Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)

Definitions

• This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists, hi particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human tumor necrosis factor delta and epsilon, sometimes hereinafter referred to as "TNF delta" and "TNF epsilon".
• TNF-alpha and beta are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol, 7:625-655, 1989).
• Tumor necrosis factor (TNF-alpha and TNF-beta) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell activation and proliferation and also as playing important roles in immune regulation and inflammation.
• the ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biologically active form being a trimeric/multimeric complex. Soluble forms of the TNF ligand superfamily have only been identified so far for TNF, LT-alpha, and FasL (for a general review, see Grass, H. and Dower, S.K., Blood, 85 (72 :3378-3404 (1995)), which is hereby incorporated by reference in its entirety.
• TNF is produced by a number of cell types, including monocytes, fibroblasts, T cells, natural killer (NK) cells and predominately by activated machrophages.
• TNF-alpha has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, resistance to parasites, producing an anti-viral response, septic shock, growth regulation, vascular endothelium effects and metabolic effects. TNF-alpha also triggers endothelial cells to secrete various factors, including PAF-1, IL-1, GM-CSF and IL-6 to promote cell proliferation. In addition, TNF-alpha up-regulates various cell adhesion molecules such as E-Selectin, ICAM-l and VCAM-l.
• the first step in the induction of the various cellular responses mediated by the members of the TNF ligand superfamily is their binding to specific cell surface receptors.
• the TNF receptor superfamily contains at present ten known membrane proteins and several viral open reading frames encoding TNFR-related molecules.
• the p75 low-affinity Nerve Growth Factor (NGF) receptor was the first cloned receptor of this family (Johnson, D. et al. Cell, 47:545 (1986). Subsequently, cloning of two specific receptors for TNF show that they were related to the NGF receptor (Loetscher, H. et al., Cell, 61:351 (1990)).
• TNF receptor superfamily includes the p75 nerve growth factor receptor, p60 TNFR-I, p80 TNFR-II, TNFR-RP/TNFR-m, CD27, CD30, CD40, 4- IBB, OX40 and FAS/APO-1.
• SFV-T2 in Shope fibroma virus (Smith, C.A. et al., Biochem. Biophys. Res.
• Inflammation which is characterized by redness, swelling, heat, and pain, is an essential immune response which occurs following tissue injury or infection. The initial event triggers an elaborate signaling cascade which results in increased local blood flow, blood clotting, and vascular permeability.
• phagocytic leukocytes can begin to neutralize pathogens and contribute to tissue repair.
• the immune cells can begin to neutralize pathogens and contribute to tissue repair.
• blood clotting factors such as histamine and bradykinin
• cell adhesion molecules such as cell adhesion molecules
• cytokines such as interleukins and chemokines
• immune system effector cells such as neutrophils, macrophages, and lymphocytes.
• Medical conditions resulting from inflammation include, but are not limited to, inflammatory bowel disease, multiple sclerosis, arthritis, asthma, allergies, sarcoidosis, septic shock, gastrointestinal cancers, pancreatitis, dermatitis, gout, systemic lupus erythematosis, and Grave's disease. Inflammation is also a potentially life-threatening complication of cardiopulmonary bypass surgery, renal ischemia-reperfusion, and traumatic injury. [011] Several steroidal and nonsteroidal drugs have been used to control inflammation or to provide symptomatic relief. However, these therapies can be accompanied by numerous side effects which limit their usefulness. Therefore, there is a continuing need for more effective and less toxic alternatives for modulating the inflammatory response.
• novel TNF delta and TNF epsilon which have been putatively identified as being tumor necrosis factor ligands by homology between the amino acid sequence set out in Figures 1 A and IB and 2 A and 2B and known amino acid sequences of other proteins in the tumor necrosis factor family such as human TNF-alpha and TNF-beta.
• polypeptides of the present invention have been identified as a novel members of the TNF ligand super-family based on structural and biological similarities.
• the polynucleotides comprise the region encoding human TNF delta and TNF epsilon in the sequences set out in Figures 1A and IB, 2A and 2B, 6A and 6B and 7A and 7B.
• isolated nucleic acid molecules encoding human TNF delta including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
• nucleic acid molecules encoding a mature human TNF delta polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97377 deposited on December 8, 1995 and a mature human TNF epsilon polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97457 deposited on on March 1, 1996, or a human TNF epsilon polypeptide expressed by the human cDNA corresponding to clone HADCA12 (SEQ ID NO:13) contained in the ATCC Deposit No.
• TNF delta polypeptides particularly human TNF delta and TNF epsilon polypeptides, that destroy some transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory response.
• novel polypeptides of human origin referred to herein as TNF delta and TNF epsilon as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
• variants of human TNF delta and TNF epsilon encoded by naturally occurring alleles of the human TNF delta and TNF epsilon gene are variants of human TNF delta and TNF epsilon encoded by naturally occurring alleles of the human TNF delta and TNF epsilon gene.
• TNF delta and TNF epsilon polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously- derived human TNF delta-encoding polynucleotide and TNF epsilon-encoding polynucleotide under conditions for expression of human TNF delta and TNF epsilon in the host and then recovering the expressed polypeptide.
• products, compositions and methods for, among other things: assessing TNF delta and TNF epsilon expression in cells by determining TNF delta and TNF epsilon polypeptides or TNF delta-encoding mRNA or TNF epsilon- encoding mRNA polypeptides; assaying genetic variation and aberrations, such as defects, in TNF delta and TNF epsilon genes; and administering a TNF delta or TNF epsilon polypeptide or polynucleotide to an organism to augment TNF delta or TNF epsilon function or remediate TNF delta or TNF epsilon dysfunction.
• polynucleotides and in particular probes that hybridize to human TNF delta or TNF epsilon sequences are provided.
• antibodies against TNF delta or TNF epsilon polypeptides are provided.
• the antibodies are highly selective for human TNF delta or TNF epsilon.
• TNF delta or TNF epsilon agonists are provided.
• preferred agonists are molecules that mimic TNF delta or TNF epsilon, that bind to TNF delta-binding molecules or receptor molecules or to TNF epsilon-binding molecules or receptor molecules , and that elicit or augment TNF delta-induced or TNF epsilon-induced responses.
• Also among preferred agonists are molecules that interact with TNF delta and TNF epsilon or TNF delta and TNF epsilon polypeptides, or with other modulators of TNF delta activities, and thereby potentiate or augment an effect of TNF delta and TNF epsilon or more than one effect of TNF delta and TNF epsilon.
• TNF delta and TNF epsilon antagonists are provided.
• preferred antagonists are those which mimic TNF delta and TNF epsilon so as to bind to TNF delta and TNF epsilon receptors or binding molecules but not elicit a TNF delta- and TNF epsilon-induced response or more than one TNF delta- and TNF epsilon-induced response.
• agonists and antagonists may be used to mimic, augment or inhibit the action of TNF delta and TNF epsilon polypeptides. They may be used, for instance, to prevent septic shock, inflammation, cerebral malaria, activation of the HIN virus, graft- host rejection, bone resorption, rheumatoid arthritis and cachexia.
• compositions comprising a TNF delta and TNF epsilon polynucleotide or a TNF delta and TNF epsilon polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism.
• the compositions comprise a TNF delta and TNF epsilon polynucleotide for expression of a TNF delta and TNF epsilon polypeptide in a host orgamsm for treatment of disease.
• Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of TNF delta and TNF epsilon.
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
• diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, po
• osteomyelodysplasia e.g., aplastic anemia, etc.
• liver disease e.g., acute and chronic hepatitis, liver injury, and cirrhosis
• autoimmune disease e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.
• cardiomyopathy e.g., dilated cardiomyopathy
• diabetes diabetic complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e.g., wounds, burns, and bone fractures).
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen, anti- viral immune responses.
• polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response.
• polynucleotides and or polypeptides of the invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
• polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders.
• polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or. autoimmune conditions, such as those described herein or are otherwise known in the art.
• FIGS 1A and IB show the nucleotide (SEQ ID NO:l) and deduced amino acid (SEQ ID NO:2) sequence of human TNF delta.
• SEQ ID NO:l nucleotide sequence
• SEQ ID NO:2 deduced amino acid sequence of human TNF delta.
• a TNF family signature sequence is found at positions Val-138 through Phe- 154 in the TNF-delta amino acid sequence shown in Figures 1A and IB. See, Peitsch M.C., et al, Int. Immunol. 5:233-38 (1993); Farrah T., et al, Nature 358:26 (1992); Bazan J.F., Curr. Biol. 3:603-606(1993).
• a potential asparagine-linked glycosylation site is marked in Figures 1 A and IB with a bolded asparagine symbol (N) in the TNF-delta amino acid sequence and a bolded pound sign (#) above the first nucleotide encoding that asparagine residue in the TNF-delta nucleotide sequence.
• N asparagine symbol
• # bolded pound sign
• a potential N-linked glycosylation sequence is found at the following location in the TNF-delta amino acid sequence: N-220 through P-223 (N-220, L-221, S-222, and P-223).
• TNF-beta (an alignment of these sequences is presented in Figure 3) are underlined in Figures 1 A and IB. These regions are not limiting and are labeled as conserveed Domain
• CD- ⁇ , CD- ⁇ , CD-m CD- ⁇ V, CD-V, CD-VI, CD-V ⁇ , CD-V ⁇ , CD-IX, CD-X, and
• Figures 2A and 2B show the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequence of human TNF epsilon.
• a TNF family signature sequence is found at positions Val-73 through Phe-89 in the TNF-epsilon amino acid sequence shown in Figures 2A and 2B. See, Peitsch M.C., et al, Int. Immunol. 5:233-38 (1993); Farrah T., et al, Nature 358:26 (1992); Bazan J.F., Curr. Biol. 3:603-606(1993). The sequence is marked in the Figures with a double underline under the corresponding amino acids.
• TNF-beta (an alignment of these sequences is presented in Figure 3) are underlined in Figures 2A and 2B. These regions are not limiting and are labeled as conserveed Domain (CD)-L CD-ID, CD-JN, CD-V, CD-VI, CD-VH, CD-Nm, CD-IX, CD-X, and CD-XI in Figures 2 A and 2B.
• CD conserveed Domain
• Figure 3 shows the regions of similarity (alignment report) between amino acid sequences of T ⁇ F alpha (SEQ ID ⁇ O:5; GenBank Accession No. Z15026), TNF beta (SEQ ID NO:6; GenBank Accession No. Z15026), TNF delta (SEQ ID NO:l), and TNF epsilon (SEQ ID NO:2), polypeptides.
• Figure 4 shows structural and functional features of TNF delta deduced by the indicated techniques, as a function of amino acid sequence.
• Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, as predicted for the amino acid sequence of SEQ ID NO:2 using the default parameters of the recited computer programs.
• the "Antigenic Index - Jameson- Wolf ' graph the indicated location of the highly antigenic regions of the TNF-delta protein, i.e., regions from which epitope-bearing peptides of the invention may be obtained.
• the data shown in Figure 4 can be easily represented in tabular format similar to the data shown in Table ⁇ . Such a tabular representation of the exact data disclosed in Figure 4 can be generated using the MegAlign component of the DNA* STAR computer sequence analysis package set on default parameters.
• Figure 5 shows structural and functional features of TNF epsilon deduced by the indicated techniques, as a function of amino acid sequence.
• Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, as predicted for the amino acid sequence of SEQ ID NO:4 using the default parameters of the recited computer programs, hi the "Antigenic Index - Jameson- Wolf graph, the indicated location of the highly antigenic regions of the TNF-epsilon protein, i.e., regions from which epitope-bearing peptides of the invention may be obtained.
• the data shown in Figure 5 can be easily represented in tabular format similar to the data shown in Table JJJ. Such a tabular representation of the exact data disclosed in Figure 5 can be generated using the MegAlign component of the DNA* STAR computer sequence analysis package set on default parameters.
• Figures 6A and 6B show the nucleotide and deduced amino acid sequence of a longer open reading frame of human TNF delta obtained from the identical nucleotide sequence shown in Figures 1 A and IB (SEQ ID NOs: 1 and 2).
• the amino acid sequence shown in Figures 6 A and 6B is also shown as SEQ ID NO: 11.
• a TNF family signature sequence is found at positions Nal-155 through Phe-171 in the T ⁇ F-delta amino acid sequence shown in Figures 6A and 6B. See, Peitsch M.C., et al, Int. Immunol. 5:233-38 (1993); Farrah T., et al, Nature 358:26 (1992); Bazan J.F., Curr. Biol. 3:603-606(1993).
• the sequence is marked in the Figures with a double underline under the corresponding amino acids.
• a potential asparagine-linked glycosylation site is marked in Figures 6A and 6B with a bolded asparagine symbol ( ⁇ ) in the T ⁇ F-delta amino acid sequence and a bolded pound sign (#) above the first nucleotide encoding that asparagine residue in the T ⁇ F-delta nucleotide sequence.
• a potential ⁇ -linked glycosylation sequence is found at the following location in the T ⁇ F-delta amino acid sequence: ⁇ -237 through P-240 (N-237, L-238, S-239, and P-240).
