JP2008194038A - HUMAN TUMOR NECROSIS FACTOR delta and epsilon - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a human TNF δ and TNF ε polypeptides and a polynucleotide encoding the polypeptide and to provide a method for producing the polynucleotide. <P>SOLUTION: The isolated polynucleotide includes a polynucleotide having at least 70% homology to a member selected from the following group of (a) a polynucleotide encoding a polypeptide including a specific amino acid sequence, (b) a polynucleotide encoding a polypeptide including a specific amino acid sequence, (c) a polynucleotide encoding a polypeptide including amino acid 39 to amino acid 233 of a specific sequence, (d) a polynucleotide complementary to the polynucleotide of (a), (b) or (c) and (e) a polynucleotide including at least 15 bases of the polynucleotide of (a), (b), (c) or (d). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to newly identified polynucleotides and polypeptides; variants or derivatives of those polynucleotides and polypeptides; processes for making these polynucleotides and polypeptides, and variants and derivatives thereof; It relates in part to the use of agonists and antagonists of these polypeptides; and their polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and other respects, the present invention relates to human tumor necrosis factor δ and ε polynucleotides and polypeptides (hereinafter sometimes referred to as “TNFδ” and “TNFε”).

    Human tumor necrosis factor α (TNF-α) and β (TNF-β or lymphotoxin) are related to a broad class of members of the polypeptide mediator, including interferons, interleukins and growth factors, collectively Called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol., 7: 625-655, 1989).

  Tumor necrosis factors (TNF-α and TNF-β) were first discovered as a result of their anti-tumor activity, but now involve mediating apoptosis, cell activation and proliferation of several transformed cell lines It is recognized as a multifaceted cytokine that allows multiple biological activities, and further plays an important role in immune regulation and inflammation.

  To date, there are nine known members of the TNF-ligand superfamily, TNF-α, TNF-β (lymphotoxin-α), LT-β, OX40L, FASL, CD30L, CD27L, CD40L and 4-1BBL. The ligands of the TNF ligand superfamily are acidic TNF-like molecules with about 20% sequence homology (range; 12-36%) in the extracellular domain and exist mainly as membrane-bound forms, biological activity The form is a trimer / multimer complex. Soluble forms of the TNF ligand superfamily have so far been identified only for TNF, LTα, and FASL (for a general review, Gruss, H. and Dower, SK, Blood, 85 (12): 3378-3404 (1995), which is hereby incorporated by reference in its entirety).

  These proteins are involved in the regulation of cell proliferation, activation and differentiation, including the control of cell death or cell death by apoptosis or cytotoxicity (Armitage, RJ, Curr. Opin. Immunol., 6 : 407 (1994) and Smith, CA, Cell, 75: 959 1994).

  TNF is produced by a number of cell types including monocytes, fibroblasts, T cells, natural killer (NK) cells, and is predominantly produced by activated macrophages. TNF-α plays a role in rapid necrosis of tumors, immune stimulation, autoimmune disease, graft rejection, resistance to parasites, production of antiviral responses, septic shock, growth regulation, vascular endothelial and metabolic effects It has been reported to have. TNF-α also triggers endothelial cells to secrete various factors, including PAF-1, IL-1, GM-CSF and IL-6, and promotes cell proliferation. Furthermore, TNF-α up-regulates various cell adhesion molecules such as E-selectin, ICAM-1 and VCAM-1.

  The first step in the induction of various cellular responses mediated by members of the TNF ligand superfamily is their binding to specific cell surface receptors. The TNF receptor superfamily currently contains several viral open reading frames encoding 10 known membrane proteins and TNFR-related molecules. The p75 low affinity nerve growth factor (NGF) receptor was the first cloned receptor in this family (Johnson, D. et al., Cell, 47: 545 (1986)). Subsequently, the cloning of two specific receptors for TNF indicates that they are related to the NGF receptor (Loetscher, H. et al., Cell, 61: 351 (1990)). In recent years, a novel type I transmembrane TNF receptor superfamily has been established. This family includes the p75 nerve growth factor receptor, p60 TNFR-1, p80 TNFR-II, TNFR-RP / TNFR-III, CD27, CD30, CD40, 4-1BB, OX40, and FAS / APO-1. In addition, several viral open reading frames encoding soluble TNF receptors have been identified (see, for example, Shop fibroma virus SFV-T2 (Smith, CA, et al., Biochem, Biophys. Res. Commun., 176: 335, 1991) and vaccinia virus Va53 or SaIF19R (Howard. ST, Virology, 180: 633, 1991)). These receptors are characterized by multiple cysteine-rich domains in the extracellular (amino terminal) domain, which have been shown to be involved in ligand binding. The average homology in the cysteine-rich extracellular region between human family members is in the range of 25-30%.

  Clearly, there is a need for factors that regulate normal and abnormal cell activation and differentiation. There is therefore a need for the identification and characterization of such factors that modulate the activation and differentiation of both normal and diseased cells. In particular, it controls apoptosis of transformed cell lines, mediates cell activation and proliferation, and is functionally associated as the primary mediator of immune regulation and inflammatory response, and, among other things, prevents, ameliorates or There is a need to isolate and characterize additional TNF ligands, similar to members of the TNF ligand superfamily, that can play a role in correcting.

  The present invention relates to human TNFδ and TNFε polypeptides, polynucleotides encoding the polypeptides, methods of producing the polypeptides (particularly by expressing the polynucleotides), and agonists of the polypeptides and Relates to antagonists. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists, and antagonists in applications. This application relates in part to research, diagnosis, and clinical technology. Accordingly, it is an object of the present invention to provide human TNFδ and TNFε polypeptides, polynucleotides encoding those polypeptides, methods for producing these polypeptides, and agonists and antagonists of those polypeptides.

In one aspect, the present invention includes an isolated polynucleotide comprising a polynucleotide having at least 70% identity to a member selected from the group consisting of:
(A) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4;
(C) a polynucleotide encoding a polypeptide comprising amino acid 39 to amino acid 233 of SEQ ID NO: 2;
(D) a polynucleotide that is complementary to the polynucleotide of (a), (b), or (c); and (e) the polynucleotide of (a), (b), (c), or (d) A polynucleotide comprising at least 15 bases is provided.

  In one embodiment, the polynucleotide of the present invention may be DNA.

  In one embodiment, the polynucleotide of the present invention can be RNA.

  In one embodiment, the polynucleotide of the present invention may be genomic DNA.

  In a preferred embodiment, the polynucleotide of the invention can encode a polypeptide comprising the amino acid of SEQ ID NO: 2.

  In a more preferred embodiment, the polynucleotide of the invention may encode a polypeptide comprising amino acid 39 to amino acid 233 of SEQ ID NO: 2.

  In a further preferred embodiment, the polynucleotide of the present invention may encode a polypeptide comprising the amino acid of SEQ ID NO: 4.

  In a still preferred embodiment, the polynucleotide of the present invention can encode a polypeptide comprising amino acids 1 to 188 of SEQ ID NO: 4.

In one aspect, the present invention includes an isolated polynucleotide comprising a polynucleotide that is at least 70% identical to a member selected from the group consisting of:
(A) a polynucleotide encoding a mature polypeptide having an amino acid sequence expressed by human cDNA contained in ATCC Deposit No. 97377;
(B) a polynucleotide encoding a mature polypeptide having an amino acid sequence expressed by the human cDNA included in ATCC Deposit No. 97457;
(C) a polynucleotide that is complementary to the polynucleotide of (a) or (b);
(D) Provided is a polynucleotide comprising at least 15 bases of the polynucleotide of (a), (b), or (c).

  In one embodiment, the polynucleotide of the invention can comprise nucleotide 447 to nucleotide 1717 of SEQ ID NO: 1.

  In a preferred embodiment, the polynucleotide of the invention may comprise nucleotide 332 to nucleotide 1717 of SEQ ID NO: 1.

  In a more preferred embodiment, the polynucleotide of the invention may comprise nucleotides 1 to 564 of SEQ ID NO: 3.

  In a further embodiment, the vector of the present invention may contain the DNA.

  In one embodiment, the host cell of the present invention may comprise the above vector.

  In one embodiment, the present invention provides a process for producing a polypeptide comprising the step of expressing a polypeptide encoded by the DNA from the host cell.

  In another embodiment, the invention includes a step of genetically engineering a cell using the vector, thereby producing a cell that expresses a polypeptide encoded by a human cDNA contained in the vector. Provide a process.

In one aspect, the invention provides a polypeptide comprising a member selected from the group consisting of:
(A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2; and (b) a polypeptide comprising amino acids 39 to 233 according to SEQ ID NO: 2;
(C) a polypeptide comprising amino acids 1 to 188 set forth in SEQ ID NO: 4; and (d) a polypeptide having at least 70% identity to the polypeptide set forth in (a), (b), or (c) I will provide a.

  In one embodiment, the polypeptide of the invention may comprise amino acid 1 to amino acid 233 of SEQ ID NO: 2.

  In a preferred embodiment, the polypeptide of the invention may comprise amino acid 39 to amino acid 233 of SEQ ID NO: 2.

  In a more preferred embodiment, the polypeptide of the invention may comprise amino acids 1 to 188 of SEQ ID NO: 4.

  In another embodiment, the present invention provides a compound that inhibits activation of the polypeptide.

  In one aspect, the present invention provides a method for the treatment of a patient having a need for TNFδ, comprising administering to the patient a therapeutically effective amount of the polypeptide.

  In one aspect, the invention provides a method for the treatment of a patient having a need for TNFε comprising administering to the patient a therapeutically effective amount of the polypeptide of claim 17. To do.

  In one embodiment, the invention provides the method, wherein the therapeutically effective amount of the polypeptide is administered by providing the patient with DNA encoding the polypeptide and expressing the polypeptide in vivo. I will provide a.

  In another embodiment, the invention provides the method, wherein the therapeutically effective amount of the polypeptide is administered by providing the patient with DNA encoding the polypeptide and expressing the polypeptide in vivo. I will provide a.

  In one aspect, the present invention provides a method for the treatment of a patient in need of inhibiting a TNFδ polypeptide, comprising administering to the patient a therapeutically effective amount of the compound.

  In one aspect, the present invention provides a method for the treatment of a patient in need of inhibiting a TNFε polypeptide, comprising administering to the patient a therapeutically effective amount of the compound.

  In one aspect, the present invention includes a process for diagnosing a disease or susceptibility to a disease associated with underexpression of the polypeptide, comprising determining a mutation in the nucleic acid sequence encoding the polypeptide. Provide a process to

  In one aspect, the present invention provides a diagnostic process comprising analyzing the presence of the polypeptide in a sample derived from a host.

In one aspect, the present invention provides a method for identifying a compound that binds to and inhibits the activation of the polypeptide, comprising:
A receptor for the polypeptide, wherein the receptor is associated with a second component capable of providing a detectable signal in response to the binding of the compound to the receptor, and the cell expressing the receptor on its surface; Contacting the detectably TNFδ polypeptide and compound under conditions capable of binding to the receptor; and detecting the absence of a signal produced from the interaction of TNFδ with the receptor Determining whether or not binds to and inhibits the receptor.

  The present invention relates to human TNFδ and TNFε polypeptides, polynucleotides encoding the polypeptides, methods of producing the polypeptides (particularly by expressing the polynucleotides), and agonists of the polypeptides and Relates to antagonists. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists, and antagonists in applications. This application relates in part to research, diagnosis, and clinical technology. Accordingly, the present invention provides human TNFδ and TNFε polypeptides, polynucleotides encoding the polypeptides, methods of producing the polypeptides, and agonists and antagonists of the polypeptides.

