Throughout this application various publications are referenced by author and year of publication. Full citations for these publications are provided following the detailed description of the invention and examples.

Research in the area of immune cell signalling has yielded a considerable amount of information about the signal transduction events that occur downstream of antigen receptor engagement. Early work concentrated on the receptors themselves and the enzymes stimulated in response to antigen binding (reviewed by Weiss & Littman, 1994; DeFranco, 1997). More recent studies have examined how these initial events are linked to secondary messenger signalling pathways such as those activated by Ras, phospholipase Cy, and phosphoinositide 3-kinase. One class of proteins that play a critical role in the integration of these events and in the regulation of signalling cascades is the adaptor molecules.

Adaptor molecules lack enzymatic function but contain motifs and domains that permit them to participate in, and mediate, protein-protein interactions.

The protein complexes formed through these interactions act as intermediaries and couple receptor activation to downstream signalling cascades. The role of individual adaptor molecules and an analysis of their interactions has been the subject of intense study (see reviews by Samelson, 1999; Peterson et al., 1998 and Rudd, 1999), although the detailed mechanism of coupling is still poorly understood.

In comparison, the components of the TCR complex, which are required for the initial signalling events, are well characterised. Some of these components have been employed in the construction of chimeric receptor proteins as a tool to elucidate the function of individual receptor sub-units or domains (Kuwana et al., 1987; Romeo et al., 1992). More recently chimeric receptors have been used to regulate the levels of cell activation (see for example published International Patent Specifications WO 97/23613 and WO 95/02686). The ability to control the biological effects of cellular activation, for example, increased cellular proliferation, increased expression of cytokines, stimulation of cytolytic activity, differentiation of other effector functions, antibody secretion, phagocytosis, tumour infiltration and/or increased cellular adhesion, with chimeric receptors has considerable therapeutic potential. Whilst currently available chimeric receptors are capable of effectively activating cells, there is room for improvement in the efficacy of signal transduction to downstream members of secondary messenger pathways.

It would be of great value to be able to tailor the level of intracellular signalling, and thus the level of effector cell activation, to a required degree.

It would also be extremely desirable to be able to activate a cell in a positive or negative way, thus inducing or inhibiting its biological functions with greater efficacy than is possible at present.

The present invention fulfils these needs by providing a cell with an adaptor receptor protein that is capable of regulating the levels of signalling through secondary messenger pathways. The unexpected finding that adaptor proteins can play an active role in immune cell signalling pathways when removed from their natural environment, has permitted us to develop adaptor receptors that are capable of highly efficient signalling. Such novel receptors employ adaptor proteins as the intracellular signalling domain of a chimeric receptor, in combination with, for example, an extracellular ligand binding domain and a transmembrane domain.

When an adaptor receptor is expressed in an effector cell, the binding of a ligand to the extracellular binding domain results in transduction of a signal through the adaptor protein component, which in turn induces cell activation.

Thus, the signalling mediated by the adaptor protein can be tailored in response to a specific stimulus, which is defined by the nature of the extracellular binding domain. By contrast, in a natural situation adaptor proteins would function in a ubiquitous manner i. e. they would be recruited to help with cell signal transduction in response to immune cell stimulation by any antigen.

Cellular activation is characterised by a number of biological responses, including the release of cytokines and cell death and it is very surprising that such biological effects are observed when signalling is mediated by an adaptor protein when it is employed as an intracellular domain of the receptor. The present invention allows for the construction of a number of adaptor receptors, which, when engaged by an extracellular ligand, are capable of either up-or down-regulating a biological response with an unexpectedly greater efficacy than the chimeric receptors that have been described to date.

The first aspect of the invention provides a nucleic acid encoding an adaptor receptor protein which comprises an extracellular ligand-binding domain, a transmembrane domain and an intracellular signalling domain, wherein the intracellular signalling domain comprises the cytoplasmic portion of at least one adaptor protein and wherein the extracellular ligand-binding domain is not CD8 or a MHC class I protein. The invention also extends to adaptor proteins encoded by the nucleic acids of the invention as described herein, as well as a method of activating a cell, which comprises providing the cell with an adaptor receptor of the invention.

