Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• Novel T-cell proteins, peptides and antibodies derived therefrom and uses thereof are novel T-cell proteins, peptides and antibodies derived therefrom and uses thereof.
• the present invention relates to polynucleotides encoding T-cell polypeptides comprising an amino acid sequence as disclosed herein, the expression of which is upregulated during the early stages of leukocyte/lymphocyte activation in response to allo-antigens, and to biologically active fragments thereof. Furthermore, the present invention relates to a nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with a polynucleotide described herein or with a complementary strand thereof.
• the present invention pertains to vectors comprising polynucleotides encoding said T-cell protein/polypeptide, (host) cells which comprise said polynucleotide(s) or said vectors, to T- cell protein/polypeptide and biologically active fragments thereof, an antibody which specifically recognizes a T-cell protein of the invention or a fragment thereof, and to antisense constructs capable of inhibiting the expression of a polynucleotide encoding a T- cell protein. Additionally this invention provides diagnostic compositions arid methods of diagnosing biological conditions.
• the invention relates to methods for identifying binding partners to T-cell proteins and to methods for identifying leukocyte activating and co-stimulating compounds or for identifying inhibitors of leukocyte activation and stimulation.
• the present invention also relates to the use of the before described polynucleotides, vectors, proteins, antisense constructs for the preparation of compositions for diagnosing or the treatment of acute and chronic diseases, involving T-cell activation and Thl, and Th2 immune response, for the treatment of acute and chronic rejection of allo- and xeno-organ transplants and bone marrow transplantation, for the treatment of rheumatoid arthritis, lupus erythematodes, multiple sclerosis, encephalitis, vasculitis, diabetes mellitus, pancreatitis, gastritis, thyrolditis, for the treatment of (maligne) disorders of T, B or NK cells, for the treatment of asthma, lepramatosis, Helicobacter pylori associated gastriti
• Immune activation is accompanied with sequential changes in the expression of various genes over several days and involves multiple signaling pathways [1]. Stimulation of T-cells is initiated by the interaction of antigen-specific T-cell receptors (TCR) with MHC bound antigenic peptides present on the surface of antigen presenting cells (APC), but full proliferative T-cell response requires additional costimulatory signals which are provided by the interaction of proteins expressed on the surface of T-cells and APC [2,3,4,5]. In addition, a number of cytokines as well as other proteins are known to augment immune activation, although many of them appear not to be essential for the basic proliferative T- cell response [3,6].
• T-cell activation a growing body of evidence indicates that the microtubule cytoskeleton of lymphocytes plays a major role in T-cell activation. Stimulation of T-cells was demonstrated to result in molecular rearrangement in the actin cytoskeleton leading to re-localization and concentration of signaling molecules in restricted areas of the cell membrane close to the bound APC [7,8,9].
• T-cell activation provides the central event in various types of inflammation as well as in autoimmune diseases and graft rejection, knowledge about the distinct steps and molecules involved in the stimulation process is of considerable biomedical importance, as they might provide targets for therapeutic modulation of the immune response.
• Therapeutic prevention of T-cell activation in organ transplantation and autoimmune diseases presently relies on panimmunosuppressive drugs interfering with downstream intracellular events.
• Alloreactive CD4 or CD8 cells or specific alloantibodies are capable of mediating, inter alia, allograft rejection.
• the following immune mechanisms cause graft rejection by different mechanisms: (a) alloactive T-cells can recruit and activate macrophages, initiating graft injury by "delayed-type" hypersensitivity response;
• alloactive cytotoxic T-cells are capable of directly lysing graft endothelial and parenchymal cells
• alloantibodies bind to endothelium, activate the complement system, and injure thereby graft blood vessels.
• lymphocyte/leukocyte cell responses i.e. to modulate T-, B-, NK-cells and/or monocyte responses during immunological processes.
• immunological processes like, inter alia, autoimmunological events. Specific modulation of the immune response remains, therefore, a longstanding goal in immunological research.
• the present invention relates to polynucleotides encoding immune response modulating proteins. Furthermore, the present invention relates to peptides and polypeptides derived therefrom as well as to antibodies. More particularly, the present invention relates to pharmaceutical compositions comprising said peptides and polypeptides as well as to pharmaceutical compositions comprising antibodies capable of inhibiting leukocyte stimulation through the immune response modulation protein. The present invention also relates to applications in the medical field that directly arise from the polynucleotides, protein, peptides, (poly)peptides, antisense constructs and antibodies described in this invention. Additionally, the present invention relates to a novel method for testing activators and inhibitors of leukocyte proliferation and/or lymphocyte activation, i.e.
• compositions, methods and uses of the invention are useful therapeutically and/or diagnostically in situations where it is desirable to modulate (antigen-specific) immune responses, e.g., inducing and maintain (antigen- specific) T-cell or B-cell non/unresponsiveness, wherein said non/unresponsiveness comprises the selective inhibition of immune cell subsets which are able to creating a response to specific antigen(s), inter alia, antigen(s) in transplanted tissue.
• the pharmaceutical compositions, methods and uses of the invention are furthermore useful to restore (antigen-specific) B- or T-cell responsiveness.
• T-cell unresponsiveness it may be necessary to induce or maintain "selective immune" unresponsiveness in a subject who has received an organ or bone marrow transplant to prevent graft rejection by inhibiting stimulation through a T-cell protein of the invention in cells of the immune system such as T-cells, B-cells, NK- cells, monocytes and/or macrophages.
• T-cell unresponsiveness can be maintained by blocking T-cell protein stimulation in a subject who has a autoimmune disease to alleviate symptoms of the autoimmune disease.
• a T-cell protein inhibitory agent is administered to the subject in an amount and over a period of time sufficient to maintain T-cell unresponsiveness.
• T-cell unresponsiveness can be reversed in a subject bearing a tumor to stimulate a tumor specific NK- and T-cell response or in a subject receiving a vaccine to enhance the efficacy of the vaccine.
• the technical problem of the invention is to provide means and methods for the modulation of immune cell responses which are particularly useful in organ transplantation and autoimmune diseases.
• the solution to this technical problem is achieved by providing the embodiments characterized in the claims, namely novel immune response modulating T-cell proteins encoded by cell immune response cDNAs encoding T-cell proteins designated "TKI7", “TLIP7”, “THY7”, “TNIM7”, “TLO ⁇ 7”, “THOM7” and “TPIN7”, respectively, (see example 2) which comprise an amino acid sequence as depicted in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and which exhibit a central role in leukocyte/lymphocyte activation and growth, wherein said leukocyte/lymphocyte activation refers to the activation of T-, B-, NK-cells and/or monocytes.
• T-cell proteins mRNA is transiently upregulated
• the T-cell protein encoding cDNAs have been cloned and characterized; see Examples 1 and 2. Furthermore, the expression pattern of t-cell proteins was investigated after allo-stimulation of human leukocytes at time points 0 and 12 h and results obtained with alloactivated T-cells revealed an upregulation of T-cell proteins 24 h after immune activation of the T-cell proteins gene. They are thus excellent markers for diagnosis of the status of immune response in a subject.
• T-cell protein expression may efficiently lead to a significant down regulation of lymphocyte activation in response to allo-antigen and mitogens.
• Antisense polynucleotides directed to the mRNA encoding T-cell protein which are able to efficiently suppress the proliferation of lymphocytes and antigen presenting cells (monocytes, dentritic cells, B-cells) in response to alloactivation in a mixed lymphocyte culture or in response to mitogens, can be designed and produced according to methods known in the art.
