Activated T lymphocyte nucleic acid sequences and polypeptides encoded by same

Abstract

The invention provides novel human polynucleotides unique to, associated with, or highly expressed activated T-lymphocytes (Regulated in Activated T Lymphocytes). The novel polynucleotides were derived from a cDNA subtraction library constructed from human peripheral blood T lymphocytes activated with antibodies against CD3 and CD28 cell surface antigens. The invention also provides for the use and production of the polynucleotides, antisense polynucleotides, amino acid sequences, and/or polypeptides, peptides, and antigenic epitopes thereof encoded by these polynucleotides, and to compositions and methods comprising the polynucleotides and polypeptides and peptides identified herein for modulation of an immune response and for the diagnosis, prevention, or treatment of disorders associated with aberrant cellular development and differentiation including cancer and inflammation, graft vs. host disease, transplantation rejection and autoimmune disease.

Description

FIELD OF THE INVENTION

[0002] The invention relates to isolated nucleic acid and amino acid sequences of novel polypeptides expressed in activated T-lymphocytes (T-cells). The invention further relates to the use and production of the polynucleotides, amino acid sequences, and/or polypeptides or peptides, encoded by the polynucleotides, and to compositions and methods comprising the polynucleotides and polypeptides and peptides identified herein for the diagnosis, prevention, or treatment of immunological disorders as well as disorders associated with aberrant cellular development and differentiation including cancer and inflammation.

BACKGROUND OF THE INVENTION

[0003] Activation of T lymphocytes is essential for the effective response of the immune system. Optimal T cell activation requires at least two stimuli. A primary stimulus is generated upon the interaction of a foreign antigen presented by an antigen presenting cell (APC) in the context of MHC class I or class II molecules, with the T cell receptor (TcR); this specific interaction between antigen and TcR is accompanied by interactions between non-polymorphic regions of the class I and class II molecules with CD8 and CD4, respectively, which function as co-receptors. A secondary stimulus is delivered through the interaction of B7.1/B7.2 molecules on the APC with CD28 or CTLA4 present on T lymphocytes. This secondary stimulus appears to be fundamental, since, in its absence, TcR stimulation fails to induce a T cell response and may lead to cellular anergy. These different interactions initiate a series of signals that are transduced from the cell membrane to the nucleus through different specific signaling pathways that regulate cytokine gene transcription (Favero, J. et al Biochem. Pharmacol Vol. 56, pp. 1539-1547, 1998). Studies have shown that antibodies raised against the T cell surface antigens, CD3 and CD28 mimic the effects of physiologic interactions with the T cell receptor and activate the signal transduction pathways and induce functional responses in T cells.

[0004] The major signal transduction pathways involved in T cell activation are the Ras pathway, the protein kinase C pathway, the calcium/calcineurin pathway and the phosphatidylinositol-3-kinase pathway. These signaling cascades have been shown to mediate the activation of transcription factors which activate the transcription of genes including those encoding interleukin 2 (IL-2), gamma interferon (□-IFN), and granulocyte macrophage colony stimulating factor (GM-CSF).

[0005] Ras is a small GTP-binding protein which plays an important role in transmitting signals from activated cell surface receptors to downstream pathways by interacting with various effector molecules. Nore1 is a protein which contains Src homology 3 (SH3) domain binding motif. The findings that Nore1 interacts with Ras in vitro in a GTP-dependent manner and that Nore1 binds Ras following epidermal growth factor (EGF) receptor activation suggest that Nore1 is a potential Ras effector.

[0006] NF-kB is a transcription factor which plays an important role in the induction of genes during T cell activation. Under normal conditions, NF-kB is bound by the IkappaB family of inhibitory proteins in the cytoplasm. Upon antigenic stimulation and T cell activation, a series of events results in the degradation of IkappaB proteins, and the translocation of free NF-kB to the nucleus where it initiates gene transcription. NF-kB binding sites have been found in a variety of cytokine genes including those encoding IL-2, IL-2 receptor, IFN-α, IFN-β, IL-3, IL-4, IL-6 and GM-CSF and a number of pro-inflammatory factors such as IL-1, IL-6, IL-8, TNF and the redox system. Thus, NF-kB is a key factor in induction of genes during T cell activation and pro-inflammatory events. BCL3 is a protein with close homology to the IkappaB family of proteins. Studies have shown that BCL3 expression is transcriptionally upregulated by IL-9 in T cells and BCL3 interacts with the NF-kB p50 homodimers.

[0007] Proteins such as IL-2 that are encoded by genes that are expressed in activated T cells play critical roles in T cell proliferation and differentiation and the development of T cell mediated immune responses. Thus, identification of additional T cell activated proteins could lead to new diagnostic or therapeutic approaches to prevent or treat immunological disorders as well as disorders associated with aberrant cellular development and differentiation including cancer and inflammation.

SUMMARY OF THE INVENTION

[0008] The invention provides novel human polynucleotides unique to, associated with, and/or highly expressed in T lymphocytes, in particular activated T lymphocytes.

[0009] Another aspect of the invention are isolated novel human polynucleotides regulated in T lymphocytes and derived from clones of a cDNA subtraction library constructed from human peripheral blood T lymphocytes activated with antibodies against CD3 and CD28 cell surface antigens.

[0010] The present invention provides isolated polynucleotides unique to, associated with or highly expressed T lymphocytes designated herein as Regulated in Activated T Lymphocytes (RATL) and encompasses RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and RATL 6f6 polynucleotides comprising at least one of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21 as shown in any one of FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14-20, or portion thereof. Another aspect of the invention is a polynucleotide sequence comprising a complementary sequence or antisense sequence to a RATL polynucleotide. The present invention features polynucleotide sequences which hybridize under moderately stringent or stringent conditions to a polynucleotide sequence of at least one of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21 or portion thereof as shown in any one of FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14-20 or portion thereof. The polynucleotide sequences may be provided in the form of a pharmaceutical composition along with a pharmaceutically acceptable carrier.

[0011] The present invention also provides expression vectors and host cells comprising a polynucleotide encoding at least one RATL protein or functional portion thereof.

[0012] The present invention comprises proteins, polypeptides, peptides and antigenic epitopes thereof encoded by an isolated human polynucleotide regulated in T lymphocytes and derived from a cDNA subtraction library constructed from human peripheral blood T lymphocytes activated with antibodies against CD3 and CD28 cell surface antigens.

[0013] The present invention comprises proteins, polypeptides, peptides and antigenic epitopes thereof unique to, associated with, regulated in, and/or highly expressed in T lymphocytes, particularly activated T lymphocytes.

[0014] The present invention also provides the polypeptides encoded by the polynucleotides of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 having SEQ ID NOS: 2, 4, 10, 13, 16, and 22 as shown in FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14-17, respectively. Another aspect of the invention are RATL proteins or polypeptides comprising the amino acid sequence, having SEQ ID NO:2, 4, 5, 6 or 7 as shown in FIG. 1B, 2B and 3 for RATL2e12, having SEQ ID NO:10 as shown in FIG. 6 for RATL 1f7, having SEQ ID NO:13 or 16 as shown in FIG. 10 or 12A for RATL 5h6, having SEQ ID NO:22 as shown in FIG. 17 for RATL 4d9, or a functional portion or analog thereof.

[0015] The present invention further provides pharmaceutical compositions comprising at least one RATL polypeptide of the invention, or a functional portion or analog thereof and a pharmaceutically acceptable carrier.

[0016] The present invention further provides methods for producing a RATL protein or a functional portion or analog thereof.

[0017] The present invention also provides an isolated and substantially purified polynucleotide which encodes a homolog of BCL3 and NFKBp100. In a particular aspect, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:1 or 3 as shown in FIG. 1A or 2A or functional portion or variant thereof for RATL 2e12. The present invention also provides an amino acid for a BCL3 and NFkBp100 homolog comprising SEQ ID NO:2 or 4 as shown in FIG. 1B, 2B or 3, or portion thereof.

[0018] The present invention provides an isolated and substantially purified polynucleotide that encodes an Nore1 homolog. In a particular aspect, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:11, 12, 14, 15, 32 or portion thereof as shown in any one of FIGS. 8, 9, 11-12B. The present invention also provides an amino acid sequence for an Nore1 homolog comprising SEQ ID NO:13, 16 or portion thereof as shown in FIG. 10 or 12A, or portion thereof.

[0019] Also provided are antibodies, and antigen binding fragments thereof, which bind specifically to a RATL polypeptide, or an epitope thereof, for use as therapeutics and diagnostic agents.

[0020] An aspect of the invention is a method of detecting T lymphocytes in a biological sample or tissue. Another aspect of the invention is a method of detecting activated T lymphocytes in a biological sample or tissue.

[0021] The present invention also provides methods for screening for agents (e.g. agonists and antagonists) which modulate expression of a RATL protein or for agents that inhibit or augment a RATL protein function.

[0022] It is another object of the present invention to use the nucleic acid sequences, polypeptide, peptide and antibodies for diagnosis of disorders or diseases associated with aberrant cellular development, immune responses and inflammation, and organ or tissue transplantation rejection.

[0023] The present invention provides methods of preventing or treating disorders associated with aberrant cellular development, methods of regulating an immune response and inflammation and methods of preventing and suppressing rejection of grafted organs or tissue in a mammal.

[0024] The present invention also provides kits for detection of a RATL protein or portion thereof or for detection of a RATL polynucleotide sequence.

[0025] The present invention provides kits for screening and diagnosis of disorders associated with aberrant cellular development, immune responses, inflammation and graft or tissue transplantation rejection.

[0026] The invention also provides for the use of antisense molecules to disrupt or inhibit expression of genomic sequences encoding a RATL protein.

[0027] The invention further provides transgenic non-human animal expressing an exogenous RATL protein or portion thereof.

[0028] The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0029] The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0030] The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0031] The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21, having biological activity.

[0032] The invention further relates to a polynucleotide which is a variant of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0033] The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0034] The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22.

[0035] The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0036] The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

[0037] The invention further relates to an isolated nucleic acid molecule of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, wherein the polynucleotide fragment comprises a nucleotide sequence encoding an RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 protein.

[0038] The invention further relates to an isolated nucleic acid molecule of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0039] The invention further relates to an isolated nucleic acid molecule of of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0040] The invention further relates to an isolated nucleic acid molecule of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.

[0041] The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the encoded sequence included in the deposited clone.

[0042] The invention further relates to a polypeptide fragment of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the encoded sequence included in the deposited clone, having biological activity.

[0043] The invention further relates to a polypeptide domain of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the encoded sequence included in the deposited clone.

[0044] The invention further relates to a polypeptide epitope of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the encoded sequence included in the deposited clone.

[0045] The invention further relates to a full length protein of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the encoded sequence included in the deposited clone.

[0046] The invention further relates to a variant of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22.

[0047] The invention further relates to an allelic variant of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22. The invention further relates to a species homologue of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22.

[0048] The invention further relates to the isolated polypeptide of of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

[0049] The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22.

[0050] The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 or the polynucleotide of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0051] The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

[0052] The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

[0053] The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 comprising the steps of (a) contacting the polypeptide of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.

[0054] The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21.

[0055] The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.

[0056] The invention further relates to a process for making polynucleotide sequences encoding gene products having altered SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NOS: 1, 3, 5, 6, 7, 8, 9, 11, 12, 14, 15, 19, 20, and/or 21, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity as compared to the activity of the gene product of said unmodified nucleotide sequence.

[0057] The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 activity.

[0058] The invention further relates to a method of identifying a compound that modulates the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9, comprising the steps of, (a) combining a candidate modulator compound with RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 having the sequence set forth in one or more of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22; and measuring an effect of the candidate modulator compound on the activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9.

[0059] The invention further relates to a method of identifying a compound that modulates the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 having the sequence as set forth in SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9.

[0060] The invention further relates to a method of identifying a compound that modulates the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9.

[0061] The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d) determining the biological activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 in the presence of the modulator compound; wherein a difference between the activity of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

[0062] The invention further relates to a compound that modulates the biological activity of human RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 as identified by the methods described herein.

[0063] The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, in addition to, its encoding nucleic acid, or a modulator thereof, wherein the medical condition is selected from the group consisting of aberrant cellular development, immune responses and inflammation, organ or tissue transplantation rejection; T-lymphocyte disorders, graft allograft rejection and graft-versus-host reactions, autoimmune disease, allergy, asthma, cancer, immunodeficiencies and the like.

[0064] The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, in addition to, its encoding nucleic acid, or a modulator thereof, wherein the medical condition is selected from the group consisting of a disorder associated with aberrant cellular differentiation. Such disorders may include, but are not limited to: hyperaldosteronisin (Conn's Syndrome), hypocortisolism (Addison's disease), hypercortisolism (Cushing's disease), and adrenogenital syndrome; and cancers, including cancers of the nervous system; cancers of glands, tissues, and organs involved in secretion or absorption such as prostate, lung, bladder, adrenal gland, liver, uterus, and kidney; and cancers of tissues of the immune and hematopoietic systems.

[0065] The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, in addition to, its encoding nucleic acid, or a modulator thereof, wherein the medical condition is selected from the group consisting of inflammation and autoimmune dysfunctions. Inflammation may be associated with, but is not limited to, allergic reactions, asthma and adult respiratory distress syndrome, rheumatoid arthritis, osteoarthritis, glomerulonephritis, osteoporosis, dermatomyositis, polymyositis, Addison's disease, Grave's disease, irritable bowel syndrome, atrophic gastritis, lupus erythematosus, myasthenia gravis, multiple sclerosis, systemic lupus erythematosis, autoimmune thyroiditis, ulcerative colitis, anemia, pancreatitis, scleroderma, Crohn's disease, ischermia/reperfusion injury, post-traumatic inflammation, myocardial inflammation, atherosclerosis, diabetes, and inflammatory complications of cancer, hemodialysis and extracorporeal circulation, infection and trauma.

BRIEF DESCRIPTION OF THE FIGURES

[0066] These and other objections, features, and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying figures:

[0067] FIG. 1A shows the partial cDNA coding region for RATL 2e12 from Incyte clone 3345840 (SEQ ID NO:1). FIG. 1B shows the deduced amino acid sequence for RATL 2e12 from Incyte clone 3345840 (SEQ ID NO:2).

