acid sequence of 109P1D4 variant 1 is transferred to any one of a variety of fusion protein partners well known in the art, such as glutathione-S-transferase (GST) tagged fusion proteins and HIS tagged fusion proteins. Fusion can be done using recombinant DNA technology. In one embodiment, amino acids 24-419 of 109P1D4 variant 1 were fused to, expressed, purified, and immunized rabbits using recombinant technology and pET43.1 expression vector. Such fusion proteins are purified from bacteria derived using an appropriate affinity matrix.

  Other recombinant bacterial fusion proteins that can be used include maltose binding protein, LacZ, thioredoxin, NusA, or immunoglobulin constant region (see section entitled “Production of 109P1D4 in Prokaryotic Systems”, as well as Current Protocols In Molecular). Biology, Volume 2, Unit 16, edited by Frederick M. Ausubul et al., 1995; Linsley, PS, Brady, W., Urnes, M., Grosmail, L., Damle, N., and Ledbetter, L. ( 1991) J. Exp. Med. 174, 561-566).

  In addition to bacterial derived fusion proteins, protein antigens expressed by mammals are also used. These antigens are expressed from mammalian expression vectors (eg, Tag5 and Fc fusion vectors) (see Examples entitled “Generation of Recombinant 109P1D4 in Eukaryotic Systems”). These antigens retain post-translational modifications (eg, glycosylation) found in native proteins. In two embodiments, the putative first extracellular loop and third extracellular loop (amino acids 59-227, amino acids 379-453, respectively) of variant 1 are each in a Tag5 mammalian secretion vector. Cloned and expressed in 293T cells (Figure 20). Each recombinant protein is then purified by metal chelate chromatography from tissue culture supernatant and / or lysate of 293T cells stably expressing the recombinant vector. The purified Tag5 109P1D4 protein is then used as an immunogen.

  During the immunization protocol, it is useful to mix or milk the antigen in an adjuvant that enhances the host animal's immune response. Examples of adjuvants include, but are not limited to, Freund's complete adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).

  In a typical protocol, rabbits are first immunized subcutaneously with up to 200 μg (typically 100-200 μg) of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every 2 weeks with up to 200 μg (typically 100-200 μg) of immunogen in incomplete Freund's adjuvant (IFA). Approximately 7-10 days after each immunization, test bleeds are collected and used to monitor antiserum titers by ELISA.

  To test the reactivity and specificity of immune sera (eg, rabbit sera from immunization with KLH-conjugated peptides encoding amino acids 1-14 of variant 1), the full-length 109P1D4 variant 1 cDNA was transformed into pCDNA3 .1 Cloning into a myc-his expression vector or a retroviral expression vector (Invitrogen) (see examples entitled “Production of recombinant 109P1D4 in eukaryotic systems”). After transfecting the construct into 293T cells or transducing PC3 with 109P1D4 retrovirus, cell lysates were probed with anti-109P1D4 serum and anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.). The specific reactivity to denatured 109P1D4 protein is then determined using Western blot technology. The antiserum specifically recognizes 109P1D4 protein in 293T cells and PC3 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunohistochemistry (FIG. 21) and immunoprecipitation against 293T cells and other recombinant 109P1D4 expressing cells to determine specific recognition of the native protein. Western blot, immunoprecipitation, fluorescence microscopy, immunohistochemistry, and flow cytometry techniques are performed using cells that endogenously express 109P1D4 to test reactivity and specificity.

  By passing an antiserum from a rabbit immunized with a 109P1D4 variant fusion protein (eg, GST fusion protein and MBP fusion protein) through an affinity column containing the fusion partner alone or in the context of an inappropriate fusion protein. Purify by removing the antibody that is reactive to the fusion partner sequence. For example, antisera derived from GST-109P1D4 fusion protein encoding amino acids 379-453 of variant 1 is first purified by passage through a column of GST protein covalently linked to an AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage through a column composed of an MBP fusion protein that also encodes amino acids 379-453 covalently linked to an Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from rabbits immunized with other His-tagged antigens and peptides, as well as fusion partner-removed sera, are affinity purified by passing through a column matrix composed of the original protein immunogen or free peptide.

(Example 11: Production of 109P1D4 monoclonal antibody (mAb))
In one embodiment, therapeutic mAbs to 109P1D4 variants are epitopes specific for each variant protein or variants that disrupt or modulate the biological function of 109P1D4 variants (eg, interactions with ligands and substrates). Ligands that disrupt or participate in interactions with epitopes specific to a common sequence among, for example, variants that disrupt or interact with a common activity) Alternatively, it includes a mAb that reacts with a protein epitope that disrupts the interaction with the protein. Immunogens for the production of such mAbs include those that are designed to encode or contain the entire 109P1D4 protein variant sequence, or 109P1D4 that is predicted to be antigenic from computer analysis of amino acid sequences. Examples include immunogens that are designed to encode or contain protein variant regions (eg, FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 and examples entitled “antigenic profiles”) See Immunogens include peptides, recombinant bacterial proteins, and Tag5 protein expressed in mammals, as well as human IgG Fc fusion proteins and mouse IgG Fc fusion proteins. In addition, cells that have been genetically engineered to express high levels of each 109P1D4 variant (eg, 293T-109P1D4 variant 1 or 300.19-109P1D4 variant 1 mouse pre-B cells) Immunize.

To generate mAbs against 109P1D4 variants, mice were first challenged with 10-50 μg of protein immunogen or 10 ⁷ 109P1D4 expressing cells mixed in complete Freund's adjuvant first by intraperitoneal (IP) immunization. To do. Thereafter, mice are typically immunized intraperitoneally (IP) every 2-4 weeks with 10-50 μg of protein immunogen or 10 ⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used for immunization. In one embodiment, mice were immunized with 300.19-109P1D4 cells in complete Freund's adjuvant and then 300.19-109P1D4 cells in incomplete Freund's adjuvant as described above. The mice were then sacrificed and spleens were collected and used for fusion and hybridoma production. As can be observed in FIG. 27, two hybridomas were generated in which the antibody specifically recognizes 109P1D4 protein expressed in 293T cells by flow cytometry.
In addition to the protein-based and cell-based immunization strategies described above, a DNA-based immunization protocol is used in which mice are immunized by direct injection of plasmid DNA using a mammalian expression vector encoding a 109P1D4 variant sequence To do. In one embodiment, mice were immunized using a Tag5 mammalian secretion vector encoding amino acids 24-812 of the variant 1 sequence. A subsequent booster immunization is then performed with the purified protein. In another example, the same amino acid is cloned into an Fc-fusion secretion vector. In that vector, the 109P1D4 variant 1 sequence is fused at the amino terminus to an IgK leader sequence and at the carboxy terminus to the coding sequence of a human IgG Fc region or a mouse IgG Fc region. This recombinant vector is then used as an immunogen. A plasmid immunization protocol is used in combination with cells expressing the above purified protein and each 109P1D4 variant.

Alternatively, mice may be immunized directly to their footpad. In this case, 10-50 μg of protein immunogen or 10 ⁷ 254P1D6B expressing cells are injected subcutaneously into the footpad of each hind leg. The first immunization is given as an adjuvant with Titermax (Sigma ^™ ) followed by an Alum-gel in combination with the CpG oligonucleotide sequence except for the final injection (given with PBS). Injected twice a week (every 3-4 days) for 4 weeks, the mice were sacrificed 3-4 days after the last injection, at which time lymph nodes were removed immediately from the footpad, and The B cells are recovered for use as an antibody that generates a fusion partner.

  During the immunization protocol, test bleeds are taken 7-10 days after injection to monitor the titer and specificity of the immune response. Once the appropriate reactivity and specificity has been determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometry analysis, the generation of fusions and hybridomas is well-established procedures well known in the art. (See, eg, Harlow and Lane, 1988).

  In one embodiment for generating 109P1D4 monoclonal antibodies, variant 1 Tag5 antigen encoding amino acids 14-812 is expressed in 293T cells and purified from conditioned media. Balb C mice are first immunized intraperitoneally with 25 μg of Tag5 109P1D4 variant 1 peptide mixed in complete Freund's adjuvant. Mice are then immunized every 2 weeks with 25 μg of antigen mixed in incomplete Freund's adjuvant for a total of 3 immunizations. The titer of sera from immunized mice is determined by ELISA using Tag5 antigen. Serum reactivity and specificity for full-length 109P1D4 variant 1 protein is monitored by Western blotting, immunoprecipitation, and flow cytometry using 293T cells transfected with an expression vector encoding 109P1D4 variant 1 cDNA. (See, eg, the example entitled “Production of Recombinant 109P1D4 in Higher Eukaryotic Systems” and FIG. 21). Other recombinant 109P1D4 variant 1 expressing cells or cells that endogenously express 109P1D4 variant 1 are also used. Mice showing the strongest reactivity are rested and final injected with antigen in PBS and sacrificed 4 days later. Spleens of sacrificed mice are harvested and fused to SPO / 2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescence microscopy, and flow cytometry to identify 109P1D4 variant 1 specific antibody producing clones.

  In order to generate monoclonal antibodies specific for the 109P1D4 variant protein, the immunogen is designed to encode a sequence unique to each variant. In one embodiment, an antigenic peptide consisting of amino acids 1-29 of 109P1D4 variant 2 is bound to KLH to induce a monoclonal antibody specific for 109P1D4 variant 2. In another embodiment, an antigenic peptide consisting of amino acids 1-23 of 109P1D4 variant 6 is conjugated to KLH and used as an immunogen to induce 109P1D4 variant 6 specific MAb. In another example, a GST fusion protein encoding amino acids 1001-1347 of variant 3 is used as an immunogen to recognize variants 3, 4, 5, and 8, and to provide variants 1, 2, 6, 7, and An antibody that distinguishes from 9 is made. The hybridoma supernatant is then screened for each antigen and further cross-screened in cells expressing other variants to induce variant-specific monoclonal antibodies.

  The binding affinity of the 109P1D4 variant specific monoclonal antibody is determined using standard techniques. By affinity measurement, the strength of the antibody to epitope binding is quantified and using that affinity measurement, which 109P1D4 variant monoclonal antibody is preferred for diagnostic or therapeutic applications as recognized by those skilled in the art To help define The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. This BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) in real time. Monitor the effect. BIAcore analysis conveniently yields association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Alternatively, affinity binding analysis of MAbs in 109P1D4 expressing cells can be used to determine affinity.

Example 12: HLA class I binding assay and HLA class II binding assay
HLA class I and HLA class II binding assays using purified HLA molecules can be performed using the disclosed protocols (eg, PCT Publications WO94 / 201527 and WO94 / 03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998). Sidney et al., J. Immunol. 154: 247 (1995); Sette et al., Mol. Immunol. 31: 813 (1994)). Briefly, purified MHC molecules (5-500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵ I radiolabeled probe peptide as described. Following incubation, the MHC-peptide complex is separated from free peptide by gel filtration and the fraction of bound peptide is measured. Typically, in preliminary experiments, each MHC preparation is titrated in the presence of a certain amount of radiolabeled peptide and the HLA molecules required to bind 10-20% of the total radioactivity. Determine the concentration of. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label] <[HLA] and IC ₅₀ ≧ [HLA], the measured IC ₅₀ value is a reasonable approximation of the true _KD value. Peptide inhibitors are typically tested at concentrations ranging from 120 μg / ml to 1.2 ng / ml and tested in 2 to 4 completely independent experiments. In order to allow comparison of the data obtained in the various experiments, the relative binding number is calculated for each peptide. This calculation, the IC ₅₀ of a positive control for inhibition, _(typically unlabeled versions of the radiolabeled probe peptide) IC 50 of each peptide was tested by dividing by. Relative binding values are tabulated for database purposes and for comparison between experiments. These values can then be converted back to IC ₅₀ nM values. This conversion is by dividing the positive control IC ₅₀ nM for inhibition by the relative binding of the peptide of interest. This data aggregation method is accurate and consistent for comparison of peptides tested on different days or peptides tested with different lots of purified MHC.

  Binding assays such as those outlined above can be used to analyze HLA supermotif and / or HLA motif-bearing peptides (see Table IV).

(Example 13: Identification of CTL candidate epitope with HLA supermotif and CTL candidate epitope with HLA motif)
The HLA vaccine composition of the present invention may comprise multiple epitopes. Those multiple epitopes can include multiple HLA supermotifs or HLA motifs to achieve a broad population range. This example demonstrates the identification and confirmation of supermotif and motif-bearing epitopes for inclusion in such vaccine compositions. Population range calculations are performed using the following strategy.

(Computer search and algorithm for identification of supermotif and / or motif-bearing epitopes)
An example entitled “Antigenic Profile” and searches performed to identify motif-bearing peptide sequences in Tables VIII-XXI and XXII-XLIX were performed using protein sequences from the 109P1D4 gene product shown in FIGS. Use the data. The specific search peptides used to generate the table are listed in Table VII.

  A computer search for epitopes carrying an HLA class I super motif or HLA class I motif or HLA class II super motif or HLA class II motif is performed as follows. All 109P1D4 protein sequences to be translated are analyzed using a string search software program to identify peptide sequences that may contain the appropriate HLA-binding motif; Considering the supermotif disclosure, it is easily created according to information in the field. Furthermore, such calculations can be done in the head.

The identified A2, A3, and DR supermotif sequences are scored using a polynomial algorithm, and these motifs bind to specific HLA-class I or HLA-class II molecules Guess the ability to do. These polynomial algorithms consider the effects of different amino acids at various positions. These polynomial algorithms show that the total affinity (or ΔG) of the peptide-HLA molecule interaction is
“ΔG” = a _1i × a _2i × a _3i . . . × a _ni
Is essentially based on the premise that it can be approximated as a linear polynomial function of the form where a _ji is the effect of the presence of a given amino acid (j) at a given position (i) along the peptide sequence of n amino acids It is a coefficient which shows. The critical assumption of this method is that the effects at each position are essentially independent of each other (ie, independent bonds of individual side chains). If residue j is present at position i in the peptide, it is assumed that it contributes a constant amount j _i to the free energy of binding of the peptide, regardless of the rest of the sequence of the peptide.

Specific algorithm coefficient derivation methods are described in Gulukota et al. Mol. Biol. 267: 1258-126, 1997 (see also Sidney et al., Human Immunol. 45: 79-93, 1996; and Southwood et al., J. Immunol. 160: 3363-3373, 1998). Briefly, for all i positions, the anchor and non-anchor as well as the geometric mean of the average relative binding (ARB) of all peptides carrying j are calculated against the rest of the group and j Used as an estimate of _i . For class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate the algorithm score for a given peptide in the test set, the ARB value corresponding to the sequence of that peptide is multiplied. If this product exceeds the selected threshold, the peptide is presumed to bind. Appropriate threshold values are selected as a function of the desired degree of stringency.

(Selection of HLA-A2 supertype cross-reactive peptide)
The protein sequence from 109P1D4 is scanned using motif identification software to identify 8-mer, 9-mer, 10-mer, and 11-mer sequences, including the HLA-A2 supermotif major anchor specificity. Typically, these sequences are then scored using the protocol described above, and peptides corresponding to sequences with positive scores are synthesized, and these in vitro purified HLA-A ^* 0201 molecules (HLA-A ^* 0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the ability to bind to additional A2 supertype molecules (A ^* 0202, A ^* 0203, A ^* 0206, and A ^* 6802). Peptides that bind to at least three of the five A2 supertype alleles tested are typically considered to be A2 supertype cross-reactive binders. Preferred peptides bind to 3 or more HLA-A2 supertype molecules with an affinity of 500 nM or less.

(Selection of epitope bearing HLA-A3 supermotif)
The 109P1D4 protein sequence scanned above is also tested for the presence of peptides with the HLA-A3 supermotif major anchor. A peptide corresponding to the HLA-A3 supermotif carrying sequence is then synthesized and into HLA-A ^* 0301 and HLA-A ^* 1101 molecules (molecules encoded by the two most prominent A3 supertype alleles) Test for binding. A peptide that binds to at least one of its two alleles with a binding affinity of ≦ 500 nM, often ≦ 200 nM, is then transferred to other common A3 supertype alleles (eg, A ^* 3101, A ^* 3301). And A ^* 6801) are identified for binding cross-reactivity to identify molecules that can bind to at least three of the five HLA-A3 supertype molecules tested.

(Selection of epitope bearing HLA-B7 supermotif)
The 109P1D4 protein scanned above is also analyzed for the presence of 8-mer, 9-mer, 10-mer, or 11-mer peptides containing the HLA-B7 supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B ^* 0702 (the molecule encoded by the most common B7 supertype allele (ie, the prototype B7 supertype allele)). Peptides that bind B ^* 0702 with an IC ₅₀ ≦ 500 nM are identified using standard methods. These peptides are then tested for binding to other common B7 supertype molecules (eg, B ^* 3501, B ^* 5101, B ^* 5301 and B ^* 5401). Thereby, peptides that can bind to more than two of the five B7 supertype alleles to be tested are identified.

(Selection of A1 motif-bearing epitope and A24 motif-bearing epitope)
To further increase the population range, HLA-A1 and HLA-A24 epitopes can also be incorporated into the vaccine composition. Analysis of 109P1D4 protein can also be performed to identify HLA-A1 motif-containing sequences and HLA-A24 motif-containing sequences.

  High affinity and / or cross-reactive binding epitopes carrying other motifs and / or supermotifs are identified using similar methodologies.

(Example 14: Confirmation of immunogenicity)
A cross-reactive candidate CTL A2 supermotif-bearing peptide identified as described herein is selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology.

