WO1993023559A1

WO1993023559A1 - Alteration of the interaction of the cd4 t-cell receptor with an raf-1-related protein

Info

Publication number: WO1993023559A1
Application number: PCT/US1992/004061
Authority: WO
Inventors: Christopher E. Rudd; Prasad V. S. Kanteti
Original assignee: Dana-Farber Cancer Institute, Inc.
Priority date: 1992-05-13
Filing date: 1992-05-13
Publication date: 1993-11-25

Abstract

Methods for screening candidate compounds to identify compounds capable of inhibiting the naturally occurring association of the anti-Raf-1 precipitable 110 Kd phosphoprotein p110 and the CD4:p56lck T cell receptor complex, or capable of inhibiting intracellular signalling through this complex; and a substantially pure preparation of p110.

Description

ALTERATION OF THE INTERACTION OF THE CD4 T-CELL i RECEPTOR WITH 2»RAF-1-RELATED PROTEIN

This work was funded in part by grant no.CA 51887- 5 02 from the U.S. government, which has certain rights in the invention.

The field of the invention is T cell receptors.

Background of the Invention In the course of exogenous antigen-induced

10 activation of a T cell, the T cell receptor/CD3 complex (TcR/CD3) on the surface of the T cell interacts with the processed antigenic peptide bound to major histocompatibility complex (MHC) antigens on the antigen- presenting cell (Rudd et al., Immunol. Rev. 111:225-266,

15 1989) . This interaction is further strengthened by the binding of the T cell co-receptors, CD4 and CD8, which interact with the nonpolymorphic regions of MHC class II and I molecules, respectively (Clevers et al., Ann. Rev. Immunol. 6:629-662, 1988; Rudd et al., Immunol. Rev.

20 111:225-266, 1989; and Schwartz, Ann. Rev. Immunol. 3:237-261). CD4 is a 55 Kd integral membrane protein with a short cytoplasmic tail. In addition to its role in interacting with the MHC II molecule, CD4 also functions as the receptor for Human Immunodeficiency

25 Virus-I (HIV-I) (Dalgeish et al.. Nature 312:763-766, 1984; Klatzmann et al., Nature 312:767-768, 1984). CD4 and CD8 receptors also synergise with the T cell receptor complex (TcR/CD3) in the stimulation of T-cell growth (Anderson et al., J. Immunol. 139:678-682, 1987; Eichmann ^•*^■ 30 et al., Eur. J. Immunol. 17:643-650, 1987). Growth signals are likely mediated by the association of CD4/CD8 ^■■" and the TcR/CD3 complex with the protein-tyrosine kinases p56^lck and p59^fyn, respectively (Rudd et al., USSN 07/759,639, herein incorporated by reference; Barber et al., Proc. Natl. Acad. Sci. (USA) 86:3277-3281, 1989; Rudd et al., Proc. Natl. Acad. Sci. (USA) 86:3277-3281, 1988; Samelson et al., Proc. Natl. Acad. Sci. (USA) 87:4358-4362, 1990; and Veillette et al. , Cell 55:301- 308, 1988). Antibody-induced crosslinking of CD4 and TcR/CD3 induces the tyrosine and serine/threonine phosphorylation of numerous substrates (Abraham et al. , Nature 350:62-66, 1991; Baniyash et al., J. Biol. Chem. 263:18225-18230, 1988; Danielian et al., Eur. J. Immunol. 19:2183-2189, 1989; Hsi et al., J. Biol. Chem. 264:10836- 10842, 1989). Rapid tyrosine phosphorylation of certain substrates precedes the activation of second messengers such as phospholipase C (June et al., J. Immunol. 144:1591-1599, 1990). Activation ultimately results in stimulation of the phosphatidyl inositol pathway leading to the activation of protein kinase C, increased free intracellular calcium, and the activation of various proto-oncogenes (Alcover et al. , Immunol. Rev. 95:5-36, 1988; Crabtree et al.. Science 243:355-361, 1989). Recent studies have indicated that protein- tyrosine kinases interact physically with the intracellular molecules which serve as substrates. This biochemical property has provided a strategy for uncovering enzyme/substrate interactions within the tyrosine kinase cascade. Much of the interaction is mediated by src-homology (SH) regions (SH2/SH3) within these proteins and is dependent on tyrosine phosphorylation (Koch et al.. Science 252:668-674, 1991). pp60^src interacts with the SH region of phosphotidyl inositol kinase (PI-3) (Cantley et al.. Cell 64:281-302, 1991) . Likewise, the platelet-derived growth factor receptor (PDGF-R) and the epithelial cell growth factor receptor (EGF-R) interact with SH2/SH3-bearing intracellular molecules such as phospholipase C (PLC-7) , GTPase activating protein (GAP) and phosphoinositol kinase (PI-3 kinase) (Cantley et al., Cell 64:281-302, 1991) . Tyrosine phosphorylation of PLC-₇ acts to regulate its enzymatic activity (Heidecker et al. , Mol. Cell. Biol. 10:2503-2512, 1990; Kim et al. , Cell 65:436- 441, 1991).

In addition to tyrosine-mediated signaling, serine and threonine kinases such as protein kinase C (PKC) play important roles in growth regulation. Various isoforms of PKC are thought to phosphorylate substrates such as CD4 and p56^lck in T cells (Acres et al., Biol. Chem.

