Compositions and methods for the identification, assessment, prevention, and therapy of human cancers

BACKGROUND OF THE INVENTION

[0002] Cancers can be viewed as a breakdown in the communication between tumor cells and their environment, including their normal neighboring cells. Growth-stimulatory and growth-inhibitory signals are routinely exchanged between cells within a tissue. Normally, cells do not divide in the absence of stimulatory signals or in the presence of inhibitory signals. In a cancerous or neoplastic state, a cell acquires the ability to “override” these signals and to proliferate under conditions in which a normal cell would not.

[0003] In general, tumor cells must acquire a number of distinct aberrant traits in order to proliferate in an abnormal manner. Reflecting this requirement is the fact that the genomes of certain well-studied tumors carry several different independently altered genes, including activated oncogenes and inactivated tumor suppressor genes. In addition to abnormal cell proliferation, cells must acquire several other traits for tumor progression to occur. For example, early on in tumor progression, cells must evade the host immune system. Further, as tumor mass increases, the tumor must acquire vasculature to supply nourishment and remove metabolic waste. Additionally, cells must acquire an ability to invade adjacent tissue. In many cases cells ultimately acquire the capacity to metastasize to distant sites.

[0004] It is apparent that the complex process of tumor development and growth must involve multiple gene products. It is therefore important to define the role of specific genes involved in tumor development and growth and identify those genes and gene products that can serve as targets for the diagnosis, prevention and treatment of cancers.

[0005] In the realm of cancer therapy it often happens that a therapeutic agent that is initially effective for a given patient becomes, over time, ineffective or less effective for that patient. The very same therapeutic agent may continue to be effective over a long period of time for a different patient. Further, a therapeutic agent that is effective, at least initially, for some patients can be completely ineffective or even harmful for other patients. Accordingly, it would be useful to identify genes and/or gene products that represent prognostic genes with respect to a given therapeutic agent or class of therapeutic agents. It then may be possible to determine which patients will benefit from particular therapeutic regimen and, importantly, determine when, if ever, the therapeutic regime begins to lose its effectiveness for a given patient. The ability to make such predictions would make it possible to discontinue a therapeutic regime that has lost its effectiveness well before its loss of effectiveness becomes apparent by conventional measures.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to the identification of markers that can be used to determine the sensitivity or resistance of cancer cells to a therapeutic agent. By examining the expression of one or more of the identified markers, whose expression correlates with sensitivity to a therapeutic agent or resistance to a therapeutic agent, in a sample of cancer cells, it is possible to determine whether a therapeutic agent or combination of agents will be most likely to reduce the growth rate of the cancer and can further be used in selecting appropriate treatment agents. The markers of the present invention whose expression correlates with sensitivity or with resistance to an agent are listed in Tables 1 and 2, respectively. Table 3 sets forth the markers of Tables 1 and 2 with their corresponding GenBank GI number.

[0007] By examining the expression of one or more of the identified markers in a sample of cancer cells, it is possible to determine which therapeutic agent or combination of agents will be most likely to reduce the growth rate of the cancer. By examining the expression of one or more of the identified markers in a sample of cancer cells, it is also possible to determine which therapeutic agent or combination of agents will be the least likely to reduce the growth rate of the cancer. By examining the expression of one or more of the identified markers, it is therefore possible to eliminate ineffective or inappropriate therapeutic agents. Moreover, by examining the expression of one or more of the identified markers in a sample of cancer cells taken from a patient during the course of therapeutic treatment, it is possible to determine whether the therapeutic treatment is continuing to be effective or whether the cancer has become resistant (refractory) to the therapeutic treatment. It is also possible to identify new anti-cancer agents by examining the expression of one or more markers when cancer cells or a cancer cell line is exposed to a potential anti-cancer agent. Importantly, these determinations can be made on a patient by patient basis or on an agent by agent (or combination of agents) basis. Thus, one can determine whether or not a particular therapeutic treatment is likely to benefit a particular patient or group/class of patients, or whether a particular treatment should be continued.

[0008] The present invention further provides previously unknown or unrecognized targets for the development of anti-cancer agents, such as chemotherapeutic compounds. The markers of the present invention can be used as targets in developing treatments (either single agent or multiple agent) for cancer, particularly for those cancers which display resistance to agents and exhibit expression of one or more of the markers identified herein, whose expression is correlated with resistance.

[0009] Other features and advantages of the invention will be apparent from the detailed description and from the claims. Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred materials and methods are described below.

DETAILED DESCRIPTION OF THE INVENTION

[0010] General Description

[0011] The present invention is based, in part, on the identification of markers that can be used to determine whether cancer cells are sensitive or resistant to a therapeutic agent. Based on these identifications, the present invention provides, without limitation: 1) methods for determining whether a therapeutic agent (or combination of agents) will or will not be effective in stopping or slowing tumor growth; 2) methods for monitoring the effectiveness of a therapeutic agent (or combination of agents) used for the treatment of cancer; 3) methods for identifying new therapeutic agents for the treatment of cancer; 4) methods for identifying combinations of therapeutic agents for use in treating cancer; and 5) methods for identifying specific therapeutic agents and combinations of therapeutic agents that are effective for the treatment of cancer in specific patients.

[0012] Definitions

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The content of all GenBank and NUC database records cited throughout this application (including the Tables) are also hereby incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0014] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0015] A “marker” is a naturally-occurring polymer corresponding to at least one of the nucleic acids, or genetic loci, listed in Tables 1 or 2. For example, markers include, without limitation, sense and anti-sense strands of genomic DNA (i.e. including any introns occurring therein), RNA generated by transcription of genomic DNA (i.e. prior to splicing), RNA generated by splicing of RNA transcribed from genomic DNA, and proteins generated by translation of spliced RNA (i.e. including proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences). As used herein, “marker” may also include a cDNA made by reverse transcription of an RNA generated by transcription of genomic DNA (including spliced RNA).

[0016] The term “probe” refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker of the invention. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.

[0017] The “normal” level of expression of a marker is the level of expression of the marker in cells of a patient not afflicted with cancer.

[0018] “Over-expression” and “under-expression” of a marker refer to expression of the marker of a patient at a greater or lesser level, respectively, than normal level of expression of the marker (e.g. at least two-fold greater or lesser level).

[0019] As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.

[0020] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell.

[0021] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0022] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0023] A “transcribed polynucleotide” is a polynucleotide (e.g. an RNA, a cDNA, or an analog of one of an RNA or cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a genomic DNA corresponding to a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the transcript.

[0024] “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0025] “Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0026] A marker is “fixed” to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the marker dissociating from the substrate.

[0027] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. encodes a natural protein).

[0028] Expression of a marker in a patient is “significantly” higher or lower than the normal level of expression of a marker if the level of expression of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess expression, and preferably at least twice, and more preferably three, four, five or ten times that amount. Alternately, expression of the marker in the patient can be considered “significantly” higher or lower than the normal level of expression if the level of expression is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal level of expression of the marker.

[0029] Cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

[0030] A cancer cell is “sensitive” to a therapeutic agent if its rate of growth is inhibited as a result of contact with the therapeutic agent, compared to its growth in the absence of contact with the therapeutic agent. The quality of being sensitive to a therapeutic agent is a variable one, with different cancer cells exhibiting different levels of “sensitivity” to a given therapeutic agent, under different conditions. In one embodiment of the invention, cancer cells may be predisposed to sensitivity to an agent if one or more of the corresponding sensitivity markers (Table 1) are expressed.

[0031] A cancer cell is “resistant” to a therapeutic agent if its rate of growth is not inhibited, or inhibited to a very low degree, as a result of contact with the therapeutic agent when compared to its growth in the absence of contact with the therapeutic agent. The quality of being resistant to a therapeutic agent is a highly variable one, with different cancer cells exhibiting different levels of “resistance” to a given therapeutic agent, under different conditions. In another embodiments of the invention, cancer cells may be predisposed to resistance to an agent if one or more of the corresponding resistant markers (Table 2) are expressed.

[0032] A kit is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting a marker of the invention, the manufacture being promoted, distributed, or sold as a unit for performing the methods of the present invention. The reagents included in such a kit comprise probes/primers and/or antibodies for use in detecting sensitivity and resistance gene expression. In addition, the kits of the present invention may preferably contain instructions which describe a suitable detection assay. Such kits can be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting symptoms of cancer.

Specific Embodiments

[0033] I. Identification of Sensitivity and Resistance Genes

[0034] The present invention provides genes that are expressed in cancer cells that are sensitive or resistant to a given therapeutic agent and whose expression correlates with sensitivity to that therapeutic agent. The present invention also provides genes that are expressed in cancer cell lines that are resistant to a given therapeutic agent and whose expression correlates with sensitivity to that therapeutic agent. Accordingly, one or more of the sensitivity or resistance genes can be used as markers (or surrogate markers) to identify cancer cells that can be successfully treated by that agent. In addition, these markers can be used to identify cancers that have become or are at risk of becoming refractory to treatment with the agent.

[0035] II. Determining Sensitivity or Resistance to an Agent

[0036] The expression level of the identified sensitivity and resistance genes, or the proteins encoded by the identified sensitivity and resistance genes, may be used to: 1) determine if a cancer can be treated by an agent or combination of agents; 2) determine if a cancer is responding to treatment with an agent or combination of agents; 3) select an appropriate agent or combination of agents for treating a cancer; 4) monitor the effectiveness of an ongoing treatment; and 5) identify new cancer treatments (either single agent or combination of agents). In particular, the identified sensitivity and resistance genes may be utilized as markers (surrogate and/or direct) to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy, and to develop new agents and therapeutic combinations.

[0037] Accordingly, the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent, can be used to reduce the growth rate of cancer cells comprising the steps of:

[0038] a) obtaining a sample of cancer cells;

[0039] b) determining whether the cancer cells express one or more markers identified in Tables 1 or 2 or both; and

[0040] c) identifying that an agent is or is not appropriate to treat the cancer based on the expression of the markers listed in Tables 1 or Table 2 or both.

[0041] In another embodiment, the invention provides a method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of:

[0042] a) obtaining a sample of cancer cells;

[0043] b) determining whether the cancer cells express one or more markers identified in Table 1; and

[0044] c) identifying that an agent is appropriate to treat the cancer when one or more markers listed in Table 1 are expressed by the cancer cells.

[0045] Alternatively, in step (c), an agent can be identified as not being appropriate to treat the cancer when one or more markers listed in Table 1 are not expressed by the cancer cells.

[0046] In another embodiment, the invention provides a method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of:

[0047] a) obtaining a sample of cancer cells;

[0048] b) determining whether the cancer cells express one or more markers identified in Table 2; and

[0049] c) identifying that an agent is appropriate to treat the cancer when one or more markers identified in Table 2 are not expressed by the cancer cells.

[0050] Alternatively, in step (c), an agent can be identified as not being appropriate to treat the cancer when one or more markers listed in Table 2 are expressed by the cancer cells.

[0051] In another embodiment, the invention provides a method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of:

[0052] a) obtaining a sample of cancer cells;

[0053] b) exposing some of the cancer cells to one or more test agents;

[0054] c) determining the level of expression in of one or more markers listed in Table 1 both in cancer cells exposed to the agent and in cancer cells that have not been exposed to the agent; and

[0055] d) identifying that an agent is appropriate to treat the cancer when the expression of the markers listed in Table 1 is increased in the presence of the agent.

[0056] Alternatively, in step (d), an agent can be identified as not being appropriate to treat the cancer when the expression of the markers listed in Table 1 is decreased in the presence of the agent.

[0057] In another embodiment, the invention provides a method for determining whether an agent can be used to reduce the growth of cancer cells, comprising the steps of:

[0058] a) obtaining a sample of cancer cells;

[0059] b) exposing some of the cancer cells to one or more test agents;

[0060] c) determining the level of expression in of one or more markers listed in Table 2 both in cancer cells exposed to the agent and in cancer cells that have not been exposed to the agent; and

[0061] d) identifying that an agent is not appropriate to treat the cancer when the expression of the markers listed in Table 2 is increased in the presence of the agent.

[0062] Alternatively, in step (d), an agent can be identified as being appropriate to treat the cancer when the expression of the markers listed in Table 2 is decreased in the presence of the agent.

[0063] In another embodiment, the invention provides a method for determining whether treatment with an anti-cancer agent should be continued in a cancer patient, comprising the steps of:

[0064] a) obtaining two or more samples of cancer cells from a patient at different times during the course of anti-cancer agent treatment;

[0065] b) determining the level of expression in the cancer cells of one or more genes which correspond to markers listed in Table 1 in the two or more samples; and

[0066] c) continuing the treatment when the expression level of the markers listed in Table 1 does not decrease during the course of treatment.

[0067] Alternatively, in step (c), the treatment is discontinued when the expression level of the markers listed in Table 1 is decreased during the course of treatment.

[0068] In another embodiment, the invention provides a method for determining whether treatment with an anti-cancer agent should be continued in a cancer patient, comprising the steps of:

[0069] a) obtaining two or more samples of cancer cells from a patient at different times during the course of anti-cancer agent treatment;

[0070] b) determining the level of expression in the cancer cells of one or more markers listed in Table 2 in the two or more samples; and

[0071] c) continuing the treatment when the expression level of one or more markers listed in Table 2 is not increased during the course of treatment.

[0072] Alternatively, in step (c), the treatment is discontinued when the expression level of one or more markers listed in Table 2 is increased during the course of treatment.

[0073] In another embodiment of the invention, the agent used in methods of the invention is a taxane compound. In another embodiment of the invention, the expression of genes which correspond to markers listed in Table 1 or Table 2 or both is detected by measuring mRNA which corresponds to the gene. In yet another embodiment of the invention, the expression of genes which correspond to markers listed in Table 1 or Table 2 or both is detected by measuring protein which corresponds to the gene. In a further another embodiment of the invention, the cancer cells or cancer cell lines used in the methods of the invention are obtained from a patient.

[0074] In another embodiment, the invention provides a method of treating a patient for cancer by administering to the patient a compound which has been identified as being effective against cancer by methods of the invention described herein.

[0075] As used herein, an agent is said to reduce the rate of growth of cancer cells when the agent can reduce at least 50%, preferably at least 75%, most preferably at least 95% of the growth of the cancer cells.

[0076] Such inhibition can further include a reduction in survivability and an increase in the rate of death of the cancer cells. The amount of agent used for this determination will vary based on the agent selected. Typically, the amount will be a predefined therapeutic amount.

[0077] As used herein, the term “agent” is defined broadly as anything that cancer cells may be exposed to in a therapeutic protocol. In the context of the present invention, such agents include, but are not limited to, chemotherapeutic agents, such as anti-metabolic agents, e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g., TAXOL, inblastine and vincristine, alkylating agents, e.g., melphanlan, BCNU and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin and DHAD, cross-linking agents, e.g., cisplatin and CBDCA, radiation and ultraviolet light. In a preferred embodiment, the agent is a taxane compound (e.g., TAXOL).

[0078] Further to the above, the language “chemotherapeutic agent” is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents are well known in the art (see e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases. The chemotherapeutic agents generally employed in chemotherapy treatments are listed below in Table A.

1TABLE ANONPROPRIETARYNAMESCLASSTYPE OF AGENT(OTHER NAMES)AlkylatingNitrogen MustardsMechlorethamine (HN2)CyclophosphaniideIfosfamideMelphalan (L-sarcolysin)ChlorambucilEthyleniminesHexamethylmelamineAnd MethylmelaminesThiotepaAlkyl SulfonatesBusulfanAlkylatingNitrosoureasCarmustine (BCNU)Lomustine (CCNU)Semustine (methyl-CCNU)Streptozocin(streptozotocin)TriazenesDecarbazine (DTIC;dimethyltriazenoimi-dazolecarboxamide)Alkylatorcis-diamminedichloroplatinumII (CDDP)Antimeta-Folic AcidMethotrexatebolitesAnalogs(amethopterin)PyrimidineFluorouracilAnalogs(′5-fluorouracil 5-FU)Floxuridine (fluorode-oxyuridine;FUdR)Cytarabine (cytosinearabinoside)Purine AnalogsMercaptopuineand Related(6-mercaptopurine;Inhibitors6-MP)Thioguanine(6-thioguanine; TG)Pentostatin (2′ - deoxycoformycin)NaturalVinca AlkaloidsVinblastin (VLB)ProductsVincristineTopoisomeraseEtoposideInhibitorsTeniposideCamptothecinTopotecan9-amino-campotothecin CPT-11AntibioticsDactinomycin(actinomycin D)AdriamycinDaunorubicin(daunomycin;rubindomycin)DoxorubicinBleomycinPlicamycin(mithramycin)Mitomycin (mitomycin C)TAXOLTaxotereEnzymesL-AsparaginaseBiologicalInterfon alfaResponseinterleukin 2ModifiersMiscella-Platinumcis-diamminedichloroplatinumneousCoordinationII (CDDP)AgentsComplexesCarboplatinAnthracendioneMitoxantroneSubstituted UreaHydroxyureaMethyl HydraxzineProcarbazineDerivative(N-methylhydrazine,(MIH)AdrenocorticalMitotaneSuppressant(o,p′-DDD)AminoglutehimideHormonesAdrenocorticosteroidsPrednisoneandProgestinsHydroxyprogesteroneAntagonistscaproateMedroxyprogesteroneacetateMegestrol acetateEstrogensDiethylstilbestrolEthinyl estradiolAntiestrogenTamoxifenAndrogensTestosterone propionateFluoxymesteroneAntiandrogenFlutamideGonadotropin-releasingLeuprolideHormone analog

[0079] The agents tested in the present methods can be a single agent or a combination of agents. For example, the present methods can be used to determine whether a single chemotherapeutic agent, such as methotrexate, can be used to treat a cancer or whether a combination of two or more agents can be used. Preferred combinations will include agents that have different mechanisms of action, e.g., the use of an anti-mitotic agent in combination with an alkylating agent.

[0080] As used herein, cancer cells refer to cells that divide at an abnormal (increased) rate. Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkins disease; and tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.

[0081] The source of the cancer cells used in the present method will be based on how the method of the present invention is being used. For example, if the method is being used to determine whether a patient's cancer can be treated with an agent, or a combination of agents, then the preferred source of cancer cells will be cancer cells obtained from a cancer biopsy from the patient. Alternatively, a cancer cell line similar to the type of cancer being treated can be assayed. For example if breast cancer is being treated, then a breast cancer cell line can be used. If the method is being used to monitor the effectiveness of a therapeutic protocol, then a tissue sample from the patient being treated is the preferred source. If the method is being used to identify new therapeutic agents or combinations, any cancer cells, e.g., cells of a cancer cell line, can be used.

[0082] A skilled artisan can readily select and obtain the appropriate cancer cells that are used in the present method. For cancer cell lines, sources such as The National Cancer Institute, for the NCI-60 cells used in the examples, are preferred. For cancer cells obtained from a patient, standard biopsy methods, such as a needle biopsy, can be employed.

[0083] In the methods of the present invention, the level or amount of expression of one or more genes selected from the group consisting of the genes identified in Table 1 and 2 is determined. As used herein, the level or amount of expression refers to the absolute level of expression of an mRNA encoded by the gene or the absolute level of expression of the protein encoded by the gene (i.e., whether or not expression is or is not occurring in the cancer cells).

[0084] Generally, it is preferable to determine the expression of two or more of the identified sensitivity or resistance genes, more preferably, three or more of the identified sensitivity or resistance genes, most preferably all of the identified sensitivity and/or resistance genes. Thus, it is preferable to assess the expression of a panel of sensitivity and resistance genes.

[0085] As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels. Expression levels are normalized by correcting the absolute expression level of a sensitivity or resistance gene by comparing its expression to the expression of a gene that is not a sensitivity or resistance gene, e.g., a housekeeping genes that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows one to compare the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancer sample, or between samples from different sources.

[0086] Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question. The expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of sensitivity or resistance.

[0087] Preferably, the samples used will be from similar tumors or from non-cancerous cells of the same tissue origin as the tumor in question. The choice of the cell source is dependent on the use of the relative expression level data. For example, using tumors of similar types for obtaining a mean expression score allows for the identification of extreme cases of sensitivity or resistance. Using expression found in normal tissues as a mean expression score aids in validating whether the sensitivity/resistance gene assayed is tumor specific (versus normal cells). Such a later use is particularly important in identifying whether a sensitivity or resistance gene can serve as a target gene. In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data.

