EP1581554A2

EP1581554A2 - Novel t-cell proteins, peptides and antibodies derived therefrom and uses thereof

Info

Publication number: EP1581554A2
Application number: EP02758310A
Authority: EP
Inventors: Nalan Utku
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-07-04
Filing date: 2002-07-04
Publication date: 2005-10-05
Also published as: AU2002325299A1; WO2004011496A3; WO2004011496A2

Abstract

Described are polynucleotides encoding novel T-cell proteins and biologically fragments thereof, which are specifically up-regulated during early events in T-cell activation. Furthermore, vectors are described comprising said polynucleotides and/or host cells transformed therewith. Additionally, antisense constructs to said polynucleotides are described. Furthermore, methods and uses for modulating immune responses through the T-cell protein as well as pharmaceutical compositions comprising agents which act on the T-cell protein are described. Also, uses of said polynucleotides, vectors, proteins or antibodies for the preparation of diagnostic and pharmaceutical compositions for use, inter alia, in organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, or for treatment of tumors are provided.

Description

Title of the invention

Novel T-cell proteins, peptides and antibodies derived therefrom and uses thereof

Field of the invention

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. In addition, 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. Also, 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 gastritis or for the treatment of skin tumors, adrenal tumors or lung tumors, wound healing, growth disorders, inflammatory and/or infectious diseases. Several documents are cited throughout the text of this specification by number in parenthesis. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference.

Background of the invention

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]. Moreover, 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].

Although considerable information on T-cell activation has been gathered in recent years, the complex molecular mechanisms of stimulation and signaling pathways are not completely understood. Since 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;

(b) alloactive cytotoxic T-cells are capable of directly lysing graft endothelial and parenchymal cells; and

(c) alloantibodies bind to endothelium, activate the complement system, and injure thereby graft blood vessels.

The various forms of (allograft) rejection imply a temporal sequence of events including hyperacute, acute vascular and acute cellular as well as chronic rejection. Hyperacute rejection for example plays an important role in xenotransplantation and is due to natural antibodies (ref: Milford, E., Utku, A, N. Guidelines for use of immunogenetic tests organ transplantation, Manual of Clinical Laboratory Immunology, ASM Press 1997).

Considering, inter alia, the temporal sequence events in allograft and xenograft rejection, it is desired to specifically modulate lymphocyte/leukocyte cell responses, i.e. to modulate T-, B-, NK-cells and/or monocyte responses during immunological processes. Furthermore, it is desired to specifically modulate immunological processes like, inter alia, autoimmunological events. Specific modulation of the immune response remains, therefore, a longstanding goal in immunological research.

Summary of the invention

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. of leukocyte activation and/or stimulation. The pharmaceutical 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. For example, 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. In addition, 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. In these cases, 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. Alternatively, 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. For example, it might be useful to induce or maintain the status of activation of the immune cells through vaccination with T-cell protein peptides in a subject who developed a tumor to orchestrate the enhancement of immune response in T-, B-, NK-cells and/or monocytes.

Detailed description of the present invention

In view of the need of therapeutic means for the diagnosis and treatment of diseases related to immune responses of the human body, 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", "TLOΝ7", "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 in the early phase of leukocyte/lymphocyte activation and in particular in T-cell activation.

In a first set of experiments, 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.

The results obtained in accordance with the present invention provide evidence for an essential role of T-cell proteins in the early events of leukocyte activation. Thus, targeting of T-cell protein and their encoding genes provides a novel therapeutic approach for modulation of the immune response. For example, the modulation of T-cell protein expression with specific antisense polynucleotides 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. For publications describing potent and selective gene inhibition using antisense oligonucleotides; see, e.g., Vassar et al., Science 286 (1999), 735-741; Yan et al, Nature 402 (1999), 533-537; Flanagan and Wagner, Molecular and Cellular Biochemistry 172 (1997), 213-225 and Taylor, Drugs 2 (1999), 777-781. In a preferred experimental set up, allo-antigen and mitogen (ConA and PHA) stimulated human lymphocytes and monocytes are incubated in the presence and absence of T-cell protein specific antisense oligonucleotides for 24, 48, 72, 96 and 168 h. 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. For activation studies early 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.

Accordingly, 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:

(I) DNA sequences encoding the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32;

(II) the DNA sequence depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31;

(III) DNA sequences encoding a fragment or derivative of the protein encoded by the DNA sequence of (I) or (II);

(IN) DΝA sequences the complementary strand of which hybridizes with and which is at least 70% identical to the polynucleotide as defined in any one of (I) to (III); and

(N) DΝA sequences the nucleotide of which is degenerate to the nucleotide sequence of a DΝA sequence of any one of (I) to (IN);

The term "T-cell protein", in accordance with the present invention, 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-, ΝK-cell and/or monocyte proliferation in response to alloactivation in a mixed lymphocyte culture when exogeneously added to the culture. In accordance with this invention, it has been surprisingly found that a few cDΝAs are differentially expressed in alloactivated lymphocytes, i.e. human T-cells. These differentially expressed cDNAs are further characterized in Example 2. Here, it has surprisingly been found that 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. The term 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. 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 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. Thus, it should be understood that the term leukocyte may also be used herein so as to refer to individual species of leukocytes such as T-cells only.

In accordance with this invention the term "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" denotes a polypeptide comprising exactly said specific sequence but also comprises polypeptides which comprise a sequence which shows at least 70% homology to said 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. It is understood that 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.

The above described 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. Such "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. It is preferred that said variants are overall (closely) similar, and, preferably, in some regions identical to the polynucleotides and (poly)peptides described herein. The term "variant" in accordance with this invention comprises, but is not limited to allelic variants, synthetically produced variants or genetically engineered variants.

The term "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.

In accordance with the present invention, 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. 12, Issue 5, May 16 2002; and BLAT: The BLAST-like Alignment Tool, Kent, Genome Research 2002 Apr; 12(4): 656-664; see Example 4. The 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.

From the above it is evident that the 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. By the provision of these nucleotide sequences, it is now possible to isolate identical or similar polynucleotides which code for proteins with the biological, immunological activity of T-cell proteins from other species or organisms. Said nucleotide sequences may be employed, in accordance with this invention, in the pharmaceutical compositions, uses and/or methods described herein. Well-established approaches for the identification and isolation of such related sequences are, for example, the isolation from genomic or cDNA libraries using the complete or part of the disclosed sequence as a probe or the amplification of corresponding polynucleotides by polymerase chain reaction using specific primers.

Thus, 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.

With respect to the DNA sequences characterized under (IN) above, the term "hybridizing" in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in 50%formamide/6xSSC/0.1%SDS/100μg/ml ssDΝA, in which temperatures for hybridization are above 37°C and temperatures for washing in 0.1xSSC/0.1%SDS are above 55°C. Most preferably, the term "hybridizing" refers to stringent hybridization conditions, for example such as described in Sambrook, supra.

Particularly preferred are 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. Thus, the pharmaceutical 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.

It is also immediately evident to the person skilled in the art that regulatory sequences may be added to the polynucleotide as defined herein and employed in the pharmaceutical composition, uses and/or methods of the invention. For example, 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).

In a preferred embodiment the 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.

In a further embodiment, 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. Of course, it may also be appropriate to use 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. On the one hand, 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. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinicke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460. Standard methods relating to antisense technology have also been described (Melani, Cancer Res. (1991), 2897-2901). Said antisense or triple helix effect as well as the construction of relevant ribozymes is/are partially useful in pharmaceutical compositions to be employed for the suppression of the immune system, e.g., in autoimmune diseases, for the treatment of rejection events during or after transplantation, etc.. Furthermore, the person skilled in the art is well aware that it is also possible to label such a 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.

