CA2348733A1 - Novel compositions and methods of screening for t-cell and b-cell activation modulators - Google Patents

Abstract

Described heron are proteins useful in T-cell and B-cell activation. In particular, a human SWAP70 homolog found in a T-cell library and a human RasGRP homolog are described. Also described is the binding of RasGRP to SWAP70 and the human homolog

Description

NOVEL COMPOSITIONS AND METHODS OF SCREENING FOR T.CELL
AND B-CELL ACTNATION MODULATORS
The invention relates to proteins useful in T-cell and B-cell activation, and more particularly to a human SWAP70 homolog found in a T-cell library and a human RasGRP homolog, and their use in methods for identifying candidate agents which modulate these activities.
BACI~'~,Q~ND OF THE INV~~(IION
Lymphocytes are the white blood cells responsible for the immune response.
Their characteristics account for the immune system's attributes of diversity, specificity, memory, and.setf/nonself recognition. Lymphocytes, which constitute 20-40% of the body's white blood cells, circulate in the blood and lymph and are capable of migrating into the tissue spaces and lymphoid organs. The lymphocytes can be broadly subdivided on the basis of function and cell-membrane components into three populations: B cells, T cells, and NK cells. Of particular interest are B cells and T cells, and more particularly of interest is the activation of lymphocytes. As used herein, lymphocytes refer to B
cells and T cells.
Activators of B cells have been identified. For example, SWAP70 was originally identified as a B cell specific protein involved in B cell isotype switching (Borggrefe et al., J.
Biol., Chem., 17025-17035 (199$), incorporated herein in its entirety).
Since activation of specific signaling pathways in lymphocytes determines the quality, magnitude and duration of immune responses, it is desirable to identify more activation proteins and modulators thereof. These proteins and modulators will find use in transplantation, acute and chronic WO i)4I1b241 PCT/US99/25333 SUM~~Y I~ THE INVENTION
The present invention provides proteins involved in activation of T and B
cells. More particularly, JEST (also sometimes called T-SWAP) has teen identified. JEST has homology to SWAP70, originatty identified as a B cell specific protein. However, it is provided herein that JEST is involved in T cell signaling. Moreover, also provided herein is that RasGRP binds to JEST
and SWAP70 (also sometimes called SWAP herein). Thus, RasGRP is provided herein as involved in B and T cell activation signaling. Also provided herein are methods for screening for modulators of B and T cell activation.
In one aspect, a recombinant nucleic acid encoding a JEST protein that is at least about 85% identical to the amino acid sequence depicted in Figure 2 is provided. Also provided is a recombinant nucleic encoding the amino acid sequence depicted in Figure 2. Further provided herein is a recombinant nucleic acid which will hybridize under high stringency conditions to the nucleic acid sequence depicted in Figure 2 or its complement. Moreover, a recombinant nucleic acid that is at least about 90°~ identical to the nucleic acid sequence depicted in Figure 2 is provided. Also provided is a recombinant nucleic acid having the nucleic acid sequence depicted in Figure 2.
In another aspect of the invention, a n3combinant JEST protein is provided that is at least about 8590 identical to the amino acid sequence depicted in Figure 2. Also provided is a JEST protein according comprising the amino acid sequence of Figure 2. Further provided herein is a JEST protein according encoded by a nucleic acid at least about 85% identical to the nucleic acid sequence depicted in Figure 2. Moreover, a JEST protein is provided herein which is encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence of Figure 2 or its complement.
In yet another aspect of the invention, an isolated polypeptide which specifically binds to a JEST
protein is provided. Preferably, the polypeptide is an antibody, more preferably, a monoclonal antibody. In one embodiment, a monoclonal antibody is provided that reduces or eliminates the biological function of JEST protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 2 or its complement.
The present invention further provides a recombinant nucleic acid encoding a human RasGRP protein that is at least about 85% identical to the amino acid sequence depicted in Figure 7B. Moreover, a recombinant nucleic acid is provided which encodes the amino acid sequence depicted in Figure 7B.
Also provided herein is a recombinant nucleic acid which will hybridize under high stringency recombinant nucleic acid that is at least about 90~o identical to the nucleic acid sequence depicted in Figure 7A. In another embodiment, a recombinant nucleic acid having the nucleic acid sequence depicted in Figure 7A is provided. Also provided are the sequences depicted in Figures 6A, 6B, 6C
and 6D, which each bind to SWAP70. In one embodiment, the sequences of Figures 6A-6D bind to JEST.
Further provided herein is a recombinant human RasGRP protein that is at least about 95% identical to the amino acid sequence depicted in Figure 7B. In one embodiment, a human RasGRP protein is provided which comprises the amino acid sequence of Figure 7B. In another embodiment, a human RasGRP protein is provided which is encoded lay a nucleic acid at least about 85°~ identical to the nucleic acid sequence depicted in Figure 7A. Also provided herein is a human RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nuGeic acid sequence of Figure 7A or its complement.
In another aspect of the invention, an isolated polypeptide which specfically binds to human RasGRP
protein is provided. In one embodiment, such a polypeptide is an antibody, preferably a monoclonal antibody. In a preferred embodiment, a monoclonal antibody that reduces or eliminates the biological function of RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 7A or its complement is provided.
Also provided herein are expression vectors comprising the nuGeic acids described herein. Further provided herein are the host cells comprising the nucleic acids and/or vectors described herein.
in a further aspect of the invention, a process for producing a human RasGRP
protein or JEST is provided. The methods comprise culturing the host cells provided herein under conditions suitable for expression of a human RasGRP protein or JEST.
In yet another aspect of the invention, a method for screening for a bioactive agent capable of binding to a JEST protein is provided. In one embodiment, said method comprises combining a JEST protein and a candidate bioactive agent, and determining the binding of said candidate agent to said JEST
protein.
Also provided herein is a method for screening for a bioactive agent capable of binding to a human RasGRP protein. In one embodiment, said method comprises combining a human RasGRP protein and a candidate bioactive agent, and determining the binding of said candidate agent to said human In yet another aspect of the invention, a method for screening for agents capable of interfering with the binding of a SWAP70 protein and RasGRP is provided. In one embodiment, said method comprises combining a SWAP70 protein, a candidate bioactive agent and a RasGRP protein and determining the binding of said SWAP70 protein and said RasGRP protein.
Also provided herein is a method for screening for agents capable of interfering with the binding of a JEST protein and RasGRP. In one embodiment, said method comprises combining a JEST protein, a candidate bioactive agent and a RasGRP protein and determining the binding of said JEST protein and said RasGRP protein.
In yet another aspect of the invention, a' method for screening for an bioactive agent capable of modulating the activity of JEST protein is provided. In one embodiment, said method comprises adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a JEST
protein and determining the effect of the candidate bioactive agent JEST
bioactivity including lymphocyte activation.
Also provided herein is a method for screening for an bioactive agent capable of modulating the activity of human RasGRP protein, said method comprising the steps of adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a human RasGRP
protein and determining the effect of the candidate bioactive agent on. RasGRP bioactivity including T-cell and B-cell activation. The methods provided herein can be performed wherein a library of candidate bioactive agents are added to a plurality of cells comprising said recombinant nucleic acid.
In yet another embodiment, a method for screening for a candidate protein capable of binding to SWAP70, JEST or RasGRP, is provided. It is understood that any one of these proteins, can be used.
Said method comprises combining a nucleic acid encoding SWAP70, JEST or RasGRP
and a nucleic acid encoding a candidate protein, wherein an identifiable marker is expressed wherein said candidate protein binds to said SWAP70, JEST or RasGRP.
Also provided herein is a complex consisting essentially of JEST or SWAP70 and RasGRP. Other aspects of the invention will be appreciated by the description that follows.
~iRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a nucleic acid sequence (cDNA) which includes an embodiment of the coding sequence of human JEST. The start colon begins at nucleotide 541 and the stop colon begins at nucteotade 2434. The start and stop colons are circled.
Figures 2A-2B depict an embodiment of the coding sequence of human JEST
wherein the amino acid sequence translation is shoum below the coding sequence.
Figure 3 depicts a schematic of where human ESTs and human RasGRP fragments provided herein alive with rat RasGRP.
Figures 4A-4B depict a nucleic acid sequence and amino acid sequence, respectively, for SWAP70.
Figures 5A-5B depict an amino acid alignment of SWAP70 and JEST, wherein the SWAP70 is shown above JEST such that SWAP70 is the query and JEST is the subject. Specific parameters utilized for the generation of the alignment are also shown.
Figures 6A-6D depict nucleic acid sequences of human RasGRP, wherein each of these nucleic acids encoded a product which binds with SWAP70. Figure 6A is shown as "swap70.14"
in Figure 3. Figure 6B is shown as "swap70.36" in Figure 3. Figure 6C is shown as "swap70.52" in Figure 3. Figure 6D is shown as "swap70.55" in Figure 3.
Figures 7A-7B depict a nucleic acid and amino acid sequence, respectively, of a consensus sequence for human RasGRP which binds to SWAP70.
The present invention provides novel lymphocyte activation prateins and nucleic acids. In a preferred embodiment, the lymphocyte activation proteins are from vertebrates and more preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
A lymphocyte activation protein of the present invention may be identified in several ways. "Protein" in this sense includes proteins, polypeptides, and peptides. A lymphocyte activation protein may be initially identified by its association with a protein known to be involved in T-cell and B-cell activation.