• TNF-beta are underlined in Figures 6 A and 6B. These regions are not limiting and are labeled as conserveed Domain (CD)-I, CD-H, CD-DJ, CD-TV, CD-N, CD-NI, CD-N ⁇ , CD-NIJJ, CD-IX, CD-X, and CD-XI in Figures 6A and 6B.
• CD conserveed Domain
• Figures 7 A and 7B show the nucleotide and deduced amino acid sequence of a longer human T ⁇ F epsilon than that shown in Figures 2A and 2B (SEQ ID ⁇ Os:3 and 4).
• the nucleotide and amino acid sequences shown in Figures 7 A and 7B are also shown as SEQ ID NO:12 and SEQ ID NO:13, respectively.
• a TNF family signature sequence is found at positions Val-139 through Phe- 155 in the TNF-epsilon amino acid sequence shown in Figures 7A and 7B. See, Peitsch M.C., et al, Int. Immunol.
• N asparagine symbol
• # bolded pound sign
• the potential N-linked glycosylation sequences are found at the following locations in the TNF-epsilon amino acid sequence: N-113 through D-l 16 (N-113, D-l 14, S-115, and D-l 16) andN-221 through P-224 (N-221, L-222, S-223, and P-224).
• TNF-beta (an alignment of these sequences is presented in Figure 3) are underlined in Figures 2 A and 2B. These regions are not limiting and are labeled as conserveed Domain (CD)-I, CD-m, CD-IV, CD-N, CD-VL CD-N ⁇ , CD-Nm, CD-DC, CD-X, and CD-XI in Figures 2A and 2B.
• CD conserveed Domain
• the following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the examples. The explanations are provided as a convenience and are not limitative of the invention.
• the term "digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
• the various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled artisan.
• 1 microgram of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 microliters of reaction buffer.
• For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 micrograms of DNA are digested with 20 to 250 units of enzyme in proportionately larger volumes.
• the term "genetic element” generally means a polynucleotide comprising a region that encodes a polypeptide or a region that regulates transcription or translation or other processes important to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
• Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within mini-chromosomes, such as those that arise during amplification of transfected DNA by methotrexate selection in eukaryotic cells. Genetic elements also may be comprised within a host cell genome; not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.
• isolated means altered “by the hand of man” from its natural state; i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
• a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
• isolated means that it is separated from the chromosome and cell in which it naturally occurs.
• a nucleic acid molecule contained in a clone that is a member of a mixed clone library e.g., a genomic or cDNA library
• a mixed clone library e.g., a genomic or cDNA library
• a chromosome isolated or removed from a cell or a cell lysate e.g., a "chromosome spread", as in a karyotype
• nucleic acid molecule contained in a preparation of mechanically or enzymatically cleaved genomic DNA is also not “isolated” for the purposes of this invention.
• polynucleotides can be joined to other polynucleotides, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance.
• the isolated polynucleotides, alone or joined to other polynucleotides such as vectors can be introduced into host cells, in culture or in whole organisms, after which such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
• polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
• a composition such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
• ligation refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs. Techniques for ligation are well known to the art and protocols for ligation are described in standard laboratory manuals and references, such as, for instance, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al., pg. 146, as cited below. [065]
• oligonucleotide(s) refers to relatively short polynucleotides.
• RNA:DNA hybrids double-stranded DNAs, among others.
• Oligonucleotides such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
• the 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide. As is well known, this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotide(s) prior to ligation.
• a ligase such as T4 DNA ligase
• Plasmids generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
• Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures.
• Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.
• those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
• polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• polynucleotides as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the strands in such regions may be from the same molecule or from different molecules.
• the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
• One of the molecules of a triple-helical region often is an oligonucleotide.
• polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
• DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
• DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
• polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, ter alia.
• polynucleotide having a nucleotide sequence at least, for example,
• nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding TNF delta or TNF epsilon.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mismatches of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• the reference (query) sequence may be the entire TNF delta-encoding nucleotide sequence shown in Figures 1 A and IB or Figures 6 A and 6B or the entire TNF epsilon-encoding nucleotide sequence shown in Figures 2A and 2B or Figures 7A and 7B or any TNF delta or TNF epsilon polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the TNF delta and TNF epsilon N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
• nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the encoding nucleotide sequence shown in Figures 1 A and IB, Figures 2A and 2B, Figures 6A and 6B or Figures 7A and 7B, or to the nucleotide sequence of the deposited cDNA clones, can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
• Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482- 489 (1981), to find the best segment of homology between two sequences.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• a sequence of the present invention and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
• 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. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
• This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
• This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. 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 FASTDB 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 FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
• 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, hi this case the percent identity calculated by FASTDB 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 made for the purposes of this embodiment.
• the present application is directed to nucleic acid molecules at least 90%
• nucleic acid sequences disclosed herein irrespective of whether they encode a polypeptide having TNF delta and/or TNF epsilon functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TNF delta and/or TNF epsilon functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having TNF delta and/or TNF epsilon functional activity include, inter alia, (1) isolating a TNF delta and/or TNF epsilon gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TNF delta and TNF epsilon genes, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TNF delta and TNF epsilon mRNA expression in specific tissues.
• FISH in situ hybridization
• nucleic acid molecules having sequences at least
• a polypeptide having TNF delta and/or TNF epsilon functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the TNF delta and/or TNF epsilon polypeptides of the present invention as measured, for example, in a particular immunoassay or biological assay.
• TNF delta and/or TNF-epsilon polypeptide functional activity can be measured by the ability of a polypeptide sequence described herein to form multimers (e.g., homodimers and homotrimers) with the complete TNF delta and/or TNF-epsilon or extracellular domain of TNF delta and/or TNF-epsilon , and to bind a TNF delta and/or TNF-epsilon receptor.
• multimers e.g., homodimers and homotrimers
• TNF delta and/or TNF-epsilon polypeptide functional activity can be measured by the ability of a polypeptide sequence described herein to form heteromultimers with Neutrokine-alpha and/or Neutrokine-alphaS V fragments or variants (e.g., International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS: 23 and 24 respectively), especially the extracellular soluble domain of Neutrokine-alpha and/or Neutrokine-alphaSV (e.g., amino acids 134-285 of SEQ ID NO: 23).
• TNF delta and/or TNF-epsilon polypeptide functional activity can be also be measured by determining the ability of a polypeptide of the invention to induce lymphocyte (e.g., B cell) proliferation, differentiation or activation and/or to extend B cell survival. These functional assays can be routinely performed using techniques described herein (e.g., see Example 6) and otherwise known in the art. Additionally, TNF delta and/or TNF-epsilon polypeptides of the present invention modulate cell proliferation, cytotoxicity, cell survival and cell death.
• lymphocyte e.g., B cell
• TNF delta and/or TNF-epsilon polypeptides of the present invention modulate cell proliferation, cytotoxicity, cell survival and cell death.
• an in vitro cell proliferation, cytotoxicity, cell survival, and cell death assay for measuring the effect of a protein on certain cells can be performed by using reagents well known and commonly available in the art for detecting cell replication and/or death.
• numerous such assays for TNF-related protein activities are described in the various references in this disclosure. Briefly, an example of such an assay involves collecting human or animal (e.g., mouse) cells and mixing with (1) transfected host cell-supernatant containing TNF delta and/or TNF epsilon protein (or a candidate polypeptide) or (2) nontransfected host cell-supernatant control, and measuring the effect on cell numbers or viability after incubation of certain period of time.
• Such cell proliferation and/or survival modulation activities as can be measured in this type of assay are useful for treating tumor, tumor metastasis, infections, autoimmune diseases, inflammation and other immune-related diseases.
• nucleic acid sequences having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA, the nucleic acid sequence shown in Figures 2A and 2B and 7A and 7B (SEQ ID NOs:3 and 12, respectively), or fragments thereof, will encode polypeptides "having TNF- epsilon functional activity.”
• polypeptides includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds.
• polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
• polypeptides are not always entirely linear.
• polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event, and events brought about by human manipulation which do not occur naturally.
• Circular, branched and branched circular polypeptides may be synthesized by non-translational natural process and by entirely synthetic methods, as well.
• Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
• blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well.
• the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing almost invariably will be N-formylmethionine.
• polypeptides made by expressing a cloned gene in a host for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell.
• Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
• variant(s) of polynucleotides or polypeptides are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail.
• a polynucleotide variant is a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. As noted below, changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference.
• a polypeptide variant is a polypeptide that differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and the variant are closely similar overall and, in many region, identical.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
• polypeptide having an amino acid sequence at least, for example, 95%
• identical to a reference amino acid sequence of a TNF delta or a TNF epsilon polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of a respective TNF delta or TNF epsilon sequence.
• up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
• 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.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
• the identity between a reference (query) sequence is set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
• a sequence of the present invention and a subject sequence are determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
• 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.
• a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. 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. 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 FASTDB 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 FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
• 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.
• receptor molecule refers to molecules which bind or interact specifically with TNF delta or TNF epsilon polypeptides of the present invention, including not only classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which also maybe referred to as “binding molecules” and “interaction molecules,” respectively and as “TNF delta binding molecules” and “TNF delta interaction molecules” or “TNF epsilon binding molecules” and “TNF epsilon interaction molecules.” Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules maybe exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it may be specific to several groups of proteins at least one of which includes polypeptides of the invention.
• the present invention relates to novel TNF delta and TNF epsilon polypeptides and polynucleotides, among other things, as described in greater detail below.
• the invention relates to polypeptides and polynucleotides which are related by amino acid sequence homology to the TNF ligand superfamily.
• the invention relates especially to TNF delta having the nucleotide and amino acid sequences set out in Figures 1A and IB, Figures 6A and 6B, and to the TNF nucleotide and amino acid sequences of the human cDNA in ATCC Deposit No. 97377.
• the invention also relates especially to TNF epsilon having the nucleotide and amino acid sequences set out in Figures 2A and 2B, Figures 7A and 7B, and to the TNF epsilon nucleotide and amino acid sequences of the human cDNA in ATCC Deposit No. 97457 or ATCC Deposit No. PTA-1543.
• the deposits are hereinafter referred to as the deposited clones or as "the cDNA of the deposited clones.” It will be appreciated that the nucleotide and amino acid sequences set out in Figures 1 A and IB, Figures 2A and 2B, Figures 6A and 6B and Figure 7A and 7B were obtained by sequencing the human cDNA of the deposited clones.
• the sequence of the deposited clone is controlling as to any discrepancies between the two and any reference to the sequences of Figures 1 A and IB, Figures 2A and 2B, Figures 6A and 6B and/or Figures 7A and 7B include reference to the sequences of the human cDNA's of the deposited clones.
• isolated polynucleotides which encode the TNF delta polynucleotides having the deduced amino acid sequences of Figures 1 A , IB, 6 A, and 6B and TNF epsilon polypeptides having the deduced amino acid sequences of Figures 2A, 2B, 7A and 7B.
• a polynucleotide of the present invention encoding human TNF delta polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA from cells of human tissue as starting material.
• standard cloning and screening procedures such as those for cloning cDNAs using mRNA from cells of human tissue as starting material.
• the polynucleotide set out in Figures 1A, IB, 6A, and 6B was discovered in a cDNA library derived from cells of human heart tissue.
• Human TNF delta of the invention is structurally related to other proteins of the TNF ligand superfamily, as shown by the results of sequencing the cDNA encoding human TNF delta in the deposited clone.
• the cDNA sequence thus obtained is set out in Figures 1 A and IB and 6A and 6B. In one embodiment, it contains an open reading frame encoding a protein of about 233 amino acid residues with a deduced molecular weight of about 25.871 kDa ( Figures 1 A and IB, SEQ ID NO:2).
• it contains an open reading frame encoding a protein of about 250 amino acid residues with a deduced molecular weight of about 27.531 kDa ( Figures 6A and 6B, SEQ ID NO: 11).
• the protein exhibits greatest homology to TNF-alpha, among known proteins.
• the entire amino acid sequence of TNF delta of Figures 1A and IB has about 38% identity to the amino acid sequence of TNF-alpha.
• a polynucleotide of the present invention encoding human TNF epsilon polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA from cells of human tissue as starting material. Illustrative of the invention, the polynucleotide set out in Figures 2 A and 2B was discovered in a cDNA library derived from cells of human heart tissue.
• Human TNF epsilon of the invention is structurally related to other proteins of the TNF ligand superfamily, as shown by the results of sequencing the cDNA encoding human TNF epsilon in the deposited clone.
• the cDNA sequence thus obtained is set out in Figures 2 A and 2B.
• the TNF epsilon sequence is nearly identical to the sequence of TNF delta as set out in Figures 1 A and IB minus the initial 50 amino acids of SEQ ID NO:2, or initial 67 amino acids of SEQ ID NO:l 1 and a region of TNF delta comprising amino acid 96-112 of SEQ ID NO:2 (or amino acids 113-129 of SEQ JO NO:l 1).