• Antisense oligo and primer design & analysis Software can be purchased, for example, from OLIGO, Molecular Biology Insights, Inc., USA.
• Oligonucleotides can be synthesized by using an automated DNA synthesizer, for example Bio Serve Biotechnologies, Laurel, MD, and purified by polyacrylamide gel electrophoresis.
• T-cell protein specific antisense oligonucleotides For proliferation, thymidine uptake is determined after 6 h which may demonstrate a significant inhibition of T-cell activation in the presence of T-cell protein specific antisense oligonucleotides whereas the presence of unrelated control antisense oligonucleotides will not exhibit any effect on T-cell proliferation.
• T-cell specific markers such as CD69 and IL2 receptor can be analyzed by flowcytometry on human peripheral blood lymphocytes in the presence and absence of T-cell protein specific antisense oligonucleotides which demonstrate significant downregulation of activation markers (like e. g. CD25- (IL2 receptor), CD69, L/CA class 11 and transferrin receptor) on immune cells. Based on such results, it can be concluded that T-cell protein molecules are directly involved in the initiating of the immune response and might be an important target molecule for modulating the immune response.
• the invention relates to a polynucleotide encoding a T-cell protein polypeptide, the expression of which is upregulated during the early stages of T-cell leukocyte/lymphocyte activation in response to allo-antigens or a biologically active fragment thereof comprising a nucleic acid sequence selected from the group consisting of:
• T-cell protein denotes a protein involved in the signal transduction of leukocyte/lymphocyte activation and/or proliferation and down-regulation which results in suppressing leukocyte/lymphocyte, preferably T-, B-, ⁇ K-cell and/or monocyte proliferation in response to alloactivation in a mixed lymphocyte culture when exogeneously added to the culture.
• leukocyte/lymphocyte preferably T-, B-, ⁇ K-cell and/or monocyte proliferation in response to alloactivation in a mixed lymphocyte culture when exogeneously added to the culture.
• a few cD ⁇ As are differentially expressed in alloactivated lymphocytes, i.e. human T-cells. These differentially expressed cDNAs are further characterized in Example 2.
• said T-cell protein/polypeptide plays an important role in the differentiation of quiescent T-cells to activate T-cells after allo-antigen stimulation and/or cell activation/proliferation processes of B-cells, Nk-cells and/or monocytes after stimulation by said allo/autoantigens or xenoantigens or by antigens from, inter alia, pathological agents, like viruses (viral agents), bacteria, etc.
• T-cell protein denotes proteins/polypeptides, in accordance with this invention, which are identical to T-cell protein/polypeptide as described herein (see SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and Figure 2a) and the term comprises, furthermore, functional homologues of said protein/polypeptide.
• T-cell protein molecules are directly involved in the initiating of the immune response and might be an important target molecule for modulating the immune response.
• the term "leukocyte/lymphocyte” generally denotes all kinds of white blood cells and preferably refers to monocytes and lymphocytes (B- T- and NK-cells), either in combination or individually.
• leukocyte may also be used herein so as to refer to individual species of leukocytes such as T-cells only.
• T-cell protein polypeptide comprising an amino acid sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32
• said amino acid molecule may also comprise alternatives, deletions, duplications, additions, substitutions and/or silent mutations. Such silent mutations may, inter alia, comprise conservative amino acid replacements.
• polynucleotide/nucleic acid molecule encoding a T-cell polypeptide as defined herein above also comprise polynucleotides/nucleic acid molecules which encode "variants" of said T-cell polypeptide.
• variants refer to polynucleotides (and/or (poly)peptides) differing from the polynucleotides and/or (poly)peptides of the invention, but retain essential properties thereof, as, inter alia, is upregulation during early stages of leukocyte/lymphocyte activation in response to xenoantigens.
• variants are overall (closely) similar, and, preferably, in some regions identical to the polynucleotides and (poly)peptides described herein.
• variant in accordance with this invention comprises, but is not limited to allelic variants, synthetically produced variants or genetically engineered variants.
• biologically active fragment thereof refers to peptides and polypeptides that are derived from said T-cell protein and that are capable of effecting the same or similar activity or at least one of said activities of T-cell protein (see SEQ ID NO: 2) such as that they are recognised by the name poly- and/or monoclonal antibody. Most preferably, said fragment comprises an immunogenic region as identified in Figure 3.
• genes induced in the early stage of T-cell activation have been identified by examining mRNA expression in xenoactivated human lymphocytes.
• Differential display-reverse transcription PCR analysis revealed several cDNA fragments which were upregulated 24 h after xenostimulation of a human T-cell line; see Example 1.
• the corresponding (complete) cDNAs have been identified with UCSC Human Genome Project Working Draft, April 2002 assembly (hgll) http://genome.cse.ucsc.edu/; The Human Genome Browser at UCSC, Kent et al., Genome Research, Vol.
• T-cell proteins of the present invention are expected to function in cell proliferation and differentiation events during T-cell and/or general leukocyte activation.
• nucleotide sequence depicted in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 encode (fragments of) novel immune response modulating proteins.
• Said nucleotide sequences may be employed, in accordance with this invention, in the pharmaceutical compositions, uses and/or methods described herein.
• the invention also relates to polynucleotides which hybridize to the above described polynucleotides and differ at one or more positions in comparison to these as long as they encode a T-cell protein as defined above.
• Such molecules comprise those which are changed, for example, by deletion(s), insertion(s), alteration(s) or any other modification known in the art in comparison to the above described polynucleotides either alone or in combination.
• Methods for introducing such modifications in the polynucleotides of the invention are well-known to the person skilled in the art; see, e.g., Sambrook et al. (Molecular cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor NY (1989)).
• the invention also relates to polynucleotides the nucleotide sequence of which differs from the nucleotide sequence of any of the above- described polynucleotides due to the degeneracy of the genetic code.
• hybridizing in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in 50%formamide/6xSSC/0.1%SDS/100 ⁇ g/ml ssD ⁇ A, in which temperatures for hybridization are above 37°C and temperatures for washing in 0.1xSSC/0.1%SDS are above 55°C.
• hybridizing refers to stringent hybridization conditions, for example such as described in Sambrook, supra.
• polynucleotides which share 70%, preferably at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above- mentioned polynucleotides and have the same biological activity.
• Such polynucleotides also comprise those which are altered, for example by nucleotide deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or modification(s) known in the art either alone or in combination in comparison to the above-described polynucleotides. Methods for introducing such modifications in the nucleotide sequence of the polynucleotide of the invention are well known to the person skilled in the art.
• composition(s), use(s) and method(s) of the present invention may comprise any polynucleotide that can be derived from the above described polynucleotides by way of genetic engineering and that encode upon expression a T-cell protein or a biologically active fragment thereof.
• regulatory sequences may be added to the polynucleotide as defined herein and employed in the pharmaceutical composition, uses and/or methods of the invention.
• promoters, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide of the invention may be employed.
• a suitable inducible system is for example tetracycline- regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
• polynucleotide of the invention encodes a polypeptide comprising an amino acid sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 or a biologically active fragment thereof.
• the invention relates to a nucleic acid molecules of at least 15 nucleotides in length hybridizing with a polynucleotide as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences encoding no or substantially different Proteins. Such nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length.
• nucleic acids of up to 100 and more nucleotides in length.
• Said nucleic acid probes are particularly useful for various pharmaceutical and/or diagnostic applications.