261:16210-16214, 1989; Blue et al., J. Immunol. 139:3949- 3954, 1987; Hurley et al., Science 245:407-409, 1989; Rudd et al., Immunol. Rev. 111:225-266, 1989; and Rudd et al., "Molecular analysis of the interaction of p56^lck with the CD4 and CD8 antigens." In: Mechanisms of Lymphocyte Activation and Immune Regulation III. Gupta et al. (eds) , Plenum Press, New York, pp 85-89, 1991). Engagement of TcR/CD3 complex on T cells (Siegel et al. J. Biol. Chem. 265:18472-18480, 1990; Zmuidzinas et al., Mol. Cell. Biol. 11:2794-2803, 1991), as well as stimulation by interleukin 2 (IL-2) (Turner et al., Proc. Natl. Acad. Sci. (USA) 88:1227-1231, 1991), has been reported to stimulate Raf-1 kinase activity in these cells, an event which appears to require protein kinase C. The 70-72 Kd Raf-1 kinase is the cellular homologue of v-raf, the transforming gene of murine sarcoma virus 3611 (Rapp et al., Proc. Natl. Acad. Sci. (USA) 80:4218- 4222, 1983). The amino terminal region of Raf-1 shares homology with protein kinase C (Stanton et al., Mol. Cell. Biol. 9:639-647, 1989). V-Raf transforms murine 3T3 fibroblasts, and transfection with a c-Raf gene having a 5' deletion results in the induction of DNA synthesis in serum-starved fibroblast cells, indicating an important role for Raf in cell cycle progression (Heidecker et al., Mol. Cell. Biol. 10:2503-2512, 1990; Kolch et al.. Nature 349:426-428, 1991; Smith et al., Mol. Cell. Biol. 10:3828-3833, 1990; Stanton et al., Mol. Cell. Biol. 9:639-647, 1989; Tahira et al., Nucl. Acids Res. 15:4809-4820, 1987). In fibroblasts, PDGF binding results in a rapid Tyr/Ser phosphorylation of Raf, increased catalytic activity of Raf, and a physical association of Raf with the PDGF-R (Morrison et al. , Proc. Natl. Acad. Sci. (USA) 85:8855-8859, 1989; Morrison et al. , Cell 58:649-657, 1989). This interaction, which is mediated by unknown residues within Raf, provides a potential link between the tyrosine kinase and serine/threonine kinase signalling pathways in fibroblasts (Li et al. , Cell 64:479-482, 1991; Morrison et al.. Cell 58:649-657, 1989). Unlike other receptors in T cells, Raf-1 does not associate physically with the

TcR/CD3 complex (Siegel et al., J. Biol. Chem. 265:18472- 18480, 1990) .

Summary of the Invention The CD4 and CD8 antigens on T cells have been previously shown to associate with the src family member p56 ^k and a novel GTP-binding, 32 Kd protein termed p32. The identification of receptor interactions with intracellular mediators is essential to the elucidation of downstream signals mediated by engagement of these receptor complexes. It has now been found that a previously undetected llOKd polypeptide, herein termed "pllO", is associated with the CD4:p56^lc complex in human peripheral blood T lymphocytes and certain leukemic T cell lines. As described in detail in the experimental section below, pllO is recognized directly by an antiserum to the C-terminal region of the 70-72 Kd serine/threonine kinase Raf-1, and appears identical to a 110 Kd protein detected in anti-Raf-1 immunoprecipitates formed using the same antiserum. Despite its association with CD4:p56^lck complex, pllO was found to be phosphorylated predominantly on serine residues. Furthermore, phorbal ester treatment of cells resulted in a transient increase in the detection of pllO associated with CD4:p56^lc , concomitant with the modulation of

CD4:p56^lck from the cell surface. This Raf-1-related pllO is therefore likely to play a role in signals generated from the CD4:p56^lck complex.

The invention features methods for screening candidate compounds to identify compounds capable of inhibiting the naturally occurring association of pllO and the CD4:p56^lck T cell receptor complex. One such method involves the steps of:

(a) providing a first and a second sample of cells expressing both pllO and the CD4:p56^lck T cell receptor complex;

(b) incubating the first sample in the presence of a candidate compound;

(c) incubating the second sample in the absence of the candidate compound;

(d) generating a first immunoprecipitate by adding to the first sample a first aliquot of an antibody capable of specifically binding pllO (i.e., which forms an immune complex directly with pllO) : for example, an anti-raf-1 antibody or antiserum, or an antibody raised against pllO or an immunogenic fragment thereof;

(e) generating a second immunoprecipitate by adding to the second sample a second aliquot of the same antibody; and (f) determining whether the amount of CD4:p56^lck T cell receptor complex present in the first immunoprecipitate is less than the amount of CD4:p56^lc T cell receptor complex present in the second immunoprecipitate, the presence of a lesser amount of lek CD4:p56 T cell receptor complex in the first immunoprecipitate than in the second immunoprecipitate indicating that the candidate compound inhibits the association. Alternatively, the assay can be carried out using an antibody which specifically binds a component of the CD4:p56^lck T cell receptor complex (e.g., an anti-CD4 or anti-p56^lck antibody) ; in such an assay, the amount of pllO present in each immunoprecipitate is determined, with the presence of a lesser amount of pllO in the first immunoprecipitate than in the second immunoprecipitate indicating that the candidate compound inhibits the association. In a variation on this method, the ability of a compound to inhibit intracellular signalling through the CD4:p56^lck T cell receptor complex is determined by

(b) incubating the first sample in the presence of a candidate compound;

(c) incubating the second sample in the absence of the candidate compound;

(d) generating a first immunoprecipitate by adding to the first sample a first aliquot of an antibody capable of specifically binding to a component of the lcl

CD4:p56 T cell receptor complex or to pllO; (e) generating a second immunoprecipitate by adding to the second sample a second aliquot of the antibody; and