Adaptor proteins are defined herein as proteins that play a positive or negative regulatory role in immune cell signal transduction pathways by mediating protein-protein interactions, and lack intrinsic enzymatic activity.

The"intrinsic enzymatic activity"of a protein or polypeptide domain means the ability of that protein or polypeptide domain to catalyse any enzymatic reaction, and thus inclues oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase activity.

Adaptor proteins for use in the invention may be sub-divided into two- classes : those that are purely cytoplasmic and those that have (or are predicted to have) a transmembrane domain as well as a cytoplasmic domain. Examples of cytoplasmic adaptor proteins include SLAP, SLP-76, SKAP55, Grap, 3BP2, Grb-2, Nck, CRKL, Shc, and Cbl. The adaptor protein Grap2, which is also known as GrbX, GrbLG, Grf40, Gads and GRID (Ellis et a/., 2000), is yet another example of a cytoplasmic adaptor protein that may be used in the invention. In contrast, LAT (also known as p36), TRIM and SIT are all adaptor proteins that are predicted to have a transmembrane domain in addition to a cytoplasmic domain, and these may also be employed in the invention.

Any complete adaptor protein (including for example, any of those mentioned above) or, optionally, in the case of transmembrane adaptor proteins, the cytoplasmic portion of any adaptor protein may be used as the intracellular signalling domain of an adaptor receptor. It would be particularly advantageous to include any cysteine residues in the juxtamembrane region of such a cytoplasmic portion, since such residues are frequently sites for palmitoylation and are likely to be involved in membrane localisation and function (Zhang et al., 1999). It is preferred that all, or the cytoplasmic part of, LAT, SLP-76, Grap, Grb2, TRIM, SIT or Cbl, are used in the invention.

The use of all, or the cytoplasmic parts, of LAT, TRIM and SIT are especially preferred.

According to the invention a further component of an adaptor receptor is the transmembrane domain. This may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived

from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i. e. comprise at least the transmembrane region (s) of) the a, ß or Ç chain of the T-cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. It is preferred that transmembrane regions derived from all or part of the a, ß or 4 chain of the T- cell receptor, CD28, CD3£, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154 are employed in the invention. Alternatively, the transmembrane domain will be derived from all or part of the transmembrane domain of any adaptor protein that has such a domain; preferably it will be derived from the transmembrane domain of LAT, SIT or TRIM. As another alternative, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine (see for example Published International Patent Specification WO00/63374). Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

A third component of the adaptor receptor is the extracellular ligand-binding domain. The incorporation of such a domain confers on the adaptor receptor the ability to exhibit specificity for a specific ligand or class of ligands. By using the specificity of the extracellular ligand-binding domain to define precise ligands or classes of ligands that are capable of activating the receptor, it may be tailored to generate a desired cellular response in the cell in which it is expressed.

The term"extracellular ligand-binding domain"as used herein, refers to any oligo-or polypeptide that is capable of binding a ligand with the exception of the CD8 and any MHC class I protein. Accordingly antibody binding domains, antibody hypervariable loops or CDRs, receptor binding domains and other ligand binding domains, examples of which will be readily apparent to the skilled artisan, are described by this term. Preferably the domain will be capable of interacting with a cell surface molecule. Example of proteins associated with binding to cell surface molecules, which are of particular use

in this invention include, antibody variable domains (VH or V), T-cell receptor variable region domains (TCR (x, TCRß, TCRy, TCR6) or the chains of CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B, CD49D, CD49E, CD49F, CD61, CD41, or CD51. Whilst it may be of benefit to use the entire domain or chain in some instances, fragments may be used where appropriate.

Particularly useful binding components are derived from antibody binding domains and include Fab'fragments or, especially single chain Fv fragments.

The choice of domain will depend upon the type and number of ligands that define the surface of a target cell. For example, the extracellular ligand binding domain may be chosen to recognise a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

Thus examples of cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells. In the latter case, specific examples of cell surface markers are the bombesin receptor expressed on lung tumour cells, carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), CD33, the folate receptor, epithelial cell adhesion molecule (EPCAM) and erb-B2. Other molecules of choice are cell surface adhesion molecules, inflammatory cells present in autoimmune disease, and T-cell receptors or antigens that give rise to autoimmunity. The potential ligands listed above are included by way of example; the list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.

