WO1994018832A1 - Cd4 mediated modulation of lipid kinases - Google Patents

Abstract

A method of inhibiting or reducing signal transduction through CD4, which method includes the step of introducing into a T cell a peptide which decreases the association of PI 3-kinase and PI 4-kinase with CD4/p56lck.

Description

CD4 MEDIATED MODULATION OF L PID INASES Field of the Invention This invention relates to signal transduction in T cells.

Background of the Invention CD4, a T cell surface antigen, serves as a receptor for MHC class II antigens and as a receptor for the Human Immunodeficiency Virus (HIV-l) viral coat protein gpl20 (Reinherz, E.L. et al. Immunol Today. 1983, vol. 4, pp. 5-8; Janeway, C. , Ann. Rev. Immunol.. 1992, vol. 10, pp. 645-674; Rudd, C.E. et al., Immunol. Reviews. 1989, vol. Ill, pp. 223-266; Dalgleish, A.G. et al, Nature, 1984, vol. 312, pp. 736-766; Bedinger, P. et al., Nature, 1984, vol. 334, pp. 162-165). It is coupled to the protein-tyrosine kinase p56^lc (Rudd, C.E. et al., Proc. Natl. Acad. Sci fUSA.. 1988, vol. 85, pp. 5190- 5194; Barber, E.K. et al, Proc. Natl. Acad. Sci fUSA..

1989, vol. 86, pp. 3277-3281; Veillette, A. et al. , Cell. 1988, vol. 55, pp. 301-308; Rudd, C.E., Immunol. Today.

1990, vol. 11, pp. 400-406) an interaction necessary for an optimal response of certain T cells to antigen (Glaichenhaus, N. et al., Cell. 1991, vol. 64, pp. 511- 520; Abraham, N. et al., Nature, 1991, vol. 350, pp. 62- 66; Sleckman, B.P. , et al., J. Immunol.. 1988, vol. 141, pp. 49-54) . CD4-p56^lck is known to associate with and functionally synergise with the TcR/CD3 complex (Burgess, K.E. et al., Eur. J. Immunol.. 1991, vol. 21, pp. 1663- 1668; Eich ann, K. et al., Eur. J. Immunol.. 1987, vol. 17, pp. 643-650; Anderson, P. et al., J. Immunol.. 1987, vol. 139, pp. 678-682).

Infection by the Human Immunodeficiency Virus (HIV) is characterised by an impaired T cell response and a loss of CD4+T cells (Fauci, A.S., Science. 1988, vol. 239, pp. 617-622). CD4 binding to HIV gpl20 may partially induce this deficiency by processes such as anergy or apoptosis ( einhold, K.J. et al., J. Immunol.. 1989, vol. 142, pp. 3091, 3097; Groux, H. et al., . Exp. Med.. 1992, vol. 175, pp. 331-340), and in the absence of activation via the T cell receptor, the association of p56^lck with CD4 has been reported to transduce a negative signal (Haughn, L. et al. , Nature. 1992, vol. 358, pp. 328-331) . Early activation events have been reported to render T cells susceptible to HIV-1-induced formation of giant multi-nucleated cells referred to as syncytia

(Monhagheghpour, N. et al., J. Biol. Chem.. 1992, vol. 358, pp. 328-331) .

Summary of the Invention It has now been found that the CD4-p56^lck complex in T cells associates with two lipid kinases: PI 3-kinase and PI 4-kinase. The level of activity of these enzymes increases dramatically when CD4 is crosslinked on the cell surface either by HIV gpl20 or anti-CD4 antibody. Since PI 3-kinase, which phosphorylates phosphoinositol on the 3-position, and PI 4-kinase, which phosphorylates phosphoinositol on the 4-position, are known to be important in intracellular signalling in cells via the platelet derived growth factor receptor (PDGF-R) (Fant, .J. et al., Cell. 1992, vol. 69, pp. 413-423) and the epidermal growth factor receptor (EGF-R) (Cochet, C. et al., Biol. Chem.. 1991, vol. 266, pp. 637-644) pathways, respectively, the finding that these lipid kinases are functionally associated with CD4-p56^lck in T cells suggests that they are also involved in intracellular signalling via the T cell receptor complex.

The invention features a method of inhibiting or reducing signalling through the CD4-T cell receptor complex by blocking the physical association of lipid kinase with CD4-p56^lck. In one embodiment, the lipid kinase is a PI 3-kinase. In another embodiment, the kinase is a PI 4-kinase. The association of the lipid kinases with the CD4/p56^lc complex of mammalian T cells can be disrupted in a number of ways. In accordance with the present invention, it has been discovered that PI 3- kinase preferentially binds to a binding region located on the CD4/p56^lc complex. Thus, in one embodiment, the physical association of PI 3-kinase with CD4-p56^lck can be disrupted by blocking formation of the CD4/p56^lc complex. Complex formation can be blocked, for example, using a reagent, such as a peptide or an antibody, that binds to the CD4-binding region of p56^lck. A preferred reagent is a peptide corresponding to a fragment of the cytoplasmic domain of CD4, which spans amino acids 396-433, RCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI (SEQ ID NO: 1) . A fragment of CD4 or a fragment of CD8 with the sequences, KKTCQCPHRFQKT (SEQ ID NO: 2) or RRVCKCPRPWKS (SEQ ID NO: 3) , respectively, can be used to block assocation with p56^lc . Peptides with the following consensus sequences can also be used to block the interaction, KKXCXCPXXXXKT (SEQ ID NO: 4) and RRXCXCPXXXXKS (SEQ ID NO: 5). Similarly, complex formation can also be disrupted using a reagent, such as a peptide or antibody that binds to the p56^lck binding region of CD4. In this embodiment, the reagent is preferably a peptide corresponding to a CD4- binding fragment of p56^lck.

The regions of p56^lck that bind to the PI 3-kinase have also been identified in accordance with the present invention and are referenced herein as the SH2 and SH3 regions. In another method of the invention, the physical association of PI 3-kinase with the CD4/p56^lc complex can be disrupted by introducing into the T cell a peptide comprising an SH2 region, FFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKI RNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKP (SEQ ID NO 6) , or SH3 region,

QDNLVIALHSYEPSHDGDLGFEKGEPLRILEQSGE KAQSLTTGQEGFIPFNFVAK ANSLEPEP (SEQ ID NO: 7) of p56^lck or both. The physical association of PI 3-kinase with the CD4/p56^lck complex can also be disrupted by introducing into a T cell a peptide corresponding to the CD4/p56^lck-binding fragment of PI 3- kinase. In a preferred embodiment, the CD4/p56^lc binding fragment includes the sequence of amino acids, PPALPPK (SEQ ID NO: 8) of p85, or APALPPK (SEQ ID NO: 9) of p85(j0), two isoforms of a PI 3-kinase subunit.