[0088] III. Isolated Nucleic Acid Molecules

[0089] One aspect of the invention pertains to isolated nucleic acid molecules that correspond to a marker of the invention, including nucleic acids which encode a polypeptide corresponding to a marker of the invention or a portion of such a polypeptide. Isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules that correspond to a marker of the invention, including nucleic acids which encode a polypeptide corresponding to a marker of the invention, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0090] An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an “isolated” nucleic acid molecule is free of sequences (preferably protein-encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0091] A nucleic acid molecule of the present invention, e.g., a nucleic acid encoding a protein corresponding to a marker listed in Tables 1 and/or 2, can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0092] A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0093] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which has a nucleotide sequence complementary to the nucleotide sequence of a nucleic acid corresponding to a marker of the invention or to the nucleotide sequence of a nucleic acid encoding a protein which corresponds to a marker of the invention. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.

[0094] Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises a marker of the invention or which encodes a polypeptide corresponding to a marker of the invention. Such nucleic acids can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.

[0095] Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more markers of the invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.

[0096] The invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein which corresponds to a marker of the invention, and thus encode the same protein.

[0097] In addition to the nucleotide sequences described in the GenBank and NUC database records described herein, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).

[0098] As used herein, the phrase “allelic variant” refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.

[0099] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

[0100] In another embodiment, an isolated nucleic acid molecule of the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid corresponding to a marker of the invention or to a nucleic acid encoding a protein corresponding to a marker of the invention. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 75% (80%, 85%, preferably 90%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). A preferred, non-limiting example of stringent hybridization conditions for annealing two single-stranded DNA each of which is at least about 100 bases in length and/or for annealing a single-stranded DNA and a single-stranded RNA each of which is at least about 100 bases in length, are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Further preferred hybridization conditions are taught in Lockhart, et al., Nature Biotechnology, Volume 14, 1996 August:1675-1680; Breslauer, et al., Proc. Natl. Acad. Sci. USA, Volume 83, 1986 June: 3746-3750; Van Ness, et al., Nucleic Acids Research, Volume 19, No. 19, 1991 September: 5143-5151; McGraw, et al., BioTechniques, Volume 8, No. 6 1990: 674-678; and Milner, et al., Nature Biotechnology, Volume 15, 1997 June: 537-541, all expressly incorporated by reference.

[0101] In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention that can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby. For example, one can make nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.

[0102] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from the naturally-occurring proteins which correspond to the markers of the invention, yet retain biological activity. In one embodiment, such a protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of one of the proteins which correspond to the markers of the invention.

[0103] An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0104] The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule corresponding to a marker of the invention or complementary to an mRNA sequence corresponding to a marker of the invention. Accordingly, an antisense nucleic acid of the invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences which flank the coding region and are not translated into amino acids.

[0105] An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0106] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide corresponding to a selected marker of the invention to thereby inhibit expression of the marker, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into an ovary-associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0107] An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

[0108] The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide corresponding to a marker of the invention can be designed based upon the nucleotide sequence of a cDNA corresponding to the marker. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

[0109] The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

[0110] In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

[0111] PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670-675).

[0112] In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5′ end of DNA (Mag et al, 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

[0113] In other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[0114] The invention also includes molecular beacon nucleic acids having at least one region which is complementary to a nucleic acid of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid of the invention in a sample. A “molecular beacon” nucleic acid is a nucleic acid comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher. When the complementary regions of the nucleic acid are not annealed with one another, fluorescence of the fluorophore is quenched to a lesser degree. Molecular beacon nucleic acids are described, for example, in U.S. Pat. No. 5,876,930.

[0115] IV. Isolated Proteins and Antibodies

[0116] One aspect of the invention pertains to isolated proteins which correspond to individual markers of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide corresponding to a marker of the invention. In one embodiment, the native polypeptide corresponding to a marker can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides corresponding to a marker of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide corresponding to a marker of the invention can be synthesized chemically using standard peptide synthesis techniques.

[0117] An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

[0118] Biologically active portions of a polypeptide corresponding to a marker of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein corresponding to the marker (e.g., the amino acid sequence listed in the GenBank and NUC database records described herein), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.

[0119] Preferred polypeptides have the amino acid sequence listed in the one of the GenBank and NUC database records described herein. Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.

[0120] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total# of positions (e.g., overlapping positions)×100). In one embodiment the two sequences are the same length.

[0121] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PAM120 weight residue table can, for example, be used with a k-tuple value of 2.

[0122] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

[0123] The invention also provides chimeric or fusion proteins corresponding to a marker of the invention. As used herein, a “chimeric protein” or “fusion protein” comprises all or part (preferably a biologically active part) of a polypeptide corresponding to a marker of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker). Within the fusion protein, the term “operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the amino-terminus or the carboxyl-terminus of the polypeptide of the invention.

[0124] One useful fusion protein is a GST fusion protein in which a polypeptide corresponding to a marker of the invention is fused to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.

[0125] In another embodiment, the fusion protein contains a heterologous signal sequence at its amino terminus. For example, the native signal sequence of a polypeptide corresponding to a marker of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

[0126] In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide corresponding to a marker of the invention is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction can be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.

[0127] Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et al, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.

[0128] A signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteolytically cleaved (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

[0129] The present invention also pertains to variants of the polypeptides corresponding to individual markers of the invention. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.

[0130] Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

[0131] In addition, libraries of fragments of the coding sequence of a polypeptide corresponding to a marker of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and internal fragments of various sizes of the protein of interest.

[0132] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering 6(3):327-331).

[0133] An isolated polypeptide corresponding to a marker of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acid residues of the amino acid sequence of one of the polypeptides of the invention, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with a marker of the invention to which the protein corresponds. Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydrophobicity sequence analysis, hydrophilicity sequence analysis, or similar analyses can be used to identify hydrophilic regions.

[0134] An immunogen typically is used to prepare antibodies by immunizing a suitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

[0135] Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The terms “antibody” and “antibody substance” as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.

[0136] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.

[0137] The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be harvested or isolated from the subject (e.g., from the blood or serum of the subject) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.

[0138] At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

[0139] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

[0140] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al (1986) Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0141] Antibodies of the invention may be used as therapeutic agents in treating cancers. In a preferred embodiment, completely human antibodies of the invention are used for therapeutic treatment of human cancer patients, particularly those having an cancer. Such antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide corresponding to a marker of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0142] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1994, Bio/technology 12:899-903).

[0143] An antibody directed against a polypeptide corresponding to a marker of the invention (e.g., a monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the marker (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the marker. The antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in an ovary-associated body fluid) as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0144] Further, an antibody (or fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0145] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0146] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982).

[0147] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0148] Accordingly, in one aspect, the invention provides substantially purified antibodies or fragments thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. In various embodiments, the substantially purified antibodies of the invention, or fragments thereof, can be human, non-human, chimeric and/or humanized antibodies.

[0149] In another aspect, the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.

[0150] In still a further aspect, the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to an amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6×SSC at 45° C. and washing in 0.2 ×SSC, 0.1% SDS at 65° C. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.

[0151] The substantially purified antibodies or fragments thereof may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain or cytoplasmic membrane of a polypeptide of the invention. In a particularly preferred embodiment, the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of the present invention.

[0152] Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.

[0153] The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.

[0154] Still another aspect of the invention is a method of making an antibody that specifically recognizes a polypeptide of the present invention, the method comprising immunizing a mammal with a polypeptide. The polypeptide used as an immnungen comprises an amino acid sequence selected from the group consisting of the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA of the nucleic acid molecules of the present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C.

[0155] After immunization, a sample is collected from the mammal that contains an antibody that specifically recognizes the polypeptide. Preferably, the polypeptide is recombinantly produced using a non-human host cell. Optionally, the antibodies can be further purified from the sample using techniques well known to those of skill in the art. The method can further comprise producing a monoclonal antibody-producing cell from the cells of the mammal. Optionally, antibodies are collected from the antibody-producing cell.

[0156] V. Recombinant Expression Vectors and Host Cells

[0157] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide corresponding to a marker of the invention (or a portion of such a polypeptide). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0158] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology vol. 185, Academic Press, San Diego, Calif. (1991). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0159] The recombinant expression vectors of the invention can be designed for expression of a polypeptide corresponding to a marker of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells {using baculovirus expression vectors}, yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0160] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0161] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., p. 60-89, In Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, Calif., 1991). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0162] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, p. 119-128, In Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, Calif., 1990. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0163] In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.).

[0164] Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).

[0165] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.

[0166] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the α-fetoprotein promoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).

[0167] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue-specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., 1986, Trends in Genetics, Vol. 1(1).

[0168] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0169] A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).

[0170] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.

[0171] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0172] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide corresponding to a marker of the invention. Accordingly, the invention further provides methods for producing a polypeptide corresponding to a marker of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the marker is produced. In another embodiment, the method further comprises isolating the marker polypeptide from the medium or the host cell.

[0173] The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide corresponding to a marker of the invention have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a marker protein of the invention have been introduced into their genome or homologous recombinant animals in which endogenous gene(s) encoding a polypeptide corresponding to a marker of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide corresponding to the marker and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0174] A transgenic animal of the invention can be created by introducing a nucleic acid encoding a polypeptide corresponding to a marker of the invention into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, 4,873,191 and in Hogan, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

[0175] To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide corresponding to a marker of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, 1987, Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al., 1992, Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

[0176] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991, Science 251:1351-1355). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0177] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

[0178] VI. Pharmaceutical Compositions

[0179] The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as “active compounds”) corresponding to a marker of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0180] The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid corresponding to a marker of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid corresponding to a marker of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid corresponding to a marker of the invention and one or more additional active compounds.

[0181] The invention also provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the marker, or (b) have a modulatory (e.g., stimulatory or inhibitory) effect on the activity of the marker or, more specifically, (c) have a modulatory effect on the interactions of the marker with one or more of its natural substrates (e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d) have a modulatory effect on the expression of the marker. Such assays typically comprise a reaction between the marker and one or more assay components. The other components may be either the test compound itself, or a combination of test compound and a natural binding partner of the marker.

[0182] The test compounds of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Test compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).

[0183] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0184] Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).

[0185] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a marker or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to a marker or biologically active portion thereof. Determining the ability of the test compound to directly bind to a marker can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to the marker can be determined by detecting the labeled marker compound in a complex. For example, compounds (e.g., marker substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0186] In another embodiment, the invention provides assays for screening candidate or test compounds which modulate the activity of a marker or a biologically active portion thereof. In all likelihood, the marker can, in vivo, interact with one or more molecules, such as but not limited to, peptides, proteins, hormones, cofactors and nucleic acids. For the purposes of this discussion, such cellular and extracellular molecules are referred to herein as “binding partners” or marker “substrate”.

[0187] One necessary embodiment of the invention in order to facilitate such screening is the use of the marker to identify its natural in vivo binding partners. There are many ways to accomplish this which are known to one skilled in the art. One example is the use of the marker protein as “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al , 1993, Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696; Brent WO94/10300) in order to identify other proteins which bind to or interact with the marker (binding partners) and, therefore, are possibly involved in the natural function of the marker. Such marker binding partners are also likely to be involved in the propagation of signals by the marker or downstream elements of a marker-mediated signaling pathway. Alternatively, such marker binding partners may also be found to be inhibitors of the marker.

[0188] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that encodes a marker protein fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a marker-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be readily detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the marker protein.

[0189] In a further embodiment, assays may be devised through the use of the invention for the purpose of identifying compounds which modulate (e.g., affect either positively or negatively) interactions between a marker and its substrates and/or binding partners. Such compounds can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof. Such compounds may also be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. The preferred assay components for use in this embodiment is an cancer marker identified herein, the known binding partner and/or substrate of same, and the test compound. Test compounds can be supplied from any source.

[0190] The basic principle of the assay systems used to identify compounds that interfere with the interaction between the marker and its binding partner involves preparing a reaction mixture containing the marker and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the marker and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the marker and its binding partner is then detected. The formation of a complex in the control reaction, but less or no such formation in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the marker and its binding partner. Conversely, the formation of more complex in the presence of compound than in the control reaction indicates that the compound may enhance interaction of the marker and its binding partner.

[0191] The assay for compounds that interfere with the interaction of the marker with its binding partner may be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the marker or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the markers and the binding partners (e.g., by competition) can be identified by conducting the reaction in the presence of the test substance, i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the marker and its interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[0192] In a heterogeneous assay system, either the marker or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non-anchored component may be labeled, either directly or indirectly. In practice, microtitre plates are often utilized for this approach. The anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the marker or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored.

[0193] In related embodiments, a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix. For example, glutathione-S-transferase/marker fusion proteins or glutathione-S-transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed marker or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound assay components, the immobilized complex assessed either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of marker binding or activity determined using standard techniques.

[0194] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a marker or a marker binding partner can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the protein-immobilized surfaces can be prepared in advance and stored.

[0195] In order to conduct the assay, the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted assay components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected.

[0196] In an alternate embodiment of the invention, a homogeneous assay may be used. This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test compound. The formed complexes are then separated from unreacted components, and the amount of complex formed is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test compounds modulate (inhibit or enhance) complex formation and which disrupt preformed complexes.

[0197] In such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, complexes of molecules may be separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sci 1993 August; 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11I:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl., 699:499-525). Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, nondenaturing gels in the absence of reducing agent are typically preferred, but conditions appropriate to the particular interactants will be well known to one skilled in the art. Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation. The bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in complex formation in both the presence and the absence of a test compound can be compared, thus offering information about the ability of the compound to modulate interactions between the marker and its binding partner.

[0198] Also within the scope of the present invention are methods for direct detection of interactions between the marker and its natural binding partner and/or a test compound in a homogeneous or heterogeneous assay system without further sample manipulation. For example, the technique of fluorescence energy transfer may be utilized (see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No. 4,868,103). Generally, this technique involves the addition of a fluorophore label on a first ‘donor’ molecule (e.g., marker or test compound) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule (e.g., marker or test compound), which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). A test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background. In this way, test substances that modulate interactions between a marker and its binding partner can be identified in controlled assays.

[0199] In another embodiment, modulators of marker expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA or protein, corresponding to a marker in the cell, is determined. The level of expression of mRNA or protein in the presence of the candidate compound is compared to the level of expression of mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of marker expression based on this comparison. For example, when expression of marker mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of marker mRNA or protein expression. Conversely, when expression of marker mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of marker mRNA or protein expression. The level of marker mRNA or protein expression in the cells can be determined by methods described herein for detecting marker mRNA or protein. In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a marker protein can be further confirmed in vivo, e.g., in a whole animal model for cellular transformation and/or tumorigenesis.

[0200] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., an marker modulating agent, an antisense marker nucleic acid molecule, an marker-specific antibody, or an marker-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0201] It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Exemplary doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 5 grams per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram). It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g. a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0202] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0203] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0204] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0205] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0206] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0207] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0208] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0209] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0210] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0211] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0212] For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the epithelium). A method for lipidation of antibodies is described by Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193.

[0213] The nucleic acid molecules corresponding to a marker of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0214] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0215] VII. Monitoring the Effectiveness of an Anti-Cancer Agent

[0216] As discussed above, the identified sensitivity and resistance genes can also be used as markers to assess whether a tumor has become refractory to an ongoing treatment (e.g., a chemotherapeutic treatment). When a tumor is no longer responding to a treatment the expression profile of the tumor cells will change: the level of expression of one or more of the sensitivity genes will be reduced and the level of expression of one or more of the resistance genes will increase.

[0217] In such a use, the invention provides methods for determining whether an anti-cancer treatment should be continued in a cancer patient, comprising the steps of:

[0218] a) obtaining two or more samples of cancer cells from a patient undergoing anti-cancer therapy;

[0219] b) determining the level of expression of one or more genes selected from the group consisting of the sensitivity genes (Table 1) and the resistance genes (Table 2) in the sample exposed to the agent and in a sample of cancer cells that is not exposed to the agent; and

[0220] c) discontinuing or altering treatment when the expression of one or more sensitivity genes decreases or when the expression of one or more resistance genes increases.

[0221] As used here, a patient refers to any subject undergoing treatment for cancer. The preferred subject will be a human patient undergoing chemotherapy treatment.

[0222] This embodiment of the present invention relies on comparing two or more samples obtained from a patient undergoing anti-cancer treatment. In general, it is preferable to obtain a first sample from the patient prior to beginning therapy and one or more samples during treatment. In such a use, a baseline of expression prior to therapy is determined and then changes in the baseline state of expression is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pretreatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of a particular gene is increasing or decreasing.

[0223] In general, when monitoring the effectiveness of a therapeutic treatment, two or more samples from the patient are examined. Preferably, three or more successively obtained samples are used, including at least one pretreatment sample.

[0224] VIII. Detection Assays

[0225] An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample involves obtaining a biological sample (e.g. an ovary-associated body fluid) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide corresponding to a marker of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide corresponding to a marker of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0226] A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

[0227] For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

[0228] There are many established methods for anchoring assay components to a solid phase. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.

[0229] Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

[0230] In order to conduct assays with the above mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

[0231] In a preferred embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.

[0232] It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0233] In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0234] Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter 11 (1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 October 10;699(1-2):499-525). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

[0235] In a particular embodiment, the level of mRNA corresponding to the marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).

[0236] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.

[0237] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.

[0238] An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0239] For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.

[0240] As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancer sample, or between samples from different sources.

[0241] Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.

[0242] Preferably, the samples used in the baseline determination will be from cancer or from non-cancer cells of tissue. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is specific (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from cells provides a means for grading the severity of the cancer state.

[0243] In another embodiment of the present invention, a polypeptide corresponding to a marker is detected. A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide corresponding to a marker of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[0244] Proteins from cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0245] A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express a marker of the present invention.

[0246] In one format, antibodies, or antibody fragments, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

[0247] One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

[0248] The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample (e.g. an ovary-associated body fluid such as a urine sample). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing cancer. For example, the kit can comprise a labeled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for interpreting the results obtained using the kit.

[0249] For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.

[0250] For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[0251] IX. Electronic Apparatus Readable Media and Arrays

[0252] Electronic apparatus readable media comprising a marker of the present invention is also provided. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon a marker of the present invention.

[0253] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[0254] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the markers of the present invention.

[0255] A variety of software programs and formats can be used to store the marker information of the present invention on the electronic apparatus readable medium. For example, the nucleic acid sequence corresponding to the markers can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of dataprocessor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the markers of the present invention.

[0256] By providing the markers of the invention in readable form, one can routinely access the marker sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the present invention in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[0257] The invention also includes an array comprising a marker of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[0258] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0259] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of breastcancer, progression of cancer, and processes, such a cellular transformation associated with cancer.

[0260] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0261] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes that could serve as a molecular target for diagnosis or therapeutic intervention.

SPECIFIC EXAMPLES

[0262] At least some of the examples set forth below relate to sensitivity or resistance to TAXOL. TAXOL is a chemical compound within a family of taxane compounds which are art-recognized as being a family of related compounds. The language “taxane compound” is intended to include TAXOL, compounds which are structurally similar to TAXOL and/or analogs of TAXOL. The language “taxane compound” can also include “mimics”. “Mimics” is intended to include compounds which may not be structurally similar to TAXOL but mimic the therapeutic activity of TAXOL or structurally similar taxane compounds in vivo. The taxane compounds of this invention are those compounds which are useful for inhibiting tumor growth in subjects (patients). The term taxane compound also is intended to include pharmaceutically acceptable salts of the compounds. Taxane compounds have previously been described in U.S. Pat. Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, 5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference.

[0263] The structure of TAXOL, shown below, offers many groups capable of being synthetically functionalized to alter the physical or pharmaceutical properties of TAXOL.

[0264] For example, a well known semi-synthetic analog of TAXOL, named Taxotere (docetaxel), has also been found to have good anti-tumor activity in animal models. Taxotere has t-butoxy amide at the 3′ position and a hydroxyl group at the C10 position (U.S. Pat. No. 5,840,929).

[0265] Other examples of TAXOL derivatives include those mentioned in U.S. Pat. No. 5,840,929 which are directed to derivatives of TAXOL having the formula:

[0266] wherein R1 is hydroxy, —OC(O)Rx, or —OC(O)ORx; R2 is hydrogen, hydroxy, —OC(O)Rx, or —OC(O)ORx; R2′ is hydrogen, hydroxy, or fluoro; R6′ is hydrogen or hydroxy or R2′ and R6′ can together form an oxirane ring; R3 is hydrogen, C1-6 alkyloxy, hydroxy, —OC(O)Rx, —OC(O)ORx, —OCONR7R11; R8 is methyl or R8 and R2 together can form a cyclopropane ring; R6 is hydrogen or R6 and R2 can together form a bond; R9 is hydroxy or —OC(O)Rx; R7 and R11 are independently C1-6 alkyl, hydrogen, aryl, or substituted aryl; R4 and R5 are independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, or —Z—R10; Z is a direct bond, C1-6 alkyl, or C2-6 alkenyl; R10 is aryl, substituted aryl, C3-6 cycloalkyl, C2-6 alkenyl, C1-6 alkyl, all can be optionally substituted with one to six same or different halogen atoms or hydroxy; Rx is a radical of the formula:

[0267] wherein D is a bond or C1-6 alkyl; and Ra, Rb and Rc are independently hydrogen, amino, C1-6 alkyl or C1-6 alkoxy.