• they may be used as PCR primers for amplification of polynucleotides encoding T-cell proteins and/or is homologues and may, thereby, serve as useful diagnostic tools.
• Another application is the use as a hybridization probe to identify polynucleotides hybridizing to the polynucleotides encoding T-cell protein by homology screening of genomic DNA libraries.
• Nucleic acid molecules employed in this preferred embodiment of the invention which are complementary to a polynucleotide as described above may also be used for repression of expression of a gene comprising such a polynucleotide, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-A1 0 291 533, EP Al 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a polynucleotide as described herein above.
• nucleic acid probe with an appropriate marker for specific (Inter alia, diagnostic) applications, such as for the detection of the presence of a polynucleotide as described herein above in a sample derived from an organism.
• nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
• said nucleic acid molecule may either contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
• Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
• Such nucleic acid molecules may further contain ribozyme sequences as described above.
• polynucleotide to be used in the invention can be employed for "gene targeting” and/or “gene replacement”, for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
• the polynucleotides as defined herein above may be employed in vaccination approaches.
• vaccination approaches may be, inter alia, useful in prevention or treatment of malignant diseases, for example in the prevention or therapy of tumors of the hematopoietic system.
• Vaccination approaches employing nucleic acid molecules are well known in the art and are described, inter alia, in Leither (2000), Vaccine 18, 765-777.
• nucleic acid molecules are labeled.
• Said labels may comprise radiolabels or fluorescence labels.
• said nucleic acid molecules may be used for the suppression of T-cell protein expression. Particularly preferred in this embodiment are the above described hybridizing nucleic acid molecules.
• the polynucleotide as employed in accordance with this invention and encoding the above described T-cell protein or (a) biologically active fragment(s) thereof may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
• said polynucleotide is part of a vector.
• Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
• the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
• Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
• Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E.
• regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
• Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription such regulatory elements may also comprise transcription termination signals, such as the SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide.
• leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art.
• the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
• the heterologous sequence can encode a fusion protein including an C- or N- terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
• suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (In-vitrogene), or pSPORTl (GIBCO BRL).
• the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
• the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the protein of the invention may follow.
• antisense constructs are made based on the polynucleotide encoding T-cell protein (or (a) biologically active fragment(s) thereof) and combined with an appropriate expression control sequence.
• the present invention relates to (a) vector(s), particularly (a) plasmid(s), cosmid(s), virus(es) and bacteriophage(s) used conventionally in genetic engineering that comprise a polynucleotide encoding a T-cell protein and/or (a) functional fragment(s) thereof (as defined herein above).
• vector(s) particularly (a) plasmid(s), cosmid(s), virus(es) and bacteriophage(s) used conventionally in genetic engineering that comprise a polynucleotide encoding a T-cell protein and/or (a) functional fragment(s) thereof (as defined herein above).
• polypeptides and vectors to be employed in accordance with this invention can be reconstituted into liposomes for delivery to target-cells of the immune system.
• the here described vectors containing the polynucleotides described herein above can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.
• the present invention provides for a vector as defined herein above, wherein said polynucleotide or nucleic acid molecule is operably linked to regulatory sequences allowing for the transcription and, optionally, expression of said acid molecules.
• the present invention relates to a cell, preferably a host cell, comprising the polynucleotide or vector described above.
• said cell is a eukaryotic, most preferably a mammalian cell if therapeutic uses of the protein are envisaged.
• yeast and less preferred prokaryotic, e.g., bacterial cells may serve as well, in particular if the produced protein is used as a diagnostic means or if said protein is employed in methods as described herein above.
• the polynucleotide or vector described herein which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
• Prokaryotic is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a protein of the invention.
• Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
• eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells.
• the protein encoded by the polynucleotide of the present invention may be glycosylated or may be non- glycosylated.
• T-cell proteins as employed in accordance with the present invention may also include an initial menthionine amino acid residue.
• a polynucleotide as described herein can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art.
• methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, NY, 1989).
• the genetic constructs and methods described therein can be utilized for expression of the T-cell protein in eukaryotic or prokaryotic hosts.
• expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
• the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
• transgenic animals preferably mammals, comprising nucleic acid molecules/polynucleotides as defined herein may be used for the large scale production of the T-cell protein and/or for the large scale production of pharmaceutical compositions described herein.
• an animal preferably mammalian cell naturally having a polynucleotide described herein present in its genome can be used and modified such that said cell expresses the endogenous gene corresponding to the polynucleotide described herein above under the control of an heterologous promoter.
• the introduction of the heterologous promoter which does not naturally control the expression of the polynucleotide of the invention can be done according to standard methods, see supra. Suitable promoter include those mentioned hereinbefore.
• a method for the production of a T-cell protein or a biologically active fragment thereof may comprise:
• the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
• the T-cell protein and/or biological active fragments thereof can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
• the protein of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "ProteinPurification", Springer- Verlag, N.Y. (1982).
• Substantially pure proteins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses.
• the proteins may then be used therapeutically (including extracorporally) or in developing and performing assay procedures.
• the present invention relates to a T-cell protein or a biological active fragment thereof encoded by polynucleotide described herein above or produced by a method of as above.
• the T- cell protein or a (biologically active) fragment thereof can be further coupled to other moieties as described above for, e.g., drug targeting and imaging applications, i.e. pharmaceutical and/or diagnostic uses.
• Such coupling may be conducted chemically after expression of the protein to site of attachment or the coupling product may be engineered into the protein of the invention at the DNA level.
• the DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured.
• T- cell protein/polypeptides may by particularly useful in (a) pharmaceutical setting(s) where specific leukocyte activation should be controlled.
• specific overexpression of T-cell proteins or (biologically active) fragments thereof may be obtained by gene therapeutic approaches.
• T-cell expression is induced by activation of cells of the immune system. Without being bound by theory, it is therefore envisaged that one function of T-cell is the control of the cell activation events in the immune system.
• T-cell protein as described herein above enables the production of T-cell protein specific antibodies.
• hybridoma technology enables production of cell lines secreting antibodies to essentially any desired substance that produces an immune response.
• RNA encoding the light and heavy chains of the immunoglobulin can then be obtained from the cytoplasm of the hybridoma.
• the 5' end portion of the mRNA can be used to prepare cDNA to be inserted into an expression vector.
• the DNA encoding the antibody or is immunoglobulin chains can subsequently be expressed in cells, preferably mammalian cells.
• renaturation techniques may be required to attain proper conformation of the antibody.
• point substitutions seeking to optimize binding may be made in the DNA using conventional cassette mutagenesis or other protein engineering methodology such as is disclosed herein.
• the present invention also relates to an antibody specifically recognizing T-cell protein or (a) fragment(s) (peptides, polypeptides) thereof.
• said antibody is a monoclonal antibody, a single chain antibody, humanized antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivate of any of these.
• Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications development by the art.
• antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
• surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97- 105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
• the production of chimeric antibodies is described, for example, in WO89/09622.
• Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or any other modification(s) known in the art either alone or in combination.
• Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. It is particularly preferred that the here described pharmaceutical compositions comprise antibodies/antibody contracts which may be employed in intracellular settings.
• Such antibody constructs/antibodies are well known in the art and are, inter alia, described in Lener (2000), Eur. J. Biochem. 267, 1196-1205, who described intracellular antibodies against p21 ras.
• the present invention relates to a cell that has been modified to express a T-cell- protein or an antibody as described herein.