(Target cell line for cell screening)
The HLA-A2.1 gene was generated by transfer to 721.221, an HLA-A, HLA-B, HLA-C null mutant human B lymphoblast cell line. The 221A2.1 cell line is used as a peptide loading target to measure the activity of HLA-A2.1 restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, non-essential amino acids, and 10% (v / v) heat inactivated FCS. Cells expressing the antigen of interest, or transfectants containing a gene encoding the antigen of interest can be used as target cells to confirm the ability of peptide-specific CTL to recognize endogenous antigens.

(Primary CTL induction culture)
(Dendritic cell (DC) production)
PBMCs were thawed in RPMI containing 30 μg / ml DNAse and washed twice with complete medium (RPMI-1640 + 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin / streptomycin) Resuspend in complete medium. The monocytes are purified by plating at 10 × 10 ⁶ PBMC / well in 6 well plates. After 2 hours at 37 ° C., non-adherent cells are removed by gently shaking the plate and aspirating the supernatant. The wells are washed a total of 3 times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Thereafter, 3 ml of complete medium containing 50 ng / ml GM-CSF and 1,000 U / ml IL-4 is added to each well. On day 6, TNFα is added to DC at 75 ng / ml and on day 7 the cells are used for CTL-induced culture.

(Induction of CTL using DC and peptide)
CD8 + T cells are isolated by positive selection using Dynal immunomagnetic beads (Dynabeads® M-450) and dechacha-bead® reagent. Typically, about 200 × 10 ⁶ to 250 × 10 ⁶ PBMC are processed to obtain 24 × 10 ⁶ CD8 ⁺ T cells (sufficient for 48-well plate culture). Briefly, PBMCs are thawed in RPMI containing 30 μg / ml DNAse and washed once with PBS containing 1% human AB serum and in PBS / 1% AB serum at a concentration of 20 × 10 Resuspend at ⁶ cells / ml. Magnetic beads are washed 3 times with PBS / AB serum, added to the cells (140 μl beads / 20 × 10 ⁶ cells) and incubated for 1 hour at 4 ° C. with continued mixing. The beads and cells were washed 4 times with PBS / AB serum to remove non-adherent cells and in PBS / AB serum containing 100 μl / ml detergent-bead® reagent and 30 μg / ml DNAse. To 100 × 10 ⁶ cells / ml (based on the original cell number). The mixture is incubated for 1 hour at room temperature with continued mixing. The beads are washed again with PBS / AB / DNAse and CD8 + T cells are collected. DCs were collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS containing 1% BSA, counted and at 20 ° C. in the presence of 3 μg / ml β ₂ -microglobulin. Pulse with 40 μg / ml peptide at a cell concentration of 1 × 10 ⁶ to 2 × 10 ⁶ / ml for 4 hours. The DC is then irradiated (4,200 rad), washed once with media and counted again.

(Induction culture setting)
0.25 ml of cytokine producing DC (1 × 10 ⁵ cells / ml) was added to 0.25 ml of CD8 + T cells (2 × 10 ⁶ cells) in each well of a 48 well plate in the presence of 10 ng / ml IL-7. / Ml). The next day, recombinant human IL-10 is added at a final concentration of 10 ng / ml, and 48 hours later rhuman IL-2 is added at 10 IU / ml.

(Restimulation of induced cultures using adherent cells pulsed with peptides)
At 7 and 14 days after primary induction, the cells are restimulated with adherent cells pulsed with peptide. Thaw the PBMC and wash twice with RPMI and DNAse. The cells are resuspended at 5 × 10 ⁶ cells / ml and irradiated at approximately 4200 rad. PBMCs are plated at 2 × 10 ⁶ in 0.5 ml complete medium per well and incubated at 37 ° C. for 2 hours. The plate was washed twice with RPMI by gentle tapping to remove non-adherent cells and 10 μg / ml in the presence of 3 μg / ml β ₂ microglobulin in 0.25 ml RPMI / 5% AB / well. Adherent cells are pulsed with 2 peptides at 37 ° C. for 2 hours. Peptide solution is aspirated from each well and the wells are washed once with RPMI. Most of the medium is aspirated from the induction culture (CD8 + cells) and made up to 0.5 ml with fresh medium. The cells are then transferred to a well containing adherent cells pulsed with the peptide. After 24 hours, recombinant human IL-10 is added at a final concentration of 10 ng / ml, and again the next day and 2-3 days later, recombinant human IL2 is added at 50 IU / ml (Tsai et al., Critical Reviews in Immunology 18 (1-2): 65-75, 1998). After 7 days, the culture is assayed for CTL activity in a ⁵¹ Cr release assay. In some experiments, the cultures are assayed for peptide specific recognition in an in situ IFNγ ELISA during the second re-stimulation, and after 7 days, an assay for endogenous recognition is performed. After growth, activity is measured in both assays for side-by-side comparison.

(Measurement of CTL dissolution activity by ⁵¹ Cr release)
Cytotoxicity is measured in a standard (5 hour) ⁵¹ Cr release assay by assaying individual wells at a single E: T 7 days after the second restimulation. Peptide pulsed targets are prepared by incubating the cells with 10 μg / ml peptide overnight at 37 ° C.

Adherent target cells are removed from the culture flask using trypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹ Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37 ° C. The labeled target cells were resuspended in 1ml per 10 ^6, with a concentration of 3.3 × ¹⁰ 6 / ml of K562 cells (NK-sensitive erythroblast line used to reduce non-specific lysis) Dilute 1:10. Target cells (100 μl) and effectors (100 μl) are plated in 96 well round bottom plates and incubated at 37 ° C. for 5 hours. At that time, 100 μl of supernatant is collected from each well and has the formula:
[(Test sample cpm−spontaneously generated ⁵¹ Cr released sample cpm) / (maximum ⁵¹ Cr released sample cpm−spontaneously generated ⁵¹ Cr released sample cpm)] × 100
Determine the percent dissolution according to:

  Maximum release and spontaneous release are determined by incubating the labeled target with 1% Triton X-100 and media alone, respectively. Cultures with specific lysis (sample-background)> 10% for individual wells and> 15% at the highest two E: T ratios when assaying grown cultures, Define a positive culture.

(In situ measurement of human IFNγ production as an indicator of peptide-specific and endogenous recognition)
Immulon 2 plates are coated overnight at 4 ° C. using mouse anti-human IFNγ monoclonal antibody (4 μg / ml 0.1 M NaHCO ₃ , pH 8.2). The plate was washed with Ca ²⁺ , Mg ²⁺ free PBS / 0.05% Tween 20, blocked for 2 hours using PBS / 10% FCS, then CTL (100 μl / well) and target (100 μl / well) Is added to each well and the standard and blank wells are left empty (with medium only). Target cells (either peptide pulses or endogenous targets) are used at a concentration of 1 × 10 ⁶ cells / ml. Plates are incubated for 48 hours at 37 ° C. in the presence of 5% CO ₂ .

Recombinant human IFN-γ is added to standard wells starting at 400 pg / 100 μl / well or 1200 pg / 100 μl / well and the plate is incubated at 37 ° C. for 2 hours. The plate is washed and 100 μl of biotinylated mouse anti-human IFN-γ monoclonal antibody (2 μg / ml in PBS / 3% FCS / 0.05% Tween 20) is added and incubated for 2 hours at room temperature. After washing again, 100 μl HRP-streptavidin (1: 4000) is added and the plate is incubated at room temperature for 1 hour. The plate is then washed 6 times with wash buffer, 100 μl / well of color development solution (TMB 1: 1) is added and the plate is allowed to develop for 5-15 minutes. The reaction is stopped using 50 μl / well 1M H ₃ PO ₄ and read at OD450. A culture is considered positive if at least 50 pg of IFN-γ / well is measured above background and is twice the background level of expression.

(CTL proliferation)
Cultures exhibiting specific lytic activity against peptide pulsed targets and / or tumor targets are grown with anti-CD3 for 2 weeks. Briefly, 5 × 10 ⁴ CD8 ⁺ cells are added to a T25 flask containing: RPMI-1640 (10% (v / v) human AB serum, nonessential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol. 1 × 10 ⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) / ml, 2 × 10 ⁵ irradiated (8,000 rad) EBV transformed cells in 1 ml containing L-glutamine and penicillin / streptomycin) / Ml, and 30 ng / ml OKT3 (anti-CD3). Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU / ml, followed by fresh medium at 50 IU / ml every 3 days thereafter. When the cell concentration exceeds 1 × 10 ⁶ / ml, the cells are split and the cultures are collected in a ⁵¹ Cr release assay at 13: 1 to 15: 1 with an E: T ratio of 30: 1, 10: 1, 3: 1 and 1: 1. Assay during the day or assay at 1 × 10 ⁶ / ml in an in situ IFNγ assay using the same target as before growth.

Cultures are grown in the absence of anti-CD3 ⁺ as follows. Cultures exhibiting specific lytic activity against peptides and endogenous targets are selected and 5 × 10 ⁴ CD8 ⁺ cells are added to a T25 flask containing: RPMI-1640 (10% (v / v) human AB serum 1 × 10 ⁶ autologous PBMC / ml (containing 10 μg / ml peptide for 2 hours at 37 ° C. in 1 ml, non-essential AA, containing sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin) And irradiated (4,200 rad)); 2 × 10 ⁵ irradiated (8,000 rad) EBV transformed cells / ml.

(Immunogenicity of peptides with A2 supermotif)
A2 supermotif cross-reactive binding peptides are tested in a cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered an epitope if it induces at least a peptide-specific CTL in the individual, and preferably also recognizes an endogenously expressed peptide.

  Immunogenicity can also be confirmed using PBMCs isolated from patients bearing tumors that express 109P1D4. Briefly, PBMCs are isolated from patients, restimulated with peptide-pulsed monocytes, and assayed for their ability to recognize peptide-pulsed target cells and transfected cells that endogenously express the antigen.

(Evaluation of A ^* 03 / A11 immunogenicity)
HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methods similar to those used to evaluate the immunogenicity of HLA-A2 supermotif peptides.

(Evaluation of B7 immunogenicity)
Immunogenicity screening of B7 supertype cross-reactive binding peptides identified as shown herein is confirmed in a manner similar to the confirmation of peptides carrying A2 and A3 supermotifs.

  Peptides carrying other supermotifs / motifs (eg, HLA-A1, HLA-A24, etc.) are also confirmed using similar methodology.

Example 15: Implementation of an extended supermotif to improve the binding ability of native epitopes by making analogs
HLA motifs and HLA supermotifs (including primary and / or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. In addition, the definition of HLA motifs and HLA supermotifs also allows manipulation of highly cross-reactive epitopes by identifying residues within the native peptide sequence. This native peptide sequence imparts certain properties to the peptide (eg, greater cross-reactivity within a group of HLA molecules including supertypes and / or greater binding affinity for some or all of those HLA molecules) To be analogized. Examples of analogized peptides that exhibit regulated binding affinity are shown in this example.

(Analogization at the primary anchor residue)
Peptide manipulation strategies are performed to further increase epitope cross-reactivity. For example, the main anchor of the A2 supermotif-bearing peptide is modified to introduce, for example, preferred L, I, V or M at position 2 and I or V at the C-terminus.

In order to analyze the cross-reactivity of the analog peptides, each engineered analog is first bound for the prototype A2 supertype allele A ^* 0201, and then A2 supertype if A ^* 0201 binding ability is maintained. Test for cross-reactivity.

  Alternatively, the peptide is confirmed to bind one or all supertype members, and then analogized to modulate the binding affinity for any one (or more) of the supertype members, and the population range Append.

Analog selection for immunogenicity in cell screening analysis typically involves parental wild-type (WT) peptides to bind at least weakly to three or more A2 supertype alleles (ie, IC ₅₀ of 5000 nM or less). Further limited by ability to combine). The principle for this requirement is that the WT peptide must be endogenously present in an amount sufficient to be biologically relevant. Analogized peptides have been shown to increase immunogenicity and cross-reactivity by T cells specific for the parent epitope (eg, Parkhurst et al., J. Immunol. 157: 2539, 1996; and Pogue et al. Proc. Natl. Acad. Sci. USA 92: 8166, 1995).

  In cell screening for these peptide analogs, it is important to confirm that analog-specific CTLs can also recognize wild-type peptides and, where possible, target cells that endogenously express this epitope. .

(Analogization of peptides containing HLA-A3 supermotif and B7 supermotif)
An analog of the epitope bearing the HLA-A3 supermotif is generated using a strategy similar to that used in analogizing the peptide bearing the HLA-A2 supermotif. For example, a peptide that binds to 3/5 of the A3 supertype molecule is engineered with a primary anchor residue to carry a preferred residue (V, S, M or A) at position 2.

The analog peptide is then tested for the ability to bind A ^* 03 and A ^* 11 (prototype A3 supertype allele). Next, a peptide showing a binding ability of ≦ 500 nM is confirmed to have A3 supertype cross-reactivity.

  Similar to A2 motif-bearing peptides and A3 motif-bearing peptides, peptides that bind more than two B7 supertype alleles can be modified to increase cross-reactivity binding or greater binding affinity or binding half-life where possible. Can be achieved. B7 supermotif-bearing peptides can be substituted with preferred residues (V, I, L or F) at the C-terminal primary anchor position, for example, as shown in Sidney et al. (J. Immunol. 157: 3480-3490, 1996). Operate to have.

  Analogization at the primary anchor residue of other motif-bearing epitopes and / or other supermotif-bearing epitopes is performed in a similar manner.

  The analog peptide is then confirmed for immunogenicity, typically in a cell screening assay. Again, it is generally important to demonstrate that analog-specific CTLs can also recognize wild-type peptides and, if possible, targets that endogenously express this epitope.

(Analogization at secondary anchor residues)
Furthermore, HLA supermotifs are valuable for manipulating highly cross-reactive peptides and / or peptides that bind HLA molecules with increased affinity, and this manipulation is associated with properties such as these. It is valuable in manipulating by identifying specific residues at the next anchor position. For example, the binding ability of a B7 supermotif-containing peptide having an F residue at position 1 is analyzed. The peptide is then analogized, eg, to replace F at position 1 with L. The analogized peptide is evaluated for increased binding affinity, binding half-life and / or increased cross-reactivity. Such a procedure identifies analogized peptides with enhanced properties.

  Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7 transgenic mice, eg, according to IFA immunization or lipopeptide immunization. Analogized peptides are further tested for the ability to stimulate a recall response using PBMC from patients with 109P1D4-expressing tumors.

(Other analog strategies)
Another form of peptide analogization involves replacing cysteine with α-aminobutyric acid, regardless of anchor position. Due to its chemical nature, cysteine forms disulfide bridges and has the property of structurally changing the peptide sufficiently to reduce binding capacity. Substitution with α-aminobutyric acid instead of cysteine has been shown not only to alleviate this problem, but in some cases also improves binding and cross-linking capabilities (see, eg, Sette et al., (See review by Persistent Viral Infections, R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

  Thus, the use of single amino acid substitutions can modulate the binding properties and / or cross-reactivity of peptide ligands to HLA supertype molecules.

(Example 16: Identification and confirmation of 109P1D4-derived sequence having HLA-DR binding motif)
Peptide epitopes with HLA class II supermotifs or HLA class II motifs are identified and confirmed as outlined below using methodologies similar to those described for HLA class I peptides.

(Selection of epitope bearing HLA-DR supermotif)
In order to identify HLA class II HTL epitopes from 109P1D4, the 109P1D4 antigen is analyzed for the presence of sequences carrying the HLA-DR motif or HLA-DR supermotif. Specifically, a 15-mer sequence containing the DR supermotif is selected (including a 9-mer core and a 3-residue N-terminal flanking region and a 3-residue C-terminal flanking region (15 amino acids total).

  Protocols have been developed to estimate peptides that bind to DR molecules (Southwood et al., J. Immunol. 160: 3363-3373, 1998). These protocols specific for individual DR molecules allow for scoring and ranking of 9-mer core regions. Each protocol not only scores the peptide sequence for the presence of DR supermotif primary anchors (ie, positions 1 and 6) within the 9mer core, but also evaluates the sequence for the presence of secondary anchors. Using allele-specific selection tables (see, eg, Southwood et al., Ibid), we found that these protocols efficiently select peptide sequences that have a high probability of binding a particular DR molecule. It was. In addition, performing these protocols in tandem has found that, in particular, implementations on DR1, DR4w4, and DR7 can efficiently select DR cross-reactive peptides.

  The 109P1D4-derived peptides identified above are tested for their ability to bind to various common HLA-DR molecules. All peptides are first tested for binding to these DR molecules in the primary panel: DR1, DR4w4 and DR7. Peptides that bind at least two of these three DR molecules are then tested for binding to DR2w2β1, DR2w2β2, DR6w19, and DR9 molecules in a secondary assay. Finally, binding peptides that bind at least two of these four secondary panel DR molecules (and thus at least four of the seven different DR molecules cumulatively) to DR4w15, DR5w11, and DR8w2 molecules Are screened in a tertiary assay. Peptides that bind at least 7 of the 10 DR molecules, including primary screening assays, secondary screening assays and tertiary screening assays, are considered cross-reactive DR binders. Of particular importance are peptides derived from 109P1D4 that have been found to bind common HLA-DR alleles.

(Selection of DR3 motif peptide)
Since HLA-DR3 is an allele that predominates in the White, Black and Latin American populations, DR3 binding ability is an appropriate criterion in the selection of HTL epitopes. Thus, peptides that have been shown to be candidates can also be assayed for their DR3 binding ability. However, given the binding specificity of the DR3 motif, peptides that bind only to DR3 can also be considered candidates for inclusion in vaccine formulations.

  In order to efficiently identify peptides that bind DR3, the target 109P1D4 antigen was selected from one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152: 5742-5748, 1994). Sequences with are analyzed. The corresponding peptide is then synthesized and identified as having the ability to bind DR3 with an affinity of 1 μM or better (ie, less than 1 μM). Peptides that meet this binding criteria and are considered HLA class II high affinity binders will be found.