WO 00/2bI41 PCT/US99/25333 Lymphocyte activation proteins may be novel or may have been known in the art to exist, but not known to bind to SWAP or JEST or related to lymphocyte activation or lymphocyte activation proteins.
Novel lymphocyte activation nucleic acids or lymphocyte activation proteins are initially identified by substantial nucleic acid and/or amino acid sequence identity or similarity to the respective sequences shown in the figures, Such sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence.
In an additional aspect, the invention provides nucleic acids encoding lymphocyte activation proteins, namely, those shown in the figures, and their homologues. Preferred embodiments include JEST and the human homologue of RasGRP.
RasGRP, guanyl nucleotide-releasing protein for the small guanosine triphosphatase Ras, has been shown to activate Ras and cause transformation in fibroblasts (Ebinu et al., Science, 280:1082-1086 (1998), incorporated herein in its entirety). Signaling of RasGRP was associated with its partitioning in the membrane fraction. Based on its expression in neurons and ability to activate Ras, it may function to promote neuron differentiation, axonal growth and synaptic plasticity. It is now appreciated as shown herein that RasGRP message is induced in activated T and B cells.
Moreover, the relationship between SWAP and RasGRP shown herein indicates a novel cell activation pathway having numerous homologs in different cell lineages.
JEST has sequence homology to a human EST (AA306449) from a Jurkat T cell library. The full length sequence was derived from an activated human peripheral blood T-B cell switching library and from a Jurkat cDNA library. JEST has different expression pattern by Northern than SWAP, being most prominent in thymus, lymphoid organs, peripheral bload leukocytes and testis. As provided herein, JEST is involved in T cell signaling as well as mediating those signaling events in part by binding to RasGRP.
As discussed above, lymphocyte activation proteins include proteins which bind to SWAP or JEST, which are themselves considered lymphocyte activation proteins. Herein is provided RasGRP as a lymphocyte activation protein. In a preferred embodiment, human RasGRP is provided.
In a preferred embodiment, a protein is a "lymphocyte activation protein" it the overall sequence identity of the protein sequence to any one of the amino acid sequences shown in the figures is about or greater than about 75°~, more preferably greater than about 80°~, even more preferably greater wo oortsumcrms99ns333 will be as high as about 93 to 95 or 98%. It is understood that each sequence identification number provides an individual embodiment which can be selected individually or with any combination of members of the group. Sequence identity will be determined using standard techniques known in the art, including, but not limited to, the kacal sequence identity algorithm of Smith & Waterman, Adv. Appl.
Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairvvise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simpli5cation of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); ~e method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol.
215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A
particularly useful BLAST
program is the WU-BLAST'-2 program which was obtained from Altschul et al., Methods in logy, ~ø: 460-480 (1996); http:llblast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span =1, overlap fraction = 0.125, word threshold (T) = 11, The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A 9~° amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the '9onger"
sequence in the aligned region. The "longer' sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
In a similar manner, "percent (9~6) nucleic acid sequence identity" with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the lymphocyte WO OOIZb?,4i1 PGT/US991Z5333 _g_ activation protein. A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein shown in the Figures, it is understood that the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, in one embodiment, sequence identity of sequences shorter than that shown in the Figures, as discussed below, will be determined using the number of amino acids in the shorter sequence.
Lymphocyte activation proteins of the present invention may be shorter or longer than the amino acid sequences shown in the Figures. Thus, in a preferred embodiment, included within the definition of lymphocyte activation proteins are portions or fragments of the sequences provided herein. In one embodiment herein, fragments of lymphocyte activation proteins are considered lymphocyte activation proteins if a) they share at least one antigenic epitope; b) have at least the indicated sequence identity;
c) and preferably have lymphocyte activation biological activity, including binding to SWAP. In some I S cases, where the sequence is used diagnostically, that is, when the presence or absence of lymphocyte activation protein nucleic acid is determined, only the indicated sequence identity is required. The nucleic acids of the present invention may also be shorter or longer than the sequences in the Figures. The nucleic acid fragments include any portion of the nucleic acids provided herein which have a sequence not exactly previously identified; fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein.
in addition, as is more fully outlined below, lymphocyte activation proteins can be made that are longer than those depicted in the Figures for example, by the addition of epitope or purification tags, the addition of other fusion sequences, etc.
Lymphocyte activation proteins may also be identified as being encoded by lymphocyte activation nucleic acids. Thus, in one embodiment, lymphocyte activation proteins are encoded by nucleic acids that will hybridize to any one of the complementary sequences to the sequences depicted in the figures. In another embodiment, any of the nucleic acid sequences in the Figures can be utilized.
Hybridization conditions are further described below.
In a preferred embodiment, when the lymphocyte activation protein is to be used to generate WO OOn6241 iPCT/US99/Z5333 full length protein shown in the figures. By "epitope" or "determinant" herein is meant a portion of a protein which will generate andlor bind an antibody. Thus, in most instances, antibodies made to a smaller lymphocyte activation protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity. The term "antibody" includes antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
In a preferred embodiment, the antibodies to lymphocyte activation are capable of reducing or eliminating the biological function of lymphocyte acfrvation proteins, as is described below. That is, the addition of anti-lymphocyte activation antibodies (either polyclonal or preferably monoclonal) to lymphocyte activation (or cells containing these proteins) may reduce or eliminate the lymphocyte activation activity. Generally, at feast a 25°~6 decrease in activity is preferred, with at least about 50%
being particularly preferred and about a 95-900°~ decrease being especially preferred.
The lymphocyte activation antibodies of the invention bind to lymphocyte activation proteins. In a preferred embodiment, the antibodies specifically bind to lymphocyte activation proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10''- 10$ M~', with a preferred range being 10'' -10'° M-'. Antibodies are further described below.
In the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and colon bias of different organisms. Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence. Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences of the figures is preferably greater than 85°~, and preferably greater than 75%, more preferably greater than about 80°~, particularly greater than about 85% and most preferably greater than 90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%.
in a preferred embodiment, a lymphocyte activation nucleic acid encodes a lymphocyte activation protein. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the lymphocyte activation proteins of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more colons in a way which does not change the amino acid sequence of the lymphocyte activation protein.
In one embodiment, the nucleic acid is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequences which complements are shown in the figures and is considered a lymphocyte activation gene. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A
Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize speci0cally at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically abou! 0.01 to 1.0 M sodium ion concentration (or other salts) at pH
7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency cond'ttions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.
The lymphocyte activation proteins and nucleic acids of the present invention are preferably recombinant. As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half life of such molecules in physiological environments.
The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded strand ("Watson') also defines the sequence of the other strand ("Crick');
thus the sequences depicted in the Figures also include the complement of the sequence. By the term "recombinant nucleic acid"
herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated lymphocyte activation nucleic acid, in a linear form, or an expression vector formed in vitrr~ by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nuGeic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantfy, i.e. using the '~~y'~ cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nuGeic acid a5 depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is nom~ally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5°~ by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90°~ being particularly preferred. The definition includes the production of a lymphocyte activation protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
Also included within the definition of lymphocyte activation proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes:
substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the lymphocyte activation protein, using cassette or PCR
mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above.
However, variant lymphocyte activation protein fragments having up to about 100-150 residues may be prepared by in predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the lymphocyte activation protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed lymphocyte activation variants screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites in DNA
having a known sequence are well known, for example, M13 primer mutagenesis and PCR
mutagenesis. Screening of the mutants is done using assays of lymphocyte activation protein activities.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.
When small alterations in the characteristics of the lymphocyte activation protein are desired, substitutions are generally made in accordance with the following chart:
Chart I
Original Residue E~cemRlary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln ,qsn Glu ~p Gly Pro His Asn, Gln wo oon6m Pcrrus99ns~

Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val lie, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structun:; the charge or hydrophobicity of the molecule at the target S site; or the bulk of the side chain. The substitutions which in general ace expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyi; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyt, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the lymphocyte activation proteins as needed. Aftematively, the variant may be designed such that the biological activity of the lymphocyte activation protein is altered. For example, glycosylation sites may be altered or removed. In an embodiment provided herein, mutations in the JEST binding domain are made so as to modulate binding characteristics.
Covalent modifications of lymphocyte activation polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a lymphocyte activation polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a lymphocyte activation polypeptide.
Derivatization with bifunctional agents is useful, for instance, for crosslinking lymphocyte activation to a water-insoluble support matrix or surface for use in the method for purifying anti-lymphocyte crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-((p-azidophenyl)dithiojpropioimi-date.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of praline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-amino groups of lysine, arginine, and histidine side chains [T.E. Greighton, Proteins: Structure and Molerw,ular Properties, W.H. Freeman 8 Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidat'ron of any C-terminal carboxyl group.
Another type of covalent modification of a lymphocyte activation polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence lymphocyte activation polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence lymphocyte activation polypeptide.
Addition of glycosylation sites to lymphocyte activation polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence lymphocyte activation polypeptide (for O-linked glycosylation sites). A lymphocyte activation amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding a lymphocyte activation polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on a lymphocyte activation polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rey B~f~em., pp. 259-306 (1987).
Removal of carbohydrate moieties present on a lymphocyte activation polypeptide may be accomplished chemically or enzymatically or by mutational subsfttution of codons encoding for amino WO 00lZ6241 PCT/US99l15333 in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem.
Bioohvs., x:52 (1987) and by Edge et al., (ipal. Bioghem., u$:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Seth. Enzvmol., y~$:350 (1987).
Another type of covalent modahcation of lymphocyte activation comprises linking a lymphocyte activation polypeptide to one of a variety of nonproteina~ceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S.
Patent Nos. 4,640,835;