(f) determining the level of serine/threonine kinase activity in the first immunoprecipitate compared to the level of said activity in the second immunoprecipitate, a lower level in the first immunoprecipitate being an indication that the candidate compound is capable of inhibiting intracellular signalling through the CD4:p56^lck T cell receptor complex. This activity can be determined by using an exogenous substrate polypeptide having a serine or threonine residue available for phosphorylation, or by measuring autophosphorylation of serine/threonine residues on the immunoprecipitated pllO itself. Candidate compounds which might be used in the above methods include, but are not limited to, peptide fragments of, or compounds which include an amino acid sequence corresponding to a portion of, pllO, p56 , or the cytoplasmic tail of CD4. A "fragment" or "peptide fragment" of a protein is defined as a peptide having a sequence corresponding to a portion of that protein. Such peptides may consist entirely of amino acids linked by peptide bonds, or they may have one or more of such peptide bonds replaced with another type of covalent bond, termed a "nonpeptide bond", which renders the peptide impervious to proteolytic cleavage at that point. Such nonpeptide bonds are widely known in the art, as they have been shown to be useful for stabilizing peptides against proteolytic attack while making little difference to the overall conformation of the peptide. Those peptides found to be capable of disrupting or otherwise interfering with the naturally occurring binding interaction between pllO and CD4:p56^lck complex could be used in a analytical or therapeutic method that would include the step of contacting the T cell receptor complex with such a peptide.

The invention also includes a substantially pure preparation of pllO (i.e., a preparation at least 50% of the protein component of which is pllO) , and a method for isolating or purifying pllO, which method includes the steps of

(a) contacting a biological sample containing pi10 with an antibody which binds to pllO to form an antigen- antibody complex; and (b) separating the antigen-antibody complex from the remainder of the biological sample. Alternatively, the method can be carried out on a sample which contains both pllO and CD4:p56^lck, using an antibody which binds to a component of the CD4rp56^lc complex.

Also within the invention are an isolated DNA encoding pllO, a plasmid containing such a DNA (which plasmid may also contain an expression control sequence capable of directing expression of pllO from the DNA encoding pllO) , and a cell containing the isolated DNA. The pllO so encoded may correspond to human, mouse, rat, pig, cow, or any other animal's pllO which can be detected by the methods disclosed herein. By "isolated DNA encoding pllO" is meant a DNA sequence that is free of the genes which, in the naturally-occurring genome of the organism from which the isolated DNA of the invention is derived, flank the pllO gene. The term therefore 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, a synthetic DNA, or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Substantially purified pllO, or antigenic fragments thereof which are not identical to any portion of 70-72 Kd Raf-1, may be used to immunize an animal and thereby generate an antibody (polyclonal or monoclonal) which binds preferentially to pllO compared to 70-72 Kd Raf-1: i.e., the antibody binds to the latter protein, if at all, with an affinity substantially less than the affinity with which it binds pllO. Such an antibody would be useful in methods for purifying pllO; for interfering with the association of pllO and the CD4:p56^lck complex in T cells by contacting such cells with the antibody; for determining the amount of pllO in a sample by contacting the sample with the antibody and determining the amount of antigen-antibody complex formation in the sample; and for determining the level of pllO kinase activity in a sample by contacting the sample with the antibody to form an immunoprecipitate, and determining the level of serine/threonine kinase activity in that immunoprecipitate. Either of the latter two methods could be employed as a diagnostic technique for certain conditions characterized by abnormal signal transduction via the CD4:p56 :pll0 pathway (e.g., a cancerous or precancerous condition, an autoimmune disease, or a human immunodeficiency virus (HIV) infection) . In such a case, the biological sample would include T cells obtained from an animal (e.g., a human) suspected of having such a condition, and the amount of pllO or the level of pllO kinase activity in the sample would be compared to the amount or level in a normal sample, with an abnormal amount or level being an indication that the animal has the condition.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description The drawings are first described. Drawings

Fig.l is an autoradiogram of proteins from each of the indicated cell lines, immunoprecipitated with the indicated antibody, phosphotransferase labelled with ³²P, and analyzed by SDS-PAGE. Cells examined were of the human cell lines HPB-ALL (lanes 1-5) , REX (lanes 9-11) , M0LT15 (lanes 12-15) , peripheral blood lymphocytes (PBL) (lanes 6-8) , and the murine T cell hybridoma F6 (DC27 transfected with CD8extra/CD4cyto) (lane 17) . Antibodies and antisera include rabbit anti-mouse (RaM) (lanes 1, 6, 9, 12, 16); 19Thy5D7 (anti-human CD4) (lanes 2, 7, 13); 21Thy2D3 (anti-human CD8) (lanes 3, 14); RW28C8 (anti- human CD3) (lane 4); anti-Raf serum (lanes 5, 8, 11, 15, 19) ; T1B-105 (anti-murine CD8) (lane 17) ; and anti-p59^fyn sera (lane 18) .

Fig. 2A is an autoradiogram of proteins from solubilized PMA-treated (100 μg/ml) HPB-ALL cells. The cells were immunoprecipitated with rabbit anti-mouse (left panel) , anti-CD4 (middle panel) , and anti-Raf sera (right panel) .

Fig. 2B is an analysis by flow cytometry of PMA- induced down regulation of CD4. An aliquot of cells (5 x 10⁵) was withdrawn at various times from the cell culture used for Fig. 2A and reacted with the monoclonal antibody 19Thy5D7, followed by fluorescein-conjugated goat anti- mouse antibody. Mean channel fluorescence intensity was used to calculate the relative density of CD4 receptors on the cell surface.

Fig. 3 is a set of two-dimensional phosphoamino acid analyses of (Fig. 3A) pllO present in Raf-1 immunoprecipetates; (Fig. 3B) pllO associated with CD4:p56^lck; and (Fig. 3C) p56^lck. Extracted gel-purified proteins were digested with 6 N HCl, and the phosphoamino acids were separated by 2D thin layer electrophoresis on cellulose plates. P-Y, P-S and P-T represent phosphotyrosine, phosphoserine and phosphothreonine respectively.