• TNF epsilon is a splicing variant of TNF delta
• TNF epsilon comprises 168 amino acid residues and the sequence of Figures 2 A and 2B show a protein of TNF epsilon without any N-terminal hydrophobic region.
• TNF epsilon comprises 234 amino acid residues and the sequence of Figures 7A and 7B show the full-length protein of TNF epsilon. The protein exhibits greatest homology to TNF-alpha.
• TNF epsilon of Figures 2 A and 2B has about 20% identity to the amino acid sequence of TNF-alpha
• HADCA12 Another clone (HADCA12) containing cDNA sequence overlapping the coding region of SEQ ID NO: 3 and which contains the complete amino terminus of TNF- epsilon has been identified.
• the polynucleotide sequence contained in HADCA12 is shown in Figures 7 A and 7B and in SEQ ID NO: 12 along with the deduced amino acid sequences of the TNF epsilon protein encoded by this polynucleotide (SEQ ID NO: 13).
• Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• the coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown in Figures 1A and IB, 2A and 2B, 6A and 6B, and 7A and 7B. It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptide of the DNA of Figures Figures 1A and IB, 2A and 2B, 6A and 6B, and 7A and 7B.
• Polynucleotides of the present invention which encode the polypeptide of
• Figures Figures 1 A and IB, 2A and 2B, 6A and 6B, and/or 7A and 7B may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
• the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• the HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al, Cell, 37:767 (1984), for instance.
• TNF-delta and/or TNF-epsilon polynucleotides of the invention are fused to a polynucleotide encoding a "FLAG" polypeptide.
• a TNF-delta-FLAG or a TNF-epsilon-FLAG fusion protein is encompassed by the present invention.
• the FLAG antigenic polypeptide may be fused to a TNF-delta or TNF-epsilon polypeptide of the invention at either or both the amino or the carboxy terminus.
• a TNF-delta-FLAG or a TNF-epsilon-FLAG fusion protein is expressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector (available from Sigma, St. Louis, MO, USA). See, Andersson, S., et al, J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al, Proc. Natl. Acad. Sci.
• a TNF-delta-FLAG or a TNF-epsilon-FLAG fusion protein is detectable by anti-FLAG monoclonal antibodies (also available from Sigma). See, e.g., Example 29.
• the TNF delta and/or TNF epsilon polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers).
• the TNF delta and TNF epsilon polypeptides of the invention are trimers.
• the present invention relates to monomers and multimers of the TNF delta and/or TNF epsilon proteins of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
• the polypeptides of the invention are monomers, dimers, trimers or tetramers.
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers.
• the term homomer refers to a multimer containing only TNF delta proteins of the invention (including TNF delta fragments, variants, and fusion proteins, as described herein). These homomers may contain TNF delta proteins having identical or different polypeptide sequences. As used herein, the term homomer, may alternatively refer to a multimer containing only TNF epsilon proteins of the invention (including TNF epsilon fragments, variants, and fusion proteins, as described herein). These homomers may contain TNF epsilon proteins having identical or different polypeptide sequences.
• a homomer of the invention is a multimer containing only TNF delta proteins having an identical polypeptide sequence
• a homomer of the invention is a multimer containing TNF delta proteins having different polypeptide sequences.
• a homomer of the invention is a multimer containing only TNF epsilon proteins having an identical polypeptide sequence.
• a homomer of the invention is a multimer containing TNF epsilon proteins having different polypeptide sequences.
• the multimer of the invention is a homodimer
• the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• heteromer refers to a multimer containing TNF delta proteins of the invention and heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequence encoded by the TNF delta gene or TNF-epsilon gene).
• heteromer refers to a multimer containing TNF epsilon proteins of the invention and heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequence encoded by the TNF delta gene or TNF-epsilon gene), hi a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• the heteromeric multimer of the invention is a heterotrimer comprising both TNF-delta polypeptides and Neutrokine-alpha and/or Neutokine-alphaSV polypeptides (e.g., International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24 respectively).
• the heteromeric multimer of the invention is a heterotrimer consisting of one TNF-delta polypeptide and two Neutrokine-alpha and/or Neutokine-alphaSV polypeptides.
• the heteromeric multimer of the invention is a heterotrimer consisting of two T ⁇ F-delta polypeptides and one ⁇ eutrokine-alpha and/or Neutokine-alphaSV polypeptide.
• the heteromeric multimer of the invention is a heterotrimer comprising T ⁇ F-delta and/or T ⁇ F-epsilon polypeptides and ⁇ eutrokine-alpha and/or Neutokine-alphaSV polypeptides (e.g., International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24 respectively).
• the heteromeric multimer of the invention is a heterotrimer consisting of one TNF-epsilon polypeptide and two Neutrokine-alpha and/or Neutokine-alphaSV polypeptides.
• the heteromeric multimer of the invention is a heterotrimer consisting of two TNF-epsilon polypeptides and one Neutrokine-alpha and/or Neutokine-alphaSV polypeptide.
• the heteromeric multimer of the invention is a heterotrimer comprising TNF-delta and/or TNF epsilon polypeptides and Neutrokine-alpha and/or Neutokine-alphaSV polypeptides (e.g., International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24, respectively).
• Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers of the invention such as, for example, homodimers or homotrimers
• multimers of the invention are formed when proteins of the invention contact one another in solution.
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
• multimers of the invention are formed by covalent associations with and/or between the TNF delta and/or TNF epsilon proteins of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 11, and.or SEQ ID NO: 13 or the polypeptides encoded by the deposited cDNA clones).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TNF delta and/or TNF epsilon fusion protein.
• covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
• the covalent associations are between the heterologous sequences contained in a TNF delta-Fc fusion protein of the invention (as described herein) or between the heterologous sequences contained in a TNF epsilon-Fc fusion protein of the invention (as described herein).
• covalent associations of fusion proteins of the invention are between the heterologous sequences contained in a TNF delta-Fc fusion protein of the invention and the heterologous sequences contained in a TNF epsilon-Fc fusion protein of the invention.
• covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
• the multimers of the invention may be generated using chemical techniques known in the art.
• proteins desired to be contained in the multimers of the invention maybe chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C- or N-termini of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers of the invention may be generated using genetic engineering techniques known in the art.
• proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N- terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polynucleotide encoding a polypeptide encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly the human TNF delta having the amino acid sequence set out in Figures 1 A and IB and/or Figures 6 A and 6B and TNF epsilon having the amino acid sequences set out in Figures 2A and 2B, and/or Figures 7A and 7B.
• the term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by introns) together with additional regions, that also may contain coding and/or non-coding sequences.
• the present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figures 1A and IB, 2A and 2B, 6 A and 6B, and/or 7 A and 7B.
• the present invention encompasses polynucleotides encoding the extracellular soluble form of the TNF delta and TNF epsilon proteins, (e.g., a polynucleotide encoding amino acid residues 88-233 of SEQ ID NO:2, a polynucleotide encoding amino acid residues 105-250 of SEQ ID NO:l 1, a polynucleotide encoding amino acid residues 39-168 of SEQ ID NO:4, and or a polynucleotide encoding amino acid residues 105-234 of SEQ ID NO: 13.
• polynucleotides encoding the extracellular soluble form of the TNF delta and TNF epsilon proteins e.g., a polynucleotide encoding amino acid residues 88-233 of SEQ ID NO:2, a polynucleotide encoding amino acid residues 105-250 of SEQ ID NO:l 1, a polynucleo
• a variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
• Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
• variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides.
• the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
• TNF delta and TNF epsilon which have the amino acid sequence of the TNF delta having the amino acid sequence set out in Figures 1 A and IB and/or Figures 6A and 6B and TNF epsilon having the amino acid sequences set out in Figures 2A and 2B, and/or Figures 7A and 7B in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
• silent substitutions, additions and deletions which do not alter the properties and activities of the TNF delta and TNF epsilon. Also especially preferred in this regard are conservative substitutions.
• polynucleotides encoding polypeptides having the amino acid sequence of Figures 1A and IB, 2A and 2B, 6A and 6B, and/or 7 A and 7B without substitutions.
• Further preferred embodiments of the invention are polynucleotides that-are at least 70% identical to a polynucleotide encoding the TNF delta polypeptide having the amino acid sequence set out in Figures 1 A and IB and/or Figures 6A and 6B and the TNF epsilon polypeptide having the amino acid sequences set out in Figures 2A and 2B, and/or Figures 7A and 7B, and polynucleotides which are complementary to such polynucleotides.
• polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding the TNF delta and TNF epsilon polypeptide and polynucleotides complementary thereto, hi this regard, polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
• Particularly preferred embodiments in this respect are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figures 1 A and IB, 2 A and 2B, and/or 7 A and 7B.
• the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
• the present invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
• stringent conditions means hybridization will occur when at least 95% and preferably at least 97% of the bases between sequences are complementary (e.g., G:C; A:T).
• stringent conditions means hybridization according to any stringent conditions recited herein.
• polynucleotide assays of the invention may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding TNF delta and TNF epsilon and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the human TNF delta and TNF epsilon gene.
• Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases.
• the coding region of the TNF delta and TNF epsilon gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
• the polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia.
• the polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
• Such sequences may play a role in processing of a protein from precursor to a mature form, may facilitate protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
• the additional amino acids may be processed away from the mature protein by cellular enzymes.
• a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
• inactive precursors When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
• a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
• a leader sequence which may be referred to as a preprotein
• a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
• the cDNA deposit is referred to herein as "the deposited clone” or as "the cDNA of the deposited clone.”
• the clones were deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, USA, on December 8, 1995, March 1, 1996, and March 21, 2000 and assigned ATCC Deposit No. 97377 97457, and PTA-1543, respectively.
• the deposited materials in Deposit Nos. 97377 ans 97457 are pBluescript SK (-) plasmids (Stratagene, La Jolla, CA) that contain TNF epsilon human cDNA and the full length TNF delta cDNA, as described above.
• the present invention further relates to human TNF delta and TNF epsilon polypeptides having the deduced amino acid sequences of Figures 1A and IB, 2A and 2B, 6A and 6B, and/or 7A and 7B.
• the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments, it is a recombinant polypeptide.
• the invention also relates to fragments, analogs and derivatives of these polypeptides.
• fragment when referring to the polypeptide of Figures 1A and IB, 2A and 2B, 6A and 6B, and/or 7A and 7B means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
• an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
• the invention further includes variations of the TNF-delta polypeptide which show substantial TNF-delta polypeptide functional activity (e.g., biological activity) or which include regions of TNF-delta polypeptide such as the protein portions discussed below.
• the invention also includes variations of the TNF-epsilon polypeptide which show substantial TNF-epsilon polypeptide functional activity (e.g., biological activity) or which include regions of TNF-epsilon polypeptide such as the polypeptide portions discussed below.
• Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity.
• 2 A and 2B, 6A and 6B, and/or 7A and 7B may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or a leader, human serum albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S.
• Patent No. 5,876,969 issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety
• a secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
• Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
• the TNF-delta and/or TNF-epsilon polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table I).
• particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of TNF delta and TNF epsilon set out in Figures Figures 1 A and IB, 2A and 2B, 6A and 6B, and/or 7A and 7B, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
• particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of the TNF delta and TNF epsilon of the human cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
• substitutions are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
• variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments having the amino acid sequence of the TNF delta as set out in Figures 1 A and IB and/or Figures 6A and 6B, and/or TNF epsilon as set out in Figures 2A and 2B, and/or Figures 7A and 7B, or of the cDNA in the deposited clones, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
• the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
• the TNF delta polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide, e.g., amino acid residues 88 - 233 of SEQ ID NO:2) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95%o identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
• the TNF delta polypeptides of the present invention include the polypeptide of SEQ ID NO: 11 (in particular the mature polypeptide, e.g., amino acid residues 105 - 250 of SEQ ID NO:l 1) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of.
• SEQ ID NO: 11 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 11 and still more preferably at least 95% similarity (still more preferably at least 95%) identity) to the polypeptide of SEQ ID NO: 11 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
• the TNF epsilon polypeptides of the present invention include the polypeptide of SEQ ID NO:4 (in particular the mature polypeptide, e.g., amino acid residues 39 - 168 of SEQ ID NO:4) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:4 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:4 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:4 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
• the TNF epsilon polypeptides of the present invention include the polypeptide of SEQ JJD NO: 13 (in particular the mature polypeptide, e.g., amino acid residues 105 - 234 of SEQ ED NO: 13) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO: 13 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 13 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO: 13 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
• Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
• a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned TNF delta and TNF epsilon polypeptides and variants or derivatives thereof.
• fragments may be "free-standing,” i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
• the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide.
• certain preferred embodiments relate to a fragment of a TNF delta and TNF epsilon polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the amino terminus of the TNF delta and TNF epsilon fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from TNF delta and TNF epsilon. [0148] As representative examples of polypeptide fragments of the invention, there may be mentioned those which have from about 30 to about 233 amino acids.
• about includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
• about 100 to 233 amino acids in this context means a polypeptide fragment of 100 plus or minus several, a few, 5, 4, 3, 2 or 1 amino acids to 233 plus or minus several a few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 100 minus several amino acids to 233 plus several amino acids to as narrow as 100 plus several amino acids to 233 minus several amino acids.