The above described nucleic acid molecules may either be DNA or RNA or a hybrid thereof. Furthermore, 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.

In this respect, it is also to be understood that the 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.

In a particular preferred embodiment of the present invention, the polynucleotides as defined herein above may be employed in vaccination approaches. Such 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.

In a preferred embodiment said nucleic acid molecules are labeled. Said labels may comprise radiolabels or fluorescence labels. In another preferred embodiment 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. Preferably 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. Preferably, 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, preferably mammalian 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. coli, and examples for 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. Furthermore, depending on the expression system used 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. Optionally, 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. In this context, 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).

Preferably, 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. 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. In one preferred embodiment of the present invention 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.

In accordance with the above, 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). Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the 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.

In a more preferred embodiment, 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.

In a still further embodiment, the present invention relates to a cell, preferably a host cell, comprising the polynucleotide or vector described above. Preferably, said cell is a eukaryotic, most preferably a mammalian cell if therapeutic uses of the protein are envisaged. Of course, 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. The term "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. The term "eukaryotic" is meant to include yeast, higher plant, insect and preferably mammalian cells. Depending upon the host employed in a recombinant production procedure, 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. Furthermore, 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. In general, 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. Furthermore, 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.

Alternatively, 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.

In this context, it should be mentioned that a method for the production of a T-cell protein or a biologically active fragment thereof may comprise:

(a) culturing a host described herein above under conditions allowing for the expression of the protein; or

(b) in vitro translation of the polynucleotide encoding a T-cell protein and/or a biologically active fragment thereof; and recovering the protein (or a fragment thereof) produced in (a) or (b).

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. Once expressed, 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. Once purified, partially or to homogeneity as desired, the proteins may then be used therapeutically (including extracorporally) or in developing and performing assay procedures.

Hence, in a further embodiment, 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. It will be apparent to those skilled in the art that 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. If necessary, T- cell protein/polypeptides may by particularly useful in (a) pharmaceutical setting(s) where specific leukocyte activation should be controlled. As mentioned herein below, specific overexpression of T-cell proteins or (biologically active) fragments thereof may be obtained by gene therapeutic approaches. As documented in the appended examples, 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.

Furthermore, the provision of the T-cell protein as described herein above enables the production of T-cell protein specific antibodies. In this respect, 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.

Depending on the host cell, renaturation techniques may be required to attain proper conformation of the antibody. If necessary, 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.

Thus, the present invention also relates to an antibody specifically recognizing T-cell protein or (a) fragment(s) (peptides, polypeptides) thereof.

In a preferred embodiment of the invention, 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. Furthermore, 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. When derivates of said antibodies are obtained by the phage display technique, 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. Methods for the production of humanized antibodies are described in, e.g., EP-Al 0 239 400 and WO90/07861. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in,e.g., WO91/10741, WO94/02602, WO96/34096 and WO96/33735. 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.

In a still further embodiment, 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 comprising the polynucleotides, nucleic acid molecules, vectors, cells, proteins, antibodies or antisense construct of the invention. Preferably, 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. Such antisense constructs/oligonucleotides are particularly useful in the down regulation of leukocyte/lymphocyte responses/activations. Therefore, the here described pharmaceutical 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. Methods and computer programs for the preparation and rational selection of antisense oligonucleotide sequences are described in the prior art; see for example Smith, Eur. J. Pharm. Sci. 11 (2000), 191-198; Toschi, Methods 22 (2000), 261-269; Sohail, Adv. Drug Deliv. Rev. 44 (2000), 23-34; Mouton, J. Comput. Biol. 7 (2000), 277-292. These procedures comprise how to find optimal hybridization sites, and on how to select sequences that bind to for example T-cell protein. These methods can include the more empirical testing of large numbers of mRNA complementary sequences to the more systematic techniques, i.e. 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.

28 (2000), 4113-4124. Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside and implications for nucleic acid analysis by hybridisation is described in Nguyen, Nucleic Acids Res. 28 (2000), 3904-3909. Preferably, 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. In a preferred embodiment 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,

29 or 31.

In yet another embodiment 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.

An example for the use of the Pharmaceutical 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. Examples of suitable pharmaceutical carriers are well known in the art and 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. Administration of the suitable 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. As is well known in the medical arts, 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 μg (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. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to lOmg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg 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 10⁶ to 10¹² 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. Examples of 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. Furthermore, 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.

Furthermore, it is envisaged by the present invention that 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. For example, 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). Subsequent to administration, said polynucleotides or vectors may be stably integrated into the gnome of the subject. On the other hand, 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.

In another embodiment the present invention relates to 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. 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.

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.

Furthermore, 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. Furthermore, 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.

In a further embodiment 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.

Furthermore, 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's DNA to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same. The detectable markers of the present invention may be labeled with commonly employed radioactive labels, such as, for example, ³²P or ³⁵S, although other labels such as biotin or mercury as well as those described above may be employed as well. Various methods well- known the person skilled in the art may be used to label the detectable markers. For example, DNA sequences and RNA sequences may be labeled with ³²P or ³⁵S using the random primer method. Once a suitable detectable marker has been obtained, 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. Various methods for the detection of nucleic acids are well-known in the art, e.g., Southern and northern blotting, PCR. primer extension and the like. Furthermore, 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 T₂ 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.

In another embodiment, the present invention relates to a pharmaceutical composition comprising an agent which stimulates a leukocyte through the T-cell protein as described herein, and optionally a pharmaceutically acceptable carrier. As is immediately evident to the person skilled in the art, the provision of the T-cell protein as an immunomodulating molecule opens up the way of alternative approaches for leukocytes stimulation and treating corresponding diseases. 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. Examples for this type of "vaccine" is described, e.g., in WO91/11194 and in the literature, e.g., referred to above. The 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. Such agents can be identified in accordance with a method of the invention described below. Such 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. Such promoter may be regulated and thus permit the controlled expression of the T-cell protein in certain cells.

Therefore, in a further aspect the present invention relates to a method for identifying a binding partner to a T-cell protein polypeptide comprising:

(a) contacting a T-cell protein polypeptide (protein) of the invention with a compound to be screened; and

(b) determining whether the compound effects an activity of the polypeptide (protein).

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. Similarly, 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. (A) potential binding partner(s) of T-cell protein is/are G-protein interacting molecule(s). Preferably, 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 (or cell membrane(s) containing the expressed polypeptide) 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. Alternatively, 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. Preferably, 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. Moreover, the assays can discover agents which may inhibit or enhance the production of T-cell protein from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compounds which bind to T-cell protein comprising the steps of:

(a) incubating a candidate binding compound with T-cell protein; and

(b) determining if binding has occurred:

Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of:

(a) incubating a candidate compound with T-cell protein;

(b) assaying a biological activity as described above, and

(c) determining if a biological activity of T-cell protein has been altered.

As mentioned hereinbefore, 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

(a) culturing leukocytes, lymphocytes or monocytes in the presence of the T-cell protein, (poly)peptide, antibody, cell and/or the antisense construct described above and, optionally, in the presence of a component capable of providing a detectable signal in response to leukocyte proliferation/activation, with a compound to be screened under conditions permitting interaction of the compound with the T-cell protein, (poly)peptide, antibody or cell(s), and

(b) detection the presence or absence of a signal generated from the interaction of the compound with the cells.

The term "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.