Fig. 4 is an autoradiogram of proteins from HPB- ALL cells treated with PMA (100 ng/ml) , lysed, immunoprecipitated with the indicated antibody, phosphotransferase labelled with ³²P, and analyzed on SDS- PAGE. Anti-CD26 (Ctl 1) (lane 1), anti-CD27 (Ctl 2) (lane 2) , and rabbit anti-mouse (Ctl) (lane 8) antibodies were used as negative controls. Other antibodies include anti-Raf (lane 3) and anti-CD4 antibodies 19Thy5D7 (lane 4), Leu3A (lane 5), T119 (lane 6), and MT151 (lane 7). An excess of recombinant soluble CD4 (2 mg/ml) successfully prevented anti-CD4 (19Thy5D7) precipitation of p56^lck and pllO (lane 9 vs lane 10) .

Fig. 5 is a two-dimensional tryptic peptide map comparison of anti-CD4- and anti-Raf-precipitated pllO. Phorbol ester-treated HPB-ALL cells were solubilized and immunoprecipitated with antiCD4 (19Thy5D7) or anti-Raf antiserum. Following kinase labeling, the samples were separated on SDS-PAGE and the radiolabeled proteins were subjected to digestion with trypsin. Peptides were then separated by two-dimensional phosphopeptide mapping.

Fig. 5A: Tryptic phosphopeptide map of the pllO present in anti-Raf-1 immunoprecipitate. Fig 5B: Tryptic phosphopeptide map of pllO associated with the CD4:p56^lc complex. Fig. 6 is an autoradiogram of proteins immunoprecipitated by anti-Raf (lanes 1-6) or an i-CD4 (lanes 7 and 8) antibodies, subjected to in vitro phosphotransferase labeling with [<₇-³²P]-ATP, denatured in 1% (wt/vol) SDS, diluted 10-fold in lysis buffer, and subjected to re-precipitation using rabbit anti-mouse (lanes 1,4,8,9) or affinity-purified anti-Raf serum (lanes 2,3,5,6,7). In some cases, reprecipitation was conducted in the presence of the Raf peptide (lmg/ml) to which the serum was raised (lanes 3,6). Also shown is an SDS-PAGE analysis of a labelled pllO cut from an anti- Raf lane, extracted from the gel, and re-precipitated with anti-Raf-1 antiserum in the absence (lane 10) or presence (lane 11) of Raf peptide.

Preparation and use of pllO As described in detail herein, an anti-Raf-1- precipitable llOKd phosphoprotein herein termed pllO has been identified by applicants as a integral component of the CD4:p56^lc complex in T cells. This information can be exploited in a number of useful ways. Substantial amounts of purified naturally occurring pllO can be obtained from T cells by appropriate modifications to the procedures described below, including the use of anti- Raf, anti-CD4, or anti-p56^lck as an immunoaffinity reagent. Once the amino acid sequence of pllO has been determined by standard sequencing techniques, pi10 or fragments thereof may be produced by chemical synthesis. Alternatively, the gene or cDNA encoding the protein may be cloned as described below, and recombinant pllO and fragments and analogs thereof produced in substantial quantities. Once either the gene encoding pllO or significant amounts of the polypeptide itself are available, pllO and fragments or analogs thereof may be analyzed for their ability to interfere with infection and/or activation of T cells. The active site and/or ligand binding site(s) on pllO can be determined by methods analogous to those utilized by Veillette et al. (Eur. J. Immunol. 20:1397-1400, 1990), Turner et al. (Cell 60:755-765, 1990), Shaw et al. (Mol. Cell. Biol. 10:1853-1862, 1990) and Glaichenhaus et al. (Cell 64:511- 520, 1991); analogs of pllO may then be produced which are deficient in either or both functions. Inhibitors of the activity of pllO can be utilized to reduce the susceptibility of T cells to viral infection via the CD4 antigen, or to reduce the susceptibility of a human to cancer formation or the development of autoimmune disease. Such inhibitors may act directly on pllO, or on CD4, p56^lck, or a kinase or phosphatase which acts on any of the three. Cloning and expression of pllO cDNA

A cDNA encoding the pllO polypeptide may be obtained as follows:

HPB-ALL cells stimulated with PMA are used to prepare a cDNA library by standard methods. The library is screened at low stringency with a hybridization probe prepared from c-Raf cDNA (Bonner et al.. Nucleic Acids Res. 14:1009, 1986). Positive clones are then expressed in an expression system such as COS cells cotransfected with CD4 cDNA and p56^lck cDNA, and anti-CD4- immunoprecipitates prepared as described below are analyzed on SDS-PAGE for the presence of a 110 Kd phosphoprotein. Verification that a given clone is pllO cDNA can be accomplished by, for example, a competition assay in which a CD4:p56^lck:pllO complex containing labelled pllO is exposed to excess unlabelled putative recombinant pllO. Alternatively, a cDNA may be obtained by screening a cDNA library with a degenerate hybridization probe based upon a partial amino acid sequence of purified pllO, which may be obtained from SDS-PAGE fractionation of anti-raf or anti-CD4 immunoprecipitates, prepared as described in the experimental section below.