• nucleotide sequence of the deposited cDNA a nucleotide sequence encoding the polypeptide sequence encoded by the deposited cDNA
• a nucleotide sequence encoding the polypeptide sequence depicted in Figures 1A and IB SEQ ED NO:2 and/or Figures 6A and 6B (SEQ ID NO:l 1)
• the nucleotide sequence shown in Figures 1A and IB (SEQ ID NO:l) is intended fragments at least 15 nt, and more preferably at least about 20 nt, still more preferably at least 30 nt, and even more preferably, at least about 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length.
• nucleotide sequence shown in Figures 7A and 7B is intended fragments at least 15 nt, and more preferably at least about 20 nt, still more preferably at least 30 nt, and
• fragments have numerous uses that include, but are not limited to, diagnostic probes and primers as discussed herein.
• larger fragments such as those of 501-1500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs (clones HLTBT71, HPDDO12, and/or HADCA12) or as shown in Figures 1 A and IB (SEQ ID NO:l), 2A and 2B (SEQ ID NO:3) 7A and 7B (SEQ ED NO: 12), respectively.
• fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from, for example, the nucleotide sequence of the deposited cDNAs, or the nucleotide sequences as shown in Figures 1 A and IB (SEQ ID NO:l), 2A and 2B (SEQ ID NO:3) ,or 7A and 7B (SEQ ID NO: 12), respectively.
• a fragment at least about 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence, hi this context "about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
• TNF-delta polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 282 to 315, 316 to 348, 349 to 380, 381 to 410, 411 to 440, 441 to 470, 471 to 500, 501 to 530, 531 to 560, 561 to 590, 591 to 620, 621 to 650, 651 to 680, 681 to 710, 711 to 740, 741 to 770, 771 to 800, 801 to 830, 831 to 860, 861 to 890, 891 to 920, 921 to 950, 951 to 980, 981 to 1010, 1011 to 1040 of SEQ ID NO:l, or the complementary strand thereto, or the cDNA contained in the deposited clone.
• “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or
• TNF-epsilon polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 2 to 31, 32 to 61, 62 to 91, 92 to 121, 122 to 151, 152 to 181, 182 to 211, 212 to 241, 242 to 271, 272 to 301, 302 to 331, 332 to 361, 362 to 391, 392 to 421, 422 to 451, 452 to 481, 482 to 511, 512 to 541, 542 to 571 of SEQ ID NO:3, or the complementary strand thereto, or the cDNA contained in the deposited clone, hi this context "about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
• TNF-epsilon polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 108 to 139, 140 to 170, 171 to 201, 202 to 232, 233 to 263, 264 to 294, 295 to 325, 326 to 356, 357 to 381, 382 to 419, 420 to 452, 453 to 485, 486 to 517, 518 to 550, 551 to 582, 583 to 615, 616 to 648, 649 to 680, 681 to 713, 714 to 746, 747 to 778, 779 to 809 of SEQ ID NO: 12, or the complementary strand thereto, or the cDNA contained in the deposited clone.
• “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
• the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TNF-delta and/or TNF-epsilon functional activity.
• a polypeptide demonstrating a TNF-delta and/or TNF-epsilon "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TNF-delta and/or TNF-epsilon protein.
• Such functional activities include, but are not limited to, biological activity (e.g., cell proliferation, differentiation, and/or growth), antigenicity, ability to bind (or compete with a TNF-delta and or TNF-epsilon polypeptide for binding) to an anti-TNF-delta and/or TNF-epsilon antibody, immunogenicity (ability to generate/induce antibody which binds to a TNF-delta and/or TNF-epsilon polypeptide), ability to form multimers with TNF-delta and/or TNF- epsilon polypeptides of the invention, ability to form heteromultimers with Neutrokine alpha-and/or Neutrokine-alphaSV polypeptides (e.g., International Publication No.
• biological activity e.g., cell proliferation, differentiation, and/or growth
• antigenicity ability to bind (or compete with a TNF-delta and or TNF-epsilon polypeptide for binding) to an
• WO 98/18921 Science. 285:260 (1999); SEQ ID NOS 23 and 24, respectively); and ability to bind to a receptor or ligand for a TNF-delta and/or TNF-epsilon polypeptide (e.g., TACI (See, von Bulow, G. U. and Bram, R.J., Science 278:138-41 (1997) and GenBank accesion number AAC51790, and or BCMA (GenBank accession number NP_001183)), and/or TR11, and/or TR11SV1, and/or TR11SV2 (See, International Publication No. WO99/22085).
• TACI See, von Bulow, G. U. and Bram, R.J., Science 278:138-41 (1997) and GenBank accesion number AAC51790, and or BCMA (GenBank accession number NP_001183)
• TR11, and/or TR11SV1, and/or TR11SV2 See, International Publication No. WO
• TNF-delta and/or TNF-epsilon polypeptides can be assayed by various methods.
• various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agg
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody, hi a further embodiment, the secondary antibody is labeled.
• Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• truncation mutants of TNF delta and TNF epsilon are truncation mutants of TNF delta and TNF epsilon.
• Truncation mutants include TNF delta and TNF epsilon polypeptides having the amino acid sequence of Figures 1 A and IB and 2 A and 2B, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally.
• fragments characterized by structural or functional attributes of TNF delta and TNF epsilon are fragments characterized by structural or functional attributes of TNF delta and TNF epsilon.
• Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta- regions”), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TNF delta and TNF epsilon.
• Certain preferred regions in these regards are set out in Figure 4 for TNF delta and Figure 5 for TNF epsilon, and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 1 A and IB and 2A and 2B.
• such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions and coil-regions, Chou-Fasman alpha-regions, beta-regions and turn-regions, Kyte-Doolittle hydrophilic regions and hydrophilic regions, Eisenberg alpha and beta amphipathic regions, Karpms-Schulz flexible regions, Emini surface-forming regions and Jameson- Wolf high antigenic index regions.
• fragments in this regard are those that comprise regions of TNF delta and TNF epsilon that combine several structural features, such as several of the features set out above.
• the regions defined by the residues following the signal peptide region of Figures 1A, IB, 2A, 2B, 4 and 5, which all are characterized by amino acid compositions highly characteristic of turn-regions, hydrophilic regions, flexible-regions, surface-forming regions, and high antigenic index-regions are especially highly preferred regions.
• Such regions may be comprised within a larger polypeptide or may be by themselves a preferred fragment of the present invention, as discussed above. It will be appreciated that the term "about” as used in this paragraph has the meaning set out above regarding fragments in general.
• the polynucleotides of the invention encode functional attributes of TNF delta and/or TNF epsilon.
• Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TNF delta and/or TNF epsilon.
• Regions of high antigenicity are determined from the data presented in columns Vm, LX, Xm, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
• Tables II and HI include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 4 and 5.
• regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 4 and 5.
• such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson- Wolf regions of high antigenic index.
• Val 89 A A 1.24 -0.29 . 0.30 0.69
• Trp 28 A A 0.58 -0.09 * 0.64 0.97
• Val 40 A B 1.24 -0.29 0.30 0.69
• Trp 56 A B -0.17 0.50 * * -0.60 0.48 Table in (continued)
• fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson- Wolf program) of complete (i.e., full-length) TNF delta (SEQ JO NO:2) and/or TNF epsilon (SEQ ID NO:4).
• Certain preferred regions are those set out in Figures 4 and 5 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in Figures 1 A and IB (SEQ ID NO:2), 2A and 2B (SEQ ID NO:4), 6A and 6B (SEQ JD NO:l 1) and 7A and 7B (SEQ JD NO: 13), such preferred regions include; Garnier- Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson- Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.
• polypeptides are also encompassed by the invention.
• highly preferred fragments in this regard are those that comprise regions of TNF delta and/or TNF epsilon that combine several structural features, such as several of the features set out above.
• polynucleotides of the invention are less than
• polynucleotides of the invention comprise at least
• polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TNF delta or TNF epsilon coding sequence, but do not comprise all or a portion of any TNF delta or TNF epsilon intron.
• the nucleic acid comprising TNF delta or TNF epsilon coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TNF delta or TNF epsilon gene in the genome), hi other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s). [0172] Further preferred regions are those that mediate activities of TNF delta and
• TNF epsilon Most highly preferred in this regard are fragments that have a chemical, biological or other activity of TNF delta and TNF epsilon, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Highly preferred in this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides, such as the related polypeptides set out in Figure 3, including human TNF-alpha and beta. Among particularly preferred fragments in these regards are truncation mutants, as discussed above.
• the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize under stringent conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode the fragments.
• preferred polynucleotides are those that correspond to the preferred fragments, as discussed above.
• the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
• Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention.
• polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation.
• the polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
• polynucleotides of the invention maybe transfected into host cells with another, separate, polynucleotide encoding a selectable marker, using standard techniques for co-transfection and selection in, for instance, mammalian cells, i this case the polynucleotides generally will be stably incorporated into the host cell genome.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• the vector construct may be introduced into host cells by the aforementioned techniques.
• a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
• Electroporation also may be used to introduce polynucleotides into a host. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells.
• a wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length in Sambrook et al. cited above, which is illustrative of the many laboratory manuals that detail these techniques.
• the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
• Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
• the vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction.
• Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
• vectors are those for expression of polynucleotides and polypeptides of the present invention.
• such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
• Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
• the vectors provide for specific expression.
• Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific.
• Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
• a variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
• the engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.
• a great variety of expression vectors can be used to express a polypeptide of the invention.
• Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SN40, vaccinia viruses, adenovirases, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
• any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
• the appropriate D ⁇ A sequence may be inserted into the vector by any of a variety of well-known and routine techniques, i general, a D ⁇ A sequence for expression is joined to an expression vector by cleaving the D ⁇ A sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 D ⁇ A ligase.
• Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein.
• the D ⁇ A sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mR ⁇ A transcription.
• appropriate expression control sequence(s) including, for instance, a promoter to direct mR ⁇ A transcription.
• promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SN40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein.
• expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
• constructs may contain control regions that regulate as well as engender expression.
• control regions that regulate as well as engender expression.
• such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
• Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
• the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
• Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.
• the vector containing the appropriate D ⁇ A sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide.
• appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
• the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above.
• the constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted.
• the sequence may be inserted in a forward or reverse orientation.
• the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.
• vectors which are commercially available, are provided by way of example.
• vectors preferred for use in bacteria are pQE70, pQE60 and pQE- 9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, ⁇ NH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233- 3, pDR540, pRIT5 available from Pharmacia.
• eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl andpSG available from Stratagene; andpSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the-present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., TNF-delta and/or TNF-epsilon coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TNF-delta and/or TNF-epsilon polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous TNF-delta and/or TNF- epsilon polynucleotides.
• endogenous genetic material e.g., TNF-delta and/or TNF-epsilon coding sequence
• genetic material e.g., heterologous polynucleotide sequences
• heterologous control regions e.g., promoter and/or enhancer
• endogenous TNF-delta and/or TNF-epsilon polynucleotide sequences via homologous recombination
• heterologous control regions e.g., promoter and/or enhancer
• endogenous TNF-delta and/or TNF-epsilon polynucleotide sequences via homologous recombination
• Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter.
• a reporter transcription unit lacking a promoter region such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter.
• CAT activity which can be detected by standard CAT assays.
• Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7.
• promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter
• bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.
• eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.
• RSV Rous sarcoma virus
• metallothionein promoters such as the mouse metallothionein-I promoter.
• polynucleotides encoding TNF-delta and/or TNF- epsilon polypeptides of the invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell.
• signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell.
• E. coli one may wish to direct the expression of the protein to the periplasmic space.
• Examples of signal sequences or proteins (or fragments thereof) to which the polypeptides of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase.
• MBP maltose binding protein
• ompA the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit
• alkaline phosphatase Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs.
• polynucleotides encoding TNF delta or TNF epsilon polypeptides of the invention maybe fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
• Examples of signal peptides that may be fused to a polypeptide of the invention in order to direct its secretion in mammalian cells include, but are not limited to, the MPIF-1 signal sequence (amino acids 1-21 of GenBank Accession number AAB51134), the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:23), and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:24).
• a suitable signal sequence that may be used in conjunction with baculoviral expression systems is the gp67 signal sequence, (amino acids 1-19 of GenBank Accession Number AAA72759).
• the present invention also relates to host cells containing the above- described constructs discussed above.
• the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
• constructs in the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. Basic Methods in Molecular Biology, (1986).
• Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
• Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.
• RNAs derived from the DNA constructs of the present invention can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
• Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
• recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
• promoters are those derived from the genes that encode glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK”), a-factor, acid phosphatase, and heat shock proteins, among others.
• PPG 3-phosphoglycerate kinase
• Selectable markers include the ampicillin resistance gene of E. coli and the trpl gene of S. cerevisiae.
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
• enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression.
• the polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site.