[0068] FIG. 2A shows the partial cDNA coding region for RATL 2e12 from Incyte clone 6758514 (SEQ ID NO:3). FIG. 2B shows the deduced amino acid sequence for RATL 2e12 from Incyte clone 6758514 (SEQ ID NO:4).

[0069] FIG. 3 shows the subtraction clone nucleic acid sequence of RATL 6f6 (SEQ ID NO:5).

[0070] FIG. 4 shows a 1247 bp partial cDNA sequence of RATL 1f7 (SEQ ID NO:8).

[0071] FIG. 5 shows a consensus sequence of RATL 1f7 (SEQ ID NO:9).

[0072] FIG. 6 shows the deduced amino acid sequence encoded by RATL 1f7 (SEQ ID NO:10).

[0073] FIG. 7 shows a Northern blot analysis of the tissue distribution of mRNA of RATL 1f7 and RATL 5h6.

[0074] FIG. 8 shows the original subtraction nucleic acid sequence of RATL 5h6 (SEQ ID NO:11).

[0075] FIG. 9 shows the contig nucleic acid sequence of RATL 5h6 (SEQ ID NO:12).

[0076] FIG. 10 shows a partial polypeptide sequence (SEQ ID NO:13) encoded by the contig nucleic acid sequence of RATL 5h6.

[0077] FIG. 11 shows the full-length nucleic acid sequence of RATL 5h6 (SEQ ID NO:14).

[0078] FIG. 12A and 12B shows the full-length nucleic acid sequence of RATL 5h6 (SEQ ID NO:15), the coding region (SEQ ID NO:29), and deduced amino acid sequence of RATL 5h6 (SEQ ID NO:16).

[0079] FIG. 13 shows the alignment between RATL 5h6 (SEQ ID NO:16), mouse Ras effector protein Nore1 (SEQ ID NO:17), and human RASFIC (SEQ ID NO:18). Identical residues between all three proteins are indicated by bold shading and residues conserved in at least two proteins are shaded gray.

[0080] FIG. 14 shows the RATL 4d9 subtraction clone nucleic acid sequence (SEQ ID NO:19).

[0081] FIG. 15 shows the RATL 4d9 contig nucleic acid sequence (SEQ ID NO:20).

[0082] FIG. 16 shows the full length nucleic acid sequence of RATL 4d9 (SEQ ID NO:21).

[0083] FIG. 17 shows the open reading frame sequence (SEQ ID NO:30) and deduced amino acid sequence (SEQ ID NO:22) of RATL 4d9.

[0084] FIG. 18 shows the Northern expression analysis of RATL 4d9 on multiple tissue and activated T-cell Northern blots.

[0085] FIG. 19 shows the subtraction clone nucleic acid sequence for RATL 2f3, 2h1 and 2g11 (SEQ ID NO:6).

[0086] FIG. 20 shows the contig nucleic acid sequence of RATL 2f3, 2h1 and 2g11 (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

[0087] The following definitions are provided to more fully describe the present invention in its various aspects. The definitions are intended to be useful for guidance and elucidation, and are not intended to limit the disclosed invention and its embodiments.

[0088] “Activated T lymphocyte” as used herein refers to a T lymphocyte treated with any number of stimuli such as cytokines, adhesion molecules, chemicals, antigens, non-specific mitogens, MHC molecules, adhesion molecules, antigen presenting cells, combinations of stimuli and the like, resulting in metabolic, functional, and phenotypic changes in comparision with a resting, non-activated T lymphocyte.

[0089] “Nucleic acid sequence”, as used herein, refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. By way of non-limiting example, fragments include nucleic acid sequences that encode a protein, peptide, or oligopeptide that are greater than 20-60 nucleotides in length, and preferably 60-100 nucleotides in length or at least 100 to 1000 nucleotides or greater in length. Nucleic acids for use as probes or primers may vary in length and are typically 5 to 10 nucleotides, 10 to 20 nucleotides in length, and 20 to 50 nucleotides in length.

[0090] Amino acid sequence” as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Amino acid fragments may be from about 5 to about 35 amino acids, preferably about 5 to about 20 amino acids, and has the biological or immunological function of a RATL protein or epitope thereof.

[0091] As will be appreciated by the skilled practitioner, should the amino acid fragment comprise an antigenic epitope, for example, biological function per se need not be maintained. The terms RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 polypeptide and RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 protein are used interchangeably herein to refer to the encoded product of the RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 nucleic acid sequence according to the present invention.

[0092] Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0093] “Peptide nucleic acid”, as used herein, refers to an oligomer of modified nucleic acid base pairs covalently linked through an amide bond. PNAs have utility in a number of antisense and anti-gene applications. These molecules typically act by inhibiting transcription (Nielsen, P. E. et al (1993) Anticancer Drug Des., 8:53-63). PNA may be pegylated to extend their lifespan in the cell where they preferentially bind to complementary single stranded DNA and RNA.

[0094] Regulated in Activated T Lymphocytes (RATL) protein, as used herein, refers to the amino acid sequences of substantially purified RATL proteins which, although isolated from a human cDNA library source according to the present invention, may be obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and non-human primates, preferably human and from a variety of sources including natural, synthetic, semi-synthetic, or recombinant.

[0095] “Consensus”, as used herein, refers to a sequence that reflects the most common choice of base or amino acid of each position among a series of related DNA, RNA or protein sequences. Areas of particularly good agreement often represent conserved functional domains.

[0096] A “variant” of a RATL protein, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.

[0097] A “deletion”, as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.

[0098] An “insertion” or “addition”, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.

[0099] A “substitution”, as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0100] The term “biologically active”, as used herein, refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic RATL protein, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[0101] The term “agonist”, as used herein, refers to a molecule which when bound to a RATL protein or functional portion thereof, increases the amount of, or prolongs the duration of, the activity of a RATL protein. Agonists may include proteins, nucleic acids, carbohydrates, organic molecules or any other molecules which bind to and modulates the effect of a RATL protein.

[0102] The term “antagonist”, as used herein, refers to a molecule which decreases or eliminates the biological or immunological activity of the RATL protein. The antagonist may or may not bind to a RATL protein or functional portion thereof and may be a competitive or non-competitive inhibitor of a RATL protein or functional portion thereof. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, organic molecules or any other molecules that decreases, reduces or eliminates the activity of a RATL protein.

[0103] It is another aspect of the present invention to provide modulators of the RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 protein and RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 peptide targets which can affect the function or activity of RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 in a cell in which RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 function or activity is to be modulated or affected. In addition, modulators of RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 can affect downstream systems and molecules that are regulated by, or which interact with, RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 in the cell. Modulators of RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 include compounds, materials, agents, drugs, and the like, that antagonize, inhibit, reduce, block, suppress, diminish, decrease, or eliminate RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 function and/or activity. Such compounds, materials, agents, drugs and the like can be collectively termed “antagonists”. Alternatively, modulators of RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 include compounds, materials, agents, drugs, and the like, that agonize, enhance, increase, augment, or amplify RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 function in a cell. Such compounds, materials, agents, drugs and the like can be collectively termed “agonists”.

[0104] As used herein the terms “modulate” or “modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein. The definition of “modulate” or “modulates” as used herein is meant to encompass agonists and/or antagonists of a particular activity, DNA, RNA, or protein.

[0105] The term “mimetic”, as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of a RATL protein or portions thereof and, as such, is able to effect some or all of the actions of the RATL protein.

[0106] The term “derivative”, as used herein, refers to the chemical modification of a nucleic acid encoding or complementary to a RATL protein. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative would encode a polypeptide which retains essential biological and/or functional characteristics of the natural molecule. A derivative polypeptide is one which is modified by glycosylation, pegylation, or any similar process that retains the biological and/or functional or immunological activity of the polypeptide from which it is derived.

[0107] The term “substantially purified”, as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% or greater free from other components with which they are naturally associated.

[0108] “Oligonucleotides” or “oligomers” refer to a nucleic acid sequence, preferably comprising contiguous nucleotides, of at least about 6 nucleotides to about 60 nucleotides, preferably at least about 8 to 10 nucleotides in length, more preferably at least about 12 nucleotides in length, e.g., about 15 to 35 nucleotides, or about 15 to 25 nucleotides, or about 20 to 35 nucleotides, which can be typically used, for example, as probes or primers, in PCR amplification assays, hybridization assays, or in microarrays. It will be understood that the term oligonucleotide is substantially equivalent to the terms primer, probe, or amplimer, as commonly defined in the art. It will also be appreciated by those skilled in the pertinent art that a longer oligonucleotide probe, or mixtures of probes, e.g., degenerate probes, can be used to detect longer, or more complex, nucleic acid sequences, for example, genomic DNA. In such cases, the probe may comprise at least 20-200 nucleotides, preferably, at least 30-100 nucleotides, and more preferably, 50-100 nucleotides.

[0109] “Microarray” is an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon, or other type of membrane; filter; chip; glass slide; or any other type of suitable solid support.

[0110] “Amplification”, as used herein, refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, D. W. and G. S. Dveksler (1995), PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0111] The term “hybridization”, as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

[0112] The term “hybridization complex”, as used herein, refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases. The hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides or any other appropriate substrate to which cells or their nucleic acids have been fixed.

[0113] The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, as well as the design and use of PNA molecules.

[0114] The term “homology”, as used herein, refers to a degree of complementarity. There may be partial homology or complete homology wherein “complete homology” is equivalent to identity i.e., 100% identity. A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term “substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

[0115] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are:

[0116] Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

[0117] The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

[0118] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

[0119] By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five-amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0120] As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in Table 1 (SEQ ID NO:2) or to the amino acid sequence encoded by cDNA contained in a deposited clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program(Vector NTI suite of programs, version 6.0).

[0121] The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N- or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N- and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

[0122] For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

[0123] In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.

[0124] As known in the art, numerous equivalent conditions may be employed to comprise either low or high stringency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

[0125] The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0126] Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

[0127] In addition, the present invention also encompasses the conservative substitutions provided in Table II below.

TABLE II
For Amino Acid	Code	Replace with any of:
Alanine	A	D-Ala, Gly, beta-Ala, L-Cys, D-Cys
Arginine	R	D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg,
		Met, Ile, D-Met, D-Ile, Orn, D-Orn
Asparagine	N	D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln
Aspartic Acid	D	D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln
Cysteine	C	D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine	Q	D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid	E	D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Glycine	G	Ala, D-Ala, Pro, D-Pro, B-Ala, Acp
Isoleucine	I	D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine	L	D-Leu, Val, D-Val, Met, D-Met
Lysine	K	D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,
		Met, D-Met, Ile, D-Ile, Orn, D-Orn
Methionine	M	D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val,
		D-Val
Phenylalanine	F	D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp,
		D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4,
		or 5-phenylproline
Proline	P	D-Pro, L-1-thioazolidine-4-carboxylic acid, D-
		or L-1-oxazolidine-4-carboxylic acid
Serine	S	D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met,
		Met(O), D-Met(O), L-Cys, D-Cys
Threonine	T	D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,
		Met(O), D-Met(O), Val, D-Val
Tyrosine	Y	D-Tyr, Phe, D-Phe, L-Dopa, His, D-His
Valine	V	D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0128] Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

[0129] Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press,N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0130] In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.

[0131] Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.

[0132] The terms “stringency” or “stringent conditions” refer to the conditions for hybridization as defined by nucleic acid composition, salt and temperature. These conditions are well known in the art and may be altered to identify and/or detect identical or related polynucleotide sequences in a sample. A variety of equivalent conditions comprising either low, moderate, or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), reaction milieu (in solution or immobilized on a solid substrate), nature of the target nucleic acid (DNA, RNA, base composition), concentration of salts and the presence or absence of other reaction components (e.g., formamide, dextran sulfate and/or polyethylene glycol) and reaction temperature (within a range of from about 5° C. below the melting temperature of the probe to about 20° C. to 25° C. below the melting temperature). One or more factors may be varied to generate conditions, either low or high stringency, that is different from but equivalent to the aforementioned conditions.

[0133] As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. As will be further appreciated by the skilled practitioner, the melting temperature, Tm, can be approximated by the formulas as known in the art, depending on a number of parameters, such as the length of the hybrid or probe in number of nucleotides, or hybridization buffer ingredients and conditions (see, for example, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982 and J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Eds. F. M. Ausubel et al., Vol. 1, “Preparation and Analysis of DNA”, John Wiley and Sons, Inc., 1994-1995, Suppls. 26, 29, 35 and 42; pp. 2.10.7-2.1 0.16; G. M. Wahl and S. L. Berger 1987 Methods Enzymol. 152:399-407); and A. R. Kimmel, 1987; Methods of Enzymol. 152:507-511). As a general guide, Tm decreases approximately 1° C.-1.5° C. with every 1% decrease in sequence homology. Also, in general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is initially performed under conditions of low stringency, followed by washes of varying, but higher stringency. Reference to hybridization stringency, e.g., high, moderate, or low stringency, typically relates to such washing conditions.

[0134] Thus, by way of non-limiting example, “high stringency” refers to conditions that permit hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at about 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at about 65° C., it will not be stable under high stringency conditions). High stringency conditions can be provided, for instance, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE (saline sodium phosphate EDTA) (1×SSPE buffer comprises 0.15 M NaCl, 10 mM Na2HPO4, 1 mM EDTA), (or 1×SSC buffer containing 150 mM NaCl, 15 mM Na3 citrate □ 2 H2O, pH 7.0), 0.2% SDS at about 42° C., followed by washing in 1×SSPE (or saline sodium citrate, SSC) and 0.1% SDS at a temperature of at least about 42° C., preferably about 55° C., more preferably about 65° C.

[0135] “Moderate stringency” refers, by nonlimiting example, to conditions that permit hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE (or SSC), 0.2% SDS at 42° C. (to about 50° C.), followed by washing in 0.2×SSPE (or SSC) and 0.2% SDS at a temperature of at least about 42° C., preferably about 55° C., more preferably about 65° C.

[0136] “Low stringency” refers, by non-limiting example, to conditions that permit hybridization in 10% formamide, 5×Denhardt's solution, 6×SSPE (or SSC), 0.2% SDS at 42° C., followed by washing in 1×SSPE (or SSC) and 0.2% SDS at a temperature of about 45° C., preferably about 50° C.