Adaptor receptors of the invention may be designed to be bi-or multi-specific i. e. they may comprise more than one ligand binding domain and therefore be capable of exhibiting specificity for more than one ligand. Such receptors may recruit cellular immune effector cells, such as T cells, B cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, or mast cells or components of the complement cascade.

We have also found surprisingly, that the inclusion of additional cytoplasmic signalling components as part of the receptor can further modulate the degree of cellular activation observed after ligand has bound to an adaptor receptor. Thus in a second aspect of the invention there is provided a nucleic acid encoding an adaptor receptor as described in the first aspect of the invention, wherein the intracellular signalling domain comprises at least one additional cytoplasmic signalling component.

The term"cytoplasmic signalling component"as used herein, refers to cytoplasmic sequences of the TCR and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement. These sequences lack intrinsic enzymatic activity. The term also encompasses any signalling sequence derived from any other immune cell receptor, derivatives or variants of these sequences, and any synthetic sequence, that has the same functional capability.

Signals generated through the TCR alone are insufficient for full activation of the T cell. Thus, T cell cytoplasmic signalling components can be sub- divided into two classes: those that initiate antigen-dependent primary activation through the TCR (primary signalling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary signalling sequences). It will be appreciated that a primary signalling sequence may contain one or more primary signalling motif responsible for signal transduction. Accordingly, cytoplasmic signalling components for use in this aspect of the invention may comprise all or part of primary (i. e. one or more primary signalling motifs) and/or secondary signalling sequences.

The term"primary signalling motif"is defined as a sequence that transduces either a stimulatory or an inhibitory signal, which regulates primary activation of the TCR complex. Examples of stimulatory primary signalling motifs include any sequence that broadly conforms to the consensus sequence Y- X2-UI-Xn-Y-X2-UI such as for example immunoreceptor tyrosine-based

activation motifs (ITAMs). Motifs that act in an inhibitory way include immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which are defined as broadly conforming to the consensus amino acid sequence of I/V-X-Y-X2- L (Burshtyn et al., 1999). It should be noted that, other than in Figure 2 and in some instances in the examples (where the standard three-letter code is used to describe amino acid sequence), the standard single letter code is used throughout this application to describe both amino acid and nucleotide sequences.

Stimulatory primary signalling motifs, for use in the invention may contain the consensus amino acid sequence Y-X2-UI-Xn-Y-X2-L/I. In this formula, X represents any amino acid, a subscripted number indicates the number of residues present at that position within the motif and the value n implies any number greater than zero. The value of n may be varied between 5 and 12, and more preferably lies in the range 6 to 9. It is intended that that the terms X2 or Xn can represent 2 or n amino acids (respectively) which may either be the same or different.

Where the value of n lies between 6 and 8, it is preferred that at least one of the additional primary signalling motifs in the adaptor receptor will be an immunoreceptor tyrosine based activation motif (ITAM) for example, all or part of TCR41, TCR2, TCR3 (i. e the first, second or third, ITAMs of the TCRÇ chain), FcRy, FcRp, CD3y, CD38, CD3E, CD5, CD22, CD79a, CD79b or CD66d, or variants thereof. Examples of primary signalling motifs derived from these molecules are shown in Table 1 below. Preferably at least one additional primary signalling motif for use in the invention will be derived from all or part of TCR41, TCR2, TCR43, FcRy, FcRp, CD3y, CD38, CD5, CD22, CD79a, CD79b or CD66d, or a variant thereof.

Alternatively, at least one of the additional primary signalling motifs will be non-natural but still conform to the consensus amino acid sequence of sequence Y-X2-UI-Xn-Y-X2-L/I. Preferred examples of such non-natural primary signalling motifs with a value of n between 6 and 8, will be SB14a or

SB15a as described herein in Table 1, or non-natural variants thereof. Where the value of n is 9 or greater, SBXa, SBQ9a, SB16a or non-natural variants thereof, may be employed as the additional motif (s), with SB16a being especially preferred (Table 1).