In yet another embodiment of the invention, the physical association of a PI 4-kinase with the CD4/p56^lck complex is disrupted. This embodiment involves introducing into a mammalian T cell a reagent, such as a peptide, antibody, or Fab fragment of an antibody, that binds to the CD4-binding region of p56^lck, a reagent that binds to the p56^lc -binding region of CD4, or a reagent, such as a peptide corresponding to the CD4/p56^lc complex- binding fragment of PI 4-kinase. A sequence of PI 4- kinase that is related to SEQ ID NO: 8 and SEQ ID NO: 9 can be used to inhibit the interaction. This peptide can be identified by a proline-rich region, and in a preferred embodiment, would contain at least two prolines in a seven amino acid fragment, and preferably 3 or more. The invention can be used to treat mammals such as humans whose immune responses have become disregulated as a result of auto-immune disease or infection with HIV. The invention can also be used to suppress T cell- mediated rejection of transplanted organs, such as kidney, liver, bone marrow and pancreatic islets.

The therapeutic method of the invention specifies the introduction of an appropriate peptide into T cells of an animal, such as a human patient. This may be accomplished by administering the peptide to a patient in solution, allowing cells to passively take up the peptide by pinocytosis. Alternatively, one could introduce a nucleic acid encoding the peptide and appropriate regulatory elements into the cell. This method would allow the transcription and translation of the sequence into the desired peptide in the cytoplasm of the cell where the peptide can readily bind to its target proteins.

Another feature of the invention is a method of screening candidate compounds for the purpose of identifying compounds that inhibit the binding of PI 3- kinase to the CD4-p56^lck complex. In this assay, a T cell or another cell that expresses CD4-p56^lc and PI 3-kinase is contacted, in the presence of a candidate compound, with anti-CD4 antibody, HIV gpl20, or another CD4 ligand. To cross-link the cell surface CD4, the cells are contacted with secondary antibody, such as anti-gpl20 or rabbit anti-mouse antiseru . The cells are gently lysed and CD4-p56^lck and associated proteins immunoprecipitated with an antibody to one or more of the components of the complex. A decrease in the amount of PI 3-kinase in the complex in the presence of the candidate compound compared to the level in its absence is indicative that the candidate compound inhibits the association of PI 3- kinase with CD4-p56^lck. Similarly, a decrease in the amount of PI 4-kinase in a similar assay system using cells that express CD4-p56^lck and PI 4-kinase indicates that the candidate compound inhibits the binding of PI 4- kinase to CD4-p56^lck.

The invention also features a method of screening for candidate compounds that inhibit the activity of a PI kinase enzyme associated with CD4-p56^lck. To identify such inhibitory compounds, a cell that expresses the CD4- p56^lck complex and PI 3-kinase or PI 4-kinase is contacted with anti-CD4 or HIV gpl20 in the presence of a candidate compound. To cross-link the cell surface CD4, the cells are contacted with secondary antibody, such as anti-gpl20 or rabbit anti-mouse antiserum. The products of the enzymatic activity of PI 3-kinase and PI 4-kinase, phosphorylated PI 3 (PI 3-P) and phosphorylated PI 4 (PI 4-P) , respectively, can be measured, for example, using high pressure liquid chromatography (HPLC) analysis and/or thin layer chromatography (TLC) . A decrease in the amount of phosphorylated substrate indicates that the candidate compound inhibits the activity of the PI kinase, supporting its utility in modulating abnormal increases in the activity of these enzymes.

Brief Description of the Drawings Pig. IA and Pig. IB are autoradiographs of TLC analysis of the PI-P formed in anti-CD4 immunoprecipitates. PI kinase activity associated with CD4 is enhanced by antibody-induced CD4 crosslinking. Pig. IA. Untreated HPB-.ALL cells were lysed in a NP40/digitonin lysis buffer (0.5% each) and subjected to immunoprecipitation with rabbit anti-mouse antiserum (RαM) (lane 1), anti-CD4 (lanes 2 and 14), anti CD26

(lane 13) and anti-p56^lc (lane 15) . Alternatively, cells were pre-treated with anti-CD29 (lane 3) , anti-CD26 (lane 4), univalent anti-CD4 (H+L)_! (lane 5), univalent anti- CD4 (H+L) plus RαM (lane 6) or bivalent anti-CD4 (lanes 7 and 10) or bivalent anti-CD4 plus RαM (lanes 8 and 11) prior to precipitation. Addition of anti-CD4 (lanes 7 and 10) for six minutes at 37°C greatly enhanced the levels of precipitable PI kinase activity. Crosslinking of either univalent anti-CD4 (lane 6) or bivalent anti- CD4 (lanes 8 and 11) with RαM increased levels of precipitable activity. Anti-p85 precipitates from cell lysates served as a positive control (lane 12; shorter term exposure of film; see below) . Pig. IB. Left Panel: Effect of RαM-induced anti-CD4 crosslinking at 37°C on precipitable CD4-associated PI kinase activity. Anti-CD4 precipitates (lanes 1-3) ; anti-p56^lck precipitates from CD4 depleted cell lysates (lanes 4-6) . Times of incubation: 0 min (lanes 1, 4); 3 min (lanes 2, 5); 6 min (lanes 3, 6). The intermediate spot in lane 6 is a contaminating hot spot unrelated to the experiment. Right Panel: Kinetic analysis of the regulation of CD4:p56^lc -associated PI kinase activity. Lane 7 represents immune complexes obtained from HPB-ALL cells treated with anti-CD29 and crosslinked with RαM (0 min at 37°C) . Cells treated with anti-CD4 alone (0 min at 37°C) (lane 8) . Anti-CD4/RαM crosslinked samples correspond to: 0 min (lane 9) , 2 min (lane 10) , 5 min (lane 11) , 7.5 min (lane 12), 10 min (lane 13) and 12.5 min (lane 14).