[0137] For additional stringency conditions, see T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). It is to be understood that the low, moderate and high stringency hybridization/washing conditions may be varied using a variety of ingredients, buffers and temperatures well known to and practiced by the skilled practitioner.

[0138] The term “antisense”, as used herein, refers to nucleotide sequences and compositions containing nucleic acid sequences, which are complementary to a specific DNA or RNA sequence. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. Antisense molecules include PNA and may be produced by any method, including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes that block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand.

[0139] The term “portion”, as used herein, with regard to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. Thus, a protein “comprising at least a portion of an amino acid sequence of a RATL protein” encompasses the full-length human RATL protein and fragments thereof.

[0140] “Transformation”, as defined herein, describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and partial bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

[0141] The term “antigenic determinant”, as used herein, refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to an antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

[0142] The terms “specific binding” or “specifically binding”, as used herein, in reference to the interaction of an antibody and a protein or peptide, mean that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words, the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope “A”, the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled “A” and the antibody will reduce the amount of labeled A bound to the antibody.

[0143] The term “sample” or “biological sample” is used in its broadest sense. A biological sample suspected of containing nucleic acid encoding a RATL protein or fragments thereof and/or the sample or biological sample is one suspected of containing a RATL protein or fragments thereof, or one or more antibodies immunoreactive with a RATL protein or antigenic portion thereof. The sample comprise a cell, an organelle, membrane isolated from a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for Northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue, and the like.

[0144] The term “correlates with expression of a polynucleotide”, as used herein, indicates that the detection of the presence of ribonucleic acid using one or more of SEQ ID NOS: 2, 4, 10, 13, 16, and 22 or portions thereof by Northern analysis is indicative of the presence of mRNA encoding a RATL protein in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.

[0145] “Alterations” in the polynucleotide of SEQ ID NOS: 2, 4, 10, 13, 16, and 22 as used herein, comprise any alteration in the sequence of polynucleotides regulated in/or associated with T lymphocytes, in particular activated T lymphocytes, which may also encode a RATL protein including deletions, insertions, and point mutations that may be detected using hybridization assays. Included within this definition is the detection of alterations to the genomic DNA sequence regulated in/or associated with T lymphocytes which may also encode a RATL protein (e.g., by alterations in the pattern of restriction fragment length polymorphisms capable of hybridizing to SEQ ID NOS: 2, 4, 10, 13, 16, and 22 as shown in any one of FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14-20, respectively), the inability of a selected fragment of SEQ ID NOS: 2, 4, 10, 13, 16, and 22 as shown in any one of FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14-20, respectively to hybridize to a sample of genomic DNA (e.g., using allele-specific oligonucleotide probes), and improper or unexpected hybridization, such as hybridization to a locus other than the normal chromomsomal locus for the polynucleotide sequence regulated in/or associated with activated T lymphocytes which may encode a RATL protein (e.g., using fluorescent in situ hybridization (FISH) to metaphase chromosome spreads).

[0146] An expressed sequence tag or EST, as used herein, is a nucleic acid sequence that represents a portion of a coding sequence, the total coding sequence of a polypeptide, or an untranslated region outside of the coding sequence.

[0147] A contig sequence as used herein is a group of small EST clones assembled into larger sequence fragments based on overlapping regions.

[0148] As used herein, the term “antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab′)2, Fv, chimeric antibody, single chain antibody which are capable of binding the epitopic or antigenic determinant. Antibodies that bind a RATL polypeptides or immunological epitope thereof can be prepared using intact polypeptides or fragments containing small peptides of interest or prepared recombinantly for use as the immunizing antigen. The polypeptide or peptide used to immunize an animal can be derived from the transition of RNA or synthesized chemically, and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, keyhole limpet homocyanin (KLH), and thyroglobulin. The coupled peptide is then used to immunize the animal (e.g, a mouse, a rat, or a rabbit).

[0149] The term “humanized antibody” refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability, such as those described in U.S. Pat. No. 5,585,089.

The Invention

[0150] The invention is a novel human polynucleotide unique to, associated with, regulated in, or highly expressed in T lymphocytes, in particular activated T lymphocytes designated herein as Regulated in Activated T Lymphocytes (RATL) as well as protein or peptides and variants thereof encoded by the RATL polynucleotides, and the use of these products for modulating immune responses, in particular T lymphocyte responses as well as for the diagnosis, prevention, or treatment of disorders associated with aberrant immune responses such as organ and tissue transplantation rejection and disorders associated with aberrant cellular development and inflammation.

[0151] Polynucleotides regulated in/or associated with T lymphocytes, in particular activated T lymphocytes, of the present invention were identified by screening clones isolated from a cDNA subtraction library constructed from human peripheral blood T lymphocytes activated using antibodies against CD3 and CD28 cell surface antigens, subtracting out clones existing in resting T lymphocytes.

[0152] The present invention comprises proteins, polypeptides, peptides and antigenic epitopes thereof encoded by an isolated human polynucleotide regulated in T lymphocytes and derived from a cDNA subtraction library constructed from human peripheral blood T lymphocytes activated with antibodies against CD3 and CD28 cell surface antigens. The proteins, polypeptides and antigenic epitopes thereof are unique to, associated with, regulated in and/or highly expressed in T lymphocytes, particularly activated T lymphocytes.

[0153] In one embodiment, the polypeptides are encoded by polynucleotides of RATL 2e12, RATL 1f7, RATL 5h6, and RATL 4d9 having SEQ ID NOS: 1, 3, 8, 9, 11, 12, 14, 15, 32, 19, 20, 21 or 33 as shown in FIGS. 1A, 2A, 4, 5, 8, 9, 11-12B or 14, respectively.

[0154] In one particular embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4 as shown in FIG. 1B and 2B designated as RATL 2e12. In another embodiment, the invention encompasses a polypeptide encoded by SEQ ID:1 or 3, or portion or variant thereof, as shown in FIG. 1A and 2A, respectively. RATL 2e12 polypeptide shares chemical and structural homology with the BCL3 and NFkBp100. The RATL 2e12 polypeptide of the present invention shares about 31 to about 35% identity with BCL3 and NFkBp100. The RATL 2e12 polypeptide of the present invention comprises at least one ankryin repeat, more preferably comprises multiple ankryin repeats. In one embodiment the RATL 2e12 polypeptide comprises five ankryin repeats. Functional portions of the polypeptide of RATL 2e12 are an ambit of the invention and may include one or more ankryin repeats as shown in FIG. 3 and combinations thereof.

[0155] Electronic Northern analysis showed that RATL 2e12 is expressed predominantly in the hemic and immune system.

[0156] Northern analysis showed the expression of RATL 2e12 in activated normal human T lymphocytes, and down-regulated in activated tumor-derived human T lymphocyte cell line. This, along with the homology with BCL3 and NFkBp100 indicates a functional role for RATL 2e12 in regulating activation and differentiation of normal T lymphocytes and tumorigenic events. This provides a rationale for its use in the treatment of T cell mediated diseases such as autoimmune disease, RA, inflammatory bowel disease, psoriasis, multiple sclerosis, solid organ transplantation rejection, and the like.

[0157] In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:16 as shown in FIG. 12A, designated herein as RATL 5h6. In another embodiment, the invention encompasses a polypeptide encoded by SEQ ID NO:15 or 32, portion or variant thereof, as shown in FIG. 12A. RATL 5h6 polypeptide shares chemical and structural homology with the putative ras effector Nore1 and with mouse MaxP1. The RATL 5h6 polypeptide shares about 90% or greater identity with Nore1 and MaxP1.

[0158] Electronic Northern analysis showed that RATL 5h6 is expressed predominately in the hemic and immune system, in particular in the spleen.

[0159] Northern analysis showed the expression of RATL 5h6 in activated and non-activated normal human T lymphocytes as well as activated and non-activated tumor-derived T lymphocytes. This expression, along with the homology with Nore1 and MaxP1, indicates a functional role for RATL 5h6 in treatment of T cell medicated diseases.

[0160] In yet another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:10 as shown in FIG. 6, designated herein as RATL1f7 and functional portions thereof. The invention further encompasses a polypeptide encoded by SEQ ID NO:8 or 9 or portion or variant thereof. RATL1f7 polypeptide does not share homology with any known mammalian protein. Functional portions of a RATL1f7 protein may contain one or more of the following sites: a potential cyclic adenosine monophosphate, a casein kinase II site, a protein kinase C site and phosphorylation sites. RATL1f7 shares 26% homology with a Plasmodium falciparum surface protein.

[0161] Northern analysis showed that RATL1f7 expression is upregulated in normal human peripheral blood T lymphocytes upon activation using anti CD3 and CD28 antibody.

[0162] Electronic Northern analysis showed expression of RATL1f7 in several tumor-tissue libraries as well as in an asthmatic lung library.

[0163] Based on the tissue expression and potential cyclic adenosine monophosphate, casein kinase II, protein kinase C and phosphorylation sites, RATL1f7 may play a role in tumorigenesis, activation of oncogenes and autoimmune diseases. Antagonists or inhibitors of RATL 1f7 may be used to treat T cell mediated diseases such as autoimmune diseases and antagonists or to treat cancer.

[0164] In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:22 as shown in FIG. 17, designated herein as RATL4d9. The invention further encompasses a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:19, 20, 21, 33 or portion or variant thereof shown in FIGS. 14-17. The RATL4d9 polypeptide does not share homology with any known protein.

[0165] Electronic Northern analysis showed expression of RATL4d9 predominately in hemic and immune tissue libraries, in particular in thymus.

[0166] Northern analysis showed expression of RATL4d9 in heart, thymus, testis and thyroid. It is upregulated in normal T lymphocytes with activation.

[0167] Based on the detection and isolation of RATL4d9 in an activated T lymphocyte subtraction library, and expression in thymus tissues, RATL4d9 may play a role in modulating T lymphocyte functions.

[0168] Features of the Polypeptide Encoded by RATL4d9

[0169] The determined nucleotide sequence of the RATL4d9 cDNA in FIG. 17 (SEQ ID NO:21) contains an open reading frame encoding a protein of about 328 amino acid residues, with a deduced molecular weight of about 25.1 kDa. The amino acid sequence of the predicted RATL4d9 polypeptide is shown in FIG. 17 (SEQ ID NO:22).

[0170] The coding region of the RATL4d9 polynucleotide is predicted to be from nucleotide 1024 to nucleotide 1677 of SEQ ID NO:21 as shown in FIG. 17, and the polypeptide corresponding to amino acids 1 thru 218 of SEQ ID NO:22. The present invention encompasses the polynucleotide encompassing the entire coding region of RATL4d9.

[0171] Alternatively, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of RATL4d9. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 1027 thru 1677 of SEQ ID NO:21, and the polypeptide corresponding to amino acids 2 thru 218 of SEQ ID NO:22. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

[0172] Alternatively, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of RATL4d9. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 1030 thru 1677 of SEQ ID NO:21, and the polypeptide corresponding to amino acids 3 thru 218 of SEQ ID NO:22. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

[0173] In preferred embodiments, the following N-terminal RATL4d9 deletion polypeptides are encompassed by the present invention: M1-V218, M2-V218, R3-V218, R4-V218, E5-V218, D6-V218, E7-V218, E8-V218, E9-V218, E10-V218, G11-V218, T12-V218, M13-V218, M14-V218, K15-V218, A16-V218, K17-V218, G18-V218, D19-V218, L20-V218, E21-V218, M22-V218, K23-V218, E24-V218, E25-V218, 26-V218, E27-V218, I28-V218, S29-V218, E30-V218, T31-V218, G32-V218, E33-V218, L34-V218, V35-V218, G36-V218, P37-V218, F38-V218, V39-V218, S40-V218, A41-V218, M42-V218, P43-V218, T44-V218, P45-V218, M46-V218, P47-V218, H48-V218, N49-V218, K50-V218, G51-V218, T52-V218, R53-V218, F54-V218, S55-V218, E56-V218, A57-V218, W58-V218, E59-V218, Y60-V218, F61-V218, H62-V218, L63-V218, A64-V218, P65-V218, A66-V218, R67-V218, A68-V218, G69-V218, H70-V218, H71-V218, P72-V218, N73-V218, Q74-V218, Y75-V218, A76-V218, T77-V218, C78-V218, R79-V218, L80-V218, C81-V218, G82-V218, R83-V218, Q84-V218, V85-V218, S86-V218, R87-V218, G88-V218, P89-V218, G90-V218, V91-V218, N92-V218, V93-V218, G94-V218, T95-V218, T96-V218, A97-V218, L98-V218, W99-V218, K100-V218, H101-V218, L102-V218, K103-V218, S104-V218, M105-V218, H106-V218, R107-V218, E108-V218, E109-V218, L110-V218, E111-V218, K112-V218, S113-V218, G114-V218, H115-V218, G116-V218, Q117-V218, A118-V218, G119-V218, Q120-V218, R121-V218, Q122-V218, D123-V218, P124-V218, R125-V218, P126-V218, H127-V218, G128-V218, P129-V218, Q130-V218, L131-V218, P132-V218, T133-V218, G134-V218, I135-V218, E136-V218, G137-V218, N138-V218, W139-V218, G140-V218, R141-V218, L142-V218, L143-V218, E144-V218, Q145-V218, V146-V218, G147-V218, T148-V218, M149-V218, A150-V218, L151-V218, W152-V218, A153-V218, S154-V218, Q155-V218, R156-V218, E157-V218, K158-V218, E159-V218, V160-V218, L161-V218, R162-V218, R163-V218, E164-V218, R165-V218, A166-V218, V167-V218, E168-V218, W169-V218, R170-V218, E171-V218, R172-V218, A173-V218, V174-V218, E175-V218, K176-V218, R177-V218, E178-V218, R179-V218, A180-V218, L181-V218, E182-V218, E183-V218, V184-V218, E185-V218, R186-V218, A187-V218, I188-V218, L189-V218, E190-V218, M191-V218, K192-V218, W193-V218, K194-V218, V195-V218, R196-V218, A197-V218, E198-V218, K199-V218, E200-V218, A201-V218, C202-V218, Q203-V218, R204-V218, E205-V218, K206-V218, E207-V218, L208-V218, P209-V218, A210-V218, A211-V218, and/or V212-V218 of SEQ ID NO:22. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal RATL4d9 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