The cDNA encoding pllO may then be incorporated into an expression system such as the pGex-2T expression system (Smith et al.. Gene 67:31, 1988) or a baculo expression system using the vector Xpress System vector (Invitrogen Corp, CA) in SF9 cells, in order to produce relatively large quantities of recombinant pllO polypeptide. After purification of the recombinant protein by standard methods using, for example, heparin and DEAE chromatography, or an affinity column (e.g., an anti-Raf affinity column) , the purified polypeptide may be incorporated into an in vitro assay to screen for potential agonists and antagonists of pllO. Alternatively, the recombinant pllO may be proteolytically digested (e.g., with trypsin) or treated with CNBr to form discrete fragments that can then be purified (by, for example, high performance liquid chromatography [HPLC]) and assayed for their ability to inhibit the interaction of pllO and the CD4:p56^lck complex. Such an assay may be carried out as an in vitro reconstitution assay, in which the ability of purified or semi-purified, natural or recombinant CD4, p56^lck and pllO to associate in a complex is determined both in the presence and in the absence of a given pllO fragment. Such an assay may require the use of membrane-bound CD4 to properly mimic cellular conditions. Alternatively, a CD4 peptide which corresponds to either the cytoplasmic tail of CD4, or CD4 minus only its membrane-spanning amino acid residues, may be substituted for holo CD4 in this assay. The assay for active fragments of pllO may also be carried out in cells which express either natural or recombinant CD4, p56^lck, and pllO. Such cells may be rendered permeable to the peptide fragments by standard methods.

Other means for producing the pllO peptides of the invention include chemical synthesis and genetic engineering of pllO cDNA: e.g., deleting appropriate portions of the coding sequence, or inserting a nonsense codon at an appropriate site, and expressing the modified cDNA in a standard expression system. The desired polypeptide fragment is then purified by standard means and tested in the assay described above. Alternatively, the pllO cDNA may be engineered to encode an analog of pllO which binds to the CD4:p56^lck complex in a manner that blocks wildtype pllO, but which is incapable of carrying out the intracellular signalling function of wildtype pllO. Such a mutant, when transfected into a T cell, would compete with the T cell's naturally occurring pllO, and would diminish the cell's ability to transmit signals through the CD4:p56^lc :pllO pathway. These mutants would be generated by standard means, such as by site-directed mutagenesis or restriction enzyme digestion, and coexpressed with CD4 and p56^lck in pllO-minus cells. The cells could then be assayed for changes in patterns of serine, threonine and/or tyrosine phosphorylation; alterations in proliferative ability or lymphokine production; or effects on the helper function of CD4+ cells.

Structure/function information published with respect to two related molecules, c-raf and v-raf, would provide guidance to one of ordinary skill in the art as to what residues of pllO to target for mutagenesis. pllO cDNA may be co-expressed at high levels with CD4 cDNA and p56^lck cDNA in an app: priate cellular expression system, such as COS cells or baculoviral SF^* cells, and preparations containing all three recombinε.rfc proteins then utilized in a cell-based or cell-free assay for agents which inhibit the association of the three proteins, or the phosphorylation or dephosphorylation of any of the three. Such a cell-based assay might be designed to detect changes in the modulation of CD4 from the cell surface, which may influence the ability of CD4 to associate with adjacent antigens, cause syncytia formation relating to HIV-1 infection, and/or alter epitope expression of CD4, thereby affecting binding by anti-CD4 antibodies or gpl20 of HIV-1. The assay would involve detection of such changes by the use of a technology such as immunofluorescence, enzyme-linked immunoadsorbent assay (ELISA) , or radioimmunoassay, or by measuring cell-cell adhesion caused by the binding of CD4 to MHC antigens. Alternatively, a cell-free, in vitro assay may be used, such as those described herein with or without cellular membranes. The level of expression of pllO in T cells may correlate with a particular disease state, and thus be a diagnostic indicator for that disease state. The normal level of expression of pllO, and the levels in T cells affected with such conditions as HIV infection, autoimmune disease, and T cell leukemias, can be measured by any standard method, including antibody-based technologies and Northern blots. For the latter, a hybridization probe consisting of a segment of pllO cDNA at least 20 nucleotides (preferably at least 40, and more preferably at least 80 nucleotides) in length would be used under stringent conditions.

Experimental Data MATERIALS AND METHODS Chemicals

Nonidet P-40 (NP-40) , digitonin, phorbol 12- myristate 13-acetate (PMA) , phenyl methyl sulfonyl fluoride (PMSF) and TPCK-trypsin were obtained from Sigma (St. Louis, MO, USA) and [₇-³²P]-ATP (specific activity 3000 Ci/mol) was from NEN (Billerica, MA, USA) . SDS— polyacrylamide gels were prepared from pre-mixed acrylamide and bisaσrylamide from Protogel, National Diagnostics (Manville, NJ, USA) . Protein A Sepharose CL beads and Ficoll-Paque were obtained from Pharmacia (Piscataway, NJ, USA) , Staphylococcus aureus (SAC;

Pansorbin) was purchased from Calbiochem (San Diego, CA, USA) . Recombinant soluble CD4 (RsCD4) (extracellular region) was obtained from Biogen (Boston, MA, USA) . All other reagents used were of analytical grade quality. Monoclonal antibodies and antisera

Rabbit anti-mouse was obtained from Dako Corporation (Carpinteria, CA, USA) . Details regarding 19Thy5D7, a monoclonal antibody against CD4, have been described (Reinherz et al. , Immunol. Rev. 74:83-112, 1983). The other anti CD4 antibodies used (T119, Leu 3A, and MT151) were obtained from the Fourth International Workshop on Human Leucocyte Differentiation Antigens (Vienna, 1989) . 4B4 (anti-CD29) and 1F7 (anti-CD26) have been previously reported (Morimoto et al., J. Immunol. 134:3762-3769, 1985; Morimoto et al., J. Immunol. 143:3430, 1989; Rudd et al., J. Exp. Med. 166:1758-1773, 1987) . Rabbit anti-Raf antiserum was generated against a synthetic peptide (CTLTTSPRLPVF) (SEQ ID NO:l) corresponding to amino.acid residues 315-326 of v-Raf, and was affinity-purified according to Schultz et al. (Virology 146:78-89, 1985). Cells

Cells used included the T lymphoblastoid cell lines HPB-ALL, REX and MOLT-15. F6 is a mouse hybridoma cell line derived from DC 27 cells that were transfected with DNA encoding the CD8α/CD4 chimeric molecule (CD8α extracellular/transmembrane regions linked to CD4 cytoplasmic tail) (Zamoyska et al.. Nature 342:278-281, 1989) . The cells were cultured in RPMI-1640 medium containing fetal bovine serum (10%) , L-glutamine (2 mM) , penicillin (50 U/ml), and streptomycin (50 mg/ml) at 37°C and 5% C0₂. F6 cells were growth-selected in RPMI medium supplemented with G418 at 1 mg/ml. Peripheral blood lymphocytes were isolated by Ficoll-Paque centrifugation from leucopaks of normal human donors (Dana-Farber Cancer Institute blood bank) .