Fig. 1C (left and middle) are immunoblots; Fig.lC (right) is a histogram of flow cytometry analysis. Pig. 1C. Left Panel: anti-p56^lc immunoblotting of CD4 precipitates during the time course of CD4 crosslinking with anti-CD4 and RαM. RαM control (lane 1) . Anti-CD4 and RαM (lanes 2-4) . Times of incubation: 0 min (lane 2) ; 3 min (lane 3) ; 6 min (lane 4) . Middle Panel: Anti- p85 immunoblotting of CD4 immunoprecipitates during the time course of incubation, as in Left Panel (lanes 5-9) . RαM control (lane 5) . Anti-CD4 plus RαM (lanes 6-8) . Times of incubation: 0 min (lane 6) ; 3 min (lane 7) ; 6 min (lane 8) . Anti-p85 (lane 9) . Right Panel: Flow cytometric analysis of the expression of CD4 receptor after anti-CD4 crosslinking. Fig. 2 is a graph of an HPLC analysis of deacylated lipids. Both PI 3- and PI 4-kinase activities associate with the CD4:p56^lck complex and are upregulated upon anti-CD4 antibody crosslinking. HPB-ALL cells at a density similar to that described for anti-CD4 crosslinking experiment (Fig. 1) were exposed to a mixture of anti-CD4 antibody (20μg/ml; 19Thy5D7; IgG2A) and rat anti-mouse antibody (0.2 μg/ml; Zymed, CA; IgG2a) at 37°C for various times. In contrast to the experiment described in Fig. 1, cells were shifted to 37°C at the same time as the addition of anti-CD4 and RαM. For the control, comparable amounts of anti-CD29 (4B4) and rat anti-mouse were used. Cells were lysed at the indicated times and analysed for CD4-associated PI kinase activity by TLC as described in Fig. 1. The corresponding PI-P spots were extracted, deacylated and subjected to HPLC analysis as described (Whitman, M. et al., Nature. 1985, Vol. 315, pp. 239-242; Auger, K.R. et al., Cell. 1989, Vol. 57, pp. 167-175). Control (+-+); anti-CD4 and RαM: 0.5 min (o-o) and 15 min (o-o) . Pi-p analysis corresponding to anti-p85 showed only the presence of PI- 3P; Lower panel (o-0) and the position of ³H-gPI-4P standard is indicated (o-o) .

Pig. 3A is a graph of a binding analysis of HIV-1 gpl20 as detected by anti-gpl20. Competition analysis of soluble HIV-gpl20 by anti-gpl20 antisera.

Pig. 3B is an autoradiograph of TLC analysis of CD4 precipitable PI kinase activity after gpl20 mediated crosslinking. Cells were treated for various periods of time with the following antibodies: goat anti-rabbit (lane 1); rabbit anti-gpl20 (lane 2); HIV-1 gpl20 and rabbit anti-gpl20 (lane 3); HIV-1 gpl20, rabbit anti- gpl20 and goat anti-rabbit (lane 4-8) Time of incubation: 0 min (lanes 4 and 6) ; 3 min (lanes 5 and 7) ; 6 min (lane 8) . Lanes 6 to 8 are from a separate experiment.

Fig. 3C is a graph of HPLC analysis of the deacylated reaction products. PI-P produced by immune complexes from cells treated with anti-gpl20 alone (♦-♦) : or HIV-1 gpl20, rabbit anti-gpl20 plus goat anti-rabbit (o-o) were deacylated and analysed by HPLC; [³H]-gPI-4P standard is indicated ( ) . CD4-associated PI kinase activity is also enhanced by HIV-1 anti-gpl20-mediated crosslinking of CD4 receptors. Pig 4A is an autoradiograph of TLC analysis of PI kinase activity associated with glutathione-S-transferase (GST) fusion proteins from HPB-ALL lysates. GST alone (lane 1); GST p56^lc SH2 (lane 2); GST p56^lck SH3 (lane 3); GST p56^lck SH2/SH3 (lane 4) and anti-p85 (lane 5). Pig. 4B is a graph of HPLC analysis of deacylated PI-P formed from precipitates of various GST fusion proteins. GST (OOO) , GST p56^lck SH2 (•••), GST p56^lc SH3 (o-o), GST p56^lck SH2/SH3 (o-o) . [³H]-gPI-P standard as indicated.

Detailed Description

In accordance with the present invention, it has been shown that the CD4:p56^lc complex of T cells associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis demonstrated that both PI 3-P and PI 4-P were formed in the lipid kinase reaction carried out on CD4-p56^lc -containing immune complexes, indicating that PI 3- and PI 4-kinases associate with CD4-p56^lck. Furthermore, the p85 subunit of PI 3-kinase was detected in the CD4-p56^lck-containing immune complex by immunoblotting with anti-p85 antiserum. CD4-p56^lck-associated PI 4-kinase activity was five-fold greater than PI 3-kinase activity. The association of these lipid kinases with the CD4-p56^lck complex was dependent on p56^lc binding to CD4, since precipitation of the kinase from CD4-depleted samples failed to show detectable PI kinase activity. Significantly, crosslinking with HIV gpl20 plus anti-HIV gpl20 and anti- CD4 plus RαM induced a 10-20-fold increase in levels of PI 3 and PI 4-kinase activity in anti-CD4 precipitates. These findings implicate the stimulation of CD4-p56^lc - linked PI kinases in HIV-induced defects and immune regulatory disorders.

Immunoprecipitation:

HPB-ALL (widely available cell line) cells (20 x 10⁶/ml) were solubilised in NP-40/Digitonin (0.5% (v/v) ) in 20mM Tris HC1, pH 8.3 containing 150mM NaCl, lmM PMSF (phenyl methlysulphonyl fluoride) , and immunoprecipitated with an excess of anti-CD4 antibody (19Thy5D7, 20 μg/ml) (Reinherz E. et al. Immunol. Rev. 1983 vol. 74, pp.83- 112) , as described (Rudd, C.E et al, Proc. Natl. Acad. Sci fUSA.. 1988, vol. 85, pp. 5190-5194; Barber, E.K. et al., Proc. Natl. Acad. Sci fUSA.. 1989, vol. 86, pp. 3277-3281) . Alternatively, the same number of cells in cold RPMI containing 2% fetal calf serum (FCS) were incubated with an excess of anti-CD4 antibody, or anti- CD26 (1F7, lOμg/ml) (Morimotto et al., J. Immunol. 1985, vol.134, pp.3762-3769, Morimoto et al. , J. Immunol. 1089, vol. 143, pp. 3430-3439, Rudd, C.E. , J. Exp. Med. 1987, vol. 166, pp. 1758-1773)or anti-CD29 (4B4, 10μg/mL) (Coulter Immunology, Florida) for 30 min followed by washing and incubation with rabbit anti-mouse antiserum (RαM) (2μg/ml) (DAKO Corp. Carpinteria, CA) for the same time. Cells were either lysed or further incubated with RαM (6μg/mL) for 30 min at the same cell density. After washing twice with ice cold medium, cells were resuspended in warm RPMI with 2% FCS and incubated for 5 min at 37°C. Activation through receptor crosslinking was arrested by diluting the cells with ice cold RPMI. Cells were centrifuged, washed twice with ice cold RPMI and solubilised in NP-40/Digitonin lysis buffer, as described (Rudd, C.E et al, Proc. Natl. Acad. Sci (USA.. 1988, vol. 85, pp. 5190-5194; Barber, E.K. et al., Proc. Natl. Acad. Sci (USA.. 1989, vol. 86, pp. 3277-3281). Lysates were centrifuged for 10 min at 15,000 x g. The cleared lysate was then incubated with Protein A Sepharose beads (Pharmacia) for 2 hours at 4°C under constant rotation. For a positive control, immunoprecipitations from cell lysates were carried out using anti-p85 rabbit antisera raised against the 85Kd subunit of PI 3-kinase. Immune complexes were washed thrice with PBS/1% NP-40, twice with lOOmM Tris/0.5M LiCl and twice with TNE (lOmM Tris-Hcl, pH 7.5, 150mM NaCl and lmM EGTA) . Lipid Kinase Assay:

The lipid kinase reaction was carried out on the incubated beads using phosphatidyl inositol and [³²Pγ]-ATP (20μCi) (Auger, K.R. et al., Cell. 1989, Vol. 57, pp. o 167-175) as described herein incorporated by reference.

Lipids were then extracted using chloroform and methanol

(1:1) and separated by thin layer chromatography on a silica gel plate precoated with potassium oxalate using a basic system (chloroform, methanol, water, ammonium hydroxide (60: 47, 11.3, 2). The plates were then removed dried, wrapped in a plastic wrap, and exposed to an x-ray film overnight at

-70°C, except for lane 12 which is a l/2h exposure.

Flow Cytometry:

HPB-ALL cells were treated with anti-CD4 plus RαM, as described. Cells were then suspended in pre-warmed RPMI and incubated at 37°C for the indicated times. Aliquots of the cells at the different time points were centrifuged, incubated with goat anti-rabbit-FITC

(Fisher) and assayed for CD4 surface expression by flow cytometry using an EPICS cell sorter (Coulter) . Immunoblot ing:

Cells were treated and immunoprecipitations carried out using Protein A Sepharose beads as outlined above. Cell lysates were further depleted of CD4 by sequential precipitation (3 times) using anti-CD4 antibody, followed by two preclearing steps using Protein A Sepharose. Depleted lysates were then subjected to precipitation using anti-p56^lc sera. The antisera were raised in rabbits against an amino acid terminal peptide (residues 39-64), RNGSEVRDPLVTYEGSNPPASPLQDN (SEQ ID NO: 10) , of p56^lc coupled to key hole limpet haemocyanin (KLH) . Lipid kinase assay was performed on the precipitates. Following transfer of the proteins to nitrocellulose, membranes were blocked with gelatin (2% w/v) and were probed with the anti-p56^lck rabbit antisera (1:1000). Immunoblots were visualized using goat anti- rabbit alkaline phosphatase system (Promega) . p85 of PI- 3 kinase was visualized by immunoblotting with anti-p85 rabbit sera (1:4500) and enhanced by chemiluminescence (ECL) Western blotting (Amersham) .

High Performance Liquid Chromatography (HPLC) Analysis.

Crosslinking of cell surface CD4 was conducted with anti-CD4 plus RαM for 6 min at 37°C. The immunoprecipitations with different antibodies and the lipid kinase assay were performed as described.

Deacylation of phospholipids and HPLC analysis were carried out as described herein incorporated by reference (Whitman, M. et al., Nature, 1985, Vol. 315, pp. 239-242; Auger, K.R. et al.. Cell. 1989, Vol. 57, pp. 167-175). [³H]-gPI-4-F standard was co-injected with the [³²P]- labelled samples in each HPLC analysis. Effects of gpl20 Binding:

Conditions of HIV gpl20 binding were established as previously described (Kaufmann, R. et al., J. AIDS. 1992, vol. 15, pp. 760-770). HIV gpl20 (ABT, Cambridge, MA) was radiolabelled with ¹²⁵I-Bolton Hunter reagent (NEN, Boston, MA) and incubated with 2 x 10⁵ cells for 2 hours. Free and bound ligand were separated by centrifugation of cells through silicon oil (specific density: 1.011 g/ml) . The bottom of the 300μl vials (Sarstedt) was cut off from measurement of bound radioligand. Non-specific binding was determined by performing the experiment in the presence of lOOnM soluble CD4. Anti-gpl20 serum blocks the binding of soluble gpl20, and binds, but does not dissociate CD4- bound gpl20. Computation of the binding parameters (equilibrium dissociation constant (Kd) , etc.) were determined as described (Kaufmann, R. et al., J. AIDS. 1992, vol. 15, pp. 760-770). For lipid kinase analysis, HPB-ALL cells were harvested and suspended at a density of 20 x 10⁶ cells/ml in ice cold RPMI (2% FCS) and rotated with recombinant gpl20 (ABT, Cambridge) derived from a baculovirus Sf/9 cell system at 4°C for 2 hours [concentration of native pure protein-1.0 x 10^-8M; Kd 10.06 x 10"⁸M] . Based on the measurement of the association kinetics, this procedure results in gpl20 binding to 10 percent of surface CD4 molecules. The cells were washed twice with ice cold RPMI (2% FCS) and treated with anti-gpl20 rabbit sera (1:100; ABT, Cambridge) for 1 h at 4°C. ^• A 1/100 dilution of rabbit anti-gpl20 results in antibody binding at 45-50% of gpl20-CD4 complexes on the cell surface. The cells were washed and treated with saturating amounts of goat anti- rabbit antibody (Sigma, 1:200) for 0.5 hour at 4°C. Cells were then incubated at 37°C for the indicated times. Following cell lysis, immunoprecipitations were carried out and subjected to the lipid kinase reaction. The reaction products were deacylated and analysed by HPLC as described above. GST fusion proteins.