[0268] Further examples of Rx include methyl, hydroxymethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, chloromethyl, 2,2,2-trichloroethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethenyl, 2-propenyl, phenyl, benzyl, bromophenyl, 4-aminophenyl, 4-methylaminophenyl, 4-methylphenyl, 4-methoxyphenyl and the like. Examples of R4 and R5 include 2-propenyl, isobutenyl, 3-furanyl (3-furyl), 3-thienyl, phenyl, naphthyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, ethenyl, 2-propenyl, 2-propynyl, benzyl, phenethyl, phenylethenyl, 3,4-dimethoxyphenyl, 2-furanyl (2-furyl), 2-thienyl, 2-(2-furanyl)ethenyl, 2-methylpropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl and the like.

[0269] TAXOL derivatives can be readily made by following the well established paclitaxel chemistry. For example, C2, C6, C7, C10, and/or C8 position can be derivatized by essentially following the published procedure, into a compound in which R3, R8, R2, R2′, R9, R6′ and R6 have the meanings defined earlier. Subsequently, C4-acetyloxy group can be converted to the methoxy group by a sequence of steps. For example, for converting C2-benzoyloxy to other groups see, S. H. Chen et al, Bioorganic and Medicinal Chemistry Letters, Vol. 4, No. 3, pp 479-482 (1994); for modifying C10-acetyloxy see, J. Kant et al, Tetrahedron Letters, Vol. 35, No. 31, pp 5543-5546 (1994) and U.S. Pat. No. 5,294,637 issued Mar. 15, 1994; for making C10 and/or C7 unsubstituted (deoxy) derivatives see, European Patent Application 590 267A2 published Apr. 6, 1994 and PCT application WO 93/06093 published Apr. 1, 1993; for making 7β,8β-methano, 6,7-α,α-dihydroxy and 6,7-olefinic groups see, R. A. Johnson, Tetrahedron Letters, Vol. 35, No 43, pp 7893-7896 (1994), U.S. Pat. No. 5,254,580, issued Oct. 19, 1993, and European Patent Application 600 517A1 published Jun. 8, 1994; for making C7/C6 oxirane see, U.S. Pat. No. 5,395,850 issued Mar. 7, 1995; for making C7-epi-fluoro see, G. Roth et al, Tetrahedron Letters, Vol 36, pp 1609-1612 (1993); for forming C7 esters and carbonates see, U.S. Pat. No. 5,272,171 issued Dec. 21, 1993 and S. H. Chen et al., Tetrahedron, 49, No. 14, pp 2805-2828 (1993).

[0270] In U.S. Pat. No. 5,773,464, TAXOL derivatives containing epoxides at the C10 position are disclosed as antitumor agents. Other C-10 taxane analogs have also appeared in the literature. Taxanes with alkyl substituents at C-10 have been reported in a published PCT patent application WO 9533740. The synthesis of C-10 epi hydroxy or acyloxy compounds is disclosed in PCT application WO 96/03394. Additional C-10 analogs have been reported in Tetrahedron Letters 1995, 36(12), 1985-1988; J. Org. Chem. 1994, 59, 4015-4018 and references therein; K. V. Rao et. al. Journal of Medicinal Chemistry 1995, 38 (17), 3411-3414; J. Kant et. al. Tetrahedron Lett. 1994, 35(31), 5543-5546; WO 9533736; WO 93/02067; U.S. Pat. No. 5,248,796; WO 9415929; and WO 94/15599.

[0271] Other relevant TAXOL derivatives include the sulfenamide taxane derivatives described in U.S. Pat. No. 5,821,263. These compounds are charachterized by the C3′ nitrogen bearing one or two sulfur substiuents. These compounds have been useful in the treatment of cancers such as ovarian, breast, lung, gastic, colon, head, neck, melanoma, and leukemia.

[0272] U.S. Pat. No. 4,814,470 discusses TAXOL derivatives with hydroxyl or acetyl group at the C10 position and hydroxy or t-butylcarbonyl at C2′ and C3′ positions.

[0273] U.S. Pat. No. 5,438,072 discusses TAXOL derivatives with hydroxyl or acetate groups at the C10 position and a C2′ substitutuent of either t-butylcarbonyl or benzoylamino.

[0274] U.S. Pat. No. 4,960,790 discusses derivatives of TAXOL which have, at the C2′ and/or C7 position a hydrogen, or the residue of an amino acid selected from the group consisting of alanine, leucine, isoleucine, saline, phenylalanine, proline, lysine, and arginine, or a group of the formula:

[0275] wherein n is an integer of 1 to 3 and R2 and R3 are each hydrogen on an alkyl radical having one to three carbon atoms or wherein R2 and R3 together with the nitrogen atom to which they are attached form a saturated heterocyclic ring having four to five carbon atoms, with the proviso that at least one of the substituents are not hydrogen.

[0276] Other similar water soluble TAXOL derivatives are discussed in U.S. Pat. Nos. 4,942,184, 5,433,364, and in 5,278,324.

[0277] Many TAXOL derivatives may also include protecting groups such as, for example, hydroxy protecting groups. “Hydroxy protecting groups” include, but are not limited to, ethers such as methyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl, methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl, dialkylsilylethers, such as dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether, and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl, benzyl, and p-nitrophenyl. Additional examples of hydroxy protecting groups may be found in standard reference works such as Greene and Wuts, Protective Groups in Organic Synthesis, 2d Ed., 1991, John Wiley & Sons, and McOmie; and Protective Groups in Organic Chemistry, 1975, Plenum Press. Methods for introducing and removing protecting groups are also found in such textbooks.

[0278] A. Generation of Subtracted Libraries

[0279] Subtracted libraries are generated using a PCR based method that allows the isolation of clones expressed at higher levels in one population of mRNA (tester) compared to another population (driver). Both tester and driver mRNA populations are converted into cDNA by reverse transcription, and then PCR amplified using the SMART PCR kit from Clontech. Tester and driver cDNAs are then hybridized using the PCR-Select cDNA subtraction kit from Clontech. This technique results in both subtraction and normalization, which is an equalization of copy number of low-abundance and high-abundance sequences. After generation of the subtractive libraries, a group of 96 or more clones from each library is tested to confirm differential expression by reverse Southern hybridization.

[0280] RNA was generated and pooled from two groups of cancer cell lines shown in Tables B and C. One group of nine cell lines was determined to be sensitive to TAXOL (Table C), the other group of nine cell lines was determined to be resistant to TAXOL (Table B). Sensitivity to TAXOL was based of known GI50 values for these cells, which for this study was defined as the concentration of TAXOL required to inhibit growth of the cell line by 50%. More precisely, the quantity used in the calculation is the potency measure −log{GI50}. Pooled RNA from TAXOL sensitive cancer cell lines was used as tester against driver RNA pooled from TAXOL resistant cancer cell lines. The results of this subtractive library are shown in Table 1. Pooled RNA from TAXOL resistant cancer cell lines was used as tester against driver RNA pooled from TAXOL sensitive cancer cell lines. The results of this subtractive library are shown in Table 2.

[0281] Tables 1 and 2 show the accession number (“Accession #”) of the markers of the present invention. The accession number is the identification number assigned to the marker in the relevant database (see, e.g. http://www.ncbi.nlm.nih.gov/genbank/guery form.html). Table 3 shows the accession number (“Acc. No.”) of the markers of the present invention with the corresponding GenBank GI number (“GI No.”) The GenBank GI number is the identification number assigned the marker in the GenBank database (see supra). One skilled in the art can thus obtain from the Tables of the present invention both the GenBank accession number as well as the GenBank GI number for a marker of the present invention, thereby identifying the nucleotide and/or polypeptide sequence of that marker.

2TABLE BTAXOLResistantLog Gl 50 forTissue of OriginCell LineTAXOLNon-small cell lungEKVX−6.6carcinomaNon-small cell lungHOP-92−7.2carcinomaColonHCT-15−6.7MelanomaMALME-3M−6.8MelanomaSK-MEL-28−7.1OvarianOVCAR-4−6.3RenalACHN−5.8BreastMCF-−5.57/AdrResBreastT-47D−6.9−6.5(Mean)

[0282]

3

TABLE C

TAXOL

Sensitive
Log GI 50 for

Tissue of Origin
Cell Line
TAXOL

Non-small cell lung
NCl-H460
−8.5

carcinoma

Non-small cell lung
NCl-H522
−8.5

carcinoma

Colon
HT-29
−8.6

Melanoma
SK-MEL-2
−8.3

Melanoma
SK-MEL-5
−8.4

Ovarian
OVCAR-3
−8.5

Renal
SN12C
−8.5

Breast
MCF-7
−8.5

Breast
MDA-MB-
−8.6

435

−8.5

(Mean)

[0283] B. Sensitivity Assays and Identification of Therapeutic and Drug Screening Targets

[0284] A sample of cancerous cells with unknown sensitivity to a given drug is obtained from a patient. An expression level is measured in the sample for a gene corresponding to one of the markers identified in either Table 1 and/or in Table 2. If the gene is expressed, and the marker of the invention to which the gene corresponds is listed among the markers of Table 1, then the drug will be effective against the cancer. Accordingly, if the gene is not expressed, and the marker of the invention to which the gene corresponds is listed among in the markers of Table 1, then the drug will not be effective against the cancer. If the gene is expressed, and the marker of the invention to which the gene corresponds is listed among the markers of Table 2, then the drug will not be effective against the cancer. Accordingly, if the gene is not expressed, and the marker of the invention to which the gene corresponds is listed among the markers of Table 2, then the drug will be effective against the cancer.

[0285] Thus, by examining the expression of one or more of the identified markers in a sample of cancer cells, it is possible to determine which therapeutic agent(s), or combination of agents, to use as the appropriate treatment agents.

[0286] By examining the expression of one or more of the identified markers in a sample of cancer cells taken from a patient during the course of therapeutic treatment, it is also possible to determine whether the therapeutic agent is continuing to work or whether the cancer has become resistant (refractory) to the treatment protocol. For example, a cancer patient receiving a treatment of paclitaxel would have cancer cells removed and monitored for the expression of a marker. If the expression level of a marker remains substantially the same, the treatment with paclitaxel would continue. However, a significant change in marker expression would suggest that the cancer may have become resistant to paclitaxel and another chemotherapy protocol should be initiated to treat the patient.

[0287] Importantly, these determinations can be made on a patient by patient basis or on an agent by agent (or combinations of agents). Thus, one can determine whether or not a particular therapeutic treatment is likely to benefit a particular patient or group/class of patients, or whether a particular treatment should be continued.

[0288] The identified markers further provide previously unknown or unrecognized targets for the development of anti-cancer agents, such as chemotherapeutic compounds, and can be used as targets in developing single agent treatment as well as combinations of agents for the treatment of cancer.

[0289] Other Embodiments

[0290] The present invention is not to be limited in scope by the specific embodiments described that are intended as single illustrations of individual aspects of the invention and functionally equivalent methods and components are within the scope of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0291] All references cited herein, including journal articles, patents, and databases are expressly incorporated by reference.

4TABLE 1Sequence IDAccession #Sequence 1AA001696Sequence 2AA002128Sequence 3AA009923Sequence 4AA010575Sequence 5AA010689Sequence 6AA010893Sequence 7AA011002Sequence 8AA022748Sequence 9AA022943Sequence 10AA025349Sequence 11AA028882Sequence 12AA034237Sequence 13AA036750Sequence 14AA037181Sequence 15AA040929Sequence 16AA043103Sequence 17AA043137Sequence 18AA045176Sequence 19AA046473Sequence 20AA046810Sequence 21AA046888Sequence 22AA047054Sequence 23AA054771Sequence 24AA056334Sequence 25AA058936Sequence 26AA069078Sequence 27AA069560Sequence 28AA069850Sequence 29AA071084Sequence 30AA074035Sequence 31AA074291Sequence 32AA074845Sequence 33AA075527Sequence 34AA081348Sequence 35AA082884Sequence 36AA083270Sequence 37AA083410Sequence 38AA085444Sequence 39AA085511Sequence 40AA088344Sequence 41AA088758Sequence 42AA095772Sequence 43AA099904Sequence 44AAI00707Sequence 45AAI01783Sequence 46AAI02138Sequence 47AAI02721Sequence 48AAI02853Sequence 49AAI13420Sequence 50AAI15218Sequence 51AAI15838Sequence 52AAI21574Sequence 53AAI25927Sequence 54AAI27186Sequence 55AAI28091Sequence 56AAI28878Sequence 57AAI28965Sequence 58AAI30823Sequence 59AAI31227Sequence 60AAI32844Sequence 61AAI36789Sequence 62AAI42909Sequence 63AAI43438Sequence 64AAI43746Sequence 65AAI47080Sequence 66AAI49963Sequence 67AAI56443Sequence 68AAI56615Sequence 69AAI56616Sequence 70AAI57300Sequence 71AAI60517Sequence 72AAI61269Sequence 73AAI66632Sequence 74AAI66675Sequence 75AAI67700Sequence 76AAI67814Sequence 77AAI73279Sequence 78AAI74034Sequence 79AAI76813Sequence 80AAI79439Sequence 81AAI81153Sequence 82AAI81811Sequence 83AAI81858Sequence 84AAI87817Sequence 85AAI88680Sequence 86AAI88832Sequence 87AAI91341Sequence 88AAI95178Sequence 89AAI96515Sequence 90AAI99684Sequence 91AA203284Sequence 92AA205412Sequence 93AA211509Sequence 94AA211584Sequence 95AA213580Sequence 96AA215584Sequence 97AA216094Sequence 98AA223381Sequence 99AA224124Sequence 100AA224163Sequence 101AA225289Sequence 102AA226279Sequence 103AA235063Sequence 104AA235365Sequence 105AA251312Sequence 106AA256442Sequence 107AA262249Sequence 108AA278456Sequence 109AA279497Sequence 110AA280091Sequence 111AA281007Sequence 112AA293450Sequence 113AA295872Sequence 114AA300065Sequence 115AA305272Sequence 116AA305566Sequence 117AA305591Sequence 118AA305876Sequence 119AA305954Sequence 120AA306620Sequence 121AA306660Sequence 122AA306692Sequence 123AA306696Sequence 124AA307818Sequence 125AA308126Sequence 126AA308230Sequence 127AA308374Sequence 128AA308443Sequence 129AA308570Sequence 130AA308801Sequence 131AA309594Sequence 132AA309832Sequence 133AA311573Sequence 134AA311896Sequence 135AA312002Sequence 136AA313534Sequence 137AA313688Sequence 138AA314188Sequence 139AA314196Sequence 140AA314584Sequence 141AA314961Sequence 142AA315889Sequence 143AA318817Sequence 144AA325285Sequence 145AA325809Sequence 146AA333358Sequence 147AA344846Sequence 148AA347752Sequence 149AA348032Sequence 150AA350063Sequence 151AA350719Sequence 152AA354709Sequence 153AA355003Sequence 154AA356654Sequence 155AA359705Sequence 156AA361393Sequence 157AA361953Sequence 158AA362701Sequence 159AA367082Sequence 160AA371964Sequence 161AA375228Sequence 162AA377891Sequence 163AA384315Sequence 164AA384731Sequence 165AA400974Sequence 166AA401759Sequence 167AA411068Sequence 168AA416628Sequence 169AA421632Sequence 170AA424661Sequence 171AA425212Sequence 172AA425260Sequence 173AA425795Sequence 174AA427816Sequence 175AA428014Sequence 176AA428889Sequence 177AA443112Sequence 178AA445966Sequence 179AA447302Sequence 180AA447645Sequence 181AA448559Sequence 182AA449337Sequence 183AA453555Sequence 184AA453632Sequence 185AA453719Sequence 186AA454129Sequence 187AA455807Sequence 188AA458628Sequence 189AA459544Sequence 190AA460383Sequence 191AA460941Sequence 192AA463563Sequence 193AA464471Sequence 194AA467869Sequence 195AA469319Sequence 196AA476568Sequence 197AA476980Sequence 198AA477822Sequence 199AA478382Sequence 200AA478397Sequence 201AA479044Sequence 202AA479490Sequence 203AA480144Sequence 204AA481078Sequence 205AA488519Sequence 206AA493269Sequence 207AA503327Sequence 208AA505810Sequence 209AA506026Sequence 210AA506542Sequence 211AA507244Sequence 212AA514617Sequence 213AA515132Sequence 214AA521134Sequence 215AA521377Sequence 216AA521478Sequence 217AA527168Sequence 218AA527704Sequence 219AA534504Sequence 220AA552146Sequence 221AA557336Sequence 222AA558876Sequence 223AA559209Sequence 224AA572791Sequence 225AA581467Sequence 226AA582612Sequence 227AA587269Sequence 228AA594137Sequence 229AA599533Sequence 230AA609167Sequence 231AA612898Sequence 232AA625833Sequence 233AA626477Sequence 234AA628322Sequence 235AA634277Sequence 236AA639123Sequence 237AA639223Sequence 238AA641277Sequence 239AA643063Sequence 240AA652845Sequence 241AA663986Sequence 242AA679329Sequence 243AA682527Sequence 244AA682861Sequence 245AA687499Sequence 246AA701625Sequence 247AA703094Sequence 248AA703179Sequence 249AA704155Sequence 250AA704856Sequence 251AA705590Sequence 252AA707255Sequence 253AA714838Sequence 254AA716568Sequence 255AA720598Sequence 256AA723130Sequence 257AA766044Sequence 258AA770043Sequence 259AA773324Sequence 260AA773727Sequence 261AA805504Sequence 262AA807426Sequence 263AA808186Sequence 264AA812594Sequence 265AA827097Sequence 266AA829474Sequence 267AA829769Sequence 268AA853584Sequence 269AA854927Sequence 270AA863276Sequence 271AA863446Sequence 272AA868174Sequence 273AA868640Sequence 274AA872126Sequence 275AA877795Sequence 276AA902103Sequence 277AA903253Sequence 278AA917448Sequence 279AA933679Sequence 280AA933684Sequence 281AA938929Sequence 282AA962515Sequence 283AA973392Sequence 284AA974390Sequence 285AA977809Sequence 286AA993601Sequence 287AF017688Sequence 288AF038251Sequence 289AI002420Sequence 290AI005164Sequence 291AI015844Sequence 292AI025782Sequence 293AI028404Sequence 294AI039096Sequence 295AI061159Sequence 296AI061422Sequence 297AI075189Sequence 298AI079233Sequence 299AI097371Sequence 300AI125755Sequence 301AI142257Sequence 302AI143987Sequence 303AI147744Sequence 304AI148558Sequence 305AI150088Sequence 306AI186525Sequence 307AI199904Sequence 308AI199936Sequence 309AI201570Sequence 310AI201576Sequence 311AI203647Sequence 312AI214250Sequence 313AI216862Sequence 314AI239435Sequence 315AI241369Sequence 316AI245345Sequence 317AI250290Sequence 318AI266663Sequence 319AI267185Sequence 320AI267425Sequence 321AI267612Sequence 322AI275090Sequence 323AI275233Sequence 324AI278769Sequence 325AI278776Sequence 326AI279562Sequence 327AI290088Sequence 328AI290518Sequence 329AI298973Sequence 330AI301926Sequence 331AI307771Sequence 332AI308800Sequence 333AI312603Sequence 334AI342411Sequence 335AI347826Sequence 336AI347995Sequence 337AI349645Sequence 338AI350809Sequence 339AI362793Sequence 340AI366549Sequence 341AI366974Sequence 342AI375141Sequence 343AI376613Sequence 344AI376640Sequence 345AI379614Sequence 346AI400282Sequence 347AI421827Sequence 348AI433157Sequence 349AI436202Sequence 350AI469427Sequence 351AI475758Sequence 352AI525199Sequence 353AI525579Sequence 354AI568908Sequence 355AI620381Sequence 356AI650799Sequence 357Al660145Sequence 358AI670903Sequence 359AI684386Sequence 360AI702062Sequence 361AI743061Sequence 362AL047763Sequence 363C03852Sequence 364C06289Sequence 365D54540Sequence 366D58432Sequence 367D82436Sequence 368F22086Sequence 369F27502Sequence 370H05168Sequence 371H08641Sequence 372H11991Sequence 373H13883Sequence 374H15247Sequence 375H24046Sequence 376H49462Sequence 377H52955Sequence 378H73244Sequence 379H84532Sequence 380H87703Sequence 381H95493Sequence 382N28826Sequence 383N34398Sequence 384N78296Sequence 385R45371Sequence 386P55887Sequence 387R59147Sequence 388R63481Sequence 389R73183Sequence 390P76057Sequence 391R88149Sequence 392R91325Sequence 393T24595Sequence 394T29724Sequence 395T30494Sequence 396T35410Sequence 397T84755Sequence 398W05639Sequence 399W19700Sequence 400W24250Sequence 401W39447Sequence 402W45419Sequence 403W52961Sequence 404W60209Sequence 405Z21669Sequence 406Z25220Sequence 407Z45221Sequence 408AB002368Sequence 409AB018315Sequence 410AB018346Sequence 411AF026548Sequence 412AF037439Sequence 413AF038650Sequence 414AF064491Sequence 415AF070598Sequence 416AF081280Sequence 417AF086336Sequence 418AF131826Sequence 419AL021683Sequence 420D00422Sequence 421D26181Sequence 422D84307Sequence 423D88532Sequence 424L40391Sequence 425M28211Sequence 426U01184Sequence 427U14658Sequence 428U40282Sequence 429U71598Sequence 430U94855Sequence 431Q32353Sequence 432Q32355Sequence 433Q46540Sequence 434T47520Sequence 435V59657Sequence 436X19548