[0174] In preferred embodiments, the following C-terminal RATL4d9 deletion polypeptides are encompassed by the present invention: M1-V218, M1-F217, M1-H216, M1-F215, M1-P214, M1-H213, M1-V212, M1-A211, M1-A210, M1-P209, M1-L208, M1-E207, M1-K206, M1-E205, M1-R204, M1-Q203, M1-C202, M1-A201, M1-E200, M1-K199, M1-E198, M1-A197, M1-R196, M1-V195, M1-K194, M1-W193, M1-K192, M1-M191, M1-E190, M1-L189, M1-I188, M1-A187, M1-R186, M1-E185, M1-V184, M1-E183, M1-E182, M1-L181, M1-A180, M1-R179, M1-E178, M1-R177, M1-K176, M1-E175, M1-V174, M1-A173, M1-R172, M1-E171, M1-R170, M1-W169, M1-E168, M1-V167, M1-A166, M1-R165, M1-E164, M1-R163, M1-R162, M1-L161, M1-V160, M1-E159, M1-K158, M1-E157, M1-R156, M1-Q155, M1-S154, M1-A153, M1-W152, M1-L151, M1-A150, M1-M149, M1-T148, M1-G147, M1-V146, M1-Q145, M1-E144, M1-L143, M1-L142, M1-R141, M1-G140, M1-W139, M1-N138, M1-G137, M1-E136, M1-I135, M1-G134, M1-T133, M1-P132, M1-L131, M1-Q130, M1-P129, M1-G128, M1-H127, M1-P126, M1-R125, M1-P124, M1-D123, M1-Q122, M1-R121, M1-Q120, M1-G119, M1-A118, M1-Q117, M1-G116, M1-H115, M1-G114, M1-S113, M1-K112, M1-E111, M1-L110, M1-E109, M1-E108, M1-R107, M1-H106, M1-M105, M1-S104, M1-K103, M1-L102, M1-H101, M1-K100, M1-W99, M1-L98, M1-A97, M1-T96, M1-T95, M1-G94, M1-V93, M1-N92, M1-V91, M1-G90, M1-P89, M1-G88, M1-R87, M1-S86, M1-V85, M1-Q84, M1-R83, M1-G82, M1-C81, M1-L80, M1-R79, M1-C78, M1-T77, M1-A76, M1-Y75, M1-Q74, M1-N73, M1-P72, M1-H71, M1-H70, M1-G69, M1-A68, M1-R67, M1-A66, M1-P65, M1-A64, M1-L63, M1-H62, M1-F61, M1-Y60, M1-E59, M1-W58, M1-A57, M1-E56, M1-S55, M1-F54, M1-R53, M1-T52, M1-G51, M1-K50, M1-N49, M1-H48, M1-P47, M1-M46, M1-P45, M1-T44, M1-P43, M1-M42, M1-A41, M1-S40, M1-V39, M1-F38, M1-P37, M1-G36, M1-V35, M1-L34, M1-E33, M1-G32, M1-T31, M1-E30, M1-S29, M1-I28, M1-E27, M1-E26, M1-E25, M1-E24, M1-K23, M1-M22, M1-E21, M1-L20, M1-D19, M1-G18, M1-K17, M1-A16, M1-K15, M1-M14, M1-M13, M1-T12, M1-G11, M1-E10, M1-E9, M1-E8, and/or M1-E7 of SEQ ID NO:22. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal RATL4d9 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

[0175] The RATL4d9 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the RATL4d9 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the RATL4d9 polypeptide to associate with other polypeptides, particularly cognate ligand for RATL4d9, or its ability to modulate certain cellular signal pathways.

[0176] The RATL4d9 polypeptide was predicted to comprise two PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985); which are hereby incorporated by reference herein.

[0177] In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: PNQYATCRLCGRQ (SEQ ID NO:31), and/or MALWASQREKEVL (SEQ ID NO:32). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the RATL4d9 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

[0178] The RATL4d9 polypeptide was predicted to comprise two casein kinase II phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Casein kinase II (CK-2) is a protein serine/threonine kinase whose activity is independent of cyclic nucleotides and calcium. CK-2 phosphorylates many different proteins. The substrate specificity [1] of this enzyme can be summarized as follows: (1) Under comparable conditions Ser is favored over Thr.; (2) An acidic residue (either Asp or Glu) must be present three residues from the C-terminal of the phosphate acceptor site; (3) Additional acidic residues in positions +1, +2, +4, and +5 increase the phosphorylation rate. Most physiological substrates have at least one acidic residue in these positions; (4) Asp is preferred to Glu as the provider of acidic determinants; and (5) A basic residue at the N-terminal of the acceptor site decreases the phosphorylation rate, while an acidic one will increase it.

[0179] A consensus pattern for casein kinase II phosphorylations site is as follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and S or T is the phosphorylation site.

[0180] Additional information specific to casein kinase II phosphorylation sites may be found in reference to the following publication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284(1990); which is hereby incorporated herein in its entirety.

[0181] In preferred embodiments, the following casein kinase II phosphorylation site polypeptide is encompassed by the present invention: GPQLPTGIEGNWGR (SEQ ID NO:33), and/or MALWASQREKEVLR (SEQ ID NO:34). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this casein kinase II phosphorylation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

[0182] Specifically, the RATL4d9 polypeptide was predicted to comprise one tyrosine phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977(1982); Hunter T., J. Biol. Chem. 257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.

[0183] In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: HNKGTRFSEAWEYFHLAP (SEQ ID NO:17). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these RATL4d9 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

[0184] The RATL4d9 polypeptide has been shown to comprise one glycosaminoglycan attachment site according to the Motif algorithm (Genetics Computer Group, Inc.). Proteoglycans are complex glycoconjugates containing a core protein to which a variable number of glycosaminoglycan chains (such as heparin sulfate, chondroitin sulfate, etc.) are covalently attached. The glycosaminoglycans are attached to the core proteins through a xyloside residue which is in turn linked to a serine residue of the protein. A consensus sequence for the attachment site seems to exist and follows the following pattern: S-G-x-G, wherein ‘S’ represents the attachment site, and ‘x’ represents any amino acid. Additional information relating to glycosaminoglycan attachment sites may be found in reference to the following publications, which are hereby incorporated by reference herein: Hassel J. R., Kimura J. H., Hascall V. C., Annu. Rev. Biochem. 55:539-567(1986); and/or Bourdon M. A., Krusius T., Campbell S., Schwarz N. B., Proc. Natl. Acad. Sci. U.S.A. 84:3194-3198(1987).

[0185] In preferred embodiments, the following glycosaminoglycan attachment site polypeptide is encompassed by the present invention: EELEKSGHGQAGQR (SEQ ID NO:35). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RATL4d9 glycosaminoglycan attachment site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

[0186] The RATL4d9 polypeptide was predicted to comprise three N-myristoylation sites using the Motif algorithm (Genetics Computer Group, Inc.). An appreciable number of eukaryotic proteins are acylated by the covalent addition of myristate (a C14-saturated fatty acid) to their N-terminal residue via an amide linkage. The sequence specificity of the enzyme responsible for this modification, myristoyl CoA:protein N-myristoyl transferase (NMT), has been derived from the sequence of known N-myristoylated proteins and from studies using synthetic peptides. The specificity seems to be the following: i.) The N-terminal residue must be glycine; ii.) In position 2, uncharged residues are allowed; iii.) Charged residues, proline and large hydrophobic residues are not allowed; iv.) In positions 3 and 4, most, if not all, residues are allowed; v.) In position 5, small uncharged residues are allowed (Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) In position 6, proline is not allowed.

[0187] A consensus pattern for N-myristoylation is as follows: G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid, and G is the N-myristoylation site.

[0188] Additional information specific to N-myristoylation sites may be found in reference to the following publication: Towler D. A., Gordon J. I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); and Grand R. J. A., Biochem. J. 258:625-638(1989); which is hereby incorporated herein in its entirety.

[0189] In preferred embodiments, the following N-myristoylation site polypeptides are encompassed by the present invention: MPHNKGTRFSEAWEYF (SEQ ID NO:36), VSRGPGVNVGTTALWK (SEQ ID NO:37), and/or PQLPTGIEGNWGRLLE (SEQ ID NO:38). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these N-myristoylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

[0190] The RATL4d9 polypeptide was predicted to comprise a binding-protein-dependent transport system inner membrane component site using the Motif algorithm (Genetics Computer Group, Inc.). Bacterial binding protein-dependent transport systems are multicomponent systems typically composed of a periplasmic substrate-binding protein, one or two reciprocally homologous integral inner-membrane proteins and one or two peripheral membrane ATP-binding proteins that couple energy to the active transport system.

[0191] The integral inner-membrane proteins translocate the substrate across the membrane. Proteins belonging to this superfamily are as follows:

Name(s)	Transport system	Family
amiC	amiD	Oligopeptides (Streptococcus	oppBC
		pneumoniae)
amyC	amyD	Starch degradation products	malFG
appB	appC	Oligopeptides (Bacillus subtilis)	oppBC
araH		Arabinosa	araH
artM	artQ	Arginine	hisMQ
braD	braE	Branched-chain amino acids	livHM
btuC		Vitamin B12	fecCD
cysT	cysW	Sulfate/thiosulfate	cysTW
dppB	dppC	Dipeptides	oppBC
fatC	fatD	Ferric anguibactin	fecCD
fecC	fecD	Iron(III) dicitrate	fecCD
fepD	fepG	Ferric enterobactin	fecCD
feuB	feuC	Iron uptake	fecCD
glnP		Glutamine	hisMQ
gltJ	gltK	Glutamate/aspartate	hisMQ
gluC	gluD	Glutamate (Corynebacterium	hisMQ
		glutamicum)
hisM	hisQ	Histidine	hisMQ
lacF	lacG	Lactose	malFG
livH	livM	Branched-chain amino acids	livHM
malF	malG	Maltose	malFG
malC	malD	Maltodextrin	malFG
mglC		Galactosides	araH
modB		Molybdenum	cysTW
msmF	msmG	Melibiose/raffinose/isomaltotriose	malFG
nifC		Molybdenum (7)	cysTW
nikB	nikC	Nickel	oppBC
nocM	nocQ	Nopaline	hisMQ
nrtB		Nitrate (Synechococcus PCC 7942)	cysTW
occM	occQ	Octopine	hisMQ
oppB	oppC	Oligopeptides	oppBC
opuB		Glycine betaine (Bacillus	cysTW
		subtilis)
phnE		Phosphonates
potB	potC	Spermidine/putrescine	cysTW
potH	potI	Putrescine	cysTW
proW		Glycine betaine/L-proline	cysTW
pstA	pstC	Phosphate	cysTW
rbsC		Ribose	araH
sapB	sapC	Peptides	oppBC
afuB		Iron(III)	cysTW
ugpA	ugpE	sn-glycerol-3-phosphate	malFG
xylH		Xylose	araH
mbpY		Unknown, from plastid of	cysTW
		Marchantia polymorpha
yaeE		Unknown, from Escherichia coli	cysTW
yhdX	yhdY	Unknown, from Escherichia coli	hisMQ
yehW	yehY	Unknown, from Eseherichia coli	cysTW
yejB	yejE	Unknown, from Eseherichia coli	oppBC
yjcV		Unknown, from Eseherichia coli	araH
yjfF		Unknown, from Escherichia coli	araH
ytfT		Unknown, from Escherichia coli	araH
lplB		Unknown, from Bacillus subtilis	malfG
lplC		Unknown, from Bacillus subtilis	cysTW
yckA	yckJ	Unknown, from Bacillus subtilis	hisMQ
yzeB		Unknown, from Bacillus subtilis	cysTW
yzmC	yzmD	Unknown, from Bacillus subtilis	cysTW
yzyA		Unknown, from Bacillus subtilis	cysTW

[0192] It has been shown that most of these proteins contain a conserved region located about 80 to 100 residues from their C-terminal extremity. This region seems to be located in a cytoplasmic loop between two transmembrane domains. Apart from the conserved region, the sequence of these proteins is quite divergent, however they can be classified into seven families which have been respectively termed: araH, cysTW, fecCD, hisMQ, livHM, malFG and oppBC.

[0193] A consensus pattern for binding-protein-dependent transport system inner membrane component sites are as follows: [LIVMFY]-x(8)-[EQR]-[STAGV]-[STAG]-x(3)-G-[LIVMFYSTAC]-x(5)-[LIVMFYSTA]-x(4)-[LIVMFY]-[PKR].

[0194] Additional information specific to binding-protein-dependent transport system inner membrane component sites may be found in reference to the following publications: Ames G. F. -L., Annu. Rev. Biochem. 55:397-425(1986); Higgins C. F., Hyde S. C., Mimmack M. M., Gileadi U., Gill D. R., Gallagher M. P., J. Bioenerg. Biomembr. 22:571-592(1990); Dassa E., Hofnung M., EMBO J. 4:2287-2293(1985); Saurin W., Koster W., Dassa E., Mol. Microbiol. 12:993-1004(1994); and Pearce S. R., Mimmack M. L., Gallagher M. P., Gileadi U., Hyde S. C., Higgins C. F., Mol. Microbiol. 6:57-57(1992); which are hereby incorporated herein by reference in their entirety.

[0195] In preferred embodiments, the following binding-protein-dependent transport system inner membrane component site polypeptide is encompassed by the present invention: HHPNQYATCRLCGRQVSRGPGVNVGTTALWKHLKSMHRE (SEQ ID NO:18). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this binding-protein-dependent transport system inner membrane component site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

[0196] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2330 of SEQ ID NO:21, b is an integer between 15 to 2344, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a+14.

[0197] The invention also encompasses a RATL polypeptide variants. A preferred RATL polypeptide variant is one having at least 80%, preferably 90% or greater, amino acid identity to the amino acid sequence of SEQ ID NOS: 2, 4, 5, 6, 13, 16, 10 or 22 for RATL 2e12, RATL 5h6, RATL 1f7, and RATL 4d9, respectively. A more preferred RATL variant is one having at least 95% amino acid sequence identity to SEQ ID NO:2, 4, 5, 6, 13, 16, 10 or 22 for RATL 2e12, RATL 5h6, RATL 1f7, or RATL 4d9.