4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Lymphocyte activation polypeptldes of the present invention may also be modified in a way to form chimeric molecules comprising a lymphocyte activation polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of a lymphocyte activation polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-or carboxyhterminus of a lymphocyte activation polypeptide. The presence of such epitope-tagged forms of a lymphocyte activation polypeptide can be detected using an antibody against the tag polypeptide.
Also, provision of the epitope tag enables a lymphocyte activation polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative, embodiment, the chimeric molecule may comprise a fusion of a lymphocyte activation polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule as discussed further below.
Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag poiypeptide and its antibody 12CA5 [Field et al., MoI. Cell. 8iol., $:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Bioloav, $:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, $(6):547-553 (1990)]. Other tag polypeptides include the Fiag-peptide [Hopp et al., BioTechnoloav, $:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., ~jg~~g, x:192-194 (1992)]; tubulin epitope peptide [Skinner et al., ,I. Bi2. Chem., 2$ø:15163-15166 (1991 )]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci.
USA, ~:6393~397 (1990)].
In an embodiment herein, lymphocyte activation proteins of the lymphocyte activation family and wo oon6m pcrms~ns333 Thus, probe or degenerate poiymerase chain n~action (PCR) primer sequences may be used to end other related lymphocyte activation proteins from humans or other organisms.
As will be appreciated by those in the art, particularly useful probe andlor PCR primer sequences include the unique areas of a lymphocyte activation nucleic acid sequence. As is generally known in the art, preferred PCR
primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR
reaction are well known in the art. It is therefore also understood that provided along with the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred. The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length.
Once a lymphocyte activation nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form an entire full length or mature lymphocyte activation nucleic acid.
Wherein the full length nucleic acid has a signal peptide and/or transmembrane region(s), it can be modified to exclude one or more of these regrons so as to encode a peptide in its mature soluble form.
Once isolated from its natural source, e.g., contained within a piasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant lymphocyte activation nucleic acid can be further-used as a probe to identify and isolate other lymphocyte activatron nucleic acids. It can also be used as a "precursor" nucleic acid to make modified or variant lymphocyte activation nucleic acids and proteins. The skilled artisan understands that wherein two or more nucleic acids overlap, the overlapping portions) of one of the overlapping nucleic acids can be omitted and the nucleic acids combined for example by ligation to form a longer linear lymphocyte activation nucleic acid so as to, for example, encode the full length or mature peptide. The same applies to the amino acid sequences of lymphocyte activation polypeptides in that they can be combined so as to form one contiguous peptide.
Using the nucleic acids of the present invention which encode a lymphocyte activation protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably (inked to the nucleic acid encoding a lymphocyte activation protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA
for a polypeptide if it is expressed as a pneprotein that participates in the secretion of the polypeptide;
a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA
sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptionai and translational regulatory nucleic acid will generally be appropriate to the host cell used to express a lymphocyte activation protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express a lymphocyte activation protein in Bacillus.
Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
In general, the transcriptionai and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters, Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression anti in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specfic locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art. Preferred methods to effect homologous recombination are described in PCT US93103868 and PCT US98105223, hereby incorporated by reference.

WO 110n6241 IPGT/US99l25333 In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97I01019 and PCTIUS97/01048, both of which are hereby expressly incorporated by reference.
The lymphocyte activation proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encx~ding a lymphocyte activation protein, under the appropriate conditions to induce or cause expression of a lymphocyte activation protein. The conditions appropriate for lymphocyte activation protein express'ron will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangasfer cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, neuronal cells, and those from the spleen, thymus, prostate, testes, uterus, colon, small intestine, PBL, lymph nodes, bone marrow and liver, and all cells involved in the immune system.
in a preferred embodiment, the lymphocyte activation proteins are expressed in mammalian cells.
Mammalian expression systems are also known in the art, and include retroviral systems. A
mammalian promoter is any ONA sequence capable of binding mammalian RNA
polymerise and initiating the downstream (3') transcription of a coding sequence for lymphocyte activation protein into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA
polymerise II to begin RNA
synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus l_TR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translat'ron stop colon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenytation. Examples of transcription terminator and polyadenlytion signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as weft as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the poiynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
In a preferred embodiment, lymphocyte activation proteins are expressed in bacterial systems.
Bacterial expression systems are well known in the art.
A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA
polymerise and initiating the downstream (3') transcription of the coding sequence of lymphocyte activation protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerise binding site and a transcription initiation site.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan.
Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tic promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerise and initiate transcription.
In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E.

colon and a sequence 3-9 nuGeotides in length located 3 -11 nucleotides upstream of the initiation colon.
The expression vector may also include a signal peptide sequence that provides for secretion of a lymphocyte activation protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Stnsptococcus lividans, among others.
The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
In one embodiment, lymphocyte activation proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
In a preferred embodiment, lymphocyte activation proteins are produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymoipha, Kluyveromyces fragilis and K.
lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
Preferred promoter sequences for expression in yeast include the inducibte GAI_1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerale mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, phosphotransferase gene, which confers resistance to 6418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.
A lymphocyte activation protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the lymphocyte activation protein may be fused to a carrier protein to form an immunogen.
Alternatively, a lymphocyte activation protein may be made as a fusion protein to increase expression, or for other masons. For example, when a lymphocyte activation protein is a lymphocyte activation peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes. Similarly, lymphocyte activation proteins of the invention an be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), etc.
In one embodiment, the lymphocyte activation nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes;
b) immune labels, which may be antibodies yr antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position.
In a preferred embodiment, a lymphocyte activation protein is purified or isolated after expression.
lymphocyte activation proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, a lymphocyte activation protein may be purified using a standard anti-lymphocyte activation antibody column. Ultrafiltration and diaflltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Pur~cation, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the lymphocyte activation protein.
In some instances no purification will be necessary.
Once expressed and purified if necessary, the lymphocyte activation proteins and nucleic acids are useful in a number of applications.