DNA sequences encoding the SH2 (residues 127-234) , SH3 (residues 62-126) and SH2/SH3 (residues 62-234) of p56^lck tyrosine kinase were amplified by polymerase chain reaction (PCR) from a plasmid containing full length p56^lc cDNA (Koga Y. et al., Eur. J. Immunol. 1986, vol. 16, pp. 1643-1646), using specific 3' and 5' primers which included restriction sites for subcloning into the pGEX-2T vector (Pharmacia, Uppsala, Sweden) cut with Bam HI and Eco Rl. E. coli DH5α bacteria were used for transformation and expression of the fusion proteins (Smith, D.B. et al., Gene. 1988, vol. 67, pp. 31-40). Purity of individual preparations was confirmed by SDS- PAGE. Additional bacteria containing a plasmid encoding the GST p56^lck SH2 fusion protein was obtained (gift from Dr. Christopher Walsh; Dana-Farber Cancer Institute, Boston, MA) . HPB-ALL cell lysates were prepared as described above and incubated with the GST and GST fusion proteins (50 μg/ml of lysate) in the presence of fatty acid-free bovine serum albumin (BSA) (2.0 mg/ml) for 1 hour at 4°C, and 100 μl of a 50% suspension of glutathione beads (Pharmacia) pre-equilibrated in lysis buffer was added to these samples. The tubes were then rotated at 4°C for 10-15 min. The beads were washed thrice as described above. The lipid kinase reaction was carried out and lipids separated on TLC. PI-P were detected by autoradiography. PI-P spots were cut, extracted, deacylated and analyzed using HPLC. Results: To determine whether the CD4-p56^lc complex can associate with PI kinases, anti-CD4 precipitates from the leukemic T cell line HPB-ALL (Fig. IA) were assayed for the ability to generate monophosphorylated phosphoinositides (PI-P) from exogenously added lipids. A five minute incubation of intact HPB-ALL cells with either univalent (H+L) -_ or bivalent anti-CD4 antibody followed by immunoprecipitation resulted in the detection of significant amounts of PI kinase activity (Fig. IA, lanes 5-11) . As negative controls, incubation of cells with RαM (lanes 1 and 9), anti-CD29 (4B4) (lane 3) or anti-CD27 (1F7) (lanes 4 and 13) failed to co-precipitate PI kinase enzymatic activity. An antibody to the 85kDa subunit of PI 3-kinase served as a positive control (lane 12) . Anti-CD4 precipitation from cells that had not been pre-incubated with anti-CD4 precipitated some PI kinase activity (lane 2 and 14) . Anti-p56^lck precipitated low levels of activity (lane 15) , although consistently less than precipitated by anti-CD4 (lane 14) . Crosslinking of either univalent anti-CD4 (lane 6) or bivalent anti-CD4 (lanes, 8, 11) with RαM further increased levels of precipitable activity. Similar results were obtained using ether T cells lines and peripheral T cells.

We next attempted to determine changes in PI kinase activity over a time-course of anti-CD4 binding. Anti-CD4 crosslinking resulted in a significant time- dependent increase in precipitable lipid kinase activity, as demonstrated by the increase in PI-P formed (Fig. IB, lanes 1-3 and 7-14) . Kinetic analysis showed a transient increase in activity with maximal precipitable kinase activity at about 5 min. , followed by a gradual decrease (Fig. IB, lanes 9-14) ; under the same conditions, elevated activity was still detected at 15 min. Under the same conditions, surface CD4 underwent, if anything, a slight decrease in expression (Fig. 1C, right panel) . Similarly, the amount of co-precipitated p56^lck underwent no detectable change, as monitored by anti-p56^lc immunoblotting (Fig. 1C, left panel, lanes 1-4) . Immunoblotting using an antibody to the p85 subunit of PI-3 kinase positively identified co-precipitated p85 associated with CD4-p56^lck (Fig. 1C, middle panel, lanes 5-9) . Over the time-course of anti-CD4 crosslinking, there was no detectable increase in p85 binding to the CD4-p56^lck complex. Taken together, these data indicate that receptor ligation increases the activity of PI 3- kinase associated with CD4.

The small amount of activity precipitated by the anti-p56^lck serum (Fig. IA, lane 15) was readily lost by depletion of lysates using anti-CD4 antibody (Fig. IB, lanes 4-6) . These data indicate that PI kinase preferentially binds to the CD4-p56^lck complex.

HPLC analysis of the deacylated products of the anti-CD4 associated PI kinase confirmed the presence of PI 3-P and surprisingly, anti-CD4-p56^lc crosslinking also revealed the generation of significant amounts of PI 4-P (Fig. 2, upper panel). PI 4-P formation exceeded PI 3-P by some 2 to 5 fold. Anti-p85 precipitates showed exclusive labelling of the PI 3-P product (Fig. 2, lower panel) . Cross-linking of CD4-p56^lc complexes resulted in time-dependent increase in both PI 3 and PI 4 kinase activities (Fig. 2, upper panel). Both kinases showed a two to three fold increase from 0.5 to 15 min. of crosslinking (see legend to Fig. 2) .

A central finding of this study is that crosslinking of CD4-p56^lck induced by HIV gpl20 also increased associated PI kinase enzyme activity. Purity of HIV-1 gpl20 from Baculovirus Sf/9 cells (>80%) and native protein (15.7%) were assessed as previously described (Kaufmann, R. et al., J. AIDS. 1992, vol. 15, pp. 760-770) . Displacement studies showed gpl20 binding to a single binding site with Kd=1.06 x 10"⁸M (Kaufmann, R. et al., J. AIDS. 1992, vol. 15, pp. 760-770). Studies were conducted using concentrations of gpl20 designed to bind to a relatively low percent (approximately 10 percent) of CD4 surface receptors. This was followed by exposure of cells to a 1/100 dilution of rabbit anti- gpl20 designed to bind 50 percent of gpl20-CD4 complexes (Fig. 3A) . The combined exposure to gpl20 and rabbit anti-gpl20 precipitated moderate levels of PI kinase activity (Fig. 3B, lane 3) . Further crosslinking of the immune complexes with a saturating concentration of goat anti-rabbit serum (1/200 dilution) demonstrated that HIV- 1 gpl20 aggregation induced a time-dependent increase in precipitable PI kinase activity (Fig. 3B, lanes 4-8) . As controls, neither goat anti-rabbit, nor rabbit anti-gpl20 alone precipited activity (lanes 1,2). HPLC analysis of the products revealed the presence of increased levels of PI 3-P and PI 4-P compared to controls (Fig. 3C) . Although previous studies have raised controversy about the effects of HIV-1 gpl20-mediated CD4 crosslinking on p56^lc activity (Juszczak, R.J. et al., Biol. Chem.. 1991, vol. 266, pp. 11176-11183; Horak, I.D. et al., Nature. 1990, vol. 348, pp. 557-560; Kaufmann, R. et al. , J. AIDS. 1992, vol. 15, pp. 760-770), this data indicates that the crosslinking of HIV-1 gpl20 has a marked stimulatory effect on the activity of PI 3- and PI 4- kinases associated with the receptor.

