CA2222823A1

CA2222823A1 - Composition of slap-130, a slp-76 associated protein and methods of use therefor

Info

Publication number: CA2222823A1
Application number: CA 2222823
Authority: CA
Inventors: Gary A. Koretzky; Michael A. Musci; Ranee L. Hendricks-Taylor; David G. Motto
Original assignee: University of Iowa Research Foundation UIRF
Current assignee: University of Iowa Research Foundation UIRF
Priority date: 1996-12-23
Filing date: 1997-12-22
Publication date: 1998-06-23

Abstract

COMPOSITIONS OF SLAP-130, A SLP-76 ASSOCIATED PROTEIN, AND METHODS OF USE THEREFOR

Isolated nucleic acid molecules encoding a novel phosphoprotein, SLAP-130, that interacts with the SH2 domain of the leukocyte protein SLP-76, are disclosed. In addition to isolated nucleic acids molecules encoding SLAP-130, the invention provides antisense nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals carrying a SLAP-130 transgene. The invention further provides isolated SLAP-130 proteins and peptides, SLAP-130 fusion proteins and anti-SLAP-130 antibodies. Methods of using the SLAP-130 compositions ofthe invention are also disclosed, including methods for detecting SLAP-130 protein or mRNA in a biological sample, methods of modulating SLAP-130 activity in a cell, and methods for identifying agents that modulate an interaction between SLAP-130 and SLP-76.

Description

COMPOSITIONS OF SLAP-130, A SLP-76 ASSOCIATED PROTEIN, AND METHODS OF USE THEREFOR

5 Back~round of the Invention Engagement of the T cell antigen receptor (TCR) results in the activation of protein tyrosine kinases (PTK) and the subsequent tyrosine phosphorylation of numerous proteins (Howe, L.R. and Weiss, A. (1995) Trends Biochem. Sci. 20:59-64; see also Perlmutter, R.M.
et al. (1993) ,4nnu. Rev. Immunol. 11:451-499; and Chan, A.C. et al. (1994) Annu. Rev.
Immunol. 12:555 592). Efforts to characterize substrates ofthe TCR induced PTK activity led to the cloning of a 76 kDa protein termed SLP-76 (for SH2-domain-containing Leukocyte Protein of 76 kDa). SLP-76 was originally identified based upon its ability to interact with the protein Grb2, an adaptor molecule involved in coupling signal transduction pathways (Motto, D. et al. (1994) J. Biol. Chem. 269:21608-21613; Reif, K. et al. (1994) J. Biol. Chem.
269:14081-14087; Buday, L. etal. (1994)J Biol. Chem. 269:9019-9023; and Sieh, M. etal.
(1994) Mol. Cell. Biol. 14:4435 4442).
Molecular cloning of SLP-76 cDNAs (human and mouse) revealed that the SLP-76 protein comprises an acidic amino-terminal region, a proline-rich central region and a carboxy-terminal SH2 domain (Jackman J.K. et al. (1995) J. Biol. Chem. 270:7029-7032).
Northern analysis demonstrated that SLP-76 mRNA is expressed exclusively in peripheral blood leukocytes, spleen and thymus (Jackman, J.K et al. (1995) supra). Insight into the function of SLP-76 in T cells came from experiments showing that overexpression of SLP-76 augments TCR-mediated signals that lead to the induction of IL-2 gene promoter activity (Motto, D.G. et al. (1996) J. Exp. Med. 183:1937-1943; Wu, J. et al. (1996) Immunity _:593-602). Interestingly, three distinct regions of SLP-76 that are responsible for protein-protein interactions in T cells are required for the augmentation of IL-2 promoter activity by overexpression of SLP-76 (Fang, N. et al. (1996) J. Immunol M 57:3769-3773; Wardenburg, J.B. et al. (1996) J. Biol. Chem. 271 :19641-19644). These data suggest that SLP-76 functions as a link between proteins that regulate signals generated by TCR ligation.
Certain SLP-76-associated proteins that participate with SLP-76 in transducing signals from the TCR to the nucleus have been identified. Examples include the protooncogene Vav, which associates with the amino-terminal acidic region of SLP-76 in a phosphotyrosine dependent manner (Wu, J. et al. (1996) Immunity _:593-602; Onodera, H. et al. (1996) J. Biol. Chem. 271:22225-22230; Tuosto, L. et al. (1996) J. Exp. Med. 184:1161-1167). Identification and characterization of other proteins capable of interacting with SLP-76 will be important for understanding the role of SLP-76 in T cell activation and, accordingly, for designing approaches to modulate this process.

Summary of the Invention Nucleic acid molecules encoding a novel protein, termed SLAP-130, that interactswith the leukocyte protein SLP-76, have now been isolated and characterized. The nucleotide sequence of a SLAP-130 cDNA, and predicted amino acid sequence of SLAP-130 protein, are shown in Figure 1 (and in SEQ ID NOs: 1 and 2, respectively). SLAP-130 is predomin~ntly expressed in hematopoietic cells, is a substrate for the TCR-stimulated protein tyrosine kinases and was identified based upon its ability to interact with the src homology 2 (SH2) domain of SLP-76. Overexpression of SLAP-130 (limini~hes TCR induced activation of a promoter containing three NFAT sites in a T cell line and blocks the augmentation of 10 activity of this promoter that is seen when SLP-76 is overexpressed in these T cells, indicating that SLAP-130 can function as a negative regulator of signals that activate IL-2 gene transcription. This invention pertains to isolated compositions of SLAP-130 protein and isolated nucleic acid sequences encoding SLAP-130, other compositions related thereto and methods of use thereof.
One aspect of the invention pertains to isolated nucleic acid molecules encodingSLAP-130, or fragments thereof. In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding SLAP-130 protein. In another embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein, wherein the protein (i) comprises an amino acid 20 sequence at least 60 % homologous (more preferably 70%, 80%, 90% or 95% homologous) to the amino acid sequence of SEQ ID NO: 2 and (ii) associates with the SH2 domain of SLP-76 or modulates T cell receptor mediated sign~ling. In yet another embodiment, the invention provides an isolated nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
25 In yet another embodiment, the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or encoding the amino acid sequence of SEQ ID NO: 2. Isolated nucleic acid molecules encoding SLAP-130 fusion proteins and isolated antisense nucleic acid molecules are also encompassed by the invention.Another aspect of the invention pertains to vectors, such as recombinant expression 30 vectors, containing an nucleic acid molecule of the invention and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce SLAP-130 protein by culturing the host cell in a suitable medium. If desired, SLAP-1 ~0 protein can be then isolated from the host cell or the medium.
Still another aspect of the invention pertains to isolated SLAP-130 proteins, or35 portions thereof. In one embodiment, the invention provides an isolated SLAP-130 protein, or a portion thereof that interacts with SLP-76. In another embodiment, the invention provides an isolated protein that comprises an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2 and associates with the SH2 domain of SLP-76 or modulates T cell receptor mediated sign~ling In still other embodiments, the invention provides an isolated protein comprising the amino acid sequence of SEQ ID NO: 2. SLAP-130 fusion proteins are also encompassed by the invention.
The SLAP-130 proteins of the invention, or fragments thereof, can be used to prepare anti-SLAP-130 antibodies. Accordingly, the invention further provides an antibody that specifically binds SLAP-130 protein. In one embodiment, antibodies ofthe invention are polyclonal antibodies. In another embodiment, antibodies of the invention are monoclonal antibodies. In yet another embodiment, the antibodies are labeled with a detectable substance.
The SLAP-130-encoding nucleic acid molecules of the invention can be used to 10 prepare nonhuman transgenic ~nim~l~ which contain cells carrying a transgene encoding SLAP-130 protein or a portion of SLAP-130 protein. Accordingly, such transgenic ~nim~l~
are also provided by the invention. In one embodiment, a SLAP-130 transgene is integrated randomly into the genome of an animal. Alternatively, the SLAP-130-encoding nucleic acid molecules of the invention also can be used to make homologous recombinant ~nim~l~ (e.g, 15 "knockout ~nimzll~"), in which a SLAP-130 transgene (or portion thereof) is integrated at a specific location within the genome of the animal by homologous recombination (e.g., to alter or disurpt an endogenous gene encoding endogenous SLAP-130 protein).
Another aspect of the invention pertains to methods for detecting the presence of SLAP-130 activity in a biological sample. To detect SLAP-130 activity, the biological 20 sample is contacted with an agent capable of detecting SLAP-130 activity, such as SLAP- 130 protein (such as a labeled anti-SLAP-130 antibody) or SLAP-130 mRNA (such as a labeled nucleic acid probe capable of hybridizing to SLAP-130 mRNA) such that the presence of SLAP-130 activity is detected in the biological sample.
Still another aspect of the invention pertains to methods for modulating SLAP-130 25 activity in a cell. To modulate SLAP-130 activity in a cell, the cell is contacted with an agent that modulates SLAP-130 activity such that SLAP-130 activity in the cell is modulated. In one embodiment, the agent inhibits SLAP-130 activity. In another embodiment, the agent stimulates SLAP-130 activity. In one embodiment, the agent modulates the activity of SLAP-130 protein (e.g., the agent can be an antibody that specifically binds to SLAP-130 30 protein). In another embodiment, the agent modulates transcription of a SLAP-130 gene or translation of a SLAP-130 mRNA (e.g, the agent can be a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the SLAP- 130 mRNA or the SLAP-130 gene).
Still another aspect of the invention pertains to methods for identifying agents that 35 modulate an interaction between SLAP-130 and SLP-76. In these methods, SLAP-130 (or a SLP-76-interacting portion thereof) is combined with SLP-76 (or a SLAP-130-interacting portion thereof, such as the SLP-76 SH2 domain) in the presence and absence of a test compound. The degree of interaction between SLAP-130 and SLP-76 is determined in the presence and absence of the test compound. A modulatory agent is identified based upon the ability of the test compound to increase or decrease (e.g., stimulate or inhibit) the degree of interaction between SLAP-130 and SLP-76 (as compared to the degree of interaction in the absence of the test compound).

S Brief Description of the Drawings Figure 1 shows the cDNA sequence and deduced amino acid sequence of human SLAP-130 (SEQ ID NOs: 1 and 2, respectively). The coding region corresponds to nucleotides 31-2379. The region encompassing a peptide having the amino acid sequence PPNVDLTK (SEQ ID NO: 4)iS indicated by an underline.
Figure 2 is a photograph of a Northern blot analysis of polyA+ RNA from the indicated tissues hybridized to a SLAP-130 nucleic acid probe, demonstrating expression of SLAP-130 mRNA in the lymphoid co~llpalllllent.
Figure 3A is a photograph of an immunoprecipitation/Western blot experiment demonstrating the Jurkat T cells transiently transfected with pEF/SLAP-130 (encoding a FLAG epitope-tagged SLAP-130 fusion protein) express a 130 kDa protein reactive with anti-FLAG antibody.
Figure 3B a photograph of an immunoprecipitation/Western blot experiment demonstrating the Jurkat T cells transfected with pEF/SLAP-130 (encoding a FLAG epitope-tagged SLAP-130 fusion protein) and stimulated with pervanadate express a 130 kDa protein that can be immunoprecipitated by the SLP-76 SH2 domain.
Figure 4 is a photograph of an immunoprecipitation/Western blot experiment demonstrating that SLAP-130 and SLP-76 associate in Jurkat T cells. Lysates from Jurkat cells were subjected to immunoprecipitation with anti-SLP-76 antiserum and then immunoblotted with both anti-SLP-76 and anti-SLAP-13 antiserum.
Figure SA is a bar graph depicting the luciferase reporter gene activity in Jurkat T
cells cotransfected with an NFAT luciferase reporter construct and either pEF (control vector), pEF/SLP-76 (a SLP-76 expression vector), pEF/SLAP-130 (a SLAP-130 expression vector) or both pEF/SLP-76 and pEF/SLAP-130, demonstrating that overexpression of SLAP-130 ~limini~hes transcriptional activation through the NFAT response element.
Figure SB is a photograph of an immunoblot experiment depicting the expression of FLAG epitope-tagged constructs in the transfected Jurkat cells of Figure SA.
Immunoblotting was performed with anti-FLAG antibodies.