• the ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
• initiation codon usually AUG
• translation stop codon at the end of the polypeptide
• polyadenylation signal and a transcription termination signal appropriately disposed at the 3' end of the transcribed region.
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
• a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
• region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
• the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
• Suitable prokaryotic hosts for propagation, maintenance or expression of polynucleotides and polypeptides in accordance with the invention include Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various species of Pseudomonas, Streptomyces, and Staphylococcus are suitable hosts in this regard. Moreover, many other hosts also known to those of skill maybe employed in this regard.
• useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
• Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.
• mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al, Cell, 23:175 (1981).
• Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.
• Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expression.
• DNA sequences derived from the SV40 splice sites, and the S V40 polyadenylation sites are used for required non-transcribed genetic elements of these types.
• polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
• HPLC high performance liquid chromatography
• Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
• N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TNF delta ligand (e.g., TACI and/or BCMA and/or TR11, and/or TR11SV1, and/or TR11SV2)) may still be retained.
• TNF delta ligand e.g., TACI and/or BCMA and/or TR11, and/or TR11SV1, and/or TR11SV2
• the ability of shortened TNF delta muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
• Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TNF delta mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TNF delta amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TNF delta amino acid sequence shown in Figures 1A and IB (i.e., SEQ ID NO:2), up to the Leucine residue at position number 228 and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising the amino acid sequence of residues n 1 -233 of Figures 1 A and IB (SEQ ID NO:2), where n 1 is an integer from 2 to 228 corresponding to the position of the amino acid residue in Figures 1 A and IB (SEQ ID NO:2).
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following amino acid sequences: G-2 to L-233; G-3 to L-233; P-4 to L-233; V-5 to L-233; R-6 to L-233; E-7 to L-233; P-8 to L-233; A-9 to L-233; L-10 to L-233; S-ll to L- 233; V-12 to L-233; A-13 to L-233; L-14 to L-233; W-15 to L-233; L-16 to L-233; S-17 to L-233; W-18 to L-233; G-19 to L-233; A-20 to L-233; A-21 to L-233; L-22 to L-233; G-23 to L-233; A-24 to L-233; V-25 to L-233; A-26 to L-233; C-27 to L-233; A-28 to L-233; M- 29 to L-233; A-30 to L
• Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the TNF-delta polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the mature form of TNF delta comprises, or alternatively consists of Ala-88 to Leu-233 of the TNF delta sequence shown in Figures 1A and IB (SEQ JD NO:2).
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mature TNF-delta polypeptides described above.
• the present invention also encompasses polynucleotides encoding the above polypeptide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the mature form of TNF delta comprises, or alternatively consists of Ala-105 to Leu-250 of the TNF delta sequence shown in Figures 6A and 6B (SEQ ID NO:l 1).
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mature TNF-delta polypeptides described above.
• the present invention also encompasses polynucleotides encoding the above polypeptide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides - comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TNF delta mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TNF delta amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TNF delta polypeptide shown in Figures 1 A and IB (SEQ ID NO:2), up to the arginine residue at position number 6, and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 1 of Figures 1 A and IB (i.e., SEQ ID NO:2), where m 1 is an integer from 6 to 232 corresponding to the position of the amino acid residue in Figures 1 A and IB (SEQ ID NO:2).
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following amino acid sequences: M-1 to K-232; M-1 to V-231; M-1 to F-230; M-1 to G-229; M-1 to L-228; M-1 to F-227; M-1 to T-226; M-1 to G-225; M-1 to H-224; M-1 to P-223; M-1 to S-222; M-1 to L-221; M-1 to N-220; M-1 to L-219; M-1 to K-218; M-1 to A-217; M-1 to R-216; M-1 to A-215; M-1 to R-214; M-1 to P-213; M-1 to 1-212; M-1 to I- 211; M-1 to V-210; M-1 to S-209; M-1 to L-208; M-1 to 1-207; M-1 to D-206; M-1 to G- 205; M-1 to Q-204; M-1 to
• Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the TNF-delta polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TNF delta polypeptide, which may be described generally as having residues n'-m 1 of Figures 1 A and IB (i.e., SEQ ID NO:2), where n 1 and m 1 are integers as described above.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TNF epsilon amino acid sequence shown in Figures 2A and 2B (i.e., SEQ ID NO:4), up to the leucine residue at position number 163 and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising the amino acid sequence of residues n 2 -168 of Figures 2 A and 2B (SEQ ID NO:4), where n 2 is an integer from 2 to 163 corresponding to the position of the amino acid residue in Figures 2A and 2B (SEQ JD NO:4).
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following amino acid sequences: T-2 to L-168; G-3 to L-168; G-4 to L-168; P-5 to L-168; S-6 to L-168; Q-7 to L-168; N-8 to L-168; G-9 to L-168; E-10 to L-168; G-ll to L- 168; Y-12 to L-168; P-13 to L-168; W-14 to L-168; Q-15 to L-168; S-16 to L-168; L-17 to L-168; P-18 to L-168; E-19 to L-168; Q-20 to L-168; S-21 to L-168; S-22 to L-168; D-23 to L-168; A-24 to L-168; L-25 to L-168; E-26 to L-168; A-27 to L-168; W-28 to L-168; E-29 to L-168; S
• Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the TNF-epsilon polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the mature form of TNF epsilon comprises, or alternatively consists of, amino acid residues Ala-39 to Leu-168 of the TNF epsilon sequence shown in Figures 2 A and 2B (SEQ ID NO:4).
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mature TNF-epsilon polypeptides described above.
• the present invention also encompasses polynucleotides encoding the above polypeptide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the mature form of TNF epsilon comprises, or alternatively consists of Ala- 105 to Leu-234 of the TNF epsilon sequence shown in Figures 7A and 7B (SEQ JD NO: 13).
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mature TNF-epsilon polypeptides described above.
• the present invention also encompasses polynucleotides encoding the above polypeptide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TNF epsilon mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TNF epsilon amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TNF epsilon polypeptide shown in Figures 2A and 2B (SEQ ID NO:4), up to the serine residue at position number 6, and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 2 of Figures 2A and 2B (i.e., SEQ ID NO:4), where m 2 is an integer from 6 to 167 corresponding to the position of the amino acid residue in Figures 2A and 2B (SEQ ID NO:4).
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following amino acid sequences: G-l to K-167; G-l to V-166; G-l to F-165; G-l to G-164; G-l to L-163; G-l to F-162; G-l to T-161; G-l to G-160; G-l to H-159; G-l to P-158; G-l to S-157; G-l to T -i « • G-l to N-155; G-l to L-154; G-l to K-153; G-l to A- 152; G-l to R-151; G-l toM50; G-l to R-149; G-l to P-148; G-l to 1-147; G-l to 1-146; G-l to V-145; G-l to S-144; G-l to L-143; G-l to 1-142;
• Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the TNF- delta polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
• Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%) or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.
• the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TNF epsilon polypeptide, which maybe described generally as having residues n 2 -m 2 of Figures 2A and 2B (i.e., SEQ ID NO:2), where n 2 and m 2 are integers as described above.
• TNF-delta and/or TNF-epsilon proteins of the invention comprise fusion proteins as described above wherein the TNF-delta and/or
• TNF-epsilon polypeptides are those described as n -m and/or n -m , herein, hi preferred embodiments, the application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• TNF-delta polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown in Figures 1 A and IB (SEQ ID NO: 1) or the complementary strand thereto.
• TNF-epsilon polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:4, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown in Figures 2A and 2B (SEQ ID NO:3) or the complementary strand thereto.
• Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
• the present invention also encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone, or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:l or contained in the deposited clone under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:l), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• the present invention also encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:4, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone, or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:3 or contained in the deposited clone under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:3), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• epitope of a polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID NO:3
• polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• epitope of a polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID NO:3
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)).
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes nonspecific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids.
• Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
• Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
• Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
• a preferred immunogenic epitope includes the secreted protein (e.g., amino acid residues 88-233 of SEQ ID NO:2, amino acid residues 105-250 of SEQ JD NO: 11 , amino acid residues 39-168 of SEQ JD NO:4, and/or amino acid residues 105-234 of SEQ ID NO: 13).
• the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
• immunogemc epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
• Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
• polypeptides of the present invention maybe fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) or albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides.
• fusion proteins may facilitate purification and may increase half-life in vivo.
• Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
• an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
• HA hemagglutinin
• a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
• the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
• the tag serves as a matrix- binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
• alteration of polynucleotides corresponding to SEQ ID NO:l and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence
• polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• Proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, et al, Nature 310:105-111 (1984)).
• a peptide corresponding to a fragment of the TNF delta and or TNF epsilon polypeptides of the invention can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TNF delta and/or TNF epsilon polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalamne, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
• the amino acid can be D (dextrorotary) or L (levorotary).
• the invention additionally, encompasses TNF delta and/or TNF epsilon polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include biotin, 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 a radioactive metal ion, e.g., alpha-emitters such as, for example, 213 Bi, or other radioisotopes such as, for example, iodine ( 131 1, 125
• TNF delta and/or TNF epsilonmpolypetides of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ⁇ h ⁇ , 177 Lu, 90 Y, 166 Ho, and 153 Sm, to polypeptides.
• the radiometal ion associated with the macrocyclic chelators attached to TNF delta and/or TNF epsilon polypeptides of the invention is l ⁇ h ⁇ .
• the radiometal ion associated with the macrocyclic chelator attached to TNF delta and/or TNF epsilon polypeptides of the invention is 90 Y.
• the macrocyclic chelator is l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA).
• DOTA is attached to the TNF delta and/or TNF epsilon polypeptide of the invention via a linker molecule.
• linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.
• TNF delta and/or TNF epsilon polypeptides of the invention may be labeled with biotin.
• biotinylated TNF delta and/or TNF epsilon polypeptides of the invention maybe used, for example, as an imaging agent or as a means of identifying one or more TNF delta and/or TNF epsilon receptor(s) or other coreceptor or coligand molecules.
• the TNF delta and/or TNF epsilon polypeptides of the invention can be used as an agent to target and kill cells expressing a TNF delta and/or TNF epsilon receptor (e.g., TACI and or BCMA).
• a TNF delta and/or TNF epsilon receptor e.g., TACI and or BCMA
• the polypeptides of the invention are associated, e.g., by covalent association or through protein-protein interactions, to a cytotoxin or cytotoxic agent.
• 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 homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (IT) (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.
• paclitaxel TAXOL
• doxetaxel TAXOTERE
• Rh6ne-Poulenc Rorer Antony, France
• gemcitabine ifosfamide, vinorelbine, navelbine, novantrone, teniposide, aminopterin, xeloda, ibandronate, CPT-1 1, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicins, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• DMFO difluoromethylornithine
• anti-hormonal agents that act to regulate or inhibit hormone action on tumors
• anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprohde, and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• 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.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, ATM I (See, International Publication No.
• CD40 Ligand 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 (“JL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM- CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-1 interleukin-1
• JL-2 interleukin-2
• IL-6 interleukin-6
• GM- CSF granulocyte macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• chemically modified derivatives of TNF delta and/or TNF epsilon which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.
• the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• the polyethylene glycol may have a branched structure.
• polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 75:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference. [0250]
• the polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
• Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
• polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• pegylation of the proteins of the invention may be accomplished by any number of means.
• polyethylene glycol may be attached to the protein either directly or by an intervening linker.
• Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. P:249-304 (1992); Francis et al, Intern. J. of Hematol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO CH 2 CF 3 ).
• MPEG monmethoxy polyethylene glycol
• ClSO CH 2 CF 3 tresylchloride
• polyethylene glycol is directly attached to amine groups of the protein.
• the invention includes protein- polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S.
• Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with l, -carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives.
• the number of polyethylene glycol moieties attached to each protein of the invention may also vary.
• the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
• the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13- 15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. P:249-304 (1992).
• TNF delta and/or TNF epsilon proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given TNF delta and/or TNF epsilon polypeptide.
• TNF delta and/or TNF epsilon polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
• Cyclic, branched, and branched cyclic TNF delta and/or TNF epsilon polypeptides may result from postfranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation
• polynucleotides and polypeptides of the present invention may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties TNF delta and TNF epsilon.
• applications in apoptosis of transformed cell lines, mediation of cell activation and proliferation and primary mediators of immune regulation antimicrobial, antiviral and inflammatory response susceptibility to pathogens. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms.
• proteins of the invention can also be expressed in transgenic animals.
• mice Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
• techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
• transgene i.e., nucleic acids of the invention
• transgene i.e., nucleic acids of the invention
• Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al, Appl. Microbiol Biotechnol. 40:691-698 (1994); Carver et al, Biotechnology (NY) 11:1263-1270 (1993); Wright et al, Biotechnology (NY) P:830-834 (1991); and Hoppe et al, US Patent Number 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al, Proc. Natl. Acad.
• transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al, Nature 380:64-66 (1996); Wihnut et al, Nature 555:810-813 (1997)), each of which is herein incorporated by reference in its entirety).
• the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals.
• the transgene maybe integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
• the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)).
• the regulatory sequences required for such a cell- type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
• gene targeting is preferred.
• vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
• the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Science 265:103- 106 (1994)).
• the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
• the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
• founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
• breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
• Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TNF delta and/or TNF epsilon polypeptides, studying conditions and/or disorders associated with aberrant TNF delta and/or TNF epsilon expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
• cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention are administered to a patient in vivo.
• Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc.
• the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
• the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
• the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. US Patent Number 5,399,349; and Mulligan & Wilson, US Patent Number 5,460,959, each of which is incorporated by reference herein in its entirety).
• the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
• the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
• This invention is also related to the use of the polynucleotides of the present invention to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of a polypeptide of the present invention associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of polypeptide of the present invention, such as, for example, neoplasia such as tumors.
• Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. PCR (Saiki et al, Nature, 324: 163-166 1986).
• RNA or cDNA may also be used in the same ways.
• PCR primers complementary to the nucleic acid encoding TNF delta or TNF epsilon can be used to identify and analyze TNF delta or TNF epsilon expression and mutations.
• deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
• Point mutations can be identified by hybridizing amplified DNA to radiolabeled TNF delta or TNF epsilon RNA or alternatively, radiolabeled TNF delta or TNF epsilon antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
• Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing.
• cloned DNA segments may be employed as probes to detect specific DNA segments.
• the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
• a sequencing primer is used with double-stranded PCR product or a single- stranded template molecule generated by a modified PCR.
• the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
• DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences maybe distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science, 230:1242 1985). [0272] Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc.
• the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
• restriction enzymes e.g., restriction fragment length polymorphisms ("RFLP")
• RFLP restriction fragment length polymorphisms
• Southern blotting of genomic DNA In addition to more conventional gel-elecfrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
• sequences of the present invention are also valuable for chromosome identification.
• the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
• Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
• the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
• the cDNA herein disclosed is used to clone genomic DNA of a gene of the present invention. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially.
• the genomic DNA the is used for in situ chromosome mapping using well known techniques for this purpose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
• PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
• sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
• Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
• Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
• This technique can be used with cDNA as short as 50 or 60.
• Verma et al Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
• the present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of a protein in the present invention in cells and tissues, including determination of normal and abnormal levels.
• a diagnostic assay in accordance with the invention for detecting over-expression of TNF protein of the present invention compared to normal control tissue samples maybe used to detect the presence of neoplasia, for example.
• Assay techniques that can be used to determine levels of a protein, such as a protein of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
• An ELISA assay initially comprises preparing an antibody specific to a protein of the present invention, preferably a monoclonal antibody.
• a reporter antibody generally is prepared which binds to the monoclonal antibody.
• the reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic, which in this example is horseradish peroxidase enzyme.
• a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
• a non-specific protein such as bovine serum albumin.
• the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any protein of the present invention attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer.
• the reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to a protein of the present invention.
• Unattached reporter antibody is then washed out.
• Reagents for peroxidase activity including a colorimetric substrate are then added to the dish.
• Immobilized peroxidase, linked to protein of the present invention through the primary and secondary antibodies, produces a colored reaction product.
• the amount of color developed in a given time period indicates the amount of protein of the present invention present in the sample.
• Quantitative results typically are obtained by reference to a standard curve.
• a competition assay may be employed wherein antibodies specific to protein of the present invention attached to a solid support and labeled protein of the present invention and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of protein of the present invention in the sample.
• polypeptides of the invention include antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide (e.g. TNF delta and/or TNF epsilon), preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding), hi specific embodiments, antibodies of the invention bind homomeric, especially homotrimeric, TNF delta and/or TNF epsilon polypeptides.
• TCR T-cell antigen receptors
• antibodies of the invention bind heteromeric, especially heterotrimeric, TNF delta and/or TNF epsilon polypeptides such as a heterotrimer contaimng two TNF delta and/or TNF epsilon polypeptides and one Neutrokine-alpha and/or Neutokine-alphaSV polypeptides (e.g., International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24 respectively) or a heterotrimer containing one TNF delta and/or TNF epsilon polypeptide and two Neutrokine-alpha and/or Neutokine-alphaSV polypeptides.
• heteromeric, especially heterotrimeric, TNF delta and/or TNF epsilon polypeptides such as a heterotrimer contaimng two TNF delta and/or TNF epsilon polypeptides and one Neutrokine-alpha and/or Neutokine-
• the antibodies of the invention bind homomeric, especially homotrimeric, TNF delta polypeptides, wherein the individual protein components of the multimers consist of the mature form of TNF delta (e.g., amino acids residues 88-233 of SEQ ID NO:2, or amino acids residues 105-250 of SEQ ID NO: 11.)
• the antibodies of the invention bind homomeric, especially homotrimeric, TNF epsilon polypeptides, wherein the individual protein components of the multimers consist of the mature form of TNF epsilon (e.g., amino acids residues 39-168 of SEQ ID NO:4, or amino acids residues 105-234 of SEQ ID NO: 13.)
• antibodies of the invention bind heteromeric, especially heterotrimeric, TNF delta and/or TNF epsilon polypeptides such as a heterotrimer containing two TNF delta and/or TNF epsilon polypeptides and one Neutrokine-alpha polypeptide or a heterotrimer containing one TNF delta and/or TNF epsilon polypeptide and two Neutrokine-alpha polypeptides, and wherein the individulal protein components of the TNF delta and/or TNF epsilon heteromer consist of the mature extracellular soluble portion of either TNF delta and/or TNF epsilon or (e.g., amino acids residues 88-233 of SEQ ID NO:2, amino acids residues 105-250 of SEQ ID NO:ll, amino acids residues 39-168 of SEQ ID NO:4, or amino acids residues 105-234 of SEQ ID NO: 13.) or the mature extracellular soluble portion Neut
• the antibodies of the invention bind conformational epitopes of a TNF delta and/or TNF monomeric protein, hi specific embodiments, the antibodies of the invention bind conformational epitopes of a TNF delta and or TNF epsilon multimeric, especially trimeric, protein.
• antibodies of the invention bind conformational epitopes that arise from the juxtaposition of TNF delta and or TNF epsilon with a heterologous polypeptide, such as might be present when TNF delta and/or TNF epsilon forms heterotrimers (e.g., with Neutrokine-alpha and/or Neutrokine-alpha SV polypetides polypeptides (see, e.g, (International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS, 23 and 24), or in fusion proteins between TNF delta and/or TNF epsilon and a heterologous polypeptide.
• heterotrimers e.g., with Neutrokine-alpha and/or Neutrokine-alpha SV polypetides polypeptides (see, e.g, (International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS, 23 and 24),
• Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, 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.
• antibody 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 (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• immunoglobulin molecules of the invention are N-(2-89] in specific embodiments.
• immunoglobulin molecules of the invention are IgG4.
• 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, CHI, 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, CHI, CH2, and CH3 domains.
• the antibodies of the invention may be from any animal origin including birds and mammals.
• the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
• "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. Patent No. 5,939,598 by Kucherlapati et al.
• the antibodies of the present invention may be monospecific, bispecif ⁇ c, trispecific or of greater multispecificity.
• Multispecific antibodies maybe specific for different epitopes of a polypeptide of the present invention or maybe 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.
• a heterologous epitope such as a heterologous polypeptide or solid support material.
• 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 that 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 that 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.
• 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 homolog 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.
• antibodies of the present invention cross react with Neutrokine-alpha (also known as B lymphocyte stimulator (BLyS)) and/or Neufrokine alpha splice variant (International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24, respectively).
• Neutrokine-alpha also known as B lymphocyte stimulator (BLyS)
• Neufrokine alpha splice variant International Publication No. WO 98/18921; Science. 285:260 (1999); SEQ ID NOS:23 and 24, respectively.
• antibodies of the present invention cross-react with murine, rat and/or rabbit homologs 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.
• antibodies of the present invention do not cross react with Neutrokine-alpha (also known as B lymphocyte stimulator (BLyS)) and/or Neufrokine alpha splice variant (Science. 285:260 (1999)).
• 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.
• antibodies that 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 5X10 "2 M, 10 "2 M, 5X10 “3 M, 10 " 3 M, 5X10 " , 10 “4 M, 5X10 "5 M, 10 "5 M.
• binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 "6 M, 5X10 “7 M, 10 “7 M, 5X10 “ 8 M, 10 "8 M, 5X10 “9 M, 10 "9 M, 5X10 “10 M, 10 “10 M, 5X10 “n M, 10 " ⁇ M, 5X10 "12 M, 10 "12 M, 5X10 "13 M, 10 "13 M, 5X10 "14 M, 10 "14 M, 5X10 "15 M, and 10 "15 M.
• 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.
• 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%.
• Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
• the present invention includes antibodies which disrupt binding of polypeptides of the invention with TACI and/or BCMA and/or TR11, and/or TR11SV1, and/or TR11SV2.
• 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.
• receptor activation can be determined by detecting stimulation of the transcription factor NF-AT by measuring the expression of an NF- AT regulatory element linked to a SEAP, beta-gal, CAT, or other reporter element.
• 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).
• antibodies are provided that inhibit ligand 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.
• 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.
• 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.
• 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.
• antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation.
• 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. Patent 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. hnmunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.
• 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.
• 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).
• 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.
• 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, radionuclides, metal ion chelating agents, drugs, or toxins, or any combination thereof.
• a preferred radionuclide is 90 Y.
• Another preferred radionuclide is ⁇ In.
• 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.
• the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, 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.
• the antibodies of the present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
• 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.
• 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.
• 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.
• 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.
• a “monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
• 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 Example 9. Briefly, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
• 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.
• 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.
• Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV).
• EBV Epstein Barr Virus
• Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference herein.
• the source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
• EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones.
• polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines.
• suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4).
• the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
• Antibody fragments that recognize specific epitopes may be generated by known techniques.
• 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 CHI domain of the heavy chain.
• the antibodies of the present invention can also be generated using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
• 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 Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene flT or gene Vm 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. hnmunol. Methods 184:177-
• 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.
• chimeric, humanized, or human antibodies For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it maybe 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.
• 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 framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• 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. Patent No.
• 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. Patent 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. Patent No. 5,565,332).
• 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. Patent 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.
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
• 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, hi 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 that 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.
• 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)).
• 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.
• 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.
• anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
• 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.
• Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al, Hum. Gene Ther. 5:595-601 (1994); Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol 57:257-283 (1997); Proba et al, J. Mol. Biol 275:245-253 (1998); Cohen et al, Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al, J. Mol. Biol.
• 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 NO:2.
• the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
• 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 ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• 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 a suitable source (e.
• nucleotide sequence and corresponding amino acid 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.
• 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 known 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.
• CDRs complementarity determining regions
• 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.
• the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
• 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.
• 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.
• 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.
• 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.
• Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
• an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody or single chain antibody of the invention)
• 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.
• the invention 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. Patent 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.
• 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.
• the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter, hi 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.
• 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, NSO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from
• 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.
• 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., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), 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; pDSf vectors (Inouye & friouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
• GST glutathione S- transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a 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.
• 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).
• 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 El 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, 1984, Proc.
• 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., 1987, Methods in Enzymol. 153:51-544).
• 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.
• 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, NSO, 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.
• breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D
• normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• stable expression is preferred.
• cell lines which stably express the antibody molecule may be engineered.
• 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.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• 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.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci.
• 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)).
• 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)).
• 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, 1983, Mol. Cell. Biol. 3:257).
• Vectors which use glutamine synthase (GS) or DHFR as the selectable marker can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
• An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
• Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
• glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein.
• glutamine synthase expression vectors that may be used according to the present invention are- commercially available from suplliers, including, for example Lonza Biologies, h e. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al, Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entirities by reference herein.
• 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.
• a single vector may be used which encodes both heavy and light chain polypeptides. hi such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• an antibody molecule of the invention 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.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
• 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.
• 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.
• 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.
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
• 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, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• 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. Patent Nos.
• polypeptides of the present invention 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 of the present invention 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).
• polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dirneric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• 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.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5
• Fc portions for the purpose of high-throughput screening assays to identify antagonists of hJL-5.
• the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification.
• 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, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• 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.
• 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.
• 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. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• 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;
• suitable radioactive material include iodine ( 131 1, 125 1, 123 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 1 I5m In, 1 I3m In, I I2 In, m h ⁇ ), and technetium ( 99
• antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to,
• the radiometal ion associated with the macrocyclic chelator attached to antibodies of the invention is ⁇ In. In another preferred embodiments, the radiometal ion associated with the macrocyclic chelator attached to antibodies of the invention is 90 Y.
• the macrocyclic chelator is l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA).
• DOTA is attached to the TNF delta and/or TNF epsilon polypeptide of the invention via a linker molecule.
• linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al, Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety.
• U.S. Patents 5,652,361 and 5,756,065 which disclose chelating agents that maybe conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties.
• Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T reaction).
• bifunctional conjugating agents see e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety
• 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.
• 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 homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (11) (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., vincri
• 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.