If the 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). Preferably 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. Furthermore, the person skilled in the art will readily recognize which further compounds and/or cells may be used in order to perform the methods of the invention, for example, B-cells, interleukins, or enzymes, if necessary, that, e.g., convert a certain compound into the precursor which in turn stimulates or suppresses lymphocytes or monocyte activation or that provide for (co)stimulatory signals. Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.

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. Furthermore, 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, Furthermore, 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). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033- 1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used in combination with the method of the invention for e.g., optimizing known leukocyte activators or inhibitors. Appropriate peptidomimetics can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein and in the appended examples. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of inhibitors or activators of leucocyte stimulation can be used for the design of peptidomimetic inhibitors or activators of leukocyte activation to be tested in the method of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

In summary, 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 compounds identified or obtained according to the method of the present invention are thus expected to be very useful in diagnostic and in particular for therapeutic applications. Hence, in a further embodiment 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. Hence, 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. Once the transacting factor is identified, 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. In addition, if 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. Furthermore, upon identification of the transacting factor, further components in the pathway leading to activation (e.g. signal transduction) or repression of a gene encoding the T-cell protein described herein can then be identified. Modulation of the activities of the components can the be pursued, in order to develop additional drugs and methods for modulating the expression or activity of the T-cell protein. In yet another embodiment 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.

Beside the above described possibilities to use the polynucleotides according to the invention for gene therapy and their use to identify homologous molecules, 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. Thus, if 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. In this way the polynucleotide according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with T-cell proteins. Other methods for identifying compounds which interact with the T-cell protein according to the invention or nucleic acid molecules encoding such molecules are, for example, the in vitro screening with the phage displays system as well as filter binding assays or "real time" measuring of interaction using, for example, the BIAcore apparatus (Pharmacia); see references cited supra.

Furthermore, 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 tumors, adrenal tumors or lung tumors, wound healing, growth disorders, inflammatory and/or infectious diseases. It is particularly preferred that 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. Thus, as described before, the present invention provides a novel route of therapeutic intervention via modulating, preferably inhibiting the activity of the above-described T-cell protein. Therefore, 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. Preferably, said suppressing of 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.

In a further embodiment 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. In a preferred embodiment said leukocyte is a B-cell, T-cell, NK- cell and/or monocyte.

These and other embodiments are disclosed and encompassed by the description and Examples of the present invention. Further literature concerning any one of the antibodies, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized which is available on the Internet, for example under http://www.ncbi.nim.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nim.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.cli biology/research_tools.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

BRIEF DESCRIPTION OF DRAWINGS

This disclosure may best be understood in conjunction with the accompanying drawings, incorporated herein be references, which show:

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.

Figure 3 a: Antigenicity of TKI7 protein: The most immunogenic region is localized between amino acid 15 to 26.

Figure 3 b: Antigenicity of TLIP7 protein: The most antigenic region includes amino acids

1-15 and 28-50 starting atN-Terminus of TLIP7.

Figure 3 c: Antigenicity of THY7 protein: The most antigenic regions are between amino acids 5-17and 28-33 and 38-43.

Figure 3 d: Antigenicity of TVIM7 protein: The most antigenic regions are between amino acids 5-58.

Figure 3 e: Antigenicity of TLON7 protein: The most antigenic region includes amino acids

1-28 starting atN-Terminus of TLON7.

Figure 3 f: Antigenicity of THOM7 protein: The most antigenic regions are between amino acids 4-28 and 29-62.

Figure 3 g: Antigenicity of TPIN7 protein: The most antigenic regions are between amino acids 5-25 and 30-41.

EXAMPLES

A better understanding of the present invention and of is many advantages will be had the following examples, given by way of illustration.

Example 1: Identification of novel cDNA fragments encoding T-cell proteins that are differentially expressed in alloactivated human T cell lines

To identify novel genes induced during the early stages of T cell activation in response to allo-antigens, differential display RT-PCR (DDRT-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. Cells were co-cultured in tissue flasks at an initial concentration of 10⁶ cells/ml restimulated with stimulator cells three times in 10 day intervals prior to RNA isolation. Total 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. Briefly, 2μg of total 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 μl was performed by using lμg of cDNA, l,25mM MgCl₂, 50 mM KC1, 10 mM Tris-HCl (pH 8,3), 2,5 nM primer, 5 μCl ³⁵ S-dATP, and 0,3 U Taq polymerase. The primers for the PCR amplification were: 5'- TGCTTCAGCACTGCC -3' and - 5'TTATTGTATTTGAAGTAA-3'. The 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.

Example 2: Characterisation of cloned T-cell protein cDNAs

Analysis of the cDNAs showed several genes at 24 hours.

T-cell protein 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 :

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 Considering the homology of the TKI7 cDNA to IFR3 molecule, it may be supposed (without being bound by theory) that TKI7 is a potential regulatory molecule during the course of immune response to different antigens inducing immune deviation towards Th2 response.

T-cell protein 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. 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.

T-cell protein 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.

Like most normal cells, resting lymphocytes contain very low levels of phospho tyrosine which is followed by dramatic increase in intracellular tyrosine phosphorylation that follows a brief treatment of T cells with membrane-permeable phosphothyrosine phosphate (PTPase) inhibitors, such as phenylarsine oxide or pervanadate.

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. Using homologues recombination, transgenic mice, and mutant cell lines, it has been demonstrated that two members of the Src family of PTKs, Lck and Fyn, are important in T cells, although their relative contribution and redundancy remains unclear. In B cells, the corresponding Src family PTKs are Lyn, Blk and Fyn, while natural killer (NK) cells also utilize c-Fgr. The two Syk family PTKs ZAP-70 and Syk are also activated within the first minute and lack of a functional zap gene is known to cause a severe immunodeficiency in humans. TCR crosslinking also transiently activates the Csk kinase, which negatively regulates Lck and Fyn. Finally, the 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. As described above, thyrosine kinases are essential for signalling events of cells transducing signals from membrane receptors to transcription factors which regulates specific gene expression.

T-cell protein 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. 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.

T-cell protein 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. 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.

Several mammalian mitochondrial 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).

Ion proteases are localized on mitochondrial membranes binding TG reach single strand DNA elements. In E. coli, such regions are responsible for replication and transcription of mitochondrial DNA. This indicates that 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.

T-cell protein THOM7

Another one of the upregulated transcripts, THOM7, was reamplified, subcloned and 201 base pairs were sequenced (SEQ ID NO. 11). 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). 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. 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.

Specialized blood cells of all lineages originate from the pluripotent stem cell by hematopoesis. Blood cells have a limited life-span, and new cells are generated continuously throughout life. The ancestral pluripotent stem cell must therefore ensure its own survival by proliferation before differentiating into all lineages of blood cells. This balance of proliferation and differentiation is highly dynamic, and pluripotent stem cells and progenitor cells are produced in relation to physiological stresses of the organism (such as bleeding, or infection). The intriguing question arises how this process of blood cell development is controlled, and which genes are involved in this complex process. Cytochrome like molecules are involved in the process of electron transportation which is induced upon cell activation. T-cell protein 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

Peripheral blood lymphocytes (PBMC) are isolated from human volunteers using standard Ficoll centrifugation methods and diluted into RPMI 1640 containing 10% FKS. Number of cells is determined with Neubauer hemocytometer. PBMC are exposed to phytohemagglutinin (PHA) (1 μg/ml). 20.000 cells in a final volume of 0,1 ml of complete RPMI are added to individual wells of a 96-well microtiter plate.