Detailed Description of the Invention This invention pertains to compositions related to the SLP-76 associated proteinSLAP-130, and methods of use thereof. A cDNA encoding SLAP-130 was isolated based on the ability of the SLAP-130 protein to interact with the SH2 domain of SLP-76 (see Example 1). Analysis ofthe tissue distribution of SLAP-130 revealed that SLAP-130 mRNA is expressed in peripheral blood Iymphocytes, thymus and spleen but not in a variety of non-lymphoid tissues (see Example 2). SLAP-130 protein has been expressed recombinantly in m~mm~ n cells as a fusion protein with an epitope tag, and this fusion protein can be precipitated by the SH2 domain of SLP-76 (see Example 3). Native SLAP-130 associates with SLP-76 in vivo, as demonstrated by coimmunoprecipitation of SLAP-130 and SLP-76 with either anti-SLAP-130 antiserum or anti-SLP-76 antiserum (see Example 4).
Overexpression of SLAP-130 in a T cell line inhibits TCR-induced activation of a promoter containing Nuclear Factor of Activated T cell (NFAT) binding sites and, furthermore, blocks the augmentation of NFAT-cont~ining promoter activity that is seen when SLP-76 is overexpressed in these cells, indicating that at least under certain conditions SLAP-130 can 10 function as a negative regulator of TCR-mediated signaling (see Example 5).
The invention encompasses, for example, isolated SLAP-130 proteins, as well as fragments and fusion proteins thereof, antibodies that bind to SLAP-130, isolated nucleic acid molecules encoding SLAP-130, as well as fragments thereof and antisense nucleic acid molecules, SLAP-130 vectors and host cells, transgenic ~nim~l~ carrying a SLAP-130 15 transgene, methods of detecting or modulating SLAP-130 activity in a cell and methods of identifying agents that modulate the interaction between SLAP-130 and SLP-76.
So that the invention may be more readily understood, certain terms are first defined.
As used herein, the term "SLP-76" refers to a 76 kDa, leukocyte-specific protein, the human and mouse forms of which have the amino acid sequences disclosed in Jackman, J.K.
20 etal.(l995) J. Biol. Chem. 270:7029-7032.
As used herein, the term "src homology 2 domain"(abbreviated as SH2 domain) refers to a protein domain, typically of about 100 amino acids in length and conserved among a variety of cytoplasmic signaling proteins (including SLP-76), that binds phosphotyrosine containing peptides. For a review article on SH2 domains, see Koch, C.A. et al. (1991) 25 Science 252:668-674 (which also discloses and compares the amino acid sequences of many different SH2 domains). The SH2 domain of human SLP-76 comprises approximately the region encompassing amino acid residues 420 to 514 (as disclosed in Jackman, J.K. et al.
(199~) supra), the amino acid sequence of which is shown in SEQ ID NO: 3.
As used herein, the term "nucleic acid molecule" is intended to include DNA
30 molecules (e.g, cDNA or genomic DNA) and RNA molecules (e.g, mRNA). The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
As used herein, an "isolated nucleic acid molecule" refers to a nucleic acid molecule that is free of gene sequences which naturally flank the nucleic acid in the genomic DNA of 35 the organism from which the nucleic acid is derived (i.e., gene sequences that are located adjacent to the isolated nucleic molecule in the genomic DNA of the organism from which the nucleic acid is derived). For example, in various embodiments, the isolated SLAP-130 nucleic acid molecule may contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be free of other cellular material.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60 % homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that at least sequences at least 65 %, more preferably at least 70 %, and even more preferably at least 75 % homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A
preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X
sodium chloride/sodium citrate (SSC) at about 45~C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65~C.
As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural 1 5 protein).
As used herein, an "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g, complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
As used herein, the term "coding region" refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term "noncoding region" refers to regions of a nucleotide sequence that are not translated into amino acids (e.g, 5' and 3' untranslated regions).
As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector, wherein additional DNA
segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g, bacterial vectors having a bacterial origin of replication and episomal m~mm~ n vectors). Other vectors (e.g, non-episomal m:~mm~ n vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host g~nJme. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.
Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
As used herein, the term "host cell" is intended to refer to a cell into which a nucleic acid of the invention, such as a recombinant expression vector of the invention, has been 5 introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the 10 term as used herein.
As used herein, a "transgenic animal" refers to a non-human animal, preferably amAmmAI, more preferably a mouse, in which one or more of the cells of the animal includes a "transgene". The term "transgene" refers to exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome 15 of the mature animal, for example directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
As used herein, a "homologous recombinant animal" refers to a type of transgenicnon-human animal, preferably a mAmmAl, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an 20 exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
As used herein, an "isolated protein" refers to a protein that is substantially free of cellular material or culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In 25 one embodiment ofthe invention, an isolated SLAP-130 protein is prepared by expressing the protein in non-mAmmAlian cells (e.g, yeast or bacterial host cells) such that the isolated SLAP-130 protein is substantially free of other mAmmAIiAn cellular material.
As used herein, the term "antibody" is intended to include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e., molecules that 30 contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as Fab and F(ab')2 fragments. The terms "monoclonal antibody" and "monoclonal antibody composition", as used herein, refer to a population of antibody molecules that contain only one species of an antigen binding ~lt~ capable of immunoreacting with a particular epitope of an antigen. A monoclonal antibody composition thus typically displays a single binding 35 affinity for a particular antigen with which it immunoreacts.

Various aspects of the invention are described in further detail in the following subsections:

I. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode SLAP-130, or fragments thereof. Most preferably, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO: 1. Nucleotides 31 -2379 5 ofthe sequence of SEQ ID NO: 1 correspond to the coding region ofthe human SLAP-130 cDNA. Nucleotides 1-30 correspond to a 5' untranslated (5' UT) region, whereas nucleotides 2380 to 2400 correspond to a 3' untranslated (3' UT) region. In certain embodiments, an isolated nucleic acid fragment of the invention is at least 1100 nucleotides in length. More preferably the fragment is at least 1200, 1300, 1400, 1500, 1600, 1800, 1900, 2000, 2100, 2200 or 2300 nucleotides in length. The invention further encompasses nucleic acid molecules that differ from SEQ ID NO: 1 (and fragments thereof) due to degeneracy of the genetic code and thus encode the same SLAP-130 protein as that encoded by SEQ ID NO: 1.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID
1~ NO:2.
U.S. Patent Application Serial No. 08/774,061, to which this application claims priority, also discloses SLAP-130 cDNA and deduced protein sequences as SEQ ID NOs: 1 and 2, respectively. The sequences in USSN 08/774,061 differ slightly from those in the instant application due to a minor sequencing error. More specifically, a stretch of three 20 guanines was read as four guanines, which altered the deduced amino acid sequence at the C-terminus ofthe SLAP-130 protein (the last 13 amino acids of SEQ ID NO: 2 of USSN08/774,061 are replaced with the last 30 amino acids of SEQ ID NO: 2 of the instant application). Resequencing also revealed two additional amino acid sequence differences between SEQ ID NO: 2 of USSN 08/774,061 and SEQ ID NO: 2 of the instant application, at 2~ position 273 (a proline to leucine change) and position 526 (an asparagine to lysine change), which likely represent polymorphisms between cell types. All such polymorphisms are encompassed by the invention. The sequences of SEQ ID NOs: 1 and 2 of the instant application represent human T cell cDNA and protein, respectively.
A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or a 30 portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human SLAP-130 cDNA can be isolated from a cDNA library (e.g., prepared from human blood cells (commercially available fronl Stratagene) or from human T Iymphocytes or the human T cell line Jurkat) using all or portion of SEQ ID NO: 1 as a hybridization probe and standard hybridization techniques 3~ (e.g., as described in Sambrook, J., et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1. For example, mRNA can be isolated from human cells (e.g, by the _ 9 _ guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemis~ry 18:
5294-5299) and cDNA can be prepared using reverse transcriptase (e.g, Moloney MLV
reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic 5 oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO: 1. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropl;ate vector and characterized by DNA sequence analysis. Furthermore, 10 oligonucleotides corresponding to a SLAP-130 nucleotide sequence can be prepared by standard synthetic techniques, e.g, using an automated DNA synthesizer.
In addition to the human SLAP-130 nucleotide sequence shown in SEQ ID NO: 1, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of SLAP-130 may exist within a population (e.g, the 15 human population). Such genetic polymorphism in the SLAP-130 gene may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1 -5 % variance in the nucleotide sequence of the a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SLAP-130 that are the result of natural allelic variation and that do not alter the functional activity of SLAP- 130 are 20 intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding SLAP-130 proteins from other species, and thus which have a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1 but that is related to the human sequence, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and nonhuman homologues ofthe human SLAP-130 cDNA ofthe25 invention can be isolated based on their homology to the human SLAP-130 nucleic acid molecule disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention hybridizes under stringent conditions to the nucleic acid molecule comprising the 30 nucleotide sequence of SEQ ID NO: 1. In certain embodiment, the nucleic acid is at least 1100, 1200, 1300, 1400, 1500, 1600, 1800, 1900,2000,2100,2200Or2300nucleotidesinlength. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally-occurri~lg nucleic acid molecule. In on embodiment, the nucleic acid encodes natural human SLAP-130 35 protein. In another embodiment, the nucleic acid molecule encodes a natural murine homologue of human SLAP-130 protein, such as mouse SLAP-130 protein.
In addition to naturally-occurring allelic variants of the SLAP-130 sequence that may exist in the population, the skilled artisan will further appreciate that changes may be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, thereby leading to changes in the amino acid sequence of the encoded protein, without altering the functional activity ofthe SLAP-130 protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made in the sequence of SEQ ID NO: 1. A "non-essential" amino acid residue is a residue that can be altered from the 5 wild-type sequence of SLAP-130 (e.g, the sequence of SEQ ID NO: 2) without altering the functional activity of SLAP-130, such as its ability to associate with SLP-76 or its ability to modulate T cell receptor mediated sign~ling, whereas an "essential" amino acid residue is required for functional activity. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SLAP-130 proteins that contain changes in amino acid residues that are not essential for SLAP-130 activity. Such SLAP-130 proteins differ in amino acid sequence from SEQ ID NO: 2 yet retain SLAP-130 activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 and associates with the SH2 domain of SLP-76 or 15 modulates T cell receptor mediated signaling. Preferably, the protein encoded by the nucleic acid molecule is at least 70 % homologous to SEQ ID NO: 2, more preferably at least 80 %
homologous to SEQ ID NO: 2, even more preferably at least 90 % homologous to SEQ ID
NO: 2, and most preferably at least 95 % homologous to SEQ ID NO: 2.
To determine the percent homology of two amino acid sequences (e.g, SEQ ID NO: 220 and a mutant form thereof), the sequences are aligned for optimal comparison purposes (e.g, gaps may be introduced in the sequence of one protein for optimal alignment with the other protein). The amino acid residues at corresponding amino acid positions are then compared.
When a position in one sequence (e.g, SEQ ID NO: 2) is occupied by the same or a similar amino acid residue as the corresponding position in the other sequence (e.g, a mutant form of 25 SLAP-130), then the molecules are homologous at that position (i.e., as used herein amino acid "homology" is equivalent to amino acid identity or similarity). As used herein, an amino acid residue is "similar" to another amino acid residue if the two amino acid residues are members of the same family of residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains 30 (e.g, lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucir.e, isoleucine, proline, pheny1~1~nine, methionine, tryptophan), beta-branched si~e chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine).
35 The percent homology between two sequences, therefore, is a function of the number of identical or similar positions shared by two sequences (i. e., % homology = # of identical or similar positions/total # of positions x 100).
An isolated nucleic acid molecule encoding a SLAP-130 protein homologous to the protein of SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID NO: 1 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined above. Thus, a nonessential amino acid residue in SLAP-130 protein is preferably replaced with another amino acid residue from the same side chain family.
10 Alternatively, in another embodiment, mutations can be mtroduced randomly along all or part of a SLAP-130 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for their ability to interact with SLP-76 (e.g., using a GST-SLP-76-SH2 fusion protein) to identify mutants that retain SLP-76-interacting ability.
Following mutagenesis of SEQ ID NO: 1, the encoded mutant protein can be 15 expressed recombinantly in a host cell and the ability of the mutant protein to interact with SLP-76 can be determined using an in vitro interaction assay. For example, a recombinant SLAP-130 protein (e.g, a mutated or truncated form of SEQ ID NO: 2) can be radiolabeled and incubated with a GST-SLP-76-SH2 fusion protein. Glutathione-sepharose beads are then added to the mixture to precipitate the SLAP-130-GST-SLP-76-SH2 complex, if such a 20 complex is formed. After washing the beads to remove non-specific binding, the amount of radioactive protein associated with the beads is determined and compared to the amount of radioactive protein rem~ining in the eluate to thereby determine whether the SLAP-130 protein is capable of interacting with the SLP-76 SH2 domain.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of 25 the coding region of SEQ ID NO: 1, for example a fragment encoding a biologically active portion of SLAP-130. The term "biologically active portion of SLAP-130" is intended to include, for example, portions of SLAP-130 that retain the ability to associate with SLP-76 or modulate T cell receptor signaling. The ability of a portion of SLAP-130 to interact with SLP-76 can be determined using an assay described in further detail in Example 3. Briefly, 30 to determine the ability of a portion of SLAP-130 to associate with the SLP-76 SH2 domain, a nucleic acid molecule encoding the portion of SLAP-130 can be cloned into an expression vector, the expression vector can be introduced into Jurkat T cells, the T cells can be stimulated with an activ~.c. of a protein tyrosine kinase, such as pervanadate, and imml]noprecipitations can be carried out using a SLP-76 SH2 domain fusion protein (e.g, a 35 fusion protein comprising the SLP-76 SH2 domain, the amino acid sequence of which is shown in SEQ ID NO: 3, and glutathione-S-transferase (GST)). The ability ofthe SLAP-130 protein, or portion thereof, to be immunoprecipitated by the SLP-76 SH2 domain fusion protein (e.g, SLP-76 SH2/GST) indicates that the SLAP-130 protein, or portion thereof, associates with the SH2 domain of SLP-76.