[0198] The present invention encompasses fragments of a RATL protein, in particular a fragment of RATL 2e12, RATL 5h6, RATL 1f7 and RATL 4d9. In one embodiment, a RATL2e12 fragment comprises at least one ankryin repeat.

[0199] The present invention provides isolated RATL polypeptides, peptides, homologs, variants and agonists thereof having immune and inflammation regulatory activity. Such proteins are substantially free of contaminating endogenous materials. Derivatives of the RATL polypeptides within the scope of the invention also include various structural forms of the primary protein which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, an RATL protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.

[0200] The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives are prepared by linking particular functional groups to amino acid side chains or at the N- or C-termini.

[0201] The present invention further encompassed fusion proteins comprising the amino acid sequence of an RATL polypeptide or portion thereof linked to an immunoglobulin Fc region. An exemplary Fc region is a human IgG1 which comprises the hinge region, CH2 and CH3 domains of an Fc region of human IgG1. Depending on the portion of the Fc region used, a fusion protein may be expressed as a dimer, through formation of interchain disulfide bonds.

[0202] The invention also encompasses polynucleotides or oligonucleotides that are unique to, regulated in and/or associated with T lymphocytes, in particular activated T lymphocytes. These polynucleotides or oligonucleotides include ESTs, contigs, open-reading frames, genes, antisense and the like.

[0203] In one embodiment, the polynucleotide or oligonucleotides of the present invention encompass an untranslated region of a nucleic acid sequence. Such sequences are associated with human T lymphocytes, in particular T lymphocytes activated using antibody against CD28 and CD3. Such sequences are useful in detecting activated T lymphocytes, and monitoring therapeutics that modulate T cell activation. One example of such a polynucleotide is RATL2f3 comprising the nucleic acid sequence of SEQ ID NO:6 or portion thereof as shown in FIG. 19 or SEQ ID NO:7 or portion thereof as shown in FIG. 20. Another example of such a polynucleotide is RATL6f6 comprising the nucleic acid sequence of SEQ ID NO:21 as shown in FIG. 21.

[0204] The invention also encompasses polynucleotides which encode a RATL protein, polypeptide, functional portion or analog thereof. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of a RATL protein can be used to generate recombinant molecules which express a RATL protein or peptide. In a particular embodiment, the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NOS. 1, 3, 8, 9, 11, 12, 14, 15, 32, 19, 20, 21, 33 or portion or variant thereof as shown in FIGS. 1A, 2A, 4-5, 8, 9, 11-12B, 14-17, which encode RATL 2e12, RATL 1f7, RATL 5h6 and RATL 4d9, respectively.

[0205] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a RATL protein, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of a naturally occurring RATL protein, and all such variations are to be considered as being specifically disclosed.

[0206] Although nucleotide sequences which encode a RATL protein and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring RATL protein under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding a RATL protein or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding a RATL protein and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

[0207] The invention also encompasses production of DNA sequences, or portions thereof, which encode a RATL protein and its derivatives, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding a RATL protein or any portion thereof.

[0208] Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequences of SEQ ID NOS: 1, 3, 8, 9, 11, 12, 14, 15, 19, 32, 20, 21, 23, 24, 25, 33 or portion or variant thereof as shown in FIGS. 1A, 2A, 4-5, 8, 9, 11-12B, and 14-20 for RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3, and RATL 6f6, under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods of Enzymol. 152:507-511), and may be used at a defined stringency. In one embodiment, sequences include those capable of hybridizing under moderately stringent conditions (prewashing solution of 2×SSC, 0.5% SDS, 1.0 mM MEDTA, pH 8.0) and hybridization conditions of 50° C., 5×SSC, overnight, to the sequences encoding a RATL protein and other sequences which are degenerate to those which encode the RATL protein.

[0209] Altered nucleic acid sequences encoding a RATL protein which are encompassed by the invention include deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent RATL protein. The encoded protein may also contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent RATL protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of RATL protein is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

[0210] Also included within the scope of the present invention are alleles of the genes encoding a RATL protein. As used herein, an “allele” or “allelic sequence” is an alternative form of the gene which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or poypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0211] Methods for DNA sequencing which are well known and generally available in the art may be used to practice any embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of of DNA polymerase I, SEQUENCE (US Biochemical Corp. Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and th ABI 377 DNA sequencers (Perkin Elmer).

[0212] The nucleic acid sequences encoding a RATL protein may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed, “restriction-site” PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

[0213] Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Mn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

[0214] Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR.

[0215] Another method which may be used to retrieve unknown sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

[0216] When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into the 5′ and 3′ non-transcribed regulatory regions.

[0217] Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

[0218] In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode a RATL protein or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a RATL protein in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a RATL protein.

[0219] As will be understood by those of skill in the art, it may be advantageous to produce a RATL protein encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0220] The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter a RATL protein encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.

[0221] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding a RATL protein may be ligated to a heterologous sequence to encode a RATL-fusion protein. For example, to screen peptide libraries for inhibitors of a RATL protein biological activity, it may be useful to encode a RATL-chimeric protein that can be recognized by a commercially available antibody. A RATL-fusion protein may also be engineered to contain a cleavage site located between the RATL protein encoding sequence and the heterologous protein sequence, so that the RATL protein may be cleaved and purified away from the heterologous moiety.

[0222] In another embodiment, sequences encoding a RATL protein may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a RATL protein, or a functional portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer).

[0223] The newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, W H Freeman and Co., New York, N.Y), by reversed-phase high performance liquid chromatography, or other purification methods as are known in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionally, the amino acid sequence of a RATL protein, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

[0224] In order to express a biologically active RATL protein the nucleotide sequences encoding the RATL protein or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

[0225] Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

[0226] A variety of expression vector/host systems may be utilized to contain and express sequences encoding a RATL protein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus. TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0227] In one embodiment a vector useful in cloning and expression of a RATL polynucleotide is pcDNA3, having the restriction sites 5′ Eco RI and 3′ Not I (Invitrogen Corporation, Carlsbad, Calif.) or an equivalent vector.

[0228] The “control elements” or “regulatory sequences” are those non-translated regions of the vector-enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BILL) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a RATL protein, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

[0229] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for a RATL protein. For example, when large quantities of are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding a RATL protein may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides, as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0230] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0231] In cases where plant expression vectors are used, the expression of sequences encoding a RATL protein may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0232] An insect system may also be used to express a RATL protein. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding a RATL protein may be cloned into a non-essential region of the virus such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of a RATL nucleic acid sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the RATL protein may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0233] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding a RATL protein may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing a RATL protein in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous, sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0234] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a RATL protein. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a RATL protein, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

[0235] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

[0236] For long-term, high-yield production of recombinant proteins, stable expression is-preferred. For example, cell lines which stably express a RATL protein may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

[0237] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk− or aprt− cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisd), which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β glucuronidase and its substrate GUS, and liciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0238] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a RATL protein is inserted within a marker gene sequence, recombinant cells containing sequences encoding can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a RATL protein under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0239] Alternatively, host cells which contain the nucleic acid sequence encoding a RATL protein and express the RATL protein may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

[0240] The presence of polynucleotide sequences encoding a RATL protein can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of polynucleotides encoding the RATL protein. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding the RATL protein to detect transformants containing DNA or RNA encoding the RATL protein. As used herein “oligonucleotides” or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.

[0241] A variety of protocols for detecting and measuring the expression of a RATL protein, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the RATL protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

[0242] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding a RATL protein include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding a RATL protein, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kit (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio)). Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0243] Host cells transformed with nucleotide sequences encoding a RATL protein may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode a RATL protein may be designed to contain signal sequences which direct secretion of the RATL protein through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding the RATL protein to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). (Porath, J et al. (1992, Prot. Exp. Purif. 3:263-281). (Kroll, D. J. et al. 993; DNA Cell Biol. 12:441-453).

[0244] In addition to recombinant production, fragments of a RATL protein may be produced by direct peptide synthesis using solid-phase techniques (Merrifiel J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments of a RATL protein be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

[0245] Certain host cells of the invention can also be used to produce non-human 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 RATL-coding sequence has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous RATL sequences have been introduced into their genome or homologous recombinant animals in which endogenous RATL sequences have been altered. Such animals are useful for studying the function and/or activity of RATL and for identifying and/or evaluating modulators of RATL activity. Accordingly, another aspect of the invention pertains to non-human transgenic animals which contain cells carrying a transgene encoding a RATL protein or portion thereof. In one embodiment, the transgene alters an endogenous gene encoding an endogenous RATL protein (e.g. homologous recombinant animals in which the endogenous RATL gene has been functionally disrupted or “knocked out” or the nucleotide sequence of the endogenous RATL gene has been mutated or the transcriptional regulatory region of the endogenous RATL gene has been altered).

[0246] A transgenic animal of the invention can be created by introducing RATL-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g. by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human RATL nucleic acid sequence of SEQ ID NO:1, 3, 8, 9, 11, 12, 14, 15, 19, 20, 21, 23, 24, 25, 32 or 33 can be introduced as a transgene into the genome of a non-human animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.

[0247] A tissue-specific regulatory sequence(s) can be operably linked to the RATL transgene to direct expression of RATL protein 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; 4,870,009 and 4,873,191.

Therapeutics

[0248] A RATL protein, functional portion, analog, agonist or antagonist thereof (collectively used herein as a RATL therapeutic) may be used to modulate immune function, in particular immune function of T lymphocytes, modulate inflammation, modulate cell growth and differentiation and modulate cancer development. In such uses a particular RATL therapeutic can be administered for the purpose of inhibiting or suppressing immune responses in particular T lymphocyte proliferation, or for inhibiting T lymphocyte activation for suppressing immune responses in mammals, preferably humans. A soluble RATL therapeutic may be administered for preventing or treating organ or graft allograft rejection and graft-versus-host reactions, autoimmune disease, allergy, asthma, cancer, immunodeficiencies and the like.

[0249] In another embodiment, a RATL therapeutic may be administered to a subject to treat or prevent a disorder associated with aberrant cellular differentiation. Such disorders may include, but are not limited to: hyperaldosteronisin (Conn's Syndrome), hypocortisolism (Addison's disease), hypercortisolism (Cushing's disease), and adrenogenital syndrome; and cancers, including cancers of the nervous system; cancers of glands, tissues, and organs involved in secretion or absorption such as prostate, lung, bladder, adrenal gland, liver, uterus, and kidney; and cancers of tissues of the immune and hematopoietic systems. In one aspect, antibodies which are specific for a RATL protein may be used directly as a therapeutic, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express one or more RATL proteins or peptides.

[0250] In another embodiment, a vector expressing the complementary or antisense sequence of the polynucleotide encoding a RATL protein may be administered to a subject to treat or prevent a disorder associated with aberrant cellular differentiation including those listed above.

[0251] In another embodiment, a RATL therapeutic may be administered to a subject to treat or prevent inflammation and autoimmune dysfunctions. Inflammation may be associated with, but is not limited to, allergic reactions, asthma and adult respiratory distress syndrome, rheumatoid arthritis, osteoarthritis, glomerulonephritis, osteoporosis, dermatomyositis, polymyositis, Addison's disease, Grave's disease, irritable bowel syndrome, atrophic gastritis, lupus erythematosus, myasthenia gravis, multiple sclerosis, systemic lupus erythematosis, autoimmune thyroiditis, ulcerative colitis, anemia, pancreatitis, scleroderma, Crohn's disease, ischermia/reperfusion injury, post-traumatic inflammation, myocardial inflammation, atherosclerosis, diabetes, and inflammatory complications of cancer, hemodialysis and extracorporeal circulation, infection and trauma. In one aspect, antibodies which are specific for a RATL protein may be used directly to treat or prevent inflammation and autoimmune dysfunctions.

[0252] In another embodiment a vector expressing the complementary or antisense sequence of a polynucleotide encoding a RATL protein may be administered to a subject to treat or prevent inflammation associated with expression of the RATL protein including those conditions listed above.

[0253] In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, antisense sequences or vectors described above may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0254] A RATL protein, functional portion, analog, agonist or antagonist thereof may be produced using methods which are generally known in the art. In particular, a purified RATL protein, portion, analog, agonist or antagonist thereof may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind a RATL protein, portion, analog, agonist or antagonist. Such libraries are available through Sigma-Aldrich Co.

[0255] Antibodies specific for a RATL protein, portion, analog, agonist or antagonist thereof may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.

[0256] For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Ribi adjuvant R700 (Ribi, Hamilton, Mont.), incomplete Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacillus Calmette Guérin) and Corynebacterium parvum are especially preferable.

[0257] It is preferred that the peptides, fragments, or oligopeptides used to induce antibodies to a RATL protein have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. The peptides, fragments or oligopeptides may comprise a single epitope or antigenic determinant or multiple epitopes. Short stretches of RATL amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.

[0258] Monoclonal antibodies to a RATL protein may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42, Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120).

[0259] In addition, techniques developed for the production of “chimeric antibodies,” the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce a RATL protein specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0260] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0261] Antibody fragments which contain specific binding sites for a RATL protein may also be generated. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 254.1275-1281).

[0262] Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between a RATL protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering RATL epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).

[0263] In another embodiment of the invention, the polynucleotides encoding a RATL protein, or any fragment thereof or antisense molecules, may be used for therapeutic purposes. In one aspect, antisense to the polynucleotide encoding a RATL protein may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding a RATL protein. Thus, antisense molecules may be used to modulate T lymphocyte activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligomers or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding a RATL protein.

[0264] Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express antisense molecules complementary to the polynucleotides of the gene encoding a RATL protein. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).

[0265] Genes encoding a RATL protein can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes a RATL protein. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.

[0266] As mentioned above, modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA, or PNA, to the control regions of the gene encoding a RATL protein, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. 1. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y). The antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

[0267] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a RATL protein.

[0268] Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0269] Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding a RATL protein. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

[0270] RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

[0271] Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient as disclosed in U.S. Pat. Nos. 5,399,493 and 5,437,994. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art.

[0272] Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

[0273] An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of at least one RATL protein, antibodies to a RATL protein, mimetics, agonists, antagonists, or inhibitors of a RATL protein. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, hormones, or biological response modifiers.