The functional domains of p56^lck were evaluated for binding to lipid kinases. Mutations within the SH2 and SH3 regions of pp60^sro had previously been reported to influence PI 3-kinase binding to that protein (O'Brien, M.C. et al., Mol. Cell Biol.. 1990, vol. 10, pp. 2855- 2862) . GST fusion proteins containing the _g56^lck→ SH2 and SH3 domains were used to precipitate PI 3-kinase activity from cell lysates. Under these conditions, the SH3 domain precipitated high amounts of activity, with little observed in GST-SH2 precipitates (Fig.4A, lanes 3 and 2, respectively) . However, combined SH2/SH3 domains precipitated the greatest amount of activity (lane 4) . The adjacent SH2 domain increased the level of precipitable PI kinase activity from 3 to 8-fold. HPLC analysis demonstrated that SH3 and SH2/SH3 domains precipitated exclusively PI 3-kinase activity (Fig. 4B) . These data clearly show that PI 3-kinase bound to the SH3 domain of p56^lck and while the GST-p56^lck SH2 domain bound very little PI 3-kinase, adjacent SH2/SH3 domains precipitated the highest levels of activity, suggesting that the SH2 domain may cooperate with the SH3 domain, facilitating greater recognition of the kinase. The CD4-p56^lck-PI 3/PI 4-kinase interaction may influence the pathogenesis of HIV infectivity. HIV infection is characterised by the impaired function and loss of CD4+ T cells, an event that may be mediated by apoptosis or syncytia formation (Fauci, A.S., Science. 1988, vol. 239, pp. 617-622; Weinhold, K.J. et al. , J. Immunol.. 1989, vol. 142, pp. 3091-3097; Groux, H. et al., J. Exp. Med.. 1992, vol. 175, pp. 331-340). The yeast homolog of PI 3-kinase, Vps34 kinase, regulates membrane sorting and morphogenesis in that species (Hiles, I.D. et al., Cell. 1992, vol. 70, pp. 419-429;

Herman, P.K. et al., Mol. Cell. Biol.. 1990, vol. 10, pp. 6742-6754) . Crosslinking by HIV-1 gpl20 of a small percent of CD4 molecules on the cell surface (about 5%) was sufficient to allow the detection of a marked increase in associated PI 3 and PI 4-kinase activity in contrast to the observed effect of such crosslinking CD4- p56^lck activity (Horak, I.D. et al., Nature, 1990, vol. 348, pp. 557-560; Kaufmann, R. et al., J. AIDS. 1992, vol. 15, pp. 760-770). By altering lipid metabolism, these kinases may facilitate viral entry and inappropriately activate pathways involved in syncytium formation, or otherwise be detrimental to T cell signalling. The above results suggest a number of practical applications for the invention, discussed below in Examples 1-3.

Example 1: Peptide Therapy. The abnormal formation of syncytia and the inhibition of T cell growth that contribute to the depletion of T cells in patients infected with HIV may be treated by administering a peptide to block the interaction of a lipid kinase, such as PI 3-kinase or PI 4-kinase, with CD4-p56^lc . This peptide may be a fragment of the cytoplasmic domain of CD4 (e.g SEQ ID NO: 1,2,3,4, or 5) , a fragment of p56^lck (e.g SEQ ID NO: 6 or 7) , a fragment of PI 3-kinase (e.g. SEQ ID NO: 8 or 9) or a fragment of PI 4-kinase.

In the consensus sequences having the sequence of SEQ ID NO: 4 and SEQ ID NO: 5, "X" represents any amino acid, but is preferably an amino acid that is a conservative substitution of the corresponding amino acid in peptides having the sequence of SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Peptide sequences that can be used to block the association of PI 3-kinase to the CD4/p56^lc complex include peptides having the sequence of SEQ ID NO: 8 and SEQ ID NO: 9; however, other proline-rich peptides that bind to SH3 binding sequences can be used, such as a fragment of the 3BP1 protein that binds to the SH3 of the Abl kinase (Cicchetti et al, Science 1992, vol.257, pp. 803-806) , PPPLPPV (SEQ ID NO: 11) , or a sequence found in the SOS protein, PPPIPPRLA (SEQ ID NO: 12), or as sequence found in the Ras GAP protein, PPVPP (SEQ ID NO: 13) . Other such proline-rich regions can be readily ascertained by scanning the amino acid sequences of other appropriate protein tyrosine kinase-binding polypeptides. The term "fragment", as applied to a polypeptide, will ordinarily be at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering.

Also within the invention are analogs of the above peptides. Analogs can differ from the native peptides of CD4, p56^lck, PI 3-kinase and PI 4-kinase by amino acid sequence, or by modifications which do not affect the sequence, or by both. Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivitization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. The invention includes analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases. Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as a therapeutic. Such mimetics, and methods of incorporating them into polypeptides, are well known in the art. Similarly, the replacement of an L- amino acid residue is a standard way of rendering the polypeptide less sensitive to proteolysis. Also useful are aminc-terminal blocking groups such as t- butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, bensyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,- dinitrophenyl. Blocking the charged amino- and carboxy- termini of the peptides would have the additional benefit of enhancing passage of the peptide through the hydrophobic cellular membrane and into the cell.

Since the formation of T cell syncytia in HIV- infected patients has also been observed in the brain, modification of these peptides to improve penetration of the blood-brain barrier would also be useful. p . . . Polypeptides may be altered to increase lipophilicity

(e.g. by esterification to a bulky lipophilic moiety such as cholesteryl) or to supply a cleavable "targetor" moiety that enhances retention on the brain side of the barrier (Bodor et al., Science 1992, vol. 257, pp. 1698- 1700) . Alternatively, the polypeptide may be linked to an antibody specific for the transferrin receptor, in order to exploit that receptor's role in transporting iron across the blood-brain barrier (Friden et al., Science. 1993, vol. 259, pp. 373-377).

Peptides may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used, e.g. with liposomes. Such methods are well known to those of ordinary skill in the art. It is expected that an intravenous dosage of approximately 1 to 100 μmcles of the peptide of the invention would be administered per kg of body weight per day. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.