The ability of a portion of SLAP-130 to modulate T cell receptor signaling can be determined using an assay described in further detail in Example 5. Briefly, to determine the ability of a portion of SLAP-130 to modulate T cell receptor sign~ling, a nucleic acid molecule encoding the portion of SLAP-130 can be cloned into an expression vector, the S expression vector can be cotransfected into Jurkat T cells with an ~plop~iate reporter gene construct for measuring T cell receptor signaling (e.g., an IL-2 promoter reporter gene construct or a reporter gene construct cont~ining NFAT sites), the T cells can be stimulated with an activator of a protein tyrosine kinase, such as pervanadate, and reporter gene activity in the presence and absence ofthe portion of SLAP-130 can be evaluated. The ability ofthe 10 SLAP-130 protein, or portion thereof, to be modulate reporter gene activity indicates that the SLAP-130 protein, or portion thereof, to modulate T cell receptor signaling. In view of the foregoing, the invention encompasses isolated nucleic acid fragments encoding biologically active fragments of SLAP-130, such as fragments of the nucleic acid molecule of SEQ ID
NO: 1 and nucleic acid molecules encoding fragments of the protein of SEQ ID NO: 2.
Another aspect of the invention pertains to isolated nucleic acid molecules that are antisense to the coding strand of a SLAP-130 mRNA or gene. An antisense nucleic acid of the invention can be complementary to an entire SLAP-130 coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a coding region of the coding strand of a nucleotide sequence encoding SLAP-130. In another embodiment, the antisense nucleic acid molecule is antisense to a noncoding region of the coding strand of a nucleotide sequence encoding SLAP-130. In certain embodiments, the antisense nucleic acid is at least 1100, 1200, 1300, 1400, 1500, 1600, 1800, 1900, 2000, 2100, 2200 or 2300 nucleotides in length.
Given the coding strand sequences encoding SLAP-130 disclosed herein (e.g., nucleotides 31-2379 of SEQ ID NO: 1), antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule may be complementary to the entire coding region of SLAP-130 mRNA, or alternatively can be an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SLAP-130 mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation start site of SLAP-130 mRNA. An antisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g, phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
In another embodiment, an antisense nucleic acid of the invention is a ribozyme.5 Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. A ribozyme having specificity for a SLAP-130-encoding nucleic acid can be designed based upon the nucleotide sequence of a SLAP-130 cDNA disclosed herein (i.e., SEQ ID NO: 1). For example, a derivative of a Tetrahymena L-19 IVS RNA can be 10 constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a SLAP-130-encoding mRNA. See for example Cech et al. U.S.
Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, SLAP-130 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261:
1411-1418.
Yet another aspect of the invention pertains to isolated nucleic acid molecules encoding SLAP-130 fusion proteins. Such nucleic acid molecules, comprising at least a first nucleotide sequence encoding a SLAP-130 protein, polypeptide or peptide operatively linked to a second nucleotide sequence encoding a non-SLAP-130 protein, polypeptide or peptide, 20 can be prepared by standard recombinant DNA techniques. SLAP-130 fusion proteins are described in further detail below in subsection III.

II. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably recombinant expression 25 vectors, cont~ining a nucleic acid encoding SLAP-130 (or a portion thereof). The expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
30 Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The telm "regulatory sequence" is intended to includes promoters, enhancers and other expression 35 control elements (e.g, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g, tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or 5 peptides, encoded by nucleic acids as described herein (e.g, SLAP-130 proteins, mutant forms of SLAP-130 proteins, SLAP-130 fusion proteins and the like).
The recombinant expression vectors of the invention can be designed for expression of SLAP-130 protein in prokaryotic or eukaryotic cells. For example, SLAP-130 can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) 10 yeast cells or m~mm~lian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA
(1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors 15 cont~ining constitutive or inducible promotors directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors can serve one or more purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; 3) to aid in the purification of the recombinant 20 protein by acting as a ligand in affinity purification; 4) to provide an epitope tag to aid in detection and/or purification of the protein; and/or 5) to provide a marker to aid in detection of the protein (e.g., a color marker using ,B-galactosidase fusions). Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the 25 fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.; Smith, D.B. and Johnson, K.S.
(1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or 30 protein A, respectively, to the target recombinant protein. Recombinant proteins also can be expressed in eukaryotic cells as fusion proteins for the same purposes discussed above.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and ~Er 1 ld (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-35 89). Target gene expression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target gene expression from the pET
1 ld vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ~ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant 5 protein (Gottesm~n, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nuc. Acids Res. 20:2111 -2118). Such alteration of nucleic acid sequences of the 10 invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the SLAP-130 expression vector is a yeast expression vector.
Examples of vectors for expression in yeast S. cerivisae include pYepSec 1 (Baldari. et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San 15 Diego, CA).
Alternatively, SLAP-130 can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g, Sf 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in m~mm~lian cells using a m~mm~lian expression vector. Examples of m~mm~lian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (K~llfm~n et al. (1987), EMBO J. 6:187-195). When used in m~mm~lian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
A preferred m~mm~ n expression vector for expressing SLAP-130 is pEF-BOS
(Mi7ll~him~, S. et al. (1990) Nucl. Acids Res. 18:5322) (discussed further in the Examples).
In another embodiment, the recombinant m~mm~ n expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g, tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include lymphold specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), neuron-specific promoters (e.g, the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and m~mm:~ry gland-specific promoters (e.g, milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No.
264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
S Moreover, inducible regulatory systems for use in m~mm~ n cells are known in the ar~, for example systems in which gene expression is regulated by heavy metal ions (see e.g, Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al.
(1985) Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g, Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton, FL, ppl67-220), hormones (see e.g, Lee et al.
10 (1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042;
Klock et al. (1987) Nature 329:734-736; Israel & K~llfm~n (1989) Nucl. Acids Res. 17:2589 2604; and PCT Publication No. WO 93/23431), FK506-related molecules (see e.g, PCT
Publication No. WO 94/18317) or tetracyclines (Gossen, M. and Bujard, H. (1992) Proc.
Natl. ~lcad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCT
15 Publication No. WO 94/29442; and PCT Publication No. WO 96/01313). Accordingly, in another embodiment, the invention provides a recombinant expression vector in which SLAP-130 DNA is operatively linked to an inducible eukaryotic promoter, thereby allowing for inducible expression of SLAP-130 in eukaryotic cells.
The invention further provides a recombinant expression vector comprising a DNA
20 molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to SLAP-130 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense 25 RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a reeom~inant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be 30 ~letermined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.
Another aspect of the invention pertains to recombinant host cells into which a vector, preferably a recombinant expression vector, of the invention has been introduced. A host cell 3~ may be any prokaryotic or eukaryotic cell. For example, SLAP-130 protein may be expressed in bacterial cells such as E. coli, inseet eells, yeast or m~mm~ n eells (sueh as Jurkat T cells, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, CA 02222823 l997-l2-22 the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or 5 transfecting host cells can be found in Sambrook et al. (Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
For stable transfection of m~mm:~lian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate 10 the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g, resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that 15 encoding SLAP-130 or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g, cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) SLAP-130 protein. Accordingly, the invention further 20 provides methods for producing SLAP-130 protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding SLAP-130 has been introduced) in a suitable medium until SLAP-130 is produced. In another embodiment, the method further comprises isolating SLAP-130 from the medium or the host cell. In its native form SLAP-130 protein is 25 thought to be an intracellular protein and, accordingly, recombinant SLAP-130 protein can be expressed intracellularly in a recombinant host cell and then isolated from the host cell, e.g, by lysing the host cell and recovering the recombinant SLAP-130 protein from the lysate.
Alternatively, recombinant SLAP-130 protein can be prepared as a extracellular protein by operatively linking a heterologous signal sequence to the amino-terminus of the protein such 30 that the protein is secreted from the host cells. In this case, recombinant SLAP-130 protein can be recovered from the culture medium in which the cells are cultured.
Certain host cells of the invention can also be used to produce nonhuman transgenic ~nim~l~ For example, in one embodiment, a host cell of the inven[iJn is a fertilized oocyte or an embryonic stem cell into which SLAP-130-coding sequences have been introduced.
35 Such host cells can then be used to create non-human transgenic ~nim~l~ in which exogenous SLAP-130 sequences have been introduced into their genome or homologous recombinant ~nim~ in which endogenous SLAP-130 sequences have been altered. Such ~nim~l~ areuseful for studying the function and/or activity of SLAP-130 and for identifying and/or evaluating modulators of SLAP-130 activity. Accordingly, another aspect ofthe invention pertains to nonhuman transgenic ~nim~ls which contain cells carrying a transgene encoding a SLAP-130 protein or a portion of a SLAP-130 protein. In a subembodiment, of the transgenic animals of the invention, the transgene alters an endogenous gene encoding an endogenous SLAP-130 protein (e.g, homologous recombinant ~nim~l~ in which the endogenous SLAP-130 gene has been functionally disrupted or "knocked out", or the nucleotide sequence of the endogenous SLAP-130 gene has been mutated or the transcriptional regulatory region of the endogenous SLAP- 130 gene has been altered).
- A transgenic animal ofthe invention can be created by introducing SLAP-130-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g, by microinjection, 10 and allowing the oocyte to develop in a pseudopregnant female foster animal. The human SLAP-130 cDNA sequence of SEQ ID NO: 1 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of the human SLAP-130 gene, such as a mouse SLAP-130 gene, can be isolated based on hybridization to the human SLAP-130 cDNA and used as a transgene. Intronic sequences and polyadenylation 15 signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the SLAP-130 transgene to direct expression of SLAP-130 protein to particular cells. Methods for generating transgenic ~nim~l~ via embryo manipulation and microinjection, particularly ~nim~ls such as mice, have become conventional in the art and are described, for example, in 20 U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder e~ al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe SLAP-130 transgene in its genome and/or expression of SLAP-130 mRNA in 25 tissues or cells of the ~nim~ls A transgenic founder animal can then be used to breed additional :~lnim~l~ carrying the transgene. Moreover, transgenic ~nim:~ls carrying a transgene encoding SLAP-130 can further be bred to other transgenic ~nim~l~ carrying othertransgenes.
To create a homologous recombinant animal, a vector is prepared which contains at 30 least a portion of a SLAP-130 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g, functionally disrupt, the endogenous SLAP-130 gene. The SLAP-130 gene may be a human gene (e.g, from a human genomic clone isolated from a human genomic library screened wl.n .he cDNA of SEQ ID NO: 1), but more preferably, is a non-human homologue of a human SLAP-130 gene. For example, a mouse SLAP-130 gene35 can be isolated from a mouse genomic DNA library using the human SLAP-130 cDNA of SEQ ID NO: 1 as a probe. The mouse SLAP-130 gene then can be used to construct ahomologous recombination vector suitable for altering an endogenous SLAP-130 gene in the mouse genome. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous SLAP-130 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous SLAP-130 gene is mutated or otherwise altered but still encodes functional protein (e.g, the upstream regulatory region can be altered to thereby alter the expression of 5 the endogenous SLAP-130 protein). In the homologous recombination vector, the altered portion ofthe SLAP-130 gene is flanked at its 5' and 3' ends by additional nucleic acid ofthe SLAP-130 gene to allow for homologous recombination to occur between the exogenous SLAP-130 gene carried by the vector and an endogenous SLAP-130 gene in an embryonic stem cell. The additional fl~nking SLAP-130 nucleic acid is of sufficient length for 10 successful homologous recombination with the endogenous gene. Typically, several kilobases of fl~nking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced SLAP-130 gene has homologously recombined with the endogenous SLAP-130 gene are selected (see e.g, Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g, a mouse) to form aggregation chimeras (see e.g, Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp.
113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed ~nim~l~ in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant ~nim~l.s are described further in Bradley, A. (1991) Current Opinion in Biotechnology _:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO
93/04169 by Berns et al.
In addition to the foregoing, the skilled artisan will appreciate that other approaches known in the art for homologous recombination can be applied to the instant invention.
Enzyme-assisted site-specific integration systems are known in the art and can be applied to integrate a DNA molecule at a predetermined location in a second target DNA molecule.
Examples of such enzyme-assisted integration systems include the Cre recombinase-lox targe. ~ystem (e.g., as described in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res.
21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g., as described in Dang, D.T. and Perrimon, N. (1992) Dev. Genet. 13:367 375; and Fiering, S. et al. (1993) Proc. Natl. Acad.
Sci. USA 90:8469-8473). Tetracycline-regulated inducible homologous recombination systems, such as described in PCT Publication No. WO 94/29442 and PCT Publication No.
WO 96/01313, also can be used.

III. Isolated SLAP-130 Proteins and Anti-SLAP-130 Antibodies Another aspect of the invention pertains to isolated SLAP-130 proteins, and portions thereof, such as biologically active portions, as well as peptide fragments suitable as immunogens to raise anti-SLAP-130 antibodies. In one embodiment, the invention provides an isolated preparation of SLAP-130 protein. Preferably, the SLAP-130 protein has an amino acid sequence shown in SEQ ID NO: 2. In other embodiments, the SLAP-130 protein is substantially homologous to SEQ ID NO: 2 and retains the functional activity of the protein of SEQ ID NO: 2 yet differs in amino acid sequence due to natural allelic variation or 10 mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the SLAP-130 protein is a protein which comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 and that interacts with SLP-76. Preferably, the protein is at least 70 % homologous to SEQ ID NO: 2, more preferably at least 80 % homologous to SEQ ID NO: 2, even more preferably at least 90 %
15 homologous to SEQ ID NO: 2, and most preferably at least 95 % homologous to SEQ ID
NO: 2.
The invention further provides a portion of a SLAP-130 protein that interacts with SLP-76. The SLAP-130 protein interacts with the SH2 domain of SLP-76 and it is known that SH2 domains recognize phosphotyrosine-containing binding sites. Based on analysis of 20 the amino acid sequence of SLAP-130, tyrosine residues can be identified as potential SLP-76 SH2 domain binding sites (when the tyrosine residue of SLAP-130 is phosphorylated).
Accordingly, peptides encompassing tyrosine-cont~ining regions of SLAP-130 are provided by the invention and can be prepared by standard peptide synthesis techniques. Preferably, the tyrosine-cont~ining peptide is at least 5 amino acids in length and more preferably at least 25 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length. An in vitro interaction assay (such as an assay l]tili7ing a GST-SLP-76-SH2 fusion protein) can be used to determine the ability of such peptides, when phosphorylated on tyrosine, to interact with the SLP-76 SH2 domain.
SLAP-130 proteins are preferably produced by recombinant DNA techniques. For 30 example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the SLAP-130 protein is expressed in the host cell. The SLAP-130 protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a SLAP-130 polypeptide can 35 be synthesized chemically using standard peptide synthesis techniques. Moreover, native SLAP-130 protein can be isolated from cells (e.g, human T cells or the human T cell line Jurkat), for example using an SLP-76 SH2 fusion protein to precipitate SLAP-130 from cell lysates (described further in the Examples) or by immunoprecipitation using an anti-SLAP-130 antibody.