To study the influence of T-cell proteinantisense oligonucleotides on proliferative response of PHA-stimulated lymphocytes, cells 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 μM oligos (diluted into 1 x TE-Puffer, pH 7.2). The 96-well plates are incubated at 37°C and 5% CO₂ 24 hr, 48 hr, 72 hr, 96 hr and 168 hr, pulsed with 1 μCi of [³H] thymidine per well and harvested 6 hrs later. Incorporated radioactivity is determined in a scintillation counter and the datas are evaluated with StatView.

The creation of immune response is analyzed in the presence and absence of 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. 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.

Protein analysis with program PSORT revealed that 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.

Example 4: Gene identification

Further analysis was performed in order to identify human genomic clones corresponding to the cDNAs cloned in Example 1.

For identification of genomic DNA and full-length cDNA corresponding to the T-cell protein encoding sequences SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 13 publicly available bioinformatics have been used. UCSC Human Genome Project Working Draft, April 2002 assembly (hgl l) http://genome.cse.ucsc.edu/; The Human Genome Browser at UCSC, Kent et al., Genome Research, Vol. 12, Issue 5, May 16 2002; and BLAT: The BLAST-like Alignment Tool, Kent, Genome Research 2002 Apr; 12(4): 656-664 have been used to identify chromosome and genomic DNA sequences. Independently, a search has been performed by running SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 13 with the FASTA similarity program in the database of EMBL ALL. Similarity searches have been done using a word size of 6 (ktup=6), with a gap penalty set to -12, extension of the gap -2. Genscan and the Acembly program, which reconstructs gene models solely from mRNA and EST evidence, have been used to identify cDNAs. For a description of the Genscan program and the model that underlies it, reference is made to Burge and Karlin, Prediction of complete gene structures in human genomic DNA, J. Mol. Biol. 268 (1997), 78-94. The splice site models used are described in more detail in Burge, Modeling dependencies in pre-mRNA splicing signals. In Salzberg, S., Searls, D. and Kasif, S., eds. Computational Methods in Molecular Biology, Elsevier Science, Amsterdam (1998), 127-163.

T-cell protein TKI7:

Location chromosome 14:89242214-89242452 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: 1 (2 - 237 : 114585 - 114822). Predicted cDNA and encoded amino acid sequence:

SIZE IDENTITY CHROMOSOME STRAND START END cDNA START END TOTAL

234 98.3% 14 - 89242215 89242452 YourSeq 2 237 237 aagccccagctgactgaggtccgcaagcatctgaccgccgccctggaccgagggaaccttaagtcagaattcctacaagaat ccaatctgatcatggccaagttgaattatgtggaaggtgattataaagaagctctgaacatttacgcccgggtgggcctgga cgatctgccactgacagctgtcccgccctacaggctgcgggtgatcgcagaagcctacgctaccaaaggtgatgtttggaga agctgcctatttcttcttctaccagtaatctccatgtggaccgggaacaggatgtcatcacctgttatgagaaagcagggga catcgcactcctgtatctccaagagatagaaagggtaatactttctaatattcaaaacagaagccctaagcctggccctgct ccccacgatcaagaactaggttttttcctagaaacaggacttcagagagcccatgtcctctatttcaaaa (SEQ ID NO:

15)

KPQLTEVRKHLTAALDRGNLKSEFLQESNLIMAKLNYVEGDYKEALNIYARVGLDDLPLTAVPPYRLRVIAEAYATKGDVWR

SCLFLLLPVISMWTGNRMSSPVMRKQGTSHSCISKR (SEQ ID NO: 16)

T-cell protein TLIP7:

Location chromosome 12:114273255-114273467 corresponding to accession number EMBL: AC002979 Homo sapiens clone RP3-440L2, WORKING DRAFT SEQUENCE, 3 unordered pieces; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 99,1 % identity in an 212 nt overlap with SEQ ID NO: 3 (8 - 218 : 18229 - 18440). Predicted cDNA:

SIZE IDENTITY CHROMOSOME STRAND START END cDNA START END TOTAL

211 99.6% 12 - 114273256 114273467 YourSeq 8 218 221

ATGGGCGAGCCCAAGACCTCGGTGGCCCCGCTGAGCATCGCCAACGGCACGACCCCCGCCAGCACCTCGGAGGACGCCATCA

AGAGCATCCTGGAGCAGGCACGCCGTGAGATGCAGGCGCAACAGCAGGCGCTGCTGGAGATGGAGGTGGCGCCCAGGGGCCG

CTCGGTGCCCCCCTCGCCCCCGGAGCGGCCATCACTGGCCACCGCGAGCCAGAACGGGGCCCCGGCCTTGGTGAAGCAGGAG

GAGGGCAGCGGGGGCCCCGCGCAGGCGCCGCTCCCGGTCCTGTCCCCCGCCGCCTTCGTGCAGAGCATCATCCGCAAGGTCA AGTCCGAGATCGGCGACGCCGGCTACTTCGACCACCACTGGGCCTCCGACCGCGGCCTGCTCAGCCGCCCCTACGCCTCCGT GTCGCCCTCGCTGTCCTCCTCCTCCTCCTCTGGCTACTCTGGCCAGCCCAACGGCCGCGCCTGGCCCCGCGGGGACGAGGCC CCTGTGCCCCCCGAGGACGAGGCGGCGGCAGGGGCGGAGGACGAACCCCCCAGGACGGGCGAGCTCAAGGCTGAGGGCGCGA CGGCCGAGGCGGGCGCGCGGCTGCCCTACTACCCGGCCTACGTGCCGCGCACCCTGAAGCCCACCGTGCCGCCGCTGACCCC CGAGCAGTACGAGCTGTACATGTACCGTGAGGTAGACACGCTGGAGCTCACCCGCCAGGTCAAGGAGAAGCTGGCCAAGAAC GGCATCTGCCAGAGGATCTTCGGGGAGAAGGTGCTGGGCCTGTCACAGGGCAGCGTGAGCGACATGCTGTCCCGGCCGAAGC CATGGAGCAAGCTGACGCAGAAGGGGCGGGAGCCCTTCATCCGCATGCAGCTGTGGCTCTCTGACCAGCTCGGCCAGGCAGT GGGCCAGCAGCCTGGTGCCTCCCAGGCCAGTCCCACAGAACCAAGGTCCTCACCATCCCCACCCCCCAGCCCCACAGAGCCT GAGAAGAGCTCCCAGGAGCCGTTGAGCCTGTCCCTGGAGAGCAGCAAGGAGAACCAGCAGCCAGAGGGCCGCTCCAGCTCCT CGTTGAGCGGGAAGATGTACTCAGGCAGCCAGGCCCCAGGGGGCATCCAGGAGATCGTGGCCATGTCCCCCGAGCTGGACAC GTACTCCATCACCAAGAGGGTGAAGGAGGTCCTCACAGACAACAATCTAGGGCAGCGGCTGTTTGGGGAAAGCATCCTGGGT CTGACACAGGGCTCCGTGTCTGACCTGCTGTCCCGGCCCAAACCCTGGCACAAGCTGAGCCTGAAGGGGCGGGAGCCTTTTG TCCGCATGCAGCTGTGGCTCAATGACCCCCATAACGTGGAGAAGCTGAGGGATATGAAGAAGCTGGAGAAGAAAGCCTACCT GAAACGTCGCTATGGCCTCATCAGCACCGGCTCAGACAGTGAGTCCCCGGCCACCCGCTCAGAGTGCCCCAGCCCCTGCCTG CAGCCCCAGGACCTGAGCCTCCTGCAGATCAAGAAGCCCCGGGTGGTGCTGGCACCCGAGGAGAAGGAGGCACTGCGGAAGG CCTATCAGCTGGAACCCTACCCCTCGCAGCAGACCATCGAGCTCCTCTCCTTCCAGCTCAACCTCAAGACCAACACCGTCAT CAACTGGTTCCACAACTACAGGTCCCGGATGCGCCGGGAGATGTTGGTGGAGGGGACCCAGGATGAGCCAGACCTTGATCCA AGCGGGGGTCCTGGAATCCTACCGCCAGGCCACTCCCACCCAGACCCCACCCCGCAGAGCCCTGACTCTGAGACTGAGGACC AGAAGCCAACCGTGAAGGAACTGGAGCTTCAGGAGGGCCCTGAGGAGAACAGCACACCCCTGACCACCCAGGACAAGGCCCA AGTGAGGATCAAGCAGGAACAGATGGAGGAGGATGCTGAGGAAGAGGCAGGCAGCCAGCCCCAGGACTCAGGGGAGCTGGAC AAAGGCCAAGGTCCCCCCAAAGAGGAGCATCCCGACCCTCCGGGTAATGATGGACTCCCAAAAGTGGCTCCCGGGCCCCTCC TTCCAGGTGGATCCACCCCAGACTGTCCCTCACTTCATCCCCAACAGGAGAGTGAGGCCGGGGAGCGACTTCACCCGGACCC TTTAAGTTTTAAGTCAGCCTCAGAGTCCTCACGCTGCAGCCTGGAGGTGTCACTGAACTCGCCCTCGGCCGCCTCCTCACCA GGCCTCATGATGTCTGTGTCACCTGTCCCCTCCTCCTCAGCTCCCATCTCCCCATCCCCACCTGGCGCCCCCCCTGCCAAAG TGCCGAGTGCCAGCCCCACTGCTGACATGGCTGGAGCCTTGCACCCCAGTGCCAAGGTGAACCCCAACTTGCAGCGGCGGCA TGAGAAGATGGCCAATCTGAACAACATCATTTACCGAGTAGAGCGGGCTGCCAATCGGGAGGAGGCCCTGGAGTGGGAGTTC TGA(SEQ ID NO: 17)