  The DR3 binding epitope thus identified is included in the vaccine composition along with the DR supermotif-bearing peptide epitope.

  As with HLA class I motif-bearing peptides, class II motif-bearing peptides are analogized to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimized residue for DR3 binding, and substitution for this residue often improves DR3 binding.

(Example 17: Immunogenicity of 109P1D4-derived HTL epitope)
This example determines the immunogenic DR supermotif and DR3 motif-bearing epitopes from those identified using the methodology described herein.

  In a manner similar to the determination of immunogenicity of CTL epitopes by assessing the immunogenicity of HTL epitopes, by assessing their ability to stimulate HTL responses, and / or by using appropriate transgenic mouse models, Check. Immunogenicity is determined by screening for the following: 1.) In vitro primary induction using normal PBMC or ) Recall response from patients with 109P1D4-expressing tumors.

Example 18: Calculation of phenotypic frequencies of HLA supertypes in various racial backgrounds to determine population range width
This example shows an evaluation of the breadth of the population range of vaccine compositions composed of multiple supermotifs and / or multiple epitopes containing multiple motifs.

To analyze population range, determine the gene frequency of the HLA allele. The gene frequency of each HLA allele is calculated from the antigen frequency or allele frequency using the binomial distribution gf = 1- (SQRT (1-af)) (eg, Sidney et al., Human Immunol. 45:79). -93, 1996). To obtain the overall phenotypic frequency, the cumulative gene frequency is calculated and the cumulative antigen frequency is derived by using the inverse equation [af = 1- (1-Cgf) ² ].

If frequency data is not available at the level of DNA typing, an equivalent to serologically defined antigen frequency is assumed. In order to obtain the entire potential supertype population range, no connection imbalance is assumed and only those alleles identified as belonging to each of the supertypes are included (minimum estimate). An assessment of the overall potential range achieved by the combination between loci is made by adding to the A range the percentage of non-A inclusion populations that can be expected to be covered by the considered B allele (eg, total = A + B ^* (1-A)). The confirmed members of the A3-like supertype are A3, A11, A31, A ^* 3301, and A ^* 6801. The A3-like supertype may also include A34, A66, and A ^* 7401, but these alleles were not included in the overall frequency calculation. Similarly, identified members of the A2-like supertype family are A ^* 0201, A ^* 0202, A ^* 0203, A ^* 0204, A ^* 0205, A ^* 0206, A ^* 0207, A ^* 6802 and A ^* 6901. Finally, confirmed alleles of the B7-like supertype are B7, B ^* 3501-03, B51, B ^* 5301, B ^* 5401, B ^* 55001-2, B ^* 5601, B ^* 6701, and B ^*. 7801 (B ^* 1401, B ^* 3504-06, B ^* 4201, and B ^* 5602 are also potentially members).

  The population range achieved by combining the A2, A3 and B7 supertypes is approximately 86% in the five major racial groups. The range can be expanded by including peptides carrying the A1 and A24 motifs. On average, A1 is present in 12% of the population spanning five different major ethnic groups (white, North American black, Chinese, Japanese, and Latino), and A24 is 29% of these populations. % Present. Collectively, these alleles are represented with an average frequency of 39% in these same racial populations. When A1 and A24 are combined with a range of A2 supertype alleles, A3 supertype alleles and B7 supertype alleles, the total range across major races is over 95%. See, for example, Table IV (G). A similar approach can be used to assess the population range achieved by a combination of class II motif-bearing epitopes.

  Immunogenicity studies in humans (eg Bertoni et al., J. Clin. Invest. 100: 503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159: 1648, 1997) We have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in vaccines that are immunogenic in diverse populations.

  By using a sufficient number of epitopes (as disclosed herein and according to the prior art), the average population range is estimated to be greater than 95% in each of the five major racial populations. Monte Carlo simulation analysis, which is the theory of games (this is well known in the art (see, for example, Osborne, MJ and Rubinstein, A. “A course in game theory” MIT Press, 1994)) Estimate the percentage of individuals in the population consisting of ethnic groups of Caucasian, North American Black, Japanese, Chinese, and Latino that recognize the vaccine epitopes described herein Can be used to A preferred percentage is 90%. A more preferred percentage is 95%.

(Example 19: CTL recognition of endogenously processed antigen after priming)
In this example, the CTL induced by a native peptide analog or an analogized peptide epitope identified and selected as described herein is an endogenously synthesized antigen (ie, a native antigen). Make sure you recognize.

Effector cells isolated from transgenic mice immunized with peptide epitopes (eg, epitopes bearing the HLA-A2 supermotif) are restimulated in vitro using peptide-coated stimulator cells. After 6 days, effector cells are assayed for cytotoxicity and cell lines containing peptide-specific cytotoxic activity are further restimulated. After an additional 6 days, these cell lines were tested for cytotoxic activity against ⁵¹ Cr-labeled Jurkat-A2.1 / ^Kb target cells in the presence or absence of peptides and were also synthesized endogenously. ⁵¹ Cr-labeled target cells (ie, cells stably transfected with the 109P1D4 expression vector) having the same antigen.

This result demonstrates that CTL lines obtained from animals primed with peptide epitopes recognize endogenously synthesized 109P1D4 antigen. The choice of transgenic mouse model used for such analysis depends on the epitope being evaluated. In addition to HLA-A ^* 0201 / ^Kb transgenic mice, there are several other including human A11 alleles (which can also be used to assess A3 epitopes) and mice with human B7 alleles. Transgenic mouse models have been characterized and others (eg, transgenic mice for HLA-A1 and A24) have been developed. Mouse models of HLA-DR1 and HLA-DR3 have also been developed and can be used to assess HTL epitopes.

Example 20: Activity of CTL-HTL conjugated epitopes in transgenic mice
This example demonstrates the induction of CTL and HTL in transgenic mice by use of 109P1D4-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprises a peptide to be administered to a patient having a 109P1D4 expressing tumor. The peptide composition can comprise multiple CTL epitopes and / or HTL epitopes. These epitopes are identified using the methodology described herein. This example also shows that enhanced immunogenicity can be achieved by including one or more HTL epitopes in the CTL vaccine composition; such peptide compositions are conjugated to the CTL epitope. HTL epitopes may be included. The CTL epitope can be an epitope that binds to multiple HLA family members or an analog of that epitope with an affinity of 500 nM or less. These peptides can be lipidated if desired.

Immunization procedure: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159: 4753-4761, 1997). For example, to confirm the immunogenicity of an A2 / ^Kb mouse (which is transgenic for the human HLA A2.1 allele and carries the HLA-A ^* 0201 motif or the HLA-A2 supermotif. Used) in Freund's incomplete adjuvant, or DMSO / saline if the peptide composition is a lipidated CTL / HTL conjugate, or PBS or if the peptide composition is a polypeptide. Prime subcutaneously (base of the tail) with 0.1 ml peptide in Freund's incomplete adjuvant. Seven days after priming, splenocytes from these animals are restimulated with syngeneic irradiated LPS activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays are transduced using the HLA-A2.1 / ^Kb chimeric gene (eg, Vitiello et al., J. Exp. Med. 173: 1007, 1991). The infected Jurkat cells.

CTL activation in vitro: primed after 1 week, spleen cells (30 × 10 ⁶ cells / flask) in the culture medium / T25 flask 10 ml, syngeneic irradiated at 37 ° C. (3000 rad) peptide-coated lymph Co-culture with blasts (10 × 10 ⁶ cells / flask). After 6 days, effector cells are harvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0-1.5 × 10 ⁶ ) are incubated at 37 ° C. in the presence of 200 μl ⁵¹ Cr. After 60 minutes, the cells are washed three times and resuspended in R10 medium. Peptides are added at a concentration of 1 μg / ml if necessary. For the assay, 10 ^{4 51} Cr labeled target cells are added to different concentrations of effector cells (final volume 200 μl) in U-bottom 96-well plates. After 6 hours incubation at 37 ° C., a 0.1 ml aliquot of the supernatant is removed from each well and the radioactivity is measured with a Micromedic automatic gamma counter. The% specific lysis is determined by the following formula:% specific release = 100 × (experimental release−spontaneous release) / (maximum release−spontaneous release). To facilitate comparison between separate CTL assays performed under the same conditions, the ⁵¹ Cr release% data is expressed as lytic units / 10 ⁶ cells. One lysis unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour ⁵¹ Cr release assay. In order to obtain specific lytic units / 10 ⁶ , the lytic units / 10 ⁶ obtained in the absence of peptide are subtracted from the lytic units / 10 ⁶ obtained in the presence of peptide. For example, 30% ⁵¹ Cr release is 50: 1 effector (E): target (T) ratio in the absence of peptide (ie 5 × 10 ⁵ effectors for 10,000 targets). Cell) and in the presence of peptide at 5: 1 (ie 5 × 10 ⁴ effector cells for 10,000 targets), the specific lytic unit is [(1 / 50,000) − (1 / 500,000)] × 10 ⁶ = 18 LU.

  The results were analyzed to assess the magnitude of the CTL response in animals injected with the immunogenic CTL / HTL conjugate vaccine preparation and as outlined above in the example entitled “Verification of immunogenicity”. Compare, for example, the magnitude of a CTL response achieved using a CTL epitope. A similar analysis can be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and / or multiple HTL epitopes. According to these procedures, it is found that a CTL response is induced and at the same time an HTL response is induced upon administration of such a composition.

Example 21: Selection of CTL and HTL epitopes for inclusion in a 109P1D4-specific vaccine
This example shows the procedure for selecting peptide epitopes for the vaccine composition of the invention. The peptides in this composition can be in the form of a nucleic acid sequence, either a single sequence encoding the peptide or one or more sequences (ie, a minigene), or of a single epitope and / or polyepitope. It can be a peptide.

  The following principles are utilized when selecting multiple epitopes for inclusion in a vaccine composition. Each of the following principles are balanced to make a selection.

  Upon administration, an epitope is selected that mimics the immune response that correlates with 109P1D4 clearance. The number of epitopes used depends on patient observations that spontaneously exclude 109P1D4. For example, if a patient who spontaneously eliminates 109P1D4 expressing cells is observed to produce an immune response against at least three epitopes from the 109P1D4 antigen, at least three epitopes for HLA class I should be included. A similar principle is used to determine HLA class II epitopes.

Often, an epitope with a binding affinity of HLA class I The following _{IC 50} 500 nM for molecules or _{IC 50} of less 1000nM for class II,; high from or BIMAS web site (URL bimas.dcrt.nih.gov/) HLA class I peptides with binding scores are selected.

  In order to achieve a wide range of vaccines across diverse populations, a sufficient population of supermotifs or sufficient arrays of allele-specific motif-bearing peptides can be selected to provide a broad population range. In one embodiment, the epitope is selected to provide a population range of at least 80%. Monte Carlo analysis (statistical evaluation methods known in the art) can be used to assess the breadth of the population range, ie redundancy.

  When making a polyepitope composition, or minigene encoding it, it is typically desirable to produce the smallest possible peptide containing the epitope of interest. The principle used is similar if not the same as that used when selecting peptides containing a nested epitope. For example, the protein sequence for a vaccine composition is selected because this sequence has the maximum number of epitopes contained within the sequence (ie, has a high concentration of epitopes). Epitopes may be nested or overlapping (ie, frame shifted from each other). For example, if overlapping epitopes are used, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and is bound by an HLA molecule upon administration of such a peptide. Multi-epitope peptides can be generated synthetically, recombinantly, or via cleavage from a native source. Alternatively, analogs can be generated from the native sequence, whereby one or more epitopes contain substitutions that alter the cross-reactivity and / or binding affinity properties of the polyepitope peptide. Such vaccine compositions are administered for therapeutic or prophylactic purposes. This embodiment has the potential to apply an undiscovered aspect of immune system processing to native nested sequences, thereby facilitating the generation of therapeutic or prophylactic immune response inducing vaccine compositions I will provide a. Furthermore, such embodiments provide the possibility of motif-bearing epitopes for currently unknown HLA structures. Furthermore, this embodiment (which does not create any analog) directs the immune response to multiple peptide sequences that are actually present in 109P1D4, thus avoiding the need to evaluate any linked epitopes. Finally, this embodiment provides economies of scale when producing nucleic acid vaccine compositions. In connection with this embodiment, a computer program can be derived according to the principles of the art, which identifies the maximum number of epitopes per sequence length in the target sequence.

  Vaccine compositions composed of selected peptides are safe and effective when administered and have a magnitude similar to an immune response that controls or eliminates cells having or overexpressing 109P1D4 Elicits an immune response.

Example 22: Construction of “minigene” multi-epitope DNA plasmid
This example discusses the construction of a minigene expression plasmid. The minigene plasmid can of course contain B cell epitopes, CTL epitopes and / or HTL epitopes or B cell epitope analogs, CTL epitope analogs and / or HTL epitope analogs of the various configurations described herein.

  Minigene expression plasmids typically include multiple CTL peptide epitopes and HTL peptide epitopes. In this example, HLA-A2 supermotif-bearing peptide epitope, HLA-A3 supermotif-bearing peptide epitope, HLA-B7 supermotif-bearing peptide epitope, HLA-A1 motif-bearing peptide epitope, and HLA-A24 motif-bearing peptide epitope are defined as DR Used in combination with supermotif-bearing epitopes and / or DR3 epitopes. HLA class I supermotifs derived from 109P1D4 or peptide epitopes carrying HLA class I motifs are selected such that multiple supermotifs / motifs are presented to ensure a broad population range. Similarly, HLA class II epitopes are selected from 109P1D4 to provide a broad population range. That is, both the HLA DR-1-4-7 supermotif-bearing epitope and the HLA DR-3 motif-bearing epitope are selected for inclusion in the minigene construct. Selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

  Such a construct may further comprise a sequence that directs the HTL epitope to the endoplasmic reticulum. For example, an Ii protein (Ii protein) can be fused to one or more HTL epitopes as described in the art, where the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence As a result, an HLA class II epitope is directed to the endoplasmic reticulum and this epitope binds to an HLA class II molecule.

  This example illustrates the method used for the construction of a minigene-bearing expression plasmid. Other expression vectors that can be used for minigene compositions are available and known to those of skill in the art.

  The minigene DNA plasmid of this example comprises a consensus Kozak sequence and a consensus mouse κIg light chain signal sequence followed by a CTL epitope and / or an HTL epitope selected according to the principles disclosed herein. This sequence encodes an open reading frame fused to the Myc and His antibody epitope tags encoded by the pcDNA3.1 Myc-His vector.

  For example, overlapping oligonucleotides that can be on average about 70 nucleotides long, with 15 nucleotides overlapping, are synthesized and HPLC purified. These oligonucleotides encode selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequences, and signal sequences. The final multi-epitope minigene is assembled by extending overlapping oligonucleotides in three sets of reactions using PCR. A Perkin / Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95 ° C. for 15 seconds at an annealing temperature (5 ° C. lower than the lowest Tm calculated for each primer pair). 30 seconds and 1 minute at 72 ° C.

For example, a minigene is prepared as follows. In the first PCR reaction, each 5 μg of two oligonucleotides are annealed and extended. In an example using 8 oligonucleotides (ie, 4 pairs of primers), oligonucleotide 1 and oligonucleotide 2, oligonucleotide 3 and oligonucleotide 4, oligonucleotide 5 and oligonucleotide 6, and oligonucleotide 7 and oligo Nucleotide 8 was added to Pfu polymerase buffer (1 × = 10 mM KCL, 10 mM (NH 4) ₂ SO ₄ , 20 mM Tris-chloride (pH 8.75), 2 mM MgSO ₄ , 0.1% Triton X-100, 100 μg / ml. BSA), 0.25 mM of each dNTP, and 2.5 U of Pfu polymerase in a 100 μl reaction. The full length dimer product is gel purified and the two reactants including 1 and 2 and 3 and 4 products, 5 and 6 and 7 and 8 products are mixed, annealed, and over 10 cycles Elongate. The halves of the two reactions are then mixed and adjacent primers are added to perform 5 cycles of annealing and extension before amplifying the full length product. This full length product is gel purified, cloned into pCR-blunt (Invitrogen), and individual clones are screened by sequencing.

Example 23: Plasmid construct and extent to which the plasmid construct induces immunogenicity
To a degree that a plasmid construct (eg, a plasmid constructed according to the previous examples) can induce immunogenicity, transducing or transfecting APC with the epitope-expressing nucleic acid construct, followed by epitope presentation by this APC. Confirm in vitro by determining. Such studies determine “antigenicity” and allow the use of human APC. By quantifying the density of epitope-HLA class I complexes on the cell surface by this assay, the ability of the epitope presented by APC in a state recognized by T cells is determined. Quantification can be performed by directly measuring the amount of peptide eluted from the APC (see, eg, Sijts et al., J. Immunol. 156: 683-692, 1996; Demotz et al., Nature 342: 682-684, 1989). Or the number of peptide-HLA class I complexes is determined by measuring the amount of lysis or lymphokine release induced by the affected or transfected target cells, and then equal levels of lysis or lymphokine Can be assessed by determining the concentration of peptide required to obtain a release of (see, eg, Kageyama et al., J. Immunol. 154: 567-576, 1995).

  Alternatively, immunogenicity can be determined by in vivo injection into mice, followed by CTL activity and HTL activity (these activities can be determined by cytotoxicity and cell proliferation assays (eg, Alexander et al., Immunity 1: 751-761 respectively). , As described in detail in 1994).

For example, to confirm the ability of a DNA minigene construct having at least one HLA-A2 supermotif peptide to induce CTL in vivo, for example, HLA-A2.1 / ^Kb transgenic mice were treated with 100 μg of naked Immunize intramuscularly with cDNA. As a means of comparing the level of CTL induced by cDNA immunization, the control group of animals also contains multiple epitopes synthesized as a single polypeptide, since multiple epitopes are encoded by minigenes. Immunize with the peptide composition.