The invention also provides SLAP-130 fusion proteins. As used herein, a SLAP-130"fusion protein" comprises a SLAP-130 polypeptide operatively linked to a non-SLAP-130 polypeptide. A "SLAP-130 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to SLAP-130 protein, or a peptide fragment thereof, whereas a "non-S SLAP-130 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to another protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the SLAP-130 polypeptide and the non-SLAP-130 polypeptide are fused in-frame to each other. The non-SLAP-130 polypeptide may be fused to the N-terminus or C-terminus of the SLAP-130 polypeptide. Examples of fusion proteins include epitope-tagged SLAP-130 proteins, such as SLAP-130 that has been fused in frame to the FLAGTM epitope (see Example 3) and glutathione-S-transferase fusion proteins in which the SLAP-130 sequence (or a portion thereof) is fused to the C-terminus ofthe GST sequences.
Such fusion proteins can facilitate the detection and/or purification of recombinant SLAP-130. A fusion protein of the invention may comprise the entire SLAP-130 protein or only a 15 portion ofthe SLAP-130 protein.
Preferably, a SLAP-130 fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, 20 restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary 25 overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). A SLAP-130-encoding nucleic acid can be cloned into such an 30 expression vector such that the fusion moiety is linked in-frame to the SLAP-130 protein.
An isolated SLAP-130 protein, or fragment thereof, can be used as an immunogen to generate antibodies that bind SLAP-130 using standard techniques for polyclonal and monoclonal antibody preparation. The SLAP-130 protem ~an be used to generate antibodies or, alternatively, an antigenic peptide fragment of SLAP-130 can be used as the immunogen.
35 An antigenic peptide fragment of SLAP-130 typically comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and encompasses an epitope of SLAP-130 such that an antibody raised against the peptide forms a specific immune complex with SLAP-130. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of SLAP-130 that are located on the surface of the protein, e.g., hydrophilic regions. A standard hydrophobicity analysis of the SLAP-130 protein sequence shown in SEQ ID NO: 2 can be performed to identify such 5 hydrophilic regions.
A SLAP-130 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other m~mm~l) with the immunogen. An appropriate immunogenic preparation can contain, for examples, recombinantly expressed SLAP-130 protein or a chemically synthesized SLAP-130 peptide. The preparation can 10 further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic SLAP-130 preparation induces a polyclonal anti-SLAP-130 antibody response.
Accordingly, another aspect ofthe invention pertains to anti-SLAP-130 antibodies.
Polyclonal anti-SLAP-130 antibodies can be prepared as described above by immunizing a suitable subject with a SLAP-130 immunogen. The anti-SLAP-130 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized SLAP-130. If desired, the antibody molecules directed against SLAP-130 can be isolated from the m~mm~l (e.g, from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-SLAP-130 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) JBiol Chem 255:4980-83;Yeh etal. (1976) PNAS 76:2927-31;andYeh etal. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R.
H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell line (typically a myeloI.la) is fused to lymphocytes (typically splenocytes) from a m~mm:~l immunized with a SLAP-130 immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds SLAP-130.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-SLAP-130 monoclonal antibody (see, e.g, G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g, a myeloma cell line) is derived from the same m~mm~ n species as the lymphocytes.
5 For example, murine hybridomas can be made by fusing Iymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines.
These myeloma lines are available from the American Type Culture Collection (ATCC), - Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fusedmyeloma cells (unfused splenocytes die after several days because they are not transformed).
Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supern~t~nt~ for antibodies that bind SLAP-130, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-SLAP-130 antibody can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage display library) with SLAP-130 to thereby isolate immunoglobulin library members that bind SLAP-130. Kits for generating and screening phage display libraries are commercially available (e.g, the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Sur~ZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al.
International Publication No. WO 92/18619; Dower et al. International Publication No. WO
91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288;
McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809;
Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9: l 373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137;
Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-SLAP-130 antibodies, such as chimeric and hllm:~ni7ed monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and hllm~ni7ed monoclonal antibodies can be produced by5 recombinant DNA techniques known in the art, for example using methods described in 3~obinson et al. International Patent Publication PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT Application WO 86/01533;
Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023;
Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al.
(1987)J. Immunol. 139:3521-3526; Sunetal. (1987)PNAS84:214-218;Nishimuraetal.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446 449; and Shaw et al.
(1988)J. Natl CancerInst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207;
Oi et al. (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J.
Immunol. 141:4053-4060.
An anti-SLAP-130 antibody (e.g., monoclonal antibody) can be used to isolate SLAP-130 protein by standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-SLAP-130 antibody can facilitate the purification of natural SLAP-130 from cells and of recombinantly-produced SLAP-130 expressed in host cells. Moreover, an anti-SLAP-130 antibody can be used to detect SLAP-130 protein (e.g., in a cellular Iysate or cell supernatant). Detection may be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Accordingly, in one embodiment, an anti-SLAP-130 antibody of the invention is labeled with a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ~-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I 131I 35S or 3H.

IV. Pharmaceutical Compositions SLAP-130 modulators ofthe invention (e.g., SLAP 130 inhibitory or stimulatory agents, including SLAP-130 proteins and antibodies) can be incorporated into pharmaceutical compositions suitable for a~mini.~tration. Such compositions typically comprise the modulatory agent and a ph~rm~ceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical ~lmini~tration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the 5 compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of ~tlmini~tration. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile 10 diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with 15 acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Ph~ çeutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous 20 plepalation of sterile injectable solutions or dispersion. For intravenous ~(lministration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the cont~min~ting 25 action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the 30 use of surfactants. Prevention of the action of microorg~ni.~m~ can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mamtol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by 35 including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic ~-lmini~tration, the active compound can be incorporated with excipients and used in the form 10 of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar 15 nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch;
a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide;
a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimin~tion from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of 25 such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in 30 U.S. PatentNo. 4,522,811.

V. Methods of the Invention Anothel a,pect of the invention pertains to methods of using the various SLAP- 130 compositions of the invention. For example, the invention provides a method for detecting 35 the presence of SLAP-130 activity in a biological sample. The method involves contacting the biological sample with an agent capable of detecting SLAP-l 30 activity, such as SLAP-130 protein or SLAP-130 mRNA, such that the presence of SLAP-130 activity is detected in the biological sample. A preferred agent for detecting SLAP-130 mRNA is a labeled nucleic acid probe capable of hybridizing to SLAP-130 mRNA. The nucleic acid probe can be, for example, the SLAP-130 cDNA of SEQ ID NO: 1, or a portion thereof sufficient to specifically hybridize under stringent conditions to SLAP-130 mRNA. A preferred agent for detecting SLAP-130 protein is a labeled antibody capable of binding to SLAP-130 protein.
Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g, Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a 10 fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample"
is intended to include tissues, cells and biological fluids. For example, techniques for detection of SLAP-130 mRNA include Northern hybridizations and in situ hybridizations.
Techniques for detection of SLAP-130 protein include enzyme linked immunosorbent assays 15 (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
The invention further provides methods for identifying agents that modulate an interaction between SLAP-130 and SLP-76. In one embodiment, the method comprises:
(a) combining:
(i) a SLAP-130 protein, or SLP-76-interacting portion thereof; and (ii) SLP-76, or a SLAP-130-interacting portion thereof;
in the presence and absence of a test compound;
(b) determining the degree of interaction between (i) and (ii) in the presence and absence of the test compound; and (c) identifying an agent that modulates an interaction between SLAP-130 and SLP-76.
25 Isolated SLAP-130 and/or SLP-76 proteins may be used in the method, or, alternatively, only portions of SLAP-130 and/or SLP-76 may be used. For example, an isolated SLP-76 SH2 domain (or a larger subregion of SLP-76 that includes the SH2 domain) can be used as the SLAP-130-interacting portion of SLP-76. Likewise, an isolated SH2 binding domain of SLAP-130 (e.g., a peptide comprising a phosphotyrosine that interacts with the SLP-76 SH2 30 domain) can be used as the SLP-76-interacting portion of SLAP-130. In a preferred embodiment, one or both of (i) and (ii) are fusion proteins, such as GST fusion proteins (e.g., GST-SLP-76-SH2 can be used as the SLAP-130-interacting portion of Fyn). The degree of interaction between (i) and (ii) can be determined, for example, by labeling one of the proteins with a detectable substance (e.g, a radiolabel), isolating the non-labeled protein and 35 qu~~ Lillg the amount of detectable substance that has become associated with the non-labeled protein. The assay can be used to identify agents that either stimulate or inhibit the interaction between SLAP-130 and SLP-76. An agent that stimulates the interaction between SLAP-130 and SLP-76 is identified based upon its ability to increase the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence of the agent, whereas an agent that inhibits the interaction between SLAP-130 and SLP-76 is identified based upon its ability to decrease the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence of the agent. Assays systems for identifying agents that modulate SH2 domain-ligand interactions that can be adapted to SLAP-130/SLP-76 in accordance with the present invention are described further in U.S.
Patent No. 5,352,660 by Pawson.
Yet another aspect of the invention pertains to methods of modulating SLAP-130 activity in a cell. The modulatory methods of the invention involve contacting the cell with an agent that modulates SLAP-130 activity such that SLAP-130 activity in the cell is 10 mo~ te~l The agent may act by modulating the activity of SLAP-130 protein in the cell or by mocll~ ing transcription of the SLAP-130 gene or translation of the SLAP-130 mRNA.
As used herein, the term "modlll~tin~" is intended to include inhibiting or decreasing SLAP-130 activity and stimulating or increasing SLAP-130 activity. Accordingly, in one embodiment, the agent inhibits SLAP-130 activity. An inhibitory agent may function, for 15 example, by directly inhibiting SLAP-130 activity, by inhibiting an interaction between SLP-76 and SLAP-130, by inhibiting SLP-76/SLAP-130-mediated sign~ling, and/or by inhibiting TcR/CD3/SLP-76/SLAP-130-mediated signaling. In another embodiment, the agent stimulates SLAP-130 activity. A stimulatory agent may function, for example, by directly stimulating SLAP-130 activity, by promoting an interaction between SLP-76 and SLAP-130, 20 by promoting SLP-76/SLAP-130-mediated sign~ling, and/or by promoting TcR/CD3/SLP-76/SLAP- 130-mediated signaling .

A. Inhibitory Agents According to a modulatory method of the invention, SLAP-130 activity is inhibited in a cell by contacting the cell with an inhibitory agent. Inhibitory agents of the invention can be, for example, intracellular binding molecules that act to inhibit the expression or activity of SLAP-130. As used herein, the term "intracellular binding molecule" is intended to include molecules that act intracellularly to inhibit the expression or activity of a protein by 30 binding to the protein itself, to a nucleic acid (e.g, an mRNA molecule) that encodes the protein or to a second protein with which the first protein normally interacts (e.g., molecules that bind to SLP-76 to thereby inhibit the interaction between SLP-76 and SLAP-130).
Examples of intracellular binding molecules, described in further de~all below, include antisense SLAP-130 nucleic acid molecules (e.g., to inhibit translation of SLAP-130 mRNA), 35 intracellular anti-SLAP-130 antibodies (e.g., to inhibit the activity of SLAP-130 protein), molecules that mimic an SH2 binding site of SLAP-130 (e.g, to inhibit the interaction of SLAP-130 with the SH2 domain of SLP-76) and dominant negative mutants ofthe SLAP-130 protein.

In one embodiment, an inhibitory agent of the invention is an antisense nucleic acid molecule that is complementary to a gene encoding SLAP-130, or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule. The use of antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986; Askari, F.K. and McDonnell, W.M. (1996) N. Eng J. Med. 334:316-318; Bennett, M.R. and Schwartz, S.M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen, J.S. (1995) Cancer Gene Ther. 2:47-59;
Rossi, J.J. (1995) Br. Med. Bull. 51 :217-225; Wagner, R.W. (1994) Nature 372:333-335).
10 An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (e.g., an mRNA sequence) and accordingly is capable of hydrogen bonding to the coding strand of the other nucleic acid molecule. Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5' or 3'15 untranslated region of the mRNA or a region bridging the coding region and an untranslated region (e.g., at the junction of the 5' untranslated region and the coding region). Furthermore, an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element. Preferably, an antisense nucleic acid is designed so as to be complementary to a 20 region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA. An antisense nucleic acid for inhibiting the expression of SLAP-130 protein in a cell can be designed based upon the nucleotide sequence encoding the SLAP-130 protein (e.g., SEQ ID NO: 1, or a portion thereof), constructed according to the rules of Watson and Crick base pairing.
An antisense nucleic acid can exist in a variety of different forms. For example, the antisense nucleic acid can be an oligonucleotide that is complementary to only a portion of a SLAP-130 gene. An antisense oligonucleotides can be constructed using chemical synthesis procedures known in the art. An antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase 30 the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used. To inhibit SLAP-130 expression in cells in culture, one or more antisense oligon.~ eotides can be added to cells in culture media, typically at about 200 llg oligonucleotide/ml.
Alternatively, an antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA. For example, for inducible expression of antisense RNA, an inducible eukaryotic regulatory system, such as the Tet system (e.g, as described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 82:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCT Publication No. WO 94/29442; and PCT Publication No. WO
96/01313) can be used. The antisense expression vector is prepared as described above for recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation. The antisense expression vector can be in the form of, for 10 example, a recombinant plasmid, phagemid or attenuated virus. The antisense expression vector is introduced into cells using a standard transfection technique, as described above for recombinant expression vectors.
In another embodiment, an antisense nucleic acid for use as an inhibitory agent is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are 15 capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region (for reviews on ribozymes see e.g., Ohkawa, J. et al. (1995) J. Biochem.
118:251 258; Sigurdsson, S.T. and Eckstein, F. (1995) Trends Biotechnol. 13:286 289; Rossi, J.J. (1995) Trends Biotechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med. 73:65-71).
A ribozyme having specif1city for SLAP-130 mRNA can be designed based upon the nucleotide 20 sequence of the SLAP-130 cDNA. For example, a derivative of a Tetrahymena L-l 9 IVS RNA
can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a SLAP-130 mRNA. See for example U.S. Patent Nos. 4,987,071 and 5,116,742, both by Cech et al. Alternatively, SLAP-130 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example 25 Bartel, D. and Szostak, J.W. (1993) Science 261: 1411 -1418.
Another type of inhibitory agent that can be used to inhibit the expression and/or activity of SLAP-130 in a cell is an intracellular antibody specific for the SLAP-130 protein. The use of intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g, Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 2:101-108; Werge, 30 T.M. et al. (1990) FEBSLetters 274:193-198; Carlson, J.R. (1993) Proc. Natl. Acad. Sci. USA
90:7427-7428; Marasco, W.A. et al. (1993) Proc. Natl. Acad. Sci. USA 2_:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen, S-Y. et al. (1994) Human Gene Therapy 5:595 6' 1; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA 21 :5075-5079; Chen, S-Y. et al.
(1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R.R. et al. (1994) J. Biol. Chem.
35 262:23931 -23936; Beerli, R.R. et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672;
Mh:~shilk~r, A.M. et al. (1995) EMBO J. 14:1542 1551; Richardson, J.H. et al. (1995) Proc.
Natl. Acad. Sci. USA 22:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al., and PCT Publication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, a recombinant expression vector is prepared which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell. For inhibition of SLAP-130 activity according to the 5 inhibitory methods of the invention, an intracellular antibody that specifically binds the SLAP-130 protein is expressed in the cytoplasm ofthe cell. To prepare an intracellular antibody expression vector, antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest, e.g, SLAP-130, are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the SLAP-130 protein.Hybridomas secreting anti-SLAP-130 monoclonal antibodies, or recombinant anti-SLAP-130 monoclonal antibodies, can be prepared as described above. Once a monoclonal antibody specific for SLAP-130 protein has been identified (e.g, either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library), DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques. For hybridoma derived antibodies, light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening. For recombinant antibodies, such as from a phage display library, cDNA encoding the light and heavy chains can be recovered from the display package (e.g, phage) isolated during the library screening process. Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the "Vbase" human germline sequencedatabase.
Once obtained, the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods. To allow for cytoplasmic expression of the light and heavy chains, the nucleotide sequences encoding the hydrophobic leaders of the light and heavy chains are removed. An intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly. In the most preferred embodiment, the ~rector encod~,s a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker (e.g, (Gly4Ser)3) and expressed as a single chain molecule.
To inhibit SLAP-130 activity in a cell, the expression vector encoding the anti-SLAP-130 intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.