[0274] The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0275] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0276] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[0277] Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth, and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrohdone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0278] Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[0279] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[0280] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyloleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0281] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0282] The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0283] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0284] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of a RATL protein, such labeling would include amount, frequency, and method of administration.

[0285] Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

[0286] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0287] A therapeutically effective dose refers to that amount of active ingredient, for example a RATL protein or biologically active fragments thereof, antibodies of a RATL protein, agonists, antagonists or inhibitors of a RATL protein which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0288] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0289] Normal dosage amounts may vary from 0.1 to 100,000 microgram, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. In one embodiment, dosages of a RATL protein or fragment thereof from about 1 ng/kg/day to about 10 mg/kg/day, and preferably from about 500 ug/kg/day to about 5 mg/kg/day are expected to induce a biological effect. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

[0290] In a specific embodiment, formulations of the present invention may further comprise antagonists of P-glycoprotein (also referred to as the multiresistence protein, or PGP), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). P-glycoprotein is well known for decreasing the efficacy of various drug administrations due to its ability to export intracellular levels of absorbed drug to the cell exterior. While this activity has been particularly pronounced in cancer cells in response to the administration of chemotherapy regimens, a variety of other cell types and the administration of other drug classes have been noted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations in the PGP gene significantly reduces PGP function, making it less able to force drugs out of cells. People who have two versions of the mutated gene—one inherited from each parent—have more than four times less PGP than those with two normal versions of the gene. People may also have one normal gene and one mutated one. Certain ethnic populations have increased incidence of such PGP mutations. Among individuals from Ghana, Kenya, the Sudan, as well as African Americans, frequency of the normal gene ranged from 73% to 84%. In contrast, the frequency was 34% to 59% among British whites, Portuguese, Southwest Asian, Chinese, Filipino and Saudi populations. As a result, certain ethnic populations may require increased administration of PGP antagonist in the formulation of the present invention to arrive at the an efficacious dose of the therapeutic (e.g., those from African descent). Conversely, certain ethnic populations, particularly those having increased frequency of the mutated PGP (e.g., of Caucasian descent, or non-African descent) may require less pharmaceutical compositions in the formulation due to an effective increase in efficacy of such compositions as a result of the increased effective absorption (e.g., less PGP activity) of said composition.

[0291] Moreover, in another specific embodiment, formulations of the present invention may further comprise antagonists of OATP2 (also referred to as the multiresistance protein, or MRP2), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). The invention also further comprises any additional antagonists known to inhibit proteins thought to be attributable to a multidrug resistant phenotype in proliferating cells.

[0292] Preferred antagonists that formulations of the present may comprise include the potent P-glycoprotein inhibitor elacridar, and/or LY-335979. Other P-glycoprotein inhibitors known in the art are also encompassed by the present invention.

[0293] Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to increase the efficacy of a pharmaceutical composition, either directly or indirectly. Such a use may be administered in simultaneous conjunction with said pharmaceutical, or separately through either the same or different route of administration (e.g., intravenous for the polynucleotide or polypeptide of the present invention, and orally for the pharmaceutical, among others described herein.).

Diagnostics

[0294] Antibodies which specifically bind a RATL protein or epitope thereof may be used for the diagnosis of conditions or diseases characterized by expression of a RATL protein, or in assays to monitor patients being treated with a RATL protein, agonists, antagonists or inhibitors. The antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for a RATL protein include methods which utilize the antibody and a label to detect it in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described above.

[0295] A variety of protocols including ELISA, RIA, and FACS for measuring a RATL protein are known in the art and provide a basis for detection and/or diagnosing altered or abnormal levels of RATL expression. Normal or standard values for RATL expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to RATL under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of the RATL protein expressed in subject samples, control samples and test samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing T lymphocyte activation, for diagnosing diseases such as autoimmune disease and solid organ transplant rejection and for monitoring therapeutic treatment with compounds that modulate the immune system.

[0296] In another embodiment of the invention, the polynucleotides encoding a RATL protein may be used for detection and diagnostic, purposes. The polynucleotides which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of a RATL protein may be correlated with T lymphocyte activation, with immune or inflammatory diseases or with cancer. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of a RATL protein, and to monitor regulation of RATL protein levels during therapeutic intervention

[0297] In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding a RATL protein or closely related molecules, may be used to identify nucleic acid sequences which encode a RATL protein. The specificity of the probe, whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5′ regulatory region, or a less specific region, e.g., especially in the 3′ coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding a RATL protein, alleles, or related sequences.

[0298] Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the RATL encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NOS: 1, 3, 8, 9, 11, 12, 14, 15, 19, 20, 21, 23, 24, 25, 32 or 33 or from genomic sequence including promoter; enhancer elements, and introns of the naturally occurring RATL.

[0299] Means for producing specific hybridization probes for DNAs encoding a RATL protein include the cloning of nucleic acid sequences encoding a RATL protein or RATL derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

[0300] Polynucleotide sequences encoding a RATL protein may be used for detection of T lymphocytes, and for some polynucleotide sequences, detection of activated T lymphocytes. Such detection is useful in determining the immune status or functioning of an individual and for monitoring immune response to vaccination. Polynucletide sequences encoding a RATL protein may also be used for diagnosis of disease such as cancer and diseases associated with T cell activation such as autoimmunedisease. Polynucleotide sequences encoding a RATL protein may also be used for the diagnosis of disorders or inflammation associated with expression of a RATL protein. Examples of such disorders or conditions include hyperaldosteronism (Conn's Syndrome), hypocortisolism (Addison's disease), hypercortisolism (Cushing's disease), adrenogenital syndrome; cancers of the nervous system, cancers of glands, tissues, and organs involved in secretion or absorption such as prostate, lung, bladder, adrenal gland, liver, uterus, and kidney, and cancers of tissues of the immune and hematopoietic systems; allergic reactions, asthma and adult respiratory distress syndrome, rheumatoid arthritis, osteoarthritis, glomerulonephritis, osteoporosis dermatomyositis, polymyositis, Addison's disease, Grave's disease, irritable bowel syndrome, inflammatory bowel disease, psoriasis, atrophic gastritis, lupus erythematosus, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, ulcerative colitis, anemia, pancreatitis, scleroderma, Crohn's disease, ischemia/reperfusion injury, post-traumatic inflammation, myocardial inflammation, atherosclerosis, multiple sclerosis, and inflammatory complications of cancer, hemodialysis and extracorporeal circulation, infection and trauma, and tissue and organ transplantation rejection. The polynucleotide sequences encoding a RATL protein may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered RATL expression. Such qualitative or quantitative methods are well known in the art.

[0301] In a particular aspect, the nucleotide sequences encoding a RATL protein may be useful in assays that detect activation or induction of various cancers or precancer, particularly breast cancer, colon cancer, prostate cancer and the like. The nucleotide sequences encoding a RATL protein may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding the RATL protein in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

[0302] In order to provide a basis for the diagnosis of disease associated with expression of a RATL protein, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes a RATL protein, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.

[0303] Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

[0304] With respect to cancer, the presence of an abnormal amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0305] Additional diagnostic uses for oligonucleotides designed from the sequences encoding a RATL protein may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5′ to 3′) and another with antisense (3′ to 5′), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.

[0306] Methods which may also be used to quantitate the expression of a RATL protein include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P. C. et al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

[0307] In another embodiment of the invention, the nucleic acid sequences of RATL from a coding or non-coding region may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. A RATL sequence, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen for drugs that either augment or inhibit a biological function. Such techniques include FISH, FACS, or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

[0308] FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques Pergamon Press, New York, N.Y.) may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981 f). Correlation between the location of the gene encoding a RATL protein on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.

[0309] In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580), any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.

[0310] In another embodiment of the invention, a RATL protein, its catalytic or immunogenic fragments or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. A RATL protein, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen for drugs that either augment or inhibit a biological function. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the RATL protein, its catalytic or immunogenic fragments or oligopeptides thereof, and the agent being tested, may be measured.

[0311] Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, as applied to a RATL protein, large numbers of different small test compounds are synthesized on a solid. substrate, such as plastic pins or some other surface. The test compounds are reacted with RATL or fragments thereof, and washed. Bound RATL is then detected by methods well known in the art. Purified RATL can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

[0312] In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding a RATL protein or immunogenic epitope thereof specifically compete with a test compound for binding RATL. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a RATL protein.

[0313] In additional embodiments, the nucleotide sequences which encode a RATL protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

Antibodies

[0314] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (erg., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

[0315] Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

[0316] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multi specificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715;WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0317] Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

[0318] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

[0319] The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[0320] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

[0321] The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111 (Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

[0322] Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

[0323] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

[0324] The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[0325] The antibodies of the present invention may be generated by any suitable method known in the art.

[0326] The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988); and Current Protocols, Chapter 2; which are hereby incorporated herein by reference in its entirety). In a preferred method, a preparation of the RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.

[0327] Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

[0328] The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

[0329] In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0330] The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Preferably, the immunizing agent consists of an RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 polypeptide or, more preferably, with a RATL 2e12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0331] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. More preferred are the parent myeloma cell line (SP2O) as provided by the ATCC. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0332] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).

[0333] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra, and/or according to Wands et al. (Gastroenterology 80:225-232 (1981)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0334] The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0335] The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0336] The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

[0337] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0338] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples described herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[0339] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

[0340] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

[0341] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0342] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

[0343] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

[0344] In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0345] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).

[0346] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice 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, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0347] Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).

[0348] 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 mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

[0349] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

[0350] Such anti-idiotypic antibodies capable of binding to the RATL 2e 12, RATL 1f7, RATL 5h6, RATL 4d9, RATL 2f3 and/or RATL 6f6 polypeptide can be produced in a two-step procedure. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

[0351] The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, Preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

[0352] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0353] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).

[0354] Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

[0355] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22.

[0356] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0357] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0358] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

[0359] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

[0360] In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, 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, e.g., humanized antibodies.

[0361] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

[0362] More preferably, a clone encoding an antibody of the present invention may be obtained according to the method described in the Example section herein.

Methods of Producing Antibodies

[0363] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[0364] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

[0365] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

[0366] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

[0367] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0368] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0369] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0370] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

[0371] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[0372] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

[0373] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0374] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0375] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[0376] The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies -specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

[0377] The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570;

[0378] Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).

[0379] As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NOS: 2, 4, 10, 13, 16, and/or 22 may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light-chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fe portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fe portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem . . . 270:9459-9471 (1995).

[0380] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

[0381] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., 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, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidinibiotin; 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, 111In or 99Tc.

[0382] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, 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 homologues 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).

[0383] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or 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, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0384] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0385] 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).

[0386] 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, which is incorporated herein by reference in its entirety.

[0387] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

[0388] The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins'with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.

[0389] During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent “super” MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.

[0390] Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.

[0391] MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).

[0392] A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby incorporated by reference in their entirety herein.

Uses for Antibodies Directed against Polypeptides of the Invention

[0393] The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of-antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.

[0394] Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), ppl47-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).

[0395] Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.

Immunophenotyping

[0396] The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0397] These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays for Antibody Binding

[0398] The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

[0399] Imnunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0400] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0401] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

[0402] The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

[0403] The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

[0404] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

[0405] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

[0406] The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

[0407] It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

[0408] Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.

[0409] Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.

[0410] Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

[0411] In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.

[0412] In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).

Antibody-based Gene Therapy

[0413] In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

[0414] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

[0415] For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0416] In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

[0417] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[0418] In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem . . . 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

[0419] In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

[0420] Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

[0421] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

[0422] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

[0423] In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

[0424] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

[0425] Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0426] In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

[0427] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0428] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

[0429] The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

[0430] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

[0431] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

[0432] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0433] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

[0434] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

[0435] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0436] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

[0437] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[0438] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0439] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0440] The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0441] The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0442] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0443] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0444] Diagnosis and Imaging with Antibodies

[0445] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

[0446] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0447] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0448] One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

[0449] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from 5 about to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

[0450] Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

[0451] In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0452] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

[0453] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

[0454] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

[0455] In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

[0456] In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

[0457] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

[0458] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

[0459] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

[0460] Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

EXAMPLES

[0461] The examples described below are provided to illustrate the present invention and are not included for the purpose of limiting the invention.

Example 1

[0462] Methods

[0463] T Cell Preparation:

[0464] Human T cells were prepared by standard rosetting protocols with sheep red blood cells (SRBCs). Briefly, peripheral blood mononuclear cells (PBMCs) from 225 ml of heparinized blood from each of 2 donors were prepared by centrifugation over ficoll. T cells (E+ fraction) were isolated by resetting with SRBCs. Messenger RNA (mRNA) from one half of the unstimulated T cells (approximately 2.25×108 cells) was prepared according to the manufacturer's instructions with a FAST-TRACK mRNA isolation kit (Invitrogen). The remaining T cells were diluted to 1.25×106/ml in RPMI/10%FBS containing the costimulatory anti-CD28 mAb 2E12 at 5 □g/ml and added (20 ml/plate) to 10 cm tissue culture plates (Corning) that had been coated with anti-CD3 mAb GI9-4. Plates were coated by incubating 5 ml of 5 ug/ml mAb diluted in PBS for 7hours at 37° C. followed by washing 3 times with PBS. The plates were cultured under normal conditions of cell culture for 18 hours. Activated cells were harvested by vigorous pipetting and scraping to obtain both suspension and adherent cells, pelleted by centrifugation, and processed for mRNA isolation as described above.

[0465] Subtraction Library Construction:

[0466] A cDNA subtraction library was made using the CLONTECH PCR-Select™ cDNA Subtraction Kit (Clontech, Palo Alto, Calif.). Manufacturer's protocols were followed for 500 ng of anti-CD3/anti-CD28 activated peripheral blood T cell poly A+ RNA (tester) and 500 ng of unactivated, resting peripheral blood T cell poly A+ RNA (driver). Five secondary PCR reactions were combined and run on a 1.2% agarose gel. Fragments ranging from approximately 0.3 kb-1.5 kb were gel purified using the QIAgen gel extraction kit (QIAgen Inc., Valencia, Calif.) and inserted into the TA cloning vector, pCR2.1 (Invitrogen). TOP10F′ competent E. Coli (Invitrogen) were transformed and plated on Lauria-Bertani (LB) plates containing 50 micrograms/ml ampicillin. Approximately 600 clones were isolated and grown in LB broth containing similar concentrations of ampicillin. Plasmids were isolated using QIAgen miniprep spin (QIAgen) and sequenced using ABI cycle sequencers (ABI Prism, PE Applied Biosystems).