Spleen cells from immunized animals are stimulated twice with each composition (peptide epitope or polyepitope peptide encoded in the minigene), respectively, and then peptide specific cytotoxic activity in a ⁵¹ Cr release assay Assay for. These results indicate the magnitude of the CTL response to A2-restricted epitopes and thus the in vivo immunogenicity of minigene vaccines and polyepitope vaccines.

  Thus, it is found that the minigene elicits an immune response against the HLA-A2 supermotif peptide epitope, such as that induced by a polyepitope peptide vaccine. Similar analyzes were also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, Thereby, it is also found that this minigene elicits an appropriate immune response directed against the provided epitope.

To confirm the ability of minigenes encoding class II epitopes to induce HTL in vivo, for epitopes that cross-react with DR transgenic mice or with appropriate mouse MHC molecules, see, for example, IA ^b Restricted mice are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTL induced by DNA immunization, a group of control animals is also immunized with the actual peptide composition emulsified in Freund's complete adjuvant. CD4 + T cells (ie HTL) are purified from spleen cells of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³ H-thymidine incorporation proliferation assay (see, eg, Alexander et al., Immunity 1: 751-761, 1994). This result indicates the magnitude of the HTL response and thus demonstrates the in vivo immunogenicity of this minigene.

  A DNA minigene constructed as described in the previous example can also be identified as a vaccine in combination with a boost agent using a prime boost protocol. This boosting agent can be a recombinant vaccine (eg, Barnett et al., Aids Res. And Human Retroviruses 14, Addendum 3: S299-S309, 1998) or a recombinant vaccine that expresses a minigene or DNA encoding the complete protein of interest, for example. (See, for example, Hanke et al., Vaccine 16: 439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95: 7648-53, 1998; Hanke and McMichael, Immunol. Letters 66: 177-181, 1999; Robinson et al., Nature Med. 5: 526-34, 1999).

For example, the effectiveness of the DNA minigene used in the prime boost protocol is first evaluated in transgenic mice. In this example, A2.1 / ^Kb transgenic mice are IM immunized with 100 μg of a DNA minigene encoding an immunogenic peptide (including at least one HLA-A2 supermotif-bearing peptide). After an incubation period (ranging from 3 to 9 weeks), the mice are boosted with IP using 10 ⁷ pfu / mouse recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding this minigene but without vaccinia boost. After an additional incubation period of 2 weeks, splenocytes from the mice are assayed immediately for peptide specific activity in an ELISPOT assay. In addition, splenocytes are stimulated in vitro with A2 restricted peptide epitopes encoded in the minigene and recombinant vaccinia and then assayed for peptide specific activity in αIFN ELISA, βIFN ELISA, and / or γIFN ELISA .

  It is found that the minigene utilized in the prime boost protocol elicits a higher immune response than the DNA alone against the HLA-A2 supermotif peptide. Such analysis is also performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. obtain. The use of the prime boost protocol in humans is described in the following example entitled “Induction of CTL Response Using Prime Boost Protocol”.

Example 24: Peptide composition for prophylactic use
The vaccine composition of the present invention can be used to prevent 109P1D4 expression in humans at risk for tumors carrying this antigen. For example, a polyepitope peptide epitope composition (or a nucleic acid comprising a polyepitope peptide epitope) comprising a plurality of CTL epitopes and HTL epitopes, such as the epitopes selected in the examples above (which also comprises more than 80% of the population Is selected to be targeted) to individuals at risk for 109P1D4-related tumors.

  For example, a peptide-based composition is provided as a single polypeptide comprising multiple epitopes. Typically, the vaccine is administered in a physiological solution containing an adjuvant (eg, Freund's incomplete adjuvant). The peptide dose for the first immunization is about 1 μg to about 50,000 μg for a 70 kg patient, generally 100 μg to 5,000 μg. Following the initial administration of the vaccine, boosters are administered at 4 weeks, followed by an assessment of the magnitude of the immune response in the patient by a technique that determines the presence of an epitope-specific CTL population in the PBMC sample. Additional booster doses are administered as necessary. This composition is found to be safe and effective as a prophylactic for 109P1D4-related diseases.

  Alternatively, a composition that typically includes a transfected agent is used for the administration of nucleic acid based vaccines according to methodologies known in the art and disclosed herein.

Example 25: Polyepitope vaccine composition derived from native 109P1D4 sequence
Computers preferably defined for each class I and / or class II supermotif or motif to identify the native 109P1D4 polyprotein sequence as a “relatively short” region of a polyprotein containing multiple epitopes Analyze using algorithm. Preferably, this “relatively short” region is shorter than the full length native antigen. This relatively short sequence containing multiple distinct or overlapping “nested” epitopes can be used to create a minigene construct. This construct is engineered to express a peptide corresponding to the native protein sequence. This “relatively short” peptide is generally less than 250 amino acids long, often less than 100 amino acids long, preferably less than 75 amino acids long, and more preferably less than 50 amino acids long. Since the protein sequence of the vaccine composition has the maximum number of epitopes contained in the sequence (ie, it has a high concentration of epitopes), the protein sequence of the vaccine composition is selected. As shown herein, epitope motifs may be nested or overlapping (ie, frame shifted relative to each other). For example, using overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such vaccine compositions are administered for therapeutic or prophylactic purposes.

  Examples of the vaccine composition include a plurality of CTL epitopes derived from 109P1D4 antigen and at least one HTL epitope. The polyepitope native sequence is administered either as a peptide or as a nucleic acid sequence encoding the peptide. Alternatively, an analog can be made from its native sequence, whereby one or more epitopes contain substitutions that alter the cross-reactivity and / or binding affinity properties of the polyepitope peptide.

  The embodiment of this example applies the undiscovered aspects of immune system processing to native nested sequences, thereby producing a vaccine composition that induces a therapeutic or prophylactic immune response. Provides the possibility to make it easier. Furthermore, such embodiments provide the possibility of motif-bearing epitopes for currently unknown HLA structures. Furthermore, this embodiment (except similar embodiments) directs the immune response to multiple peptide sequences that are actually present in native 109P1D4, thereby eliminating the need to evaluate any linking epitopes . Finally, this embodiment provides economies of scale when producing peptide vaccine compositions or nucleic acid vaccine compositions.

  For this embodiment, computer programs are available in the art that can be used to identify the largest number of epitopes per sequence length in the target sequence.

Example 26: Polyepitope vaccine composition derived from multiple antigens
The 109P1D4 peptide epitopes of the present invention are used in combination with epitopes derived from other target tumor associated antigens to create vaccine compositions useful for the prevention and treatment of 109P1D4 antigen and cancers expressing such other antigens. . For example, the vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 109P1D4 and a tumor-associated antigen that is often expressed in a target cancer associated with 109P1D4 expression, or one or more separate Can be administered as a composition comprising a cocktail of these epitopes. Alternatively, the vaccine can be administered as a minigene construct or as a dendritic cell loaded with peptide epitopes in vitro.

Example 27: Use of peptides to assess immune response
The peptides of the invention can be used to analyze the immune response for the presence of specific antibodies, CTL or HTL to 109P1D4. Such analysis can be performed in the manner described in Ogg et al., Science 279: 2103-2106, 1998. In this example, the peptide according to the invention is used as a reagent (not as an immunogen) for diagnostic or prophylactic purposes.

In this example, a highly sensitive human leukocyte antigen tetramer complex (“tetramer”), eg, a post-immunization HLA A ^* 0201 comprising a 109P1D4 peptide containing a different stage of disease or an A ^* 0201 motif. Used for cross-sectional analysis of 109P1D4 HLA-A ^* 0201-specific CTL frequencies from positive individuals Tetrameric complexes are described (Musey et al., N. Engl. J. Med. 337: 1267, 1997). Briefly, purified HLA heavy chain (A ^* 0201 in this example) and β2-microglobulin are synthesized by a prokaryotic expression system, which is synthesized in a transmembrane-cytosolic tail. Deletion and modification by COOH terminal addition of the sequence containing the BirA enzyme biotinylation site, heavy chain, β2-microglobulin, The 45 kD refold product is isolated by high performance protein liquid chromatography and then in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5 ′ triphosphate and magnesium. Biotinylated with Bir A. Streptavidin-phycoerythrin conjugate is added at a molar ratio of 1: 4 and the tetramer product is concentrated to 1 mg / ml, and the resulting product is referred to as tetramer-fecoerythrin.

For analysis of patient blood samples, approximately 1 million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate buffered saline. Three-color analysis is performed using tetramer-phycoerythrin with anti-CD8-tricolor, and anti-CD38. PBMC are incubated with tetramer and antibody on ice for 30-60 minutes and then washed twice prior to formaldehyde fixation. Apply a gate that contains more than 99.98% control samples. Controls for the tetramer include both A ^* 0201-negative individuals and A ^* 0201-positive unaffected donors. The percentage of cells stained with this tetramer is then determined by flow cytometry. This result indicates the number of epitope-restricted CTL-containing cells in the PBMC sample, thereby the extent of the immune response to the 109P1D4 epitope, and thus the state of exposure to 109P1D4, or a vaccine that elicits a prophylactic or therapeutic response Easily indicate the state of exposure to

Example 28: Use of peptide epitopes to assess recall responses
The peptide epitopes of the invention are used as reagents for evaluating T cell responses (eg, acute or recall responses) in patients. Such an analysis may be performed in patients who have recovered from 109P1D4-related disease or who have been vaccinated with 109P1D4 vaccine.

  For example, vaccinated human class I restricted CTL responses can be analyzed. This vaccine may be any 109P1D4 vaccine. PBMC are collected from the vaccinated individuals and HLA typed. Then, an appropriate peptide epitope of the present invention, which optionally possesses a supermotif for providing cross-reactivity to a plurality of HLA supertype family members, is analyzed for samples derived from individuals possessing that HLA type. Used for.

  PBMCs from vaccinated individuals were separated with Ficoll-Histopaque density gradient (Sigma Chemical Co., St. Louis, Mo.), washed 3 times in HBSS (GIBCO Laboratories) and heat inactivated 10% human Resuspended in RPMI-1640 (GIBCO Laboratories) (complete RPMI) supplemented with L-glutamine (2 mM), penicillin (50 U / ml), streptomycin (50 μg / ml) and Hepes (10 mM) with AB serum Plate using culture format. Synthetic peptides containing the epitopes of the invention are added to each well at 10 μg / ml and the HBV core 128-140 epitope is added to each well as a source of T cell help during the first week of stimulation. Add 1 μg / ml.

In a microculture format, 4 × 10 ⁵ PBMCs are stimulated with peptides in 8 replicate cultures in 100 μl / well complete RPMI 96 well round bottom plates. On days 3 and 10, 100 μl complete RPMI and 20 U / ml final concentration of rIL-2 are added to each well. On day 7, the culture was transferred to a flat bottom 96-well plates and peptide are restimulated with self feeder cells irradiated (3,000 rad) of rIL-2 and 10 ^5. The culture is tested for cytotoxic activity on day 14. A positive CTL response was determined as previously described (Rehermann et al., Nature Med. 2: 1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97: 1655-1665, 1996; and Rehermann et al., J. Biol. Clin. Invest. 98: 1432-1440, 1996) Based on a comparison with unaffected control subjects, two or more of eight replicate cultures show more than 10% specific ⁵¹ Cr release. I need.

  The target cell line is autologous and allogeneic EBV-transformed B-LCL, both of which are purchased or described from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) (Guihot et al., J. Virol. 66: 2670-2678, 1992) either from a pool of patients.

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-compatible B lymphoblast cell lines or autologous EBV transformed B lymphoblast cell lines, which are overnight with 10 μM of the synthetic peptide epitope of the present invention. Incubate and label with 100 μCi of ⁵¹ Cr (Amersham Corp., Arlington Heights, IL) for 1 hour, after which they are washed 4 times with HBSS.

Cytotoxic activity is determined in a standard 4-hour, separate well ⁵¹ Cr release assay using a U-bottom 96-well plate containing 3,000 targets / well. Stimulated PBMC are tested on day 14 at an effector / target (E / T) ratio of 20-50: 1. The% cytotoxicity is determined from the following formula: 100 × [(experimental release−spontaneous release) / (maximum release−spontaneous release)]. Maximum release is determined by lysis of the target with surfactant (2% Triton X-100; Sigma Chemical Co., St. Louis, MO). Spontaneous release is less than 25% of maximum release for all experiments.

  The results of such an analysis indicate the extent to which the HLA-restricted CTL population was stimulated by previous exposure to 109P1D4 or 109P1D4 vaccine.

Similarly, class II restricted HTL responses can also be analyzed. Purified PBMC are cultured in 96-well flat bottom plates at a density of 1.5 × 10 ⁵ cells / well and stimulated with 10 μg / ml of the synthetic peptide of the invention, 109P1D4 whole antigen, or PHA. Cells are routinely plated as 4-6 well replicas for each condition. After 7 days of culture, the medium is removed and replaced with fresh medium containing 10 U / ml IL-2. Two days later, 1 μCi of ³ H thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then collected on a glass fiber mat and analyzed for ³ H thymidine incorporation. Antigen-specific T cell proliferation, to calculate the ^{3 H-thymidine} incorporation in the presence of an antigen as divided by the ratio with ^{3 H-thymidine} incorporation in the absence of antigen.

Example 29: Induction of specific CTL responses in humans
Human clinical trials for immunogenic compositions comprising CTL and HTL epitopes of the present invention are set up as an IND Phase I, dose escalation study and conducted as a randomized, double-blind placebo controlled trial. Such a test is designed, for example, as follows:
A total of about 27 individuals are enrolled and divided into three groups;
Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of the peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg of the peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of the peptide composition.

  Four weeks after the first injection, all subjects were boosted at the same dose.

  The endpoint measured in this study is related to the safety and tolerability of the peptide composition and its immunogenicity. The cellular immune response to the peptide composition is an indicator of the intrinsic activity of the peptide composition and can therefore be seen as a measure of biological effectiveness. The following summarizes clinical and laboratory data regarding safety and efficacy endpoints.

  Safety: The incidence of adverse events is monitored in the placebo and drug treatment groups and evaluated with their extent and reversibility.

  Assessment of vaccine efficacy: Blood is drawn from the subject before and after injection for assessment of vaccine efficacy. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted into freezing media and stored frozen. Samples are assayed for CTL activity and HTL activity.

  Vaccines are found to be safe and effective.

Example 30: Phase II study in patients expressing 109P1D4
A phase II study is conducted to study the effect of administration of the CTL-HTL peptide composition to patients with cancer that express 109P1D4. The primary objective of this study is to determine the effective dose and regimen to induce CTL in cancer patients expressing 109P1D4, and to establish the safety of inducing CTL and HTL responses in these patients And how much CTL activation improves the clinical picture of these patients (eg, as evidenced by lesion reduction and / or atrophy). Such a study is designed as follows, for example.

  This study will be conducted at multiple centers. The study design is an open-label, uncontrolled dose escalation protocol, where the peptide composition is administered as a single dose followed by a single booster injection of the same dose 6 weeks later . This dose is 50 μg, 500 μg, and 5,000 μg per injection. Record adverse drug effects (severity and reversibility).

  There are three patient groups. The first group is injected with 50 μg of this peptide composition, and the second and third groups are injected with 500 μg and 5,000 μg of peptide composition, respectively. Patients within each group range from 21 years to 65 years and show different racial population backgrounds. All of them have tumors that express 109P1D4.

  Clinical expression or antigen-specific T cell responses are monitored to evaluate the effects of administering this peptide composition. This vaccine composition is found to be safe and effective in the treatment of 109P1D4-related diseases.

Example 31 Induction of CTL Response Using Prime Boost Protocol
Underlying the protocol used to confirm the efficacy of DNA vaccines in transgenic mice, as described above in the example entitled "Plasmid construct and the extent to which the plasmid construct induces immunogenicity" Prime boost protocols with similar principles can also be used to administer vaccines to humans. Such a vaccine regimen can include, for example, an initial administration of naked DNA followed by a boost using a recombinant virus encoding the vaccine or a recombinant protein / polypeptide or peptide mixture administered in an adjuvant. .

For example, the first immunization is administered with an expression vector (eg, IM (or SC or ID) in an amount of 0.5 mg to 5 mg at multiple sites in the example entitled “Gene” Multi-Epitope DNA Plasmid Construction ”. Expression vector as constructed in the form of a naked nucleic acid). This nucleic acid (0.1 μg to 1000 μg) can also be administered using a gene gun. After a 3-4 week incubation period, a booster dose is then administered. The booster can be a recombinant fowlpox virus administered at a dose of 5 × 10 ⁷ pfu to 5 × 10 ⁹ pfu. Alternative recombinant viruses (eg, MVA virus, canarypox virus, adenovirus, or adeno-associated virus) can also be used for boosters, or polyepitope proteins or mixtures of these peptides can be administered. For assessment of vaccine efficacy, patient blood samples are obtained at intervals before immunization and after administration of the first vaccine and vaccine booster dose. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted into frozen media and stored frozen. Samples are assayed for CTL activity and HTL activity.

  Analysis of these results indicates that a response is generated that is strong enough to achieve therapeutic or protective immunity to 109P1D4.

(Example 32: Administration of vaccine composition using dendritic cells (DC))
Vaccines comprising the peptide epitopes of the invention may be administered using APC or “professional” APC (eg, DC). In this example, peptide-pulsed DC is administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising a peptide CTL epitope and a peptide HTL epitope of the present invention. These dendritic cells are injected back into the patient to elicit CTL and HLT responses in vivo. The induced CTL and HTL then destroy or promote the destruction of the target cells carrying the 109P1D4 protein from which the epitope in this vaccine is derived, respectively.