Other inhibitory agents that can be used to inhibit the activity of a SLAP-130 protein are chemical compounds that inhibit the interaction between SLAP-130 and SLP-76. Such compounds can be identified using screening assays that select for such compounds, as described in detail above. Additionally or alternatively, compounds that inhibit the S interaction of SLAP-130 with the SLP-76 SH2 domain can be designed using approaches known in the art. SH2 domains are known to interact with phosphotyrosine-containing peptides, with the specificity of a particular SH2 domain for a target binding site being influenced by the amino acid residues surrounding the phosphotyrosine residue (see e.g, Songyang, Z. et al. (1993) Cell 72:767-778). A consensus motif for the binding site of the 10 SLP-76 SH2 domain can be determined using methods known in the art (see Songyang, Z. et al. (1993) Cell 72:767-778). Based on the amino acid sequence of a consensus motif for the SLP-76 SH2 domain binding site, potential SH2 binding sites within SLAP-130 can be identified.
A competitive inhibitor of SLAP-130/SLP-76 SH2 interactions can be designed based 15 on the amino acid sequence(s) of an SH2 binding site(s) of SLAP-130 or the amino acid sequence of a consensus SH2 binding motif for SLP-76. In one embodiment, such aninhibitory molecule comprises a nonhydrolyzable phosphonopeptide having an appropriate amino acid sequence for recognition by the SLP-76 SH2 domain. In this compound, the tyrosine residue within the SH2 binding site is replaced with phosphonomethyl-phenylalanine 20 (Pmp), a nonnatural analogue of phosphotyrosine that is resistant to hydrolysis by phosphatases. Nonhydrolyzable phosphonopeptide inhibitors of SH2 domain interactions can be prepared as described in Domchek, S.M. et al. (1992) Biochemistry 31 :9865-9870. Such nonhydrolyzable phosphonopeptides can competitively inhibit the interaction between the SLP-76 SH2 domain and its target phosphotyrosine-containing binding site within SLAP-130 25 and, moreover, are proteolytically stable (i. e., the phosphonopeptide is resistant to the action of phosphatases). In other embodiments, an inhibitory molecule can comprise a peptidomimetic of the SH2 binding site, such as a benzodiazepine mimetic of a dipeptidyl amide backbone or a boronotyrosine-containing analogue of the phosphotyrosine-containing SH2 binding site (e.g., as described in PCT Publication WO 95/25118 by Bachovchin).
30 These peptidomimetics can competitively inhibit the interaction between the SLP-76 SH2 domain and its target phosphotyrosine-containing binding site within SLAP-130 yet are resistant to degradation.
Yet another form of an inhibitory agent of the inv~;n.lon is an inhibitory form of a SLAP-130 protein, also referred to herein as a dominant negative inhibitor. A dominant 35 negative inhibitor can be a form of a SLAP-130 protein that retains the ability to interact with the SH2 domain of SLP-76 but that lacks one or more other functional activities such that the dominant negative form of SLAP-130 cannot participate in normal signal transduction. This dominant negative form of a SLAP-130 protein may be, for example, a mutated form of SLAP-130 in which the SH2 binding site that interacts with the SH2 domain of SLP-76 is conserved but in which one or more amino acid residues elsewhere in the protein are mutated.
Such dominant negative SLAP-130 proteins can be expressed in cells using a recombinant expression vector encoding the mutant SLAP-130 protein, which is introduced into the cell by standard transfection methods. Mutation or deletion of specific codons within the SLAP-5 130-encoding cDNA can be performed using standard mutagenesis methods. The mutated cDNA is inserted into a recombinant expression vector, which is then introduced into a cell to allow for expression ofthe mutated SLAP-130 protein. The ability ofthe mutant SLAP-130 protein to interact with SLP-76 can be assessed using standard in vitro interaction assays, such as that using GST-SLP-76-SH2 described above. The effect ofthe mutant SLAP-130 10 protein on normal T cell signal transduction can be assessed, for example, by expressing the mutant SLAP-130 protein in T cells in culture (e.g., peripheral blood T cells or Jurkat cells), stimulating the T cells (e.g, using anti-CD3 antibodies) and measuring at least one indicator of T cell activation (e.g, calcium flux, tyrosine phosphorylation, IL-2 production). A mutant form of SLAP-130 that retains the ability to interact with SLP-76 but that interferes with 15 normal T cell signal transduction when expressed in the T cell can be selected as a dominant negative inhibitor of SLAP-130 activity.

B. Stimulatory Agents According to a modulatory method ofthe invention, SLAP-130 activity is stimulated in a cell by contacting the cell with a stimulatory agent. Examples of such stimulatory agents include active SLAP-130 protein and nucleic acid molecules encoding SLAP-130 that are introduced into the cell to increase SLAP-130 activity in the cell. A preferred stimulatory agent is a nucleic acid molecule encoding a SLAP-130 protein, wherein the nucleic acid molecule is introduced into the cell in a form suitable for expression ofthe active SLAP-130 protein in the cell. To express a SLAP-130 protein in a cell, typically a SLAP-130 cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques, as described herein. A SLAP-130 cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library as described herein. Following isolation or amplification of SLAP-130 cDNA, the DNA fragment is introduced into an expression vector and transfected into target cells by standard methods, as described herein.
Other stimulatory ~gt,i1ts that can be used to stimulate the activity of a SLAP-130 protein are chemical compounds that stimulate SLAP-130 activity in cells, such as compounds that promote the interaction between SLAP-130 and SLP-76. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.

In addition to use of an agent that modulates the expression or activity of SLAP-130 protein, the modulatory methods of the invention can involve the use of one or more additional agents that modulate T cell activation. For example, the modulatory methods of the invention can involve the use of an agent that modulates SLAP-130 activity in 5 combination with an agent that modulates tyrosine phosphorylation in T cells (e.g, an agent that inhibits protein tyrosine kinase activity, such as herbimycin A, or a derivative or analogue thereofl, an agent that modulates intracellular calcium levels in T cells (e.g., a calcium ionophore), a phorbol ester (e.g, PMA), a cytokine that modulates T cell activation (e.g, IL-2 and/or IL-4) and the like. Various agents that modulate T cell activation are 10 known in the art.

The modulatory methods of the invention can be performed in vitro (e.g, by culturing the cell with the agent or by introducing the agent into cells in culture) or, alternatively, in vivo (e.g., by a~mini.stering the agent to a subject or by introducing the agent into cells of a 15 subject, such as by gene therapy). For practicing the modulatory method in vitro, cells can be obtained from a subject by standard methods and incubated (i.e., cultured) in vitro with a modulatory agent of the invention to modulate SLAP-130 activity in the cells. For example, peripheral blood mononuclear cells (PBMCs) can be obtained from a subject and isolated by density gradient centrifugation, e.g, with Ficoll/Hypaque. Specific cell populations can be 20 depleted or enriched using standard methods. For example, monocytes/macrophages can be isolated by adherence on plastic. T cells can be enriched for example, by positive selection using antibodies to T cell surface markers, for example by incubating cells with a specific primary monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using magnetic beads coated with a secondary antibody that binds the primary mAb. Specific cell 25 populations (e.g, T cells) can also be isolated by fluorescence activated cell sorting according to standard methods. Monoclonal antibodies to T cell-specif1c surface markers known in the art and many are commercially available. If desired, cells treated in vitro with a modulatory agent of the invention can be re~(lministered to the subject. For a~1ministration to a subject, it may be preferable to first remove residual agents in the culture from the cells before 30 ~(lministering them to the subject. This can be done for example by a Ficoll/Hypaque gradient centrifugation of the cells. For further discussion of ex vivo genetic modification of cells followed by rea(lministration to a subject, see also U.S. Patent No. 5,399,346 by W.F.
Anderson et al.
For practicing the modulatory method in vivo in a subject, the modulatory agent can 35 be ~ministered to the subject such that SLAP-130 activity in cells ofthe subject is mod~ te-l The term "subject" is intended to include living or~nisms in which an immune response can be elicited. Preferred subjects are m~mm~ls. Examples of subjects include hllm~n~, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.

For stimulatory or inhibitory agents that comprise nucleic acids (including recombinant expression vectors encoding SLAP-130 protein, antisense RNA, intracellular antibodies or dominant negative inhibitors), the agents can be introduced into cells of the subject using methods known in the art for introducing nucleic acid (e.g, DNA) into cells in 5 vivo. Examples of such methods encompass both non-viral and viral methods, including:
Direct Injection: Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). For example, a delivery apparatus (e.g, a "gene gun") for injecting DNA into cells in vivo can be used. Such an apparatus is commercially 10 available (e.g., from BioRad).
Cationic Lipids: Naked DNA can be introduced into cells in vivo by complexing the DNA with cationic lipids or encapsulating the DNA in cationic liposomes. Examples of suitable cationic lipid formulations include N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride (DOTMA) and a 1: 1 molar ratio of 1,2-dimyristyloxy-propyl-3-15 dimethylhydroxyethylammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine(DOPE) (see e.g., Logan, J.J. et al. (1995) Gene Therapy _:38-49; San, H. et al. (1993) Human Gene Therapy 4:781-788).
Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for 20 a cell-surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem.
263:14621, Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320).
Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to 25 avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci.
USA 90:2122-2126).
Retrovir2~ses: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A recombinant 30 retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper Vil.l~ by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro 35 or in vivo with such viruses can be found in Current Protocols in Molecular Biolo~y, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include ~Crip, ~Cre, ~2 and ~Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, Iymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145;
Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl.
Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu 10 etal. (1993)J. Immunol. 150:4104-4115; U.S. PatentNo. 4,868,116; U.S. PatentNo.

4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it 15 may be necessary to stimulate replication of the target cell.
Adenoviruses: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616;
Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
20 Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g, Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.
Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells 25 (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al.
(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of 30 insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g, retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al.
cited supra; Haj-Ahmand and Graham (1986) ~. I irol. 57:267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral E1 and E3 genes but 35 retain as much as 80 % of the adenoviral genetic material.
Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al.
Curr. TopicsinMicro. andImmunol. (1992) 158:97 129). Itisalsooneofthefewviruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration(seeforexampleFlotteetal. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;
Samulski et al. (1989) J. Virol. 63:3822-3828, and McT ~llghlin et al. (1989) J. Virol.
62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and 5 can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. ~ndocrinol. 2:32-39;Tratschinetal. (1984)~ Virol. 51:611 619;andFlotteetal.(1993)J. Biol. Chem. 268:3781-3790).
The efficacy of a particular expression vector system and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g, Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected by an appropfiate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product.
A modulatory agent, such as a chemical compound that modulates the SLAP-130/SLP-76 interaction, can be ~(lmini~tered to a subject as a pharrnaceutical composition.
Such compositions typically comprise the modulatory agent and a pharmaceuticallyacceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical ~mini~tration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be prepared as described above in subsection IV.