• This embodiment may be well suited for, e.g., restoring B- and/or T-cell responsiveness to an antigen, in particular if the antibody of the invention capable of stimulating T-cell proliferation is expressed in a form suitable to be presented on the cell surface.
• the invention also relates to an antisense construct capable of inhibiting the expression of a polynucleotide encoding a T-cell protein of the invention.
• the invention furthermore relates to a pharmaceutical composition
• a pharmaceutical composition comprising the polynucleotides, nucleic acid molecules, vectors, cells, proteins, antibodies or antisense construct of the invention.
• the pharmaceutical composition comprises an antisense construct capable of inhibiting the expression of a polynucleotide encoding T-cell protein (and/or (a) biologically active fragment(s) thereof) as defined herein above.
• antisense constructs/oligonucleotides are particularly useful in the down regulation of leukocyte/lymphocyte responses/activations.
• compositions comprising (specific) antisense constructs which are capable of inhibiting the expression of T-cell may protein may be particularly useful in the treatment and/or prevention of pathological or medical situations where an immunoactivation is not desired.
• These situations comprise, but are not limited to, treatment of acute and chronic rejections of allo- and xeno(organ)transplants or bone marrow transplantations, inflammation processes and/or allergies.
• the use of antisense oligonucleotides/constructs is well known in the art and described, inter alia, in Irizawa (1995), Clin. Exp. Immunology 100, 383- 389 or Boeve (1994), J. Leukocyte Biol. 55, 169-174.
• RNase H mapping use of combinatorial arrays and prediction of secondary structure of mRNA by computational methods.
• Structures that bind to structured RNA i.e. aptastrucs and tethered oligonucleotide probes, and foldback triplex-forming oligonucleotides can also be employed for the purpose of the present invention.
• Relating to selection of antisense sequences by aid of computational analysis, valuable www addresses are given in the above-identified prior art.
• Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes is described for example in Amarzguioui, Nucleic Acids Res.
• the antisense molecules comprise at least 14 or 15, more preferably about 17 to 20 or more, and most preferably about at least 20, 25 or 30 or more consecutive nucleotides (including nucleotide analogs) of or complementary to any one of the above described polynucleotides encoding T-cell protein or corresponding genomic sequences, including 5'- and 3 '-untranslated regions, introns, transcriptional regulatory sequences and the like.
• the antisense molecule comprises said at least 14 or 15 nucleotides complementary to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
• the present invention relates to a pharmaceutical composition(s) described herein for use in cell or organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, for the treatment of tumors or for the improvement of allograft or xenograft tolerance.
• composition of the Invention for improving allograft or xenograft tolerance is described with respect to administration of an LFA-3 and CD2 binding protein, respectively, in WO93/06852.
• the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
• suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
• Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
• compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
• the dosage regimen will be determined by the attending physician and clinical factors.
• dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
• a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
• the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to lOmg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 12 copies of the DNA molecule.
• the composition of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
• Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsion.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsion or suspensions, including saline and buffered media.
• Parental vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
• Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
• the pharmaceutical composition of the invention may comprise further agents such as T-cell, B- cell, NK-cell or monocyte costimulatory molecules or cytokines known in the art, or their inhibitors or activators depending on the intended use of the pharmaceutical composition.
• the various polynucleotides and vectors encoding the above described peptides or polypeptides are administered either alone or in any combination using standard vectors and/or gene delivery systems, and optionally together with a pharmaceutically acceptable carrier or excipient.
• the polynucleotide of the invention can be used alone or as part of a vector to express the (poly)peptide described herein in cells, for, e.g., gene therapy or diagnostics of diseases related to disorders of the immune system.
• the polynucleotides or vectors described herein are introduced into the cells which in turn produce the T-cell protein (or (a) fragment(s) thereof).
• said polynucleotides or vectors may be stably integrated into the gnome of the subject.
• viral vectors may be used which are specific for certain cells or tissues and persist in said cells.
• Suitable pharmaceutical carriers and excipients are well known in the art.
• the pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of diseases, which are related to leukocyte, lymphocyte and/or monocyte related immunodeficiencies and malignancies such as multiple myeloma, T-, B-cell leukemia, infectious diseases related to T-, B-, NK-cell and monocyte proliferation, immune activation in refection of transplants, autoimmune disorders, allergy.
• the present invention relates to a diagnostic composition
• a diagnostic composition comprising any one of the above described proteins, antibodies, (poly)peptides, polynucleotides, vectors or cells, and optionally suitable means for detection.
• the (poly)peptides and antibodies described above are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay.
• RIA radioimmunoassay
• sandwich immunometric assay
• Western blot assay the Western blot assay.
• the (poly)peptides and antibodies can be bound in many different carriers and used to isolate cells specifically bound to said polypeptides.
• Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides. agaroses, and magnetite.
• the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
• labels there are many different labels and method of labeling known to those of ordinary skill in the art.
• Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and biolun inescent compounds.
• the here described diagnostic compositions are particularly useful for the detection of an activated status of the immune system, in particular to detect activation of T-cells, B-cells, NK-cells and/or monocytes.
• Said diagnostic compositions may also be used for methods for detecting expression of a polynucleotide encoding T-cell protein (or its homologues) by detecting the presence of mRNA coding for a T-cell protein which comprises obtaining mRNA from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a polynucleotide encoding T- cell protein (or its homologues) under suitable hybridizing conditions (see also supra), detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the T-cell protein (or its homologues) by the cell.
• the invention comprises methods of detecting the presence of a T-cell protein in a sample, for example, a cell sample, which comprises obtaining a cell sample from a subject, contacting said sample with one of the aforementioned antibodies under conditions permitting binding of the antibody to the T-cell protein, and detecting the presence of the antibody so bound, for example, using immuno assay techniques such as radio- immunoassay or enzyme-immunoassay.
• one skilled in the art may specifically detect and distinguish polypeptides which are functional T-cell proteins from a mutated forms which have lost or altered their leukocyte (T-cell, B-cell, etc.) stimulatory activity by using an antibody which either specifically recognizes a (poly)peptide which has T-cell protein activity but does not recognize an inactive form thereof or which specifically recognizes an in inactive form but not the corresponding polypeptide having T-cell activity.
• the antibodies as described in the present invention may also be used in affinity chromatography for purifying the T-cell protein or above described (poly)peptides and isolating them from various sources. Said purified proteins/(poly)peptides may be employed in the pharmaceutical compositions, uses an/or method of the present invention.
• the invention relates to a method for diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to a disorder in the immune system comprising the steps of determining the presence or absence of a mutation in the polynucleotide if the indention and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of an mutation in the polynucleotide of the invention.
• the indention relates to a method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject related to a disorder in the immune system comprising the steps of determining the presence or amount of expression of the protein of the invention in a biological sample and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the protein.
• the invention also encompasses a method for diagnosing in a subject a predisposition (susceptibility) to a disorder associated with the expression of a T-cell protein allele which comprises isolating DNA from victims of the disorder associated with the under- or over- expression of a T-cell protein; digesting the isolated DNA with at least one restriction enzyme; electrophoretically separating the resulting DNA fragments on a sizing gel; contacting the resulting gel with a nucleic acid probe as described above capable of specifically hybridizing to DNA encoding a T-cell protein and labeled with a detectable marker; detecting labeled bands on the gel which have hybridized to the labeled probe to create a band pattern specific to the DNA of victims of the disorder associated with the expression of a T-cell protein; preparing the subject's DNA according to the above- mentioned steps to produce detectable labeled bands on a gel; and comparing the band pattern specific to the DNA of victims of the disorder associated with the expression of a T- cell protein and the subject'
• the detectable markers of the present invention may be labeled with commonly employed radioactive labels, such as, for example, 32 P or 35 S, although other labels such as biotin or mercury as well as those described above may be employed as well.