For example, a cocktail of peptides containing an epitope is administered ex vivo to PBMC or DC isolated therefrom. Medicaments (eg, Progenipoietin ^™ (Monsanto, St. Louis, MO) or GM-CSF / IL-4) to facilitate DC recovery may be used. After pulsing the DC with the peptide and prior to reinfusion into the patient, the DC is washed to remove unbound peptide.

The number of DCs reinjected into the patient can vary (eg, Nature Med. 4: 328, 1998; Nature, as clinically understood and readily determined by those skilled in the art based on clinical results). Med. 2:52, 1996 and Prostate 32: 272, 1997). Typically 2-50 × 10 ⁶ DCs are administered per patient, although higher numbers of DCs (eg, 10 ⁷ or 10 ⁸ ) may also be provided. Such cell populations typically contain between 50% and 90% DC.

In some embodiments, peptide loaded PBMCs are injected into a patient without DC purification. For example, PBMC produced after treatment with a drug (eg, Progenipoietin ^™ ) is injected into a patient without purifying the DC. The total number of PBMCs administered often ranges from 10 ⁸ to 10 ¹⁰ . In general, the cell dose injected into a patient is based on the percentage of DC in each patient's blood, as determined, for example, by immunofluorescence analysis using specific anti-DC antibodies. Thus, for example, if Progenipoietin ^™ mobilizes 2% DC in the peripheral blood of a given patient and that patient is receiving 5 × 10 ⁶ DC, this patient will receive a total of 2.5 × 10 ⁸ Inject PBMC loaded with peptide. The DC% mobilized by an agent (eg, Progenipoietin ^™ ) is typically estimated to be between 2-10%, but can vary as will be appreciated by those skilled in the art.

(Activation of CTL / HTL response ex vivo)
Alternatively, an ex vivo CTL response or HTL response to the 109P1D4 antigen, together with a source of APC (eg, DC) and an immunogenic peptide, is combined with a patient or genetically compatible CTL precursor cell or HTL precursor cell. Can be induced by incubation in tissue culture. After an appropriate incubation time (typically about 7-28 days), the progenitor cells are activated and expanded into effector cells. The effector cells are injected into the patient and destroy (CTL) or promote destruction (HTL) of specific target cells (ie, tumor cells).

(Example 33: Alternative method for identification and confirmation of motif-bearing peptide)
Another way to identify and confirm a motif-bearing peptide is to elute the peptide from cells that carry a defined MHC molecule. For example, EBV-transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases, these cells express only a single type of HLA molecule. These cells can be transfected with a nucleic acid that expresses the antigen of interest (eg, 109P1D4). The peptides produced by endogenous antigen processing of the peptides produced as a result of transfection then bind to intracellular HLA molecules and are transported and presented on the cell surface. The peptides are then eluted from the HLA molecules by exposure to mild acidic conditions, and their amino acid sequences are determined, for example, by mass spectrometry (eg, Kubo et al., J. Immunol. 152: 3913, 1994). Since most of the peptides that bind to a particular HLA molecule carry a motif, this is an alternative way to obtain a motif-bearing peptide associated with a particular HLA molecule expressed on a cell.

  Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct that encodes a single HLA allele. These cells can then be used as described (ie, these cells are transduced with a nucleic acid encoding 109P1D4 in order to isolate the peptide corresponding to 109P1D4 displayed on the cell surface. Can be effected). Peptides obtained from such analyzes possess a motif that corresponds to binding to a single HLA allele expressed in cells.

  As will be appreciated by those skilled in the art, a similar analysis can be performed in cells carrying more than one HLA allele and subsequent specific peptides can be determined for each expressed HLA allele. . Furthermore, those skilled in the art will also recognize that means other than transfection, such as loading with protein antigens, can be used to provide a source of antigen to the cells.

(Example 34: Complementary polynucleotide)
A sequence complementary to the 109P1D4 coding sequence or any portion thereof is used to detect, reduce or inhibit the expression of naturally occurring 109P1D4. Although the use of oligonucleotides comprising about 15-30 base pairs is described, substantially the same means are used with smaller or larger sequence fragments. Appropriate oligonucleotides are designed using, for example, OLIGO 4.06 software (National Biosciences) and 109P1D4 coding sequence. In order to inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and the complementary oligonucleotide is used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosome binding to the transcript encoding 109P1D4.

Example 35: Purification of naturally occurring 109P1D4 or recombinant 109P1D4 using 109P1D4 specific antibodies
Naturally occurring 109P1D4 or recombinant 109P1D4 is substantially purified by immunoaffinity chromatography using an antibody specific for 109P1D4. An immunoaffinity column is constructed by covalently attaching an anti-109P1D4 antibody to an activated chromatography resin (eg, CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech)). After this bonding, the resin is blocked and washed according to the manufacturer's instructions.

  The medium containing 109P1D4 is passed through an immunoaffinity column and the column is washed under conditions that preferentially adsorb 109P1D4 (eg, a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that interfere with antibody / 109P1D4 binding (eg, pH 2 to pH 3 buffer, or high concentration of chaotrope (eg, urea or thiocyanate ions)) and GCR. Collect P.

Example 36: Identification of molecules that interact with 109P1D4
109P1D4 or a biologically active fragment thereof is labeled with 121 1 Bolton-Hunter reagent. (See, eg, Bolton et al. (1973) Biochem. J. 133: 529.) Candidate molecules pre-ordered in the wells of a multi-well plate are incubated with labeled 109P1D4, washed, and labeled Assay any wells containing the 109P1D4 complex. Data obtained using different concentrations of 109P1D4 are used to calculate the number, affinity, and association values of 109P1D4 with candidate molecules.

Example 37: In vivo assay for promoting 109P1D4 tumor growth
The effect of 109P1D4 protein on tumor cell growth is assessed in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously into each flank with either tkNeo empty vector or 1 × 10 ⁶ PC3 cells, DU145 cells, or 3T3 cell lines containing the nucleic acid sequences of the present invention. At least two strategies may be used: (1) polyomavirus, fowlpox virus (UK 2,211,504 (published July 5, 1989)), adenovirus (eg, adenovirus 2), bovine Constitutive promoters derived from the genomes of viruses such as papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), or heterologous mammalian promoters (eg, actin promoter or immunity) Constitutive expression under the control of a promoter, such as a constitutive promoter obtained from a globulin promoter), provided that such promoter is compatible with the host cell system, and (2) an inducible vector system (eg, Under the control of ecdysone, tet, etc.) Regulated expression, provided that such promoters are compatible with the host cell system. Tumor volume is then monitored for the appearance of obvious tumors and followed over time to see if cells expressing the gene of the invention proliferate at a faster rate and of 109P1D4 protein expressing cells. It is determined whether the tumor exhibits characteristics of altered aggressiveness (eg, enhanced metastasis, angiogenesis, decreased responsiveness to chemotherapeutic drugs).

In addition, mice can be transplanted with 1 × 10 ⁵ identical cells at the same location, and the protein of the present invention is capable of local proliferation or cell metastasis in prostate cancer (particularly in lung, lymph nodes, and bone marrow). Determine whether it has an effect on the ability to metastasize.

  This assay is also useful for determining the inhibitory effects of candidate therapeutic compositions (eg, 109P1D4 protein-related intracellularly expressed antibodies, 109P1D4 gene-related antisense molecules and ribozymes).

Example 38: 109P1D4 monoclonal antibody-mediated inhibition of tumors in vivo
The marked expression of 109P1D4 protein in cancer tissues in Table I and the restricted expression in normal tissues, as well as the predicted cell surface expression of 109P1D4 protein, make 109P1D4 protein an excellent target for antibody therapy. Similarly, 109P1D4 protein is a target for T cell-based immunotherapy. Thus, for example, for the 109P1D4 gene expressed in prostate cancer, the therapeutic efficacy of anti-109P1D4 protein mAb in a human prostate cancer xenograft mouse model has been demonstrated to be androgen-independent LAPC-4 and LAPC-9 xenografts ( Craft, N., et al., Cancer Res, 1999.59 (19): p. 5036-6) and 109P1D4 androgen-independent recombinant cell line PC-3 (e.g., Kaighn, ME, et al., Invest Urol, 1979.17 (1): see pages 16-23); similar models are used for other cancers.

  The efficacy of antibodies against tumor growth and metastasis formation is studied, for example, in mouse orthotopic prostate cancer xenograft models and mouse kidney xenograft models. The antibody can be unconjugated, as discussed in this example, or can be conjugated to a therapeutic modality as is understood in the art. The anti-109P1D4 protein mAb inhibits the formation of both androgen-dependent LAPC-9 and androgen-independent PC3-109P1D4 protein tumor xenografts. Anti-109P1D4 protein mAb also delays the growth of established orthotopic tumors and prolongs survival of tumor-bearing mice. These results demonstrate the utility of anti-109P1D4 protein mAb in the treatment of local prostate cancer and advanced stage prostate cancer (eg (Saffran, D. et al., PNAS 10: 1073-1078 or World Wide Web URL www.pnas.org/cgi/doi.10.1073/pnas.051624698).

  Administration of anti-109P1D4 protein mAb leads to established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant increase in survival of tumor-bearing mice. These studies show that the proteins of the invention are attractive targets for immunotherapy and the therapeutic potential of 109P1D4 protein mAb as a treatment for local and metastatic cancers. This example shows that non-conjugated 109P1D4 protein-related monoclonal antibodies are effective in inhibiting the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice; It is also effective.

(Tumor suppression using multiple unbound mAbs)
(Materials and methods)
(109P1D4 monoclonal antibody):
Monoclonal antibodies are raised against the proteins of the invention as described in the example of the title “Generation of 109P1D4 Monoclonal Antibodies”. This antibody is characterized for their ability to bind to the respective protein of the invention by ELISA, Western blot, FACS, and immunoprecipitation. Epitope mapping data (eg, epitope mapping data for anti-109P1D4 mAb as measured by ELISA and Western analysis) indicates that this antibody recognizes an epitope on each 109P1D4 protein. Immunohistochemical analysis of prostate cancer tissue and prostate cells is performed using these antibodies.

  The monoclonal antibody is purified from ascites or hybridoma tissue culture supernatant by Protein G Sepharose chromatography, dialysis against PBS, filter sterilization, and storage at -20 ° C. Protein measurements are performed by Braford assay (Bio-Rad, Hercules, CA). A cocktail containing a therapeutic monoclonal antibody, or a mixture of individual monoclonal antibodies, is prepared and used for the treatment of LAPC-9 prostate tumor xenografts subcutaneously or orthotopically injected mice.

(Cancerous xenografts and cancer cell lines)
LAPC-9 xenografts express wild-type androgen receptor and produce prostate specific antigen (PSA). The graft is cultured in 6-8 week old severe combined immunodeficient (SCID) mice (Tacnic Farms) with subcutaneous trocar implants (Craft, N et al., Supra). This prostate carcinoma cell line PC3 (Amrican Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS.

  Recombinant PC3 cells and recombinant 3T3 cell populations expressing the proteins of the present invention were obtained from Hubert, R .; S. STEAP: a prosthetic-specific cell-surface antigen highly expressed in human prosthetic tutors. Proc. Natl. Acad. Sci. USA, 1999.96 (25) p. Produced by retroviral gene transfer as described in 1425-8. The anti-protein staining of the present invention is detected using FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates), followed by Coulter Epis-XL flow cytometry.

(Xenograft mouse model)
Subcutaneous (sc) tumors are generated by injecting 1 × 10 ⁶ cancer cells mixed with Matrigel (Collaborative Research) at a 1: 1 dilution into the right flank of male SCID mice. To test antibody efficacy in tumorigenesis, i. p. Antibody injection begins on the same day as tumor cell injection. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen that is not expressed in human cells. Tumor size is determined by caliper measurements, and tumor volume is calculated from length x width x height. S. Of diameter greater than 1.5 cm. c. Mice with tumors are sacrificed. PSA levels were determined by using the PSA ELISA kit (Anogen, Mississauga, Ontario). For example, circulating levels of anti-109P1D4 protein mAbs were determined by a capture ELISA (Bethyl Laboratories, Montgomery, TX) (eg, Saffran, D. PNAS 10: 1073-1078 or www.pnas.org/cgi/doi/10. 1073 / pnas.0551624698).

Orthotopic injections are performed under anesthesia by using ketamine / xylazine. For prostate orthotopic studies, an incision is made through the abdomen to expose the bladder and seminal vesicles, which are removed through the incised site to expose the ventral prostate. LAPC-9 cells or PC3 cells (5 × 10 ⁵ ) were mixed with Matrigel and injected into the ventral leaves in a volume of 10 μl. In order to monitor tumor growth, blood is drawn from mice every other week to measure PTA levels. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, and an incision is delivered to expose the back of the prostate. Tumor cells such as LAPC-9 cells (5 × 10 ⁵ ) mixed with Matrigel are injected into the prostate in a volume of 10 μl (Yoshida Y et al., Anticancer Res. 1998, 18: 327; Ahn et al., Tumor Biol. 2001, 22: 146). To monitor tumor growth, mice are bled every other week to measure PSA levels. These mice are separated into groups for appropriate treatment and anti-protein or control mAbs are injected intraperitoneally.

(Anti-109P1D4 protein mAb inhibits growth of representative 109P1D4 protein-expressing xenograft cancer tumors)
The effect of anti-109P1D4 mAb on tumor formation is tested using the orthotopic LAPC-9 and PC3 proteins of the present invention. Compared to the subcutaneous tumor model, these orthotopic models require injection of tumor cells directly into the prosthesis or kidney of each mouse, and local tumor growth, metastasis to distant sites Development, deterioration of mouse health, and subsequent death (Saffran, D. et al., PNAS (supra); Fu, X. et al., Int J Cancer, 1992.52 (6): p. 987-90; Kubota, T., J Cell Biochem, 194.56 (1): p.4-8). This feature allows the orthotopic model to be a more representative model of human disease progression and to follow the therapeutic effects of mAbs at clinically relevant endpoints.

  Therefore, tumor cells are injected into the organs of mice and after 2 days the mice are divided into two groups and treated with either: a) 200-500 μg anti-109P1D4 Ab, or b) PBS (2 3 times a week for 5 weeks).

  A major advantage of the orthotopic prostate cancer model is the ability to study the occurrence of metastases. Metastasis formation in mice bearing established orthotopic tumors, IHC analysis in lung sections using antibodies against prostate-specific cell surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, RS et al. Proc. Natl. Acad. Sci. USA, 1999, 96 (25): p14523-8).

  Mice bearing established orthotopic LAPC-9 or recombinant PC3-1091D4 protein tumors are administered 1000 μg injections of either anti-109P1D4 mAb or PBS for 4 weeks. Both groups of mice can establish high systemic tumor mass (PSA levels greater than 300 ng / ml for IAPC-9), ensuring that metastasis formation in the lungs of mice is frequent. The mice are then sacrificed and their prostate and lungs are analyzed for the presence of tumor cells by IHC analysis.

  These studies show the broad anti-tumor efficacy of anti-109P1D4 antibodies against prostate cancer development and progression in a xenograft mouse model. Anti-109P1D4 protein antibodies inhibit tumor formation, delay the growth of already established tumors and prolong the survival of treated mice. Furthermore, anti-109P1D4 mAb shows a dramatic inhibitory effect on the spread of local bladder and prostate tumors to distal sites, even in the presence of large systemic tumor tissue mass. Thus, anti-109P1D4 mAb is effective against major clinically relevant endpoints (tumor growth), prolonged survival and health.

Example 39: Therapeutic and diagnostic uses of anti-109P1D4 antibodies in humans
Anti-109P1D4 monoclonal antibodies are safe and are effectively used for diagnostic, prophylactic, prognostic and / or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts using anti-109P1D4 mAb shows a strong and extensive coloration in cancer, but significantly lower or undetectable in normal tissues Show the level. Detection of 109P1D4 in cancer and metastatic disease demonstrates the usefulness of mAbs as diagnostic and / or prognostic indicators. Anti-109P1D4 antibodies are therefore used in diagnostic applications (eg, immunohistological methods of renal biological specimens to detect cancer from suspect patients).

  As measured by flow cytometry, anti-109P1D4 mAb specifically binds to cancer cells. Accordingly, anti-109P1D4 antibodies are used in diagnostic whole body imaging applications (eg, radioimmunoscintigraphy and radioimmunotherapy) for the detection of local cancers and metastases that exhibit 109P1D4 expression (eg, Potamianos S). Et al., Anticancer Res 20 (2A): 925-948 (2000)). The divergence or release of the extracellular domain of 109P1D4 into the extracellular environment (e.g. found in alkaline phosphatase B10 (Meerson, N, R, Hepatology 27: 563-568 (1998)) is associated with serum from suspected patients and / or Enables diagnostic detection of 109P1D4 with anti-109P1D4 antibody in urine samples.

  Anti-109P1D4 antibodies that specifically bind 109P1D4 are used in therapeutic applications for the treatment of cancers that express 109P1D4. Anti-109P1D4 antibodies are in a non-conjugated manner and in a conjugated form in which the antibody is conjugated to one of various therapeutic or imaging modalities well known in the art (eg, prodrugs, enzymes or radioisotopes) used. In preclinical studies, unbound and bound anti-109P1D4 antibodies are tested for efficacy in tumor prevention and growth inhibition in SCID mouse cancer xenograft models (eg, kidney cancer models AGS-K3 and AGS-K6) ( See, for example, the example entitled “109P1D4 Monoclonal Anti-Body Mediated of Blader and Lung Tumors In Vivo”). Either conjugated anti-109P1D4 antibody or unconjugated anti-109P1D4 antibody is used either as a therapeutic modality in human clinical trials, either alone or in combination with other therapies as described in the examples below.