As demonstrated in Example 5, overexpression of SLAP-130 in a T cell line inhibits TCR-induced ac.i~ ation of a promoter containing NFAT sites and, furthermore, blocks the augmentation of activity of this promoter that is seen when SLP-76 is overexpressed in these cells, indicating that at least under certain conditions SLAP-130 can function as a negative regulator of TCR-mediated signaling. Accordingly, modulation of SLAP-130 activity may be beneficial in a variety of clinical situations in which is desirable to modulate T cell immune responses, including immunodeficiencies, infectious diseases (e.g, viral infections), cancer, autoimmune diseases, transplantations (e.g, graft rejection or graft-versus-host disease) and allergies, as discussed further below. The overexpression experiments implicate SLAP-130 as a negative regulator of TCR-mediated sign~ling, suggesting that, under al~plopl;ate conditions, downregulation of SLAP-130 activity would stimulate TCR-mediated si~;n~ling, whereas upregulation of SLAP-130 would inhibit TCR-mediated sign~ling.
Accordingly, in preferred modulatory methods ofthe invention, a SLAP-130 inhibitory agent is used to stimulate T cell activation, whereas a SLAP-130 stimulatory agent is used to inhibit T cell activation. It should be appreciated however, that under different conditions or in different cell environments, SLAP-130 may also have positive effects and, therefore, modulatory methods in which a SLAP-130 stimulatory agent is used to stimulate T cell 10 activation or a SLAP-130 inhibitory agent is used to inhibit T cell activation are also encompassed by the invention.
Immunodeficiencies: Stimulation of T cell activation through the use of a modulatory agent that modulates SLAP-130 activity may be beneficial in a variety of clinical disorders characterized by general or specific immunodeficiency, including human immunodeficiency 15 virus infection and congenital immunodeficiency diseases.
~nfectious Diseases: Stimulation of T cell activation through the use of a modulatory agent that modulates SLAP-130 activity may be beneficial in a variety of infectious disease, as a means to promote a T cell response against the infectious agent. Such infectious diseases include bacterial, viral, fungal and parasitic infections.
Cancer: Stimulation of T cell activation through the use of a modulatory agent that modulates SLAP-130 activity may be beneficial in a variety of malignancies, as a means to promote a T cell response against malignant cells. Alternatively, for T cell leukemias and lymphomas, inhibition of T cell activation through use of a modulatory agent that modulates SLAP-130 activity may be beneficial, as a means to inhibit growth or progression of these 25 malignancies.
Autoimmune Diseases: Inhibition of T cell activation through the use of a modulatory agent that modulates SLAP-130 activity may be beneficial in a variety of autoimmune disorders, as a means to downregulate T cell response against autoantigens. It is well known in the art that many autoimmune disorders are the result of inappropriate activation of T cells 30 that are reactive against self tissue and that promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Non-limiting examples of autoimmune diseases and disorders having an autoimmune component that may be treated according to the modulatory methods of the invention include diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic 35 arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral 5 progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.
The efficacy of a modulatory agent in ameliorating autoimmune diseases can be tested in an animal models of human diseases. Such animal models include experimental allergic encephalomyelitis as a model of multiple sclerosis, the NOD mice as a model for diabetes, the mrl/lpr/lpr mouse as a model for lupus erythematosus, murine collagen-induced arthritis as a model for rheumatoid arthritis, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). A modulatory (i. e., stimulatory or inhibitory) agent of the invention is ~mini~tered to test ~nim~l~ and the course of the disease in the test :~nim~l~ is then monitored by the standard methods for the particular model being used. Effectiveness of the modulatory agent is evidenced by amelioration of the disease condition in ~nim~ treated with the agent as compared to untreated animals (or 20 ~nim~l~ treated with a control agent).
Transplantation: Inhibition of T cell activation through the use of a modulatory agent that modulates SLAP-130 activity may be beneficial in transplantation, as a means to downregulate T cell responses against an allograft or to inhibit graft-versus-host disease.
Accordingly, the modulatory methods of the invention can be used both in solid organ 25 transplantation and in bone marrow transplantation.
Allergies: Allergies are mediated through IgE antibodies whose production is regulated by the activity of T cells and the cytokines produced thereby. Accordingly, the modulatory methods of the invention can be used to inhibit T cell activation as a means to downregulate allergic responses. A modulatory agent may be directly ~lmini~tered to the 30 subject or T cells may be obtained from the subject, contacted with an modulatory agent ex vivo, and re~(lmini~tered to the subject. Moreover, in certain situations it may be beneficial to co~mini~ter to the subject the allergen together with the modulatory agent or cells treated with the modulatory agent to desensitize the allergen-specific respollse.

3~ In addition to the foregoing disease situations, the modulatory methods of the invention may be used for other purposes. For example, the modulatory methods that result in increased T cell activation can be used in the production of T cell cytokines in vitro.
Furthermore, the modulatory methods of the invention may be applied to vaccinations to promote T cell responses to an antigen of interest in a subject. That is, a modulatory agent of the invention may be used in combination with a vaccine to promote T cell responses against the vaccinating antigen.

This invention is further illustrated by the following examples which should not be 5 construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLE 1: Cloning and Characterization of a SLAP-130 cDNA

To facilitate purification of molecules that associates with the SH2 domain of SLP-76, a variant of the Jurkat T cell line, JA2/SLP-SH2, was established which expresses a chimeric surface protein consisting of the extracellular and transmembrane domains of the HLA-A2 molecule in frame with the SH2 domain of SLP-76. The SH2 domain of SLP-76 was amplified by PCR using the oligonucleotides GGGAGATCTGA
15 GAATTCATTAAATGAAGAG (SEQ ID NO: S) and CCCAGATCTGCACTGGTATC
TGGAACCTCG (SEQ ID NO: 6) cont:~ining Bgl II restriction sites for litigation of this fragment in frame with the cDNA of HLA-A2 present in pcDNA3/A2/CD45. In this and all subsequent examples, Jurkat T cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (1000 U/ml), streptomycin (1000 U/ml), and glutamine 20 (20 mM), whereas the JA2/SLP-SH2 Jurkat variant was maintained in the above medium suppiemented with 2 mg/ml geneticin, (GIBCO, Gaithersburg, MD).
The A2 epitope of the JA2/SLP-SH2 chimeric protein enabled the isolation of proteins associated with the SLP-76 SH2 domain by large scale immunoprecipitation with anti-A2 mAb CR11 -351 (a gift of C. Lutz, University of Iowa, Iowa City, IA). JA2/SLP-25 SH2 cells were stimulated with pervanadate for m~im~l tyrosine phosphorylation ofnumerous proteins in T cells and lysed in NP40 lysis buffer. A large scale anti-A2 immunoprecipitation of the pervanadate stimulated JA2/SH2 cells was subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA), and visualized by Ponceau S staining. A single major species of approximately 130 kDa was 30 excised and subjected to tryptic digestion and reverse phase high performance liquid chromatography for protein sequencing. Individual peptides were sequenced using a Procise 492 Protein Sequencer (Perkin Elmer, Foster City, CA).
One peptide sequence, PPNVLJL rK (SEQ ID NO: 4), was represented in the dbEST
database by an Expressed Sequence Tag (EST) clone that was obtained from Genome Systems, Inc., St. Louis, MO (I.M.A.G.E. consortium ID# 241254). This clone was then sequenced completely, revealing an open reading frame of 1074 base pairs. A region of this clone was amplified with primers CCACCAAATGTTGACCTGA CGAAATTC (SEQ ID
NO: 7) and TCTGGGAGGTAGGCTTGGGAC (SEQ ID NO: 8), and then used to screen a human thymus ~gtlO cDNA library (#NL1127a, Promega, Madison, WI).

A cDNA clone cont~inin~; 370 base pairs of the EST sequence and an additional 1008 base pairs of 5' coding sequence was isolated and found to contain a putative start site, 27 bases downstream of a stop codon, suggesting that the clone contained the 5' coding sequence of ppl30. The rem~ining 3' cDNA was amplified from Jurkat cDNA by 3' RACE (rapidamplification of cDNA ends) using a SLAP- 130 specific primer (GATGCTGATGATGGTT
TCCCTGCTCCTC; SEQ ID NO: 9) and the universal primer included in the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). The nucleotide and predicted amino acid sequence ofthe entire isolated SLAP-130 cDNA is shown in Figure 1 (and SEQ ID
NOs: 1 and 2, respectively). The region encompassing the peptide cont~ining the sequence 10 PPNVDLTK is indicated by an underline. Independent amplification of this region, as well as the entire coding sequence of SLAP-130 from Jurkat total RNA, was performed by reverse transcription-polymerase chain reaction(RT-PCR) using the GeneAmp kit (Perkin Elmer, Norwalk, CT) to confirm the sequence of the cDNA.
The complete open reading frame consists of 2349 bp (nucleotides 31 -2379 of SEQ1~ ID NO: 1), translating to a protein of 783 amino acids with an abundance of proline (13%), acidic (15%), and basic (15%) residues. This protein was designated SLAP-130 for SLP-76 associated ~hosphoprotein of 130 kDa.

EXAMPLE 2: Tissue Distribution of SLAP-130 mRNA
Northern blot analysis of human mRNA from multiple tissues was performed to determine the tissue distribution of SLAP-130. Northern blot analysis was performed following the protocols included with the Human Multiple Tissue Northern (MTN) Blots I
and II (Clontech). The PCR fragment utilized to screen the ~gtlO cDNA library was labeled 25 with [a-32P]dCTP (Amersham, Arlington Heights, IL) by random priming using anOligolabeling Kit (Pharmacia Biotech) and utilized for hybridization to poly A+ RNA of tissues represented in the MTN blots. Hybridization with SLAP-130 mRNA was detected by autoradiography .
The results of the Northern blot analysis are shown in Figure 2. The results 30 demonstrate that SLAP-130 mRNA is expressed in hematopoietic tissues (peripheral blood mononuclear cells, spleen, and thymus) but not in non-lymphoid tissues (colon, small intestines, ovary, testis, prostate, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, .le rt). Additionally, the entire coding sequence was amplified as a single product from total Jurkat RNA, demonstrating expression in this human T cell line.
3~
EXAMPLE 3: Immunoprecipitation of Epitope-Tagged Recombinant SLAP-130 In this example, the isolated cDNA described in Example 1 was expressed recombinantly in m:~mm;~ n cells as an epitope-tagged fusion protein and immunoprecipated, via the epitope-tag, to analyze the size of the expressed protein.
Translation of the open reading frame of the isolated SLAP-130 clone predicted a protein with an appal~lll molecular mass of only 86 kDa (i.e., smaller than the native 130 kDa SLAP-130 protein). However, the presence of multiple stop codons fl~nking either side of the coding sequence suggested that we had isolated the full length cDNA encoding the native 130 kDa, SLP-76 associated protein. As described further below, this was confirmed by generating an epitope (FLAG) tagged version of SLAP-130 cDNA for transient expression in Jurkat T cells (pEF/SLAP-130) and immunoprecipation ofthe fusion protein, which demonstrated that a 130 kDa protein was produced in m~mm~ n cells by expression of the 10 isolated cDNA.
The pEF-BOS expression vector containing the full length cDNA of SLAP-130 with an amino-terminal FLAG-tag (pEF/SLAP-130) was generated by PCR amplification ofthe amino-terminal 1350 nucleotides of SLAP-130 with primers CGGGATCCGCGA
AATATAACACGGGGGGC (SEQ ID NO: 10) and CGGGATCCGTCTATCCTTGA
15 CTCATCTCTGCTG (SEQ ID NO: 11). A BamHI site in the 5' primer and an internal XbaI
site at position 1350 were utilized for ligation ofthis portion of SLAP-130 in frame with the FLAG epitope tag in pEF/SLP-76 (Motto, D.G. et al. (1996) J. Exp. Med M83:1937 1943).
The rem~inin~; 3' cDNA was joined to the FLAG tagged 5' sequence by overlap extension PCR to generate the full length SLAP-130 cDNA (pEF/SLAP-130).
Jurkat T cells were transiently transfected with pEF/SLAP-130 and the FLAG
epitope-tagged SLAP-130 protein was precipitated using either an anti-FLAG mAb M2 (International Biotechnologies Inc., New Haven, CT).or a SLP-76 SH2 domain GST fusion protein. The cells were either left unstimulated or stimulated with pervanadate (Secrist, J.P.
et al. (1993) J. Biol Chem. 268:5886-5893) for 1 min. and then lysed in NP40 lysis buffer 25 (1% NP40, 150 mM NaCI, 10 mM Tris, pH 7.4) including protease inhibitors (50 ~lg/ml leupeptin, 50 ~lg/ml pepstatin A, 1 mM PMSF) and phosphatase inhibitors 400 IlM sodium v~n~ te, 10 mM sodium fluoride, 10 mM sodium pyrophosphate). For immuno-precipitations, antibodies were conjugated to GammaBind Plus Sepharose (Pharmacia Biotech, Uppsala, Sweden) for 2 h at 4~C and washed extensively in lysis buffer. Lysates 30 were subjected to precipitation with the antibodies or GST fusion protein for 2 h at 4~C.
Immune complexes were washed 4 times in high salt Iysis buffer (500 mM NaCI), resolved by SDS-PAGE, and subjected to immunoblot analysis with the indicated antibody and the applol)liate horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratorie~, Hercules, CA) for detection with ECL reagent (Amersham Corp. Arlington Heights, IL).
The results of the anti-FLAG mAb immunoprecipitation experiment are shown in Figure 3A. In this experiment, whole cell lysates of 1 x 106 Jurkat cells transfected with pEF/SLAP-130 (lane 2) or vector control (pEF) (lane 1) were subjected to western blot analysis with anti-FLAG mAb (2,ug/ml) followed by goat anti-mouse HRP conjugate (1:10,000). The data demonstrate that transfection of Jurkat T cells with pEF/SLAP-130 results in the expression of a protein reactive with anti-FLAG mAb which migrates with an a~pale,ll molecular mass of 130 kDa (lane 2). This protein does not appear in lysates of cells transfected with control vector DNA (lane 1).
The results of the precipitation experiment using the SLP-76 SH2 domain GST fusion 5 protein are shown in Figure 3B. In this experiment, Jurkat T cells transfected with pEF/SLAP-130 were left unstimulated (lanes 1, 3, 5, 6) or stimulated (lanes 2, 4, 7) for 1 min with pervanadate (PV). Whole cell lysates prepared from 3 x 107 cells were incubated with GST fusion protein encoding the SH2 domain of SLP-76 (GSTSH2; lanes 3, 4) or an analogous fusion protein containing a loss of function SLP-76 SH2 domain (GSTR448K;
lanes 1, 2). Precipitation ofthe epitope tagged SLAP-130 was determined by immunoblotting with anti-FLAG mAb. The results show that the SLP-76 SH2 domain fusion protein precipitates the epitope tagged SLAP- 130 from pervanadate stimulated Jurkat cells (lane 4), but not from resting cells (lane 3) transfected with pEF/SLAP-130. A loss of function mutant of the SLP-76 SH2 domain when expressed as a GST fusion protein (GSTR448K) fails to associate with FLAG-SLAP-130 in either resting or pervanadate stimulated cells (lane 1 and 2). The protein precipitated by the wild type SLP-76 SH2 domain migrates identically to a protein detected by anti-FLAG immunoblot analysis of whole cell lysates (lane 6) or an anti-FLAG immunoprecipitate (lane 5) from Jurkat cells transfected with pEF/SLAP-130. Additionally, anti-phosphotyrosine western blot of a GSTSH2 precipitate from untransfected PV stimulated Jurkat cells demonstrates that the 130 kDa protein associated with the SH2 domain of SLP-76 migrates with the same electrophoretic mobility as SLAP-130 (lane 7).