Table 1. Source and amino acid sequences of primary signalling motifs of particular use in the invention. The position of the consensus amino acid sequence is emphasised in bold. Figure 4 includes further examples of primary signalling motifs for use in the invention (SB1, SB2, SB3, SB4, SB4*, SB5, SB6, SB7, SB8, SB9, SB10, SB11, SB12, SB13, SB14, SB15, SB16, SBX and SBQ9), which correspond to the primary signalling motifs shown below with GS linkers incorporated at each end of the motif to facilitate cloning.

Source Primary Amino Acid Sequence Signalling Motif TCF1 SB1"GQNQLYNELNLGRREEYDVLDKRRGRDPEM TCR2 SB2a RKNPQEGLYNELQKDKMAEAYSEIGMKGER TCR3 SB3a RGKGHDGLYQGLSTATKDTYDALHMQA FcRy SB4a YEKSDGVYTGLSTRNQETYETLKHEKP FcRp SB5a GNKBPEDRVYEELNIYSATYSELEDPGEMSP CD3y SB6a KQTLLPNDQLYQPLKDREDDQYSHLQGNQLR <BR> <BR> CD38,SB7a ALLRNDQVYQPLRDRDDAQYSHLGGNWARNK<BR> CD3£ SB8a QNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI CD5 SB9a HVDNEYSQPPRNSRLSAYPALEGVLHRS CD22 SBlOa PPRTCDDTVTYSALHKRQVGDYENVIPDFPEDE CD79a SBlla CD79b SB12a KAGMEEDHTYEGLDIDQTATYEDIVTLRTGEV CD66d SB13a PLPNPRTAASIYEELLKHDTNIYCRMDHKAEVA FcRy SB4*a YEKSDGVYTGLSTRNQETYDTLKHEKP Non-SB14a GQDGLYQELNTRSRDEYSVLEGRKAR natural Non-SB15a GQDGLYQELNTRSRDEAYSVLEGRKAR natural Non-SB16a GQDGLYQELNTRSRDEAAYSVLEGRKAR natural Non-SBXa RKNPQEGLYNELQKDKMAEDTYDALHMQA natural Non-SBQ9a GQNQLYNELQQQQQQQQQYDVLRRGRDPEM natural Primary signalling motifs that have the capacity to inhibit cellular activation, such as ITIMs, may optionally be employed as additional components of an

adaptor receptor. Examples of ITIMs for use in the present invention include those derived from FcyR (e. g. FcyRIIB), CD22, EPOR, IL-2ßR or IL-3ßR.

The term"secondary signalling sequence"is defined as a sequence that imparts secondary or co-stimulatory signalling capacity to a molecule in T cells. Molecules containing such sequences include CD2, CD4, CD8, CD28, CD134 and CD154 (see Finney et al., 1998). Preferred secondary signalling sequences for use in the invention are those derived from CD28, CD134 and CD154, for example, SB28a RLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FA, SB29a MIETYNQTSPRSAATGLPISMK and SB34a RRDQRLPPDAHKP PGGGSFRTPIQEEQADAHSTLAKI. Further examples of secondary signalling sequences are shown in Figure 4 as SB28, SB29 and SB34, where GS linkers have been incorporated at each end of the sequence to facilitate cloning.

According to yet another aspect, cellular activation may be effected through an adaptor receptor as described in any previously described aspect of the invention, wherein the adaptor receptor comprises an additional domain that is not a primary signalling motif and not a secondary signalling sequence.

This additional domain may be, for example, any enzymatic domain except for a hydrolase. The additional domain is preferably included as a cytoplasmic element of the receptor. Where the additional domain does exhibit enzymatic activity, transferase, and more specifically protein tyrosine kinase, activity is preferred. Especially preferred examples are members of the src and syk families of protein tyrosine kinases.

Where the additional domain lacks enzymatic activity, it may be derived from the cytoplasmic part of an immune cell receptor other than the TCR or its associated co-receptor.

Cytoplasmic signalling and adaptor components may be linked to each other, or to the transmembrane domain, in a random or specified order. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids

in length may form the linkage between component parts of the receptor. A glycine-serine doublet provides a particularly suitable linker.