Example 40: Human clinical trial for the treatment and diagnosis of human carcinomas through the use of human anti-109P1D4 antibody in vivo
An antibody that recognizes an epitope on 109P1D4 is used in accordance with the present invention, and this antibody is used in the treatment of specific tumors as listed in Table 1. Based on a number of factors (including 109P1D4 expression levels), tumors as listed in Table 1 are currently preferred indicators. In connection with each of these indicators, three clinical approaches are successfully implemented.

  I. ) Adjunctive therapy: In adjunct therapy, patients are treated with anti-109P1D4 antibodies in combination with chemotherapeutic or anti-neoplastic agents and / or radiation therapy. Primary cancer targets as listed in Table I are treated under standard protocols by adding anti-109P1D4 antibody to the standard therapy of the first and second systems. The protocol design addresses the effectiveness as assessed by tumor mass reduction and the ability to reduce normal doses of standard chemotherapy. These dosage reductions allow for further and / or prolonged treatment by reducing the dose-related toxicity of the chemotherapeutic agent. Anti-109P1D4 antibodies are chemotherapeutic or anti-neoplastic agents adriamycin (advanced prostate carcinoma), cisplatin (advanced head and neck carcinoma, and lung carcinoma), taxol (breast cancer) and doxorubicin ( Used in some ancillary clinical trials in combination with (preclinical).

  II. Monotherapy: In connection with the use of anti-109P1D4 antibodies in tumor monotherapy, these antibodies are administered to patients without chemotherapeutic or anti-neoplastic agents. In one embodiment, monotherapy is performed clinically in end-stage cancer patients with a wide range of metastatic disease. Patients show some disease stabilization. The study demonstrates efficacy in treatment-resistant patients with cancerous tumors.

III. ) Contrast agent: Radiolabeled antibodies are utilized as diagnostic and / or contrast agents by binding radionuclides (eg iodine or yttrium (I ¹³¹ , Y ⁹⁰ )) to anti-109P1D4 antibodies. In such a role, the labeled antibody is localized in both solid tumors as well as metastatic lesions of 109P1D4-expressing cells. In connection with the use of anti-109P1D4 antibodies as contrast agents, these antibodies are useful for solid tumor surgical as both pre-operative screening and post-operative follow-up to determine which tumors remain and / or revert. Used as a treatment aid. In one ^embodiment, a (111 In) 109P1D4 antibodies in patients with carcinomas expressing 109P1D4, as used to (analogy as contrast agent in a Phase I human clinical trials, e.g., Divgi et al., J. Natl. Cancer Inst. 83: 97-104 (1991)). The patient is tracked with a standard front and rear gamma camera. These results indicate that primary and metastatic lesions are identified.

(Dose and route of administration)
As will be appreciated by those skilled in the art, administration considerations can be determined relative to similar products present in the clinic. Thus, anti-109P1D4 antibody can be administered at dosages ranging from 5 to 400 mg / m ² , eg, used at lower dosages for safety testing. The affinity of an anti-109P1D4 antibody compared to the affinity of a known antibody for its target is one parameter used by those skilled in the art to determine a similar dosage regimen. Further, anti-109P1D4 antibodies that are fully human antibodies have slower clearance when compared to chimeric antibodies; therefore, dosing in patients using such anti-109P1D4 antibodies that are fully human antibodies is likely In the range of 50-300 mg / m ² it can be lower and still remain effective. Contrary to conventional volume measurements in mg / kg, dosing in mg / m ² is a surface area based measurement and convenient dosing designed to include patients of all sizes from infants to adults It is a measurement.

  Three separate delivery approaches are useful for delivery of anti-109P1D4 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, for intraperitoneal tumors (eg, tumors of the ovary, bile duct, other ducts, etc.) intraperitoneal administration is advantageous to obtain high doses of antibody in the tumor and also to minimize antibody clearance You can prove that. In a similar manner, certain solid tumors have vasculature that is appropriate for local perfusion. Local perfusion allows high doses of antibody at the tumor site and minimizes short term clearance of the antibody.

(Clinical Development Plan (CDP))
Overview: CDP follows and develops treatment of anti-109P1D4 antibodies for adjuvant therapy, monotherapy and as a contrast agent. The study will first demonstrate safety and then confirm efficacy with repeated dosing. The study is open label comparing standard chemotherapy with standard therapy plus anti-109P1D4 antibody. As will be appreciated, one criterion that can be utilized for patient enrollment is the expression level of 109P1D4 in the patient's tumor, as determined by biopsy.

  As with any protein or antibody injection-based therapy, safety considerations are primarily related to: (i) cytokine release syndrome (ie, hypotension, fever, shaking, chills) (Ii) the generation of an immune response to the substance (ie, the patient, the generation of a human antibody to antibody therapy, or the HAHA response); and (iii) toxicity to normal cells expressing 109P1D4. Each of these safety considerations is utilized to monitor: Anti-109P1D4 antibodies are found to be safe upon human administration.

Example 41 Human Clinical Trial Adjuvant Treatment Using Human Anti-109P1D4 Antibody and Chemotherapeutic Agent
Phase I human clinical trials will be initiated to evaluate the safety of six intravenous doses of human anti-109P1D4 antibody for the treatment of solid tumors (eg, cancer of tissues listed in Table I). In this study, it is used as an adjunct treatment for antineoplastic or chemotherapeutic agents as defined herein (eg, but not limited to cisplatin, topotecan, doxorubicin, adriamycin, taxol, etc.) If so, the safety of a single dose of anti-109P1D4 antibody is evaluated. Test design includes delivery of six single doses of an anti-109P1D4 antibody, the dosage of the antibody over a course of treatment according to the following schedule, increases to approximately about 25 mg / ^{m 2} ~ about 275 mg / ^{m 2:}

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275
mg / m ² mg / m ² mg / m ² mg / m ² mg / m ² mg / m ²
Chemotherapy + + + + + +
(Standard administration)
Patients are followed closely for one week after each administration of antibody and chemotherapeutic agent. In particular, the above safety issues: (i) cytokine release syndrome (ie, hypotension, fever, tremor, chills); (ii) occurrence of immunogenic response to the substance (ie, against human antibody therapeutics, The patient is evaluated for the generation of human antibodies by the patient, or HAHA response); and (iii) toxicity to normal cells expressing 109P1D4. Standard and follow-up tests are utilized to monitor each of these safety issues. Patients are also evaluated for clinical outcome and in particular for tumor mass reduction as evidenced by MRI or other imaging.

  This anti-109P1D4 antibody has proven to be safe and effective, and phase II clinical trials confirm this efficacy and make the optimal dosing more accurate.

(Example 42: Human clinical trial: Monotherapy using human anti-109P1D4 antibody)
Anti-109P1D4 antibodies are safe with respect to the adjunct studies discussed above, and phase II human clinical trials confirm efficacy and optimal dosing for monotherapy. Such a test is achieved and encompasses the same safety and analysis of results as the adjunct test described above, except that the patient receives a dose of anti-109P1D4 antibody and does not receive chemotherapy at the same time.

Example 43 Human Clinical Trial: Diagnostic Imaging Using Anti-109P1D4 Antibody
Again, if the adjuvant therapy discussed above is safe within the safety criteria discussed above, human clinical trials will be conducted regarding the use of anti-109P1D4 antibody as a diagnostic contrast agent. This protocol is designed in a manner substantially similar to that described in the art (eg, Divgi et al., J. Natl. Cancer Inst. 83: 97-104 (1991)). This antibody is found to be both safe and effective when used as a diagnostic modality.

Example 44: 109P1D4 Functional Assay
(I. Phosphorylation of 109P1D4 on tyrosine residues)
One characteristic of the cancer cell phenotype is that active signaling of surface-bound receptor molecules (eg, EGF receptors) via tyrosine phosphorylation of their cytoplasmic domains and subsequent interaction with cytoplasmic signaling molecules. It is. In order to investigate the possibility that 109P1D4 is phosphorylated at its cytoplasmic tyrosine residue, 293T cells were transfected with the 109P1D4 gene in an expression plasmid, whereby 109P1D4 was fused with a Myc / His tag and then Stimulated with pervanadate (a 1: 1 mixture of Na ₃ VO ₄ and H ₂ O ₂ ). After solubilizing these cells with Triton X-100, 109P1D4 protein was immunoprecipitated with anti-His polyclonal antibody (pAb), subjected to SDS-PAGE, and Western blotted with anti-phosphotyrosine. Equivalent immunoprecipitates were Western blotted with anti-His antibody. In FIG. 22, 109P1D4 shows tyrosine phosphorylation only upon cell treatment with pervanadate and not without treatment. This suggests that pervanadate, which indicates intracellular protein tyrosine phosphorylation (PTP), allows phosphotyrosine accumulation at 109P1D4 (tyrosine kinase activity). In addition, large amounts of 109P1D4 protein are trapped in the insoluble fraction upon pervanadate activation. This suggests the association of this protein with cytoskeletal compounds. Similar effects of partial insolubility in Triton X-100 were observed for cadherins (proteins associated with protocadherins based on the homology of these extracellular domains). Cadherins are known to interact with cytoskeletal proteins (including actin) that are not readily soluble in the wash conditions used in this study. Together, these data indicate that 109P1D4 can be phosphorylated in tyrosine and a signaling molecule (phospholipase-Cγ1, Brb2, Shc, Crk, PI-3-kinase that possesses an SH2 binding domain or a PTB binding domain. p85 subunit, rasGAP, Sro family kinase and abi family kinase, including but not limited to surface receptors. Such interactions are important for downstream signaling through 109P1D4, resulting in altered adhesion, proliferation, migration or finishing of secreted factors. Furthermore, the 109P1D4 protein interacts with cytoskeletal components (eg actin) that facilitate its cell adhesion function. These phenotypes are enhanced in 109P1D4-expressing tumor cells and contribute to increasing their ability to metastasize and proliferate in vivo.

  Thus, when 109P1D4 plays a role in cell signaling and phosphorylation, 109P1D4 is used as a target for diagnostic, prognostic, prophylactic, and / or therapeutic purposes.

(Example 45: 109P1D4 RNA interference (RNAi))
RNA interference (RNAi) technology is performed for various cellular assays related to oncology. RNAi is a post-translational gene silencing mechanism that is activated by double-stranded RNA (dsRNA). RNAi includes specific mRNA degradation that results in changes in protein expression and subsequent changes in gene function. In mammalian cells, these dsRNAs, called short interfering RNAs, have the correct composition to activate RNAi pathways that target degradation, specifically for some mRNAs. checking ... For example, Elbashir S. et al. M.M. , Et. al. Duplexesof 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411 (6836): 494-8 (2001). Thus, RNAi technology is well used in mammalian cells to perform targeted gene silencing.

  Loss of cell growth control is a hallmark of cancer cells; therefore, it can assess the role of 109P1D4 in cell viability and / or cell proliferation assays. Therefore, RNAi was used to examine the function of the 1091D4 antigen. In order to generate siRNA for 109P1D4, it shows important molecular parameters (G: C content; melting point, etc.) and the ability to significantly reduce the expression level of 1091D4 protein when they are introduced into cells An algorithm was used to predict oligonucleotides with Thus, the three targeting sequences for 1091D4 siRNA are the following: 5′AAGAGGATACTGGTGAGATCT 3 ′ (SEQ ID NO: 57) (Oligo 109P1D4.a), 5 ′ AAGAGCAATGGTGCTCGTAAA 3 ′ (SEQ ID NO: 58) (Oligo 109P1D4.c) ), And 5 ′ AACACCAGAAGGAGACAAGAT3 ′ (SEQ ID NO: 59) (oligo 109P1D4.d). According to this example, a 109P1D4 siRNA composition comprising siRNA corresponding to the ORF of 109P1D4 protein or a partial sequence thereof (siRNA that is double-stranded) is used. Thus, siRNA subsequences are generally used in this manner, and siRNA subsequences are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 consecutive RNA nucleotides. These siRNA sequences are complementary to at least a portion of the mRNA coding sequence and are non-complementary to at least a portion thereof. In preferred embodiments, the subsequence is 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In a preferred embodiment, these siRNAs achieve a knockdown of 109P1D4 antigen in cells expressing the protein and have a functionally functional effect as follows.

  These selected siRNAs (109P1 D4.a oligo, 109P1D4.c oligo, 109P1D4.d oligo) were tested on LNCaP cells in a 3H thymidine incorporation assay (which measures cell proliferation). In addition, these oligonucleotides knock down the 109P1D4 antigen in cells expressing that protein and have functional effects as described below using protocols such as:

(Mammalian siRNA transfection :) On the day prior to siRNA transfection, various cell lines were cultured in 2 × 10 ³ medium (without antibiotics) at 80 μ (96-well format) for proliferation assays. 10% FBSRPMI 1640). In parallel with the 109P1D4-specific siRNA oligo, the following sequences were included in all experiments as controls: a) Transformed in mock using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and annealing buffer (without siRNA). B) luciferase 4 specific siRNA (targeting sequence: 5′-AAGGGAGAAGACGAACACUCUTT-3 ′) (SEQ ID NO: 60); and c) Eg5 specific siRNA (targeting sequence: 5′-AACTGAAGACCCTGAAGAATAATA-3 ′) (SEQ ID NO: 61). SiRNA was used at 10 nM and the final concentration of Lipofectamine 2000 was μg / ml.

  The procedure is as follows: These siRNAs are diluted in OPTIMEM (serum-free transfection medium; Invitrogen) at 0.1 μM (10-fold concentration) and at room temperature for 5-10 minutes Incubated. Lipofectamine 2000 was diluted with 10 μg / ml (10-fold concentration) for the total number of transfections. Lipofectamine 2000 diluted to an appropriate amount to 10-fold concentration was mixed 1: 1 with diluted 10-fold concentrated siRNA and incubated at room temperature (RT) for 20-30 minutes (5-fold concentration). Transfection solution). 20 μl of 5 × concentrated transfection solution was added to each sample and incubated at 37 ° C. for 96 hours prior to analysis.

( ³ H thymidine incorporation assay :)
This proliferation assay is a 3H thymidine incorporation method for determining the proliferation of viable cells by incorporating a label and incorporating it into DNA.

The procedure is as follows: Cells in logarithmic growth phase are trypsinized, washed, counted and plated in 96-well plates at 1000-4000 cells / well in 10% FBS. To do. After 4-8 hours, the medium is replaced. These cells are incubated for 24-72 hours, ³ H-Thy is pulsed at 1.5 pCi / ml for 14 hours, collected on a filter mat, and Microbetairlux or others Count with a scintillation cocktail at the counter.

  To study the function of 109P1D4 in cells, 109P1D4 endogenously expressed 1091D4 cell line (LNCaP), negative control siRNA (Luc4, targeting sequence that does not appear in the human genome), positive control siRNA (target And 109P1D4 specific siRNA (109P1D4.a, 109P1D4.c, and 109P1D4.d) together with a modified Eg5) and siRNA oligo (LF2K) were subjected to silencing by transfecting (FIG. 23). These results indicate that when these cells are treated with a siRNA positive control that specifically targets 1091D4 mRNA, the resulting “1091D4 deficient cells” can be expressed as measured by this assay. Proliferation was shown to decrease (see for example 1091D4.a treated cells).

  These data indicate that 109P1 D4 plays an important role in cancer cells, and deficiency of 109P1D4 clearly reduces the viability of these cells. 109P1D4, constitutively expressed in many tumor cell lines, plays an important role in malignant tumors; its expression is a major indicator of disease, and such diseases are often uncontrollable It should be noted that it is characterized by a modest cell growth rate and decreased apoptosis. It is important to correlate cell phenotype with gene knockdown after RNAi treatment, which can draw reasonable conclusions and control toxicity and other non-specific effects of these reagents Is done. For this purpose, assays that measure the expression levels of both proteins and mRNAs against RNAi-treated targets are important, and these assays include Western blotting, FACS staining with antibodies, immunoprecipitation , Northern blotting or RT-PCR (Taqman method or standard method). The effect on siRNA phenotype in these assays correlates with protein and / or mRNA knockout levels in the same cell line. 109P1D4 protein decreases after treatment with siRNA oligos such as those described above (eg, 109P1 D4).

A method for analyzing 109P1D4-related cell proliferation is to measure DNA synthesis as a marker for proliferation. Labeled DNA precursors (ie, ³ H thymidine) are used and the amount of these precursors incorporated into the DNA is quantified. The amount of these labeled precursors taken up by DNA is proportional to the amount of cell division occurring in the medium. Another method used to measure cell proliferation is to perform a clonogenic assay. In these assays, a defined number of cells are plated on an appropriate matrix and the number of colonies formed after the growth phase after siRNA treatment is counted.

  In identifying the 109P1D4 cancer cell target, it is considered to complement its cell survival / cell proliferation analysis with apoptosis and cell cycle profile studies. Prominent biochemical features of the apoptotic process are genomic DNA fragmentation, an irreversible reduction that leads to cell death. The method of observing the fragmentation of DNA in cells is based on the immunoassay that detects increased concentrations of histone complexed DNA fragments (ie, mononucleosomes and oligonucleosomes) in the cytoplasm of apoptotic cells. The integrated DNA fragment is detected immunochemically (ie, an ELISA that detects cell death). This assay does not require prior labeling of those cells and can detect DNA degradation in cells that do not proliferate in vitro (newly isolated tumor cells).