T-cell protein THY7:

Location chromosome 2:241254390-241254679 corresponding to accession number EMBL: AC112715 Homo sapiens BAC clone RP11-370L10 from 2, complete sequence; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 96,3 % identity in an 295 nt overlap with SEQ ID NO: 5 (9 - 303 : 26341 - 26049). Predicted cDNA:

SIZE IDENTITY CHROMOSOME STRAND START END cDNA START END TOTAL

289 95.9% 2 + 241254391 241254679 YourSeq 13 303 303

ATGGCAATGTCGGAACAAGATGATGAGACAGACCGCCGAGTGCTCCAGGCTTCAAATCCTAAAGAAGTCAACAGATGGAGGA

AGGACCATGATACACTCAACAAGGAATTGAGCATCAGCATCCTTGAAGCCTCTGTCACAGCATCCTCTGCAAGAGAGGATGA

AAGGCAAATTCCAGGCTCCTTTGGAAGAATGACAATGCTACAGAAAAGCACATGGATTTGCAGCTTCGTGGACATCACCTGT

GAGGGTGACAGCTCTTTACTCTCAGTGAGTTCCCCAGCCTGGCCCAGAAGCACACTGGAACACAGAAGTCATTCAATCAGGC

AGAAGCTGTGCCCACCAGACAGACTTATTAAGGAGTTCAGTTCACATCCAGGGCAACATGTCAACAAGGACGCGTGTGTCAC TTTTTCTGTCCAGAAGAAAACAGCCTGTGCTCGGGACCTTCGACCGATGTACAGTGCGGTGCTTACACAGTCTGGACTCAAA TGCATCTGGAATGGAGAACCACCCCCAGTGCTGCAGAGCCACCTCATTTTCTATCATCTACCCACCTGGTTTTCGGCCCGCA TGGGACTGAATAAGACCTGGAGTGTTTTGGGAAATGAAGATGGCTTTGCTCTCGCTTCCCCTTCCCTTTTCATCAACCTCAC AAGACGTCAAGTGTTTGCACGTGCACAAGCACTCACCGTGCAGAAACGTCTCATCCTTTCAGCCCTACTGAAATGCCAGCCC TGCTACTCAGTCTCCCCAGGTGCTGTTGAGTAG (SEQ ID NO: 19)

T-cell protein TVIM7:

Location chromosome 12:45992207-45992385 corresponding to accession number EMBL: AC026125 Homo sapiens chromosome 12 clone RP11-234P5; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 98,4 % identity in an 185 nt overlap with SEQ ID NO: 7 (7 - 190 : 65661 - 65845). Predicted cDNA:

ATGAGCCACCACCCGTCGGGCCTCCGGGCCGGCTTCAGCTCCACCTCATACCGCCGTACCTTCGGTCCACCGCC CTCACTATCCCCCGGGGCCTTCTCCTACTCGTCCAGCTCCCGCTTCTCCAGCAGCCGCCTGCTGGGCTCCGCGT CCCCGAGCTCCTCGGTGCGCCTGGGCAGCTTCCGTAGCCCCCGAGCGGGAGCGGGCGCCCTCCTGCGCCTGCCC TCGGAGCGCCTCGACTTCTCCATGGCCGAGGCCCTCAACCAGGAGTTCCTGGCCACGCGCAGCAACGAGAAGCA GGAGCTGCAGGAGCTCAACGACCGCTTCGCCAACTTCATCGAGAAGGTACGCTTTCTGGAGCAGCAGAACGCGG CCCTGCGCGGGGAGCTGAGCCAAGCCCGGGGCCAGGAGCCGGCGCGCGCCGACCAGCTGTGCCAGCAGGAGCTG CGCGAGCTGCGGCGAGAGCTGGAGCTGTTGGGCCGCGAGCGTGACCGGGTGCAGGTGGAGCGCGACGGGCTGGC GGAGGACCTGGCGGCGCTCAAGCAGAGGTTGGAGGAGGAGACGCGCAAGCGGGAGGACGCGGAGCACAACCTCG TGCTCTTCCGCAAGGACGTGGACGATGCCACTCTGTCCCGCCTGGAACTAGAGCGCAAGATTGAGTCTCTGATG GATGAGATTGAGTTCCTCAAGAAGCTGCACGAGGAGGAGCTGCGAGACCTGCAGGTGAGTGTGGAGAGCCAGCA GGTGCAGCAGGTGGAGGTGGAAGCCACGGTGAAGCCCGAGCTGACGGCAGCGCTGAGGGACATCCGCGCGCAGT ACGAGAGCATCGCCGCGAΆGAACCTGCAGGAGGCGGAGGAGTGGTACAΆGTCCAΆGTACGCGGACCTGTCCGAC GCTGCCAACCGGAACCACGAGGCCCTGCGCCAGGCCAAGCAGGAGATGAACGAGTCCCGACGCCAGATCCAGAG TCTAACGTGCGAGGTGGACGGGCTGCGCGGCACGAACGAGGCGCTGCTCAGGCAGTTGAGAGAGCTGGAGGAGC AGTTCGCCCTGGAGGCGGGGGGCTACCAGGCGGGCGCTGCGCGGCTCGAGGAGGAGCTGCGACAGCTAAAAGAG GAGATGGCGCGGCACCTGAGGGAGTACCAGGAGCTCCTCAACGTCAAGATGGCCCTGGACATCGAGATCGCCAC CTACCGCAAGCTGCTGGAGGGCGAGGAGAGCCGGATCTCCGTGCCCGTCCATTCTTTTGCCTCCTTAAATATAA AGACGACTGTGCCTGAGGTGGAGCCTCCCCAGGACAGCCACAGCCGGAAGACGGTTCTGATCAAGACCATTGAG ACCCGGAATGGGGAGGTGGTGACAGAGTCCCAGAAGGAGCAGCGCAGTGAGCTGGACAAGTCTTCTGCCCACAG TTACTGA(SEQ ID NO: 21)