The mixing and matching of positive-and negative-regulatory adaptors with stimulatory and inhibitory primary signalling motifs, and/or with different secondary signalling sequences, and/or further, potential enzymatic, domains thus provides a multiplicity of adaptor receptors, each capable of regulating cellular activation to a different degree.

Between the extracellular ligand-binding domain and the transmembrane domain, or between the cytoplasmic signalling components and the transmembrane domain, there may be incorporated a spacer domain. As used herein, the term"spacer domain"generally means any oligo-or polypeptide that functions to link the transmembrane domain to, either the extracellular ligand-binding domain or, the cytoplasmic signalling components in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

Spacer domains may derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, or CD28; all or part of an antibody constant region; all or part of natural spacer components between functional parts of cytoplasmic signalling components, for example spacers between ITAMs may be used. Alternatively, the spacer may be a synthetic sequence that corresponds to a naturally occurring spacer sequence, or may be an entirely synthetic spacer sequence.

Spacer domains may be designed in such a way that they, either minimise the constitutive association of adaptor receptors, thus reducing the incidence of constitutive activation in the cell or, promote such associations and enhance the level of constitutive activation in the cell. Either possibility may be achieved artificially by deleting, inserting, altering or otherwise modifying amino acids and naturally occurring sequences in the transmembrane and/or

spacer domains, which have side chain residues that are capable of covalently or non-covalently interacting with the side chains of amino acids in other polypeptide chains. Particular examples of amino acids that can normally be predicted to promote association include cysteine residues, charged amino acids or amino acids such as serine or threonine within potential glycosylation sites.

Adaptor receptors may be designed in such a way that the spacer and transmembrane components have free thiol groups, thereby providing the receptor with multimerisation, and particularly dimerisation, capacity. Such multimeric receptors are preferred, especially dimers. Adaptor receptors with transmembrane and spacer domains derived from CD28 components, the zeta chain of the natural T cell receptor, adaptor transmembrane domains and/or antibody hinge sequences are especially preferred.

Nucleic acid coding sequences for use in the preparation of the nucleic acids of this invention are widely reported in the scientific literature and are also available in public databases (e. g. Genbank, EMBL etc.) DNA may be commercially available, may be part of cDNA libraries, or may be generated using standard molecular biology and/or chemistry procedures as will be clear to those of skill in the art. Particularly suitable techniques include the polymerase chain reaction (PCR), oligonucleotide-directed mutagenesis, oligonucleotide-directed synthesis techniques, enzymatic cleavage or enzymatic filling-in of gapped oligonucleotide. Such techniques are described by Sambrook & Fritsch, 1989, and in the examples contained hereinafter.

The nucleic acids of the invention may be used with a carrier. The carrier may be a vector or other carrier suitable for the introduction of the nucleic acids ex-vivo or in-vivo into target cell and/or target host cells. Examples of suitable vectors include viral vectors such as retroviruses, adenoviruses, adeno-associated viruses (AAVs), Epstein-Barr virus (EBV) and Herpes simplex virus (HSV). Non-viral vector may also be used, such as liposomal

vectors and vectors based on condensing agents such as the cationic lipids described in International patent application numbers W096/10038, W097/18185, W097/25329, W097/30170 and W097/31934. Where appropriate, the vector may addition include promoter and regulatory sequences and/or replication functions from viruses, such as retrovirus long terminal repeats (LTRs), AAV repeats, SV40 and human cytomegalovirus (hCMV) promoters and/or enhancers, splicing and polyadenylation signals and EBV and BK virus replication functions. Tissue-specific regulatory sequences such as the TCR-a promoter, E-selectin promoter and the CD2 promoter and locus control region may also be used. The carrier may be an antibody.

The invention also includes cloning and expression vectors containing a nucleic acid according to any of the above-described aspects of the invention. Such expression vectors will incorporate the appropriate transcriptional and translation control sequences, for example, enhancer elements, promoter-operator regions, termination stop sequence, mRNA stability sequences, start and stop codons or ribosome binding sites, linked where appropriate in-frame with the nucleic acid molecules of the invention.

Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses). Many expression systems suitable for the expression of heterologous proteins are well known and documented in the art. For example, the use of prokaryotic cells such as Escherichia coli to express heterologous polypeptides and polypeptide fragments is well established (see for example, Sambrook & Fritsch, 1989, Glover, 1995a). Similarly, eukaryotic expression systems have been well developed and are commonly used for heterologous protein expression (see for example, Glover, 1995b and O'Reilly et al., 1993). In eukaryotic cells, apart from yeasts, the vectors of choice are virus-based. Particularly suitable viral vectors include baculovirus-, adenovirus-, and vaccinia virus-based vectors.

Vectors containing the relevant regulatory sequences (including promoter, termination, polyadenylation, and enhancer sequences, marker genes) can either be chosen from those documented in the literature, or readily constructed for the expression of the receptors of this invention using standard molecular biology techniques. Such techniques, and protocols for the manipulation of nucleic acids, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, DNA transformation and gene expression, as well as the analysis of proteins, are described in detail in Ausubel et al., 1992 or Rees et al., 1993.

Suitable host cells for the in vitro expression of high levels of adaptor receptor protein include prokaryotic cells e. g. E. coli, eukaryotic yeasts e. g Saccharomyces cerevisiae, Pichia species, Schizosaccharomyces pombe, mammalian cell lines and insect cells. Alternatively adaptor receptors may be expressed in vivo, for example in insect larvae, plant cells, or in particular in mammalian tissues.

Introduction of the nucleic acid into a host cell may employ any available technique. In eukaryotic cells suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, particle bombardment, liposome-mediated transfection or transduction using retrovirus, adenovirus or other viruses, such as vaccinia or, for insect cells, baculovirus. In bacterial cells, suitable techniques may include calcium chloride transformation, electroporation or transfection using bacteriophage.

The nucleic acid may remain in an episomal form within the cell, or it may integrate into the genome of the cell. If the latter is desired, sequences that promote recombination with the genome will be included in the nucleic acid.

Following introduction of the nucleic acid into host cells, the cells may be cultured under conditions to enhance or induce expression from the adaptor receptor gene as appropriate.

Thus, further aspects of the invention provide host cells containing a nucleic acid encoding an adaptor receptor, and host cells expressing an adaptor receptor protein.

According to still further aspects, the nucleic acids of the invention can be employed in either the ex-vivo or in-vivo treatment of target cells and/or target host cells.

For ex-vivo use, the nucleic acid may be introduced into effector cells, removed from the target host, using methods well known in the art e. g. transfection, transduction (including viral transduction), biolistics, protoplast fusion, calcium phosphate mediated DNA transformation, electroporation, cationic lipofection, or targeted liposomes. The effector cells are then reintroduced into the host using standard techniques. Examples of suitable effector cells for the expression of the adaptor receptors of the present invention include cells associated with the immune system such as lymphocytes e. g. cytotoxic T-lymphocytes, tumour infiltrating lymphocytes, neutrophils, basophils, or T-helper cells, dendritic cells, B-cells, haematopoietic stem cells, macrophages, monocytes or NK cells. The use of cytotoxic T-lymphocytes is especially preferred.

The nucleic acid according to this aspect of the invention is particularly suitable for in vivo administration. In order to achieve this, the DNA may be in the form of a targeted carrier system in which a carrier as described above is capable of directing DNA to a desired effector cell. Examples of suitable targeted delivery systems include targeted naked DNA, targeted liposomes encapsulating and/or complexe with the DNA, targeted retroviral or adenoviral systems and targeted condensed DNA such as protamine and polylysine-condensed DNA.

Targeting systems are well known in the art and include, for example, using antibodies or fragments thereof against cell surface antigens expressed on target cells in vivo such as CD8, CD16, CD4, CD3, selecting (e. g. E-selectin),

CD5, CD7, CD24, and activation antigens (e. g. CD69 an dIL-2R.

Alternatively other receptor-ligand interactions can be used for targeting e. g.

CD4 to target HIVgp160-expressing target cells.

In general, the use of antibody-targeted DNA is preferred, particularly antibody-targeted naked DNA, antibody-targeted condensed DNA and especially antibody-targeted liposomes. Types of liposomes that may be used include for example pH-sensitive liposomes, where linkers that are cleaved at low pH may be used to link the antibody to the liposome. The nucleic acids of the present invention may also be targeted directly to the cytoplasm by using cationic liposomes, which fuse with the cell membrane.