  The most important effector molecule for targeting apoptotic cells is caspase. When activated, caspases are proteases that cleave many substrates with aspartic acid residues at the C-terminal site, thereby affecting the immediate early stages of activated apoptosis. All caspases are synthesized as pro-enzymes and are associated with cleavage of aspartate residues upon activation. In particular, caspase 3 appears to play a central role in the initiation of apoptotic cytoreduction. Early events of apoptosis can be detected by assays to determine caspase 3 activation. Following RANi treatment, Western blot detection of the presence of active caspase 3 or cleavage of the product found in apoptotic cells (ie, RARP) by proteases supports the induction of apoptotic activity. Because the intracellular mechanisms that cause apoptosis are complex, each has its advantages and limitations. Considering other thresholds / endpoints (eg, cell morphogenetic methods, chromatin condensation, membrane blebbing, apoptotic bodies) further support that cell death is apoptotic. Since not all gene targets that control cell growth are apoptotic, the DNA content of permeabilized cells is measured to obtain a DNA content profile or cell cycle profile. The nucleus of apoptotic cells contains less DNA because it flows into the cytoplasm (sub-G1 population). In addition, DNA dyes (ie, propidium iodide (PI)) are also present in various cell cycles in the cell population due to the presence of various amounts of DNA in G0 / G1, S and G2 / M. In these studies, the subpopulation can be quantified.

  For the 109P1D4 gene, RNAi studies make it easier to understand the gene's contribution in the cancer pathway. Such active RNAi molecules are used in identification assays to screen for mAbs that are active anti-tumor therapeutics. Furthermore, siRNA is administered as a therapeutic agent to cancer patients to reduce the malignant growth of several types of cancer. These are listed in Table I. When 109P1D4 plays a role in cell survival, cell proliferation, tumorigenesis or apoptosis, it is used as a target for diagnostic, prophylactic and / or therapeutic purposes.

  Throughout this application, various website data content, publications, patent applications, and patents are incorporated (websites are incorporated with their URL Resource Uniform URL).

  The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as an illustration of individual aspects of the invention and are not intended to have any functional equivalents. Things are also within the scope of the present invention. In addition to the modifications described herein, various modifications to the models and methods of the present invention will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly within the scope of the present invention. Intended. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

A 192 nucleotide 109P1D4 SSH sequence. The cDNA and amino acid sequence of 109P1D4 variant 1 (also referred to as “109P1D4 v.1” or “109P1D4 variant 1”) is shown in FIG. 2A. The starting methionine is underlined. This open reading frame spans nucleic acids 846-3911, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 1 (also referred to as “109P1D4 v.1” or “109P1D4 variant 1”) is shown in FIG. 2A. The starting methionine is underlined. This open reading frame spans nucleic acids 846-3911, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 1 (also referred to as “109P1D4 v.1” or “109P1D4 variant 1”) is shown in FIG. 2A. The starting methionine is underlined. This open reading frame spans nucleic acids 846-3911, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 1 (also referred to as “109P1D4 v.1” or “109P1D4 variant 1”) is shown in FIG. 2A. The starting methionine is underlined. This open reading frame spans nucleic acids 846-3911, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 2 (also referred to as “109P1D4 v.2”) is shown in FIG. 2B. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 503 to 3667 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 2 (also referred to as “109P1D4 v.2”) is shown in FIG. 2B. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 503 to 3667 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 2 (also referred to as “109P1D4 v.2”) is shown in FIG. 2B. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 503 to 3667 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 2 (also referred to as “109P1D4 v.2”) is shown in FIG. 2B. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 503 to 3667 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 3 (also referred to as “109P1D4 v.3”) is shown in FIG. 2C. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4889, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 4 (also referred to as “109P1D4 v.4”) is shown in FIG. 2D. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4859, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 5 (also referred to as “109P1D4 v.5”) is shown in FIG. 2E. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 846-4778 including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 6 (also referred to as “109P1D4 v.6”) is shown in FIG. 2F. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 614-3727, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 6 (also referred to as “109P1D4 v.6”) is shown in FIG. 2F. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 614-3727, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 6 (also referred to as “109P1D4 v.6”) is shown in FIG. 2F. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 614-3727, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 6 (also referred to as “109P1D4 v.6”) is shown in FIG. 2F. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 614-3727, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 7 (also referred to as “109P1D4 v.7”) is shown in FIG. 2G. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-3881, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 7 (also referred to as “109P1D4 v.7”) is shown in FIG. 2G. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-3881, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 7 (also referred to as “109P1D4 v.7”) is shown in FIG. 2G. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-3881, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 7 (also referred to as “109P1D4 v.7”) is shown in FIG. 2G. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-3881, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 8 (also referred to as “109P1D4 v.8”) is shown in FIG. 2H. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 735-4757, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 9 (also referred to as “109P1D4 v.9”) is shown in FIG. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 514-3627, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 9 (also referred to as “109P1D4 v.9”) is shown in FIG. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 514-3627, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 9 (also referred to as “109P1D4 v.9”) is shown in FIG. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 514-3627, including the stop codon. The cDNA and amino acid sequence of 109P1D4 variant 9 (also referred to as “109P1D4 v.9”) is shown in FIG. The codon for the initiating methionine is underlined. This open reading frame spans nucleic acids 514-3627, including the stop codon. 109P1D4 v. 1 SNP variant, 109P1D4 v. 2 SNP variants and 109P1D4 v. 3 SNP variant. Although these SNP variants are shown separately, they can also appear in any combination and in any of the transcription variants listed above. 109P1D4 v. 1 SNP variant, 109P1D4 v. 2 SNP variants and 109P1D4 v. 3 SNP variant. Although these SNP variants are shown separately, they can also appear in any combination and in any of the transcription variants listed above. 109P1D4 v. 6 SNP variant, 109P1D4 v. 7 SNP variants and 109P1D4 v. 8 SNP variant. Although these SNP variants are shown separately, they can also appear in any combination and in any of the transcription variants listed above. 109P1D4 v. The amino acid sequence of 1 is shown in FIG. 3A; this sequence has 1021 amino acids. 109P1D4 v. The amino acid sequence of 2 is shown in FIG. 3B; this sequence has 1054 amino acids. 109P1D4 v. The amino acid sequence of 3 is shown in Figure 3C; this sequence has 1347 amino acids. 109P1D4 v. The amino acid sequence of 3 is shown in Figure 3C; this sequence has 1347 amino acids. 109P1D4 v. The amino acid sequence of 4 is shown in FIG. 3D; this sequence has 1337 amino acids. 109P1D4 v. The amino acid sequence of 4 is shown in FIG. 3D; this sequence has 1337 amino acids. 109P1D4 v. The amino acid sequence of 5 is shown in FIG. 3E; this sequence has 1310 amino acids. 109P1D4 v. The amino acid sequence of 6 is shown in FIG. 3F; this sequence has 1037 amino acids. 109P1D4 v. The amino acid sequence of 6 is shown in FIG. 3F; this sequence has 1037 amino acids. 109P1D4 v. The amino acid sequence of 7 is shown in FIG. 3G; this sequence has 1048 amino acids. 109P1D4 v. The amino acid sequence of 8 is shown in FIG. 3H; this sequence has 1340 amino acids. 109P1D4 v. The amino acid sequence of 8 is shown in FIG. 3H; this sequence has 1340 amino acids. 109P1D4 v. The 9 amino acid sequence is shown in FIG. 3I; this sequence has 1037 amino acids.

As used herein, reference to 109P1D4 includes all of its variants (unless the contents are otherwise expressly indicated otherwise those shown in FIGS. 2, 3, 10, 11 and 12). Including).
109P1D4 v. 1 protein and protocadherin-11 alignment. 109P1D4 v. 1 protein and protocadherin-11 alignment. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. Hopp and Woods methods (Hopp T.P., Woods K., Ltd.) accessed on the ProtScale website located on the world wide web (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server. R., 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828), 109P1D4 v. 1-v. 9 hydrophilic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. The Kyte and Doolittle method (Kyte J., Doolittle R.F., accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 1982. J. Mol.Biol.157: 105-132), 109P1D4 v. 1-v. 9 Hydropathic amino acid profiles. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Janin's method (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using 1-v. Nine percent accessible residue amino acid profile. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. Bhaskaran and Ponwaswamy's method (Bhaskaran R., and Ponwaswamy P.K.) accessed on the ProtScale website located on the World Wide Web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. , 1988. Int. J. Pept. Protein Res.32: 242-255), 109P1D4 v. 1-v. Average mean amino acid profile of 9. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The Delage and Roux methods (Deleage, G., Rox B. 1987 Protein) accessed on the ProtScale website located on the world wide web (.expasy.ch / cgi-bin / protscale.pl) through the ExPasy molecular biology server. 109P1D4 v. Determined by computer algorithm sequence analysis using Engineering 1: 289-294). 1-v. 9 β-turn amino acid profiles. The structure of 109P1D4 transcriptional variant. Variant 109P1D4 v. 2 to v. 9 is v. 1 transcriptional variant. Variant v. 2 is v. The central part of one sequence (the 3 ′ part of exon 1 and the 5 ′ part of exon 2) was shared. Variant v. 6 is v. 2 but similar to v. An extra exon was added between exon 1 and exon 2 of 2. v. 3 represents the 5 ′ portion of exon 1 and exon 2, v. 1 and 5 additional exons downstream. v. 3 compared to v. 4 is v. 3 exon 4 was deleted while v. 5 is v. Three exons 3 and 4 were deleted. Variant v. 5 lacked exon 3 and exon 4. This gene (referred to as PCD11) is located in sex chromosomes X and Y. The ends of exons in the transcript are colored on the box. Potential exons of this gene are shown in order on the human genome. Poly A tail and single nucleotide differences are not shown in the figure. Intron and exon lengths are not proportional. The structure of 109P1D4 transcriptional variant. Variant 109P1D4 v. 2 to v. 9 is v. 1 transcriptional variant. Variant v. 2 is v. The central part of one sequence (the 3 ′ part of exon 1 and the 5 ′ part of exon 2) was shared. Variant v. 6 is v. 2 but similar to v. An extra exon was added between exon 1 and exon 2 of 2. v. 3 represents the 5 ′ portion of exon 1 and exon 2, v. 1 and 5 additional exons downstream. v. 3 compared to v. 4 is v. 3 exon 4 was deleted while v. 5 is v. Three exons 3 and 4 were deleted. Variant v. 5 lacked exon 3 and exon 4. This gene (referred to as PCD11) is located in sex chromosomes X and Y. The ends of exons in the transcript are colored on the box. Potential exons of this gene are shown in order on the human genome. Poly A tail and single nucleotide differences are not shown in the figure. Intron and exon lengths are not proportional. Schematic alignment of 109P1D4 protein variants. Variant 109P1D4 v. 2 to v. 9 was a protein translated from the corresponding transcriptional variant. All of these protein variants shared the common part of the sequence (ie, 3-1011 of v.1) except for a few amino acids that differ in this segment from the transcript SNP. Variant v. 6 and v. 9 was the same except for the two amino acids at 906 and 1001. Variant v. 8 is v. Except for the N-terminus and the 2aa deletion at 1117-8. It was almost the same as 5. No single amino acid difference was shown. Numbers in parentheses indicate variant v. Corresponds to the position in FIG. Schematic alignment of 109P1D4 protein variants. Variant 109P1D4 v. 2 to v. 9 was a protein translated from the corresponding transcriptional variant. All of these protein variants shared the common part of the sequence (ie, 3-1011 of v.1) except for a few amino acids that differ in this segment from the transcript SNP. Variant v. 6 and v. 9 was the same except for the two amino acids at 906 and 1001. Variant v. 8 is v. Except for the N-terminus and the 2aa deletion at 1117-8. It was almost the same as 5. No single amino acid difference was shown. Numbers in parentheses indicate variant v. Corresponds to the position in FIG. Intentionally omitted. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol.25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. 109P1D4 protein variants 1-9 (v.1- (SEQ ID NO: 31); v.2- (SEQ ID NO: 32); v.3- (SEQ ID NO: 33); v.4- (SEQ ID NO: 34); 5- (SEQ ID NO: 35); v.6- (SEQ ID NO: 36); v.7- (SEQ ID NO: 37); v.8- (SEQ ID NO: 38); v.9- (SEQ ID NO: 39)) Secondary structure prediction and transmembrane domain prediction. The secondary structure of the 109P1D4 protein variant was accessed on the ProtScale website located on the World Wide Web (.expasy.ch / tools /) from the Expasy molecular biology server (NPS @: Network). Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]: 147-150 Combet C., Blanket C., Georgon C. and Release G., http: //pbil.ibcp.bcp. pl? page = npsa_nn.html). This method predicts the presence and location of α-helices, extended strands, and random coils from primary structural protein sequences. The percentage of protein variants of a given secondary structure has also been described. Fig. 13J Upper Panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. TMBASE-A database of membrane spanning protein segments Biol. Chem. Schematic representation of the possible presence of the transmembrane region of the variant. 13J lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Hejne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A: Schematic representation of the possible presence of the transmembrane region of 109P1D4 variant based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). 13K upper panel: Hofmann and Stoffel's TMpred algorithm utilizing TMBASE (based on K. Hofmann, W. Stoffel. TMBASE-A database of membranes scanning protein biol. Schematic representation of the possible presence of the transmembrane region of the variant. Figure 13K lower panel: Sonnhammer, von Heijne, and Krog's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A. Schematic representation of the possibility of the presence of a 109P1D4 variant transmembrane region, based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). 13K upper panel: Hofmann and Stoffel's TMpred algorithm utilizing TMBASE (based on K. Hofmann, W. Stoffel. TMBASE-A database of membranes scanning protein biol. Schematic representation of the possible presence of the transmembrane region of the variant. FIG. 13L lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik LL Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A. Schematic representation of the possibility of the presence of a 109P1D4 variant transmembrane region, based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). Fig. 13M Upper Panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. Schematic representation of the possible presence of the transmembrane region of the variant. FIG. 13M lower panel: Sonnhammer, von Heijne, and Krog's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A: Schematic representation of the possible presence of the transmembrane region of 109P1D4 variant based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). Fig. 13N Upper Panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. Schematic representation of the possible presence of the transmembrane region of the variant. FIG. 13N lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A. Schematic representation of the possibility of the presence of a 109P1D4 variant transmembrane region, based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). FIG. 13O upper panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. Schematic representation of the possible presence of the transmembrane region of the variant. FIG. 13O lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A. Schematic representation of the possibility of the presence of a 109P1D4 variant transmembrane region, based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). Fig. 13P Upper Panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (4 based on K. Hofmann, W. Stoffel. TMBASE-A database of membranes spanning protein segments Biol. Cheml. Schematic representation of the possible presence of the transmembrane region of the variant. Fig. 13P lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik LL Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A: Schematic representation of the possible presence of the transmembrane region of 109P1D4 variant based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). Fig. 13Q Upper panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. TMBASE-A database of membranes spanning protein segments Biol. Cheml, 109, P9, P19, P93, Dp4, Chem. Schematic representation of the possible presence of the transmembrane region of the variant. Figure 13Q lower panel: Sonnhammer, von Heijne, and Krog's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A: Schematic representation of the possible presence of the transmembrane region of 109P1D4 variant based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). Fig. 13R upper panel: Hofmann and Stoffel's TMpred algorithm using TMBASE (based on K. Hofmann, W. Stoffel. TMBASE-A database of membranes spanning protein segments Biol. Chem9, P4, Chem. 4P, 19). Schematic representation of the possible presence of the transmembrane region of the variant. Figure 13R lower panel: Sonnhammer, von Heijne, and Klogh's TMHMM algorithm (Erik L. L. Sonnhammer, Gunnar von Heinne, and Anders Krogh: A hidden Markov model. Conf. On Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littletown, F. Major, R. Lathrop, D. Sankoff, and C. Sensen A: Schematic representation of the possible presence of the transmembrane region of 109P1D4 variant based on Press, 1998). The TMpred algorithm and the TMHMM algorithm are accessed from an Expasy molecular biology server located on the world wide web (.expasy.ch / tools /). FIG. 14 shows the expression of 109P1D4 in lymphoma cancer patient specimens. RNA was extracted from peripheral blood lymphocytes and umbilical cord blood isolated from cancer specimens of normal individuals and lymphoma patients. The 109P1D4 sequence was probed by Northern blot using a total amount of 10 μg RNA. Set a kilo-base size standard next to it. The results show that 109P1D4 expression is shown in lymphoma patient specimens, but 109P1D4 expression is not shown in the tested normal blood cells. FIG. 15 shows the expression of 109P1D4 by RT-PCR. First-strand cDNA is derived from biological pool 1 (liver, lung and kidney), biological pool 2 (pancreas, colon and stomach), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, kidney cancer pool Ovarian cancer pool, breast cancer pool, cancer metastasis pool, and pancreatic cancer pool. Normalization was performed by PCR using primers for actin and GAPDH. Thirty cycles of amplification were performed by semi-quantitative PCR using primers for 109P1D4. The results show strong expression of 109P1D4 in all tested cancer pools. Very little expression was detected in the biological pool. FIG. 16 shows the expression of 109P1D4 in normal tissues. Multiple tissues were probed with 109P1D4 SSH fragment by two Northern blots (Clontech, both using 2 μg mRNA / lane). The kilobase (kb) size standard is shown next to it. The results show the expression of about 10 kb 109P1D4 transcript in the ovary. Weak expression was also detected in placenta and brain, but not in other normal tissues tested. FIG. 17 shows the expression of 109P1D4 in human cancer cell lines. RNA was extracted from many human prostate cancer cell lines and bone cancer cell lines. A total amount of 10 μg RNA / lane Northern blot was probed with 109P1D4 SSH fragment. The kilobase (kb) size standard is shown next to it. Results show 109P1D4 expression in LAPC-9AD, LAPC-9AI, LNCaP prostate cancer cell lines, and bone cancer cell lines SK-ES-1 and RD-ES. FIG. 18A shows 109P1D4 expression in normal human tissue. Analysis was performed by cDNA dot blot containing 76 different samples from human tissue using a 109P1D4 SSH probe. Expression was detected only in multiple regions of the brain, placenta, ovary and fetal brain among all tissues tested. FIG. 18B shows the expression of 109P1D4 in cancer patient specimens. The expression of 109P1D4 was assayed in a panel of human cancers on a dot blot of RNA (T) and in each corresponding normal tissue panel (N). Compared to normal tissue, up-regulated expression of 109P1D4 in tumors was observed in uterus, lung and stomach. It is detected in normal adjacent tissue isolated from diseased tissue, but not detected in normal tissue isolated from healthy donors. This may indicate that these tissues are not completely normal and 109P1D4 can be expressed in early stage tumors. FIG. 19 shows 109P1D4 expression in lung cancer patient specimens. RNA was extracted from normal lung, prostate cancer xenograft LAPC-9AD, bone cancer cell line RD-ES, and lung cancer patient tumors. A Northern blot of 10 μg total RNA was probed with 109PD4. A kilo-base size standard is set aside. The results showed strong expression of 109PD4 in lung tumor tissue and RD-ES cell line, but no expression in normal lung. FIG. 20 shows the expression of soluble secreted Tag5 109P1D4 in 293T cells. 293T cells were transfected with either an empty vector or a Tag5 secretion vector encoding the extracellular domain of 109P1D4 variant 1 (ECD; amino acids 24-812) fused to a Myc / His epitope tag. Two days later, cells and media were harvested and analyzed for recombinant Tag5 109P1D4 protein expression by SDS-PAGE followed by Western blotting of anti-His epitope tags. Arrows indicate immunoreactive bands corresponding to 109P104 ECD present in the media and lysates from Tag5 109P1D4 transfected cells. FIG. 21 shows the expression of 109P1D4 protein in 293T cells. 293T cells were transfected with either an empty vector or a pCDNA3.1 vector encoding the full length cDNA of 109P1D4 variant 1 fused to a Myc / His epitope tag. Two days later, cells were harvested and analyzed for expression of 109P1D4 variant 1 protein by SDS-PAGE followed by Western blotting of anti-His epitope tags. The arrow indicates an immunoreactive band corresponding to the full length 109P104 variant 1 protein that is expressed in cells transfected with the 109P1D4 vector but not expressed in control cells. FIG. 22 shows tyrosine phosphorylation of 109P1D4 after pervanadate treatment. 293T cells were transfected with the neomycin resistance gene alone or with 109P1D4 in the pSRμ vector. Twenty-four hours after transfection, the cells were left overnight in 10% serum or grown in 0.1% serum. These cells are then left untreated or 200 μM pervanadate (Na ₃ VO ₄ And H ₂ O ₂ For a period of 30 minutes. These cells were lysed in Triton X-100 and 109PD4 protein was immunoprecipitated with anti-His monoclonal antibody. The immunoprecipitate was subjected to SDS-PAGE followed by Western blot using either anti-phosphotyrosine (upper panel) or anti-His (lower panel). 109P1D4 protein was tyrosine phosphorylated in response to pervanadate treatment, and following pervanadate-induced activation, large amounts of protein migrated to the insoluble fraction. FIG. 23 shows the effect of 109P1D4 RNAi on cell proliferation. LNCaP cells were transfected with Lipofectamine 2000 alone or with SiRNA oligonucleotides. The siRNA oligonucleotides include Luc4 specific for luciferase as a negative control, Eg5 specific for mitochondrial spindle protein Eg5, or 109P1D4 protein (109P1D4.a, 109P1D4.c, and 109P1D4) as a negative control at a concentration of 20 nM. Three siRNAs specific to .d) were included. 24 hours after transfection, these cells were ³ Incorporation was measured after 72 hours with H-thymidine. All three siRNAs for 109P1D4 indicate that 109P1D4 expression, which inhibited the growth of LNCaP cells, is important for the growth pathway of these cancer cells.