T-cell proteinTLON7:

Location chromosome 14:89242138-89242452 corresponding to accession number EMBL: CNSOIDSV (AL122020) Human chromosome 14 DNA sequence BAC C-3035D6 of library CalTech-D from chromosome 14 ofHomo sapiens (Human); see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 99,1 % identity in an 322 nt overlap with SEQ ID NO: 9 (6 - 327 : 114901 - 114580). Predicted cDNA and amino acid sequence:

SIZE IDENTITY CHROMOSOME STRAND START END cDNA START END TOTAL 314 100.0% 14 + 89242139 89242452 YourSeq 9 322 365 aagccccagctgactgaggtccgcaagcatctgaccgccgccctggaccgagggaaccttaagtcagaattcctacaagaat ccaatctgatcatggccaagttgaattatgtggaaggtgattataaagaagctctgaacatttacgcccgggtgggcctgga cgatctgccactgacagctgtcccgccctacaggctgcgggtgatcgcagaagcctacgctaccaaaggtgatgtttggaga agctgcctatttcttcttctaccagtaatctccatgtggaccgggaacaggatgtcatcacctgttatgagaaagcagggga catcgcactcctgtatctccaagagatagaaagggtaatactttctaatattcaaaacagaagccctaagcctggccctgct ccccacgatcaagaactaggttttttcctagaaacaggacttcagagagcccatgtcctctatttcaaaa (SEQ ID NO: 23)

KPQLTEVRKHLTAALDRGNLKSEFLQESNLIMAKLNYVEGDYKEALNIYARVGLDDLPLTAVPPYRLRVIAEAYATKGDVWR SCLFLLLPVISMWTGNRMSSPVMRKQGTSHSCISKR (SEQ ID NO: 24)

T-cell protein THOM7:

Location chromosome 1:151045681-151045817 corresponding to accession number EMBL: AL139010 Human DNA sequence from clone RP11-444M10 on chromosome 1; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 98 % identity in an 101 nt overlap with SEQ ID NO: 11 (97-197 : 67068 - 66969). Predicted full-length cDNA:

ATGGTGTCAGCTGAACAGCAGCAGCAGCGCAACCTGTTGGGAAAACTTGTAAGGTTTATGCTGAAGCAGGAGAAACCGAAGG TAGACATGAAGAAGAACTTCCTGCTCTTAAGTTTTCAAGAATCCAGAAAAAGTAGGATTTTGGCCAGAAAAGATGAAAACCA TAAGCATCAGGAGGCCTCAGGTATAATCCAGCCCAAGCCCTTCACTGTAGACTGGAGGACATCAGACCCAGGGTCAATCAGC CTCAGGAATTTGCTGCCAGGGCACAGAATGCTTCCAGAAGTCGCCCAGCCATTTGGACACAGCAGGTTAACAGTGGTGGGTC CTCCACGGGGCCTAGAGGATGTCCAGTCAGTGAGCCCAGATAAGCAAGTCTACAGTGAAGATATACTAGACCTAGCTTCTAT ACATTACCTGGCAGGACAGGAAGAGGGAAGAAGCCACACCGGCCAGAGGCTGGAATTCCACCTACCCCTGCACATCAATCGA TGGCTGGATACCAGGGCAGGCCAGGGGAGCTCCGGGGATAAAGCCTGTGCCAGCAGCCAGCACTGTGGACCAGACACAGAGA AGACTGGAGACGTGTTCAGTTACAATACCCAATCAGACAATTTCCATGCAGGAGCAGGGAAGGTTCAGGCTAGACACAAAGA AGAACTTCCTGCTTGCCAGGGAGTATTTGAAATCAAAGTCAAAGAACCCTCCAGGAGATTGCCAAGTAAACTGGTCCAGAGG CCCTGGGAAGAAACCAGACAGGAACTGCCTTCCACCCATCCCGAAGGAGAAGGTAGCCAACTGGATGGCTTTGAAGGGCCAA GACCTGGCCTCACCTCCCTGAATGCAAGCCAGAGGAGAAGACAGCCACCAGTCAGATCTCTAAGCACGCTGGAGTTTGAGAT CTTTTGGGAGCAAAGAAGATATTCCAAGGAAGCCCAGATCCCTGATACATTAAGACATGAGGATGCCAGTAAGACACGCAAC AGAAACTAG (SEQ ID NO: 25)

Location chromosome 20:20905633-20905700 corresponding to accession number EMBL: HSDJ746H2 (ALl 09953) Human DNA sequence from clone RP4-746H2 on chromosome 20; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 95,9 % identity in an 73 nt overlap with SEQ ID NO: 11 (30 - 102 : 147599 - 147671). Predicted full-length cDNA:

ATGGTGACAAAATCAAGCATGGTGCTGTCAGGCAAAGTCATGAAGAAGGTTACTAGAGTGGGCTCCAGCAGCATAGGGCAGT TCTCTGGGAAAGAGGCAGGTTCTTTTGGTCCAGCCCTGACGTCTGGGGTCATTGTAAGGCTCTGCTTCCACGTGAGCTTCAC GAACAAAGGGAAGGGGGTGCTGGACATGAGCAGTCAGCCCCAAAATCGTGGCTGTCCACGGTCTTTAGTCACGGGGGAAACA ATAGTAACATCTGCTGAAGAGTTTTGCATAAGGGTGAAAATGAGGCCATCTGGAAGAAGCAGCAGGGAACTGGCTAAAGCCC AGGCCACTGCCCAGGTCACAGCAGTATCGCAGGTACTTCCTCGGCCATGCACTTCTTGTTTACTTTCTGACTCAGAGGAATT GCAAATCATAGATGATTGGCTGGAAGAAAAGCATCCAACTCAAACTAGCTCAGTCAAAAAACAAGGACCTCTCACAGTGTTT CCCAGAGCCACCTGCCAGGGAACCTGGCTCACCCACATCTTTATTCTGGCCCCACTGGAGCATCGAATTGTAACCGAGTACG CTCATTTTTCTAGAGGAATGGGCTTGAATTGCTACGTGGTAGACCATGGCACACTGTCATTGGAGGGAACAGCCCCCGGTAA GCATGGGCATCTTGGGGATGATATGCCCACCTGGTACCTTCTTCCCTTGGACCATGCTCAGTCATTGAGTAGAATAAGAATA GCTCTTTCATGCTTTGCTAGAGAAGGAGAGCTGTCCACCAAGGAGCCCGCAAATGTAGCACGCGTGGATCTTGCCCAGAGAT GTTGCTGCAGGATGGGTGCTGGCCCTTGTGCTGTCCTGAGTCCGGTGAACACCAGCCCCAAGCAAGCGAGGAAGGGAGGAAG CCTGGTGACTGCTCAGTACTCCCCTCTGGGAAAGTCCTTCCACGTCTCCATGGCTGGAGGCTTAAATTCAAATCGAAGTAGC AAAGCAGACTACTATTTGAAGGCTGCTCTCAGGGAAAAGGAGCACTTCAGGTATGTCCCCCTGTGGGCCAAAAAATGCCCCA GCAAAGAGGAGTGGGCACTTGTAGATGGAAACCATCAAGCCGTCCCAGCTGCTTGTCGCCATTCTTTTAAAGAAGCGTTAAC TCATCACTTTAATCATAAGCAGCAATTTGGAATTTCAAGTGGTCGTTCCAGCAGACACCTCCTCCCCAAAACACCTCGAAGT GTTGGATGTTTCCCCCTGTGGTGTGTCCCTCCTCTCCTGAGTGTCACTGCAGCCACACTCAAGGATACCGAGGACACATTTT GGAAACCACCACGACAAATTCCATGGGCACACCCAGTGTGGCAGCTGGAGCTCTGTTTTGAAAGATATTTGCCAGAGGAAAC ACAGCCTGCTGTTTGGGTTGGAAGCAGCACGATGGACTATTTCAACACTGCCCAGCTACAGTTGTTCCCGTCAATATCCCCT GCGCGGCCGCTTTCCGGGGGCCTTCTCAATCTGCTGTCAACTCGCCTTCGCGGAGCTTCACGAGAGGGTAGGCAGGGCTCGG GGGGAGGCCTGCTGGCAGGACTGAATGTCTTGGGTGGTGGCCCATTCACACCCCTAACGAGATCATTACCACTAACTAAATA CAGGCCTGGCCTTGTGACCCTCTGGGGAATCGAGTGGGCTGGTGCGGGACATCAGTATCGTCTCCAACTCCCACTGCACTGC CTTCCAGCACTGAAAGCTAAGCCCGGGATCAAAAGGTCTCTACGCCCTGGTCTGGCCTCGCTGTCCCCAAGCCTCGAGCCCC TCGGTGACATCCCTGATGACACTGTCAAGTCTTCCCACAGCTCTCCACGGGGCCCAGAGACAAGCTGGATGAACCTTGGTCA CAAGTCCGGGCCCAGCATCATCCCAGATTGTAACATCAAAGATGAGGACTTTGTTGTTGCTGTTTCAGATGCCACTGACACC TTGAAGAAGGTGACCAGCTGGAAACTAATTTACAAGGATCAAAACTCTGAACCTCTTCCAGAAAACAGCAGGGCCCTCATGG AAATAGGCGGCAAATCATTTCAGTTTTTTCCATTTTCTCCTCATGCTGGGCGCAGGACTCAGCATGACTTAGGGCACCCCAG GGGCTGGGGGCCAGGACTAGCTAAAGTCGTACCTGCTGGAGCAATGTCTTCTGCTCTGCGAAGGAGAGATGGCTCCGTGCTC AGTGAAGCAGAAATGGTGGAGTGCTTGGTCTCCTGTGCGCTCCTCAGCTCTCCTACAAACCCCAGCAGCGAGCATGCCCATG AGGGTGTCTTGACTCTTAGGGGCTCTGCCTACACCACAGAGCAGCCTTTCCCCCTCAGGCCTGAGCTTGTGCTGGAACCTTT TGGCTAA (SEQ ID NO: 27)

T-cell protein TPIN7:

Location chromosome 16cl:91281268-91281403 corresponding to accession number EMBL: AC010536 Homo sapiens chromosome 16 clone RP11-278A23; see EBI's public pages under http//www.ebi.ac.uk. This genomic sequence shows 97,8 % identity in an 135 nt overlap with SEQ ID NO: 13 (6-140 : 17869 - 17735). Predicted full-length cDNA: ATGCAGCAGAGAAGGCTAGTCCGAGGCCCCCGAGCCCGCTGCCGAAGACGGAGCTCCAAGACCGAACCTCGTAAAGGGCCCA GCCGCACCTCTACCCGGAAGCGGCTCGAACTCGGGGCCGGAAGTGACTCCATTTCTGTGCGCCGAGCTCCGCCCCACGAGCA CCTGTTTCCGAGCGGAGAGCGCGGGCCGTTTTCTTTCCTGGTGTCCCGTCGCGGCTTGGGACCCGGCAAGATGGGCAAGAAG GGCAAGAAGGAGAAGAAGGGCCGCGGCGCGGAGAAGACGGCCGCCAAGATGGAGAAGAAGGTGTCTAAGCGCTCGCGGAAGG AGGAGCCCGGGGTCTGCAGGAGGATCCGTCCTGACCTTGGGGTTGAGTGTTCTGCGGCTGCTGCTCATGGATTCCCTAACCG GGGCCAGTTTTATGGCCTGCCGTGCGCCAGGCACAGCTCCCTGGGCTTTACTCCTCACGAGCAGTCACCCAATGATGATGAG GTTCAGCCAAGGTCAAAGACCGCTCTAAGTAGCAGCAGCAAAGCTTTCGCGGAAGACCTGGAAGCGCTCATAGCCCATTTCC AGACACTCGATGCCAAGAGGACTCAGACTGTGGAACTTCCGTGCCCCCCACCCTCACCAAGGAAGTGTAGAGGCAGCCAGTT TGTGGTGACAGAGGATTCTGTGAGGAAGGGGACAGCGCTCAGGTTAAATGCCTCCCTCTCGGTTCATCCTGAGAAAGATGAG TTAATCCTTTTTGGAGGTGAATATTTCAACGGCCAAAAAACTTTTTTGTATAACGAGCTCTATGTCTACAATACCAGAAAGG ACACCTGGACCAAAGTTGACATCCCCAGTCCACCTCCGAGGCGCTGTGCTCACCAGGCGGTGGTAGTGCCTCAAGGTGGCGG ACAGCTGTGGGTCTTTGGAGGGGAGTTTGCCTCTCCCAACGGAGAGCAGTTCTACCACTACAAGGATCTCTGGGTCCTGCAT TTGGCCACCAAGACCTGGGAACAAGTCAAGCCGGGAATGTTAACCAGTCAAATGAAGAGTCTGCTGTGGGTGGGGAGGGTGC AGACAGATTCCCTGCCCTGGGCCCACCTGCTGGAGGAGGACATGACCATAGCCTTACAGTGGGACCCGGAAGGTGCTGTGGT GTGGGGCTTTGCTGGTTCTGTCACTCGGATCGTGGGTATCTGTTCCAAGGGAATAGGGATGCACTGTGGGTACCTGGTCACT TTGGGAGTGGCCCTGGCCCCTCTCTTGGGCCGGCAGCTGCGGATGGCAGCCCCTACCTCGTGTGCAGCTCCAGCTGCATCCC ACACTGCAGGCTCGGTCGTACGCGGGACATCCGTGGCAGCTGCCCTGGGTCTGGATTGTCTGATAAACAGACACCTCATTCC TCGAATGCTTGCTAATGGTGCTCCTGTTTCACAGTTTTCCTCAAGTGTTCTCCGTGCTCGTCGTCACTTCATACCTGTGGCA GACACTGGACTACAGCTGCGGCGCCTTGCAGAATGA (SEQ ID NO: 29)

Relationship between TKI7 and TLON7

During the course of analysis of the T-cell protein encoding sequences of TKI7 (SEQ ID NO: 1) and TLON7 (SEQ ID NO: 9) with the Human Genome Browser at UCSC, it turned out that both sequences are related to each other such that they represent their respective antisense sequence, with the nucleotide sequence of TKI7 being contained in that of TLON7; see the above analysis. Furthermore, it appears as if the nucleotide sequence of TLON7 is more accurate than that of TKI7. Therefore, the nucleotide sequence of TKI7 has been corrected and revised through alignment with the corresponding genomic sequences. This version of 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).