Immunoprecipitation and kinase assay

50xl0⁶ cells were lysed using a mixture of non- ionic detergents [NP-40 (0.5% wt/vol), digitonin (0.5% wt/vol), 20 mM Tris HCl pH 8.3, 150 mM NaCl, l mM PMSF] for 30 min at 4°C (Rudd et al., Proc. Natl. Acad. Sci. (USA) 86:3277-3281, 1988). The lysate was then centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was precleared with 100 μl of SAC (10% suspension in the lysis buffer) . Immunoprecipitation was then carried out by incubation of the lysate with the antibody for 2 h at 4°C, followed by further incubation with 50 μl of protein A-Sepharose beads (10% wt/vol) for 1.0 h. Im unoprecipitates were washed 3 times with ice cold lysis buffer, suspended in 30 μl of kinase buffer (lOmM HEPES, pH 7.0-7.2, 3.5 mM MgCl₂, 3.5 mM MnCl_j) containing 10-20 μCi of [₇-³²P]-ATP, and incubated at room temperature for 10 min. The samples were then subjected to SDS-PAGE analysis (Laemmli, Nature 227:680-685, 1970), and the phosphorylated proteins were visualized by autoradiography. Certain gels were treated with 1 M KOH for 2 h at 55^βC, to remove the alkali-labile phosphate groups from ser/thr phosphorylated proteins (Cooper et al., Meth. Enzy. 99:387-405, 1983) .

For re-precipitations, SDS (1% wt/vol) and β- mercaptoethanol (0.1% vol/vol) were added to the samples, denatured by boiling for 5 min, diluted to 0.1% SDS with lysis buffer, and subjected to immunoprecipitations (Rudd et al., Proc. Natl. Acad. Sci. (USA) 86:3277-3281, 1988). In some experiments, a specific protein band was excised from the polyacrylamide gel and the protein extracted in NH₄HC0₃ (50 mM) , SDS (0.1% wt/vol) , >3-mercaptoethanol (0.1% vol/vol), BSA (0.1%). The extract was lyophilized, resuspended in a 10 fold dilution of lysis buffer, and subjected to immunoprecipitation.

Phosphoamino acid analysis and phosphotryptic peptide mapping The extracted protein was subjected to acid hydrolysis (6 M HCl) and the phosphoamino acids were separated by two dimensional thin layer electrophoresis according to the procedure of Cooper et. al. (Meth. Enzy. 99:387-405, 1983). For two-dimensional tryptic peptide mapping, eluted proteins were exhaustively digested with TPCK-trypsin, and the resulting peptides were separated on a 0.1 mm thick cellulose plate (E. Merck, Germany) by electrophoresis (pH 8.9, 1000 volts for 30 min), and then by ascending chromatography (butanol, pyridine, acetic acid, water: 75, 50, 15, 60) (Hunter et al., Proc. Natl. Acad. Sci. (USA) 77:1311-1315, 1980; Pallas et al.. Cell 30:407-414, 1982).

RESULTS To ascertain whether the CD4:p56^lck complex is associated with any intracellular proteins, immunoprecipitates formed from peripheral blood lymphocytes and a variety of T lymphoblastoid cells (HPB- ALL, REX and MOLT 15) by anti-CD4 antibody 19Thy5D7 were analyzed by phosphotransferase labelling. As seen in Fig. 1, a 110 Kd protein (referred to herein as pllO protein) was often co-precipitated with the CD4:p56^lck complexes. Under the same labelling conditions, pllO was not detected in negative controls which included rabbit anti-mouse (lanes l, 6, 9, 12, 16), anti-CD3 (lane 4) and anti-fyn (lane 18) . In order to establish the identity of the pllO protein, immunoprecipitates were compared to precipitates formed by antiserum to the Raf-1 kinase. Previous studies have reported that in fibroblasts, a 110 Kd subunit is associated with anti-Raf immunoprecipitates, but is not itself bound by anti-Raf antibody. This 110 Kd subunit is the primary target of kinase labelling in these fibroblast anti-Raf immunoprecipitates (Siegel et al., J. Biol. Chem. 265:18472-18480, 1990; Zmuidzinas et al., Mol. Cell.