EXAMPLE 4: Association of SLAP-130 and SLP-76 in T cells To determine whether endogenous SLAP-130 and SLP-76 associate in vivo in T cells, coimmunoprecipitation experiments were performed. Immunoprecipitations and immunoblots were carried out as described in Example 3, except that an anti-SLAP-130 sheep antiserum was generated by immunization of sheep with a GST fusion proteincont~ining amino acids 1-340 of human SLAP-130 and anti-SLP-76 sheep antiserum also was generated by inoculation of sheep with a GST fusion protein containing amino acids 136-235 of SLP-76 (Motto, D.G. et al. (1996) J. Exp. Med. 183: 1937-1943). The results of this experiment is shown in Figure 4.
To determine if SLAP-130 and SLP-76 associate within cells, lysates were prepared from resting and pervanadate stimulated Jurkat cells. Lysates from 3 x 107 Jurkat T cells were subjected to immunoprecipitation with either pre-immune serum (lane l) or anti-SLAP-130 antiserum (lane 2). These immune complexes, in addition to whole cell lysates prepared from 1 x 106 Jurkat cells (lane 3), were subjected to western blot analysis with anti-SLAP-130 antiserum (1 :250) followed by rabbit anti-sheep HRP conjugate (1: 10,000).

Additionally, whole cell Iysates were prepared from 5x 107 unstimulated (lane 4) or pervanadate stimulated (lane 5) Jurkat T cells and were subjected to immunoprecipitation with anti-SLP-76 antiserum and then immunoblotted with both anti-SLP-76 and anti-SLAP-76 antiserum.
The results demonstrate that the anti-SLAP-130 antiserum, but not pre-immune serum, precipitates a protein of 130 kDa from Jurkat T cells (see Fig. 4, lanes 1 and 2).
There is some SLAP-130 found in SLP-76 immunoprecipitates from resting Jurkat cells (lane 4). This is consistent with our finding of low levels of a tyrosine phosphorylated protein which migrates at 130 kDa associating with SLP-76 in unstimulated Jurkat (Motto, D.G. et 10 al. (1996) J. Exp. Med. 183:1937 1943). The amount of SLAP-130 which associates with SLP-76 increases following stimulation of Jurkat with pervanadate (lane 5). Together the data from figure 3 (discussed in Example 3) and figure 4 (discussed in this Example) support the notion that SLAP-130 associates with SLP-76 in cells, in a phosphotyrosine dependent fashion, through the SLP-76 SH2 domain.
EXAMPLE 5: Overexpression of SLAP-130 in T cells Overexpression of SLP-76 has been shown to augment TCR signaling cascades leading to IL-2 promoter activity (Motto, D.G. et al. ( 1996) J Exp. Med. 183:1937-1943;
20 Wu, J. et al. (1996) Immunity 4:593-602). A functional SH2 domain of SLP-76 is required for its activity in Jurkat T cells (Motto, D.G. et al. (1996) J. Exp. Med. 183:1937 1943;
Wardenburg, J.B. et al. (1996) J. Biol. Chem. 271 :19641-19644). To determine the effect of SLAP-130 on signals generated by TCR ligation, Jurkat cells were transiently transfected with pEF/SLAP-130 (described above in Example 3) or a control vector and a luciferase 25 reporter construct driven by the NFAT response element.
For the transfections, the cells were washed twice in PBS, suspended in cytomix (120 mM KCI; 0.15 mM CaC12; 10 mM K2HPO4/KH2PO4, pH 7.6; 25 mM Hepes, pH 7.6; 2 mM
EGTA, pH 7.5; 5 mM MgC12; 2 mM ATP; and 5 mM glutathione) (van den Hoff, M.J. et al.
(1992) Nucl. Acids. Res. 20:2902) at a concentration of 2 x 107 cells per 400 ,ul cytomix per 30 cuvette with the plasmid DNAs discussed below, and transfected at 250 mV, 960 ~lF using Gene Pulser (Bio-Rad). Transfected cells were allowed to recover for 24 h prior to manipulation in each experiment.
NFAT reporter gene .Is~ays were performed by cotransfecting 2 x 107 cells with 40 llg ofthe parental control vector pEF-BOS (Mi71lshim~, S. et al. (1990) Nucl. Acids Res.
35 18:5322) or the SLP-76 expression vector pEF/SLP-76 or the SLAP-130 expression vector pEF/SLAP- 130 plus 20 llg of the reporter gene construct NFAT-luc (Northrop, J.P. et al.
(1993) J. Biol. Chem. 268:2917-2923), which contains a triplicate ofthe nuclear factor of activated T cells (NFAT) response element upstream of the luciferase gene (gift of G.
Crabtree, Stanford University, Palo Alto, CA). Following transfection, triplicate samples of 5 x 105 cells were stimulated for 10 h with the indicated stimuli. Each sample was lysed in 100 ~g of lysis buffer (1% Triton X-100, 110 mM K2HPO4, 15 mM KH2PO4, 5 mM DTT, pII
7.8) for 10 min at room temperature and added to 100 ,ul of 2 x luciferase assay buffer (200 mM K2HPO4, 30 mM KH2PO4, 20 mM MgCl2, 10 mM ATP, pH 7.8). Samples were mixed 5 with 100 ~1 of 1 mM luciferin (Sigma Chemical Co., St. Louis, MO) and immediately assayed for luciferase activity using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
The results of the cotransfection experiments are shown in Figures 5A. For this experiment, 24 hours following transfection, the cells were stimulated with media, anti-TCR
mAb C305 (ascites 1: 1000) (gift of A. Weiss, UCSF, San Francisco, CA), anti-TCR mAb plus PMA (50 ng/ml), or PMA plus ionomycin (1 ~lM) for 10 h and then assayed forluciferase activity using a luminometer. Data are presented in the bar graph as light units of luciferase activity after treatment with the indicated stimuli. The results shown in Figure 5A
demonstrate that, as previously reported, SLP-76 alone augments T cell receptor signaling.
In contrast to the effect of SLP-76 on T cell sign~ling, however, overexpression of SLAP-130 results in (limini~hed NFAT activity following TCR ligation. Furthermore, co-transfection of SLAP-130 and SLP-76 reveals that overexpression of SLAP-130 blocks the augmentation of TCR stimulated promoter activity by SLP-76.
Expression of the FLAG-tagged cDNAs in the transfected cells was confirmed by immunoblotting whole cell lysates with anti-FLAG mAb, the results of which are shown in Figure 5B. Expression of the epitope tagged constructs in the transfected cells was determined by lysing 1 x 106 transfected cells in NP40 lysis buffer and immunoblotting with anti-FLAG mAb as described above in Example 3.
Several reports demonstrate that overexpression of SLP-76 markedly augments TCR
derived signals leading to activation of transcription factors for the IL-2 gene (Motto, D.G. et al. (1996) J. Exp. Med. 183: 1937-1943; Wu, J. et al. (1996) Immunity _:593-602; Fang, N. et al. (1996)J. Immunol. 157:3769 3773; Wardenburg, J.B. etal. (1996)J. Biol. Chem.271 :19641-19644). Since this effect of SLP-76 has been shown to require a functional SH2 domain (Motto, D.G. et al. (1996) J. Exp. Med. 183:1937-1943; Wardenburg, J.B. et al.
(1996) J. Biol. Chem. 271 :19641-19644), we initially hypothesized that molecules associating with the SLP-76 SH2 domain would also act as positive regulators of TCR
sign~ling. It is, therefore, a surprising and unexpected discovery that overexpression of SLAP-130 appears to interfere with TCR-induced NFAT activation in Jurkat cells and, additionally, blocks the ability or transfected SLP-76 to augment TCR responses.
EQUIVALENTS
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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Koretzky, G.A. et al.
(ii) TITLE OF INVENTION: Compositions of SLAP-130, a SLP-76 Associated Protein, and Methods of Use Therefor (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD
(B) STREET: 28 State Street (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: USA
(F) ZIP: 02109-1875 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/832,222 (B) FILING DATE: 3-APR-1997 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/774,061 (B) FILING DATE: 23-DEC-1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kara, Catherine J.
(B) REGISTRATION NUMBER: 41,106 (C) REFERENCE/DOCKET NUMBER: BBI-067CPCA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)227-7400 (B) TELEFAX: (617)227-5941 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2400 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

( ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31. .2379 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TAGGATGGAA AGGCAGATGT A~AGTCCCTC 30 Met Ala Lys Tyr Asn Thr Gly Gly Asn Pro Thr Glu Asp Val Ser Val Asn Ser Arg Pro Phe Arg Val Thr Gly Pro Asn Ser Ser Ser Gly Ile Gln Ala Arg Lys Asn Leu Phe Asn Asn Gln Gly Asn Ala Ser Pro Pro Ala Gly Pro Ser Asn Val Pro Lys Phe Gly Ser Pro Lys Pro Pro Val Ala Val Lys Pro Ser Ser Glu Glu Lys Pro Asp Lys Glu Pro Lys Pro Pro Phe Leu Lys Pro Thr Gly Ala Gly Gln Arg Phe Gly Thr Pro Ala Ser Leu Thr Thr Arg Asp Pro Glu Ala Lys Val Gly Phe Leu Lys Pro GTA GGC CCC AAG CCC ATC AAC TTG CCC AAA GAA GAT TCC A~A CCT ACA 414 Val Gly Pro Lys Pro Ile Asn Leu Pro Lys Glu Asp Ser Lys Pro Thr Phe Pro Trp Pro Pro Gly Asn Lys Pro Ser Leu His Ser Val Asn Gln Asp His Asp Leu Lys Pro Leu Gly Pro Lys Ser Gly Pro Thr Pro Pro Thr Ser Glu Asn Glu Gln Lys Gln Ala Phe Pro Lys Leu Thr Gly Val Lys Gly Lys Phe Met Ser Ala Ser Gln Asp Leu Glu Pro Lys Pro Leu Phe Pro Lys Pro Ala Phe Gly Gln Lys Pro Pro Leu Ser Thr Glu Asn Ser His Glu Asp Glu Ser Pro Met Lys Asn Val Ser Ser Ser Lys Gly Ser Pro Ala Pro Leu Gly Val Arg Ser Lys Ser Gly Pro Leu Lys Pro Ala Arg Glu Asp Ser Glu Asn Lys Asp His Ala Gly Glu Ile Ser Ser TTG CCC TTT CCT GGA GTG GTT TTG A~A CCT GCT GCG AGC AGG GGA GGC 846 Leu Pro Phe Pro Gly Val Val Leu Lys Pro Ala Ala Ser Arg Gly Gly Leu Gly Leu Ser Lys Asn Gly Glu Glu Lys Lys Glu Asp Arg Lys Ile Asp Ala Ala Lys Asn Thr Phe Gln Ser Lys Ile Asn Gln Glu Glu Leu Ala Ser Gly Thr Pro Pro Ala Arg Phe Pro Lys Ala Pro Ser Lys Leu Thr Val Gly Gly Pro Trp Gly Gln Ser Gln Glu Lys Glu Lys Gly Asp Lys Asn Ser Ala Thr Pro Lys Gln Lys Pro Leu Pro Pro Leu Phe Thr Leu Gly Pro Pro Pro Pro Lys Pro Asn Arg Pro Pro Asn Val Asp Leu Thr Lys Phe His Lys Thr Ser Ser Gly Asn Ser Thr Ser Lys Gly Gln Thr Ser Tyr Ser Thr Thr Ser Leu Pro Pro Pro Pro Pro Ser His Pro Ala Ser Gln Pro Pro Leu Pro Ala Ser His Pro Ser Gln Pro Pro Val Pro Ser Leu Pro Pro Arg Asn Ile Lys Pro Pro Phe Asp Leu Lys Ser Pro Val Asn Glu Asp Asn Gln Asp Gly Val Thr His Ser Asp Gly Ala Gly Asn Leu Asp Glu Glu Gln Asp Ser Glu Gly Glu Thr Tyr Glu Asp Ile Glu Ala Ser Lys Glu Arg Glu Lys Lys Arg Glu Lys Glu Glu Lys Lys Arg Leu Glu Leu Glu Lys Lys Glu Gln Lys Glu Lys Glu Lys Lys Glu Gln Glu Ile Lys Lys Lys Phe Lys Leu Thr Gly Pro Ile Gln Val Ile His Leu Ala Lys Ala Cys Cys Asp Val Lys Gly Gly Lys Asn Glu Leu Ser Phe Lys Gln Gly Glu Gln Ile Glu Ile Ile Arg Ile Thr Asp Asn Pro Glu Gly Lys Trp Leu Gly Arg Thr Ala Arg Gly Ser Tyr Gly Tyr Ile Lys Thr Thr Ala Val Glu Ile Asp Tyr Asp Ser Leu Lys Leu Lys Lys Asp Ser Leu Gly Ala Pro Ser Arg Pro Ile Glu Asp Asp Gln Glu Val Tyr Asp Asp Val Ala Glu Gln Asp Asp Ile Ser Ser His Ser Gln Ser Gly Ser Gly Gly Ile Phe Pro Pro Pro Pro Asp Asp Asp Ile Tyr Asp Gly Ile Glu Glu Glu Asp Ala Asp Asp Gly Phe Pro Ala Pro Pro Lys Gln Leu Asp Met Gly Asp Glu Val Tyr Asp Asp Val Asp Thr Ser Asp Phe Pro Val Ser Ser Ala Glu Met Ser Gln Gly Thr Asn Phe Gly Lys Ala Lys Thr Glu Glu Lys Asp Leu Lys Lys Leu Lys Lys Gln Glu Lys Glu Glu Lys Asp Phe Arg Lys Lys Phe Lys Tyr Asp Gly Glu Ile Arg Val Leu Tyr Ser Thr Lys Val Thr Thr Ser Ile Thr Ser Lys Lys Trp Gly Thr Arg Asp Leu Gln Val Lys Pro Gly Glu Ser Leu Glu Val Ile Gln Thr Thr Asp Asp Thr Lys Val Leu Cys Arg Asn Glu Glu Gly Lys Tyr Gly Tyr Val Leu Arg Ser Tyr Leu Ala Asp Asn Asp Gly Glu Ile Tyr Asp Asp Ile Ala Asp Gly Cys Ile Tyr Asp Asn Asp *