• 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, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, ATM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apop
• VEGI See, International Publication No. WO 99/23105
• CD40 Ligand 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 ('TL-6"), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-1 interleukin-1
• IL-2 interleukin-2
• 'TL-6 interleukin-6
• GM-CSF granulocyte macrophase colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• 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
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
• 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.
• 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. Patent 5,985,660; and Morrison et al, Cell, 96:737-49 (1999)).
• 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.
• Immunoprecipitation 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.
• a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxy
• 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).
• 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.
• 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 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
• ELIS 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.
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
• ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, hie, New York at 11.2.1.
• 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.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) 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.
• the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
• 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 described disorders.
• 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 as described herein).
• the antibodies of the invention can be used to treat, inhibit or prevent diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention and/or a receptor for the polypeptide of the invention (e.g., transmembrane activator and CAML interactor (TACI, GenBank accesion number AAC51790), and B-cell maturation antigen (BCMA, GenBank accession number NP_001183)), including, but not limited to, any one or more of the diseases, disorders, or conditions described herein (e.g., autoimmune diseases, disorders, or conditions associated with such diseases or disorders, including, but not limited to, autoimmune hemolytic anemia (including but not limited to cryoglobinemia or Coombs positive anemia), autoimmune neonatal thrombocytopema, idiopathic thrombocytopema purpura, autoimmunocytopenia, autoimmune neutropenia, hemolytic anemia, antiphospholipid syndrome, dermatitis (e.g., atopic dermatitis
• autoimmune thyroiditis i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmuniti.es such as, for example, (a) Graves' Disease , (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura , rheumatoid arthritis, schleroderma with anti- collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia (Addison's disease), idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis, IgA glomerulonephritis, and IgA nephropathy, bull
• antibodies of the invention are be used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis. In a another embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent advanced rheumatoid arthritis.
• antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis.
• an antibody, or antibodies, of the present invention are used to treat patients with clinical diagnosis of rheumatoid arthritis (RA). The patient treated will not have a B cell malignancy.
• the patient is optionally further treated with any one or more agents employed for treating RA such as salicylate; nonsteroidal anti-inflammatory drugs such as indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g.
• ibuprofen and fenoprofen naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac and diflunisal; antimalarials such as chloroquine; gold salts; penicillamine; or immunosuppressive agents such as methotrexate or corticosteroids in dosages known for such drugs or reduced dosages.
• the patient is only treated with an antibody, or antibodies, of the present invention.
• Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art.
• the primary response is determined by the Paulus index (Paulus et al. Athritis Rheum. 33:477-484 (1990)), i.e. improvement in morning stiffness, number of painful and inflamed joints, erythrocyte sedimentation (ESR), and at least a 2-point improvement on a 5-point scale of disease severity assessed by patient and by physician.
• Administration of an antibody, or antibodies, of the present invention will alleviate one or more of the symptoms of RA in the patient treated as described above.
• antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent hemolytic anemia.
• patients diagnosed with autoimmune hemolytic anemia e.g., cryoglobinemia or Coombs positive anemia
• AIHA autoimmune hemolytic anemia
• AHJA is an acquired hemolytic anemia due to auto-antibodies that react with the patient's red blood cells. The patient treated will not have a B cell malignancy.
• adjunct therapies such as glucocorticoids, prednisone, azathioprine, cyclophosphamide, vinca-laden platelets or Danazol
• glucocorticoids such as prednisone, azathioprine, cyclophosphamide, vinca-laden platelets or Danazol
• Antibodies of the present invention are administered to the hemolytic anemia patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art.
• Overall response rate is determined based upon an improvement in blood counts, decreased requirement for transfusions, improved hemoglobin levels and/or a decrease in the evidence of hemolysis as determined by standard chemical parameters.
• an antibody, or antibodies of the present invention will improve any one or more of the symptoms of hemolytic anemia in the patient treated as described above.
• the patient treated as described above will show an increase in hemoglobin and an improvement in chemical parameters of hemolysis or return to normal as measured by serum lactic dehydrogenase and/or bilirubin.
• antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent adult immune thrombocytopenic purpura.
• Adult immune thrombocytopenic purpura is a relatively rare hematologic disorder that constitutes the most common of the immune-mediated cytopenias. The disease typically presents with severe thrombocytopema that may be associated with acute hemorrhage in the presence of normal to increased megakaryocytes in the bone marrow.
• Splenectomy has been recommended as standard second-line therapy for steroid failures, and leads to prolonged remission in nearly 60% of cases yet may result in reduced immunity to infection.
• Splenectomy is a major surgical procedure that may be associated with substantial morbidity (15%) and mortality (2%).
• IVIG has also been used as second line medical therapy, although only a small proportion of adult patients with ITP achieve remission.
• Therapeutic options that would interfere with the production of autoantibodies by activated B cells without the associated morbidities that occur with corticosteroids and/or splenectomy would provide an important treatment approach for a proportion of patients with ITP.
• Patients with clinical diagnosis of ITP are treated with an antibody, or antibodies of the present invention, optionally in combination with steroid therapy.
• Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art.
• Overall patient response rate is determined based upon a platelet count determined on two consecutive occasions two weeks apart following treatments as described above. See, George et al. "Idiopathic Thrombocytopenic Purpura: A Practice Guideline Developed by Explicit Methods for The American Society of Hematology", Blood 88:3-40 (1996), expressly incorporated herein by reference.
• antibody agonists of the invention are be used to treat, inhibit or prevent immunodeficiencies, and/or disorders, or conditions associated with immunodeficiencies.
• immunodeficiencies include, but are not limited to, severe combined immunodeficiency (SCJD)- X linked, SCJD-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (SCJD)- X
• antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent CVTD, or a subgroup of individuals having CVID.
• antibody agonists of the invention are used as an adjuvant to stimulate B cell proliferation, immunoglobulin production, and/or to enhance B cell survival.
• the treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention and/or a receptor for the polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• the antibodies of the invention may also be used to target and kill cells expressing TNF delta and/or TNF epsilon on their surface and/or cells having TNF delta and/or TNF epsilon bound to their surface.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• 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.
• 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.
• 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, anti-tumor agents, antibiotics, and immunoglobulin therapy). 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.
• human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10-6 M, 10- 6 M, 5 X 10-7 M, 10-7 M, 5 X 10-8 M, 10-8 M, 5 X 10-9 M, 10-9 M, 5 X 10-10 M, 10-10 M, 5 X 10-11 M, 10-11 M, 5 X 10-12 M, 10-12 M, 5 X 10-13 M, 10- 13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, and 10-15 M.
• antibodies may act as antagonists to the TNF-delta and/or TNF-epsilon polypeptides of the invention.
• Such an antagonist is useful in reducing, preventing, and/or eliminating specific T cell activity mediated and/or induced by TNF- delta and/or TNF-epsilon polypeptides of the invention. Examples of such activities include autoimmune diseases, graft-versus-host disorders, allergic diseases, T cell regulated immune responses, and diseases and/or disorders related to T cell stimulation. Additional examples of T cell and general immune diseases and/or disorders that maybe reduced, prevented, and/or treated by using antagonistic antibodies directed against TNF-delta and/or TNF-epsilon polypeptides of the invention as recited herein.
• anti-TNF-delta and/or anti-TNF-epsilon antibodies of the present invention maybe used to treat, diagnose, prevent, and/or prognose acute myelogenous leukemia.
• anti-TNF-delta and/or anti-TNF- epsilon antibodies of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat, diagnose, prevent, and/or prognose acute myelogeneous leukemia.
• anti-TNF-delta and/or anti- TNF-epsilon antibodies of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat, diagnose, prevent, and/or prognose chronic myelogeneous leukemia, multiple myeloma, non-Hodgkins lymphoma, and/or Hodgkins disease.
• 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.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect.
• 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, hi particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
• 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 nucleic acids (Koller and Smithies, 1989, Proc. Natl.
• 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.
• 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.
• 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. Patent No.
• microparticle bombardment e.g., a gene gun; Biolistic, Dupont
• coating 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, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.
• 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.
• 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 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)).
• the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlsfra et al., 1989, Nature 342:435-438).
• viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
• a refroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599).
• refroviral vectors have been to delete refroviral sequences that are not necessary for packaging of the viral genome and integration into 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 refroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a refroviral 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 refroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,
• 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, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al.,
• adenovirus vectors are used.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No. 5,436,146).
• 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.
• 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.
• the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
• 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, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol.
• 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.
• 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
• 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.
• 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.
• the cell used for gene therapy is autologous to the patient.
• 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.
• 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, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
• 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
• 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.
• 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 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.
• the invention provides methods of freatment, 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.
• 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.
• 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.
• 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, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a refroviral 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 admimstered 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 maybe 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.
• a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; 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.)
• the compound or composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neural. 25:351; Howard et al., 1989, J.Neurosurg. 71:105).
• 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)).
• 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 refroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier, h 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.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is admimstered.
• 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.
• 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.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
• 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.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• 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.
• 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.
• Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.1 mg/kg to
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 and/or disorders 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.
• the invention provides a diagnostic assay for diagnosising 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.
• a diagnostic assay for diagnosising 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.
• 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
• 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, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, JVL, 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 (RJA).
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• 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
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 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., "hnmunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.
• 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, hi another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• 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.
• 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.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (TVIRI).
• kits that can be used in the above methods.
• kits comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
• 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.
• the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest, hi another specific embodiment, the kits of the present invention comprise two or more antibodies (monoclonal and/or polyclonal) that recognize the same and/or different sequences or regions of the polypeptide of the invention.
• 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).
• 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.
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
• a kit may include a control antibody that does not react with the polypeptide of interest.
• a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
• 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).
• 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.
• the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
• a kit may also include a non-attached reporter-labeled anti-human antibody, hi this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
• 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.
• the antibody is attached to a solid support, hi 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.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
• the reagent 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.
• 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).
• 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 nonspecific 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). [0421]
• 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.
• polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
• These antibodies can be, for example, polyclonal or monoclonal antibodies.
• the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
• Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature, 256:495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today, 4:72 (1983) and the EBN-hybridoma technique to produce human monoclonal antibodies (Cole et al, pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985). [0425] Techniques described for the production of single chain antibodies (U.S.
• Patent No. 4,946,778 can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
• antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
• Antisense and Ribozymes (Antagonists)
• antagonists according to the present invention are nucleic acids corresponding to the sequences contained in TNF-delta and/or TNF-epsilon, or the complementary strand thereof, and/or to nucleotide sequences contained in the deposited clones.
• antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, Okano H. et al, J. Neurochem. 55:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation.
• Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 10-1573 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
• the TNF-delta and/or TNF-epsilon antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
• a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the TNF-delta and/or TNF-epsilon antisense nucleic acid.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology methods standard in the art.
• Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding TNF- delta and/or TNF-epsilon, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive.
• Such promoters include, but are not limited to, the S V40 early promoter region (Bernoist and Chambon, Nature 2 :304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 75:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al, Nature 296:39-42 (1982)), etc.
• the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a TNF-delta and or TNF- epsilon gene.
• absolute complementarity although preferred, is not required.
• a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded TNF-delta and/or TNF-epsilon antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
• the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a TNF-delta and/or TNF-epsilon RNA it may contain and still form a stable duplex (or triplex as the case may be).
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• Oligonucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
• sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature 572:333-335 (1994).
• oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions of the TNF-delta and TNF-epsilon shown in Figures 1 A and IB, and 2A and 2B, respectively could be used in an antisense approach to inhibit translation of endogenous TNF-delta and TNF-epsilon mRNA.
• Oligonucleotides complementary to the 5' unfranslated region of the mRNA should include the complement of the AUG start codon.
• Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
• antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length, hi specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• the polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
• the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci. U.S.A.
• the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylammomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannos
• the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
• the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• the antisense oligonucleotide is an alpha- anomeric oligonucleotide.
• An alpha-anomeric oligonucleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual alpha-units, the strands run parallel to each other (Gautier et al, Nucl Acids Res. 75:6625-6641 (1987)).
• the oligonucleotide is a 2 alpha-O-methylribonucleotide or a 2 beta-O-methylribonucleotide (Inoue et al, Nucl. Acids Res. 75:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al, FEBS Lett. 215:327-330 (1987)).
• Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
• an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
• phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl Acids Res. 16:3209 (1988))
• methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Natl Acad. Sci. U.S.A. 55:7448-7451 (1988)), etc.
• antisense nucleotides complementary to the TNF-delta and/or TNF- epsilon coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
• Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy TNF-delta and/or TNF-epsilon mRNAs, the use of hammerhead ribozymes is preferred.- Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
• hammerhead ribozymes The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of TNF-delta ( Figures 1A and IB (SEQ JD NO:l)) and TNF-epsilon ( Figures 2A and 2B (SEQ JD NO:3) and Figures 7A and 7B (SEQ JD NO: 12)).
• the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the TNF-delta and TNF-epsilon mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express TNF-delta and/or TNF-epsilon in vivo.
• DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
• a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol UI or pol U promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous TNF-delta and/or TNF-epsilon messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
• Endogenous gene expression can also be reduced by inactivating or
• TNF-delta and/or TNF-epsilon gene and/or its promoter using targeted homologous recombination, (e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 57:503-512 (1987); Thompson et al, Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety).