(Summary of the Invention)
For these and other purposes, the amino acid sequences shown in FIGS. 1 and 2 and known amino acid sequences of other proteins in the tumor necrosis factor family such as human TNFα and TNFβ, referred to as novel TNFδ and TNFε. It is an object of the present invention to provide novel polypeptides that have been hypothetically identified as tumor necrosis factor ligands due to their homology.

  The polypeptides of the present invention have been identified as novel members of the TNF ligand superfamily based on structural and biological similarities.

  Furthermore, it is a further object of the invention to provide polynucleotides encoding TNFδ and TNFε, in particular polynucleotides encoding the polypeptides designated herein as TNFδ and TNFε.

  In a particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises regions encoding human TNFδ and TNFε in the sequences shown in FIGS.

  According to this aspect of the invention, there is provided an isolated nucleic acid molecule encoding human TNFδ, including mRNA, cDNA, genomic DNA, and in a further embodiment of this aspect of the invention, biological, diagnostic, Clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof (including fragments of variants, analogs and derivatives) are provided.

  Particularly preferred embodiments of this aspect of the invention include naturally occurring allelic variants of human TNFδ and TNFε.

  In accordance with this aspect of the invention, the mature human TNFδ polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97377 deposited on December 8, 1995, and the ATCC deposit deposited on March 1, 1996. An isolated nucleic acid molecule encoding a mature human TNFε polypeptide expressed by the human cDNA included in number 97457 is provided.

  Destroys several transformed cell lines, provides cell activation and proliferation, and provides functionally related TNFδ polypeptides, particularly human TNFδ and TNFε polypeptides, as primary mediators of immune regulation and inflammatory responses It is also an object of the present invention.

  In accordance with this aspect of the invention, novel polypeptides of human origin, referred to herein as TNFδ and TNFε, and biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, fragments thereof And variants and derivatives thereof, and analogs thereof, are provided.

  In particularly preferred embodiments of this aspect of the invention there are variants of human TNFδ and TNFε encoded by naturally occurring alleles of the human TNFδ and TNFε genes.

  It is another object of the present invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of variants and derivatives, and analogs thereof. In a preferred embodiment of this aspect of the invention, there are provided methods for producing the aforementioned TNFδ and TNFε polypeptides. This method comprises culturing a host cell having exogenously derived human TNFδ-encoding polynucleotide and TNFε-encoding polynucleotide operably incorporated therein under conditions for expression of human TNFδ and TNFε in the host. And then recovering the expressed polypeptide.

  In accordance with another object of the invention, there are provided products, compositions, processes, and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, diagnostic, and therapeutic purposes, among others. .

  In accordance with certain preferred embodiments of this aspect of the invention, products, compositions and, inter alia, the following methods are provided: cells by determining TNFδ and TNFε polypeptides or TNFδ encoding mRNA or TNFε encoding mRNA polypeptides A method for assessing TNFδ and TNFε expression in a subject; a method for assaying genetic mutations and abnormalities such as defects in the TNFδ and TNFε genes; A method of administering a TNFδ or TNFε polypeptide or polynucleotide to an organism.

  In accordance with certain preferred embodiments of this and other aspects of the invention, polynucleotides and particularly probes are provided that hybridize to human TNFδ or TNFε sequences.

  In certain further preferred embodiments of this aspect of the invention there are provided antibodies against TNFδ or TNFε polypeptides. In certain particularly preferred embodiments in this regard, the antibody is highly selective for human TNFδ or TNFε.

  In accordance with another aspect of the invention, a TNFδ or TNFε agonist is provided. Preferred agonists include molecules that mimic TNFδ or TNFε that bind to TNFδ binding or receptor molecules or TNFε binding molecules or receptor molecules, and molecules that elicit or increase TNFδ or ε-induced responses. In addition, preferred agonists interact with TNFδ and TNFε or TNFδ and TNFε polypeptides, or other modulators of TNFδ activity, thereby enhancing or increasing the effects of TNFδ and TNFε or more than one TNFδ and TNFε. There are molecules that do.

  In accordance with yet another aspect of the invention, TNFδ and TNFε antagonists are provided. Preferred antagonists include those that bind to TNFδ and TNFε receptors or binding molecules but mimic TNFδ and TNFε so as not to elicit TNFδ and TNFε-induced responses or more than one TNFδ and TNFε-induced responses. In addition, preferred antagonists include molecules that bind to or interact with TNFδ and TNFε to inhibit the effects of TNFδ and TNFε or more than one TNFδ and TNFε, or prevent the expression of TNFδ and TNFε. is there.

  Agonists and antagonists can be used to mimic, increase or inhibit the action of TNFδ and TNFε polypeptides. They can be used, for example, to prevent septic shock, inflammation, cerebral malaria, HIV virus activation, graft host rejection, bone resorption, rheumatoid arthritis, and cachexia.

  In a further aspect of the invention, compositions containing TNFδ and TNFε polynucleotides or TNFδ and TNFε polypeptides for administration to cells in vitro, ex vivo and in vivo, or administration to multicellular organisms are provided. In certain particularly preferred embodiments of this aspect of the invention, the composition contains TNFδ and TNFε polynucleotides for expression of TNFδ and TNFε polypeptides in a host organism for the treatment of disease. Particularly preferred in this regard is expression in human patients for the treatment of dysfunction associated with abnormal endogenous activity of TNFδ and TNFε.

  Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description. However, it should be understood that the following description and specific examples, while indicating preferred embodiments of the invention, are given for purposes of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the disclosure.

  The following illustrative descriptions are provided herein to facilitate understanding of certain terms frequently used herein, particularly in the examples. This description is provided for convenience and does not limit the invention.

  The term “digestion” of DNA refers to the catalytic cleavage of DNA with restriction enzymes that act only on specific sequences of DNA. The various restriction enzymes referred to herein are commercially available and the reaction conditions, cofactors and other requirements for use are known to those skilled in the art and are routine.

  For analytical purposes, typically 1 μg of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 μl of reaction buffer. For the purpose of isolating DNA fragments for plasmid construction, typically 5-50 μg of DNA is digested with 20-250 units of enzyme in a proportionally larger volume.

  Appropriate buffers and substrate weights for specific restriction enzymes are described in standard laboratory manuals such as those cited below, and they are specified by commercial suppliers.

  Incubation times of about 1 hour at 37 ° C. are usually used, but conditions can vary according to standard procedures, supplier instructions and reaction specificities. After digestion, the reaction can be analyzed and the fragments can be purified by electrophoresis through agarose or polyacrylamide gels using well-known methods routine to those skilled in the art.

  The term “genetic factor” generally refers to a polynucleotide comprising a region encoding a polypeptide, or a polynucleotide comprising a region that regulates transcription or translation or other processes important for expression of the polypeptide in a host cell, or It means a polynucleotide comprising both a peptide coding region and a region that regulates expression operably linked thereto.

  Genetic factors can be contained in vectors that replicate as episomal factors; that is, molecules that are physically independent of the host cell genome. They can be contained within a minichromosome, such as occurs during amplification of transfected DNA by methotrexate selection in eukaryotic cells. Genetic factors are also included within the host cell genome; rather than in their native state, rather in purified DNA after manipulation (eg, isolation, cloning and introduction into a host cell) or especially in the form of a vector Can be.

  The term “isolated” means altered “by the hand of man” from its natural state; that is, if it exists in nature, it is altered or removed from its original environment. Or both. As the term is used herein, for example, a naturally occurring polynucleotide or polypeptide that is naturally occurring in a living animal is not “isolated” but coexists in its native state. The same polynucleotide or polypeptide separated from the material is “isolated”. For example, with respect to a polynucleotide, the term isolated means that it is separated from naturally occurring chromosomes and cells.

  As part of or after isolation, such polynucleotides can be linked to other polynucleotides, e.g., for mutagenesis, to form fusion proteins, and for propagation or expression in the host. obtain. An isolated polynucleotide (alone or combined with another polynucleotide such as a vector) can be introduced into a host cell of a culture or whole organism, as the term is used herein, Even after this, such DNA is still isolated. This is because they are not naturally occurring forms or environments.

  Similarly, polynucleotides and polypeptides are not naturally occurring compositions and remain an isolated polynucleotide or polypeptide within the meaning of that term as used herein. Can be present in a composition such as a medium formulation, solution, eg, a chemical or enzymatic reaction, eg, a composition or solution for introduction of a polynucleotide or polypeptide into a cell .

  The term “ligation” refers to the process of forming a phosphodiester bond between two or more polynucleotides (often double stranded DNA). Techniques for ligation are well known in the art, and protocols for ligation are described in standard laboratory manuals and references (eg, Sambrook et al., Molecular Cloning, a Laboratory Manual, No. 1, cited below). 2nd edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al., Page 146).

  The term “oligonucleotide” refers to a relatively short polynucleotide. Often the term refers to single-stranded deoxyribonucleotides. However, it can likewise mean inter alia single-stranded or double-stranded ribonucleotides, RNA: DNA hybrids and double-stranded DNA.

  Oligonucleotides (eg, single stranded DNA probe oligonucleotides) are often synthesized by chemical methods such as those performed in automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods (including in vitro recombinant DNA-mediated techniques) and expression of DNA in cells and organisms.

  Initially, chemically synthesized DNA is typically obtained without a 5 'phosphate. The 5 'end of such oligonucleotides is not a substrate for phosphodiester bond formation by a ligation reaction using DNA ligase typically used to form recombinant DNA molecules. If ligation of such oligonucleotides is desired, phosphate can be added by standard techniques such as using kinases and ATP.

  The 3 ′ end of a chemically synthesized oligonucleotide generally has a free hydroxyl group, and in the presence of a ligase (eg, T4 DNA ligase), 5 of another polynucleotide (eg, another oligonucleotide). 'Easily forms phosphodiester bonds with phosphate. As is well known, this reaction can be selectively inhibited, if desired, by removing the 5 'phosphate of other polynucleotides prior to ligation.

  Plasmids are generally named herein according to standard naming conventions familiar to those skilled in the art, by the lowercase letter p and / or followed by uppercase letters and / or numbers. The starting plasmids disclosed herein are commercially available, are publicly available on a non-limiting basis, or are available by routine application of well-known published procedures Either can be constructed from a plasmid. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known to those of skill in the art and are readily available. In addition, one skilled in the art can readily construct any number of other plasmids suitable for use in the present invention. The characteristics, construction and use of such plasmids, and other vectors in the present invention will be readily apparent to those skilled in the art from this disclosure.

  The term “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide. This can be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, a polynucleotide as used herein includes, inter alia, 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 a mixture of single stranded and double stranded regions, single strand or more typically a hybrid molecule comprising DNA and RNA that can be double stranded or a mixture of single stranded and double stranded regions. . Furthermore, a polynucleotide as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions can be from the same molecule or different molecules. These regions may include all of one or more molecules, but more typically include only regions of some molecules. One of the molecules in the triple helix region is often an oligonucleotide.

  As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Furthermore, to name just two examples, DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases are polynucleotides as the term is used herein. is there.

  It will be appreciated that various modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as used herein is characterized by such chemically, enzymatically or metabolically modified forms of polynucleotides, and especially viruses and cells (including single and multicellular). Chemical forms of DNA and RNA.

  The term “polypeptide” as used herein includes all of the polypeptides as described below. The basic structure of a polypeptide is well known and described in a myriad of textbooks and other publications in the field. In this context, the term is used herein to mean any peptide or protein comprising two or more amino acids joined together in a linear fashion by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and long chains (which are commonly referred to in the art as proteins. , There are many types).

  Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and many amino acids (including terminal amino acids) are naturally processed (eg, processing and other translations). It will be understood that a given polypeptide can be modified by any of post-modification), as well as chemical modification techniques well known in the art. Even the common modifications that occur naturally in polypeptides are too numerous to be fully listed here, but they are found in basic textbooks and more detailed papers as well as numerous research literature. They are well described and are well known to those skilled in the art. Among the known modifications that may be present in the polypeptides of the invention, a few examples include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, Covalent attachment, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, covalent Bonding bridge formation, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorus Oxidation, prenylation, racemization, selenoylation, sulfate, Transfer RNA-mediated addition of amino acids to Park protein (e.g., arginylation), and there ubiquitination.

Such modifications are well known to those skilled in the art and are described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation are most basic textbooks (eg, Proteins-Structure). and Molecular Properties, 2nd edition, T.E. Creighton, WH Freeman and Company, New York (1993)). Many detailed reviews are available under this title (see, for example, Wald, F., Posttranslational Protein Modifications: Perspectives and Prospects, pages 1-12, Postrational Covalent Modification of C.
Press, New York (1983): Seifter et al., Analysis for protein modifications and
nonprotein cofactors. Meth. Enzymol. , 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663: 48-62 (1992)).

  It is well known and as described above, it is understood that polypeptides are not necessarily completely linear. For example, polypeptides can be branched as a result of ubiquitin binding, and they generally branch as a result of post-translational events (natural processing events and events caused by non-naturally occurring human manipulation). It can be cyclic with or without. Cyclic, branched, and branched cyclic polypeptides can be synthesized by untranslated natural processes and fully synthetic methods as well.

  Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chain, and the amino or carboxyl terminus. Indeed, the interference of amino groups or carboxyl groups or both in a polypeptide by covalent modifications is common to naturally occurring and synthetic polypeptides, and such modifications are also similar to those of the present invention. It can be present in a polypeptide. For example, E.I. The amino terminal residue of polypeptides made in E. coli is almost always N-formylmethionine prior to proteolytic processing.

  The modifications present in a polypeptide are often correlated with how it is made. For polypeptides made by expressing a cloned gene in a host, for example, the nature and extent of the majority of the modification depends on the post-translational modification capacity of the host cell and the modification signal present in the polypeptide amino acid sequence. It is determined. For example, as is well known, glycosylation often results in E. coli. It is not present in bacterial hosts such as E. coli. Thus, if glycosylation is desired, the polypeptide should be expressed in the glycosylating host (generally eukaryotic cells). Insect cells often perform the same post-translational glycosylation as mammalian cells. For this reason, insect cell expression systems have been developed, among other things, to efficiently express mammalian proteins with the natural pattern of glycosylation. Similar considerations apply to other modifications. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. In addition, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide includes all such modifications, particularly those present in polypeptides synthesized by expressing the polynucleotide in a host cell.

  As used herein, the term “variant” of a polynucleotide or polypeptide is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and are described in great detail elsewhere in this disclosure.

  A polynucleotide variant is a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. In general, the differences are limited so that the nucleotide sequences of the reference and variant are closely similar throughout and are identical in many regions. As described below, changes in the nucleotide sequence of the variant can be silent. That is, they cannot change the amino acid encoded by the polynucleotide. If the change is limited to this type of silent change, the variant encodes a polypeptide having the same amino acid sequence as the reference. As described below, changes in the nucleotide sequence of the variant may change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Such nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.

  A polypeptide variant is a polypeptide that differs in amino acid sequence from another, reference polypeptide. In general, the differences are limited such that the reference and variant sequences are closely similar throughout and are identical in many regions. 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.

  As used herein, the term “receptor molecule” refers to a molecule that specifically binds or interacts with the TNFδ or TNFε polypeptide of the present invention, including not only the classical receptors (which are preferred). Also included are other molecules that specifically bind to or interact with the polypeptides of the invention (these are also “binding molecules” and “interacting molecules”, and “TNFδ binding molecules” and “TNFδ interactions, respectively. Called “acting molecule” or “TNFε binding molecule” and “TNFε interacting molecule”). Can the binding between a polypeptide of the invention and such a molecule (including a receptor or binding or interacting molecule) be exclusive to the polypeptide of the invention (which is very highly preferred) Or can be highly specific for a polypeptide of the invention (which is highly preferred) or highly specific for a group of proteins comprising a polypeptide of the invention (which is preferred) Or may be specific for several groups of proteins, wherein at least one group comprises a polypeptide of the invention.

(Detailed description of the invention)
The present invention relates to, among other things, novel TNFδ and TNFε polypeptides, as described in greater detail below. In particular, the present invention relates to polypeptides and polynucleotides related to the TNF ligand superfamily by amino acid sequence homology. The invention particularly relates to the TNF δ having the nucleotide and amino acid sequence shown in FIG. 1 and the TNF nucleotide and amino acid sequence of the human cDNA of ATCC Accession No. 97377. The present invention also particularly relates to TNFε having the nucleotide and amino acid sequences shown in FIG. 2 and the TNFε nucleotide and amino acid sequences of the human cDNA of ATCC Accession No. 97457. These deposits are referred to hereinafter as deposit clones or “cDNA of the deposited clone”. It is understood that the nucleotide and amino acid sequences shown in FIGS. 1 and 2 were obtained by sequencing the human cDNA of the deposited clone. Thus, for any discrepancies between these two, the sequence of the deposited clone dominates and any reference to the sequence of FIGS. 1 and 2 includes a reference to the sequence of the human cDNA of the deposited clone.

  In accordance with one aspect of the present invention, there are provided isolated polypeptides encoding TNFδ and TNFε polypeptides having the deduced amino acid sequences of FIGS.

  Using the information provided herein, such as the polynucleotide sequence shown in FIG. 1, a polynucleotide of the invention encoding a human TNFδ polypeptide can be synthesized using standard cloning and screening procedures (eg, starting materials). As a procedure for cloning cDNA using mRNA derived from human tissue cells. As an illustration of the present invention, the polynucleotide shown in FIG. 1 was discovered in a cDNA library derived from cells of human heart tissue.

  The human TNFδ of the present invention is structurally related to other proteins of the TNF ligand superfamily, as shown by the results of sequencing the cDNA encoding human TNFδ in the deposited clone. The cDNA sequence thus obtained is shown in FIG. This includes an open reading frame encoding a protein of about 233 amino acid residues with a predicted molecular weight of about 25.871 kDa. This protein has the highest homology to TNFα among known proteins. The entire amino acid sequence of TNFδ in FIG. 1 has about 38% identity to the amino acid sequence of TNFα.

  A polynucleotide of the invention encoding a human TNFε polypeptide can be obtained using standard cloning and screening procedures (eg, for cloning cDNA using mRNA from cells of human tissue as starting material). Can be obtained. As an illustration of the present invention, the polynucleotide shown in FIG. 2 was discovered from a cDNA library derived from cells of human heart tissue.

  The human TNFε of the present invention is structurally related to other proteins of the TNF ligand superfamily, as shown by the results of sequencing the cDNA encoding human TNFε in the deposited clone. The cDNA sequence thus obtained is shown in FIG. The TNFε sequence is almost identical to the TNFδ sequence shown in FIG. Accordingly, TNFε is a splicing variant of TNFδ. TNFε contains 168 amino acid residues, and the sequence of FIG. 2 shows the mature protein of TNFε without any N-terminal hydrophobic region. This protein shows the highest homology to TNFα. 2 has about 20% identity to the amino acid sequence of TNFα.

  The polynucleotides of the present invention are in the form of RNA, such as mRNA, or in the form of DNA (eg, including cDNA and genomic DNA obtained by cloning or produced by chemical synthesis techniques, or combinations thereof) It can be. DNA can be double-stranded or single-stranded. Single-stranded DNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also referred to as the antisense strand).

  The coding sequence encoding the polypeptide may be identical to the coding sequence of the polynucleotide shown in FIGS. This can also be a polynucleotide having a different sequence encoding the polynucleotide of the DNA of FIGS. 1 and 2 as a result of the degeneracy of the genetic code.

  The polynucleotides of the invention that encode the polypeptides of FIGS. 1 and 2 may include, but are not limited to: the coding sequence of the mature polypeptide itself; the coding sequence of the mature polypeptide and additional coding sequences ( For example, a sequence encoding a leader or secretory sequence such as a pre- or pro- or prepro-protein sequence); a coding sequence of a mature polypeptide with or without the additional coding sequence described above, as well as a further non-coding sequence (Eg, introns and non-coding 5 ′ and 3 ′ sequences such as transcribed, untranslated sequences that play a role in transcription, mRNA processing (eg, including splicing and polyadenylation signals), ribosome binding, and mRNA stability. Provide additional functionality Additional coding sequence which encode additional amino acids, such as amino acids.

  Thus, for example, a polypeptide can be fused to a marker sequence such as a peptide, thereby facilitating purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc.), many of which are commercially available. Gentz et al., Proc. Natl. Acad. Sci. , USA, 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. The HA tag corresponds, for example, to an epitope derived from the influenza hemagglutinin protein described in Wilson et al., Cell, 37: 767 (1984).

  In accordance with the above, as used herein, the term “polynucleotide encoding a polypeptide” refers to a sequence encoding the polypeptides of the present invention, particularly human TNFδ and TNFε having the amino acid sequences shown in FIGS. A polynucleotide comprising The term is a polynucleotide comprising a single contiguous or non-contiguous region encoding a polypeptide (eg, interrupted by an intron), along with additional regions that may also include coding and / or non-coding sequences. Is included.

  The present invention further relates to variants of the polynucleotides herein above that encode fragments, analogs, and derivatives of the polypeptides having the deduced amino acid sequence of FIGS. A variant of the polynucleotide can be a naturally occurring variant such as a naturally occurring allelic variant, or it can be a variant that is not known to occur naturally. Such non-naturally occurring polypeptide variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.

  Variants in this regard include variants that differ from the above polynucleotides in nucleotide substitutions, deletions or additions. A substitution, deletion, or addition can include one or more nucleotides. A variant may be a change in the coding or non-coding region or both. Changes in the coding region can produce conservative or non-conservative amino acid substitutions, deletions, or additions.

  Particularly preferred embodiments of the invention in this regard include polynucleotides encoding polypeptides having the amino acid sequences of TNFδ and TNFε shown in FIGS. 1 and 2; variants, analogs, derivatives, and fragments, and variants , Analog, and derivative fragments.

  Even more particularly preferred in this regard are several, several, 5-10, 1-5, 1-3, 1, 1 or 0 amino acid residues. Is a polynucleotide encoding TNFδ and TNFε having the amino acid sequences of the TNFδ and TNFε polypeptides of FIGS. 1 and 2, substituted, deleted, or added in any combination. Particularly preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of TNFδ and TNFε. Also particularly preferred in this regard are conservative substitutions. Most highly preferred are polynucleotides that encode polypeptides having the amino acid sequences of FIGS. 1 and 2 without substitution. A further preferred embodiment of the present invention is a polynucleotide that is at least 70% identical to a polynucleotide encoding a TNFδ and TNFε polypeptide having the amino acid sequence shown in FIGS. 1 and 2, and is complementary to such a polynucleotide. It is a polynucleotide. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical to polynucleotides encoding TNFδ and TNFε polypeptides, and polynucleotides complementary thereto. In this regard, polynucleotides that are at least 90% identical to polynucleotides encoding TNFδ and TNFε polypeptides are particularly preferred, and of these particularly preferred polynucleotides, those having at least 95% identity are particularly preferred. Further, among those having at least 95% identity, those having at least 97% identity are highly preferred, and those having at least 98% and at least 99% identity are particularly highly preferred, at least 99% Those having% identity are more particularly preferred.

  Particularly preferred embodiments in this regard are further polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIGS. .

  The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention particularly relates to polynucleotides that hybridize to the polynucleotides herein above under stringent conditions. As used herein, the term “stringent conditions” refers to hybridization in which at least 95% and preferably at least 97% of the bases between sequences are complementary (eg, G: C; A: T ) Means what happens.