[0467] Database Mining for Overlapping EST Clones:

[0468] Over 600 hundred clone inserts were analyzed using BLAST2 (Basic Local Alignment Search Tool). Clones with ESTs (Expressed Sequence Tags) of known genes were removed using the non-redundant nucleotide database maintained by NCBI. A number of clones proved to be novel, at least, unpublished with NCBI. A further search was performed using the geneseq nucleotide patent database (also available through BLAST2). Clones containing EST's of patented sequences were eliminated to some extent (some files only contained a portion of our clone or are of low quality sequence or have no associated utility). Once sequences were analyzed for novelty; virtual cloning was performed using the D2 clustered EST database (also available on BLAST2). This D2 clustered database was designed by the Bristol-Myers Squibb Bioinformatics department. It contains both public and proprietary (Incyte) EST's assembled into contigs. A contig is a collection of smaller EST clones assembled into larger sequence fragments. The subtraction clone sequences were used to query this clustered database. Cluster sequences were assembled with the subtraction clone sequence using the sequence analysis program Sequencher (Gene Codes). The larger contig sequence was then back-searched against the non-redundant nucleotide (NRN), geneseq nucleotide patent (GNP), non-redundant protein (NRP), and geneseq peptide patent (GPP) databases.

[0469] Northern Analysis Methods

[0470] Peripheral blood T cells (PBTs) were isolated by SRBC rosetting methods as described. The T cell lymphoma cell line Jurkat and PBTs were stimulated with either a combination of PMA and ionomycin at 100 ng/ml and 1 ug/ml, respectively, or soluble anti-CD3 and anti-CD28 monoclonal antibodies at 1 ug/ml and 4 ug/ml, respectively, for 8 h, 24 h, or 48 h. Total RNA from unactivated and activated cells was isolated with Trizol reagent (Life Technologies, Baltimore, Md.). RNA,(25 ug) from each sample was diluted in RNA sample loading buffer (Sigma Chem. Co., St. Louis, Mo.) and electrophoresed on a 1.2% denaturing formaldehyde gel (Fourney et al 1988, Focus 10:1,5-7) RNA size standards (Life Technologies, Baltimore, Md.) were run in a parallel lane for estimation of hybridizing mRNA sizes. Following electrophoresis, the samples were transferred overnight to a Gene Screen Plus nylon membrane (NEN, Boston, Mass.) and subsequently cross-linked by UV irradiation. Alternatively, multiple tissue Northern blots (MTN) were obtained from Clontech (Palo Alto, Calif.). The MTN used were human MTN (#7760-1: heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas), human MTN II (#7759-1: spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood), human cancer cell line MTN (#7757-1: promyelocytic leukemia HL-60, HeLa S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitts lymphoma Raji, colorectal adenocarcinoma SW840, lung carcinoma A549, melanoma G-361), and human immune system MTN (#7768-1: spleen, lymph node, thymus, peripheral blood leukocyte, bone marrow, and fetal liver).

[0471] Membranes were prehybridized with ExpressHyb hybridization solution (Clontech, Palo Alto, Calif.) for three hours at 68° C. and then hybridized overnight at 68° C. with 32P labeled probes. Probes for each of the clones were generated by the following methods:

[0472] 1. RATL 1f7: a˜600bp EcoRI/Not1 fragment from Incyte clone 2791509 was gel purified using a Qiagen gel extraction kit and labeled with 32P-dATP using a random prime labeling kit (Roche Biochemicals, Indianapolis, Ind.) according to the manufacturer's instructions.

[0473] 2. RATL 2e12: a˜1040bp Pst1 fragment, isolated from Incyte clone 3345840, was gel purified and labeled as above.

[0474] 3. RATL 6f6: a 391bp PCR was generated using the subtraction library clone RATL 6f6 as a template and oligonucleotides JNF120 (5′-CCAACGCTAGCTATCTTGCC-3′) (SEQ ID NO:23) and JNF121 (5′-GTCGAAGCTGTCTTCTTGCG-3′) (SEQ ID NO:24) as primers. Briefly, a fragment was amplified in 50 ul reaction volume using Taq polymerase (Roche Biochemicals), 10 ng of template, 2 uM final concentration of each primer, with the following cycling conditions: 1 cycle of 94° C. 3 minutes, 55° C. 1 minute, 72° C. 1 minute, 35 cycles of 94° C. 1 minute, 57° C. 1 minute, 72° C. 1 minute; one final extension at 72° C. minutes. The PCR product was gel purified and labeled as above.

[0475] 4. RATL 5 h6: a˜593bp SaII/BamHI fragment was isolated from Incyte clone #2432375 and gel purified and labeled as described above.

[0476] 5. RATL 2f3: a 696bp PCR product was generated using cDNA from 8 hour anti-CD3 ×CD28 activated T cell cDNA as a template and oligonucleotides JNF114 (5′-CAAAGAAGCATGAGTCACCC-3′) (SEQ ID NO:25) as primers. Cycle conditions were: 1 cycle of 94° C. 3 minutes, 55° C. 1 minute, 72° C. 1 minute, cycles of 94° C. 1 minute, 57° C. 1 minute, 72° C. 1 minute; one final extension at 72° C. for 5 minutes. The PCR product was gel purified and labeled as above.

[0477] After hybridization, the membranes were washed by shaking for 30-45 minutes with low stringency solution (2×SSC/0.1%SDS) at 42° C. with 2-3 changes of solution, and then with high stringency wash solution (0.2×SSC/0.1%SDS) with 2-4 changes of solution, for 20-30 minutes each wash, at 48° C. to 60° C. The membranes were then exposed with intensifying screens to X-ray film at −70° C. for varying amounts of time. For analysis with additional probes, membranes were stripped of hybridizing probe by shaking for 15-30 minutes at 78° C. with freshly boiled 0.5% SDS.

[0478] 6. RATL 4d9: Multiple tissue Northern blots were purchased from Clontech Laboratories and hybridized with P32-labeled RATL4d9. Briefly, a RATL4d9 PCR product was run on an agarose gel, and purified using the QIAgen Gel Extraction Kit (QIAgen). Approximately 30 ng of RATL4d9 was radiolabeled (6000 Ci/mmol P32-dCTP) using the Random Primed DNA Labeling Kit (Roche, Indianapolis, Ind.). Unincorporated nucleotides were removed using NucTrap Probe Purification Columns (Stratgene, La Jolla, Calif.). Radiolabeled RATL4d9 probe was added at a specific activity of 2.5×106 cpM/ml of ExpressHyb hybridization solution (Clontech) and incubated overnight at 65° C. Blots were washed to 0.1×SSC/0.1%SDS at 50° C. and exposed to film for 18 hours.

[0479] Electronic Northern Analysis Methods

[0480] Sequences of clones from the subtraction library were searched against the Incyte database. The relative abundance of the clone in the different libraries in the Incyte collection was used to approximate a virtual Northern blot. The Incyte collection consists of 72 cardiovascular libraries, 54 connective tissue libraries, 151 digestive system libraries, 23 embryonic structure libraries, 63 exocrine system libraries, 64 exocrine glad libraries, 113 female genitalia libraries, 118 male genitalia libraries, 5 germ cell libraries, 166 hematopoietic and immune system libraries, 34 liver libraries, 50 muscoloskeletal libraries, 221 nervous system libraries, 25 pancreas libraries, 95 respiratory system libraries, 10 sense organ libraries, 15 skin libraries, 11 stomatognathic system libraries, 19 unclassified/mixed libraries, and 66 urinary tract libraries.

Example 2

[0481] Isolation and Characterization of RATL 2e12

[0482] The original clone isolated from the subtraction library was 1887-bp in length. This sequence was not present in the NRN database, however, a 307-bp portion of the sequence was found in the GNP database. This sequence coordinates are 1007-1313-bp of the subtraction clone. A search of the D2 clustered EST database revealed a contig of ESTs (Gene ID 978220). This contig only extends the sequence in the 3′ direction by 555-bp for a consensus sequence of 2442-bp. Another contig (Gene ID 208312) was identified which overlays with the 5′-end of the subtraction clone. This extended the sequence 450-bp for a consensus sequence of 2661-bp. A further analysis found yet another contig (Gene ID 123725). From the assembled contig of ESTs the coding sequence of 1491-bp encodes a 497 amino acid protein.

[0483] Two clones isolated from the subtraction library encode peptides having ankyrin repeats, as are found in the NFkB family proteins such as NFkB and BCL3 and found in karyopherins.

[0484] FIG. 1A shows the partial cDNA coding region for RATL 2e12 from Incyte clone 3345840. FIG. 1B shows the deduced amino acid sequence for RATL 2e12 from Incyte clone 3345840.

[0485] FIG. 2A shows the partial cDNA coding region for RATL2e12 from Incyte clone 6758514. FIG. 2B shows the deduced 497 amino acid sequence for RATL 2e12 from Incyte clone 6758514.

[0486] Clones 3345840 and 675514 are splice variants of the same gene. The gene is on chromosome 19. FIG. 3 shows the peptide alignment of the two splice variants. Five ankyrin repeats are boxed and delineated by arrows. The repeats are located at amino acid 268 through 300; 301 through 333; 336 through 388; 390-421 and 427-463.

[0487] RATL 2e12 shares 31% identity to BCL3 and 34% identity with NFkBp100.

[0488] Table 1 shows that RATL 2e12 mRNA was not detectable in non-activated T cells, or in non-activated Jurkat cells, but was upregulated in P/I and CD3×28 activated T cells and in P/I activated Jurkat cells. This strongly supports a role for RATL 2e12 in modulating T cell function, in particular, in modulating activation of T cells.

TABLE 1
Regulation of novel gene mRNAs in T cells and Jurkat cells
	Unact.	P/I	CD3 × 28	Unact.	P/I	CD3 × 28
Gene	T cell	T cell	T cell	Jurkat	Jurkat	Jurkat
1f7	+	+/−	++	+	++	++
2e12	−	++	++	−	++	−
2f3	−	++	++	−	++	+/−
5h6	+	++	++	+	++	++
6f6	+	++	+	−	−	−
Key
− = no message detected
+ = message detected
++ = message upregulated
+/− = message barely detectable

Example 3

[0489] Isolation and Characterization of RATL1f7

[0490] The RATL1f7 clone isolated from the subtraction library encoded a 1,247 bp partial cDNA sequence shown in FIG. 4 (SEQ ID NO:8). A consensus sequence, shown in FIG. 5, was derived from the overlapping and/or extended nucleic acid sequences encoded by the Incyte clone 5795468 (PLACFET04) (SEQ ID NO:9).

[0491] FIG. 6 shows the deduced 388 amino acid sequence of the polypeptide encoded by RATL1f7 (SEQ ID NO:10). RATL1f7 has 26% identity over 214 amino acids with a Plasmodium falciparum surface protein. RATL1F7 shares no homology with other known mammalian proteins. RATL1F7 contains potential cyclic adenosine monophosphate (position 62), casein kinase II (positions 35, 113, 115), and protein kinase C (positions 28, 141, 164, 228) phosphorylation sites.

[0492] Northern analysis shown in FIG. 7 reveals that RATL1F7 is expressed in spleen, thymus, small intestine, peripheral blood leukocytes, MOLT4 cell line, and Jurkat cell line. RATL1F7 is upregulated upon stimulation in the Jurkat T-cell line and human peripheral blood T lymphocytes with antibodies against CD3 and CD28 antigens (Table 1). In addition, the Incyte electronic northern suggests expression in several tumor tissues including bone, penis, uterus, ovary, colon, lung, gall bladder, esophageal, breast, bladder, pancreatic and brain. Expression was also seen in the asthmatic lung.

Example 4

[0493] Isolation and Characterization of RATL5h6

[0494] The original clone isolated from the subtraction library was 299-bp in length. This sequence is shown in FIG. 8. This sequence was not present in NRN or GNP databases. A search of the D2 clustered database revealed a contig of overlapping clones (Cluster 71748—1). After an assembly with the subtraction clone, a contig of 3389-bp was formed. The contig nucleic acid sequence is shown in FIG. 9. A further search of the GNP database identified human gene signature HUMGS08510. This filed sequence is 357-bp, contains 10 ambiguous bases, and lies in the 3′-URT. A partial polypeptide sequence of 352 amino acids, as depicted in FIG. 10, was used to query the NRP database.

[0495] RATL5h6 was found to be 91% identical to the putative ras effector Nore1 (AF053959) and 93% identical to mouse MaxP1 (AF002251). The ras associated (RA) domain is at amino acids 206-298. A weak ATP synthase is at amino acid 140-151.

[0496] Northern analysis shown in FIG. 7 reveals that RATL5h6 is expressed in spleen, thymus, small intestine, peripheral blood leukocytes, and Jurkat cell line and in the tumor cell lines: HL60, K562, MOLT4 cell line and SW480. RATL5h6 is upregulated upon stimulation in the Jurkat T-cell line and human peripheral blood T lymphocytes with antibodies against CD3 and CD28 antigens. In addition, the Incyte electronic Northern suggests expression predominately in the hemic and immune system, in particular, in the spleen.

[0497] The full-length cloning experiments were performed using Gene Trapper (LifeTechnologies, MD.). Briefly, PCR primers PY1010 (5′-GTAGATGACCGTGGACAGCA-3′) (SEQ ID NO:26) and PY1011 (5′-TGTAGCTGTCGATCTTCTGC-3′) (SEQ ID NO:27) were used to screen a panel of human cDNA libraries (LifeTechnologies). A strong positive PCR product was identified in human leukocyte cDNA library. The double-stranded DNA plasmid library was converted to single-stranded DNA using Gene II and Exonuclease III. Hybrids between the biotinylated oligonucleotide (PY1012: 5′-CAT-GACAGTGGGTACTGCAGCCTGGACGAG-3′) (SEQ ID NO:28) and single-stranded DNA were formed and then captured-on-paramagnetic beads. After washing, the single-stranded DNA was released and converted to double-stranded DNA by DNA polymerase. Following transformation and plating, positive clones were identified by PCR analysis. The plasmids were prepared and subjected to sequencing.