[0292]

5

TABLE 2

Sequence ID
Accession #

1/56

Sequence 1
A1039871

Sequence 2
AA778432

Sequence 3
Al092886

Sequence 4
AA082314

Sequence 5
AA862355

Sequence 6
Al659859

Sequence 7
AL047713

Sequence 8
Al189911

Sequence 9
AA853117

Sequence 10
AA424894

Sequence 11
Al366549

Sequence 12
AA558957

Sequence 13
AA313534

Sequence 14
Al139211

Sequence 15
Al131084

Sequence 16
AA601051

Sequence 17
Al267615

Sequence 18
R71549

Sequence 19
Al373754

Sequence 20
AA609618

Sequence 21
AA122401

Sequence 22
AA335862

Sequence 23
Al085616

Sequence 24
Al291072

Sequence 25
AA206225

Sequence 26
Al654989

Sequence 27
AA160532

Sequence 28
AA451923

Sequence 29
AA022750

Sequence 30
AA299694

Sequence 31
AA292009

Sequence 32
AA476770

Sequence 33
AA436859

Sequence 34
AA706420

Sequence 35
AA923755

Sequence 36
AA923755

Sequence 37
AA100982

Sequence 38
AA470067

Sequence 39
AA418433

Sequence 40
AA126150

Sequence 41
AA151805

Sequence 42
AA393176

Sequence 43
AA134909

Sequence 44
W29031

Sequence 45
AA482432

Sequence 46
AA115271

Sequence 47
AA219390

Sequence 48
AA232172

Sequence 49
AA159064

Sequence 50
AA358940

Sequence 51
AA041369

Sequence 52
AA063638

Sequence 53
AA233221

Sequence 54
AA609837

Sequence 55
AA130076

Sequence 56
AA307735

Sequence 57
AA063638

2/56

Sequence 58
AA032275

Sequence 59
AA437301

Sequence 60
AA977661

Sequence 61
AA977661

Sequence 62
AA436315

Sequence 63
AA453784

Sequence 64
Al282538

Sequence 65
AA335551

Sequence 66
AA335977

Sequence 67
Z44898

Sequence 68
AA186404

Sequence 69
AA845426

Sequence 70
AA406233

Sequence 71
AA026625

Sequence 72
AA323696

Sequence 73
Al084071

Sequence 74
Al084071

Sequence 75
AA308585

Sequence 76
H96671

Sequence 77
Al082695

Sequence 78
AA215319

Sequence 79
AA047729

Sequence 80
AA437301

Sequence 81
AA081269

Sequence 82
AA148523

Sequence 83
AA148523

Sequence 84
AA054564

Sequence 85
AA037260

Sequence 86
U89188

Sequence 87
AA307058

Sequence 88
AA486182

Sequence 89
Al347167

Sequence 90
AA034975

Sequence 91
AA461052

Sequence 92
W31059

Sequence 93
AA576065

Sequence 94
Al346050

Sequence 95
Z44898

Sequence 96
U69188

Sequence 97
W90000

Sequence 98
Al267379

Sequence 99
AA429418

Sequence 100
AA152275

Sequence 101
AA609837

Sequence 102
AA037260

Sequence 103
AA121249

Sequence 104
AA133375

Sequence 105
AA081204

Sequence 106
AA401204

Sequence 107
AA063074

Sequence 108
Al148825

Sequence 109
AA127645

Sequence 110
AA025006

Sequence 111
Z44898

Sequence 112
AA194362

Sequence 113
AA515031

Sequence 114
AA429418

3/56

Sequence 115
Al148825

Sequence 116
W27631

Sequence 117
AA373030

Sequence 118
Al628638

Sequence 119
AA831467

Sequence 120
AA443231

Sequence 121
AA443231

Sequence 122
Al308800

Sequence 123
AA857509

Sequence 124
AA632557

Sequence 125
AA150653

Sequence 126
AA148885

Sequence 127
AA318422

Sequence 128
Al580104

Sequence 129
AA227945

Sequence 130
AA429418

Sequence 131
AA428362

Sequence 132
AA229611

Sequence 133
Al261664

Sequence 134
AA788933

Sequence 135
AA504389

Sequence 136
AA788933

Sequence 137
AA233122

Sequence 138
AA192168

Sequence 139
AA452930

Sequence 140
AA523928

Sequence 141
W27631

Sequence 142
R14684

Sequence 143
AA452335

Sequence 144
AA291003

Sequence 145
AA443231

Sequence 146
AA443231

Sequence 147
Al241358

Sequence 148
AA070540

Sequence 149
AA581144

Sequence 150
AA581144

Sequence 151
AA928420

Sequence 152
AA291003

Sequence 153
AA227124

Sequence 154
AA969067

Sequence 155
AA760854

Sequence 156
AA580691

Sequence 157
AA770300

Sequence 158
AA770300

Sequence 159
AA083836

Sequence 160
AA045464

Sequence 161
AA167325

Sequence 162
AA115676

Sequence 163
AA187741

Sequence 164
AA352347

Sequence 165
AA375384

Sequence 166
AA136215

Sequence 167
AA318422

Sequence 168
AA244162

Sequence 169
AA211509

Sequence 170
AA430507

Sequence 171
AA515132

4/56

Sequence 172
AA284355

Sequence 173
AA167246

Sequence 174
AA083836

Sequence 175
AA113359

Sequence 176
AA669126

Sequence 177
Al683279

Sequence 178
AA296452

Sequence 179
AA765577

Sequence 180
AA086276

Sequence 181
AA214176

Sequence 182
AA476889

Sequence 183
Al016888

Sequence 184
AA600214

Sequence 185
AA375384

Sequence 186
AA453784

Sequence 187
AA453784

Sequence 188
W76487

Sequence 189
AA247690

Sequence 190
AA737182

Sequence 191
AA129463

Sequence 192
AA129463

Sequence 193
H04522

Sequence 194
AA169457

Sequence 195
AA010849

Sequence 196
AA156616

Sequence 197
AA431321

Sequence 198
AA558281

Sequence 199
AA581144

Sequence 200
AA935526

Sequence 201
AA088373

Sequence 202
AA453445

Sequence 203
AA968726

Sequence 204
AA770537

Sequence 205
AA330007

Sequence 206
AA330007

Sequence 207
AA853539

Sequence 208
T30659

Sequence 209
AA501373

Sequence 210
AA935526

Sequence 211
Al672524

Sequence 212
Al274797

Sequence 213
Al129827

Sequence 214
Al478372

Sequence 215
AA665560

Sequence 216
AA788933

Sequence 217
AA161518

Sequence 218
AA041467

Sequence 219
Al074333

Sequence 220
AA044787

Sequence 221
AA825917

Sequence 222
AA825917

Sequence 223
F27264

Sequence 224
H97360

Sequence 225
AA313688

Sequence 226
AA024982

Sequence 227
Al693441

Sequence 228
Al351167

5/56

Sequence 229
AA398886

Sequence 230
AA621740

Sequence 231
Al539679

Sequence 232
AA426083

Sequence 233
AA399022

Sequence 234
AA412184

Sequence 235
AA788933

Sequence 236
AA376374

Sequence 237
Al690181

Sequence 238
AA213902

Sequence 239
AA514564

Sequence 240
AA701870

Sequence 241
AA486954

Sequence 242
AA284462

Sequence 243
AA465528

Sequence 244
AA171834

Sequence 245
Al081515

Sequence 246
Al267532

Sequence 247
AL048644

Sequence 248
AA281739

Sequence 249
AA570181

Sequence 250
Al660919

Sequence 251
AA088764

Sequence 252
AA903285

Sequence 253
AA283080

Sequence 254
AA465285

Sequence 255
AA159916

Sequence 256
AA026215

Sequence 257
AA043908

Sequence 258
AA375325

Sequence 259
Al052317

Sequence 260
AA194535

Sequence 261
AA846439

Sequence 262
AA492032

Sequence 263
Al026767

Sequence 264
Al004557

Sequence 265
AA335502

Sequence 266
AA001794

Sequence 267
AA333713

Sequence 268
AA730829

Sequence 269
AA127626

Sequence 270
AA101212

Sequence 271
AA303085

Sequence 272
Al673189

Sequence 273
Al086254

Sequence 274
AA035773

Sequence 275
AA916110

Sequence 276
AA166874

Sequence 277
AA422177

Sequence 278
AA262878

Sequence 279
AA282134

Sequence 280
AA156237

Sequence 281
AA425378

Sequence 282
AA150409

Sequence 283
AA045417

Sequence 284
Al052317

Sequence 285
W01842

6/56

Sequence 286
AA425292

Sequence 287
AA985622

Sequence 288
AA722855

Sequence 289
AA426216

Sequence 290
AA788933

Sequence 291
Al138898

Sequence 292
AA778250

Sequence 293
W78198

Sequence 294
AA788933

Sequence 295
Al342607

Sequence 296
AA426216

Sequence 297
AA927120

Sequence 298
AA788933

Sequence 299
Al079871

Sequence 300
Al741829

Sequence 301
AA418715

Sequence 302
AA150636

Sequence 303
Al433965

Sequence 304
AA088373

Sequence 305
AA471070

Sequence 306
Al673422

Sequence 307
AA203123

Sequence 308
AA203627

Sequence 309
AA193592

Sequence 310
AA206064

Sequence 311
R31249

Sequence 312
AA783933

Sequence 313
AA282369

Sequence 314
AA149594

Sequence 315
AA347635

Sequence 316
AA293172

Sequence 317
AA832167

Sequence 318
AA053133

Sequence 319
AA908731

Sequence 320
AA808830

Sequence 321
AA779747

Sequence 322
AA084038

Sequence 323
Al365500

Sequence 324
Al609252

Sequence 325
H08650

Sequence 328
H92511

Sequence 327
AA088373

Sequence 328
H66289

Sequence 329
W73086

Sequence 330
AA035773

Sequence 331
AA481432

Sequence 332
AA082084

Sequence 333
AA490665

Sequence 334
AA429541

Sequence 335
AA568231

Sequence 336
AA426216

Sequence 337
AA738430

Sequence 338
AA627132

Sequence 339
AA402714

Sequence 340
AA025585

Sequence 341
AA493305

Sequence 342
AA278542

7/56

Sequence 343
AA278542

Sequence 344
AA442936

Sequence 345
AA779747

Sequence 346
Al283155

Sequence 347
Al263446

Sequence 348
AA013042

Sequence 349
AA081426

Sequence 350
AA484061

Sequence 351
AA315535

Sequence 352
AA399022

Sequence 353
Al127825

Sequence 354
AA715828

Sequence 355
Al243931

Sequence 356
Al024753

Sequence 357
AA086070

Sequence 358
AA625159

Sequence 359
AA426216

Sequence 360
AA426216

Sequence 361
Al628734

Sequence 362
Al358846

Sequence 363
AA013042

Sequence 364
Al273856

Sequence 365
AA493647

Sequence 366
AA450075

Sequence 367
AA115777

Sequence 368
AA494342

Sequence 369
AA181331

Sequence 370
AA418715

Sequence 371
AA662648

Sequence 372
AA086070

Sequence 373
AA487250

Sequence 374
AA099101

Sequence 375
AA093957

Sequence 376
AA136215

Sequence 377
AA256459

Sequence 378
Al760421

Sequence 379
AA082675

Sequence 380
AA180746

Sequence 381
AA534365

Sequence 382
W28567

Sequence 383
AA069657

Sequence 384
AA178880

Sequence 385
H23090

Sequence 386
AA130285

Sequence 387
Al052317

Sequence 388
AA453784

Sequence 389
AA312917

Sequence 390
AA521024

Sequence 391
AA188744

Sequence 392
AA593011

Sequence 393
N78223

Sequence 394
AA669106

Sequence 395
AA548445

Sequence 396
AA046827

Sequence 397
AA618310

Sequence 398
AA426216

Sequence 399
AA281412

8/56

Sequence 400
AA532835

Sequence 401
AA809070

Sequence 402
Al608739

Sequence 403
AA977802

Sequence 404
H68725

Sequence 405
AA125809

Sequence 406
Al299874

Sequence 407
AA453784

Sequence 408
AA101236

Sequence 409
AA491219

Sequence 410
AA149853

Sequence 411
AA151345

Sequence 412
Al273856

Sequence 413
AA405425

Sequence 414
AA599864

Sequence 415
AA411436

Sequence 416
AA131439

Sequence 417
AA706347

Sequence 418
Al349772

Sequence 419
AA488463

Sequence 420
AA083482

Sequence 421
AA083482

Sequence 422
AA149853

Sequence 423
AA255613

Sequence 424
AA627448

Sequence 425
AA453784

Sequence 426
AA022788

Sequence 427
AA635003

Sequence 428
AA576419

Sequence 429
AA459880

Sequence 430
AA131053

Sequence 431
AA496640

Sequence 432
Al582629

Sequence 433
Al582629

Sequence 434
Al023912

Sequence 435
AA977802

Sequence 436
W00823

Sequence 437
W00823

Sequence 438
Al674397

Sequence 439
AA700350

Sequence 440
AA745614

Sequence 441
Al302190

Sequence 442
AA393518

Sequence 443
N27985

Sequence 444
AA468753

Sequence 445
AA653775

Sequence 446
AA653775

Sequence 447
AA088373

Sequence 448
AA088373

Sequence 449
AA130783

Sequence 450
AA461143

Sequence 451
R78778

Sequence 452
AA031550

Sequence 453
AA461143

Sequence 454
AA303484

Sequence 455
AA301297

Sequence 456
AA744503

9/56

Sequence 457
T67813

Sequence 458
Al564319

Sequence 459
Al564319

Sequence 460
Al138898

Sequence 461
Al138898

Sequence 462
AA301297

Sequence 463
Al302436

Sequence 464
H27251

Sequence 465
AA418715

Sequence 466
001749

Sequence 467
AA083471

Sequence 468
Al264186

Sequence 469
T66196

Sequence 470
H29445

Sequence 471
Al469093

Sequence 472
AA136215

Sequence 473
Al267185

Sequence 474
AA935526

Sequence 475
AA026157

Sequence 476
AA001277

Sequence 477
AA552588

Sequence 478
AA099921

Sequence 479
AA380495

Sequence 480
R77384

Sequence 481
AA024924

Sequence 482
Al309007

Sequence 483
AA278434

Sequence 484
AA303150

Sequence 485
AA460060

Sequence 486
AA169355

Sequence 487
Al201573

Sequence 488
AA779747

Sequence 489
AA468753

Sequence 490
AA091431

Sequence 491
AA464646

Sequence 492
AA083836

Sequence 493
AA151506

Sequence 494
AA069195

Sequence 495
AA007137

Sequence 496
AA178912

Sequence 497
AA225164

Sequence 498
Al565125

Sequence 499
AA483181

Sequence 500
AA470909

Sequence 501
AA650493

Sequence 502
AA059309

Sequence 503
AA677142

Sequence 504
AA447503

Sequence 505
AA252216

Sequence 506
F33257

Sequence 507
AA576667

Sequence 508
AA320124

Sequence 509
AA082569

Sequence 510
AA573200

Sequence 511
AA178880

Sequence 512
AA176433

Sequence 513
AA788933

10/56

Sequence 514
Al033037

Sequence 515
AA533283

Sequence 516
Al267185

Sequence 517
AA280123

Sequence 518
C17183

Sequence 519
AA779747

Sequence 520
Al283155

Sequence 521
AA308736

Sequence 522
H52011

Sequence 523
AA034975

Sequence 524
AA034908

Sequence 525
AA564296

Sequence 526
AA515031

Sequence 527
AA083471

Sequence 528
AA088373

Sequence 529
AA088373

Sequence 530
Al218204

Sequence 531
N46402

Sequence 532
AA099023

Sequence 533
AA600987

Sequence 534
Al267185

Sequence 535
N46402

Sequence 536
N46402

Sequence 537
AA131053

Sequence 538
R19004

Sequence 539
AA099878

Sequence 540
Al608720

Sequence 541
Al131214

Sequence 542
Al139097

Sequence 543
AA089857

Sequence 544
AA047849

Sequence 545
AA058822

Sequence 546
AA147978

Sequence 547
AA199850

Sequence 548
Al167659

Sequence 549
Al167659

Sequence 550
AA574054

Sequence 551
W37586

Sequence 552
AA088373

Sequence 553
AA157619

Sequence 554
AA013042

Sequence 555
AA300170

Sequence 556
AA430088

Sequence 557
AA136096

Sequence 558
AA136756

Sequence 559
W07393

Sequence 560
AA143577

Sequence 561
AA642766

Sequence 562
AA554920

Sequence 563
AA452335

Sequence 564
AA532484

Sequence 565
AA609837

Sequence 566
AA152275

Sequence 567
Al192851

Sequence 568
AA228082

Sequence 569
Al093501

Sequence 570
AA905277

11/56

Sequence 571
AA465301

Sequence 572
AA172248

Sequence 573
AA129819

Sequence 574
AA428120

Sequence 575
Al291105

Sequence 576
Al750962

Sequence 577
AA011024

Sequence 578
AA873159

Sequence 579
AA873159

Sequence 580
AA846266

Sequence 581
AA652080

Sequence 582
Al339481

Sequence 583
AA609172

Sequence 584
AA609172

Sequence 585
AA908731

Sequence 586
Al147200

Sequence 587
AA864497

Sequence 588
AA083482

Sequence 589
Al279718

Sequence 590
AA188375

Sequence 591
AA088373

Sequence 592
AA280110

Sequence 593
AA744712

Sequence 594
AA936089

Sequence 595
AA076368

Sequence 596
AA774663

Sequence 597
AA527389

Sequence 598
AA007495

Sequence 599
AA045264

Sequence 600
AA523252

Sequence 601
AA552588

Sequence 602
AA173924

Sequence 603
AA465301

Sequence 604
AA599533

Sequence 605
AA614105

Sequence 606
AA384078

Sequence 607
AA872507

Sequence 608
AA045936

Sequence 609
AA081269

Sequence 610
AA572758

Sequence 611
AA007271

Sequence 612
Z78349

Sequence 613
AA626469

Sequence 614
AA551215

Sequence 615
AA621740

Sequence 616
AA304249

Sequence 617
AA877288

Sequence 618
AA483182

Sequence 619
N78223

Sequence 620
N78268

Sequence 621
AA365398

Sequence 622
AA131231

Sequence 623
P55380

Sequence 624
Al078529

Sequence 625
Al767123

Sequence 626
Al700226

Sequence 627
AA487586

12/56

Sequence 628
Al033304

Sequence 629
AA436895

Sequence 630
W01842

Sequence 631
AA864853

Sequence 632
AA444134

Sequence 633
Al700226

Sequence 634
AA853130

Sequence 635
H52011

Sequence 636
AA618484

Sequence 637
AA101484

Sequence 638
AA649746

Sequence 639
H01401

Sequence 640
AA375933

Sequence 641
AA166905

Sequence 642
AA151918

Sequence 643
AA845573

Sequence 644
AA013363

Sequence 645
Al096880

Sequence 646
AA905781

Sequence 647
Al052317

Sequence 648
AA513827

Sequence 649
Al393144

Sequence 650
AA788933

Sequence 651
AA788933

Sequence 652
AA311098

Sequence 653
AA542829

Sequence 654
AA166622

Sequence 655
Al054163

Sequence 656
AA164243

Sequence 657
Al288783

Sequence 658
Al186605

Sequence 659
C18682

Sequence 660
AA340224

Sequence 661
AA171834

Sequence 662
AA196793

Sequence 663
Al423079

Sequence 664
Al423079

Sequence 665
A1521128

Sequence 666
AA526226

Sequence 667
AA165306

Sequence 668
AA878771

Sequence 669
AA195865

Sequence 670
W16508

Sequence 671
AA995597

Sequence 672
Al042002

Sequence 673
AA936566

Sequence 674
AA936566

Sequence 675
AA977802

Sequence 676
AA913590

Sequence 677
AA913590

Sequence 678
Al360219

Sequence 679
AA804436

Sequence 680
AA548082

Sequence 681
T66196

Sequence 682
AA199802

Sequence 683
Al040294