Biol. 11:2794-2803, 1991). In the present study using T cell-related cell lines, anti-Raf-1 immunoprecipitates revealed the presence of a 110 Kd band from all the cell lines examined (Fig. 1, lanes 5, 8, 11, 15, 19). Little if any of the p72 Raf-1 kinase was observed in these cells. This anti-Raf-1-precipitated pllO co-migrated with the pllO associated with CD4:p56^lck, raising the possibility that the two are identical. Alkali treatment of gels resulted in the complete loss of the radiolabel from the pllO bands, suggesting phosphorylation on Ser and Thr residues. Due to some variability in the detection of pllO from experiment to experiment and also due to the fact that pllO is phoshorylated mainly on Ser/Thr, the effects of stimulating HPB-ALL cells with phorbol ester (PMA, 100 ng/ml) prior to analysis were investigated. PMA activates serine/threonine kinases, including protein kinase C (Nishizuka, Nature 334:661-665, 1988). As shown in Fig. 2B, exposure of cells to PMA resulted in a time- dependent decrease in the density of CD4 on the surface of cells as detected by flow cytometry. Consistent with this loss was an observed time-dependent shift in the Mr of CD4-associated p56^lck from 56 Kd to 60 Kd, as well as a decrease in the overall intensity of labelled p56^lc (Fig. 2A) . This has been previously noted (Rudd et al. , Immunol. Rev. 111:225-266, 1989; Rudd et al. , "Molecular analysis of the interaction of p56^lck with the CD4 and CD8 antigens." In: Mechanisms of Lymphocyte Activation and Immune Regulation III. Gupta et al. (eds) , Plenum Press, New York, pp 85-89, 1991; Hurley et al.. Science 245:407- 409, 1989). However, despite the reduction in detectable CD4 and p56^lc , the appearance of CD4-associated pllO increased markedly in PMA-treated T cells (Fig. 2A, t=0-60'). Similarly, there was an increase in the intensity of pllO phosphorylation in anti-Raf immunoprecipitates (Fig. 2A, 0-60'). As observed in untreated samples, p72 Raf-1 was not visualized under this labelling regime. 24 h treatment with PMA resulted in the complete loss of CD4 from the cell surface (Fig. 2B) , and pllO could not be detected in the CD4 immunoprecipitate (Fig. 2A, 24 h) . In subsequent experiments, cells were routinely treated with PMA prior to experimental analysis. Two-dimensional phosphoamino acid analysis revealed that the phosphorylated amino acid residues on pllO associated with CD4:p56^lck (Fig. 3, panel B) were predominantly serine residues. Only prolonged autoradiographic exposures allowed for the detection of small amounts of phosphorylation on tyrosine residues. Similarly, pllO derived from anti-Raf immunoprecipitates was found to be labelled on serine residues (panel A) . By contrast, p56^lck was phosphorylated predominantly on tyrosine residues (panel C) . This observation indicates that pllO acts as a poor in vitro substrate for p56^lck. It also supports the notion that pllO is specifically associated with the CD4:p56^lck complex, and is not a non¬ specific co-precipitated protein labelled by virtue of phosphorylation by p56^lck. The association of pllO with CD4 was then probed using other monoclonal antibodies to the receptor. Three anti-CD4 monoclonal antibodies (T119, MT151 and Leu3a) immunoprecipitated pllO with the CD4:p56^lc complex (Fig. 4, lanes 4-7). These data indicate that co-precipitation of pllO is not dependent on the recognition of any particular epitope on the CD4 antigen. 19Thy5D7 and Leu3a recognise distinct epitopes at the N-terminal domain, while MT151 reacts with the second domain (Clayton et al.. Nature 339:548-551, 1989; Landau et al., Nature 334:159-162, 1988; Qureshi et al., J. Biol. Chem. 266:20594-20597, 1991; and Sattentau et al.. Science 234:1120-1123, 1986). Significantly, it was found that adding to the lysate an excess of recombinant soluble CD4 (2.0 mg/ml), which lacks the cytoplasmic domain of naturally occuring CD4 and thus cannot associate with p56^lc or any other cytoplasmic entity, effectively competed p56^lc and the vast majority of pllO from anti- CD4 immunoprecipitates, further supporting the specificity of the pllO association (Fig. 4, lanes 9- 10) .

The possible identity of pllO associated with CD4:p56^lc and pllO associated with anti-Raf precipitates was then investigated. Initially, two-dimensional tryptic phosphopeptide maps (pH 8.0) of anti-CD4-precipitated pllO and anti-Raf-precipitated pllO were compared. The analysis showed an array of some six different spots common to both Raf-1-related pllO and CD4-associated pllO, including a prominent and intensely phosphorylated phosphopeptide (labelled #2) and three minor peptides, ##1, 3 and 5 (Figs. 5A and B) . Two additional phosphopeptides (##4 and 6) appeared to be specific to pllO associated with CD4:p56^lck complex. These data indicate that there is a marked similarity between the pllOs associated with anti-Raf and anti-CD4 precipitates. We next investigated whether pllO could be directly recognized by the anti-Raf antisera, or whether pllO is an unrelated co-precipitated polypeptide. Labelled anti-Raf or anti-CD4 immunoprecipitates were denatured by boiling in 1% SDS, followed by dilution in excess lysis buffer and re-precipitation. This approach has been used to demonstrate the association of p56^lck with CD4 and p32 with the CD4:p56^lek complex (Rudd et al., Proc. Natl. Acad. Sci. (USA) 86:3277-3281, 1988; Telfer et al.. Science 254:439-441, 1991). As shown in Fig. 6, the anti-Raf antiserum specifically re-precipitated the pllO protein from anti-Raf immunoprecipitates (lanes 2, 5) , a precipitation that was blocked by the presence of peptide against which the anti-serum was raised (lanes 3, 6) . Similarly, the anti-Raf serum was also capable of recognizing pllO from anti-CD4 immunprecipitates (lane 7) . Furthermore, eluted labelled pllO from gels was specifically re-precipitated by the anti-Raf serum (lane 10) , a precipitation which was effectively competed by relevant peptide (lane 11) . Neither pre-immune serum nor anti-CD4 antibody was capable of reprecipitating pllO (data not shown) . These results clearly demonstrate the novel observation that the pllO phosphoprotein possesses a Raf-1-related epitope.