[0498] Preliminary sequence analysis indicated that two of the clones contained the potential full-length coding region. Additional primers were synthesized and used to sequence the entire clones. The vector for these cDNA inserts is pCMVSPORT2 with cloning sites SaII (5′-end) and NotI (3′-end). The full-length nucleic acid sequence for one clone is shown in FIG. 11. The coding region and corresponding deduced amino acid sequence of RATL 5h6 is provided in FIG. 12.

[0499] RATL5h6 was aligned with mouse Ras effector protein Nore1 (mNore1) and human RASSF1C, a putative tumor suppressor gene identified in a deleted region of 3p21.3 that is commonly found in both non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) tumor isolates. Identical residues between all three proteins are indicated by bold shading and residues conserved in at least two proteins are shaded in gray. The Ras association domain identified in RATL5h6 through a search of the Pfam database (http://pfam.wustl.edu/index.html), encompassing residues 119-211, is boxed (FIG. 13). Although RATL5h6 appears to be the human orthologue of mNore1 and rat MaxP1 (not shown), the N-termini of these proteins do not share the high degree of homology that the C-terminal regions share. This could be due to alternative splicing of mRNA transcripts encoding the N-terminal regions or because these genes have diverged in humans and mice. RASSF1C, interestingly, is an alternatively spliced product from a gene that has at least three alternative splicing products deriving from differential exon usage in regions encoding the 5′ end. Experimental evidence suggests that the RASSF1C proteins encoded by the splice variants have different functions. Likewise if splice variants of RATL5h6 do exist, they could have different properties. For example, mouse Nore1 has a particularly proline rich N-terminus, while RATL5h6 does not. Proline rich regions like those found in mNore1 often serve as SH3 recognition motifs which allow interactions with adaptor proteins involved in intracellular signaling cascades.

[0500] Based on this homology, RATL5h6 is the human homolog to rat MaxP1 and is similar to Mus musculus putative ras effector Nore1. Nore1 directly interacts with ras in vitro in a GTP-dependent manner, and the interaction requires an intact ras effector domain. The novel human RATL5h6 may be pivoted in transmitting growth and differentiation signals downstream of cell surface receptors. In light of the prevalence of the RATL5h6 may be used to modulate growth and differentiation signals in any cell that is expressed in, in particular, in immune cells, more particularly in the T lymphocytes.

Example 5

[0501] Isolation and Characterization of RATL4d9

[0502] The original clone isolated from the subtraction library was 188 bp in length (FIG. 14). This nucleic acid sequence is not present in NRN or GNP databases. A search of the D2 clustered EST database revealed a contig of overlapping clones (Cluster 833701-1). After assembly with the subtraction clone, a 794-bp contig was formed (FIG. 15). This contig nucleic acid sequence is not present in NRN or GNP databases.

[0503] Using the RATL4d9 contig assembly sequence shown in FIG. 15 as a query for Incyte database searching, several EST clones that matched RATL4d9 were identified. Based upon the estimated insert sizes, three longest EST clones (#1353392, #1318291, and #2213533) were subjected to sequence analysis. Preliminary analysis indicated that Incyte#1353392 contained the full-length coding sequence and was sequenced in entirety (FIG. 16).

[0504] FIG. 17 shows the coding region (SEQ ID NO:30) and deduced amino acid sequence of the polypeptide encoded by RATL4d9 (SEQ ID NO:22). RATL4d9 shows no homology to other known proteins.

[0505] Electronic Northern analysis indicates that RATL4d9 is restricted to hemic and immune tissue and is specifically found in thymus. Standard Northern analysis revealed expression of RATL4d9 in heart, thymus, testis, thyroid, and activated T cells. (FIG. 18).

Example 6

[0506] Isolation and Characterization of RATL2f3

[0507] Three clones were isolated from the subtraction library which make up two different parts of the same gene (FIG. 19). Clones 2f3 and 2h1 are different clones of the same sequence. Clone 2g11 is an adjacent partial cDNA clone lying 3′ to clone 2f3, 2h1. Clones 2f3 and 2h1 are 748-bp in length. Clone 2g11 is 684-bp in length. Together clones 2f3, 2h1 and 2g11 make a partial sequence of 1,432 bp. This 1,432 bp sequence is not present in the NRN database, except as a genomic clone from Chromosome 21q22.1 (Accession #AP000085). However, a sequence pertaining to the 3′-untranslated region is claimed as human secreted protein cDNA encoding gene 22. This sequence is 943-bp and pertains to coordinates 488-1,427 bp of the 1,432-bp subtraction clones. A search of the D2 clustered EST database revealed a contig of 1,259-bp (FIG. 20). To learn more about this gene, IMAGE clone 1283513 (AA743773) has been ordered. This clone contains 2.3 kb insert which may give us more information about the coding sequence.

[0508] Electronic Northern analysis of various tissue libraries indicates that RATL2f3 is predominately expressed in hemic and immune system and expressed in abundance in CD4+T-lymphocytes treated with CD3 and CD8 antibodies as well as allogeneic anergic T lymphocytes treated with 40-50 M OKT3 for 3 days. It was not found in the tumor libraries screened such as neuroblastoma bone marrow tumor line, a leukemia mast cell line, lung squamous cell carcinoma, lymphoma, metastatic melanoma, Hodgkin's disease lymph node tumor.

[0509] Standard Northern analysis of mRNA expression of RATL2f3 showed high levels of expression in PMA/I stimulated peripheral blood T cells and in CD3×28 stimulated T cells but not in untreated T cells (Table 1). No expression was detected in a stimulated or non-stimulated Jurkat cell line. This provides support for the involvement of the RATL2f3 gene in the activation of normal T lymphocytes. Detection of mRNA expression of RATL 2f3 is useful in determining the state of activation of T lymphocytes.

Example 7

[0510] Isolation and Characterization of RATL6f6

[0511] The original clone isolated from the subtraction library is 635-bp in length (FIG. 21). This sequence is not present in either the NRN or GNP database. This clone is unique with no overlapping public or Incyte EST's.

[0512] Electronic Northern analysis revealed expression predominantly in the hemic and immune system with lower expression in the musculoskeletal system and connective tissue. Within the hemic and immune system, the expression was predominately in non-adherent peripheral blood mononuclear cells. RATL6f6 was not associated with any tumor cell libraries tested. Messanger RNA expression was found in unstimulated T-cells, as well as PMA/I and CD3×28 stimulated T cells (Table 1). Expression was not detected in the Jurkat cell line. Based on these results RATL6f6 nucleic acid sequences have utility in identifying and distinguishing normal T cells from tumorigenic T cells and may act to repress genes associated with tumorigenic events.

Example 8

[0513] Method of Enhancing the Biological Activity/Functional Characteristics of Invention through Molecular Evolution.

[0514] Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, pharmaceutical, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in gene therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.

[0515] Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.

[0516] Directed evolution is comprised of several steps. The first step is to establish a library of variants for the gene or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.

[0517] Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.

[0518] Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as descibed by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.

[0519] While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (W P C, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, “in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.

[0520] DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments—further diversifying the potential hybridation sites during the annealing step of the reaction.

[0521] A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

[0522] Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.

[0523] Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2-4 ug of the DNA substrate(s) would be digested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resulting fragments of 10-50bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatman) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cuttoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10-50bp fragments could be eluted from said paper using 1M NaCL, followed by ethanol precipitation.

[0524] The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10-30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 30-45 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primeness product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8 um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and 72 C for 30s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).

[0525] The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.

[0526] Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailered to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6): 1307-1308, (1997).

[0527] As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436-438, (1997).

[0528] DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.

[0529] A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.

[0530] Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.

[0531] DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host, particularly if the polynucleotides and polypeptides provide a therapeutic use. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the gene sequence for a xenobiotic ortholog of the native protein in with the gene sequence of the novel variant gene in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel varient that provided the desired characteristics.

[0532] Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucletotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the gene template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homolog sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.

[0533] In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in gene therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438, (1997), respectively.

[0534] Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832; PCT Application No. The forgoing are hereby incorporated in their entirety herein for all purposes.

[0535] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

[0536] References:

[0537] 1. Vavvas, D., Li., X, Avruch, J. and Zhang, X. F. (1998) Identification of nore1 as a potential ras effector. J. Biol. Chem. 273(10, 5439-5442.

[0538] 2. Dammann, R., Li, C., Yoon, J -H., Chin, P. L., Bates S., and Pfeifer, G. P. (2000) Epigenic inactivaton of a RAS association domain family protein from the lung tumour suppreor locus 3p21.3. Nature Genetics 25:315-319.

Claims

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:21 or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No: XXXXX, which is hybridizable to SEQ ID NO:21; (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:22 or a polypeptide fragment encoded by the cDNA sequence included in ATCC Deposit No: XXXXX, which is hybridizable to SEQ ID NO:21; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:22 or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No: XXXXX, which is hybridizable to SEQ ID NO:21; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:22 or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No: XXXXX, which is hybridizable to SEQ ID NO:21; (e) a polynucleotide encoding a polypeptide of SEQ ID NO:22 or the cDNA sequence included in ATCC Deposit No: XXXXX, which is hybridizable to SEQ ID NO:21, having immune modulatory activity; (f) an isolated polynucleotide comprising nucleotides 1030 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 3 to 218 of SEQ ID NO:22 minus the first and second amino acids; (g) an isolated polynucleotide comprising nucleotides 1027 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 2 to 218 of SEQ ID NO:22 minus the start methionine; (h) an isolated polynucleotide comprising nucleotides 1024 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 1 to 218 of SEQ ID NO:22 including the start methionine; (i) a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:21; and (j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment consists of a nucleotide sequence encoding a human protein capable of modulating an immune response.

3. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

4. A recombinant host cell comprising the vector sequences of claim 3.

5. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:22 or the encoded sequence included in ATCC Deposit No: XXXXX; (b) a polypeptide fragment of SEQ ID NO:22 or the encoded sequence included in ATCC Deposit No: XXXXX, having immune modulatory activity; (c) a polypeptide domain of SEQ ID NO:22 or the encoded sequence included in ATCC Deposit No: XXXXX; (d) a polypeptide epitope of SEQ ID NO:22 or the encoded sequence included in ATCC Deposit No: XXXXX; (e) a full length protein of SEQ ID NO:22 or the encoded sequence included in ATCC Deposit No: XXXXX; (f) a polypeptide comprising amino acids 3 to 218 of SEQ ID NO:22, wherein said amino acids 3 to 218 comprising a polypeptide of SEQ ID NO:22 minus the first and second amino acids; (g) a polypeptide comprising amino acids 2 to 218 of SEQ ID NO:22, wherein said amino acids 2 to 218 comprising a polypeptide of SEQ ID NO:22 minus the start methionine; and (h) a polypeptide comprising amino acids 1 to 218 of SEQ ID NO:22.

6. The isolated polypeptide of claim 5, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

7. An isolated antibody that binds specifically to the isolated polypeptide of claim 5.

8. A recombinant host cell that expresses the isolated polypeptide of claim 5.

9. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 8 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.

10. The polypeptide produced by claim 9.

11. A method for preventing, treating, or ameliorating a medical condition, comprising the step of administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 5, or a modulator thereof.

12. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

13. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or amount of expression of the polypeptide of claim 5 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

14. An isolated nucleic acid molecule consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a polynucleotide encoding a polypeptide of SEQ ID NO:22; (b) an isolated polynucleotide consisting of nucleotides 1030 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 3 to 218 of SEQ ID NO:22 minus the first and second amino acids; (c) an isolated polynucleotide consisting of nucleotides 1027 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 2 to 218 of SEQ ID NO:22 minus the start methionine; (d) an isolated polynucleotide consisting of nucleotides 1024 to 1677 of SEQ ID NO:21, wherein said nucleotides encode a polypeptide corresponding to amino acids 1 to 218 of SEQ ID NO:22 including the start methionine; and (e) a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:21.

15. The isolated nucleic acid molecule of claim 14, wherein the polynucleotide comprises a nucleotide sequence encoding a human protein capable of modulating an immune response.

16. A recombinant vector comprising the isolated nucleic acid molecule of claim 15.

17. A recombinant host cell comprising the recombinant vector of claim 16.

18. An isolated polypeptide consisting of an amino acid sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:22 having immune modulatory activity; (b) a polypeptide domain of SEQ ID NO:22 having immune modulatory activity; (c) a full length protein of SEQ ID NO:22; (d) a polypeptide corresponding to amino acids 3 to 218 of SEQ ID NO:22, wherein said amino acids 3 to 218 consisting of a polypeptide of SEQ ID NO:22 minus the first and second amino acids; (e) a polypeptide corresponding to amino acids 2 to 218 of SEQ ID NO:22, wherein said amino acids 2 to 218 consisting of a polypeptide of SEQ ID NO:22 minus the start methionine; and (f) a polypeptide corresponding to amino acids 1 to 218 of SEQ ID NO:22.

19. The method for preventing, treating; or ameliorating a medical condition of claim 11; wherein the medical condition is selected from the group consisting of aberrant cellular development; immune responses and inflammation; organ or tissue transplantation rejection; T-lymphocyte disorders; graft allograft rejection and graft-versus-host reactions; autoimmune disease; allergy; asthma; cancer; immunodeficiencies; a disorder associated with aberrant cellular differentiation; hyperaldosteronisin (Conn's Syndrome); hypocortisolism (Addison's disease); hypercortisolism (Cushing's disease); and adrenogenital syndrome; cancers of the nervous system; cancers of glands; tissues; and organs involved in secretion or absorption such as prostate; lung; bladder; adrenal gland; liver; uterus; and kidney; and cancers of tissues of the immune and hematopoietic systems; inflammation and autoimmune dysfunctions; allergic reactions; asthma and adult respiratory distress syndrome; rheumatoid arthritis; osteoarthritis; glomerulonephritis; osteoporosis; dermatomyositis; polymyositis; Addison's disease; Grave's disease; irritable bowel syndrome; atrophic gastritis; lupus erythematosus; myasthenia gravis; multiple sclerosis; systemic lupus erythematosis; autoimmune thyroiditis; ulcerative colitis; anemia; pancreatitis; scleroderma; Crohn's disease; ischermia/reperfusion injury; post-traumatic inflammation; myocardial inflammation; atherosclerosis; diabetes; and inflammatory complications of cancer; hemodialysis and extracorporeal circulation; infection and trauma.