Sequence 684
AA402901

13/56

Sequence 685
AA426653

Sequence 686
AA001794

Sequence 687
AA514564

Sequence 688
D54004

Sequence 689
AA211272

Sequence 690
AA161003

Sequence 691
AA232705

Sequence 692
AA020855

Sequence 693
AA081395

Sequence 694
AA885514

Sequence 695
T71404

Sequence 696
W01842

Sequence 697
AA094609

Sequence 698
AA062957

Sequence 699
AA026215

Sequence 700
AA158724

Sequence 701
Al243555

Sequence 702
AA026719

Sequence 703
AA036944

Sequence 704
AA076262

Sequence 705
AA120908

Sequence 706
AA161066

Sequence 707
Al391479

Sequence 708
AA343324

Sequence 709
N77582

Sequence 710
AA083471

Sequence 711
AA872507

Sequence 712
Al093740

Sequence 713
AA731306

Sequence 714
AA305494

Sequence 715
AA699927

Sequence 716
AA701448

Sequence 717
Al554245

Sequence 718
AA132605

Sequence 719
AA825917

Sequence 720
W78198

Sequence 721
Al609252

Sequence 722
AA022788

Sequence 723
AA026625

Sequence 724
AA206519

Sequence 725
AA256335

Sequence 726
AA256335

Sequence 727
AA013363

Sequence 728
Al300590

Sequence 729
AA069733

Sequence 730
AA182841

Sequence 731
Al342480

Sequence 732
AA451894

Sequence 733
AA915959

Sequence 734
AA147833

Sequence 735
AA229993

Sequence 736
AA199821

Sequence 737
Al198431

Sequence 738
AA928420

Sequence 739
AA256320

Sequence 740
Al267185

Sequence 741
AA528456

14/56

Sequence 742
Al567965

Sequence 743
AA101299

Sequence 744
AA618484

Sequence 745
Al278872

Sequence 746
Al278872

Sequence 747
AA708712

Sequence 748
AA398892

Sequence 749
N52168

Sequence 750
AA827758

Sequence 751
R56747

Sequence 752
AA854014

Sequence 753
AA904266

Sequence 754
AA770557

Sequence 755
AL048446

Sequence 756
AA062817

Sequence 757
AA464646

Sequence 758
AA323097

Sequence 759
AA102168

Sequence 760
AA528121

Sequence 761
AA526498

Sequence 762
AA877213

Sequence 763
AA864690

Sequence 764
AA864690

Sequence 765
AA788933

Sequence 766
AA235835

Sequence 767
AA558066

Sequence 768
AA149151

Sequence 769
AA583526

Sequence 770
AA013363

Sequence 771
AA013363

Sequence 772
Al141567

Sequence 773
AA187406

Sequence 774
Al499986

Sequence 775
AA488398

Sequence 776
AA418408

Sequence 777
AA418408

Sequence 778
AA147312

Sequence 779
AA581130

Sequence 780
AA582851

Sequence 781
AA075393

Sequence 782
AA737562

Sequence 783
Al538172

Sequence 784
N57174

Sequence 785
AA453479

Sequence 786A
A442561

Sequence 787
AA448973

Sequence 788
Al079871

Sequence 789
AA487467

Sequence 790
AA442948

Sequence 791
AA078387

Sequence 792
AA620980

Sequence 793
AA195128

Sequence 794
Al126689

Sequence 795
AA446578

Sequence 796
AA649064

Sequence 797
AA938757

Sequence 798
AA806532

15/56

Sequence 799
AA316566

Sequence 800
H98646

Sequence 801
AA635393

Sequence 802
AA191045

Sequence 803
Al198431

Sequence 804
AA595562

Sequence 805
AA532383

Sequence 806
AA430059

Sequence 807
AA609837

Sequence 808
AA126418

Sequence 809
Al083558

Sequence 810
AA465584

Sequence 811
Al129865

Sequence 812
AA316089

Sequence 813
AA341011

Sequence 814
W60762

Sequence 815
R24022

Sequence 816
R25322

Sequence 817
Al249905

Sequence 818
AA126798

Sequence 819
AA551607

Sequence 820
AA418783

Sequence 821
AA151600

Sequence 822
AA600173

Sequence 823
Al245990

Sequence 824
Al056158

Sequence 825
AA009816

Sequence 826
R91618

Sequence 827
AA977802

Sequence 828
Al741118

Sequence 829
AA039810

Sequence 830
AA357956

Sequence 831
AA431278

Sequence 832
Al096925

Sequence 833
AA400833

Sequence 834
Al309380

Sequence 835
AA330640

Sequence 836
AA169457

Sequence 837
Al079871

Sequence 838
AA236339

Sequence 839
Al301952

Sequence 840
W03618

Sequence 841
W03618

Sequence 842
AA543080

Sequence 843
AA303085

Sequence 844
AA779747

Sequence 845
AA127826

Sequence 846
AA364229

Sequence 847
AA364229

Sequence 848
AA747475

Sequence 849
F20791

Sequence 850
F20791

Sequence 851
AA814278

Sequence 852
N39195

Sequence 853
Al700982

Sequence 854
AA136215

Sequence 855
Al089782

16/56

Sequence 856
AA827570

Sequence 857
AA983883

Sequence 858
AA192168

Sequence 859
Al097410

Sequence 860
AA742474

Sequence 861
W69615

Sequence 862
Al051843

Sequence 863
R60403

Sequence 864
AA101010

Sequence 865
AA932039

Sequence 866
AA496669

Sequence 867
AA283707

Sequence 868
AA088344

Sequence 869
AA654827

Sequence 870
Al246125

Sequence 871
Al079871

Sequence 872
Al079871

Sequence 873
N88286

Sequence 874
Al738508

Sequence 875
AA070469

Sequence 876
AA256335

Sequence 877
AA256335

Sequence 878
AA010044

Sequence 879
AA091053

Sequence 880
AA000981

Sequence 881
AA148023

Sequence 882
AA622574

Sequence 883
AA936960

Sequence 884
Al201573

Sequence 885
Al344021

Sequence 886
Al127179

Sequence 887
AA026454

Sequence 888
AA026454

Sequence 889
AA136756

Sequence 890
AA129410

Sequence 891
Al032188

Sequence 892
Al032188

Sequence 893
Al741829

Sequence 894
Al214206

Sequence 895
AA314222

Sequence 896
AA099592

Sequence 897
AA765228

Sequence 898
AA831729

Sequence 899
AA190559

Sequence 900
W01842

Sequence 901
N31402

Sequence 902
Al267185

Sequence 903
AA492465

Sequence 904
Al611173

Sequence 905
Al311440

Sequence 906
AA447503

Sequence 907
AA412359

Sequence 908
AA700540

Sequence 909
Al332755

Sequence 910
AA115271

Sequence 911
AA009997

Sequence 912
AA150445

17/56

Sequence 913
AA741015

Sequence 914
AA397741

Sequence 915
AA723209

Sequence 916
AA773607

Sequence 917
Al309007

Sequence 918
AA977584

Sequence 919
AA143311

Sequence 920
W95589

Sequence 921
AAS14638

Sequence 922
Al267148

Sequence 923
Al311440

Sequence 924
Al621272

Sequence 925
Al559470

Sequence 926
AA325222

Sequence 927
AA325222

Sequence 928
Al369447

Sequence 929
AA773804

Sequence 930
AA490210

Sequence 931
AA253384

Sequence 932
AA652436

Sequence 933
AA983883

Sequence 934
AA323560

Sequence 935
R21821

Sequence 936
AA182841

Sequence 937
AA255567

Sequence 938
AA610706

Sequence 939
AA306015

Sequence 940A
A565996

Sequence 941
Al049770

Sequence 942
AA199644

Sequence 943
AA224371

Sequence 944
AA913590

Sequence 945
AA523526

Sequence 946
Al144108

Sequence 947
AA377383

Sequence 948
AA436438

Sequence 949
AA788933

Sequence 950
Al025517

Sequence 951
AA447250

Sequence 952
AA722512

Sequence 953
AA447590

Sequence 954
AA155915

Sequence 955
AA412059

Sequence 956
AA375325

Sequence 957
AA082684

Sequence 958
N47128

Sequence 959
AA227610

Sequence 960
R85339

Sequence 961
R85339

Sequence 962
AA521024

Sequence 963
AA069611

Sequence 964
AA303084

Sequence 965
AA426216

Sequence 966
AA706730

Sequence 967
R24799

Sequence 968
AA129744

Sequence 969
AA436541

18/56

Sequence 970
AA524721

Sequence 971
Al492230

Sequence 972
AA209340

Sequence 973
AA974657

Sequence 974
AA281412

Sequence 975
AA368906

Sequence 976
AA101820

Sequence 977
AA302450

Sequence 978
N49477

Sequence 979
AA399022

Sequence 980
Al453471

Sequence 981
AA699666

Sequence 982
AA372894

Sequence 983
Al217001

Sequence 984
AA328393

Sequence 985
Al249705

Sequence 986
Al090943

Sequence 987
AA126836

Sequence 988
Al536912

Sequence 989
AA935134

Sequence 990
AA994818

Sequence 991
Al342714

Sequence 992
AA916068

Sequence 993
AA375384

Sequence 994
AA310694

Sequence 995
AA037823

Sequence 996
AA190559

Sequence 997
AA332853

Sequence 998
AA827899

Sequence 999
AA065319

Sequence 1000
AA573238

Sequence 1001
AA573238

Sequence 1002
N99772

Sequence 1003
AA192253

Sequence 1004
Al017852

Sequence 1005
AA203140

Sequence 1006
AA345827

Sequence 1007
Al129468

Sequence 1008
AA151675

Sequence 1009
AA157619

Sequence 1010
Al204088

Sequence 1011
AA232691

Sequence 1012
AA232691

Sequence 1013
Al267454

Sequence 1014
AA464646

Sequence 1015
AA496961

Sequence 1016

AA496961

Sequence 1017
AA779747

Sequence 1018
Al433157

Sequence 1019
AA464646

Sequence 1020
Al311440

Sequence 1021
Al127835

Sequence 1022
AA279553

Sequence 1023
AA227876

Sequence 1024
AA455007

Sequence 1025
AA169457

Sequence 1026
AA169457

19/56

Sequence 1027
AA766864

Sequence 1028
AA026454

Sequence 1029
AA026454

Sequence 1030
Al074333

Sequence 1031
Al074333

Sequence 1032
AA448332

Sequence 1033
F27264

Sequence 1034
AA188481

Sequence 1035
Al738508

Sequence 1036
Al738508

Sequence 1037
AA928420

Sequence 1038
Al742056

Sequence 1039
AA489412

Sequence 1040
Al066421

Sequence 1041
AA368063

Sequence 1042
Z45013

Sequence 1043
AA490731

Sequence 1044
AA316071

Sequence 1045
AA872059

Sequence 1046
AA083836

Sequence 1047
AA199821

Sequence 1048
AA187824

Sequence 1049
AA913590

Sequence 1050
AA913590

Sequence 1051
Al074572

Sequence 1052
AA101077

Sequence 1053
AA668553

Sequence 1054
AA165471

Sequence 1055
AA286751

Sequence 1056
Al440495

Sequence 1057
AA526893

Sequence 1058
Al278160

Sequence 1059
Al242593

Sequence 1060
AA622574

Sequence 1061
Al591008

Sequence 1062
AA365398

Sequence 1063
AA173885

Sequence 1064
T08682

Sequence 1065
AA074869

Sequence 1066
AA357299

Sequence 1067
AA203140

Sequence 1068
AA190559

Sequence 1069
Al168018

Sequence 1070
AA451894

Sequence 1071
AA024859

Sequence 1072
AA412184

Sequence 1073
AA207216

Sequence 1074
AA463446

Sequence 1075
AA135572

Sequence 1076
Al364978

Sequence 1077
Al281972

Sequence 1078
AA418408

Sequence 1079
AA418408

Sequence 1080
AA074098

Sequence 1081
AA029567

Sequence 1082
AA747547

Sequence 1083
Al753431

20/56

Sequence 1084
AA232691

Sequence 1085
Al079871

Sequence 1086
Al300541

Sequence 1087
AA417006

Sequence 1088
AA490731

Sequence 1089
Z45013

Sequence 1090
Al066421

Sequence 1091
AA621714

Sequence 1092
AA813524

Sequence 1093
AA515261

Sequence 1094
AA040300

Sequence 1095
AA040300

Sequence 1096
Al241763

Sequence 1097
AA722512

Sequence 1098
Al034147

Sequence 1099
Al703111

Sequence 1100
Al079871

Sequence 1101
Al079871

Sequence 1102
AA485781

Sequence 1103
AA216667

Sequence 1104
AA216667

Sequence 1105
Al267282

Sequence 1106
AA485488

Sequence 1107
AA295804

Sequence 1108
AA601147

Sequence 1109
H06792

Sequence 1110
AA283707

Sequence 1111
AA101077

Sequence 1112
Al076532

Sequence 1113
Al076532

Sequence 1114
AA609534

Sequence 1115
AA232691

Sequence 1116
R60403

Sequence 1117
AA928676

Sequence 1118
AA807960

Sequence 1119
AA573742

Sequence 1120
AA463547

Sequence 1121
Al086042

Sequence 1122
AI017852

Sequence 1123
Al267185

Sequence 1124
AA167794

Sequence 1125
Al253335

Sequence 1126
AA005232

Sequence 1127
AA581144

Sequence 1128
Al204127

Sequence 1129
AA420989

Sequence 1130
AA877877

Sequence 1131
AA364275

Sequence 1132
AA455007

Sequence 1133
AA091539

Sequence 1134
AA088373

Sequence 1135
AA088373

Sequence 1136
AA595217

Sequence 1137
AA642691

Sequence 1138
Al417520

Sequence 1139
AA133375

Sequence 1140
Al472536

21/56

Sequence 1141
AA772548

Sequence 1142
AA772548

Sequence 1143
AA161488

Sequence 1144
AA609253

Sequence 1145
AA412184

Sequence 1146
AA679015

Sequence 1147
AA280235

Sequence 1148
Al080011

Sequence 1149
Al091013

Sequence 1150
AA905362

Sequence 1151
AA521069

Sequence 1152
AA226674

Sequence 1153
AA226674

Sequence 1154
AA037506

Sequence 1155
AA522790

Sequence 1156
AA375384

Sequence 1157
AA788933

Sequence 1158
Al418922

Sequence 1159
AA420989

Sequence 1160
AA323097

Sequence 1161
Al432949

Sequence 1162
Al432949

Sequence 1163
AA226914

Sequence 1164
AA442097

Sequence 1165
AA316566

Sequence 1166
H98646

Sequence 1167
Al281417

Sequence 1168
AA283707

Sequence 1169
AA505080

Sequence 1170
AA505080

Sequence 1171
AA789020

Sequence 1172
AA789020

Sequence 1173
N30234

Sequence 1174
N98658

Sequence 1175
AA323560

Sequence 1176
AA719473

Sequence 1177
AA446943

Sequence 1178
AA419395

Sequence 1179
AA419395

Sequence 1180
Al718403

Sequence 1181
AA115025

Sequence 1182
AA789013

Sequence 1183
AA830249

Sequence 1184
AA830249

Sequence 1185
AA282848

Sequence 1186
AA136215

Sequence 1187
AA336515

Sequence 1188
AA854982

Sequence 1189
AA533727

Sequence 1190
AA121207

Sequence 1191
AA424541

Sequence 1192
AA040300

Sequence 1193
AA923750

Sequence 1194
AA173885

Sequence 1195
AA352308

Sequence 1196
AA233105

Sequence 1197
H71437

22/56

Sequence 1198
Al076532

Sequence 1199
AA650333

Sequence 1200
AA702428

Sequence 1201
AA531613

Sequence 1202
AA255584

Sequence 1203
AA255584

Sequence 1204
Al141699

Sequence 1205
AA481432

Sequence 1206
AA305599

Sequence 1207
H10703

Sequence 1208
AA121879

Sequence 1209
Al249797

Sequence 1210
AA482853

Sequence 1211
Al141567

Sequence 1212
H01413

Sequence 1213
AA148508

Sequence 1214
AA729727

Sequence 1215
AA789158

Sequence 1216
AA679848

Sequence 1217
AA679848

Sequence 1218
Al221967

Sequence 1219
AA838613

Sequence 1220
Al223887

Sequence 1221
W57570

Sequence 1222
H23090

Sequence 1223
Al203342

Sequence 1224
AA056334

Sequence 1225
Al088115

Sequence 1226
AA373243

Sequence 1227
Al359989

Sequence 1228
A

A247180

Sequence 1229
AA838613

Sequence 1230
AA136215

Sequence 1231
AA876375

Sequence 1232
AA876375

Sequence 1233
AA905495

Sequence 1234
Al344928

Sequence 1235
AA769315

Sequence 1236
F37855

Sequence 1237
Al093165

Sequence 1238
AA478787

Sequence 1239
AA088344

Sequence 1240
AA766044

Sequence 1241
Al339481

Sequence 1242
AA829473

Sequence 1243
020248

Sequence 1244
AA412015

Sequence 1245
AA420989

Sequence 1246
AA259065

Sequence 1247
Al345847

Sequence 1248
AA235453

Sequence 1249
AA486335

Sequence 1250
Al129482

Sequence 1251
Al690374

Sequence 1252
Al034481

Sequence 1253
Al312562

Sequence 1254
Al312562

23/56

Sequence 1255
Al092921

Sequence 1256
AA570668

Sequence 1257
AA489032

Sequence 1258
AA047729

Sequence 1259
AA662918

Sequence 1260
Al078151

Sequence 1261
AA233672

Sequence 1262
AA157424

Sequence 1263
Al078151

Sequence 1264
AA043258

Sequence 1265
AA126623

Sequence 1266
AA062936

Sequence 1267
AA062936

Sequence 1268
AA147951

Sequence 1269
AA148100

Sequence 1270
AA281556

Sequence 1271
AA482303

Sequence 1272
Al276690

Sequence 1273
AA303709

Sequence 1274
Al004675

Sequence 1275
AA039354

Sequence 1276
AA563987

Sequence 1277
AA488634

Sequence 1278
AA399628

Sequence 1279
Al267185

Sequence 1280
AA226674

Sequence 1281
AA469121

Sequence 1282
AA652436

Sequence 1283
Al671174

Sequence 1284
AA446040

Sequence 1285
Al309007

Sequence 1286
Al201573

Sequence 1287
016203

Sequence 1288
AA035003

Sequence 1289
AA838431

Sequence 1290
AA838431

Sequence 1291
AA007569

Sequence 1292
AA853131

Sequence 1293
AA190559

Sequence 1294
AA155915

Sequence 1295
Al023372

Sequence 1296
Al276690

Sequence 1297
Al199515

Sequence 1298
Al199515

Sequence 1299
Al025517

Sequence 1300
AA127565

Sequence 1301
AA526498

Sequence 1302
AA114138

Sequence 1303
AA765228

Sequence 1304
AA765228

Sequence 1305
Al090077

Sequence 1306
AA044748

Sequence 1307
AA044748

Sequence 1308
Al267502

Sequence 1309
AA344583

Sequence 1310
AA029043

Sequence 1311
H52011

24/56

Sequence 1312
AA700350

Sequence 1313
AA483053

Sequence 1314
AA679015

Sequence 1315
M430369

Sequence 1316
Al309007

Sequence 1317
AA005232

Sequence 1318
AA102563

Sequence 1319
H98646

Sequence 1320
AA548889

Sequence 1321
AI267185

Sequence 1322
W19131

Sequence 1323
AA847560

Sequence 1324
AA279747

Sequence 1325
AA025721

Sequence 1326
AA679015

Sequence 1327
AA428184

Sequence 1328
Al273856

Sequence 1329
AA514959

Sequence 1330
AA452335

Sequence 1331
AA165463

Sequence 1332
AA514542

Sequence 1333
F32233

Sequence 1334
AA400840

Sequence 1335
AA385602

Sequence 1336
Al075412

Sequence 1337
AA772548

Sequence 1338
AA772548

Sequence 1339
AA679015

Sequence 1340
Al267282

Sequence 1341
AA075148

Sequence 1342
Al624304

Sequence 1343
Al436387

Sequence 1344
AA166622

Sequence 1345
AA916110

Sequence 1346
AA047065

Sequence 1347
AA399022

Sequence 1348