WO 00!'16241 PGT/US99/25333 The nucleotide sequences (or their complement) encoding lymphocyte activation proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA.
lymphocyte activation protein nucleic acids wilt also be useful for the preparation of lymphocyte activation protein polypeptides by the recombinant techniques described herein.
A full-length native sequence lymphocyte activation protein gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate a full-length lymphocyte activation protein gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of a lymphocyte activation protein or a lymphocyte activation protein from other species) which have a desired sequence identity to a lymphocyte activation protein coding sequence.
Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived hom the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and introns of native sequences as provided herein. By way of example, a screening method will comprise isolating the coding region of a lymphocyte activation protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as ~P or ~S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of a lymphocyte activation protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or rnRNA to determine to which members of such libraries the probe hybridizes.
The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related lymphocyte activation protein coding sequences.
Nucleotide sequences encoding a lymphocyte activation protein can also be used to construct hybridization probes for mapping the gene which encodes that lymphocyte activation protein and for the genetic analysis of individuals with genetic disorders. The nuGeotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
Nucleic acids which encode lymphocyte activation proteins or their modified forms can also be used to generate either transgenic animals or "knock out' animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A non-human transgenic animal (e.g., a mouse or or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A
transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops.
In one embodiment, cDNA encoding a lymphocyte activation protein can be used to clone genomic DNA
encoding a lymphocyte activation protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for a lymphocyte activation protein transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding a lymphocyte activation protein introduced into the germ fine of the anima! at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. fn accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of a lymphocyte activation protein can be used to construct a lymphocyte activation protein "knock out" animal which has a defective or altered gene encoding a lymphocyte act'rvatron protein as a result of homologous recombination between the endogenous gene encoding a lymphocyte activation protein and altered genomic DNA encoding a lymphocyte activation protein introduced into an embryonic cell of the animal. For example, cDNA
encoding a lymphocyte activation protein can be used to clone genomic DNA encoding a lymphocyte activation protein in accordance with established techniques. A portion of the genomic DNA encoding a lymphocyte activation protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, fig[, x:503 (1987) for a description of homologous recombination vectorsj. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., II, X9:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to farm aggregation chimeras [see e.g., Bradley, in Terafocan:inomas and Embryonic Stem Cells: A
Practical Approach, ~. 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 to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their WO OOI26241 PCT/US99i25333 animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of a lymphocyte activation protein polypeptide.
Nucleic acids encoding lymphocyte activation polypeptides, antagonists or agonists may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. 'Gene therapy' includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. it has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., eroc. ~I~,O1~[,_Acad. Sci. USA ~, 4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the catcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau ef al., Trends in Biotechnoloav y1, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where iiposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting andlor to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. ~. 4429-4432 (1987); and Wagner ef al., Proc. Natl.
Acad. Sci. USA ~7, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science Z~ø, 808-813 (1992).

In a preferred embodiment, the lymphocyte activation proteins, nucleic acids, modified proteins and cells containing the native or modified lymphocyte activation proteins are used in screening assays.
Identification of this important T-cell and B-cell activation protein permits the design of drug screening assays for compounds that modulate lymphocyte activation activity.
Screens may be designed to first find candidate agents that can bind to lymphocyte activation proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate lymphocyte activation activity. Thus, as will be appreciated by those in the art, there are a number of different assays which may be run; binding assays and activity assays.
Thus, in a preferred embodiment, the methods comprise combining a lymphocyte activation protein and a candidate bioactive agent, and determining the binding of the candidate agent to the lymphocyte activation protein. Preferred embodiments utilize a human lymphocyte activation protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. These latter embodiments may be preferred in the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative Lymphocyte activation proteins may be used, including deletion lymphocyte activation proteins as outlined above.
The term "candidate bioactive agent" or "exogeneous compound" as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., with the capability of directly or indirectly altering the bioactivity of Lymphocyte activation protein.
Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures andlor aromatic or poiyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Particularly preferred are WO 00l2624I PCT/US99lZ5333 Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Aftematively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modfied through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical mod~cations, such as acylation, alkylation, esterification, amidification to produce structural analogs.
In a preferred embodiment, the candidate bioactive agents are proteins. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as praline and hydroxyproline. The side chains may be in either the (R} or the (S} configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration.
If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
In a preferred embodiment, the candidate bioactive agents are naturally occuring proteins or fragments of naturally occuring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening against Lymphocyte activation protein.
Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occuring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the WO 00/26241 )pGT/US991Z5333 _27_ formation of all or most of the possible combinations over tkte length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines; for cross-linking, pralines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the candidate bioactive agents are nucleic acids.
By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides cavalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970);
Sprinzl et al., Eur. J.
Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett.
805 (1984), Letsinger et al., J. Am. Chem. Soc_ 110:4470 (1988); and Pauwels et al., Chemica Scripts 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991 ); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methyiphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues:
A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J.
Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference).
Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci.
USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5.386.023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &
Nucleotide 13:1597 (1994);
Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic 8 Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett.
37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modfications in Antisense Research', Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also WO 00!26241 PCT/US99/x5333 176). Several nucleic acid analogs are described in Rawls, C 8~ E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made.
Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occuring nucleic acids and analogs may be made. The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cONA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occuring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
The assays herein utilize lymphocyte activation proteins as defined herein. In one embodiment, portions of lymphocyte activation proteins are utilized, in a preferred embodiment, portions having lymphocyte activation activity are used. In addition, the assays described herein may utilize either isolated lymphocyte activation proteins or cells comprising the lymphocyte activation proteins.
Generally, in a preferred embodiment of the methods herein, the lymphocyte activation protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). It is understood that soluble assays can also be used, for example those which can be detected by fluorescent changes with binding, etc. The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teOonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic WO 00/Z6241 t'CT/US99lZ5333 long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composttion and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to °sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. In some embodiments, SWAP or JEST
can be used.
Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in this invention are screening assays wherein solid supports are not used.
In a preferred embodiment, the lymphocyte activation protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and the lymphocyte activation protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
The determination of the binding of the candidate bioactive agent to a lymphocyte activation protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of a lymphocyte activation protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.
By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
Specfic binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.
In some embodiments, only one of the components is labeled. For example, the proteins (or Alternatively, more than one component may be labeled with different labels;
using'2~1 for the proteins, for example, and a fluorophor for the candidate agents.
In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the target molecule (lymphocyte activation molecule), such as an antibody, peptide, binding partner, ligand, etc. In a preferred embodiment, the competitor is SWAP or JEST. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent. This assay can be used to determine candidate agents which interfere with binding between lymphocyte activation proteins and SWAP or JEST.
In one embodiment, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present.
Incubations may be pertormed at any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the competitor is added first, folknNed by the candidate bioactive agent.
Displacement of the competitor is an indication that the candidate bioactive agent is binding to the lymphocyte activation protein and thus is capable of binding to, and potentially modulating, the activity of the lymphocyte activation protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Aftemat'rvely, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the candidate bioactive agent is added first, wish incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the lymphocyte activation protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the lymphocyte activation protein.

wo oon6a,4>t PcrNS~ns~33 In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the lymphocyte activation proteins. In this embodiment, the methods comprise combining a lymphocyte activation protein and a competitor in a first sample. A
second sample comprises a candidate bioactive agent, a lymphocyte activation protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the lymphocyte activation protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the lymphocyte activation protein.
Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native lymphocyte activation protein, but cannot bind to modified lymphocyte activation proteins. The structure of the lymphocyte activation protein may be modeled, and used in rational drug design to synthesize agents that interact with that s'tte. Drug candidates that affect lymphocyte activation bioactivity are also identified by screening drugs for the ability tv either enhance or reduce the activity of the protein.
Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein.
Following incubation, all samples are washed free of non-spec~cally bound material and the amount of bound, generally Labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
The components provided herein for the assays provided herein may also be combined to form kits.
The kits can be based on the use of the protein and/or the nucleic acid encoding the lymphocyte activation proteins. Assays regarding the use of nucleic acids are further described below.