• a mutant, nonfunctional polynucleotide of the invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo.
• techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene.
• antagonists according to the present invention include soluble forms of TNF-delta (e.g., fragments of the TNF-delta shown in Figures 1A and IB (SEQ ID NO:2) and Figures 6A and 6B and/or TNF-epsilon (e.g., fragments of the TNF- epsilon shown in Figures 2 A and 2B (SEQ ID NO:4) and and Figures 7A and 7B (SEQ ID NO:13) that include the receptor binding domain from the extracellular region of the full length ligand).
• TNF-delta e.g., fragments of the TNF-delta shown in Figures 1A and IB (SEQ ID NO:2) and Figures 6A and 6B
• TNF-epsilon e.g., fragments of the TNF- epsilon shown in Figures 2 A and 2B (SEQ ID NO:4) and and Figures 7A and 7B (SEQ ID NO:13) that include the receptor binding domain from the
• Such soluble forms of TNF-delta and/or TNF-epsilon which may be naturally occurring or synthetic, antagonize TNF-delta and/or TNF-epsilon mediated signaling by competing with the ligand for binding to TNF-family receptors.
• Antagonists of the present invention also include antibodies specific for TNF-family ligands and TNF- delta- and/or TNF-epsilon-Fc fusion proteins.
• anti-TNF delta antibodies, and/or anti-TNF epsilon antibodies, and/or Neutrokine-alpha, and/or Neutrokine-alphaSV, and or TACI soluble domain fused to Fc, and/or BCMA soluble domain fused to Fc function as TNF delta and/or TNF epsilon antagonists.
• the expression of IL-2 receptor messenger RNA is highly increased when cells are cultured in the presence of a TNF delta and/or TNF epsilon antagonist.
• the expression of JJ -2 receptor messenger RNA is highly decreased when cells are cultured in the presence of a TNF delta and/or TNF epsilon agonist.
• TNF-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing and/or blocking the ligand/receptor signaling pathway.
• TNF ligand family include, but are not limited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), FasL, TNF- gamma (International Publication No. WO 96/14328), AJJVI-I (International Publication No.
• WO 97/33899 International Publication No. WO 97/34911
• Neutrokine-alpha and/or Neutrokine-alphaSV International Publication No. WO98/18921
• endokine-alpha International Publication No. WO 98/07880
• CD40L CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
• TNF-alpha has been shown to protect mice from infection with herpes simplex virus type 1 (HSV-1). Rossol-Voth et al., J .Gen. Virol. 72:143-147 (1991). The mechanism of the protective effect of TNF-alpha is unknown but appears to involve neither interferons nor NK cell killing. One member of the family has been shown to mediate HSV-1 entry into cells. Montgomery et al., Eur. Cytokine Newt. 7:159 (1996). Further, antibodies specific for the extracellular domain of this block HSV-1 entry into cells.
• HSV-1 herpes simplex virus type 1
• TNF-delta and/or TNF-epsilon antagonists of the present invention include both TNF-delta and/or TNF-epsilon amino acid sequences and antibodies capable of preventing mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized for binding to virus or by directly blocking binding of virus to cell surface receptors.
• TNF-delta and/or TNF-epsilon receptor immunogens may be prepared by any of a variety of standard methods using TNF-delta and/or TNF-epsilon receptor immunogens of the present invention.
• TNF-delta and/or TNF-epsilon receptor immunogens include the TNF-delta and TNF-epsilon proteins shown in Figures 1 A and IB (SEQ ID NO:2) and Figures 2A and 2B (SEQ ID NO:4), respectively, (which may or may not include a leader sequence) and polypeptide fragments of the receptor comprising the ligand binding, extracellular, transmembrane, the intracellular domains of TNF-delta and/or TNF-epsilon, or any combination thereof.
• Polyclonal and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed herein and and/or known in the art, such as, for example, those methods described in Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO 94/09137 (the contents of each of these three applications are herein incorporated by reference in their entireties), and are preferably specific to polypeptides of the invention having the amino acid sequence of SEQ JD NO:2 and/or SEQ ID NO:4.
• DNA shuffling The techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of TNF-delta and/or TNF-epsilon thereby effectively generating agonists and antagonists of TNF-delta and/or TNF-epsilon. See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten et al, Curr. Opinion Biotechnol. 5:724-33 (1997); Harayama, Trends Biotechnol.
• alteration of TNF-delta and/or TNF-epsilon polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments into a desired TNF-delta and/or TNF-epsilon molecule by homologous, or site-specific, recombination
• TNF- delta and/or TNF-epsilon polynucleotides and corresponding polypeptides may be alterred by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination, hi another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of TNF-delta and/or TNF-epsilon may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.
• heterologous molecules are TNF-alpha, TNF-beta, lymphotoxin-alpha, lymphotoxin-beta, FAS ligand, Neutrokine-alpha, and/or Neutrokine-alphaSV.
• the heterologous molecules are any member of the TNF family.
• Non-naturally occurring variants also may be produced using art-known mutagenesis techniques, which include, but are not limited to, oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl. Acids Res. 75:4331 (1986); and Zoller et al, Nucl Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al, Gene 54:315 (1985)), and restriction selection mutagenesis (see e.g., Wells et al, Philos. Trans. R. Soc.
• the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TNF-delta and/or TNF-epsilon polypeptide an effective amount of an antagonist capable of decreasing TNF-delta and/or TNF-epsilon mediated signaling.
• TNF-delta- and/or TNF-epsilon-mediated signaling is decreased to treat a disease wherein increased apoptosis, NF-kappaB expression and or JNK expression is exhibited.
• Antagonists include, but are not limited to, soluble forms of TNF-delta and/or TNF-epsilon polypeptide and antibodies (preferably monoclonal) directed against the TNF- delta and/or TNF-epsilon polypeptide.
• the polypeptides of the present invention may be employed to inhibit neoplasia, such as tumor cell growth.
• the polypeptides of the present invention maybe responsible for tumor destruction through apoptosis and cytotoxicity to certain cells.
• the polypeptides of the present invention also induce up-regulation of adhesion cells, for example, LFA-1, therefore, maybe employed for wound-healing.
• the polypeptides of the present invention may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since the polypeptides of the present invention have proliferation effects on cells of endothelial origin.
• the polypeptides of the present invention may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.
• the polypeptides of the present invention also stimulate the activation of T- cells, and may, therefore, be employed to stimulate an immune response against a variety of parasitic, bacterial and viral infections.
• the polypeptides of the present invention may also be employed in this respect to eliminate autoreactive T-cells to treat and/or prevent autoimmune diseases.
• An example of an autoimmune disease is Type I diabetes.
• This invention also provides a method for identification of molecules, such as receptor molecules, that bind the proteins of the present invention. Genes encoding proteins that bind the proteins of the present invention, such as receptor proteins, can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting.
• RNA polyadenylated RNA
• a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to the proteins of the present invention.
• the transfected cells then are exposed to labeled the proteins of the present invention.
• the proteins of the present invention can be labeled by a variety of well- known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase. Following exposure, the cells are fixed and binding of cytostatin is determined. These procedures conveniently are carried out on glass slides.
• Pools are identified of cDNA that produced TNF delta or TNF epsilon binding cells. Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening process, one or more single clones that encode the putative binding molecule, such as a receptor molecule, can be isolated.
• a labeled ligand can be photoaffinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a receptor molecule.
• Cross-linked material is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film.
• PAGE polyacrylamide gel electrophoresis
• the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
• the amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
• Polypeptides of the invention also can be used to assess TNF delta or TNF epsilon binding capacity of TNF delta or TNF epsilon binding molecules, such as receptor molecules, in cells or in cell-free preparations.
• the polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides or TNF-delta and/or TNF-epsilon antagonists (e.g., anti-TNF-delta and/or TNF-epsilon antibodies) of the invention are used to treat, diagnose, or prognose an individual having an immunodeficiency.
• an individual having an immunodeficiency expresses aberrantly low levels of TNF-delta and/or TNF- epsilon when compared to an individual not having an immunodeficiency.
• Any means described herein or otherwise known in the art may be applied to detect TNF-delta and/or TNF-epsilon polynucleotides or polypeptides of the invention (e.g., FACS analysis or ELISA detection of TNF-delta and/or TNF-epsilon polypeptides of the invention and hybridization or PCR detection of TNF-delta and/or TNF-epsilon polynucleotides of the invention) and to determine the expression profile of TNF-delta and/or TNF-epsilon, polynucleotides and/or polypeptides of the invention in a biological sample.
• TNF-delta and/or TNF-epsilon polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used according to the methods of the invention in the diagnosis and/or prognosis of an immunodeficiency.
• a biological sample obtained from a person suspected of being afflicted with an immunodeficiency (“the subject") may be analyzed for the relative expression level(s) of TNF-delta and/or TNF-epsilon polynucleotides and/or polypeptides of the invention.
• the expression level(s) of one or more of these molecules of the invention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with an immunodeficiency.
• TNF-delta and/or TNF-epsilon polynucleotides and or polypeptides of the invention and/or agonists and/or antagonists thereof, between samples obtained from the subject and the control suggests that the subject is afflicted with an immunodeficiency.
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides or TNF-delta and/or TNF-epsilon antagonists e.g., anti-TNF-delta and/or TNF-epsilon antibodies
• CVTD common variable immunodeficiency disease
• an individual having CVJD or a subset of individuals having CVID expresses aberrant levels of TNF-delta and/or TNF-epsilon receptor on their T cells, when compared to individuals not having CVID.
• Any means described herein or otherwise known in the art may be applied to detect TNF-delta and/or TNF-epsilon receptor polynucleotides or polypeptides of the invention (e.g., FACS analysis or ELISA detection of TNF-delta and/or TNF-epsilon polypeptides of the invention and hybridization or PCR detection of TNF-delta and/or TNF-epsilon polynucleotides of the invention) and to determine differentially the expression profile of TNF-delta and/or TNF- epsilon polynucleotides or polypeptides of the invention in a sample containing at least T cells or some component thereof (e.g., RNA) as compared to a sample containing at least T
• RNA TNF-delta and or TNF-epsilon polynucleotide or polypeptide expression
• a sample containing at least B cells or a component thereof e.g., RNA
• the samples may be correlated with the occurrence of CVID (i.e., "acquired agammaglobulinemia” or "acquired hypogammaglobulinemia").
• a subject of persons afflicted with CVID are characterized by high levels of expression of both TNF-delta and/or TNF-epsilon in peripheral or circulating T cells when compared to that observed in individuals not having CVID.
• persons who are not afflicted with CVJD are typically characterized by low levels of TNF-delta and/or TNF- epsilon expression.
• TNF-delta and/or TNF-epsilon polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof maybe used according to the methods of the invention in the differential diagnosis of this subset of CVID.
• a sample of peripherial T cells obtained from a person suspected of being afflicted with CVID (“the subject") may be analyzed for the relative expression level(s) of TNF-delta and/or TNF-epsilon polynucleotides and/or polypeptides of the invention.
• the expression level(s) of one or more of these molecules of the invention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with CVID ("the control").
• TNF-delta and/or TNF-epsilon polynucleotides and/or polypeptides of the invention and/ ⁇ r agonists and/or antagonists thereof, between samples obtained from the subject and the control suggests that the subject is afflicted with this subset of CVID.
• Cunningham-Rundles and Bodian followed 248 CVID patients over a period of 1-25 years and discovered that a number of associated diseases or conditions appear with increased frequency in CVJD patients (Cunningham-Rundles and Bodian, J. Clin.
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides, or agonists or antagonists thereof are used to diagnose, prognose, treat, or prevent conditions associated with CVID, including, but not limited to, conditions associated with acute and recurring infections (e.g., pneumonia, bronchitis, sinusitis, otitis media, sepsis, meningitis, septic arthritis, and osteomyelitis), chronic lung disease, autoimmunity, granulomatous disease, lymphoma, cancers (e.g., cancers of the breast, stomach, colon, mouth, prostate, lung, vagina, ovary, skin, and melanin forming cells (i.e.
• acute and recurring infections e.g., pneumonia, bronchitis, sinusitis, otitis media, sepsis, meningitis, septic arthritis, and osteomyelitis
• chronic lung disease e.g., granulomatous disease, lympho
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides, or agonists thereof are used to treat or prevent a disorder characterized by deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction.
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides, or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVJD, other primary immune deficiencies, HJN disease, CLL, multiple myeloma, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g.,
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides or TNF-delta and/or TNF-epsilon antagonists e.g., anti-TNF-delta and/or anti- TNF-epsilon antibodies
• TNF-delta and/or TNF-epsilon polynucleotides or polypeptides or TNF-delta and/or TNF-epsilon antagonists e.g., anti-TNF-delta and/or anti- TNF-epsilon antibodies
• an individual having an autoimmune disease or disorder expresses aberrantly high levels of TNF-delta and/or TNF-epsilon when compared to an individual not having an autoimmune disease or disorder.

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