  As discussed further herein with respect to the polynucleotide assays of the invention, for example, the polynucleotides of the invention described above can be used as hybridization probes for cDNA and genomic DNA as full-length cDNAs encoding TNFδ and TNFε and It can be used to isolate genomic clones and to isolate cDNA and genomic clones of other genes with high sequence similarity to the human TNFδ and TNFε genes. Such probes generally contain at least 15 bases. Preferably, such probes have at least 30 bases and can have at least 50 bases.

  For example, the coding regions of the TNFδ and TNFε genes can be isolated by screening with known DNA sequences to synthesize oligonucleotide probes. The labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention is then used to screen a library of human cDNA, genomic DNA, or mRNA, and the probe is hybridized to any member of the library. It is decided whether to soy.

  The polynucleotides and polypeptides of the present invention can be utilized as research reagents and materials for the discovery of treatments and diagnostics for human diseases, particularly as discussed further herein with respect to polynucleotide assays.

  A polynucleotide has a polypeptide that is a mature protein and an additional amino- or carboxyl-terminal amino acid, or an amino acid that is internal to the mature polypeptide (eg, a polypeptide chain with more than one mature form) If). Such sequences can inter alia play a role in the processing of the protein from the precursor to the mature form, can facilitate protein trafficking, can increase or decrease the half-life of the protein, or can be of assay or production Can facilitate the manipulation of proteins. As is common in the case of in situ, additional amino acids can be processed away from the mature protein by cellular enzymes.

  A precursor protein having a mature form of a polypeptide fused to one or more prosequences can be an inactive form of the polypeptide. Such inactive precursors are generally activated when the prosequence is removed. Some or all of the prosequences can be removed prior to activation. In general, such precursors are called proproteins.

  In summary, a polynucleotide of the present invention comprises a mature protein, a mature protein and a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more pro sequences that are not leader sequences of the preprotein. Or a preproprotein having a leader sequence and one or more prosequences, which are generally removed during the processing step to produce an active and mature form of the polypeptide, which is a precursor to the proprotein Can be coded).

  Deposits containing human TNFδ and human TNFε cDNA have been deposited with the American Type Culture Collection as described above. Also, as described above, the cDNA deposit is referred to herein as “deposited clone” or “cDNA of the deposited clone”. The clone was deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852 USA on December 8, 1995 and March 1, 1996, and assigned ATCC Accession Nos. 97377 and 97457, respectively. It was. The deposited material is the pBluescript SK (−) plasmid (Stratagene, La Jolla, Calif.) Containing full length TNFδ and TNFε human cDNA.

  The deposit was made under the terms of the Budapest Convention on the international recognition of the deposit of microorganisms for the purpose of patent procedures. Shares are released to the public unalterable and without restrictions or conditions upon patent issuance. The deposit is provided merely as a convenience to those skilled in the art and is not an admission that the deposit is required for patentability as required under 35 USC 112. The sequence of the polynucleotide contained in the deposited material, and the amino acid sequence of the polypeptide encoded thereby, are controlled in any event of conflict with any description of the sequences herein. A license may be required to make, use, or sell the deposited material, and no such license is given here.

  The invention further relates to human TNFδ and TNFε polypeptides having the deduced amino acid sequences of FIGS. 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. The terms “fragment”, “derivative”, and “analog” when referring to the polypeptides of FIGS. 1 and 2 are polypeptides that retain essentially the same biological function or activity as such polypeptides. Means peptide. Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

  The polypeptide fragments, derivatives, or analogs of FIGS. 1 and 2 can be: (i) one or more amino acid residues are conserved or non-conserved amino acid residues ( Preferably, conserved amino acid residues) and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) one or more An amino acid residue containing a substituent, or (iii) a mature polypeptide fused to another compound such as a compound that increases the half-life of the polypeptide (eg, polyethylene glycol), or (iv) ) Additional amino acids are mature polypeptides (eg, leader sequences, or secretory sequences, or mature polypeptides or proproteins). Those fused to a sequence) that are used for purification of quality sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

  Particularly preferred embodiments of the present invention in this regard include polypeptides having the amino acid sequences TNFδ and TNFε shown in FIGS. 1 and 2, variants, analogs, derivatives and fragments thereof, and variants of the fragments, There are analogs and derivatives. Alternatively, a particularly preferred embodiment of the invention in this regard is the use of polypeptides having the amino acid sequences of TNFδ and TNFε of human cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and modifications of the fragments Bodies, analogs, and derivatives.

  Among preferred variants are those in which conservative amino acid substitutions differ from the reference. Such a substitution is a substitution that substitutes a given amino acid of a polypeptide with another amino acid having similar properties. Typically observed as conservative substitutions are substitutions between the aliphatic amino acids Ala, Val, Leu, and Ile; substitution of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, Substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  Further particularly preferred in this regard are variants, analogs, derivatives and fragments having the amino acid sequence of the TNFδ and TNFε polypeptides of FIGS. 1 and 2, or the amino acid sequence of the cDNA in the deposited clone, and fragments thereof. Variants, analogs, and derivatives, where several, several, 5-10, 1-5, 1-3, 2, 1, or 0 Amino acid residues are substituted, deleted, or added in any combination. Particularly preferred among these are silent substitutions, additions and deletions that do not alter the properties and activities of TNFδ and TNFε. Also particularly preferred in this regard are conservative substitutions. Most highly preferred is a polypeptide having the amino acid sequence of FIGS. 1 and 2 without substitution.

  The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form and are preferably purified to homogeneity.

  The TGFδ polypeptide of the present invention has a polypeptide of SEQ ID NO: 2 (especially a mature polypeptide) as well as a polypeptide of SEQ ID NO: 2 of at least 70% similarity (preferably at least 70% identity), 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 (more preferably) to the polypeptide of SEQ ID NO: 2. At least 95% identity) and also a portion of such a polypeptide, wherein such portion of the polypeptide is generally at least 30 amino acids and more preferably at least 50 Including those containing amino acids.

  The TGFε polypeptide of the present invention has a polypeptide of SEQ ID NO: 4 (especially a mature polypeptide) and 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 (more preferably to the polypeptide of SEQ ID NO: 4). At least 95% identity) and also a portion of such a polypeptide, wherein such portion of the polypeptide is generally at least 30 amino acids and more preferably at least 50 Including those containing amino acids.

  As is known in the art, “similarity” between two polypeptides is the comparison of the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. Determined by.

  Fragments or portions of the polypeptides of the invention can be used to produce the corresponding full-length polypeptide by peptide synthesis; therefore, the fragment is used as an intermediate to produce the full-length polypeptide. obtain. Fragments or portions of the polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention.

  A fragment is a polypeptide having an amino acid sequence that is entirely identical to a sequence that is part but not all of the amino acid sequence of the aforementioned TNFδ and TNFε polypeptides and variants or derivatives thereof. Such fragments can be “free standing” (ie not part of or fused to other amino acids or polypeptides) or they can be larger polys in which they form part or region. It can be contained within a peptide. When included within a larger polypeptide, the currently discussed fragments most preferably form a single continuous region. However, several fragments can be contained within a single larger polypeptide. For example, certain preferred embodiments are for expression in a host where the heterologous pre- and propolypeptide regions are fused to the amino acid termini of TNFδ and TNFε fragments, and an additional region is fused to the carboxyl terminus of the fragment. It relates to fragments of the TNFδ and TNFε polypeptides of the present invention contained within the designed precursor polypeptide. Thus, a fragment in one aspect of the meaning intended herein refers to a portion of a fusion polypeptide or fusion protein derived from TNFδ and TNFε.

  As representative examples of polypeptide fragments of the present invention, mention may be made of fragments having about 30 to about 233 amino acids. In this context, “about” is greater than the indicated range and only a few, some 5, 4, 3, 2, or 1 amino acid in either or both limits, or Includes smaller ranges. For example, about 100-233 amino acids in this context are 100 ± several, some 5, 4, 3, 2, or 1 amino acid to 233 ± several, some 5, 4, 3, 2 Or a polypeptide of one amino acid residue (ie, from 100 minus a few amino acids up to 233 plus a few amino acids up to 100 plus a few amino acids up to 233 minus a few amino acids narrow) Means a peptide fragment.

  Highly preferred in this respect is the indicated range that includes an increase or decrease of about 5 amino acids in either or both extreme values. Very particular preference is given to the indicated ranges including an increase or decrease of about 3 amino acids at either or both end values. Very particular preference is given to the indicated ranges which include an increase or decrease of one amino acid at either or both extreme values or indicated ranges without additions or deletions. Most highly preferred in this regard are fragments of about 15 to about 233 amino acids.

  Among the particularly preferred fragments of the present invention are truncated variants of TNFδ and TNFε. A truncation variant is a deletion of a contiguous series of residues including the amino terminus (ie, contiguous region, part, or portion) or a contiguous series of residues including the carboxyl terminus, or a double truncation variant TNFδ and TNFε polypeptides having the amino acids of FIG. 1 or 2, excluding the deletion of two consecutive series of residues, one containing the amino terminus and one containing the carboxyl terminus, Or a variant or derivative thereof. Fragments having the above size range are also preferred embodiments of cleavage fragments, which are generally particularly preferred among fragments.

  Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of TNFδ and TNFε. In this regard, in preferred embodiments of the present invention, the α and α helix forming regions (“α region”), β sheet and β sheet forming region (“β region”), turns and turn forming regions of TNFδ and TNFε (“ Turn region "), coil and coil forming region (" coil region "), hydrophilic region, hydrophobic region, α amphiphilic region, β amphiphilic region, movable part, surface forming region, and highly antigenic indicator region Including fragments.

Particular preferred regions in these respects include regions of the aforementioned type shown in FIG. 4 for TNFδ and FIG. 5 for TNFε and identified by amino acid sequence analysis shown in FIGS. It is not limited to. As shown in FIGS. 4 and 5, such a preferred region is Garnier-Robson.
α region, β region, turn region and coil region, Chou-Fasman α region, β region and turn region, Kyte-Doolittle hydrophilic region and hydrophilic region, Eisenberg α and β amphiphilic region, Karplus-Schulz movable part, Includes the Emini surface-forming region, as well as the Jameson-Wolf highly antigenic indicator region.

  Some highly preferred fragments in this regard include regions of TNFδ and TNFε that combine several structural features (eg, some of the features shown above). In this respect, regions defined by residues following the signal peptide region of FIGS. 1, 2, 4, and 5 (all of which are turn regions, hydrophilic regions, moving parts, surface forming regions, and high antigenicity indicators) (Characterized by the amino acid composition that is very characteristic of the region) is a particularly highly preferred region. Such a region can be contained within a larger polypeptide or, as discussed above, can be a preferred fragment of the invention itself. The term “about” as used in this paragraph is generally understood to have the above-mentioned meaning with respect to fragments.

  Further preferred regions are those that mediate TNFδ and TNFε activity. Most highly preferred in this regard are fragments having chemical, biological, or other activity of TNFδ and TNFε, which have similar or improved activity or reduced undesirable activity. Including what you have. Highly preferred in this regard are fragments comprising regions that are homologous to the active region of the sequence or position or both sequences and to the related polypeptide (eg, the related polypeptide shown in FIG. 3). Yes, this includes human TNFα and β. Among the particularly preferred fragments in this regard are truncation mutants as described above.

  The present invention also includes, inter alia, polynucleotides that encode the aforementioned fragments, polynucleotides that hybridize to polynucleotides that encode fragments (particularly those that hybridize under stringent conditions), and polynucleotides that encode fragments. Is understood to relate to a polynucleotide (eg, a PCR primer) for amplifying. In these respects, preferred polynucleotides correspond to preferred fragments as described above.