AA136215

Sequence 1349
AA039354

Sequence 1350
AA634397

Sequence 1351
AA788933

Sequence 1352
AA039354

Sequence 1353
AA323602

Sequence 1354
Al332433

Sequence 1355
AA446215

Sequence 1356
AA082498

Sequence 1357
AA464646

Sequence 1358
AA465164

Sequence 1359
Al754431

Sequence 1360
Al754431

Sequence 1361
H01401

Sequence 1362
H01401

Sequence 1363
AF001541

Sequence 1364
AA047729

Sequence 1365
AA399022

Sequence 1366
Al127825

Sequence 1367
Al089220

Sequence 1368
AA770397

25/56

Sequence 1369
AA305494

Sequence 1370
AA083004

Sequence 1371
AA151600

Sequence 1372
AA233641

Sequence 1373
AA226674

Sequence 1374
AA453784

Sequence 1375
AA853539

Sequence 1376
AA449456

Sequence 1377
Al625806

Sequence 1378
Al348661

Sequence 1379
AA608668

Sequence 1380
AA303709

Sequence 1381
AA612685

Sequence 1382
AA113235

Sequence 1383
AA576065

Sequence 1384
Al033304

Sequence 1385
Al090509

Sequence 1386
D81636

Sequence 1387
AA232447

Sequence 1388
AA608790

Sequence 1389
AA307513

Sequence 1390
Al267185

Sequence 1391
Al491897

Sequence 1392
AA983883

Sequence 1393
AA171834

Sequence 1394
Al262064

Sequence 1395
Al473830

Sequence 1396
AA126418

Sequence 1397
AA487845

Sequence 1398
AA700903

Sequence 1399
AA977584

Sequence 1400
AA091539

Sequence 1401
AA580412

Sequence 1402
AA580412

Sequence 1403
AA182766

Sequence 1404
AA053874

Sequence 1405
Al090863

Sequence 1406
Al271883

Sequence 1407
AA101862

Sequence 1408
N24636

Sequence 1409
AA112046

Sequence 1410
Al421699

Sequence 1411
Al738508

Sequence 1412
Al738508

Sequence 1413
AA464646

Sequence 1414
AA234411

Sequence 1415
AA595343

Sequence 1416
AA314225

Sequence 1417
AA706004

Sequence 1418
AA704151

Sequence 1419
Al267185

Sequence 1420
AA075148

Sequence 1421
AA026719

Sequence 1422
AA420758

Sequence 1423
Al362945

Sequence 1424
AA936566

Sequence 1425
AA063512

26/56

Sequence 1426
AA112408

Sequence 1427
Al478691

Sequence 1428
AA694186

Sequence 1429
AA026719

Sequence 1430
AA541386

Sequence 1431
T71404

Sequence 1432
T71404

Sequence 1433
AA228025

Sequence 1434
AA228025

Sequence 1435
AA493211

Sequence 1436
AA228895

Sequence 1437
AA398144

Sequence 1438
Al309007

Sequence 1439
Al127815

Sequence 1440
Al127815

Sequence 1441
AF001541

Sequence 1442
AA047729

Sequence 1443
Al262125

Sequence 1444
AA459456

Sequence 1445
AA459456

Sequence 1446
AA913590

Sequence 1447
AA913590

Sequence 1448
Al241000

Sequence 1449
AA769847

Sequence 1450
AA522903

Sequence 1451
AA725363

Sequence 1452
Al093315

Sequence 1453
Al188929

Sequence 1454
H17020

Sequence 1455
AA573742

Sequence 1456
AA307513

Sequence 1457
Al144526

Sequence 1458
Al167693

Sequence 1459
AA506302

Sequence 1460
W78198

Sequence 1461
AA258725

Sequence 1462
Al887267

Sequence 1463
AA233109

Sequence 1464
R22457

Sequence 1465
Al344311

Sequence 1466
Al344311

Sequence 1467
AA128261

Sequence 1468
Al311789

Sequence 1469
AA612685

Sequence 1470
AA316516

Sequence 1471
AA316516

Sequence 1472
AA830249

Sequence 1473
AA424829

Sequence 1474
N46402

Sequence 1475
AA946876

Sequence 1476
AA411763

Sequence 1477
AA057400

Sequence 1478
AA609169

Sequence 1479
Al310129

Sequence 1480
Al679214

Sequence 1481
AA831877

Sequence 1482
AA653693

27/56

Sequence 1483
N98658

Sequence 1484
AA580144

Sequence 1485
AA219116

Sequence 1486
D81636

Sequence 1487
R35844

Sequence 1488
Al493233

Sequence 1489
AA714531

Sequence 1490
AA531487

Sequence 1491
AA765031

Sequence 1492
AA765031

Sequence 1493
AA191088

Sequence 1494
AA193645

Sequence 1495
AA376374

Sequence 1496
AA864690

Sequence 1497
AA453784

Sequence 1498
AA453784

Sequence 1499
W44970

Sequence 1500
W44970

Sequence 1501
H52011

Sequence 1502
Al678238

Sequence 1503
Al360579

Sequence 1504
H52011

Sequence 1505
H52011

Sequence 1506
AA286693

Sequence 1507
AA233689

Sequence 1508
AA453784

Sequence 1509
AA453784

Sequence 1510
AA788933

Sequence 1511
AA349417

Sequence 1512
AA101077

Sequence 1513
Al41537

Sequence 1514
AA075451

Sequence 1515
AA258725

Sequence 1516
Al309007

Sequence 1517
AA526060

Sequence 1518
AA256330

Sequence 1519
AA156569

Sequence 1520
AA130228

Sequence 1521
D61455

Sequence 1522
AA864690

Sequence 1523
AA349978

Sequence 1524
AA446888

Sequence 1525
Al254200

Sequence 1526
AA151282

Sequence 1527
AA614671

Sequence 1528
Al494235

Sequence 1529
AA410835

Sequence 1530
AA090891

Sequence 1531
Al392999

Sequence 1532
AA604210

Sequence 1533
AA974657

Sequence 1534
AA765228

Sequence 1535
AA070635

Sequence 1536
AA974049

Sequence 1537
AA644001

Sequence 1538
AA037314

Sequence 1539
AA037314

28/56

Sequence 1540
AA214710

Sequence 1541
AA196135

Sequence 1542
AA227890

Sequence 1543
AA451869

Sequence 1544
AA769847

Sequence 1545
T25387

Sequence 1546
Al342714

Sequence 1547
Al342714

Sequence 1548
AA315103

Sequence 1549
AA453217

Sequence 1550
AA349978

Sequence 1551
Al652058

Sequence 1552
Al052317

Sequence 1553
AA082498

Sequence 1554
AA662648

Sequence 1555
AA225164

Sequence 1556
AA092923

Sequence 1557
Al193577

Sequence 1558
N78081

Sequence 1559
AA243394

Sequence 1560
AA100384

Sequence 1561
Al609252

Sequence 1562
Al609252

Sequence 1563
AA075507

Sequence 1564
AA961734

Sequence 1565
AA148011

Sequence 1566
Al077325

Sequence 1567
Al077325

Sequence 1568
AA523877

Sequence 1569
AA039354

Sequence 1570
AA101077

Sequence 1571
AA083471

Sequence 1572
AA723444

Sequence 1573
AA788933

Sequence 1574
AA552818

Sequence 1575
AA088200

Sequence 1576
AA021648

Sequence 1577
AA333713

Sequence 1578
AA702428

Sequence 1579
Al342714

Sequence 1580
Z44537

Sequence 1581
AA424680

Sequence 1582
AA252440

Sequence 1583
Al004348

Sequence 1584
AA738116

Sequence 1585
AA454514

Sequence 1586
AA082795

Sequence 1587
AA242997

Sequence 1588
AA216022

Sequence 1589
AA157054

Sequence 1590
Al278872

Sequence 1591
AA258725

Sequence 1592
AA258725

Sequence 1593
AA573742

Sequence 1594
AA165638

Sequence 1595
Al435429

Sequence 1596
AA639701

29/56

Sequence 1597
AA788933

Sequence 1598
AA707546

Sequence 1599
AA243767

Sequence 1600
AA243767

Sequence 1601
AA300170

Sequence 1602
AA678586

Sequence 1603
Al435276

Sequence 1604
AA157680

Sequence 1605
AA131338

Sequence 1606
AA704512

Sequence 1607
Al630387

Sequence 1608
AA255567

Sequence 1609
AA255567

Sequence 1610
N46402

Sequence 1611
AA531504

Sequence 1612
Al351167

Sequence 1613
AA523877

Sequence 1614
AA181734

Sequence 1615
AA608623

Sequence 1616
D81635

Sequence 1617
Al148825

Sequence 1618
AA122401

Sequence 1619
AA186758

Sequence 1620
AA074086

Sequence 1621
AA216132

Sequence 1622
AA131231

Sequence 1623
AA769937

Sequence 1624
AA376374

Sequence 1625
AA412184

Sequence 1626
AA788933

Sequence 1627
AA258116

Sequence 1628
R54092

Sequence 1629
AA306488

Sequence 1630
H52011

Sequence 1631
A
A947616

Sequence 1632
AA317903

Sequence 1633
AA079835

Sequence 1634
Al131245

Sequence 1635
AA136215

Sequence 1636
AA113359

Sequence 1637
AA375933

Sequence 1638
AA469304

Sequence 1639
AA521069

Sequence 1640
AA111853

Sequence 1641
D14533

Sequence 1642
X13111

Sequence 1643
K00799

Sequence 1644
D10522

Sequence 1645
AB007952

Sequence 1646
X04408

Sequence 1647
X04408

Sequence 1648
AB002368

Sequence 1649
X73608

Sequence 1650
X73608

Sequence 1651
M55409

Sequence 1652
D83327

Sequence 1653
M10905

30/56

Sequence 1654
M30257

Sequence 1655
AF086204

Sequence 1656
AF007216

Sequence 1657
M77830

Sequence 1658
AF052113

Sequence 1659
D38583

Sequence 1660
L13385

Sequence 1661
L13385

Sequence 1662
M17885

Sequence 1663
AF016535

Sequence 1664
AF016535

Sequence 1665
AF068302

Sequence 1666
X04098

Sequence 1667
L10612

Sequence 1668
X07549

Sequence 1669
M31470

Sequence 1670
M95724

Sequence 1671
AJ010841

Sequence 1672
Y07968

Sequence 1673
AF035286

Sequence 1674
AB024704

Sequence 1675
D00726

Sequence 1676
J02943

Sequence 1677
J02943

Sequence 1678
X87949

Sequence 1679
M10941

Sequence 1680
US1586

Sequence 1681
AF004429

Sequence 1682
E02628

Sequence 1683
D87432

Sequence 1684
X07897

Sequence 1685
U39318

Sequence 1686
J03202

Sequence 1687
J03202

Sequence 1688
U34074

Sequence 1689
U42404

Sequence 1690
AF121860

Sequence 1691
D50918

Sequence 1692
D50918

Sequence 1693
X04098

Sequence 1694
X04098

Sequence 1695
U14968

Sequence 1696
U42404

Sequence 1697
AB014577

Sequence 1698
U42404

Sequence 1699
U42404

Sequence 1700
X64707

Sequence 1701
AF127918

Sequence 1702
AF127918

Sequence 1703
E01650

Sequence 1704
M64241

Sequence 1705
M64241

Sequence 1706
E02628

Sequence 1707
X04098

Sequence 1708
AL050161

Sequence 1709
U42404

Sequence 1710
X03963

31/56

Sequence 1711
D86322

Sequence 1712
U42404

Sequence 1713
U42404

Sequence 1714
U51586

Sequence 1715
M34788

Sequence 1716
M34788

Sequence 1717
M58297

Sequence 1718
AF035286

Sequence 1719
M10905

Sequence 1720
X04098

Sequence 1721
U05598

Sequence 1722
AF083322

Sequence 1723
X04098

Sequence 1724
X77956

Sequence 1725
AJ000147

Sequence 1726
M58297

Sequence 1727
E02628

Sequence 1728
AB002533

Sequence 1729
U42404

Sequence 1730
X03963

Sequence 1731
L11066

Sequence 1732
D17268

Sequence 1733
AL050179

Sequence 1734
AB007896

Sequence 1735
U07919

Sequence 1736
K00799

Sequence 1737
U61837

Sequence 1738
AF083322

Sequence 1739
U42404

Sequence 1740
U42404

Sequence 1741
X85373

Sequence 1742
E02326

Sequence 1743
Z11695

Sequence 1744
U07919

Sequence 1745
AB014511

Sequence 1746
AB014511

Sequence 1747
X79535

Sequence 1748
X79535

Sequence 1749
U42404

Sequence 1750
U42404

Sequence 1751
M11353

Sequence 1752
AF070561

Sequence 1753
AF070561

Sequence 1754
AL021683

Sequence 1755
M27508

Sequence 1756
M22590

Sequence 1757
AF070648

Sequence 1758
AJ010841

Sequence 1759
M23254

Sequence 1760
D80005

Sequence 1761
M94345

Sequence 1762
AF070524

Sequence 1763
L42542

Sequence 1764
U42404

Sequence 1765
U42404

Sequence 1766
AF070524

Sequence 1767
AF070524

32/56

Sequence 1768
AF028832

Sequence 1769
U42457

Sequence 1770
K00799

Sequence 1771
M96982

Sequence 1772
X85373

Sequence 1773
L33930

Sequence 1774
M22590

Sequence 1775
M22590

Sequence 1776
X84407

Sequence 1777
U42404

Sequence 1778
X79535

Sequence 1779
AB002533

Sequence 1780
X84407

Sequence 1781
X13238

Sequence 1782
AF086336

Sequence 1783
AB021654

Sequence 1784
U07919

Sequence 1785
AJ223812

Sequence 1786
U42404

Sequence 1787
X07897

Sequence 1788
U42404

Sequence 1789
AB019564

Sequence 1790
M27508

Sequence 1791
AF070561

Sequence 1792
AF070561

Sequence 1793
D87930

Sequence 1794
EQ1650

Sequence 1795
U42457

Sequence 1796
D25542

Sequence 1797
AB004903

Sequence 1798
AB004903

Sequence 1799
M37583

Sequence 1800
M69181

Sequence 1801
M69181

Sequence 1802
M69181

Sequence 1803
S80562

Sequence 1804
U84573

Sequence 1805
AF028832

Sequence 1806
M33308

Sequence 1807
AL035081

Sequence 1808
AF070664

Sequence 1809
AJ011007

Sequence 1810
AB018346

Sequence 1811
D42054

Sequence 1812
M58510

Sequence 1813
K00799

Sequence 1814
E02326

Sequence 1815
M16279

Sequence 1816
U42404

Sequence 1817
AL035081

Sequence 1818
AB007862

Sequence 1819
E01650

Sequence 1820
E01650

Sequence 1821
M23254

Sequence 1822
M23254

Sequence 1823
AB003102

Sequence 1824
L25610

33/56

Sequence 1825
M16279

Sequence 1826
J02943

Sequence 1827
AL049974

Sequence 1828
AF098462

Sequence 1829
M15502

Sequence 1830
E01650

Sequence 1831
AF084260

Sequence 1832
AF120268

Sequence 1833
AJ010841

Sequence 1834
AJ010841

Sequence 1835
U42404

Sequence 1836
U90545

Sequence 1837
X07897

Sequence 1838
AB007862

Sequence 1839
AF011468

Sequence 1840
L38933

Sequence 1841
U07343

Sequence 1842
M69066

Sequence 1843
L05093

Sequence 1844
X04098

Sequence 1845
AF143324

Sequence 1846
D12676

Sequence 1847
Y11312

Sequence 1848
U14970

Sequence 1849
X81198

Sequence 1850
AF021819

Sequence 1851
J03576

Sequence 1852
M10905

Sequence 1853
U42404

Sequence 1854
U42404

Sequence 1855
U42404

Sequence 1856
AB018346

Sequence 1857
AB012083

Sequence 1858
X02761

Sequence 1859
X81198

Sequence 1860
U64898

Sequence 1861
064015

Sequence 1862
Y09565

Sequence 1863
AF007145

Sequence 1864
E01650

Sequence 1865
U57847

Sequence 1866
M69066

Sequence 1867
U41850

Sequence 1868
AJ006834

Sequence 1869
012485

Sequence 1870
AJ010841

Sequence 1871
AB019568

Sequence 1872
AB014560

Sequence 1873
X97064

Sequence 1874
U42404

Sequence 1875
J00127

Sequence 1876
AF086002

Sequence 1877
U14969

Sequence 1878
U42404

Sequence 1879
AF119386

Sequence 1880
AB002533

Sequence 1881
J03634

34/56

Sequence 1882
M74775

Sequence 1883
D26124

Sequence 1884
J03460

Sequence 1885
AF151885

Sequence 1886
D49737

Sequence 1887
U42404

Sequence 1888
AF116827

Sequence 1889
M14083

Sequence 1890
Z47087

Sequence 1891
M23254

Sequence 1892
AF054990

Sequence 1893
M22324

Sequence 1894
AF077045

Sequence 1895
U42457

Sequence 1896
AB020660

Sequence 1897
D80005

Sequence 1898
X64707

Sequence 1899
AL021683

Sequence 1900
AF151885

Sequence 1901
D32000

Sequence 1902
S82470

Sequence 1903
AL049381

Sequence 1904
X81198

Sequence 1905
D50918

Sequence 1906
D50918

Sequence 1907
AF070561

Sequence 1908
S78569

Sequence 1909
U02390

Sequence 1910
U34074

Sequence 1911
U33760

Sequence 1912
E03413

Sequence 1913
J03202

Sequence 1914
M55268

Sequence 1915
J03537

Sequence 1916
D21260

Sequence 1917
AF044671

Sequence 1918
AF044671

Sequence 1919
M22918

Sequence 1920
AF054990

Sequence 1921
M69181

Sequence 1922
AF006084

Sequence 1923
L16785

Sequence 1924
Y00052

Sequence 1925
U57847

Sequence 1926
M69181

Sequence 1927
X69970

Sequence 1928
X69970

Sequence 1929
AB018346

Sequence 1930
AF077045

Sequence 1931
AF077043

Sequence 1932
AF054179

Sequence 1933
AF054179

Sequence 1934
D63998

Sequence 1935
D63998

Sequence 1936
AB000220

Sequence 1937
E03157

Sequence 1938
M33308

35/56

Sequence 1939
L34600

Sequence 1940
AF039022

Sequence 1941
X07897

Sequence 1942
AB014548

Sequence 1943
AL021683

Sequence 1944
D86957

Sequence 1945
J04543

Sequence 1946
L13806

Sequence 1947
U12465

Sequence 1948
X87176

Sequence 1949
U42458

Sequence 1950
AB007877

Sequence 1951
AB002533

Sequence 1952
AB002533

Sequence 1953
X64707

Sequence 1954
M21551

Sequence 1955
D49737

Sequence 1956
AJ223500

Sequence 1957
D13641

Sequence 1958
L34600

Sequence 1959
AF039022

Sequence 1960
M69181

Sequence 1961
AF147319

Sequence 1962
M10119

Sequence 1963
M10119

Sequence 1964
U41515

Sequence 1965
J03202

Sequence 1966
X83218

Sequence 1967
J00127

Sequence 1968
J04543

Sequence 1969
M69181

Sequence 1970
K00799

Sequence 1971
D50918

Sequence 1972
D83198

Sequence 1973
D83198

Sequence 1974
AB011115

Sequence 1975
AB011115

Sequence 1976
AF070638

Sequence 1977
AF070638

Sequence 1978
L28010

Sequence 1979
L28010

Sequence 1980
AF031385

Sequence 1981
AF031385

Sequence 1982
AB002386

Sequence 1983
AB002386

Sequence 1984
X98248

Sequence 1985
U02032

Sequence 1986
K03195

Sequence 1987
U42404

Sequence 1988
U42404

Sequence 1989
S56985

Sequence 1990
AF077611

Sequence 1991
D49950

Sequence 1992
J03460

Sequence 1993
AF070649

Sequence 1994
M24194

Sequence 1995
X13238

36/56

Sequence 1996
X13238

Sequence 1997
M11058

Sequence 1998
037991

Sequence 1999
AL050161

Sequence 2000
AJ002030

Sequence 2001
AB002533

Sequence 2002
Y00503

Sequence 2003
087665

Sequence 2004
D87665

Sequence 2005
029992

Sequence 2006
K00799

Sequence 2007
Y00345

Sequence 2008
D50918

Sequence 2009
D50918

Sequence 2010
L05095

Sequence 2011
AF077045

Sequence 2012
M22920

Sequence 2013
AL050218

Sequence 2014
M81757

Sequence 2015
L13210

Sequence 2016
X03963

Sequence 2017
AL050018

Sequence 2018
Y00819