Liposomes for use in the invention may also have hydrophilic molecules, e. g. polyethylene glycol polymers, attached to their surface to increase their circulating half-life. There are many examples, in the art, of suitable groups for attaching DNA to liposomes or other carriers; see for example International patent application numbers W088/04924, W090/09782, W091/05545, W091/05546, W093/19738, W094/20073 and W094/22429.

The antibody or other targetting molecule may be linked to the DNA, condensed DNA or liposome using conventional linking groups and reactive functional groups in the antibody, e. g. thiols or amines, and in the DNA or DNA-containing material.

Non-targeted carrier systems may also be used. In these systems targeted expression of the protein is advantageous. This may be achieved, for example, by using T cell specific promoter systems such as the zeta promoter, CD2 promoter and locus control region, CD4, CD8 TCRa and TCRp promoters, cytokine promoters, such as the IL2 promoter, and the perforin promoter.

It is intended that adaptor receptor proteins of the present invention, or the nucleic acids encoding them, be applied in methods of therapy of mammalian, particularly human, patients. Adaptor receptor proteins generated by the present invention may be particularly useful in the treatment

of a number of diseases or disorders. Such diseases or disorders may include those described under the general headings of infectious diseases, e. g. HIV infection; inflammatory disease/autoimmunity e. g. asthma, eczema; congenital e. g. cystic fibrosis, sickle cell anaemia; dermatologic, e. g. psoriasis; neurologic, e. g. multiple sclerosis ; transplants e. g. organ transplant rejection, graft-versus-host disease; metabolic/idiopathic disease, e. g. diabetes; cancer.

For example, expression of the adaptor receptor on the surface of a T cell may initiate the activation of that cell upon binding of the ligand-binding domain to a ligand on a target cell. The ensuing release of inflammatory mediators stimulated by the activation of the signalling function of the receptor ensures destruction of the target cell.

Thus a further aspect of the invention provides a method of activating a cell which comprises providing the cell with an adaptor receptor protein as described in any one of the previously described aspects of the invention and allowing ligand to bind to the extracellular domain of the adaptor receptor.

When an effector cell of the immune system is provided with an adaptor receptor according to the present invention, binding to target will activate the effector cell ; downstream effects of this activation may also result in the destruction of the target cell. If the extracellular ligand-binding domain of the adaptor receptor exhibits specificity for a surface marker on an immune cell, effector cells may be recruited to the site of disease. Accordingly, expression of an adaptor receptor in a diseased cell will ensure its destruction.

The expression of multispecific adaptor receptor proteins, or more than one adaptor receptor (with different ligand specificities), within a single host cell, may confer dual functionality on the receptor. For example, binding of the adaptor receptor to its target may not only activate the effector cell itself, but may addition attract other immune effectors to the site of disease. The target cell may thus be destroyed by the activation of the immune system.

A further aspect of the invention provides a composition comprising a nucleic acid encoding an adaptor receptor, or an adaptor receptor protein, according to any of the aspects of the invention described above, in conjunction with a pharmaceutically acceptable excipient.

Suitable excipients will be well known to those of skill in the art and may, for example, comprise a phosphate-buffered saline (e. g. 0.01 M phosphate salts, 0.138M NaCI, 0.0027M KCI, pH7.4), a liquid such as water, saline, glycerol or ethanol, optionally also containing mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates and the like ; and the salts of organic acids such as acetates propionates, malonates, benzoates and the like.

Auxiliary substances such as wetting or emulsifying agents, and pH buffering substances, may also be present. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991). Preferably, the compositions will be in a form suitable for parenteral administration e. g. by injection or infusion, for example by bolus injection or continuous infusion or particle-mediated injection.

Where the composition is for injection or infusion, it may take the form of a suspension, solution or mulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the composition may be in dry form, for reconstitution before use with an appropriate sterile liquid. For particle- mediated administration the DNA may be coated on particles such as microscopic gold particles.