• radioactive labels such as, for example, 32 P or 35 S
• Various methods well-known the person skilled in the art may be used to label the detectable markers.
• DNA sequences and RNA sequences may be labeled with 32 P or 35 S using the random primer method.
• various methods well-known to the person skilled in the art may be employed for contacting the detectable marker with the sample of interest. For example. DNA-DNA, RNA-RNA and DNA-RNA hybridizations may be performed using standard procedures.
• nucleic acids e.g., Southern and northern blotting, PCR. primer extension and the like.
• the mRNA, cRNA, cDNA or genomic DNA obtained from the subject may be sequenced to identify mutations which may be characteristic fingerprints of T-cell protein mutations in disorders associated with the expression of T-cell protein or mutated versions thereof.
• the present invention further comprises methods, wherein such a fingerprint may be generated by RFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNA may be amplified prior to analysis, the methods of which are well known in the art.
• RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase Ti, Rnase T 2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments on PAGE as described above or in the appended examples.
• a suitable RNA-Enzyme for example RNase Ti, Rnase T 2 or the like or a ribozyme
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising an agent which stimulates a leukocyte through the T-cell protein as described herein, and optionally a pharmaceutically acceptable carrier.
• an agent that stimulates a leukocyte through the T-cell protein as described herein and optionally a pharmaceutically acceptable carrier.
• the agent that stimulates the proliferation of leukocytes or lymphocytes tlirough the T-cell protein is expected to markedly enhance the proliferation of leukocytes or lymphocytes of, e.g., (activated) T-cells and thus is capable of augmenting the immune response.
• agents to be employed in accordance with the present invention usually specifically bind and/or interact to T-cell protein in order to exert their effect.
• agents can be identified in accordance with a method of the invention described below.
• agents also comprise promoters which can be inserted in front of the coding region of the T-cell protein encoding gene, e.g., via gene transfer and homologous recombination in the 5' untranslated region of the gene, see also supra.
• promoter may be regulated and thus permit the controlled expression of the T-cell protein in certain cells.
• the present invention relates to a method for identifying a binding partner to a T-cell protein polypeptide comprising:
• T-cell protein polypeptides may be used to screen for molecules that bind to T-cell protein or for molecules to which T-cell protein binds.
• the binding of T-cell protein and the molecule may activate (agonist),increase, inhibit (antagonist), or decrease activity of the T- cell protein or the molecule bound.
• Examples of such molecules include antibodies (including single-chain antibodies), oligonucleotides, proteins (e.g., receptors), or small molecules preferably, the molecule is closely related to the natural binding partner of T-cell protein, e.g., a fragment of the binding partner, or a natural substrate, a "ligand", a structural or functional mimetic; see, e.g., Collgan, Current Protocols in Immunology 1(2) (1991); Chapter 5.
• the molecule can be closely related to the natural binding partner(s) with which T-cell protein interacts, or at least, a fragment of said binding and/or interaction partner capable of being bound by T-cell protein (e.g., active site). In either case, the molecule can be rationally designed using known techniques; see also infra.
• potential binding partner(s) of T-cell protein is/are G-protein interacting molecule(s).
• the screening for these molecules involves producing appropriate cells which express T-cell protein, either as a secreted protein of as a protein in or on the cell membrane.
• Preferred cells include cells from mammals, yeast, Drosophila, or E.coli.
• Cells expressing T-cell protein are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either T-cell protein or the molecule.
• the assay may simply test binding of a candidate compound to T-cell protein, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to T-cell protein.
• the assay can be carried out using cell-free preparations, polypeptide/molecule affixes to a solid support, chemical libraries, or natural product mixtures.
• the assay may also simply comprise the steps of mixing a candidate compound with a solution containing the T-cell protein/molecule activity or binding to a standard.
• an ELISA assay can measure T-cell protein level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
• the antibody can measure T- cell protein level or activity by either binding, directly or indirectly, to T-cell protein or by competing with T-cell protein for a substrate.
• All of these above assays can be used as diagnostic or prognostic markers.
• the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., increase of immune response) by activating or inhibiting the T-cell protein/molecule.
• the assays can discover agents which may inhibit or enhance the production of T-cell protein from suitably manipulated cells or tissues.
• the invention includes a method of identifying compounds which bind to T-cell protein comprising the steps of:
• the invention includes a method of identifying agonists/antagonists comprising the steps of:
• the polynucleotides encoding T-cell protein (or (a) fragment(s) thereof) and polypeptides representing T-cell protein (or (a) fragment(s) thereof) provide a basis for the development of mimetic compounds that may be inhibitors or activators of T- cell protein or their encoding genes. It will be appreciated that the present invention also provides cell based screening methods that allow a high-throughput-screening (HTS) of compounds that may be candidates for such inhibitors and activators. Furthermore, the invention relates to a method for indentifying leukocyte/lymphocyte activation or co-stimulating compounds or for identifying inhibitors of leukocyte/lymphocyte activation and stimulation comprising
• compound in the method of the invention includes a single substance or a plurality of substances which may or may not be identical.
• Said compound(s) may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms, Furthermore, said compounds may be known in the art but hitherto not known to be capable of inhibiting proliferation of leukocytes or not known to be useful as an immune response costimulatory factor, respectively.
• the plurality of compounds may be, e.g., added to a simple in vitro, to the vulture medium or injected into the cell.
• sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. It can then be determined whether said sample or compound displays the desired properties by methods known in the art such as described herein and in the appended examples. Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s).
• said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
• the methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art (see, e.g., EP-A-0 403 506) or by using and modifying the methods as described in the appended examples.
• Compounds which can be used in accordance with the method of the present invention include peptides, proteins, nucleic acids including cDNA expression libraries, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like, Said compounds can also be functional derivatives or analogues of known leukocyte, lymphocyte (B-, T- or NK-cell) or monocyte activators or inhibitors. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
• said derivatives and analogues can be tested for their effects according to methods known in the art or as described, for example, in the appended examples.
• peptidomimetics and/or computer aided design of appropriate activators or inhibitors of leukocytes, lymphocytes, monocytes (like T-cell, B-cell, NK-cell) activation can be used, for example, according to the methods described below.
• Appropriate computer programs can be used for the identification of interactive sites if a putative inhibitor and the T-cell protein (or its biologically active fragment(s)) by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120).
• the present invention provides methods for identifying compounds which are capable of modulating immune responses. Accordingly compounds identified in accordance with the method of the present invention to be inhibitors and activators, respectively, of immune response are also within the scope of the present invention.
• Compounds found to enhance leukocyte proliferation may be used in the treatment of cancer or infections and related diseases. In addition, it may also be possible to specifically inhibit viral diseases, thereby preventing viral infection or viral spread.
• Compound identified as suppressors of leukocyte proliferation can be used, e.g., for treating skin conditions (see, e.g., WO93/06866) or in allogenic or xenogenic cell or organ transplantation in order to avoid graft refection; see also supra.
• the invention relates to a method for the production of a pharmaceutical composition comprising formulating and optionally synthesizing the compound identified in step (b) of the above described methods of the invention in a pharmaceutically acceptable form.