Sequence 2019
M17733

Sequence 2020
U94586

Sequence 2021
U94586

Sequence 2022
J00129

Sequence 2023
U42404

Sequence 2024
U42404

Sequence 2025
X51473

Sequence 2026
029643

Sequence 2027
K00799

Sequence 2028
X79535

Sequence 2029
X79535

Sequence 2030
AF020797

Sequence 2031
AB002533

Sequence 2032
AB002533

Sequence 2033
M69181

Sequence 2034
M69181

Sequence 2035
M94345

Sequence 2036
AF029890

Sequence 2037
AF029890

Sequence 2038
X56998

Sequence 2039
X56998

Sequence 2040
087665

Sequence 2041
087665

Sequence 2042
J00127

Sequence 2043
M58569

Sequence 2044
M16247

Sequence 2045
M16247

Sequence 2046
X69392

Sequence 2047
U73778

Sequence 2048
U42457

Sequence 2049
016892

Sequence 2050
X15187

Sequence 2051
M14083

Sequence 2052
Z82022

37/56

Sequence 2053
D89667

Sequence 2054
J03460

Sequence 2055
J02611

Sequence 2056
AF145316

Sequence 2057
U42404

Sequence 2058
U42404

Sequence 2059
AF070561

Sequence 2060
AF070561

Sequence 2061
AF070649

Sequence 2062
AB019568

Sequence 2063
J03634

Sequence 2064
J03634

Sequence 2065
AJ010841

Sequence 2066
D37766

Sequence 2067
AF054990

Sequence 2068
X79535

Sequence 2069
U05598

Sequence 2070
AF007791

Sequence 2071
AF038451

Sequence 2072
J03592

Sequence 2073
X87176

Sequence 2074
U42404

Sequence 2075
U42594

Sequence 2076
D29992

Sequence 2077
AL050218

Sequence 2078
AL021683

Sequence 2079
M10119

Sequence 2080
M10119

Sequence 2081
D87454

Sequence 2082
D87454

Sequence 2083
AB019568

Sequence 2084
M22920

Sequence 2085
X07979

Sequence 2086
AL050209

Sequence 2087
AL050209

Sequence 2088
AB014577

Sequence 2089
AB014577

Sequence 2090
AJ010841

Sequence 2091
AF045606

Sequence 2092
AF086280

Sequence 2093
AB014548

Sequence 2094
AF132959

Sequence 2095
D50918

Sequence 2096
X87176

Sequence 2097
M24630

Sequence 2098
EQ1650

Sequence 2099
M69181

Sequence 2100
AJ010841

Sequence 2101
AB012664

Sequence 2102
J03202

Sequence 2103
AF017789

Sequence 2104
AF017789

Sequence 2105
X56999

Sequence 2106
AF035319

Sequence 2107
K03515

Sequence 2108
U39318

Sequence 2109
X87176

38/56

Sequence 2110
X87176

Sequence 2111
AB007896

Sequence 2112
AF001434

Sequence 2113
AB007896

Sequence 2114
AB007896

Sequence 2115
D50525

Sequence 2116
Z24725

Sequence 2117
AB000220

Sequence 2118
AB000220

Sequence 2119
D16234

Sequence 2120
D16234

Sequence 2121
M55265

Sequence 2122
J03779

Sequence 2123
U90545

Sequence 2124
AF026166

Sequence 2125
E03157

Sequence 2126
X60489

Sequence 2127
AB007883

Sequence 2128
AB007883

Sequence 2129
M10905

Sequence 2130
AF026166

Sequence 2131
X63692

Sequence 2132
K00799

Sequence 2133
X15187

Sequence 2134
X15187

Sequence 2135
X63692

Sequence 2136
K00799

Sequence 2137
M10905

Sequence 2138
AB011128

Sequence 2139
X02761

Sequence 2140
L27211

Sequence 2141
AF077043

Sequence 2142
E01797

Sequence 2143
J04031

Sequence 2144
AB011128

Sequence 2145
X76302

Sequence 2146
D89053

Sequence 2147
M24194

Sequence 2148
U64898

Sequence 2149
X93207

Sequence 2150
M10905

Sequence 2151
J03202

Sequence 2152
D89053

Sequence 2153
D13642

Sequence 2154
J03537

Sequence 2155
D64015

Sequence 2156
D64015

Sequence 2157
AF000421

Sequence 2158
AF000421

Sequence 2159
M22920

Sequence 2160
M22918

Sequence 2161
D29992

Sequence 2162
D29992

Sequence 2163
J03202

Sequence 2164
J03202

Sequence 2165
M17885

Sequence 2166
AF046025

39/56

Sequence 2167
AF046025

Sequence 2168
J02943

Sequence 2169
J02943

Sequence 2170
AL021683

Sequence 2171
J03040

Sequence 2172
AJ010841

Sequence 2173
AJ010841

Sequence 2174
U05598

Sequence 2175
AB021654

Sequence 2176
J02943

Sequence 2177
D38255

Sequence 2178
D84212

Sequence 2179
AF008551

Sequence 2180
L33930

Sequence 2181
J03202

Sequence 2182
J03202

Sequence 2183
Z22555

Sequence 2184
K00799

Sequence 2185
X02761

Sequence 2186
X63432

Sequence 2187
X51473

Sequence 2188
X81198

Sequence 2189
D13388

Sequence 2190
AB018346

Sequence 2191
X70326

Sequence 2192
M23254

Sequence 2193
AF086003

Sequence 2194
K00799

Sequence 2195
X67698

Sequence 2196
X67698

Sequence 2197
M10941

Sequence 2198
EQ1650

Sequence 2199
AF086336

Sequence 2200
AJ011007

Sequence 2201
U76421

Sequence 2202
D29992

Sequence 2203
AB001106

Sequence 2204
J00129

Sequence 2205
AF052164

Sequence 2206
U61836

Sequence 2207
K02765

Sequence 2208
AB006679

Sequence 2209
M10905

Sequence 2210
AF077045

Sequence 2211
AF089747

Sequence 2212
J03460

Sequence 2213
J03460

Sequence 2214
AB007862

Sequence 2215
AB007862

Sequence 2216
AB018346

Sequence 2217
AB019568

Sequence 2218
AF000982

Sequence 2219
J00127

Sequence 2220
D45198

Sequence 2221
M15990

Sequence 2222
M15990

Sequence 2223
AB010812

40/56

Sequence 2224
U63139

Sequence 2225
AF028832

Sequence 2226
AF145316

Sequence 2227
EQ1816

Sequence 2228
Y00281

Sequence 2229
AF035752

Sequence 2230
U94586

Sequence 2231
AB019568

Sequence 2232
J04823

Sequence 2233
L43964

Sequence 2234
L43964

Sequence 2235
M31523

Sequence 2236
AF007128

Sequence 2237
AF023611

Sequence 2238
AF031385

Sequence 2239
L37368

Sequence 2240
J03460

Sequence 2241
L13286

Sequence 2242
AL050380

Sequence 2243
AL050380

Sequence 2244
U33760

Sequence 2245
AB019564

Sequence 2246
M27504

Sequence 2247
M27504

Sequence 2248
AF030555

Sequence 2249
AF030555

Sequence 2250
M37583

Sequence 2251
AF047002

Sequence 2252
AF086513

Sequence 2253
AJ002030

Sequence 2254
U85755

Sequence 2255
L05425

Sequence 2256
L05425

Sequence 2257
X07897

Sequence 2258
X07897

Sequence 2259
K03515

Sequence 2260
K03515

Sequence 2261
X70394

Sequence 2262
X70394

Sequence 2263
M10905

Sequence 2264
U41850

Sequence 2265
L22154

Sequence 2266
AJ004913

Sequence 2267
AF064093

Sequence 2268
S80343

Sequence 2269
D13641

Sequence 2270
D13641

Sequence 2271
AF016535

Sequence 2272
S68330

Sequence 2273
L05425

Sequence 2274
AF054987

Sequence 2275
AF054987

Sequence 2276
AL049386

Sequence 2277
AB019564

Sequence 2278
AF054990

Sequence 2279
AB007931

Sequence 2280
M74524

41/56

Sequence 2281
U76764

Sequence 2282
X05908

Sequence 2283
J02943

Sequence 2284
AF117815

Sequence 2285
J03537

Sequence 2286
AF070550

Sequence 2287
J00127

Sequence 2288
U42594

Sequence 2289
J03460

Sequence 2290
X53331

Sequence 2291
M58549

Sequence 2292
M81757

Sequence 2293
M86667

Sequence 2294
L06328

Sequence 2295
L08666

Sequence 2296
AJ011007

Sequence 2297
X07897

Sequence 2298
AF035752

Sequence 2299
AF077205

Sequence 2300
D13748

Sequence 2301
D13748

Sequence 2302
X63527

Sequence 2303
014665

Sequence 2304
D14665

Sequence 2305
X64707

Sequence 2306
AF132000

Sequence 2307
AF132000

Sequence 2308
X87949

Sequence 2309
M31899

Sequence 2310
M31899

Sequence 2311
X63432

Sequence 2312
V00478

Sequence 2313
U42594

Sequence 2314
U42594

Sequence 2315
AF039656

Sequence 2316
U84573

Sequence 2317
U84573

Sequence 2318
055654

Sequence 2319
AL050161

Sequence 2320
AL050380

Sequence 2321
U34360

Sequence 2322
AJ010841

Sequence 2323
AB020684

Sequence 2324
AB020684

Sequence 2325
U63139

Sequence 2326
U63139

Sequence 2327
M80359

Sequence 2328
M80359

Sequence 2329
X97065

Sequence 2330
X97065

Sequence 2331
U94586

Sequence 2332
U05598

Sequence 2333
AF070672

Sequence 2334
AF070672

Sequence 2335
AF098865

Sequence 2336
AF098865

Sequence 2337
AF070561

42/56

Sequence 2338
AF070550

Sequence 2339
AF070550

Sequence 2340
J03537

Sequence 2341
AF069072

Sequence 2342
AF069072

Sequence 2343
AF038451

Sequence 2344
AF007791

Sequence 2345
U77085

Sequence 2346
AF070561

Sequence 2347
AF070561

Sequence 2348
X79535

Sequence 2349
M10905

Sequence 2350
AF039656

Sequence 2351
U39360

Sequence 2352
U39360

Sequence 2353
D78335

Sequence 2354
U76764

Sequence 2355
AB011108

Sequence 2356
AL049974

Sequence 2357
J03202

Sequence 2358
J03202

Sequence 2359
AF077043

Sequence 2360
AF077043

Sequence 2361
U84573

Sequence 2362
U02032

Sequence 2363
Z28407

Sequence 2364
K00799

Sequence 2365
U25766

Sequence 2366
U25766

Sequence 2367
AB017363

Sequence 2368
J03537

Sequence 2369
L31610

Sequence 2370
S60099

Sequence 2371
AB007862

Sequence 2372
AB007862

Sequence 2373
AF031385

Sequence 2374
J02814

Sequence 2375
J02814

Sequence 2376
U79291

Sequence 2377
AF077045

Sequence 2378
AF077045

Sequence 2379
M94345

Sequence 2380
AF064093

Sequence 2381
AF064093

Sequence 2382
X04098

Sequence 2383
J00129

Sequence 2384
M58569

Sequence 2385
AB020660

Sequence 2386
M94345

Sequence 2387
J04991

Sequence 2388
AB014577

Sequence 2389
AL049339

Sequence 2390
AB014512

Sequence 2391
X13709

Sequence 2392
M19961

Sequence 2393
M22920

Sequence 2394
K00799

43/56

Sequence 2395
X02761

Sequence 2396
S75169

Sequence 2397
AF085361

Sequence 2398
J00127

Sequence 2399
M24486

Sequence 2400
J03460

Sequence 2401
M10119

Sequence 2402
E02628

Sequence 2403
E01650

Sequence 2404
J03202

Sequence 2405
J03202

Sequence 2406
U41515

Sequence 2407
M24630

Sequence 2408
M24630

Sequence 2409
S59184

Sequence 2410
X69970

Sequence 2411
D50312

Sequence 2412
D50312

Sequence 2413
AB000220

Sequence 2414
AB000220

Sequence 2415
D87453

Sequence 2416
U14391

Sequence 2417
U14391

Sequence 2418
AF073298

Sequence 2419
AF086336

Sequence 2420
AF086336

Sequence 2421
AF000982

Sequence 2422
AF000982

Sequence 2423
U19252

Sequence 2424
U19252

Sequence 2425
J03799

Sequence 2426D
50371

Sequence 2427
U12404

Sequence 2428
AF086557

Sequence 2429
X56932

Sequence 2430
J02943

Sequence 2431
D50371

Sequence 2432
AB028624

Sequence 2433
M65217

Sequence 2434
U30246

Sequence 2435
U30246

Sequence 2436
X02761

Sequence 2437
AB002533

Sequence 2438
AB002533

Sequence 2439
D49737

Sequence 2440
X07897

Sequence 2441
M55268

Sequence 2442
S66431

Sequence 2443
S66431

Sequence 2444
U00947

Sequence 2445
M33308

Sequence 2446
M33308

Sequence 2447
J04543

Sequence 2448
M33308

Sequence 2449
M10905

Sequence 2450
L16785

Sequence 2451
L16785

44/56

Sequence 2452
U63139

Sequence 2453
U63139

Sequence 2454
AF088071

Sequence 2455
U15008

Sequence 2456
X04098

Sequence 2457
X04098

Sequence 2458
D63476

Sequence 2459
X79535

Sequence 2460
D50918

Sequence 2461
D50918

Sequence 2462
U42404

Sequence 2463
D13641

Sequence 2464
D13641

Sequence 2465
AB023151

Sequence 2466
AB023151

Sequence 2467
U42594

Sequence 2468
U42594

Sequence 2469
U42457

Sequence 2470
EQ1816

Sequence 2471
U42404

Sequence 2472
U42404

Sequence 2473
AF086336

Sequence 2474
AF086336

Sequence 2475
U42404

Sequence 2476
J04621

Sequence 2477
AF039656

Sequence 2478
D50918

Sequence 2479
AF001601

Sequence 2480
AF001601

Sequence 2481
AB014577

Sequence 2482
AB014577

Sequence 2483
AF054183

Sequence 2484
AF052578

Sequence 2485
M12623

Sequence 2486
AF151832

Sequence 2487
AF007170

Sequence 2488
AF007170

Sequence 2489
AF054987

Sequence 2490
AF054987

Sequence 2491
J02943

Sequence 2492
L20941

Sequence 2493
L20941

Sequence 2494
M27504

Sequence 2495
M27504

Sequence 2496
AF104419

Sequence 2497
M80359

Sequence 2498
M80359

Sequence 2499
AF039656

Sequence 2500
U57847

Sequence 2501
U57847

Sequence 2502
D13642

Sequence 2503
X02761

Sequence 2504
Y08982

Sequence 2505
AJ010841

Sequence 2506
AJ002030

Sequence 2507
AJ002030

Sequence 2508
AB019568

45/56

Sequence 2509
AB021654

Sequence 2510
U05598

Sequence 2511
AF099149

Sequence 2512
L37368

Sequence 2513
L37368

Sequence 2514
AB019564

Sequence 2515
J00128

Sequence 2516
J00127

Sequence 2517
D90209

Sequence 2518
D90209

Sequence 2519
AB007862

Sequence 2520
AB007862

Sequence 2521
E01954

Sequence 2522
E01954

Sequence 2523
AB020692

Sequence 2524
AB020692

Sequence 2525
J04973

Sequence 2526
D63486

Sequence 2527
D45906

Sequence 2528
J00127

Sequence 2529
J00127

Sequence 2530
U12465

Sequence 2531
U12465

Sequence 2532
K03515

Sequence 2533
AB020662

Sequence 2534
AB002322

Sequence 2535
K03515

Sequence 2536
K03515

Sequence 2537
U30246

Sequence 2538
U30246

Sequence 2539
AF147331

Sequence 2540
X00570

Sequence 2541
AF077599

Sequence 2542
J00129

Sequence 2543
J00129

Sequence 2544
U42404

Sequence 2545
U42404

Sequence 2546
AJ002030

Sequence 2547
AJ002030

Sequence 2548
AB023195

Sequence 2549
AB023195

Sequence 2550
D50310

Sequence 2551
U39402

Sequence 2552
X87949

Sequence 2553
M10119

Sequence 2554
M10119

Sequence 2555
AF070550

Sequence 2556
AF070550

Sequence 2557
AF131781

Sequence 2558
AF131781

Sequence 2559
U53476

Sequence 2560
M10905

Sequence 2561
X02761

Sequence 2562
Y00503

Sequence 2563
J03040

Sequence 2564
U42404

Sequence 2565
U42404

46/56

Sequence 2566
AF092128

Sequence 2567
J03796

Sequence 2568
J03796

Sequence 2569
AF070606

Sequence 2570
M17885

Sequence 2571
D63486

Sequence 2572
D63486

Sequence 2573
D87127

Sequence 2574
D87127

Sequence 2575
J02943

Sequence 2576
J02943

Sequence 2577
AB001106

Sequence 2578
M10905

Sequence 2579
D45906

Sequence 2580
AF092133

Sequence 2581
J03537

Sequence 2582
M58485

Sequence 2583
AF077205

Sequence 2584
AF077205

Sequence 2585
AF007791

Sequence 2586
AF007791

Sequence 2587
D49737

Sequence 2588
D49737

Sequence 2589
X15880

Sequence 2590
X77588

Sequence 2591
U03272

Sequence 2592
AF0086336

Sequence 2593
D49489

Sequence 2594
S78085

Sequence 2595
X00570

Sequence 2596
J03460

Sequence 2597
AF007791

Sequence 2598
U77085

Sequence 2599
U77085

Sequence 2600
X07979

Sequence 2601
D63776

Sequence 2602
D83776

Sequence 2603
AF086336

Sequence 2604
AF086336

Sequence 2605
AF026004

Sequence 2606
AF077044

Sequence 2607
AF077044

Sequence 2608
AF151856

Sequence 2609
AF151856

Sequence 2610
AB018346

Sequence 2611
AB018346

Sequence 2612
U78575

Sequence 2613
U78578

Sequence 2614
M10905

Sequence 2615
K00799

Sequence 2616
K00799

Sequence 2617
L05425

Sequence 2618
L05425

Sequence 2619
AF086336

Sequence 2620
AJ011007

Sequence 2621
L19184

Sequence 2622
X67951

47/56

Sequence 2623
AF038451

Sequence 2624
M29960

Sequence 2625
AF086336

Sequence 2626
AF086336

Sequence 2627
Y00345

Sequence 2628
Y00345

Sequence 2629
J03202

Sequence 2630
AF086183

Sequence 2631
AF086183

Sequence 2632
X77588

Sequence 2633
U14528

Sequence 2634
X17206

Sequence 2635
M81757

Sequence 2636
M81757

Sequence 2637
J03202

Sequence 2638
J03202

Sequence 2639
AF007791

Sequence 2640
AF038451

Sequence 2641
AF007791

Sequence 2642
M98479

Sequence 2643
E01650

Sequence 2644
AF114264

Sequence 2645
U42404

Sequence 2646
U42404

Sequence 2647
AB002533

Sequence 2648
Y08982

Sequence 2649
AF086172

Sequence 2650
X02761

Sequence 2651
U30521

Sequence 2652
U30521

Sequence 2653
X63527

Sequence 2654
L05092

Sequence 2655
L05092

Sequence 2656
Z24725

Sequence 2657
X54942

Sequence 2658
X54942

Sequence 2659
AB002439

Sequence 2660
X81889

Sequence 2661
X81889

Sequence 2662
AF007150

Sequence 2663
L10910

Sequence 2664
AL049339

Sequence 2665
X55740

Sequence 2666
X55740

Sequence 2667
AF143096

Sequence 2668
AF143096

Sequence 2669
AB020684

Sequence 2670
U42457

Sequence 2671
Z31696

Sequence 2672
AF098865

Sequence 2673
AF098865

Sequence 2674
U12404

Sequence 2675
S75169

Sequence 2676
AF070672

Sequence 2677
AF153329

Sequence 2678
U05040

Sequence 2679
AF086336

48/56

Sequence 2680
AF132959

Sequence 2681
U47077

Sequence 2682
U34994

Sequence 2683
J03460

Sequence 2684
U06452

Sequence 2685
U06452

Sequence 2686
M34788

Sequence 2687
M34788

Sequence 2688
021260

Sequence 2689
D21260

Sequence 2690
AF039575

Sequence 2691
D55671

Sequence 2692
X16312

Sequence 2693
J03202

Sequence 2694
J03202

Sequence 2695
AF077045

Sequence 2696
D29992

Sequence 2697
AF070606

Sequence 2698
AF070606

Sequence 2699
K00799

Sequence 2700
K00799

Sequence 2701
X07979

Sequence 2702
E02628