Since blocking the association of PI 3-kinase or PI 4-kinase with the CD4-p56^lc complex interferes with activation of T cells, this method may also be useful in downregulating the immune response in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) , type 1 diabetes, and rheumatoid arthritis. Treatment of autoimmune disease in this manner may be useful in other mammals subject to this condition, such as dogs. Suppression of the T cell-mediated immune response using this method may also be useful in the treatment of allograft or xenograft recipients to prevent rejection of a transplanted organ. Example 2: Gene Therapy. Also within the invention are isolated nucleic acid sequences that encode the peptides described above. An "isolated nucleic acid", as used herein, refers to a DNA or RNA sequence, segment, or fragment which has been purified from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g. , the sequences adjacent to the fragment in a genome in which it naturally occurs. The term includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.

The DNA or isolated nucleic acid of the invention may be introduced into target cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor- mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others.

The invention also includes cells transfected with the DNA of the invention. Standard methods for transfecting cells with isolated nucleic acid are well known to those skilled in the art of molecular biology. Preferably, the cells are T cells, and they express a peptide of the invention encoded by the nucleic acid of the invention.

A therapeutic composition is provided which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid, wherein the nucleic acid includes a promoter operatively linked to a sequence encoding a heterologous polypeptide, to generate high-level expression of the polypeptide in T cells transfected with the nucleic acid. The therapeutic composition may also include a gene delivery system as described above. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal.

As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the compounds of the invention will vary, but a preferred dosage for intravenous administration is from approximately 10⁶ to 10²² copies of the nucleic acid molecule. Example 3: Screens for therapeutically useful inhibitors. A screening method for identifying compounds capable of inhibiting the association of lipid kinases, such as PI 3-kinase and PI 4-kinase, with CD4-p56^lck can be carried out as follows:

The assay used is a two step procedure employing a cell that expresses CD4, p56^lck' and PI 3-kinase or PI 4- kinase. The cell is most preferably a T cell such as HPB-ALL, but may be any type of cell which expresses CD4 on its surface and p56^lck and the kinase of interest in its cytoplasm. The cell is incubated in the presence of a candidate compound. A reference point could be established under standard conditions and the results from any assay compared to the pre-established standard as the control. Cell surface CD4 is allowed to bind to its extracellular ligand, such as HIV gpl20 or anti-CD4 antibody, and may be crosslinked with secondary antibody such as anti-gpl20 antibody or RαM, respectively. Antibodies that can be used to bind and crosslink cell surface CD4 are widely available and include Leu3a, MT151, OKT4a, and OKT4. The complex is immunoprecipitated with Protein A Sepharose beads, subjected to SDS-PAGE under denaturing conditions and immunoblotted with antibody specific for PI 3-kinase or PI 4-kinase. Inhibition of association of PI 3-kinase or PI 4-kinase with CD4-p56^lck can be detected by the reduction of the corresponding band on the immunoblot compared to a standard or a to control assay carried out in the absence of a candidate compound. A method used to identify compounds capable of inhibiting the activity of PI 3-kinase and PI 4-kinase can be carried out as follows:

Cell surface CD4 is crosslinked with anti-CD4 antibody plus RαM, or HIV gpl20 plus anti-HIV gpl20, in the presence of a candidate compound, and immunoprecipitated as described above. The immunoprecipitated complex is assayed for lipid kinase activity using phosphotidyl inositol and γ(³²P)-ATP. Following the reaction, lipids are extracted, separated using TLC, and visualized using autoradiography. A reduction in amount of (³²P)PI-P detected on the plate compared to the amount observed in a control sample which was not exposed to the candidate compound indicates that the candidate compound inhibits the association of PI kinase with CD4-p56^lck. In order to determine whether the PI kinase activity being measured is that or PI 3-kinase or PI 4-kinase, the spots from the TLC plate may be extracted, deacylated and subjected to HPLC analysis. The candidate compounds can thus be evaluated with respect to their ability to reduce the amount of precipitatable PI-3P and PI-4P compared to a standard or control assay carried out in the absence of a candidate compound.

Other embodiments The invention also includes an ex vivo treatment of T cells from a patient, such as an HIV-infected patient. Peripheral blood T cells may be removed from the patient, transfected with a nucleic acid sequence encoding a fragment of the cytoplasmic domain of CD4, p56^lck, PI 3-kinase or PI 4-kinase, and reinfused into the patient. Cells treated in this manner would be resistant to the detrimental effects of gpl20-mediated crosslinking of CD4.

Also included is a method of treating an allograft, e.g. an organ such as a kidney or liver, by perfusing, soaking, or electroporating the organ with solution containing a nucleic acid sequence encoding a fragment of the cytoplasmic domain of CD4, or a fragment of p56^lck, PI 3-kinase or PI 4-kinase, prior to transplantation. Immunocompetent T cells in the treated organ would be suppressed, thus blocking the development of graft versus host disease in the transplant recipient. Other embodiments are within the following claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Christopher E. Rudd Prasad Kanteti Lewis Cantley

(ii) TITLE OF INVENTION: CD4 MEDIATED MODULATION OF LIPID KINASES

(iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: MS-DOS (Version 5.0)

(D) SOFTWARE: WordPerfect (Version 5.1)

( i) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

( ii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/023,915

(B) FILING DATE: February 26, 1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Janis K. Fraser

(B) REGISTRATION NUMBER: 34,819

(C) REFERENCE/DOCKET NUMBER: 00530/063001 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Arg Cys Arg His Arg Arg Arg Gin Ala Glu Arg Met Ser Gin lie Lys

1 5 10 15

Arg Leu Leu Ser Glu Lys Lys Thr Cys Gin Cys Pro His Arg Phe Gin

20 25 30

Lys Thr Cys Ser Pro lie 35

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Lys Lys Thr Cys Gin Cys Pro His Arg Phe Gin Lys Thr 1 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13

(B) TYPE: amino acid

(C) STRANDEDNESS: (D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 1 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Lys Lys Xaa Cys Xaa Cys Pro Xaa Xaa Xaa Xaa Lys Thr 1 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Arg Arg Xaa Cys Xaa Cys Pro Xaa Xaa Xaa Xaa Lys Ser 1 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 108

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Trp Phe Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gin Leu Leu