  The invention also relates to vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors of the invention, and production of the polypeptides of the invention by recombinant techniques.

  Host cells can be genetically engineered to incorporate a polynucleotide of the invention and express a polypeptide of the invention. For example, a polynucleotide can be introduced into a host cell using well known techniques of infection, transduction, transfection, transfection, and transformation. The polynucleotide can be introduced alone or in combination with other polynucleotides. Such other polynucleotides can be introduced independently to the polynucleotide of the invention, can be co-introduced, or can be introduced in conjunction. Thus, for example, a polynucleotide of the invention can be transferred to a host cell along with another separate polynucleotide encoding a selectable marker using, for example, standard techniques for cotransfection and selection in mammalian cells. Can be transfected. In this case, the polynucleotide is generally stably integrated into the host cell genome.

  Alternatively, the polynucleotide can be linked to a vector containing a selectable marker for growth in a host. Vector constructs can be introduced into host cells by the techniques described above. Generally, a plasmid vector is introduced as DNA in a precipitate (eg, calcium phosphate precipitate) or complex with a charged lipid. Electroporation can also be used to introduce a polynucleotide into a host. If the vector is a virus, it can be encapsulated in vitro or introduced into a packaging cell, and the encapsulated virus can transduce the cell. In accordance with this aspect of the invention, a wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells are well known and routine to those of skill in the art. Such techniques are fully outlined in Sambrook et al. (Supra), which is a description of many laboratory manuals detailing these techniques. According to this aspect of the invention, the vector can be, for example, a plasmid vector, a single-stranded or double-stranded phage vector, a single-stranded or double-stranded RNA or a DNA viral vector. Such vectors can be introduced into cells as polynucleotides (preferably DNA) by well-known techniques for introducing DNA and RNA into cells. Vectors (in the case of phage and viral vectors) can also be and are preferably introduced into cells as encapsulated or encapsidated virus by well-known techniques for infection and transduction. Viral vectors can be replication competent or replication defective. In the latter case, viral propagation generally occurs only in complementary host cells.

  Preferred among the vectors are in certain respects for the expression of the polynucleotides and polypeptides of the invention. In general, such vectors contain a cis-acting control region effective for expression in the host operably linked to the polynucleotide to be expressed. Suitable trans-acting factors are either supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon transduction into the host.

  In certain preferred embodiments in this regard, the vector provides specific expression. Such specific expression can be inducible expression or expression only in a particular type of cell, or both inducible and cell specific. Particularly preferred among inducible vectors are vectors whose expression can be induced by ambient factors that are easy to manipulate (eg, temperature and nutrient additives). Various vectors suitable for this aspect of the invention include constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, which are well known to those of skill in the art and used routinely. Is done.

  Engineered host cells can be cultured in conventional nutrient media, which can be modified as appropriate, particularly for activating promoters, selecting for transformants, or amplifying genes. . The culture conditions (eg, temperature, pH, etc.) previously used in the host cell selected for expression are generally appropriate for expression of the polypeptides of the invention, as will be apparent to those skilled 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 virally derived vectors. For example, from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, baculoviruses, papovaviruses such as SV40, vaccinia viruses, adenoviruses, chicken pox viruses, pseudorabies viruses, and retroviruses And vectors derived from combinations thereof (eg, from plasmids and bacteriophage genetic elements such as cosmids or phagemids). All of these can be used for expression according to this aspect of the invention. In general, any vector suitable for maintaining, propagating or expressing a polynucleotide for expressing a polypeptide in a host can be used for expression in this regard.

  The appropriate DNA sequence can be inserted into the vector by any of a variety of well known and routine techniques. In general, the DNA sequence for expression is transferred to the expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then ligating them together with a restriction fragment using T4 DNA ligase. Combined. The limitations and linking procedures that can be used for this purpose are well known and routine to those skilled in the art. Appropriate procedures in this regard, and appropriate procedures for constructing expression vectors using other techniques, which are also well known and routine to those skilled in the art, are described in Sambrook et al. (Cited elsewhere) is shown in great detail.

  The DNA sequence of the expression vector is operably linked to appropriate expression control sequence (s) including, for example, a promoter that directs mRNA transcription. Representative of such promoters are lambda phage PL promoters, E. coli. E. coli lac, trp and tac promoters, SV40 early and late promoters, and retroviral LTR promoters, and only a few of the well-known promoters are listed. A number of undescribed promoters that are suitable for use in this aspect of the invention are well known and can be readily used by those skilled in the art in the manner illustrated by the discussion and examples herein. It is understood.

  In general, expression constructs contain sites for transcription initiation and termination and in the transcription region contain ribosome binding sites for translation. The coding portion of the mature transcript expressed by the construct includes a transcription initiation AUG at the start and a stop codon appropriately located at the end of the translated polypeptide.

  In addition, the construct may include control regions that regulate as well as engender expression. In general, such regions operate by controlling transcription (eg, in particular, repressor binding sites and enhancers) according to many commonly practiced procedures.

  Vectors for propagation and expression generally contain a selectable marker. Such markers can also be suitable for amplification, or the vector can contain additional markers for this purpose. In this regard, the expression vector preferably contains one or more selectable marker genes and provides phenotypic characteristics for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and E. coli. Examples include tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.

Vectors containing appropriate DNA sequences as described elsewhere herein, as well as appropriate promoters, and other appropriate control sequences may be obtained using a variety of well-known techniques suitable for expression therein of a desired polypeptide. Can be used to introduce into a suitable host. Representative examples of suitable hosts include E. coli. bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; Drosophila S2 and Spodoptera
Insect cells such as Sf9 cells; animal cells such as CHO, COS, and Bowes melanoma cells; and plant cells. Hosts for various expression constructs are well known, and one of skill in the art can readily select a host for expressing a polypeptide according to this aspect of the invention according to this disclosure.

  More particularly, the invention also includes a recombinant construct, such as an expression construct, which includes one or more of the sequences described above. The construct includes a vector, such as a plasmid or viral vector, which is inserted into such a sequence of the invention. The sequence can be inserted in the forward or reverse direction. In a particularly preferred embodiment in this regard, the construct further comprises a regulatory sequence (eg, comprising a promoter) operably linked to the sequence. Numerous 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.

  The following vectors are commercially available and are provided as examples. Among the preferred vectors for use in bacteria are pQE70, pQE60 and pQE9 available from Qiagen; pBS vectors available from Stratagene, Pagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A; and Pharmacia There are ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. These vectors are listed alone as examples of many commercially and well-known vectors and are available to those skilled in the art for use in accordance with this aspect of the invention. For example, it is understood that any other plasmid or vector suitable for introduction, maintenance, propagation, or expression of a polynucleotide or polypeptide of the invention in a host can be used in this aspect of the invention.

  A promoter region is a restriction site for introducing a reporter transcription unit lacking a promoter region (eg, a chloramphenicol acetyltransferase (“cat”) transcription unit) into a candidate promoter fragment (ie, a fragment that can contain a promoter). The vector (s) downstream can be used to select from any desired gene. As is well known, introduction of a promoter-containing fragment of a restriction site upstream of the cat gene into a vector results in the production of CAT activity, which can be detected by standard CAT assays. Suitable vectors for this purpose are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of the polynucleotides of the present invention include not only well-known and readily available promoters, but also promoters that can be easily obtained using the reporter gene by the techniques described above.

  Among the known bacterial promoters suitable for expression of polynucleotides and polypeptides according to the invention are E. coli. There are E. coli lacI and lacZ and promoter, T3 and T7 promoter, gpt promoter, λPR, PL promoter, and trp promoter. Among the known eukaryotic promoters suitable in this regard are CMV immediate early promoters, HSV thymidine kinase promoters, early and late SV40 promoters, promoters of retroviral LTRs such as Rous sarcoma virus (“RSV”), and There are metallothionein promoters such as the mouse metallothionein-I promoter. Selection of appropriate vectors and promoters for expression in the host cell is a well-known procedure, and techniques necessary for expression of the vector construct, introduction of the vector into the host, and expression in the host are known to those of skill in the art. Is everyday.

  The present invention also relates to host cells comprising 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.

  Introduction of the construct into the host cell can be done 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 (eg, Davis et al., Basic Methods in Molecular Biology, (1986)). The construct in the host cell can be used in conventional ways to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the present invention can be produced synthetically by conventional peptide synthesizers.

  The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. Cell-free translation systems can also be used to produce such proteins using RNA from the DNA constructs of the present invention. Suitable cloning and expression vectors for use in prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).

  In general, recombinant expression vectors are selectable that allow isolation of a vector-containing cell after exposure to the origin of replication, a promoter from a highly expressed gene that directs transcription of downstream structural sequences, and the vector. Contains a marker. Among suitable promoters are those derived from genes encoding saccharifying enzymes (eg, 3-phosphoglycerate kinase (“PGK”)), a-factor, acid phosphatase, and heat shock proteins, among others. Selectable markers include E.I. E. coli ampicillin resistance gene and S. coli. It includes the cerevisiae trp1 gene.

  Transcription of a DNA encoding the polypeptide of the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are usually cis-acting elements of about 10-300 bp of DNA and act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer (located on the late side of the replication origin from bp 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

  A polynucleotide of the invention that encodes a heterologous structural sequence of a polypeptide of the invention will generally be transferred to a vector using standard techniques, such that it is operably linked to a promoter for expression. Inserted. The polynucleotide is positioned so that the transcription start site is properly located 5 'of the ribosome binding site. The ribosome binding site is 5 'of the AUG that initiates translation of the polypeptide to be expressed. In general, there are no other open reading frames that begin with an initiation codon (usually AUG) and are located between the ribosome binding site and the initiation AUG. Also, generally, there is a translation stop codon at the end of the polypeptide, and there is a polyadenylation signal and a transcription end signal appropriately positioned at the 3 'end of the transcribed region.

  Appropriate secretion signals can be incorporated into the expressed polypeptide for secretion of the translated protein into the lumen, peripheral space, or extracellular environment of the endoplasmic reticulum. The signals can be endogenous to the polypeptide or they can be heterologous signals.

  The polypeptide is expressed in a modified form (eg, a fusion protein) and can include not only secretion signals, but also additional heterologous functional regions. Thus, for example, a region of additional amino acids (especially altered amino acids) can be added to the N-terminus of a polypeptide to improve stability and persistence in the host cell during purification or subsequent handling and storage. Can be done. Regions can also be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. In particular, the addition of peptide moieties to polypeptides to cause secretion or excretion, to improve stability, and to facilitate purification is well known and routine in the art. is there.

  Prokaryotic hosts suitable for propagation, maintenance, or expression of polynucleotides and polypeptides according to the present invention include Escherichia coli, Bacillus subtilis, and Salmonella typhimurium. Various species of Pseudomonas, Streptomyces, and Staphylococcus are suitable hosts in this regard. In addition, many other hosts known to those skilled in the art can also be used in this regard.

  As a representative but non-limiting example, useful expression vectors for bacterial use include selectable markers derived from commercial plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017) and bacterial A replication origin can be included. Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 “backbone” portions are combined with a suitable promoter and the structural sequence to be expressed.

  Transformation of the appropriate host strain below and growth of the host strain to the appropriate cell density, where the selected promoter is inducible, can be accomplished by any suitable means (eg, temperature shift or chemical inducer The cells are cultured for an additional period of time. The cells are then typically collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is maintained for further purification. Microbial cells used in protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cytolytic agents, and these methods are known to those skilled in the art. Is well known.

  A variety of mammalian cell culture systems can be used for expression as well. Examples of mammalian expression systems include the monkey kidney fibroblast COS-7 strain described in Gluzman et al., Cell, 23: 175 (1981). Other cell lines that can express compatible vectors include, for example, C127, 3T3, CHO, HeLa, human kidney 293, and BHK cell lines.