Screening for agents that modulate the activity of lymphocyte activation may also be done. In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activity of lymphocyte activation comprise the steps of adding a candidate bioact'rve agent to a sample of lymphocyte activation protein, as above, and determining an alteration in the biological activity of lymphocyte activation protein. "Modulating the activity of Lymphocyte activation protein" includes an increase in activity, a decrease in activit)r, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to lymphocyte activation (although this may not be necessary), and alter its biological or biochemical activitlr as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activvity or amount of lymphocyte activation protein.
Thus, in this embodiment, the methods comprise combining a lymphocyte activation sample and a candidate bioactive agent, and evaluating the effect on T-cell and B-cell activafron. By "Lymphocyte activation activity" or grammatical equivalents herein is meant one of lymphocyte activation protein's biological activities, including, but not limited to, its ability to affect T-cell and B-cell activation. One activity herein is the capability to bind to SWAP or JEST.
In a preferred embodiment, the activity of the lymphocyte activation protein is increased; in another preferred embodiment, the activity of the lymphocyte activation protein is decreased. Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
fn a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of a lymphocyte activation protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising lymphocyte activation proteins.
Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a lymphacyte activation protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
In some embodiments, the assays include exposing the cells to an T-cell and B-cell activation agent that will induce T-cell and B-cell activation in control cells, i.e. cells of the same type but that do not contain the exogeneous nucleic acid encoding an activation protein.
Alternatively, the cells may be exposed ~ conditions that normally result in T-cell and B-cell activation, and changes in the normal T-cell and B-cell activation progression are determined. Alternatively, the cells into which the lymphocyte activation nucleic acids are introduced normally under T-cell and B-cell activation, and thus ChannPS lfnr pYamnlP inhihitinn of T-npll anri R-rpll aetivatinn~ ara eiotcrminart rlnt~r,r,~lt.. rho WO 00/26241 P~CTNS99~

cells normally do not undergo T-cell and B-cell activation, and the introduction of a candidate agent causes T-cell and B-cell activation.
Thus, the effect of the candidate agent on T-cell and B~eil activation is then evaluated.
Detection of T-cell and B-cell activation may be done as will be appreciated by those in the art. fn one embodiment, indicators of T-cell and B-cell activation are used. Accordingly, these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface, etc. and used to pull out cells that are undergoing T-cell and B-cell activation. Similarly, these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS} separation.
In this way, bioacfrve agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the lymphocyte activation protein.
The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of 1 S introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
Pharmaceutical grade organic or inorganic carriers andlor diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
Without being bound by theory, it appears that a lymphocyte activation protein is an important protein in T-cell and B-cell activation. Accordingly, disorders based on mutant or variant lymphocyte activation genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant lymphocyte activation genes comprising determining all or part of the sequence of at least one endogeneous lymphocyte activation genes in a cell. As wiH be appreciated by those in the art, this may be done using any number of sequencing techniques. in a preferred embodiment, the invention provides methods of identifying the lymphocyte activation nennhm~n of .fin indiuidmnl nnrHnrie.inn d~~nrr~inin~ .,II i,r r....r~ ~~
~lvrv r.,"-,.~......v ni..~ 1..x..1 ...... L......L__..a_ wo oon6a,41 rc~rrtrs~ns333 activation gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced lymphocyte activation gene to a known lymphocyte activation gene, i.e. a wild-type gene.
The sequence of all or part of the lymphocyte activation gene can then be compared to the sequence of a known lymphocyte activation gene to determine if any differences exist.
This can be done using any number of known sequence identity programs, such as Bestfit, etc. and others outlined herein. In a preferred embodiment, the presence of a difference in the sequence between the lymphocyte activation gene of the patient and the known lymphocyte activation gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
The present discovery relating to the role of lymphocyte activation in T-cell and B-cell activation thus provides methods for inducing or preventing T-cell and B-cell activation in cells. In a preferred embodiment, the lymphocyte activation proteins, and particularly lymphocyte activation fragments, are useful in the study or treatment of conditions which are mediated by T-cell and B-cell activation, i.e. to diagnose, treat or prevent T-cell and B-cell activation-mediated disorders.
Thus, "T-cell and B-cell activation mediated disorders" or "disease state" include conditions involving both insufficient or excessive T-cell and B-cell activation. immunological disorders are numerous and known in the art.
Thus, in one embodiment, methods of modulating T-cell and B-cell activation in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-lymphocyte activation antibody or other agent identified herein or by the methods provided herein, that reduces or eliminates the biological activity of the endogeneous lymphocyte activation protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a lymphocyte activation protein or modulator including anti-sense nucleic acids.
As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, the activity of lymphocyte activation is increased by increasing the amount of lymphocyte activation in the cell, for example by overexpressing the endogeneous lymphocyte activation or by administering a gene encoding an Lymphocyte activation protein, using known gene-therapy techniques, for example.
In a preferred embodiment, the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCTIUS93/03868, hereby incorporated by reference in its entireity.
In one embodiment, the invention provides methods for diagnosing an T-cell and B-cell activation --1-a_J _.--J:L:-.~ :_ -- :_J:..:J.....1 1'4~ ~..LL.-J.- ...-...-.--...~ ~~~-.....-.~ LL- .-..L...a.. w~ 1.....~6......L~