• the present invention generally relates to a method of making a therapeutic agent comprising synthesizing the proteins, (poly)peptides, polynucleotides, vectors, antibodies or compounds according to the invention in an amount sufficient to provide said agent in a therapeutically effective amount to the patient. Methods for synthesizing these agents are well known in the art and are described, e.g. above.
• the therapeutically useful compounds identified according to the method of the invention may be administered to a patient by any appropriate method for the particular compound, e.g., orally, intravenously, parenterally, transdermally, transmucosally, or by surgery or implantation (e.g., with the compound being in the form of a solid or semi-solid biologically compatible and resorbable matrix) at or near the site where the effect of the compound is desired.
• Therapeutic doses are determined to be appropriate by one skilled in the art, see also supra.
• Such useful compounds can be for example transacting factors which bind to the T-cell protein described herein. Identification of transacting factors can be carried out using standard methods in the art (see, e.g., Sambrook, supra and Ausubel, supra). To determine whether a protein binds to the T-cell protein, standard native gel-shift analyses can be carried out. In order to identify a transacting factor which binds to the T-cell protein, the polypeptides and peptides described in this invention can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
• the transacting factor modulation of its binding to the T-cell protein as described herein can be pursued, beginning with, for example, screening for inhibitors against the binding of the transacting factor to the T-cell protein.
• Activation or repression of T-cell protein specific genes could the be achieved in subjects by applying the transacting factor (or its inhibitor) or the gene encoding it, e.g., in a vector described in the embodiments hereinbefore.
• the active form of the transacting factor is a dimer, dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
• further components in the pathway leading to activation e.g.
• the invention relates to a method for determining the status of the immune system comprising analyzing the presence of the polynucleotide or the protein of the invention.
• the described polynucleotides may also be used for several other applications, for example, for the identification of nucleic acid molecules which encode proteins which interact with the T- cell protein described above. This can be achieved by assays well known in the art, for example, as describe in Scofield (Science 274 (1996), 2063-2065) by use of the so-called yeast "two-hybrid system". In this system the (poly)peptide encoded by the polynucleotides according to the invention or a smaller part thereof is linked to the DNA-binding domain of the GAL4 transcription factor.
• a yeast strain expressing this fusion protein and comprising a lacZ reporter gene driven by an appropriate promoter, which is recognized by the GAL4 transcription factor, is transformed with a library of cDNAs which will express animal, preferably mammal proteins or peptides thereof fused to an activation domain.
• a peptide encoded by one of the cDNAs is able to interact with the fusion protein comprising a (poly)peptide of the invention, the complex is able to direct expression of the reporter gene.
• the polynucleotide according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with T-cell proteins.
• the present invention relates to the use of the polynucleotide, the nucleic acid molecule, the vectors, peptides, polypeptides, antibodies and cells described herein as well as compounds identified in accordance with a method of the invention described herein above for the preparation of a composition for diagnosing and/or the treatment of acute and chronic diseases involving T-cell activation and associated with Thl and Th2 immune response, for the treatment of acute and chronic rejection of allo- and xeno organ transplants ad bone marrow transplantation, for the treatment of rheumatoid arthritis, lupus erythrematodes, multiple sclerosis, encephalitis, vasculitis, diabetes mellitus, pancreatitis, gastritis, thyroiditis, for the treatment of disorders (inter alia malignant disorders) of T-, B- or NK-cells, for the treatment of asthma, lepramatosis, Helicobacter pylori associated gastritis or for the treatment of skin
• the polynucleotide encoding T-cell protein (or (a) fragment(s) thereof) or the antibody as defined herein above is employed for the detection of leukocyte/lymphocyte activation and/or for the treatment of diseases linked to leukocyte/lymphocytes activation.
• the polynucleotides, vectors, cells, proteins, (poly)peptides, antibodies, inhibitors, activators, pharmaceutical and diagnosis compositions, uses described herein above and methods of the invention can be used for the treatment of all kinds of diseases hitherto unknown as being related to or dependent on the modulation of T-cell protein.
• the pharmaceutical compositions, methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.
• the present invention provides a novel route of therapeutic intervention via modulating, preferably inhibiting the activity of the above-described T-cell protein.
• the present invention generally relates to a method for the treatment of a disease, disorder or condition as above which comprises administering to a cell, tissue, organ or subject an effective amount of a compound capable of suppressing T-cell protein activity and/or expression.
• a compound capable of suppressing T-cell protein activity results in inhibiting of the proliferation of PHA activated T-cell- lymphocytes.
• This can be tested, e.g., according to the method described in Example 3.
• Compounds that may be used for the above-described methods include those identified by the methods of the present invention and comprise for example T-cell protein antisense molecules, an anti-T-cell protein antibodies, peptides or peptide mimetics of T-cell protein, ligands, substrates or binding partners of T-cell protein.
• the invention relates to the use of the polynucleotides, nucleic acid molecules and antibodies of the invention for the detection of leukocyte activation as described herein above.
• said leukocyte is a B-cell, T-cell, NK- cell and/or monocyte.
• Figure 1 a Nucleotide sequences of T-cell proteins of the invention.
• Figure 1 b Amino acid sequences of T-cell proteins of the invention.
• Figure 1 c Translation of T-cell proteins of the invention.
• Figure 2 Sequence homology between T-cell proteins and other proteins.
• Figure 3 Antigenicity plots of T-cell protein amino acid sequences.
• FIG. 3 a Antigenicity of TKI7 protein: The most immunogenic region is localized between amino acid 15 to 26.
• Figure 3 g Antigenicity of TPIN7 protein: The most antigenic regions are between amino acids 5-25 and 30-41.
• Example 1 Identification of novel cDNA fragments encoding T-cell proteins that are differentially expressed in alloactivated human T cell lines
• differential display RT-PCR differential display RT-PCR analysis of mRNA expression was performed at time 0 and 24 h after stimulation of a preconditioned human T cell line allo- antigen.
• the preconditioned T cell line was prepared as follows: In conformance with institutional policies regarding human experimentation, peripheral blood lymphocytes (PBLs) were isolated from the healthy human volunteers using standard Ficoll centrifugation methods and diluted into RPMI containing 10% fetal calf serum. Isolated human PBLs (responder PBLs) were stimulated with equal numbers of irradiated (3000 rad, 13 min) stimulator PBLs from another healthy individual.
• PBLs peripheral blood lymphocytes
• RNA was isolated from cells at 0 and 24 h after last stimulation using the RNAzol B method (Tel-Test, Inc) and differential display was performed as described previously (Kojima et al, 1996).
• DDRT-PCR is a method which yields unbiased analysis of changes in message levels from cDNA amplified with multiple sets of primers followed by parallel 6% polyacrylamide gel electrophoresis.
• RNA was reverse transcribed using an oligo-dT primer and 200 U MMLV reverse transrciptase (Gibco/BRL).
• a 40 cycle PCR amplification with a total volume of 10 ⁇ l was performed by using l ⁇ g of cDNA, l,25mM MgCl 2 , 50 mM KC1, 10 mM Tris-HCl (pH 8,3), 2,5 nM primer, 5 ⁇ Cl 35 S-dATP, and 0,3 U Taq polymerase.
• the primers for the PCR amplification were: 5'- TGCTTCAGCACTGCC -3' and - 5'TTATTGTATTTGAAGTAA-3'.
• PCR products were stored at 4°C and separated by electrophoresis in 6% polyacrylamide-urea gels, transferred to filter paper, dried, and autoradiographed.