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Met Ala Lys Tyr Asn Thr Gly Gly Asn Pro Thr Glu Asp Val Ser Val ~sn Ser Arg Pro Phe Arg Val Thr Gly Pro Asn Ser Ser Ser Gly Ile Gln Ala Arg Lys Asn Leu Phe Asn Asn Gln Gly Asn Ala Ser Pro Pro Ala Gly Pro Ser Asn Val Pro Lys Phe Gly Ser Pro Lys Pro Pro Val Ala Val Lys Pro Ser Ser Glu Glu Lys Pro Asp Lys Glu Pro Lys Pro ~ro Phe Leu Lys Pro Thr Gly Ala Gly Gln Arg Phe Gly Thr Pro Ala ~er Leu Thr Thr Arg Asp Pro Glu Ala Lys Val Gly Phe Leu Lys Pro Val Gly Pro Lys Pro Ile Asn Leu Pro Lys Glu Asp Ser Lys Pro Thr Phe Pro Trp Pro Pro Gly Asn Lys Pro Ser Leu His Ser Val Asn Gln Asp His Asp Leu Lys Pro Leu Gly Pro Lys Ser Gly Pro Thr Pro Pro ~hr Ser Glu Asn Glu Gln Lys Gln Ala Phe Pro Lys Leu Thr Gly Val ~ys Gly Lys Phe Met Ser Ala Ser Gln Asp Leu Glu Pro Lys Pro Leu Phe Pro Lys Pro Ala Phe Gly Gln Lys Pro Pro Leu Ser Thr Glu Asn Ser His Glu Asp Glu Ser Pro Met Lys Asn Val Ser Ser Ser Lys Gly Ser Pro Ala Pro Leu Gly Val Arg Ser Lys Ser Gly Pro Leu Lys Pro ~la Arg Glu Asp Ser Glu Asn Lys Asp His Ala Gly Glu Ile Ser Ser ~eu Pro Phe Pro Gly Val Val Leu Lys Pro Ala Ala Ser Arg Gly Gly Leu Gly Leu Ser Lys Asn Gly Glu Glu Lys Lys Glu Asp Arg Lys Ile Asp Ala Ala Lys Asn Thr Phe Gln Ser Lys Ile Asn Gln Glu Glu Leu Ala Ser Gly Thr Pro Pro Ala Arg Phe Pro Lys Ala Pro Ser Lys Leu ~hr Val Gly Gly Pro Trp Gly Gln Ser Gln Glu Lys Glu Lys Gly Asp ~ys Asn Ser Ala Thr Pro Lys Gln Lys Pro Leu Pro Pro Leu Phe Thr Leu Gly Pro Pro Pro Pro Lys Pro Asn Arg Pro Pro Asn Val Asp Leu Thr Lys Phe His Lys Thr Ser Ser Gly Asn Ser Thr Ser Lys Gly Gln Thr Ser Tyr Ser Thr Thr Ser Leu Pro Pro Pro Pro Pro Ser His Pro ~la Ser Gln Pro Pro Leu Pro Ala Ser His Pro Ser Gln Pro Pro Val ~ro Ser Leu Pro Pro Arg Asn Ile Lys Pro Pro Phe Asp Leu Lys Ser Pro Val Asn Glu Asp Asn Gln Asp Gly Val Thr His Ser Asp Gly Ala Gly Asn Leu Asp Glu Glu Gln Asp Ser Glu Gly Glu Thr Tyr Glu Asp Ile Glu Ala Ser Lys Glu Arg Glu Lys Lys Arg Glu Lys Glu Glu Lys ~ys Arg Leu Glu Leu Glu Lys Lys Glu Gln Lys Glu Lys Glu Lys Lys ~lu Gln Glu Ile Lys Lys Lys Phe Lys Leu Thr Gly Pro Ile Gln Val Ile His Leu Ala Lys Ala Cys Cys Asp Val Lys Gly Gly Lys Asn Glu Leu Ser Phe Lys Gln Gly Glu Gln Ile Glu Ile Ile Arg Ile Thr Asp Asn Pro Glu Gly Lys Trp Leu Gly Arg Thr Ala Arg Gly Ser Tyr Gly ~yr Ile Lys Thr Thr Ala Val Glu Ile Asp Tyr Asp Ser Leu Lys Leu ~ys Lys Asp Ser Leu Gly Ala Pro Ser Arg Pro Ile Glu Asp Asp Gln Glu Val Tyr Asp Asp Val Ala Glu Gln Asp Asp Ile Ser Ser His Ser Gln Ser Gly Ser Gly Gly Ile Phe Pro Pro Pro Pro Asp Asp Asp Ile Tyr Asp Gly Ile Glu Glu Glu Asp Ala Asp Asp Gly Phe Pro Ala Pro ~ro Lys Gln Leu Asp Met Gly Asp Glu Val Tyr Asp Asp Val Asp Thr ~er Asp Phe Pro Val Ser Ser Ala Glu Met Ser Gln Gly Thr Asn Phe Gly Lys Ala Lys Thr Glu Glu Lys Asp Leu Lys Lys Leu Lys Lys Gln Glu Lys Glu Glu Lys Asp Phe Arg Lys Lys Phe Lys Tyr Asp Gly Glu Ile Arg Val Leu Tyr Ser Thr Lys Val Thr Thr Ser Ile Thr Ser Lys ~ys Trp Gly Thr Arg Asp Leu Gln Val Lys Pro Gly Glu Ser Leu Glu ~al Ile Gln Thr Thr Asp Asp Thr Lys Val Leu Cys Arg Asn Glu Glu Gly Lys Tyr Gly Tyr Val Leu Arg Ser Tyr Leu Ala Asp Asn Asp Gly Glu Ile Tyr Asp Asp Ile Ala Asp Gly Cys Ile Tyr Asp Asn Asp (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
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Glu Glu Trp Tyr Val Ser Tyr Ile Thr Arg Pro Glu Ala Glu Ala Ala Leu Arg Lys Ile Asn Gln Asp Gly Thr Phe Leu Val Arg Asp Ser Ser Lys Lys Thr Thr Thr Asn Pro Tyr Val Leu Met Val Leu Tyr Lys Asp Lys Val Tyr Asn Ile Gln Ile Arg Tyr Gln Lys Glu Ser Gln Val Tyr Leu Leu Gly Thr Gly Leu Arg Gly Lys Glu Asp Phe Leu Ser Val Ser Asp Ile Ile Asp Tyr Phe Arg Lys Met Pro Leu Leu Leu Ile Asp (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Pro Pro Asn Val Asp Leu Thr Lys (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCACCAAATG TTGACCTGAC GA~ATTC 27 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence encodingSLAP-130, or a fragment of said isolated nucleic acid molecule at least 1100 nucleotides in length.

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein, wherein the protein: (i) comprises an amino acid sequence at least 60 %
homologous to the amino acid sequence of SEQ ID NO: 2 and (ii) associates with the SH2 domain of SLP-76 or modulates T cell receptor mediated signaling.

3. The isolated nucleic acid molecule of claim 2, wherein the protein comprises an amino acid sequence at least 70 % homologous to the amino acid sequence of SEQ ID
NO: 2

4. The isolated nucleic acid molecule of claim 2, wherein the protein comprises an amino acid sequence at least 80 % homologous to the amino acid sequence of SEQ ID
NO: 2.

5. The isolated nucleic acid molecule of claim 2, wherein the protein comprises an amino acid sequence at least 90 % homologous to the amino acid sequence of SEQ ID
NO: 2.

6. An isolated nucleic acid molecule at least 1100 nucleotides in length which hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.

7. The isolated nucleic acid molecule of claim 6 which comprises a naturally-occurring nucleotide sequence.

8. The isolated nucleic acid molecule of claim 6 which encodes human SLAP-130.

9. The isolated nucleic acid molecule of claim 6 which encodes mouse SLAP-130.

10. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 1.

11. An isolated nucleic acid molecule encoding the amino acid sequence of SEQ
ID NO: 2.

12. An isolated nucleic acid molecule encoding a SLAP-130 fusion protein.

13. An isolated nucleic acid molecule which is antisense to the coding strand ofthe nucleic acid molecule of claim 1.

14. A vector comprising the nucleic acid molecule of claim 1.

15. The vector of claim 14, which is a recombinant expression vector.

16. A host cell containing the vector of claim 14.

17. A host cell containing the vector of claim 15.

18. A method for producing SLAP-130 protein comprising culturing the host cell of claim 17 in a suitable medium until SLAP-130 protein is produced.

19. The method of claim 18, further comprising isolating SLAP-130 protein fromthe host cell or the medium.

20. An isolated SLAP-130 protein, or a portion thereof that associates with the SH2 domain of SLP-76 or modulates T cell receptor mediated signaling.

21. An isolated protein which (i) comprises an amino acid sequence at least 60 %homologous to the amino acid sequence of SEQ ID NO: 2 and (ii) associates with the SH2 domain of SLP-76 or modulates T cell receptor mediated signaling.

22. The isolated protein of claim 21, which comprises an amino acid sequence at least 70 % homologous to the amino acid sequence of SEQ ID NO: 2.

23. The isolated protein of claim 21, which comprises an amino acid sequence at least 80 % homologous to the amino acid sequence of SEQ ID NO: 2.

24. The isolated protein of claim 21, which comprises an amino acid sequence at least 90 % homologous to the amino acid sequence of SEQ ID NO: 2.

25. An isolated protein comprising the amino acid sequence of SEQ ID NO: 2.

26. A SLAP-130 fusion protein.

27. Antibodies that specifically bind SLAP-130 protein.

28. The antibodies of claim 27, which are polyclonal.

29. The antibodies of claim 27, which are monoclonal.

30. The antibodies of claim 27, which are labeled with a detectable substance.

31. A nonhuman transgenic animal which contains cells carrying a transgene encoding SLAP-130 protein or a portion of SLAP-130 protein.

32. The nonhuman transgenic animal of claim 31, wherein the transgene alters an endogenous gene encoding endogenous SLAP-130 protein.

33. A method for detecting the presence of SLAP-130 activity in a biological sample comprising contacting the biological sample with an agent capable of detecting an indicator of SLAP-130 activity such that the presence of SLAP-130 activity is detected in the biological sample.

34. The method of claim 33, wherein the agent detects SLAP-130 mRNA.

35. The method of claim 34, wherein the agent is a labeled nucleic acid probe capable of hybridizing to SLAP-130 mRNA.

36. The method of claim 33, wherein the agent detects SLAP-130 protein.

37. The method of claim 36, wherein the agent is a labeled antibody that specifically binds to SLAP-130 protein.

38. A method for modulating SLAP-130 activity in a cell comprising contacting the cell with an agent that modulates SLAP-130 activity such that SLAP-130 activity in the cell is modulated.

39 The method of claim 38, wherein the agent inhibits SLAP-130 activity.

40. The method of claim 38, wherein the agent stimulates SLAP-130 activity.

41. The method of claim 38, wherein the agent modulates the activity of SLAP-130 protein.

42. The method of claim 41, wherein the agent is an antibody that specifically binds to SLAP-130 protein.

43. The method of claim 38, wherein the agent modulates transcription of a SLAP-130 gene or translation of a SLAP-130 mRNA.

44. The method of claim 43, wherein the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the SLAP-130 mRNA or the SLAP-130 gene.

45. The method of claim 38, wherein the cell is a T cell.

46. A method for identifying an agent that modulates an interaction between SLAP-130 and SLP-76, comprising:

(a) combining:
(i) a SLAP-130 protein, or SLP-76-interacting portion thereof; and (ii) SLP-76, or a SLAP-130-interacting portion thereof;
in the presence and absence of a test compound;
(b) determining the degree of interaction between (i) and (ii) in the presence and absence of the test compound; and (c) identifying an agent that modulates an interaction between SLAP-130 and SLP-76.

47. The method of claim 46, wherein the SLAP-130-interacting portion of SLP-76comprises the src homology 2 (SH2) domain of SLP-76.

48. The method of claim 46, wherein the degree of interaction between (i) and (ii) is determined by labeling (i) or (ii) with a detectable substance, isolating non-labeled (i) or (ii) and quantitating the amount of labeled (i) or (ii) that has become associated with non-labeled (i) or (ii).

49. The method of claim 46, wherein the test compound increases the degree of interaction between (i) and (ii), as compared to the degree of interaction in the absence of the test compound, and the test compound is identified as an agent that stimulates an interaction between SLAP-130 and SLP-76.

50. The method of claim 46, wherein the test compound decreases the degree of interaction between (i) and (ii), as compared to the degree of interaction in the absence of the test compound, and the test compound is identified as an agent that inhibits an interaction between SLAP-130 and SLP-76.