activation in a tissue from the individual or patient, which may include a measurement of the amount or specific activity of Lymphocyte activation protein. This activity is compared to the acfrvity of lymphocyte activation from either a unaffected second individual or from an unaffected tissue from the first individual. When these activities are different, the first individual may be at risk for an T-cell and B-cell activation mediated disorder.
The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interacctieons of the lymphocyte activation proteins can be idenfdred. Genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, namely the "yeast two-hybrid" system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686 (1991 ); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et aL, PNAS USA
88:9578 (1991); and U.S. Patent Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463. A
preferred system is described in Serial No. 091050,863, filed March 30, 1998, entitled "Mammalian Protein Interaction Cloning System'. For use in conjunction with these systems, a particularly useful shuttle vector is described in Serial No. 091133,944, filed August 14, 1998, entitled "Shuttle Vectors".
In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the gene encoding SWAP, JEST or a portion thereof, and the other encodes a test candidate. Only if the Nuo expression products bind to one another will an indicator, such as a fluorescent protein, be expressed.
Expression of the indicator indicates when a test candidate binds to SWAP or JEST and can be identified as a lymphocyte activation protein. Using the same system and the identified lymphocyte activation proteins the reverse can be performed. Namely, the lymphocyte activation proteins provided herein can be used to identify new baits, or agents which interact with lymphocyte activation proteins. Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to SWAP or JEST and the lymphocyte activation protein encoding nucleic aads to determine agents which interfere with the bait, such as SWAP or JEST, and the lymphocyte activation protein.
RasGRP can also be used.
In one embodiment, a mammalian two-hybrid system is preferred. Mammalian systems provide post-translational modifications of proteins which may contribute significantly to their ability to interact. In addition, a mammalian two-hybrid system can be used in a wide variety of mammalian cell types to mimic the regulation, induction, processing, etc. of specific proteins within a particular cell type. For example, proteins involved in a disease state such as those described above could be tested in the --1-..~~a J:..... __11_ fv:_.:1~_L. t...~-..t:.... ~1.,.--J_.._ .__-~_.~-_.___...__ aL _.._. ..._ W _ . a1. . n wo oorw~>I pc~r~us~ns333 cellular conditions will give the highest positive results. Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hortranes, drugs, growth factors and cytokines, cellular and chemical stimuli, etc., that may contribute to conditions which can effect protein-protein interactions, particuiarfy those involved in T-cell and B-cell activation.
Expression in various cell types, and assays for lymphocyte activation activity are described above.
The activity assays, such as having an effect an T-cell and B-cell activation can be performed to confirm the acitivity of lymphocyte activation proteins which have already been identified by their sequence identitylsimilarity or binding to SWAP, JEST or RasGRP as well as to further confirm the activity of lead compounds identified as modulators of T-cell and B-cell activation.
Assays involving binding such as the two-hybrid system may take into account non-specific binding proteins (NSB).
In one embodiment, the lymphocyte activation proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to lymphocyte activation proteins, which are useful as described herein. Similarly, the lymphocyte activation proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify lymphocyte activation antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to the lymphocyte activation protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the lymphocyte activation antibodies may be coupled to standard affinity chromatography columns and used to purify lymphocyte activation proteins as further described below. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specthcally bind to the lymphocyte activation protein.
The anti-lymphocyte acfrvation protein antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent andlor adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the lymphocyte activation protein polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, 1_....:~~ ~1-....~-.W 6..c.~ -__1 __..L__~ a._.~_c_ :.-Ltu:u.._ r_.__._n__ _r _ m .. . .

include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The anti-lymphocyte activation protein antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, xø:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specficaliy bind to the immunizing agent. AltemaCrvely, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the lymphocyte activation protein polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cel! [coding, Monoclonal Antibodies: Principles and practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypaxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT
medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. mmunol., x:3001 (1984); Brodeur et al., Monoclonal Antibody Prod~lction TechniauP,~ and Apalications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of Mnnnrlnns) ~n~ihnrline rliren~nil ~n~ine~ Iv.rnhnr...~a .".~;....~:.... ..-..v...:.. n..,s..-..m,. ~~_ w:_~:__ specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELiSA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, 6 anhBi, ,~"~7:220 (1980).
After the desired hybridoma cells are identfied, the clones may be subdoned by limiting dilution procedures and grown by standard methods [coding, s_yral. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Aftematively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the sutxtones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S.
Patent No. 4,816,567; Morrison et al., suorai or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobutin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
The anti-lymphocyte activation protein antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof {such as Fv, Fab, Fab', F{ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins {recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., , x:522-525 (1986); Riechmann et al., Nature, x:323-329 {1988); and Presta, Curr. O . Struct. Biol., x:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or rrwre amino acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., N~~re, ,x:522-525 (1986);
Riechmann et al., Nature, ,x:323-327 (1988); Verhoeyen et al., , cience, ~Q:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies {U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which wo oon6za» pcrn~s~n3333 -ao-some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, ~ ol. Biol., ~j:381 (1991 ); Marks et al., ~. MQI. Biol., x:581 (1991)]. The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., I~OggJl~nal Anti iodise and Cancer Theraov, Alan R.
Lies, p. 77 (1985) and Boemer et al., J. Immunol., 147(11:86-95 (1991 )].
Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U,S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et al., BioITec r~oloav 1Q, 779-783 (1992); lonberg ef al., Nature ~$ 856-859 (1994); Morrison, Nature ,~, 812-13 (1994); Fishwild et al., Nature Biotechnoloav ~, 845-51 (1996); Neuberger, Nature Biotechnoloav ~, 826 (1996);
Lonberg and Huszar, Int~ev._I~munol. ~ 65-93 (1995).
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the lymphocyte activation protein, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, x:537-539 {1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93108829, published 13 May 1893, and in Traunecker et al., EMBO
J.,1Q:3655-3659 (1991 ).
Antibody variable domains with the desired binding speci~cities (antibody-antigen combining sites) can be fused to immunoglobuNn constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 WO 00/26241 PCT/US99lZ5333 necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in EnzXmoloav, 1:210 (1986).
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two cavalenfly joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No.
4,676,980], and for treatment of HIV infection (VllO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No. 4,676,980.
The anti-lymphocyte activation protein antibodies of the invention have various utilities. For example, anti-lymphocyte activation protein antibodies may be used in diagnostic assays for a lymphocyte activation protein, e.g., detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual r,~Teehniq~, CRC
Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable moiety.
The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
For example, the detectable moiety may be a radioisotope, such as'H, "C,'ZP,'~S, or'zsl, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, ~g:945 (1962);
David et al., Biochemistry, x:1014 (1974); Pain et al., J. Immunol. Meth., x:219 (1981 );
and Nygren, ,~
Histochem. and C, ochem., ~Q:407 (1982).
Anti-Lymphocyte activation protein antibodies also are useful for the affinity purification of lymphocyte activation protein from recombinant cell culture or natural sources. In this process, the antibodies against lymphocyte activation protein are immobilized on a suitable support, such a Sephadex resin or .... . .. .. . . .. . _. . . ... . ..