  Mammalian expression vectors contain an origin of replication, appropriate promoters and enhancers, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 ′ flanking non-transcribed sequences ( These are necessary for expression). In certain preferred embodiments in this regard, DNA sequences derived from SV40 splice sites and SV40 polyadenylation sites are used for these types of required non-transcribed genetic elements.

  The polypeptides of the present invention are recovered and well-known methods (ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and Purified from recombinant cell culture by lectin chromatography). Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. Well-known methods for protein refolding can be used to regenerate the active conformation when the polypeptide is denatured during isolation and purification.

  The polypeptides of the present invention include natural purified products, products of chemical synthesis procedures, and recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). The product produced is mentioned. Depending on the host used in the recombinant production procedure, the polypeptides of the invention may or may not be glycosylated. In addition, the polypeptides of the present invention may also include an initial modified methionine residue in some cases, such as the result of a host-mediated process.

  The polynucleotides and polypeptides of the present invention can be used in accordance with the present invention for a variety of applications, particularly applications that utilize the chemical and biological properties of TNFδ and TNFε. These include mediating apoptosis, cell activation and proliferation of transformed cell lines, and immunomodulating antibacterial agents, antiviral agents, and primary mediators of susceptibility to inflammatory responses to pathogens. Further applications relate to the diagnosis and treatment of cellular, tissue and biological disorders. These aspects of the invention are further illustrated by the following discussion.

  The invention also relates to the use (eg, as a diagnostic reagent) of the polynucleotides of the invention to detect complementary polynucleotides. Detection of mutant forms of the polypeptides of the invention associated with dysfunction may result in diseases (eg, neoplasia such as tumors) or diseases resulting from underexpression, overexpression, or altered expression of the polypeptides of the invention. Provides a diagnostic tool that can add or define a diagnosis of susceptibility.

  An individual having a mutation in the gene of the present invention can be detected at the DNA level by various techniques. Nucleic acids for diagnosis can be obtained from patient cells (blood, urine, saliva, tissue biopsy, and autopsy material). Genomic DNA can be used for direct detection or can be amplified enzymatically prior to analysis using PCR. PCR (Saiki et al., Nature, 324: 163-166 1986). RNA or cDNA can also be used as well. As an example, PCR primers that are complementary to a nucleic acid encoding TNFδ or TNFε can be used to identify and analyze TNFδ or TNFε expression and mutations. For example, deletions and insertions can be detected by a change in size when compared to the normal genotype of the amplified product. Point mutations can be identified by hybridizing amplified DNA to radiolabeled TNFδ or TNFε RNA or radiolabeled TNFδ or TNFε 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 the reference gene and genes with mutations can also be revealed by direct DNA sequencing. Furthermore, the cloned DNA segment can be used as a probe for detecting a specific DNA segment. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, sequencing primers are used with double stranded PCR products or single stranded template molecules generated by modified PCR. Sequencing is performed by conventional procedures using radiolabeled nucleotides or by automated sequencing procedures using fluorescent tags.

  Genetic testing based on DNA sequence differences can be accomplished by detecting changes in the 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. Different sequence DNA fragments can be distinguished in denaturing formamide gradient gels. Here the mobility of different DNA fragments in the gel is delayed in the gel at different positions according to their specific melting temperature or partial melting temperature (see eg Myers et al., Science, 230: 1242 (1985)). ).

  Sequence changes at specific positions may also occur in nuclease protection assays (eg, RNase protection and S1 protection or chemical cleavage methods (eg, Cotton et al., Proc. Natl. Acad. Sci., 83: 4397-4401 (1985)). )). Thus, the detection of specific DNA sequences can be achieved by hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes (eg, restriction fragment length polymorphism (RFLP)) and Southern blotting of genomic DNA. It can be achieved by such a method. In addition to more traditional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

  The sequences of the present invention are also useful for chromosome identification. A sequence can specifically target and hybridize to a particular location on an individual human chromosome. Furthermore, there is currently a need to identify specific sites on the chromosome. Currently, there are few chromosomal labeling reagents based on actual sequence data (repetitive polymorphisms) available for labeling chromosomal locations. Mapping of DNA to chromosomes according to the present invention is an important first step in correlating these sequences with genes associated with disease.

  In a particular embodiment in this regard, the cDNA disclosed herein is used to clone the genomic DNA of the gene of the present invention. This can be accomplished using a variety of well-known techniques and commonly available libraries. Genomic DNA is used for in situ chromosome mapping using well-known techniques for this purpose. Typically, several attempts and errors, following routine procedures for chromosome mapping, may be necessary to identify genomic probes that give good in situ hybridization signals.

  In some cases, further, the sequence can be mapped to a chromosome 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 and thus do not complicate 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 specific DNA to specific chromosomes. Using the same oligonucleotide primers in conjunction with the present invention, partial localization can be achieved using a panel of fragments from a particular chromosome or a pool of large genomic clones in a similar manner. Other mapping strategies that can also be used to map to chromosomes are for in situ hybridization, pre-screening with flow-sorted chromosomes, and for constructing chromosome-specific cDNA libraries. Includes pre-selection by hybridization.

Fluorescence in situ hybridization (“FISH”) to metaphase spreads of cDNA clones can be used to provide accurate chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60. For a review of this technology, see Verma et al., Human Chromosomes: A Manual of Basic Techniques,
See Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data is, for example, V.I.
Found in McKusick, Mendelian Inheritance in Man (available online from Johns Hopkins University, Welch Medical Library). The relationship between the gene mapped to the same chromosomal region and the disease is then identified by linkage analysis (co-inheritance of physically adjacent genes).

  Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all affected individuals but not in any normal individual, the mutation appears to be a causative agent of the disease.

  At the current resolution of physical and genetic mapping techniques, a cDNA that is accurately positioned in a chromosomal region associated with disease can be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and 1 gene per 20 kb).

  The invention also relates to diagnostic assays, such as quantitative and diagnostic assays for detecting levels of proteins of the invention in cells and tissues, including determining normal and abnormal levels. Thus, for example, a diagnostic assay according to the present invention for detecting overexpression of a TNF protein of the present invention compared to a normal control tissue sample can be used, for example, to detect the presence of neoplasia. Assay techniques that can be used to determine levels of a protein (eg, a protein of the present invention) in a sample derived from the 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. Of these, ELISA is often preferred. The ELISA assay first comprises the step of preparing an antibody (preferably a monoclonal antibody) specific for the protein of the present invention. In addition, reporter antibodies that bind to monoclonal antibodies are generally prepared. The reporter antibody is attached to a detection reagent (eg, radioactive, fluorescent, or enzymatic, in this example a horseradish peroxidase enzyme).

  To perform an ELISA assay, a sample is removed from the host and incubated on a solid support (eg, a polystyrene dish) that binds protein in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein (eg, bovine serum albumin). The monoclonal antibody is then incubated in the dish for the time that the monoclonal antibody attaches to any protein of the invention attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer. Reporter antibody conjugated to horseradish peroxidase is placed in the dish, resulting in binding of the reporter antibody to any monoclonal antibody conjugated to the protein of the invention. The unattached reporter antibody is then washed out. A reagent for peroxidase activity (including a colorimetric substrate) is then added to the dish. Immobilized peroxidase linked to the protein of the invention via primary and secondary antibodies produces a colored reaction product. The amount of color developed at a given time indicates the amount of protein of the present invention present in the sample. Quantitative results are typically obtained with reference to a calibration curve.

  A competitive assay may be used, wherein an antibody specific for a protein of the invention attached to a solid support and a labeled protein of the invention and a sample from the host are passed through the solid support. over), and the amount of label detected attached to the solid support can be correlated with the amount of the protein of the invention in the sample.

  Polypeptides, fragments thereof, or other derivatives, or analogs thereof, or cells expressing them can be used as immunogens to produce antibodies against them. These antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric antibodies, single chain antibodies, and humanized antibodies, as well as Fab fragments or the products of Fab expression libraries. Various methods known in the art can be used for the production of such antibodies and fragments.

  Antibodies raised against a polypeptide corresponding to a sequence of the invention can be obtained by direct injection of the polypeptide into an animal or administration of the polypeptide to an animal (preferably a non-human). The antibody so obtained then binds to the polypeptide itself. Thus, even sequences that encode only a fragment of a polypeptide can be used to generate antibodies that bind to the entire native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing the polypeptide.

  Any technique that provides antibodies produced by continuous cell line cultures can be used to prepare monoclonal antibodies. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature, 256: 495-497 (1975)), trioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today, 4:72). (1983)), and EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, pages 77-96, Alan R. Liss, Inc. (1985)).

  Techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the immunogenic polypeptide products of the invention. Also, other organisms such as transgenic mice or other animals can be used to express humanized antibodies against the immunogenic polypeptide products of the invention.

  The above antibodies may be prepared by adsorbing the antibody to a solid support for isolation or identification of clones expressing the polypeptide, or for isolation and / or purification by affinity chromatography. It can be used to purify the polypeptides of the invention.

  Thus, the polypeptides of the present invention can be used to inhibit neoplasia (eg, tumor cell growth). The polypeptides of the present invention may be responsible for tumor destruction through apoptosis and cytotoxicity to specific cells. The polypeptides of the invention also induce upregulation of adherent cells (eg, LFA-1) and can therefore be used for wound healing. The polypeptides of the invention can also be used for the treatment of diseases that require growth promoting activity (eg, restenosis). This is because the polypeptide of the present invention has a proliferative effect in cells of endothelial origin. Therefore, the polypeptides of the present invention can also be used to regulate angiogenesis in endothelial cell development.

  The polypeptides of the present invention can also be used to stimulate T cell activation and thus stimulate immune responses to various parasitic, bacterial, and viral infections. The polypeptides of the invention can also be used in this respect to eliminate self-responsive T cells to treat and / or prevent autoimmune diseases. An example of an autoimmune disease is type I diabetes.

  The invention also provides a method for identifying molecules such as receptor molecules that bind the proteins of the invention. Genes encoding proteins that bind to the proteins of the invention (eg, receptor proteins) can be identified by a number of methods known to those of skill in the art (eg, ligand panning and FACS sorting). Such methods are described in many laboratory manuals such as, for example, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

  For example, expression cloning can be used for this purpose. To this end, polyadenylated RNA is prepared from cells responsive to the protein of the invention, a cDNA library is made from this RNA, the library is divided into pools, which are responsive to the proteins of the invention. Not individually transfected into cells. The transfected cells are then exposed to the labeled protein of the present invention. The proteins of the invention can be labeled by a variety of well-known techniques, including standard methods of radioiodination or inclusion of recognition sites for site-specific protein kinases. Following exposure, the cells are fixed and cytostatin binding is determined. These procedures are simply performed on a glass slide.

  A pool of cDNA that produced TNFδ or TNFε binding cells is identified. Subpools are prepared from these positives, transfected into host cells and screened as described above. Using repeated sub-pooling and re-screening processes, one or more single clones encoding a putative binding molecule (eg, a receptor molecule) can be isolated.

  Alternatively, the labeled ligand can be photoaffinity bound to a cell extract (eg, a membrane or membrane extract) prepared from a cell that expresses the molecule to which it binds (eg, a receptor molecule). Cross-linked materials are separated by polyacrylamide gel electrophoresis (“PAGE”) and exposed to X-ray film. The labeled complex containing the ligand-receptor can be excised, broken down into peptide fragments, and subjected to protein microsequencing. Amino acid sequences obtained by microsequencing are used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding putative receptor molecules Can be done.

  The polypeptides of the invention can also be used to assess the TNFδ or TNFε binding ability of a TNFδ or TNFε binding molecule (eg, a receptor molecule) in a cell or cell-free preparation.