1 5 10 15 Ala Pro Gly Asn Thr His Gly Ser Phe Leu lie Arg Glu Ser Glu Ser

20 25 30

Thr Ala Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gin Asn Gin

35 40 45

Gly Glu Val Val Lys His Tyr Lys lie Arg Asn Leu Asp Asn Gly Gly

50 55 60

Phe Tyr lie Ser Pro Arg lie Thr Phe Pro Gly Leu His Glu Leu Val

65 70 75

80

Arg His Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg

85 90 95

Pro Cys Gin Thr Gin Lys Pro Gin Lys Pro Trp Trp 100 105

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Gin Asp Asn Leu Val lie Ala Leu His Ser Tyr Glu Pro Ser His Asp

1 5 10 15

Gly Asp Leu Gly Phe Glu Lys Gly Glu Pro Leu Arg lie Leu Glu Gin

20 25 30

Ser Gly Glu Trp Trp Lys Ala Gin Ser Leu Thr Thr Gly Gin Glu Gly

35 40 45

Phe lie Pro Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu

50 55 60

Pro 65 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Pro Pro Ala Leu Pro Pro Lys 1 5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Ala Pro Ala Leu Pro Pro Lys 1 5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Arg Asn Gly Ser Glu Val Arg Asp Pro Leu Val Thr Tyr Glu Gly Ser

1 5 10 15

Asn Pro Pro Ala Ser Pro Leu Gin Asp Asn 20 25

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Pro Pro Pro Leu Pro Pro Val 1 5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 12 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Pro Pro Pro lie Pro Pro Arg Leu Ala 1 5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Pro Pro Val Pro Pro 1 5

What is claimed is:

Claims

1. A method of inhibiting or reducing signal transduction through CD4, which method comprises introducing into a T cell a peptide which decreases the association of PI 3-kinase with CD4-p56^lck.

2. The method of claim 1, wherein said peptide comprises a p56^lc -binding fragment of the cytoplasmic tail of CD4.

3. The method of claim 1, wherein said peptide comprises SEQ ID NO: 4.

4. The method of claim 1, wherein said peptide comprises SEQ ID NO: 5.

5. The method of claim 1, wherein said peptide comprises a CD4-binding fragment of p56^lck.

6. The method of claim 1, wherein said peptide comprises the SH3 region of p56^lck (SEQ ID NO: 7) .

7. The method of claim 1, wherein said peptide comprises a fragment of PI 3-kinase that binds to the CD4/p56^lck complex.

8. The method of claim 1, wherein said peptide comprises SEQ ID NO: 8.

9. The method of claim 1, wherein said peptide comprises SEQ ID NO: 9.

10. The method of claim 1, wherein said T cell is in a mammal.

11. The method of claim 10, wherein said mammal is a human.

12. The method of claim 10, wherein said mammal has or is at risk of developing an autoimmune disease.

13. The method of claim 10, wherein said mammal is a transplant recipient.

14. The method of claim 10, wherein said mammal is infected with HIV.

15. A method of inhibiting or reducing signal transduction through CD4, which method comprises introducing into a T cell a peptide which decreases the association of PI 4-kinase with CD4-p56^lc .

16. The method of claim 15, wherein said peptide is a p56^lc -binding fragment of the cytoplasmic tail of CD4.

17. The method of claim 15, wherein said peptide comprises a CD4-binding fragment of p56^lck.

18. The method of claim 15, wherein said peptide comprises a fragment of PI 4-kinase that binds to the CD4/p56^lc complex.

19. A method of inhibiting or reducing signal transduction through CD4, which method comprises introducing into a T cell a nucleic acid encoding a peptide which decreases the association of PI 3-kinase with CD4-p56^lck.

20. The method of claim 19, wherein said peptide comprises a fragment of the cytoplasmic tail of CD4.

21. The method of claim 19, wherein said peptide comprises a fragment of p56^lc .

22. The method of claim 19, wherein said peptide comprises a fragment of PI 3-kinase.

23. A method of inhibiting or reducing signal transduction through CD4, which method comprises introducing into a T cell a nucleic acid sequence encoding a peptide which decreases the association of PI 4-kinase with CD4-p56^lc .

24. The method of claim 23 wherein said peptide comprises a fragment of the cytoplasmic tail of CD4.

25. The method of claim 23, wherein said peptide comprises a fragment of p56^lck.

26. The method of claim 23, wherein said peptide comprises a fragment of PI 4-kinase.

27. A method for screening candidate compounds to identify a compound capable of inhibiting the association of PI 3-kinase with CD4-p56^lc complex, said method comprising the steps of:

(a) providing a cell that expresses CD4-p56^lc complex and PI 3-kinase;

(b) contacting said cell with an anti-CD4 antibody or HIV gpl20 in the presence of a candidate compound;

(c) immunoprecipitating the CD4-p56^lck complex; and (d) determining the amount of PI 3-kinase in said immunoprecipitate, wherein a decrease in said amount in the presence of said candidate compound is an indication that said candidate compound inhibits the association of PI 3-kinase with CD4-p56^lck.

28. The method of claim 27 wherein said cell is a T cell.

29. A method for screening candidate compounds to identify a compound capable of inhibiting the association of PI 4-kinase with CD4-p56^lck complex, said method comprising the steps of:

(a) providing a cell that expresses CD4-p56^lc complex and PI 4-kinase;

(b) treating said cell with an anti-CD4 antibody or HIV gpl20 in the presence of a candidate compound;

(c) immunoprecipitating the CD4-p56^lck complex; and

(d) determining the amount of PI 4-kinase in said immunoprecipitate, wherein a decrease in said amount in the presence of said candidate compound is an indication that said candidate compound inhibits the association of PI 4-kinase with CD4-p56^lc .

30. A method for screening candidate compounds to identify compounds capable of inhibiting the activity of PI 3-kinase in cells, said method comprising the steps of:

(a) providing a sample comprising a cell that expresses CD4-p56^lck complex and PI 3-kinase;

(b) contacting said cell with anti-CD4 antibody or HIV gpl20 in the presence of a candidate compound; and

(c) determining the amount of phosphorylated PI 3 in said sample, wherein a decrease in said amount in the presence of said candidate compound is an indication that said compound inhibits the activity of PI 3-kinase.

31. A method for screening candidate compounds to identify compounds capable of inhibiting the activity of PI 4-kinase in cells, said method comprising the steps of:

(a) providing a sample comprising a cell that express CD4-p56^lc complex and PI 4-kinase;

(b) contacting said cell with anti-CD4 antibody or HIV gpl20 in the presence of a candidate compound; and

(d) determining the amount of phosphorylated PI 4 in said sample, wherein a decrease in said amount in the presence of said candidate compound is an indication that said compound inhibits the activity of PI 4-kinase.