• the differentially expressed cDNA fragment was excised from the gel, eluted, reamplified, cloned into pBluescriptSK + plasmid, and sequenced. Homology searches were performed using BLAST at NCBI. Alignments were performed using Gene works 2.1.1.
• TKI7 One of the upregulated transcripts, termed TKI7, Figure 1, was reamplified, subcloned and 237 base pairs were sequenced (SEQ ID NO:l). By searching GenBank, the only homologies found were to ephrin receptor ( Figure 2a) as well as to IFR3 ( Figure 2b). Most antigenic region of the TKI7 molecule is localized between amino acid 15-26 as shown in Figure 3a.
• the deduced 42 amino acid sequence of TKI7 (SEQ ID NO: 2) in ORF2 showed 39% identity (57,3% homology) to ephrin receptor of chicken and 34% homology to human IRF3 and ORF2. Due to the homology of the alloactivated protein TKI7 to ephrin receptors TKI7 might be a regulator of immune response to exogenic stimulation including xenoantigens.
• Ephrin Typ B receptors are not described in humans so far. They belong to the family of tyrosin kinases which are membrane proteins involved in signal transduction events. These receptor types are known to be regulatory proteins of the organization of cytosklett proteins, cell adhesion as well as cell proliferation.
• TKI7 is shares also homology with interferon regulator factor 3 (IRF3) in open reading frame +2, +3.
• IRF3 is member of interferon regulating molecules which act as transcription factors to regulate the expression of interferon alpha, beta and gamma.
• Interferons are key molecules in the creation of innate immune response, including :
• TKI7 Antiviral immunity immune response to bacteria immune response to inflammation activation of tyrosine kinases activation of phagocytosis by monocytes regulation of MHC class I and II molecules and induction of antigen presentation polarization of T cell differentiation towards Thl cells polarization of B cell response towards IgG2a and IgG3 subclasses activation of cytolytic function of natural killer cells activation of vascular endothelial cells for cell adhesion
• TKI7 is a potential regulatory molecule during the course of immune response to different antigens inducing immune deviation towards Th2 response.
• TLIP7 Another one of the upregulated transcripts, termed TLIP7, was reamplified, subcloned and 221 base pairs were sequenced (SEQ ID NO: 3). By searching GenBank, the most significant homologies found were to apolioprotein A-I ( Figure 2). The deduced 58 amino acid sequence of TLIP7 (SEQ ID NO: 4) showed 25% identity (46% homology) to apoprotein I molecule (GenBank accession number : AAB34570). Most antigenic region of the TLIP7 molecule is localized between amino acid 5-15 and 28-50 as shown in Figure 3.
• TLIP7 Due to the homology of the alloactivated protein TLIP7 to apolipoprotein TLIP7 might be a transport protein of lipids to create new membranes for cell proliferarion upon immune response to allogenic stimulation including xenoantigens.
• THY7 Another one of the upregulated transcripts, termed THY7, was reamplified, subcloned and 303 base pairs were sequenced (SEQ ID NO: 5). By searching GenBank, the most significant homologies found were to pineal opsin ( Figure 2). The deduced 48 amino acid sequence of THY7 (SEQ ID NO: 6) showed 27% identity (39,6% homology) to p21cdcHs Thyrosine Kinase (GenBank accession number: NM_0057781). Most antigenic region of the THY7 molecule is localized between amino acid 5-17, 28-33 and 30-41 as shown in Figure 3.
• PTPase membrane-permeable phosphothyrosine phosphate inhibitors, such as phenylarsine oxide or pervanadate.
• lymphocytes When lymphocytes are activated by ligation of their receptors for antigen or other mitogens together with appropriate co-receptors or accessory molecules, there is a very rapid, but transient, increase in Ptyr in a number of cellular proteins. Inhibition of this response by pharmacological means blocks lymphocyte activation completely gave rise to the current dogma that the first event triggered by receptor ligation is the activation of one or several PTKs (11,12). Several PTKs are currently known to participate in the initiation of lymphocyte activation.
• Tec family PTKs Btk and Itk/Emt are activated in B cells and T cells, respectively.
• the importance of these PTKs is also demonstrated by the transforming capacity of some of them and the observed changes in amount or function of them inmalignanies, e.g. in T cell lymphomas, and in T cell anergy.
• Due to the homology of the alloactivated protein THY7 to thyrosine kinase THY7 might be a signalling molecule which is induced upon immune response to allogenic stimulation including xenoantigens.
• thyrosine kinases are essential for signalling events of cells transducing signals from membrane receptors to transcription factors which regulates specific gene expression.
• TVIM7 Another one of the upregulated transcripts, termed TVIM7, was reamplified, subcloned and 191 base pairs were sequenced (SEQ ID NO: 7). By searching GenBank, the most significant homologies found were to vimentin ( Figure 2). The deduced 63 amino acid sequence of TVIM7 (SEQ ID NO: 8) showed 38% identity (52% homology) to vimentin in human (GenBank accession number: NP_003371). Most antigenic region of the TVIM7 molecule is localized between amino acid 5-58 as shown in Figure 3.
• TVIM7 Due to the homology of the alloactivated protein TVIM7 to vimentin TVIM7 might belong to the family of intermediar neuronal filaments which are expressed predominantly in lymphocytes upon immune response to allogenic stimulation including xenoantigens. These molecules are important in instruction of cytoskelett orientation upon cell activation as well as cell growth and differentiation.
• TLON7 Another one of the upregulated transcripts, termed TLON7, was reamplified, subcloned and 365 base pairs were sequenced (SEQ ID NO: 9). By searching GenBank, the most significant homologies found were to Ion protease ( Figure 2). Most antigenic region of the TLON7 molecule is localized between amino acid 15-26 as shown in Figure 3. Localization of TLON7 in the cell compartments via PSORT analysis: 56.5%: mitochondrial, 21.7%: nuclear, 13.0%: cytoplasmic, 4.3%: vacuolar, 4.3%: plasma membrane.
• TLON7 The deduced 65 amino acid sequence of TLON7 (SEQ ID NO: 10) showed 23% identity (34% homology) to human ATP dependent mitochondrial Ion protease (GenBank accession number: U02389).
• a number of physiological conditions alter the expression of the mitochondrial genome in mammalian cells. However, these alterations are not always coordinated with changes in the levels of extra-mitochondrially synthesized subunits, suggesting that expression of nuclear and mitochondrial genes is not necessarily tightly coordinated. In such cases, a question arises concerning the fate of the unassembled proteins.
• ATP-dependent proteases have been described on protease, PIMl, was purified from the matrix fraction, and the gene for the human homologue has been cloned. Recently, the inner membrane ATP-dependent, M-aaa, protease and the mitochondrial intermembrane space ATP-dependent protease, MISP1, were also characterized and purified. Although the functions of these mammalian proteases are not well understood, bacterial and yeast mitochondrial ATP-dependent proteases appear to mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. Thus, the ATP-dependent proteases may have a role in ensuring protein integrity and the overall level of organellar biogenesis (Watanabe et al, Eur. J. Biochem. 266 (1999), 811-819).
• TLON7 is able to regulate gene expression of certain proteins by binding to specific DNA elements.
• TLON7 exhibits also homology to LAT-Protein (linker for activation of T-cells) (GenBank Accession: AI922013) which is itself a member of integral membrane proteins. LAT-Protein is known to be involved in T- cell activation upon phosphorylation by ZAP70 molecule.