Sequence 2703
AF016582

Sequence 2704
AF016582

Sequence 2705
U47101

Sequence 2706
U47101

Sequence 2707
L05425

Sequence 2708
AJ010841

Sequence 2709
U70660

Sequence 2710
AF052178

Sequence 2711
M98479

Sequence 2712
AF151856

Sequence 2713
AF151856

Sequence 2714
AF147331

Sequence 2715
K00799

Sequence 2716
K00799

Sequence 2717
063486

Sequence 2718
063486

Sequence 2719
D50371

Sequence 2720
AB028624

Sequence 2721
AF020797

Sequence 2722
AB014577

Sequence 2723
AB014577

Sequence 2724
AB014512

Sequence 2725
M69181

Sequence 2726
M69181

Sequence 2727
U42404

Sequence 2728
U42404

Sequence 2729
013641

Sequence 2730
D13641

Sequence 2731
D14657

Sequence 2732
L16785

Sequence 2733
087469

Sequence 2734
X13709

Sequence 2735
M19961

Sequence 2736
AL049974

49/56

Sequence 2737
M19961

Sequence 2738
X02761

Sequence 2739
Y08982

Sequence 2740
X96484

Sequence 2741
AL049339

Sequence 2742
AF114264

Sequence 2743
AF114264

Sequence 2744
Z13009

Sequence 2745
AB002330

Sequence 2746
AF092128

Sequence 2747
AF086408

Sequence 2748
M24486

Sequence 2749
M24486

Sequence 2750
AB020657

Sequence 2751
D87667

Sequence 2752
D87667

Sequence 2753
X15187

Sequence 2754
M81757

Sequence 2755
M38188

Sequence 2756
AJ007669

Sequence 2757
M64098

Sequence 2758
J02943

Sequence 2759
AB002439

Sequence 2760
S72481

Sequence 2761
S72481

Sequence 2762
D00099

Sequence 2763
D00099

Sequence 2764
M10905

Sequence 2765
M14060

Sequence 2766
Z31696

Sequence 2767
X81109

Sequence 2768
J03202

Sequence 2769
U46194

Sequence 2770
AB018281

Sequence 2771
X59618

Sequence 2772
AF016582

Sequence 2773
X79535

Sequence 2774
L13806

Sequence 2775
U75283

Sequence 2776
Y00052

Sequence 2777
M23254

Sequence 2778
X69141

Sequence 2779
J03202

Sequence 2780
K00799

Sequence 2781
AF068706

Sequence 2782
J02814

Sequence 2783
K00799

Sequence 2784
AB021654

Sequence 2785
AF016535

Sequence 2786
M10905

Sequence 2787
AB007862

Sequence 2788
U46194

Sequence 2789
AF070550

Sequence 2790
J03796

Sequence 2791
X04098

Sequence 2792
U30246

Sequence 2793
U25789

50/56

Sequence 2794
AF086336

Sequence 2795
M22918

Sequence 2796
M10905

Sequence 2797
U25766

Sequence 2798
X79535

Sequence 2799
L16785

Sequence 2800
L16785

Sequence 2801
M22636

Sequence 2802
M22636

Sequence 2803
087735

Sequence 2804
X07979

Sequence 2805
U05598

Sequence 2806
AB021654

Sequence 2807
X17206

Sequence 2808
087735

Sequence 2809
J04088

Sequence 2810
AF046025

Sequence 2811
AF046025

Sequence 2812
AF086336

Sequence 2813
AJQ11007

Sequence 2814
X02761

Sequence 2815
X02761

Sequence 2816
Y00819

Sequence 2817
X04098

Sequence 2818
X04098

Sequence 2819
AF086205

Sequence 2820
X85373

Sequence 2821
X85373

Sequence 2822
L12387

Sequence 2823
L12387

Sequence 2824
AF054990

Sequence 2825
AL049929

Sequence 2826
K00799

Sequence 2827
U42404

Sequence 2828
AF086336

Sequence 2829
AF086336

Sequence 2830
K00799

Sequence 2831
029992

Sequence 2832
029992

Sequence 2833
U31384

Sequence 2834
U31384

Sequence 2835
AF086336

Sequence 2836
AF086336

Sequence 2837
AF077043

Sequence 2838
AF043431

Sequence 2839
AF043431

Sequence 2840
M17885

Sequence 2841
U73377

Sequence 2842
X70394

Sequence 2843
X70394

Sequence 2844
X00351

Sequence 2845
X63432

Sequence 2846
X02308

Sequence 2847
M10905

Sequence 2848
M10905

Sequence 2849
Z26317

Sequence 2850
Z26317

51/56

Sequence 2851
M10905

Sequence 2852
M22918

Sequence 2853
AF011793

Sequence 2854
AJ001309

Sequence 2855
AF068706

Sequence 2856
X67731

Sequence 2857
AF016535

Sequence 2858
AF016535

Sequence 2859
X02761

Sequence 2860
AB007883

Sequence 2861
AB011089

Sequence 2862
L13806

Sequence 2863
M10905

Sequence 2864
L05425

Sequence 2865
L05425

Sequence 2866
029992

Sequence 2867
AF007791

Sequence 2868
083776

Sequence 2869
083776

Sequence 2870
AB014577

Sequence 2871
AB014577

Sequence 2872
X55740

Sequence 2873
X55740

Sequence 2874
L05425

Sequence 2875
AB017116

Sequence 2876
AB017116

Sequence 2877
K00799

Sequence 2878
U42404

Sequence 2879
U42404

Sequence 2880
J03198

Sequence 2881
J03198

Sequence 2882
M58549

Sequence 2883
029992

Sequence 2884
AB007883

Sequence 2885
AB007883

Sequence 2886
M33308

Sequence 288T
M33308

Sequence 2888
087735

Sequence 2889
087735

Sequence 2890
AB011128

Sequence 2891
AJ010841

Sequence 2892
AJ010841

Sequence 2893
U42404

Sequence 2894
U42404

Sequence 2895
M23114

Sequence 2896
S75169

Sequence 2897
S75169

Sequence 2898
X02761

Sequence 2899
AF007791

Sequence 2900
K00799

Sequence 2901
K00799

Sequence 2902
AF072928

Sequence 2903
K00799

Sequence 2904
U76421

Sequence 2905
M58549

Sequence 2906
X02761

Sequence 2907
X02761

52/56

Sequence 2908
M69181

Sequence 2909
M69181

Sequence 2910
AB019568

Sequence 2911
AF031385

Sequence 2912
X04470

Sequence 2913
X04470

Sequence 2914
U76421

Sequence 2915
AB023191

Sequence 2916
AB023191

Sequence 2917
U34994

Sequence 2918
U34994

Sequence 2919
X84407

Sequence 2920
AJ011001

Sequence 2921
AJ011001

Sequence 2922
013748

Sequence 2923
D13748

Sequence 2924
029992

Sequence 2925
D29992

Sequence 2926
U42457

Sequence 2927
U42457

Sequence 2928
AL050282

Sequence 2929
EQ1650

Sequence 2930
J03779

Sequence 2931
K00799

Sequence 2932
K00799

Sequence 2933
AF085844

Sequence 2934
M16462

Sequence 2935
Y09565

Sequence 2936
D87667

Sequence 2937
087667

Sequence 2938
L05093

Sequence 2939
AB007883

Sequence 2940
U14971

Sequence 2941
U14971

Sequence 2942
U44754

Sequence 2943
U44754

Sequence 2944
X60656

Sequence 2945
AB018346

Sequence 2946
U85658

Sequence 2947
U85658

Sequence 2948
M69181

Sequence 2949
AF008551

Sequence 2950
AF008551

Sequence 2951
D29992

Sequence 2952
S77362

Sequence 2953
J05032

Sequence 2954
AF052178

Sequence 2955
AF052178

Sequence 2956
AJ010841

Sequence 2957
AJ010841

Sequence 2958
U02032

Sequence 2959
U02032

Sequence 2960
AB011089

Sequence 2961
AF050638

Sequence 2962
085181

Sequence 2963
AJ010841

Sequence 2964
AJ010841

53/56

Sequence 2965
U07343

Sequence 2966
U42404

Sequence 2967
U42404

Sequence 2968
X04098

Sequence 2969
X04098

Sequence 2970
AF011793

Sequence 2971
AJ001309

Sequence 2972
AL049339

Sequence 2973
AF028832

Sequence 2974
AF131797

Sequence 2975
U42404

Sequence 2976
U42404

Sequence 2977
X02761

Sequence 2978
AB028974

Sequence 2979
AB028974

Sequence 2980
U45976

Sequence 2981
L22154

Sequence 2982
AF068007

Sequence 2983
D83776

Sequence 2984
D83776

Sequence 2985
J02943

Sequence 2986
J04543

Sequence 2987
M14200

Sequence 2988
U42404

Sequence 2989
U42404

Sequence 2990
AF077208

Sequence 2991
J02943

Sequence 2992
U18062

Sequence 2993
U18062

Sequence 2994
AF010309

Sequence 2995
AF010309

Sequence 2996
AL132665

Sequence 2997
AL132665

Sequence 2998
X69181

Sequence 2999
J03015

Sequence 3000
J03015

Sequence 3001
X81889

Sequence 3002
D13641

Sequence 3003
D13641

Sequence 3004
X96484

Sequence 3005
U42404

Sequence 3006
X69970

Sequence 3007
J02943

Sequence 3008
M69181

Sequence 3009
J02943

Sequence 3010
D83776

Sequence 3011
D83776

Sequence 3012
D87667

Sequence 3013
M73792

Sequence 3014
X15187

Sequence 3015
X15187

Sequence 3016
AF081280

Sequence 3017
AF086003

Sequence 3018
AF086003

Sequence 3019
M11353

Sequence 3020
M11353

Sequence 3021
M38188

54/56

Sequence 3022
000099

Sequence 3023
M19961

Sequence 3024
M19961

Sequence 3025
EQ1816

Sequence 3026
AF100741

Sequence 3027
AF001176

Sequence 3028
X04098

Sequence 3029
U92993

Sequence 3030
A21185

Sequence 3031
D17266

Sequence 3032
AJ002030

Sequence 3033
AF054179

Sequence 3034
AF054179

Sequence 3035
X56932

Sequence 3036
X69970

Sequence 3037
X69970

Sequence 3038
U82130

Sequence 3039
U82130

Sequence 3040
M10119

Sequence 3041
M10119

Sequence 3042
L16785

Sequence 3043
AF132959

Sequence 3044
AF132959

Sequence 3045
D87667

Sequence 3046
K00799

Sequence 3047
M55268

Sequence 3048
M55268

Sequence 3049
U42404

Sequence 3050
X51473

Sequence 3051
AF016535

Sequence 3052
L10910

Sequence 3053
U42404

Sequence 3054
U42404

Sequence 3055
D49737

Sequence 3056
AF086336

Sequence 3057
X07897

Sequence 3058
AB011159

Sequence 3059
AB011159

Sequence 3060
M81182

Sequence 3061
M81182

Sequence 3062
AF046025

Sequence 3063
AF046025

Sequence 3064
AB020649

Sequence 3065
021260

Sequence 3066
021260

Sequence 3067
014530

Sequence 3068
AF038451

Sequence 3069
038255

Sequence 3070
AF008551

Sequence 3071
AF008551

Sequence 3072
AF070648

Sequence 3073
AF026166

Sequence 3074
U42404

Sequence 3075
AF047439

Sequence 3076
AL021683

Sequence 3077
J04031

Sequence 3078
M30047

55/56

Sequence 3079
Y00819

Sequence 3080
AB014577

Sequence 3081
AB014577

Sequence 3082
D29992

Sequence 3083
M11353

Sequence 3084
M11353

Sequence 3085
AF0B1280

Sequence 3086
J03202

Sequence 3087
U42404

Sequence 3088
U42404

Sequence 3089
U42404

Sequence 3090
AF086336

Sequence 3091
AJ011007

Sequence 3092
M84739

Sequence 3093
M84739

Sequence 3094
AB019568

Sequence 3095
S80990

Sequence 3096
AF035812

Sequence 3097
X02761

Sequence 3098
AF035319

Sequence 3099
X28433

Sequence 3100
N80412

Sequence 3101
Q66636

Sequence 3102
V59612

Sequence 3103
T67164

Sequence 3104
V40550

Sequence 3105
V63175

Sequence 3106
V63175

Sequence 3107
X28433

Sequence 3108
V04699

Sequence 3109
V63175

Sequence 3110
V46154

Sequence 3111
X28433

Sequence 3112
V83134

Sequence 3113
Q66636

Sequence 3114
V59663

Sequence 3115
X04408

Sequence 3116
Q70007

Sequence 3117
T15718

Sequence 3118
V89990

Sequence 3119
V32416

Sequence 3120
X28433

Sequence 3121
X28433

Sequence 3122
X40333

Sequence 3123
V59695

Sequence 3124
Q32364

Sequence 3125
Q32364

Sequence 3126
V60015

Sequence 3127
X28433

Sequence 3128
X28433

Sequence 3129
X28433

Sequence 3130
X28433

Sequence 3131
A010439

Sequence 3132
A013954

56/56

Sequence 3131: found in patent publication WO99/14328

GCACGCGGTGGCGGCGGCACACTCGTCCACATCCACACAGGC

GCCCTCGTCCAGCACCCAGCCNACTTTACAGTCGCCGCAGTC

TNTGNTGGTCAGGCCCGAGCACGTTTTGCAGGACTCGTTACA

GGNTGTGCAGATGCTGT

Sequence 3132: found in patent publication WO99/24836

AGGTACACTCATCCTGCGTATCATCACTGCCATGTCCTGATA

CCCCAGCTCTGCCATATTGCCCTTCTTTTTTGCGGTATGATG

ACCACATAGAGGCCCAACCTCTTAAACACATCAATACCAATG

ATCACATTTCAATCTAGACTTCTAAGCAACGGCTGAAATCTC

TCCAGGCCAAAGGAGAGTTTGTATCACCTTACCAGAAGCTTC

TCCGGAACAATTGGCCAGAAGCCTAGAGTTCAGAAACCCAGA

CACATGCAGTAAGCAATTTCCAGTTVCTCTATAATTTAGAAG

AGGACACCATGATATGTAATGCGGGGTCTGGGAGGTTGGAAT

GCCTCCATAAAACACCTGCCATATTTTTTGGTCCAAGCCTTA

GTGGTATAAATCAAGAAGGCTGTAAATAAGACTTCAGCTTTT

TGGCTGGTGAAGTTTGGTTCC

[0293]

6

TABLE 3

Acc no.
GI no.

AA001696
1445252

AA002128
1445144

AA009923
1470970

AA010575
1471742

AA010689
1471716

AA010893
1471990

AA011002
1472029

AA022748
1486821

AA022943
1487051

AA025349
1489317

AA028882
1496304

AA034237
1506265

AA036750
1509788

AA037181
1512290

AA040929
1515636

AA043103
1521053

AA043137
1520991

AA045176
1523378

AA046473
1526403

AA046810
1524915

AA046888
1524823

AA047054
1524952

AA054771
1545707

AA056334
1548691

AA058936
1551791

AA069078
1576438

AA069560
1576972

AA069850
1577210

AA071084
1578444

AA074035
1613975

AA074291
1614159

AA074845
1614714

AA075527
1615397

AA081348
1623162

AA082884
1624941

AA083270
1625391

AA083410
1625660

AA085444
1628674

AA085511
1628697

AA088344
1633856

AA088758
1634279

AA095772
1641357

AA099904
1646050

AA100707
1647062

AA101783
1648780

AA102138
1646213

AA102721
1648220

AA102853
1648698

AA113420
1665269

AA115218
1670047

AA115838
1670916

AA121574
1679249

AA125927
1685612

AA127186
1686546

AA128091
1687353

AA128878
1690018

AA128965
1688748

AA130823
1692515

AA131227
1692735

AA132844
1694333

AA136789
1697998

AA142909
1712370

AA143438
1712808

AA143746
1713134

AA147080
1716453

AA149963
1721247

AA156443
1728068

AA156615
1728238

AA156616
1728239

AA157300
1728926

AA160517
1735884

AA161269
1735566

AA166632
1745096

AA166675
1745130

AA167700
1744868

AA167814
1744965

AA173279
1753411

AA174034
1754363

AA176813
1757945

AA179439
1760791

AA181153
1764620

AA181811
1765337

AA181858
1765325

AA187817
1774011

AA188680
1775705

AA188832
1775877

AA191341
1780003

AA195178
1784909

AA196515
1792106

AA199684
1795594

AA203284
1799010

AA205412
1803420

AA211509
1810163

AA211584
1810247

AA213580
1812199

AA215584
1815420

AA216094
1816041

AA223381
1843906

AA224124
1844683

AA224163
1844731

AA225289
1846607

AA226279
1847596

AA235063
1859499

AA235365
1859803

AA251312
1886338

AA256442
1891980

AA262249
1898724

AA278456
1919829

AA279497
1920962

AA280091
1921565

AA281007
1923751

AA293450
1941151

AA295872
1948207

AA300065
1952416

AA305272
1957598

AA305566
1957913

AA305591
1957993

AA305876
1958206

AA305954
1958283

AA306620
1958949

AA306660
1959213

AA306692
1959020

AA306696
1959024

AA307818
1960145

AA308126
1960455

AA308230
1960559

AA308374
1960703

AA308443
1960771

AA308570
1960898

AA308801
1961131

AA309594
1961964

AA309832
1962325

AA311573
1964057

AA311896
1964546

AA312002
1964331

AA313534
1965864

AA313688
1966017

AA314188
1966517

AA314196
1966525

AA314584
1966912

AA314961
1967379

AA315889
1968217

AA318817
1971142

AA325285
1977742

AA325809
1978052

AA333358
1985601

AA344846
1997084

AA347752
1999987

AA348032
2000511

AA350063
2002402

AA350719
2003036

AA354709
2007028

AA355003
2007559

AA356654
2009197

AA359705
2012096

AA361393
2013888

AA361953
2014274

AA362701
2015041

AA367082
2019471

AA371964
2024282

AA375228
2027547

AA377891
2030229

AA384315
2036634

AA384731
2037049

AA400974
2054946

AA401759
2057243

AA411068
2070180

AA416628
2077701

AA421632
2100448

AA424661
2103614

AA425212
2106120

AA425260
2106034

AA425795
2107633

AA427816
2111651

AA428014
2111742

AA428889
2111902

AA443112
2155787

AA445966
2158631

AA447302
3025388

AA447645
2161315

AA448559
2162229

AA449337
2163186

AA453555
2167224

AA453632
2167301

AA453719
2167388

AA454129
2167798

AA455807
2178583

AA458628
2183535

AA459544
2184451

AA460383
2185596

AA460941
2186061

AA463563
2188447

AA464471
2189355

AA467869
2194403

AA469319
2195853

AA476568
2204779

AA476980
2205191

AA477822
2206456

AA478382
2207016

AA478397
2207031

AA479044
2205407

AA479490
2208046

AA480144
2208295

AA481078
2210630

AA488519
2215950

AA493269
2223110

AA503327
2238294

AA505810
2241947

AA506026
2242163

AA506542
2242297

AA507244
2243683

AA514617
2254217

AA515132
2254732

AA521134
2261677

AA521377
2261920

AA521478
2262021

AA527168
2269237

AA527704
2269773

AA534504
2278757

AA552146
2322398

AA557336
2327813

AA558876
2329643

AA559209
2329405

AA572791
2347319

AA581467
2359239

AA582612
2359972

AA587269
2398083

AA594137
2409487

AA599533
2433158

AA609167
2457595

AA612898
2463936

AA625833
2538220

AA626477
2538864

AA628322
2540709

AA634277
2557491

AA639123
2562902

AA639223
2563002

AA641277
2566527

AA643063
2568281

AA652845
2584497

AA663986
2617977

AA679329
2659851

AA682527
2669808

AA682861
2669544

AA687499
2675690

AA701625
2704790

AA703094
2706207

AA703179
2706292

AA704155
2714073

AA704856
2714774

AA705590
2715508

AA707255
2717173

AA714838
2727112

AA716568
2728842

AA720598
2736733

AA723130
2740837

AA766044
2817282

AA770043
2821281

AA773324
2824895

AA773727
2825298

AA805504
2874254

AA807426
2877002

AA808186
2877592

AA812594
2882658

AA827097
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[0294]

	Number	Date	Country
	60192100	Mar 2000	US
	60197064	Apr 2000	US

Compositions and methods for the identification, assessment, prevention, and therapy of human cancers

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

Provisional Applications (2)