Other embodiments are within the following claims. What is claimed is:

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(ii) TITLE OF INVENTION: ALTERATION OF THE INTERACTION OF THE CD4 T-CELL RECEPTOR WITH A RAF-1-RELATED PROTEIN

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Claims

CLAIMS:

1. A method for screening candidate compounds to identify compounds capable of inhibiting the naturally occurring association of pllO to CD4:p56 T cell receptor complex, said method comprising the steps of: (a) providing a first and a second sample of cells expressing both pllO and the CD4:p56^lck T cell receptor complex;

(b) incubating said first sample in the presence of a candidate compound; (c) incubating said second sample in the absence of said candidate compound;

(d) generating a first immunoprecipitate by adding to said first sample a first aliquot of an antibody capable of specifically binding said pllO; (e) generating a second immunoprecipitate by adding to said second sample a second aliquot of said antibody; and

(f) determining whether the amount of said CD4:p56^lck T cell receptor complex present in said first immunoprecipitate is less than the amount of said CD4:p56 T cell receptor complex present in said second immunoprecipitate, the presence of a lesser amount of said CD4:p56^lck T cell receptor complex in said first immunoprecipitate than in said second immunoprecipitate indicating that said candidate compound inhibits said association.

2. The method of claim 1, wherein said antibody is an anti-raf-1 antibody.

3. ' A method for screening candidate compounds to identify compounds capable of inhibiting the naturally occurring association of pllO and the CD4:p56^lck T cell receptor complex, said method comprising the steps of: (a) providing a first and a second sample of cells expressing both pllO and the CD4:p56^lc T cell receptor complex;

(b) incubating said first sample in the presence of a candidate compound;

(c) incubating said second sample in the absence of said candidate compound;

(d) generating a first immunoprecipitate by adding to said first sample a first aliquot of an antibody capable of specifically binding said CD4:p56^lck T cell receptor complex;

(e) generating a second immunoprecipitate by adding to said second sample a second aliquot of said antibody; and

(f) determining whether the amount of said pllO present in said first immunoprecipitate is less than the amount of said pllO present in said second immunoprecipitate, the presence of a lesser amount of said pllO in said first immunoprecipitate than in said second immunoprecipitate indicating that said candidate compound inhibits said association.

4. A method for isolating pllO, said method comprising the steps of:

(a) contacting a biological sample comprising pllO and CD4:p56^lck T cell receptor complex with an antibody which binds to a component of said CD4:p56^lck T cell receptor complex to form an antigen-antibody complex; and

(b) separating said antigen-antibody complex from said biological sample.

5. A method for isolating pllO, said method comprising the steps of:

(a) contacting a biological sample comprising pllO with an antibody which binds to pllO to form an antigen- antibody complex; and^' (b) separating said antigen-antibody complex from said biological sample.

6. A method for interfering with the association of pllO with the CD4:p56^lck T cell receptor complex, said method comprising contacting said T cell receptor complex with a peptide comprising a fragment of said pllO.

7. A method for screening candidate compounds to identify a compound capable of inhibiting intracellular signalling through the CD4:p56^lck T cell receptor complex, said method comprising the steps of:

(a) providing a first and a second sample of cells expressing both pllO and the CD4:p56^lc T cell receptor complex;

(b) incubating said first sample in the presence of a candidate compound;

(c) incubating said second sample in the absence of said candidate compound;

(d) generating a first immunoprecipitate by adding to said first sample a first aliquot of an antibody capable of specifically binding said CD4:p56^lck T cell receptor complex or said pllO;

(f) determining the level of serine/threonine kinase activity in said first immunoprecipitate compared to the level of said activity in said second immunoprecipitate, a lower level in said first immunoprecipitate being an indication that said candidate compound is capable of inhibiting intracellular signalling through the CD4:p56^lck T cell receptor complex.

8. The method of claim 7, wherein said level of said activity in each of said immunoprecipitates is determined by measuring phosphorylation of serine/threonine residues on said pllO in each of said immunoprecipitates.

9. The method of claim 7 wherein said level of said activity in each of said immunoprecipitates is determined by adding a substrate polypeptide to each of said immunoprecipitates and measuring phosphorylation of serine/threonine residues on said substrate polypeptide.

10. A substantially pure preparation of pllO.

11. An isolated DNA encoding pllO.

12. A plasmid comprising the DNA of claim 11.

13. The plasmid of claim 12, said plasmid further comprising an expression control sequence capable of directing expression of said pllO.

14. A cell containing the isolated DNA of claim 11.

15. A peptide capable of disrupting the naturally occurring binding interaction between pllO and a CD4:p56^lck T cell receptor complex.

16. The peptide of claim 15, wherein said peptide comprises a fragment of pllO.

17. The peptide of claim 15, wherein said peptide comprises a fragment of CD4.

18. The peptide of claim 15, wherein said peptide comprises a fragment of p56^lck.

19. The peptide of claim 15, wherein a covalent bond between two residues within said peptide is a nonpeptide bond.

20. An antibody which binds preferentially to pllO compared to 70-72 Kd Raf-1.

21. A method of generating an antibody, said method comprising immunizing an animal with pllO or a fragment thereof that is not identical to any portion of 70-72 Kd Raf-1.

22. A method of interfering with the association of pllO and the CD4:p56^lc T cell receptor complex, said method comprising contacting a cell comprising pllO and a CD4:p56^lck complex with an antibody which binds to pllO.

23. A method of determining the amount of pllO in a sample, said method comprising contacting said sample with an antibody which binds to pllO; and determining the amount of antigen-antibody complex formation in said sample.

24. A method of determining the level of pllO kinase activity in a sample, said method comprising contacting said sample with an antibody which binds to pllO to form an immunoprecipitate; and determining the level of serine/threonine kinase activity in said immunoprecipitate.

25. A diagnostic method comprising providing a biological sample comprising T cells from an animal suspected of having a condition characterized by abnormal signal transduction via the CD4:p56^lck:pllO pathway; and determining the amount of pllO or the level of pllO kinase activity in said sample, an abnormal amount of pllO or level of pllO kinase activity being an indication that said animal has said condition.

26. The method of claim 25, wherein said condition is a cancerous or precancerous condition, an autoimmune disease, or a human immunodeficiency virus (HIV) infection.