  The invention also provides methods for screening compounds to identify compounds that enhance or block the action of TNFδ or TNFε on cells (eg, interaction with TNFδ or TNFε binding molecules such as receptor molecules). . An agonist is a compound that increases the natural biological function of a polypeptide of the invention or functions in a manner similar to a polypeptide of the invention, while an antagonist reduces such function. Or eliminate.

  For example, a cell compartment (eg, a preparation thereof such as a membrane or membrane preparation) expresses a molecule that binds to TNFδ or TNFε (eg, a molecule of a signaling or regulatory pathway that is modulated by TNFδ or TNFε). Can be prepared from cells. This preparation is incubated with labeled TNFδ or TNFε in the absence or presence of candidate molecules that may be agonists or antagonists of TNFδ or TNFε. The ability of the candidate molecule to bind to the binding molecule reflects a decrease in the binding of the labeled ligand. Molecules that bind free of charge (ie, without inducing the effects of TNFδ or TNFε in binding to a TNFδ or TNFε binding molecule) are probably good antagonists. Molecules that bind well and elicit effects that are identical or closely related to TNFδ or TNFε are agonists.

  The TNFδ or TNFε-like effects of potential agonists and antagonists can be determined, for example, by measuring the activity of the second messenger system following the interaction of candidate molecules with cells or appropriate cell preparations, and this effect and TNFδ or TNFε. Alternatively, it can be measured by comparing the effects of molecules that elicit the same effect as TNFδ or TNFε. Second messenger systems that may be useful in this regard include, but are not limited to, AMP guanylate cyclase, ion channels, or phosphoinositide hydrolysis second messenger systems.

  Another example of an assay for a TFNδ or TFNε antagonist is competition that combines TFNδ or TFNε and potential antagonists with membrane-bound TFNδ or TFNε receptor molecules or recombinant TFNδ or TFNε receptor molecules under conditions appropriate for competitive inhibition assays. Assay. TFNδ or TFNε can be labeled, such as by radioactivity, so that the number of TFNδ or TFNε molecules bound to the receptor molecule can be determined and the effectiveness of potential antagonists can be accurately assessed.

  Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies that bind to and thereby inhibit or extinguish its activity. A potential antagonist also binds to the same site on a binding molecule, such as a receptor molecule, without inducing small organic molecules, peptides, TFNδ or TFNε-inducing activity, thereby effecting the action of the polypeptide of the invention at its receptor. It can be a closely related protein or polypeptide such as an antibody that prevents it from being excluded from binding.

  Another potential antagonist is a soluble form of the TFNδ or TFNε receptor that binds to TFNδ or TFNε and prevents it from interacting with membrane-bound TFNδ or TFNε receptors. In this manner, the receptor is not stimulated by its ligand.

  A potential antagonist is a small molecule that binds to and occupies the binding site of a polypeptide, thereby preventing it from binding to a cellular binding molecule, such as a receptor molecule, so that normal biological activity is prevented. Including. Examples of small molecules include but are not limited to small organic molecules, peptides, or non-peptide antagonists.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple helix formation. Antisense technology is described, for example, in Okano, J. et al. Neurochem. 56: 560, 1991; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Research,
6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991). These methods are based on the binding of polynucleotides to complementary DNA or RNA. For example, the 5 ′ coding portion of a polynucleotide encoding a mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby preventing transcription and production of TFNδ or TFNε. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of mRNA molecules into TFNδ or TFNε polypeptides. The above oligonucleotides can also be delivered to cells such that antisense RNA or DNA is expressed in vivo to inhibit production of the polypeptides of the invention.

  Antagonists can be used in compositions having a pharmaceutically acceptable carrier (eg, as described herein below).

  Antagonists can be used, for example, to treat cachexia, a lipid removal defect resulting from a systemic deletion of lipoprotein lipase that is inhibited by TFNδ or TFNε. Antagonists can also be used to treat cerebral malaria where the polypeptides of the invention appear to play a pathogenic role. Antagonists can also be used to treat rheumatoid arthritis by inhibiting the production of TFNδ or TFNε-induced inflammatory cytokines (eg, IL1 in synoviocytes). When treating arthritis, the polypeptides of the invention are preferably injected intra-articularly.

  Antagonists can also be used to prevent graft-host rejection by preventing immune system stimulation in the presence of the graft.

  Antagonists can also be used to inhibit bone resorption and thus treat and / or prevent osteoporosis.

  Antagonists can also be used as anti-inflammatory factors and to treat endotoxin shock. This important symptom results from an excessive response to bacterial and other types of infection.

  The invention also relates to a composition comprising the above-described polynucleotide or polypeptide or agonist or antagonist. Accordingly, the polypeptides of the invention are used in combination with a non-sterile or sterile carrier (s) (eg, a pharmaceutical carrier suitable for administration to a subject) for use with cells, tissues or organs. obtain. Such compositions comprise, for example, an additive vehicle or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

  The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the above-described composition of the invention. In such containers, precautionary statements in a form prescribed by a government agency that regulates the manufacture, use or sale of drugs or biological products, the manufacture, use of products for human administration, Or it may be accompanied by a note reflecting the approval by the sales agency.

  The polypeptides and other compounds of the invention can be used alone or in combination with other compounds (eg, therapeutic compounds).

  The pharmaceutical composition can be in any effective and convenient manner (eg, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes, among others) Can be administered.

  A pharmaceutical composition is generally administered in an effective amount for the treatment or prevention of a particular symptom (s). Generally, the composition is administered in an amount of at least about 10 μg / kg body weight. In most cases it will be administered in an amount not exceeding about 8 mg / kg body weight per day. Preferably, in most cases, the dose will be about 10 μg / kg to about 1 mg / kg body weight daily. It will be appreciated that optimal dosage will be determined by standard methods for each treatment modality and symptom, taking into account symptom, severity, route of administration, complexity of symptoms, and the like.

  Polynucleotides, polypeptides, agonists, and antagonists that are polypeptides of the present invention can be used according to the present invention in a mode of treatment often referred to as “gene therapy” by in vivo expression of such polypeptides.

  Thus, for example, a patient-derived cell can be manipulated ex vivo with a polynucleotide encoding a polypeptide (eg, DNA or RNA), and the engineered cell can then be provided to a patient to be treated with the polypeptide. For example, cells can be manipulated ex vivo by use of a retroviral plasmid vector containing RNA encoding a polypeptide of the invention. Such methods are well known in the art and their use in the present invention will be apparent from the teachings herein.

  Similarly, cells can be manipulated in vivo for expression of the polypeptide in vivo by procedures known in the art. For example, the polynucleotides of the invention can be engineered for expression in a replication defective retroviral vector, as described above. The retroviral expression construct can then be isolated and introduced into a transduced packaging cell with a retroviral plasmid vector comprising RNA encoding a polypeptide of the invention so that the packaging cell is Now produces infectious viral particles containing the gene of interest. These producer cells can be administered to a patient for manipulation of the cells in vivo and expression of the polypeptide in vivo. These and other methods for administering the polypeptides of the present invention by such methods will be apparent to those skilled in the art from the teachings of the present invention.

  Retroviruses from which the above retroviral plasmid vectors can be derived include retroviruses such as Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus, human These include but are not limited to immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammalian tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

  Such vectors contain one or more promoters for expressing the polypeptide. Suitable promoters that can be used include the retroviral LTR; SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques, 7: 980-990 (1989), or any other promoter (e.g. Cell promoters such as, but not limited to, eukaryotic cellular promoters, including, but not limited to, histones, RNA polymerase III, and β-actin promoters. Other viral promoters that can be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

  The nucleic acid sequence encoding the polypeptide of the present invention is placed under the control of a suitable promoter. Suitable promoters that can be used include adenovirus promoters such as the adenovirus major late promoter: or heterologous promoters such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus (RSV) promoter; MMT promoter, metallothionein promoter Heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter such as herpes simplex thymidine kinase promoter; retroviral LTR (the modified retroviral LTR described hereinabove) Including, but not limited to, the β-actin promoter; and the human growth hormone promoter. The promoter can also be a native promoter that controls the gene encoding the polypeptide.

Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected are described in Miller, A. et al. , Human Gene Therapy, 1: 5-14 (1990), PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP + E-86, Including but not limited to GP + envAm12 and DAN cell lines. The vector can be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. As one alternative, retroviral plasmid vectors can be encapsulated in liposomes or conjugated to lipids and then administered to a host.

  Producer cell lines give rise to infectious retroviral vector particles that contain a nucleic acid sequence encoding a polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. The eukaryotic cell to be transduced expresses a nucleic acid sequence encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonal carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. .

  The invention is further illustrated by the following examples. Examples are provided only to illustrate the present invention by reference to specific embodiments. These illustrations illustrate certain aspects of the invention, but do not represent a limitation or limitation on the scope of the disclosed invention.

  All examples were performed using standard techniques well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques in the following examples are described in standard research references (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, referred to herein as “Sambrook”; Cold Spring; It can be performed as described in Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

  Except as otherwise specified, all parts or amounts shown in the following examples are by weight. Unless otherwise stated, in the following examples, fragment size separation is performed by agarose and polyacrylamide gel electrophoresis in Sambrook and many other references (eg, Goeddel et al., Nucleic Acids Res., 8: 4057 (1980)). ("PAGE") standard techniques. If not stated otherwise, ligation is carried out using standard buffers, incubation temperature and time (approximately 0.5 mol of DNA fragment to be ligated and about 10 units of T4 DNA ligase (“ligase” per 0.5 μg of DNA). )) Was achieved.

(Example 1)
(Expression and purification of soluble forms of human TFNδ and TFNε using bacteria)
A DNA sequence encoding human TNFδ or TNFε in the deposited polynucleotide is used with a PCR oligonucleotide primer specific for the amino acid carboxyl terminal sequence of the human TNFδ or TNFε protein and specific for the vector sequence 3 'to the gene. Amplified. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5 ′ and 3 ′ sequences, respectively.

The 5 ′ oligonucleotide primer has the sequence 5 ′ GCG GGA TCC C AG AGC CTC ACC ACA G 3 ′, which is shown in the figure starting with the underlined restriction site followed by the 115th base of the ATG codon. Contains the 16 nucleotide coding sequence.

The 3 ′ primer contains the sequence 5 ′ CGC AAG CTT ACA ATC ACA GTT TCA CAA AC 3 ′ to the last 13 nucleotides of the coding sequence shown in FIGS. 1 and 2, including the underlined HindIII restriction site, followed by the stop codon. Contains 20 complementary nucleotides.

The restriction sites are convenient for the restriction enzyme sites of the bacterial expression vector pQE-9 and were used for bacterial expression in these examples (Qiagen, Inc. Chatsworth, CA). pQE-9 encodes ampicillin antibiotic resistance (Amp ^r ) and encodes a bacterial origin of replication (ori), an IPTG-inducible promoter, a ribosome binding site (“RBS”), a 6-His tag, and a restriction enzyme site. .

  Both amplified human TNFδ DNA and vector pQE-9 were digested with BamHI and HindIII, and the digested DNA was then ligated together. Insertion of TNFδ DNA into the pQE-9 restriction vector causes the start AUG to be operably linked to the TNFδ coding region downstream of the vector's IPTG inducible promoter and appropriately translated for translation of TNFδ. Placed in-frame.

The ligation mixture is purified using competent E. coli using standard procedures. E. coli cells were transformed. Such a procedure is described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold.
Spring Harbor Laboratory Press, Cold Spring Harbor, N.A. Y. (1989). E. coli containing multiple copies of plasmid pREP4. The E. coli strain M15 / rep4 (which expresses the lac repressor and also confers kanamycin resistance (Kan ^r )) was used in carrying out the exemplary examples described herein. This strain, which is the only of many that are suitable for expressing TNFδ, is commercially available from Qiagen. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA was isolated from resistant colonies and the identity of the cloned DNA was confirmed by restriction analysis.