WO OOIZ6141 PCTNS99n5333 sample containing the lymphocyte activation protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the lymphocyte activation protein, which is bound to the immobilized antibody.
Finally, the support is washed with another suitable solvent that will release the lymphocyte activation protein from the antibody.
The anti-lymphocyte activation protein antibodies may also be used in treatment. In one embodiment, the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the lymphocyte activation protein within the cell.
In one embodiment, a therapeutically effective dose of a lymphocyte activation protein, agonist or antagonist is administered to a patient. By "therapeutically effective dose"
herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for lymphocyte activation degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
A "patient' for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
The administration of the lymphocyte activation protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneousty, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the lymphocyte activation may be directly applied as a solution or spray.
The pharmaceutical compositions of the present invention comprise a lymphocyte activation protein, agonist or antagonist in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological ~lf~.wiim..wwww wJ<IL.w A..w L..www wwd ~L. w~ wnw ww~ L.i..lw..iwwll.. .~~
w~L.ww..lww ..~J-~:~~LI- L-_~-J __.:aL

inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nific acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malefic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts"
include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, cak:ium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calaum, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following:
carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches;
binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
Additives are well known in the art, and are used in a variety of formulations.
Ail references and sequences of accession numbers cited herein are incorporated by reference in their entireity.

Claims (45)

1. A recombinant nucleic acid encoding a JEST protein that is at least about 85% identical to the amino acid sequence depicted in Figure 2.

2. A recombinant nucleic acid according to claim 1 encoding the amino acid sequence depicted in Figure 2.

3. A recombinant nucleic acid which will hybridize under high stringency conditions to the nucleic acid sequence depicted in Figure 2 or its complement.

4. A recombinant nucleic acid that is at least about 90% identical to the nucleic acid sequence depicted in Figure 2.

5. A recombinant nucleic acid having the nucleic acid sequence depicted in Figure 2.

6. An expression vector comprising the nucleic acid of claim 2.

7. A host cell comprising the nucleic acid of claim 2.

8. A host cell comprising the vector of claim 6.

9. A process for producing a JEST protein comprising culturing the host cell of claim 8 under conditions suitable for expression of a JEST protein.

10. A process according to claim 9 further comprising recovering said JEST
protein.

11. A recombinant JEST protein that is at least about 85% identical to the amino acid sequence depicted in Figure 2.

12. A JEST protein according to claim 11 comprising the amino acid sequence of Figure 2.

13. A JEST protein according to claim 11 encoded by a nucleic acid at least about 85% identical to the nucleic acid sequence depicted in Figure 2.

14. A JEST protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence of Figure 2 or its complement.

15. An isolated polypeptide which specifically binds to JEST protein.

16. A polypeptide according to claim 15 that is an antibody,

17. A polypeptide according to claim 18 wherein said antibody is a monoclonal antibody.

18. A monoclonal antibody that reduces or eliminates the biological function of JEST protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 2 or its complement.

19. A recombinant nucleic acid encoding a human RasGRP protein that is at least about 85% identical to the amino acid sequence depicted in Figure 7B.

20. A recombinant nucleic acid according to claim 19 encoding the amino acid sequence depicted in Figure 7B.

21. A recombinant nucleic acid which will hybridize under high stringency conditions to the nucleic acid sequence depicted in Figure 7A or its complement.

22. A recombinant nucleic acid that is at least about 90% identical to the nucleic acid sequence depicted in Figure 7A.

23. A recombinant nucleic acid having the nucleic acid sequence depicted in Figure 7A.

24. An expression vector comprising the nucleic acid of claim 20.

25. A host cell comprising the nucleic acid of claim 20.

26. A host cell comprising the vector of claim 24.

27. A process for producing a human RasGRP protein comprising culturing the host cell of claim 26 under conditions suitable for expression of a human RasGRP protein.

28. A process according to claim 27 further comprising recovering said human RasGRP protein.

29. A recombinant human RasGRP protein that is at least about 95% identical to the amino acid sequence depicted in Figure 7B.

30. A human RasGRP protein according to claim 29 comprising the amino acid sequence of Figure 7B.

31. A human RasGRP protein according to claim 29 encoded by a nucleic acid at least about 85%
identical to the nucleic acid sequence depicted in Figure 7A.

32. A human RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence of Figure 7A or its complement.

33. An isolated polypeptide which specifically binds to human RasGRP protein.

34. A polypeptide according to claim 33 that is an antibody.

35. A polypeptide according to claim 34 wherein said antibody is a monoclonal antibody.

36. A monoclonal antibody that reduces or eliminates the biological function of RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 7A or its complement.

37. A method for screening for a bioactive agent capable of binding to a JEST
protein, said method comprising combining a JEST protein and a candidate bioactive agent, and determining the binding of said candidate agent to said JEST protein.

38. A method for screening for a bioactive agent capable of binding to a human RasGRP protein, said method comprising combining a human RasGRP protein and a candidate bioactive agent, and determining the binding of said candidate agent to said human RasGRP protein.

39. A method for screening for agents capable of interfering with the binding of a SWAP70 protein and RasGRP comprising:
a) combining a SWAP70 protein, a candidate bioactive agent and a RasGRP
protein; and

40. A method for screening for agents capable of interfering with the binding of a JEST protein and RasGRP comprising:
a) combining a JEST protein, a candidate bioactive agent and a RasGRP protein;
and b) determining the binding of said JEST protein and said RasGRP protein.

41. A method for screening for an bioactive agent capable of modulating the activity of JEST protein, said method comprising the steps of:
a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a JEST protein;
b) determining the effect of the candidate bioactive agent JEST bioactivity including lymphocyte activation.

42. A method for screening for an bioactive agent capable of modulating the activity of human RasGRP protein, said method comprising the steps of:
a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a human RasGRP protein;
b) determining the effect of the candidate bioactive agent on RasGRP
bioactivity including T-cell and B-cell activation.

43. A method according to claim 41 or claim 42 wherein a library of candidate bioactive agents are added to a plurality of cells comprising said recombinant nucleic acid.

44. A complex consisting essentially of JEST or SWAP70 and RasGRP.

45. A method for screening for a candidate protein capable of binding to SWAP70, JEST or RasGRP, said method comprising combining a nucleic acid encoding SWAP70, JEST or RasGRP and a nucleic acid encoding a candidate protein, wherein an identifiable marker is expressed wherein said candidate protein binds to said SWAP70, JEST or RasGRP.