• THOM7 Another one of the upregulated transcripts, THOM7, was reamplified, subcloned and 201 base pairs were sequenced (SEQ ID NO. 11).
• GenBank By searching GenBank, the most significant homologies found were to homeobox protein ( Figure 2a) as well as to cytochrom C precursor CC3 ( Figure 2b).
• the deduced 66 amino acid sequence of THOM7 (SEQ ID NO: 12) showed 18,2 % identity (39,4% homology) to homeobox protein (GeneBank accession number: P09087) and 27% identity (45,5% homology) to cytochrom precursor CC3 (GeneBank accession number : P24092).
• THOM7 Most antigenic region of the THOM7 molecule is localized between amino acid 4-28 and 29-62 as shown in Figure 3.
• Homeobox genes are transcription factors regulating specific gene expression predominantly for development and growth processes of cells. Transcription factors are sequence-specific DNA-binding proteins with a variety of functions: some are thought to help fold the DNA molecule into distinct domains; others assist in the initiation of DNA replication, and many control gene transcription. Binding affinity to DNA sequences, the concentration of transcription factors and the presence of cofactors are important determinants in gene activation. Transcription factors can be classified according to the three-dimensional structure of their DNA-binding domains.
• transcription factors More than 80% of all transcription factors are characterized by zinc finger, helix- turn-helix, helix-loop-helix, leucine zipper and winged helix motifs. Moreover, the complexity of transcriptional regulation is increased by formation of functional hetero- or homodimers of transcription factors (for instance c-fos and c-jun), or by participation in large protein complexes (for instance c-myc). Transcription of a particular gene is an event regulated by a complex network of transcription factors. The total set of transcription factors present in a cell - induced by extracellular signals - provides a specific genetic imprint that results in a distinct response. Transcription factors have a regulatory role in proliferation and in all kinds of differentiation processes, like embryogenesis, organogenesis, and also hematopoiesis.
• TPIN7 Another one of the upregulated transcripts, termed TPIN7, was reamplified, subcloned and 140 base pairs were sequenced (SEQ ID NO: 13). By searching GenBank, the most significant homologies found were to pineal opsin ( Figure 2). The deduced 46 amino acid sequence of TPIN7 (SEQ ID NO: 14) showed 33% identity (44% homology) to pineal opsin (GenBank accession number : P51476). Most antigenic region of the TPIN7 molecule is localized between amino acid 5-25 and 30-41 as shown in Figure 3. Pineal opsin is an integral membrane molecule which belongs to the family of G coupled receptors. These molecules are able to activate certain signalling pathways via G-coupled receptors. TPIN7 exhibits also homology to a tumor supressor protein MN1 (GenBank accession: Q10571) which supports the finding of a regulatory role of TPIN7 upon cellular growth and activation.
• Example 3 Effects of T-cell protein specific antisense oligonucleotids in T-cell proliferation
• PBMC Peripheral blood lymphocytes
• PHA phytohemagglutinin
• T-cell proteinantisense oligonucleotides are incubated in the presence and absence of an antisense oligonucleotide A2, a sense oligonucleotide (Al) complementary to A2 and two control oligonucleotides (control oligo Cl and C2) in a concentration of 5 ⁇ M oligos (diluted into 1 x TE-Puffer, pH 7.2).
• A2 antisense oligonucleotide
• control oligo Cl and C2 control oligonucleotides
• the 96-well plates are incubated at 37°C and 5% CO 2 24 hr, 48 hr, 72 hr, 96 hr and 168 hr, pulsed with 1 ⁇ Ci of [ 3 H] thymidine per well and harvested 6 hrs later. Incorporated radioactivity is determined in a scintillation counter and the datas are evaluated with StatView.
• T-cell protein specific antisense oligonucleotides in in vitro cultures of human lymphocytes such as in mixed lymphocyte culture including T, B, NK and monocytes and mitogen activated cells.
• the results are expected to demonstrate a significant downregulation of immune response to alloantigen and mitogens in the presence of T-cell protein specific antisense (A2), whereas sense oligonucleotide, Al, or other control oligonucleotides may not exhibit any immunomodulatory effect.
• A2 T-cell protein specific antisense
• sense oligonucleotide, Al, or other control oligonucleotides may not exhibit any immunomodulatory effect. While the initial concentration of antisense construct may vary, the use of different concentrations will reveal a dose dependent inhibition of T-cell proliferation and thus specific antisense effect of the oligonucleotides.
• T-cell protein TKI7 most likely appears to be located in the cytoplasm, TLIP7, THY7, TVIM7 and THOM7 in the nucleus, TLON7 in the mitochondrium and TPIN7 in both cytoplasm and nucleus. Therefore, in order to modulate the activity of those T-cell proteins, preferably agents are used, which act intracellular such antisense constructs or intracellular antibodies.
• T-cell protein TKI7 T-cell protein TKI7
• Predicted cDNA and encoded amino acid sequence is corresponding to accession number EMBL: CNSOIDSV (AL122020) Human chromosome 14 DNA sequence BAC C-3035D6 of library CalTech-D from chromosome 14 of Homo sapiens (Human); see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 97,1 % identity in an 238 nt overlap with SEQ ID NO:
• T-cell protein TLIP7 T-cell protein
• T-cell protein THY7 T-cell protein THY7
• T-cell protein TVIM7 T-cell protein
• T-cell proteinTLON7 T-cell proteinTLON7
• T-cell protein THOM7 T-cell protein THOM7:
• T-cell protein TPIN7 T-cell protein
• TKI7 nucleotide sequence is depicted in SEQ ID NO: 31 and the corresponding encoded amino acid sequence in SEQ ID NO: 32. Furthermore, as a result of the sequence comparison with the genomic sequence of human chromosome 14 (EMBL:CNS01DSV (AL122020); see supra), the nucleotide sequence of TKI7 has been extended (SEQ ID NO: 35) so as to encode additional 60 amino acids (SEQ ID NO: 36).
• nucleotide sequences and their encoded gene products are expected to be directly related to the initial cloning products described in Examples 1 and 2. Therefore, it is credible to assume that they are as useful as the described T-cell protein fragments. Accordingly, those nucleotide and amino acid sequences in SEQ ID NOS: 15 to 32, 35 and 36 are also encompassed by the present invention, in particular their use in the uses and methods for modulating immune response and for the diagnosis and treatment of related disorders.
• nucleic acids, proteins, peptides and antibodies of the invention described herein may be tested beforehand in accordance with the above described methods.
• the nucleic acids, proteins, peptides and antibodies of the invention are particularly useful in diagnosis of, for example, activation state of T-cells, which may correlate with a disorder of the immune system.
• the provision of the present T-cell protein encoding nucleotide sequences is particularly helpful in deciphering and functional annotation of the human genome, which - though announced to be fully sequenced - has still to be investigated for putative gene function.
• the present invention provides a step forward.
• helper T-cells A. Kupfer, S.L. Swain, S.J. Singer, The specific direct interaction of helper T-cells and antigen-presenting B cells. II. Reorientation of the microtubule organizing center and reorganization of the membrane-associated cytoskeleton inside the bound helper T-cells, J. Exp. Med. 165 (1987) 1565-1580.
• Osmotic stress protein 94 (10) R. Kojima, J. Randall, B.M. Brenner, S.R. Gullans, Osmotic stress protein 94 (Osp94): A new member of the Hspll9/SSE gene subfamily, J. Biol. Chem. 271 (1996) 12327-12332.

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