Clones containing the desired construct were grown overnight (“O / N”) in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). Large scale cultures are inoculated at a dilution of about 1: 100 to 1: 250 using O / N cultures. Cells were grown to an optical density at 600 nm (OD ₆₀₀ ) between 0.4 and 0.6. Transcription from the lac repressor sensitive promoter was then induced by adding isopropyl-BD-thiogalactopyranoside (“IPTG”) to a final concentration of 1 mM and inactivating the lacI repressor. Cells were further incubated for an additional 3-4 hours. Cells were then harvested by centrifugation and disrupted by standard methods. Inclusion bodies were purified from disrupted cells using routine collection techniques and proteins were solubilized from inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed through a PD-10 column in 2 × phosphate buffered saline (“PBS”), thereby removing the urea, replacing the buffer, and Folded again. The protein was purified by further steps of chromatography to remove endotoxin. Then sterile filtered. The sterile filtered protein preparation was stored in 2 × PBS at a concentration of 95 μg / mL.

  Analysis of the preparation of TNFδ by standard methods of polyacrylamide gel electrophoresis revealed that the preparation contained about 80% monomer with an expected molecular weight of about 20.8 kDa.

  The protein is purified by chromatography on a nickel chelate column under conditions that allow type binding by a protein containing a 6-HIS tag. Protein is eluted from the column in 6 molar guanidine HCl (pH 5.0) and regenerated.

(Example 2)
(Cloning and expression of soluble human TFNδ and TFNε in baculovirus expression system)
The cDNA sequence encoding the full length human TNFδ or TNFε protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5 ′ and 3 ′ sequences of this gene:
The 5 ′ primer has the sequence 5 ′ GCG GGA TC C CCA GAG CCT CAC CAC AG AG 3 ′ and contains the underlined BamHI restriction enzyme site followed by 16 bases of the TNFδ or TNFε sequence of FIGS. To do. When inserted into an expression vector, the 5 ′ end of the amplified fragment encoding human TNFδ or TNFε provides an efficient signal peptide, as described below. An efficient signal for initiation of translation in eukaryotic cells is described in Kozak, M .; , J. et al. Mol. Biol. 196: 947-950 (1987) appropriately located in the vector portion of the construct.

The 3 ′ primer has the sequence 5 ′ CGC TCT AGA ACA ATC ACA GTT TCA CAA AC 3 ′ and contains an underlined XbaI restriction site followed by a termination codon and is shown in FIGS. Contains nucleotides complementary to the last 13 nucleotides of the sequence.

Amplified fragments can be obtained using commercially available kits (“Geneclean” BIO
101 Inc. La Jolla, Ca. ) To isolate from a 1% agarose gel. This fragment is then digested with BamHI and Asp718 and purified again on a 1% agarose gel. This fragment is designated herein as F2.

  The vector pA2GP is used to express TNFδ or TNFε protein in a baculovirus expression system using standard methods (eg, Summers et al., A Manual of Methods for Baculoviral Vectors and Insect Cell Cultures, 198 ) Using the method described in). This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67 (including the N-terminal methionine) is located just upstream of the BamHI site. The polyadenylation site of simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant viruses, The β-galactosidase gene from E. coli is inserted in the same direction as the polyhedrin promoter, followed by a polyadenylation signal for the polyhedrin gene. The polyhedrin sequence is flanked on both ends with the viral sequence for cell-mediated homologous recombination with wild-type viral DNA, producing a viable virus that expresses the cloned polynucleotide.

  As those skilled in the art will readily appreciate, many other if the construct provides a properly positioned signal (eg, in-frame AUG and signal peptide if necessary) for transcription, translation, trafficking, etc. Baculovirus vectors can be used in place of pA2-GP. For example, pAc373, pVL941, and pAcIM1. Such vectors are described, inter alia, in Luckow et al., Virology, 170: 31-39.

  Using routine procedures known in the art, the plasmid is digested with the restriction enzymes BamHI and XbaI and then dephosphorylated using calf intestinal phosphatase. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Ca). This vector DNA is designated herein as “V2”.

  Fragment F2 and dephosphorylated plasmid V2 are ligated using T4 DNA ligase. E. E. coli HB101 cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing plasmids with human TNFδ or TNFε genes are identified by digesting DNA from individual colonies with BamHI and XhaI and then analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein as pBacTNFδ.

5 μg of plasmid pBacTNFδ was obtained according to Felgner et al. Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987) with 1.0 μg of commercially available linearized baculovirus DNA (“BaculoGold ^™ baculovirus DNA”, Pharmingen, San Diego, Calif.). Transfect simultaneously. 1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid pBacTNFδ are mixed in aseptic wells of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Then 10 μl Lipofectin and 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture is then added dropwise to Sf9 insect cells (ATCC CRL 1711) seeded in 35 mm tissue culture plates with 1 ml of serum-free Grace's medium. The plate is shaken back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace insect medium supplemented with 10% fetal calf serum is added. Return plate to incubator and continue incubation at 27 ° C. for 4 days.

  After 4 days, the supernatant is collected and a plaque assay is performed as described in Summers and Smith (supra). An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal expression clones producing blue stained plaques. (A detailed description of this type of “plaque assay” can also be found in the insect cell culture and baculovirology user guide (pages 9-10) distributed by Life Technologies Inc., Gaithersburg).

  After 4 days of serial dilution, virus is added to the cells. After appropriate incubation, pick up the blue-stained plaque with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C. Clones containing appropriately inserted TNFδ or TNFε are identified by DNA or TNFε analysis including restriction mapping and sequencing. This is designated herein as V-TNFδ.

Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. Cells are infected with recombinant baculovirus V-TNFδ at a multiplicity of infection (MOI) of about 2 (about 1 to about 3). After 6 hours, the medium is removed and replaced with SF900 II medium (available from Life Technologies Inc., Gaithersburg) without methionine and cysteine. After 42 hours, 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S cysteine (available from Amersham) are added. The cells are further incubated for 16 hours, then the cells are harvested by centrifugation, lysed and the labeled protein is visualized by SDS-PAGE and autoradiography.

(Example 3)
(Tissue distribution of TFNδ expression)
To examine the expression level of TFNδ in human tissues, Northern blot analysis was performed, inter alia, using the method described by Sambrook et al. (Supra). Total cellular RNA samples were collected from RNAzol ^™ B system (Biotecx
Laboratories, Inc. 6023 South Loop East, Houston, TX 77033).

  Approximately 10 μg of total RNA was isolated from the tissue sample. RNA was size separated by electrophoresis under strong denaturing conditions through a 1% agarose gel. RNA was blotted from the gel onto a nylon filter and the filter was then prepared for hybridization to a detectably labeled polynucleotide probe.

  As a probe for detecting mRNA encoding TFNδ, the antisense strand of the coding region of the cDNA insert of the deposited clone was labeled with high specific activity. The cDNA was labeled by primer extension using the Prime-It kit (available from Stratagene). The reaction was performed using 50 ng cDNA according to the standard reaction protocol recommended by the supplier. The labeled polynucleotide was purified by column chromatography from other labeling reaction components using a Select-G-50 column (obtained from 5-Prime-3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303). .

The labeled probe was hybridized to the filter overnight at 65 ° C. in a small dose of 7% SDS, 0.5M NaPO ₄ , pH 7.4 at a concentration of 1,000,000 cpm / ml.

  The probe solution is then discarded and the filter is washed twice at room temperature and twice with 0.5 × SSC, 0.1% SDS at 60 ° C. The filter is then dried and exposed to film overnight at -70 ° C using an intensifying screen.

  Autoradiography shows that TFNδ mRNA was detected in all 16 tissues with the highest expression in the heart and then in the placenta and kidney.

Example 4
(Gene therapeutic expression of human TFNδ or TFNε)
Fibroblasts are obtained from the subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. A small chunk of tissue is placed on the wet surface of the tissue culture flask, and about 10 chunks are placed in each flask. Turn the flask upside down, close tight, and leave at room temperature overnight. After 24 hours at room temperature, the flask is turned upside down and the tissue mass remains fixed at the bottom of the flask and fresh medium (eg, Ham's F12 medium containing 10% FBS, penicillin, and streptomycin) is added. The tissue is then incubated for about a week at 37 ° C. At this point, fresh medium is added and replaced one after another every few days. After another 2 weeks in culture, a monolayer of fibroblasts appears. The monolayer is trypsinized and transferred to a larger flask.

  The gene therapy vector is digested with restriction enzymes to clone the fragment to be expressed. The digestion vector is treated with calf intestinal phosphatase to prevent self-ligation. The dephosphorylated linear vector is fractionated on an agarose gel and purified.

A cDNA capable of expressing active TFNδ or TFNε is isolated. If necessary, the ends of the fragments are modified for cloning into a vector. For example, the 5 ′ overhang can be treated with DNA polymerase to create blunt ends. The 3 ′ overhang end can be removed using S1 nuclease. Linker with T4
It can be ligated to blunt ends with DNA ligase.
Equal amounts of Moloney murine leukemia virus linear backbone and TFNδ or TFNε fragment are mixed and ligated using T4 DNA ligase. The ligation mixture is treated with E. coli. used to transform E. coli, and the bacteria are then plated on agar containing kanamycin. Kanamycin phenotype and restriction analysis confirms that the vector has a properly inserted gene.

  Packaging cells are grown in tissue culture to confluent concentrations in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum (CS), penicillin, and streptomycin. The packaging cell is transduced with a vector containing the TFNδ or TFNε gene by standard techniques. Infectious viral particles containing the TFNδ or TFNε gene recovered from the packaging cells are referred to herein as producer cells.

  Fresh medium is added to the producer cells, and after an appropriate incubation time, the medium is recovered from the plate of confluent producer cells. The medium (containing infectious virus particles) is filtered through a Millipore filter to remove detached producer cells. This filtered medium is then used to infect fibroblasts. The medium is removed from the subconfluent plate of fibroblasts and immediately replaced with filtered medium. Polybrene (Aldrich) can be included in the medium to facilitate transduction. After appropriate incubation, the medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts are infected and no selection is required. If the titer is low, it is necessary to use a retroviral vector with a selectable marker such as neo or his to select transduced cells for expansion.

  The engineered fibroblasts can then be injected into the rat either alone or after growing to confluence on microcarrier beads such as cytodex 3 beads. The injected fibroblasts produce a TFNδ or TFNε product, and the biological action of the protein is transmitted to the host.

  It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The following drawings illustrate certain embodiments of the invention. They are merely examples and do not limit the invention otherwise disclosed herein.
FIG. 1A shows the nucleotide and deduced amino acid sequence of human TNFδ. FIG. 1B shows the nucleotide and deduced amino acid sequence of human TNFδ. FIG. 2A shows the nucleotide and deduced amino acid sequence of human TNFε. FIG. 2B shows the nucleotide and deduced amino acid sequence of human TNFε. FIG. 3A shows regions of similarity between amino acid sequences of TNFα, TNFβ, TNFδ and TNFε polypeptides (alignment report). FIG. 3B shows regions of similarity between amino acid sequences of TNFα, TNFβ, TNFδ and TNFε polypeptides (alignment report). FIG. 4A shows the structural and functional characteristics of TNFδ deduced by the indicated technique as a function of amino acid sequence. FIG. 4B shows the structural and functional characteristics of TNFδ deduced by the indicated technique as a function of amino acid sequence. FIG. 5A shows the structural and functional characteristics of TNFε deduced by the indicated technique as a function of amino acid sequence. FIG. 5B shows the structural and functional characteristics of TNFε deduced by the indicated technique as a function of amino acid sequence.

Claims (1)

Invention described in the specification.

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