Claims (58)

a) a peptide of 8, 9, 10 or 11 consecutive amino acids of the protein of FIG. 2;
b) the peptides of Tables VIII-XXI;
c) a peptide of Table XXII-XLV; or d) a composition comprising the peptide of Table XLVI-XLIX.   The composition of claim 1, wherein the composition induces an immune response.   The protein according to claim 2, wherein the protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with respect to the entire amino acid sequence shown in FIG. , 98% or 99% homologous or identical.   3. The protein of claim 2, wherein the protein is bound by an antibody that specifically binds to the protein of FIG.   3. The composition of claim 2, wherein the composition contains a cytotoxic T cell (CTL) polypeptide epitope or analog thereof from the amino acid sequence of the protein of FIG.   6. The composition of claim 5, wherein the composition is further limited by the condition that the epitope is not the entire amino acid sequence of FIG.   3. The composition of claim 2, wherein the composition is further limited by the condition that the polypeptide is not the entire amino acid sequence of the protein of FIG.   3. The composition of claim 2, wherein the composition contains an antibody polypeptide epitope from the amino acid sequence of FIG.   9. The composition of claim 8, wherein the composition is further limited by the condition that the epitope is not the entire amino acid sequence of FIG. 9. The composition of claim 8, wherein the antibody epitope comprises a peptide region of at least 5 amino acids of FIG. 2 in any and all numbers increasing to the end of the peptide:
a) amino acid positions having a value greater than 0.5 in the hydrophilic profile of FIG. 5;
b) an amino acid position having a value of less than 0.5 in the hydropathic profile of FIG. 6;
c) amino acid positions having a value greater than 0.5 in the% profile of accessible residues in FIG. 7;
d) amino acid positions having a value greater than 0.5 in the mean flexibility profile of FIG. 8;
e) an amino acid position having a value greater than 0.5 in the β-turn profile of FIG. 9;
f) a combination of at least two of a) to e);
g) a combination of at least three of a) to e);
h) A composition comprising an amino acid position selected from at least four combinations of a) to e); or i) five combinations of a) to e).   A polynucleotide encoding the protein of claim 1.   12. The polynucleotide of claim 11 wherein the nucleotide comprises the nucleic acid molecule shown in FIG.   13. The polynucleotide of claim 12, wherein the polynucleotide is further limited by the condition that the encoded protein is not the entire amino acid sequence of FIG.   12. A composition comprising a polynucleotide that is fully complementary to the polynucleotide of claim 11.   109P1D4 siRNA composition comprising siRNA (double stranded RNA) corresponding to the nucleic acid ORF sequence of 109P1D4 protein or a partial sequence thereof; the partial sequence comprising 19, 20, 21 A composition comprising 22, 23, 24, or 25 consecutive RNA nucleotides in length and comprising sequences complementary and non-complementary to at least a portion of the mRNA coding sequence.   14. The polynucleotide of claim 13, wherein the polynucleotide further comprises an additional nucleotide sequence encoding the additional peptide of claim 1. A method for generating a mammalian immune response to the protein of FIG. 2 comprising:
Exposing a mammalian immune system cell to a) a 109P1D4-related protein and / or a portion of a nucleotide sequence encoding the protein, thereby producing an immune response against the protein, Method. 18. A method for generating an immune response according to claim 17, wherein the method is:
Providing a 109P1D4-related protein comprising at least one T cell epitope or at least one B cell epitope; and contacting said epitope with a T cell or B cell of a mammalian immune system, respectively, thereby Activating a cell or B cell.   19. The method of claim 18, wherein the immune system cell is a B cell, whereby the activated B cell produces an antibody that specifically binds to the 109P1D4-related protein. .   19. The method of claim 18, wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL expresses the 109P1D4-related protein. A way to kill the cells of the self.   19. The method of claim 18, wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL is a cytotoxic T cell (CTL). A method of secreting cytokines that increase cytotoxic activity or antibody production activity of B cells. A method for detecting the presence of mRNA encoding the protein of FIG. 2 in a sample comprising:
Subjecting the sample to reverse transcription using at least one 109P1D4 cDNA primer, whereby cDNA is generated when mRNA is present in the sample;
Amplifying the cDNA so generated using 109P1D4 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 109P1D4 cDNA;
Wherein the presence of amplified 109P1D4 cDNA indicates that mRNA encoding the protein of FIG. 2 is present in the sample. A method for detecting the presence of a 109P1D4-related protein or 109P1D4-related polynucleotide in a sample, the method comprising:
Contacting said sample with a substance that specifically binds to said 109P1D4-related protein or said 109P1D4-related polynucleotide, respectively, to form a complex; and determining the presence or amount of said complex in said sample A method comprising the steps of: 24. The method of claim 23 for detecting the presence of a 109P1D4-related protein in a sample, the method comprising:
Contacting the sample with an antibody or fragment thereof, wherein either the antibody or fragment thereof specifically binds to and binds to the 109P1D4-related protein, thus forming a complex. And determining the presence or amount of the complex in the sample. 24. The method of claim 23 for monitoring one or more 109P1D4 gene products in a biological sample, the method comprising:
Determining the status of one or more 109P1D4 gene products expressed by cells in a tissue sample from an individual;
Comparing the status so determined to the status of one or more 109P1D4 gene products in a corresponding normal sample; and an abnormal expression status of 109P1D4 in the tissue sample compared to the normal sample; A method comprising the step of identifying the presence.   26. The method of claim 25, wherein the method determines whether 109P1D4 mRNA or one or more increased gene products of 109P1D4 protein are present, thereby comparing to the normal sample, The method further comprising the step wherein the presence of one or more increased gene products in the test sample indicates the presence or condition of cancer.   27. The method of claim 26, wherein the tissue is selected from the tissues listed in Table I. A composition that modulates the state of a cell that expresses the protein of FIG. 2, wherein the composition:
a) a substance that modulates the state of a cell that expresses the protein of FIG. 2, or b) a composition that contains a molecule that is controlled or produced by the protein of FIG.   30. The 109P1D4 siRNA composition of claim 28, wherein the composition comprises a siRNA (double stranded RNA) corresponding to the nucleic acid ORF sequence of 109P1D4 protein or a partial sequence thereof; wherein the partial sequence is 19, 20, 21, 22, 23, 24 or 25 consecutive RNA nucleotides in length, and the partial sequence is complementary to at least a portion of the mRNA coding sequence A composition comprising a sequence that is target and non-complementary.   30. The composition of claim 28, wherein the composition further comprises a physiologically acceptable carrier.   30. A pharmaceutical composition comprising the composition of claim 28 in human unit dosage form.   29. The composition of claim 28, wherein the substance comprises an antibody or fragment thereof that specifically binds to the protein of FIG.   33. The antibody or fragment thereof of claim 32, wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof.   33. The antibody of claim 32, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.   A non-human transgenic animal producing the antibody of claim 32.   34. A hybridoma producing the antibody of claim 33.   29. The composition of claim 28, wherein the substance reduces or inhibits the viability, proliferative state, or reproductive state of a cell expressing the protein of FIG.   29. The composition of claim 28, wherein the substance increases or promotes the viability, proliferative state, or reproductive state of cells expressing the protein of FIG. 30. The composition of claim 28, wherein the substance is:
a) an antibody or fragment thereof, wherein either the antibody or fragment thereof immunospecifically binds to the protein of FIG. 2;
b) a polynucleotide encoding an antibody or fragment thereof, wherein either the antibody or fragment thereof immunospecifically binds to the protein of FIG. 2;
c) a ribozyme that cleaves a polynucleotide having a 109P1D4 coding sequence, or a nucleic acid molecule that encodes the ribozyme; and a physiologically acceptable carrier; and d) a human T cell, wherein the T cell is a specific HLA A T cell that specifically recognizes a 109P1D4 peptide subsequence with respect to the molecule;
e) the protein of FIG. 2 or a fragment of the protein of FIG. 2;
f) nucleotides encoding the protein of FIG. 2 or nucleotides encoding fragments of the protein of FIG. 2;
g) a peptide of 8, 9, 10 or 11 consecutive amino acids of the protein of FIG. 2;
h) Peptides of Tables VIII-XXI;
i) Peptides of Tables XXII-XLV;
j) Peptides of Tables XLVI to XLIX;
k) an antibody polypeptide epitope from the amino acid sequence of FIG. 2;
1) a polynucleotide encoding an antibody polypeptide epitope from the amino acid sequence of FIG. 2; or m) a 109P1D4 siRNA composition comprising an siRNA corresponding to a nucleic acid ORF sequence of 109P1D4 protein or a partial sequence thereof ( A double-stranded RNA), wherein the partial sequence is 19, 20, 21, 22, 23, 24 or 25 consecutive RNA nucleotides in length, And the partial sequence is selected from the group comprising a composition comprising sequences complementary and non-complementary to at least a portion of the mRNA coding sequence. A method of inhibiting the viability, proliferative state or reproductive state of a cancer cell expressing the protein of FIG. 2, comprising:
30. A method comprising administering to the cell the composition of claim 28, thereby inhibiting the viability, proliferative state or reproductive state of the cell.   41. The method of claim 40, wherein the composition comprises an antibody or fragment thereof, and any of the antibody or fragment thereof specifically binds to 109P1D4-related protein. 41. The method of claim 40, wherein the composition is
A method comprising (i) a 109P1D4-related protein or (ii) a polynucleotide comprising a coding sequence of 109P1D4-related protein or a polynucleotide comprising a polynucleotide complementary to the coding sequence of 109P1D4-related protein.   41. The method of claim 40, wherein the composition comprises a ribozyme that cleaves a polynucleotide encoding the protein of FIG.   41. The method of claim 40, wherein the composition comprises human T cells against the cancer cells, the T cells specifically recognizing the peptide subsequence of the protein of FIG. The partial sequence relates to a specific HLA molecule.   41. The method of claim 40, wherein the composition comprises a vector that delivers a nucleotide encoding a single chain monoclonal antibody, whereby the encoded single chain antibody is a cancer that expresses the protein of FIG. A method wherein the expression is intracellular. A method of delivering an agent to a cell expressing the protein of FIG. 2, comprising:
35. A method comprising providing the agent conjugated to an antibody or fragment thereof according to claim 32; and exposing the antibody-drug conjugate or fragment-drug conjugate to the cell. A method of inhibiting the viability, proliferative state or reproductive state of a cancer cell expressing the protein of FIG. 2, comprising:
30. A method comprising administering to the cell a compound according to claim 28 thereby inhibiting the viability, proliferative state or reproductive state of the cell. A method of targeting information for preventing or treating a tissue cancer listed in Table I to a subject in need of the prevention or treatment, the method comprising:
Detecting the presence or absence of expression of a polynucleotide associated with cancer of the tissues listed in Table I in a sample from a subject, the expression of the polynucleotide comprising:
(A) the nucleotide sequence of FIG. 2;
(B) a nucleotide sequence encoding a polypeptide encoded by the nucleotide sequence of FIG. 2;
(C) selected from the group consisting of nucleotide sequences encoding polypeptides that are 90% or more identical to the amino acid sequence encoded by the nucleotide sequence of FIG. 2;
Directing information for preventing or treating cancers of the tissues listed in Table I to a subject in need of the prevention or treatment based on the presence or absence of expression of the polynucleotide in the sample The method of inclusion.   49. The method of claim 48, wherein the information includes a description of a detection procedure or treatment for cancer of the tissues listed in Table I. A method for identifying candidate molecules that modulate cell proliferation comprising:
(A) The following:
(I) the nucleotide sequence of SEQ ID NO: 1 (ii) a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in FIG. 3;
(Iii) a nucleotide sequence encoding a polypeptide that is 90% or more identical to the amino acid sequence described in FIG. 3; and (iv) selected from the group consisting of fragments of the nucleotide sequence of (i), (ii) or (iii) Introducing a test molecule into a system comprising a nucleic acid comprising the nucleotide sequence to be tested; or to a system comprising a protein encoded by the nucleotide sequence of (i), (ii), (iii) or (iv) And (b) determining the presence or absence of an interaction between the test molecule and the nucleotide sequence or protein,
A method whereby the presence of an interaction between the test molecule and the nucleotide sequence or protein identifies the test molecule as a candidate molecule that modulates cell proliferation.   51. The method of claim 50, wherein the system is an animal.   51. The method of claim 50, wherein the system is a cell.   51. The method of claim 50, wherein the test molecule comprises an antibody or antibody fragment that specifically binds to a protein encoded by a nucleotide sequence of (i), (ii), (iii) or (iv). Including.   51. A method for treating a cancer of a tissue listed in Table I in a subject, said method comprising subjecting a subject in need thereof to a candidate molecule identified by the method of claim 50. Administering, whereby the candidate molecule treats a cancer of the tissue listed in Table I in the subject. A method for identifying a candidate therapeutic agent for treating a cancer of a tissue listed in Table I, the method comprising:
(A) The following:
(I) the nucleotide sequence of SEQ ID NO: 1 (ii) a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in FIG. 3;
(Iii) a nucleotide sequence encoding a polypeptide that is 90% or more identical to the amino acid sequence described in FIG. 3; and (iv) selected from the group consisting of fragments of the nucleotide sequence of (i), (ii) or (iii) Introducing a test molecule into a system comprising a nucleic acid comprising the nucleotide sequence to be tested; or to a system comprising a protein encoded by the nucleotide sequence of (i), (ii), (iii) or (iv) And (b) determining the presence or absence of an interaction between the test molecule and the nucleotide sequence or protein,
Thereby, the presence of an interaction between the test molecule and the nucleotide sequence or protein identifies the test molecule as a candidate therapeutic for treating cancer in the tissues listed in Table I.   56. The method of claim 55, wherein the system is an animal.   56. The method of claim 55, wherein the system is a cell.   56. The method of claim 55, wherein the test molecule comprises an antibody or antibody fragment that specifically binds to a protein encoded by the nucleotide sequence of (i), (ii), (iii) or (iv). Including.

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