Summary

The above described 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. Depending on the intended use 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.

Furthermore, as can been from those analysis, 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.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

References

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(2) CH. June, Signal transduction in T-cells, Curr. Opin. Immunol. 3 (1991) 287-293

(3) R.H. Schwartz, Costimulation of T lymphocytes: The role of CD28, CTLA-4, and B7/BB1 in Interleukin-2 production and immunotherapy, Cell 71 (1992), 1065-1068

(4) J. Banchereau, F. Bazan, D. Blanchard, F. Briere, J. Galizzi, C. van Kooten, Y. Liu, F. Rousset, S. Seeland, The CD40 antigen and ist ligand, Annu. Rev. Immunol. 12 (1994) 881-992.

(5) DJ. Lenschow, T. Walunas, J. Bluestone, CD28/B7 system of T-cell costimulation, Annu. Rev. Immunol. 14 (1996) 233-258.

(6) P. Linsley, J. Ledbetter, The role of the CD28 receptor during T-cell responses to antigen, Annu. Rev. Immunol. 11 (1993) 191-212.

(7) 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.

(8) M.N. Parsey, G.K. Lewis, Actin polymerization and pseudopod reorganization accompany anti-CD3 -induced growth arrest in Jurkat T-cells, J. Immunol. 151 (1993) 1881-1893.

(9) Ν. Selliah, W.H. Brooks, T.L. Roszman, Proteolytic cleavage of -actinin by calpain in T-cells stimulated with anti-CD3 monoclonal antibody, J. Immunol. 156 (1996) 3215-3221.

(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.

(11) Cattaneo E, Gulisano M. Signalling from tyrosine kinases in the developing neurons and glia of the mammalianbrain. Results Probl Cell Differ. 30 (2000) 217-40.

(12) Isakov Ν, Biesinger B. Lck protein tyrosine kinase is a key regulator of T-cell activation and a target for signal intervention by Herpesvirus saimiri and other viral gene products. Eur J Biochem. 267 (2000) 3413-3421

Claims

1. A polynucleotide encoding a T-cell protein, the expression of which is upregulated during the early stages of immune activation in response to xenoantigens or a biologically active fragment thereof comprising a nucleic acid molecule selected from the group consisting of

(i) DNA sequences encoding the amino acid sequence depicted in SEQ ID NO:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32; (ii) DNA sequences comprising at least the coding region of the DNA sequence depicted in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or

31; (iii) DNA sequences encoding a fragment or derivative of the protein encoded by the DNA sequence of (i) or (ii); (iv) DNA sequences the complementary strand of which hybridizes with and which is at least 70% identical to the polynucleotide as defined in any one of

(i) to (iii); and (v) DNA sequences the nucleotide of which is degenerate to the nucleotide sequence of a DNA sequence of any one of (i) to (iv).

2. A nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with a polynucleotide of claim 1 or with a complementary strand thereof.

3. A vector comprising the polynucleotide of claim 1 or the nucleic acid molecule of claim 2.

4. The vector of claim 3, wherein said polynucleotide or nucleic acid molecule is operably linked to regulatory sequences allowing for the transcription and, optionally, expression of said nucleic acid molecule.

5. A host comprising a polynucleotide of claim 1 or the vector of claim 3 or 4.

6. A method for the production of a T-cell protein/polypetide or a biologically active fragment thereof comprising:

(a) culturing the host of claim 5 under conditions allowing for the expression of the protein; or

(b) in vitro translation of the polynucleotide of claim 1 ; and recovering the protein produced in (a) or (b).

7. A T-cell protein/polypeptide or a biologically active fragment thereof encoded by the nucleic molecule of claim 1 or produced by the method of claim 6.

8. An antibody specifically recognizing the protein of claim 7.

9. A cell that has been modified to express the protein of claim 7 or the antibody of claim 8.

10. An antisense construct capable of inhibiting the expression of a polynucleotide of claim 1.

11. A pharmaceutical composition comprising the polynucleotide of claim 1 , the nucleic acid molecule of claim 2, a vector of claim 3 or 4, the cell of claim 5, the protein of claim 7, the antibody of claim 8 or the antisense construct of claim 10 and optionally a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11 for use in cell or organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, or for the treatment of tumors or for the improvement of allograft or xenograft tolerance.

13. A diagnostic composition comprising a polynucleotide of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3 or 4, the cell of claim 5 or 9, the protein of claim 7, or the antibody of claim 8; and optionally, at least one component which is labeled.

14. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to a disorder in the immune system comprising:

(a) determining the presence or absence of a mutation in the polynucleotide of claim 1 ; and

(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation:

15. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to a disorder in the immune system comprising:

(a) determining the presence or amount of expression of the protein of claim 7 in a biological sample; and

(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the protein.

16. A method for identifying a binding partner to a TKI7 protein comprising:

(a) contacting a protein of claim 7 with a compound to be screened; and

(b) determining whether the compound effects an activity of the protein.

17. A method for identifying leukocyte/lymphocyte activating or co-stimulating compounds or for identifying inhibitors of leukocyte activation and stimulation comprising

(a) culturing leukocytes, lymphocytes or monocytes in the presence of the protein of claim 7, the antibody of claim 8, the cell of claim 5 or 9 and, optionally, in the presence of a component capable of providing a detectable signal in response to leucocyte proliferation, with a compound to be screened under conditions permitting interaction of the compound with the (poly)peptide, antibody or cell(s); and

(b) detecting the presence or absence of a signal generated from the interaction of the compound with the cells.

18. A method for the production of a pharmaceutical composition comprising the steps of the method of claim 16 or 17 and formulating and optionally synthesizing the compound identified in step (b) in a pharmaceutically acceptable form.

19. A method for determining the status of an immune response comprising analyzing the presence of the polynucleotide of claim 1 or the protein of claim 7.

20. Use of the polynucleotide of claim 1 the nucleic acid molecule of claim 2, the vector of claim 3 or 4, the protein of claim 7, the antibody of claim 8, the cell of claim 5 or 9, the antisense construct of claim 10 or the compound identified according to the method of any on of claims 14 to 17 for the preparation of a composition 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, thyroiditis, for the treatment of maligne disorders of T, B or NK cells, for the treatment of asthma, lepramatosis, Helicobacter pylori associated gastritis or for the treatment of skin tumors, adrenal tumors or lung tumors, wound healing, growth disorders, inflammatory and/or infectious diseases.

21. Use of a polynucleotide of claim 1, a nucleic acid molecule of claim 2 or the antibody of claim 8 for the detection of leucocyte/lymphocyte activation.

22. Use of claim 20, wherein said leucocyte/lymphocyte is a B cell, T cell, NK cell and/or monocyte.

23. A method for the treatment of a disease, disorder or condition as defined in claim 20 which comprises administering to a cell, tissue, organ or subject a therapeutic effective amount of a compound capable of suppressing the activity and/or expression of a T-cell protein of claim 7.

24. The method of claim 23, wherein said compound is capable of inhibiting the proliferation of PHA activated T-cell-lymphocytes.

25. The method of claim 23 or 24 wherein said compound is a T-cell protein antisense molecule, an anti-T-cell protein antibody, a peptide or peptide mimetic of T-cell protein, or a ligand, substrate or binding partner of T-cell protein.