A carrier may also be used that does not itself induce the production of antibodies harmful to the individual receiving the composition and which may be administered without undue toxicity. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Pharmaceutical compositions may also contain preservatives in order to prolong shelf life in storage.

If the composition is suitable for oral administration, the formulation may contain, in addition to the active ingredient additives such as starch (e. g. potato, maize or wheat starch, cellulose), starch derivatives such as microcrystalline cellulose, silica, various sugars such as lactose, magnesium carbonate and/or calcium phosphate. It is desirable that a formulation suitable for oral administration be well tolerated by the patient's digestive system. To this end, it may be desirable to include mucus formers and resins. It may also be desirable to improve tolerance by formulating the compositions in a capsule that is insoluble in the gastric juices. In addition, it may be preferable to include the composition in a controlled release formulation.

According to yet a further aspect of the invention there is provided the use of, or a nucleic acid encoding an adaptor receptor, or an adaptor receptor protein, or a pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment or prevention of disease in humans or in animals.

The various aspects and embodiments of the present invention will now be illustrated in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Cloning cassette for construction of adaptor receptors with multiple signalling components.

Figure 2: Nucleotide and amino acid sequence of h. CD28 extracellular spacer and the human CD28 transmembrane region used in the construction of the cloning cassette describe in Figure 1.

Figure 3: Oligonucleotide sequences used in the construction of adaptor receptors.

Figure 4: Amino acid sequences of primary signalling motifs and secondary signalling sequences employed in the construction of adaptor receptors.

Figure 5: Antigen specific stimulation of the adaptor receptor, p67scFv/h. CD28/LATtm/LAT.

Figure 6: Provision of primary signalling to the adaptor receptor p67scFv/h. CD28/LATtm/LAT by stimulation with OKT3 Figure 7: Provision of primary signalling to the adaptor receptor p67scFv/h. CD28/LATtm/LAT by addition of a primary signalling motif SB14a.

Figure 8: Provision of primary signalling to the adaptor receptor p67scFv/h. CD28/LATtm/LAT by addition of a primary signalling motif SB14a.

Figure 9: Provision of further primary signalling to the adaptor receptor p67scFv/h. CD28/LATtm/LAT. SB14 by additional stimulation with OKT3.

Figure 10: Provision of secondary (costimulatory) signalling to the adaptor receptor p67scFv/h. CD28/LATtm/LAT. SB14 by stimulation with anti-CD28 antibody.

Figure 11: Transient transfection analysis in Jurkat of LAT adaptor receptors with various additional primary signalling motifs and secondary signalling sequences.

Figure 12: Transient transfection analysis in Jurkat of SIT adaptor receptors with various additional primary signalling motifs and secondary signalling sequences.

Figure 13: Transient transfection analysis in Jurkat of TRIM adaptor receptors with various additional primary signalling motifs and secondary signalling sequences.

EXAMPLES Example 1: Construction of the cloning vector, pHMF393 To facilitate construction of adaptor receptors with different binding, extracellular spacer, transmembrane and signalling components, a cloning cassette system

was devised in pBluescript SK+ (Stratagene). This is a modification of our cassette system described in published International Patent Specification number WO 97/23613.

This new cassette system is shown in Figure 1. The binding component has 5' (relative to coding direction) Not I and Hind III restriction sites and a 3' (again relative to coding direction) Spe I restriction site. The extracellular spacer is flanked by a Spe I site (therefore encoding Thr, Ser at the 5'end) and a Nar I site (therefore encoding Gly, Ala at the 3'end). The transmembrane component is flanked by a Nar I site at its 5'end (therefore encoding Gly, Ala) and by Mlu I (therefore encoding Thr, Arg) and BamH I sites (therefore encoding Gly, Ser) at the 3'end. The signalling component may be cloned in-frame into the BamH I site. Following this BamH I site there is a stop codon for transcription termination and there is also an EcoR I site situated downstream of this to facilitate the subsequent rescue of whole constructs.

To generate the cassette, a 200bp fragment was assemble by PCR, using the following oligos ; S0146, A6081, A6082 and A6083 (Figure 3). The nucleotide and amino acid sequences of this fragment are shown in Figure 2. It starts with a Spel site and consists of the extracellular spacer h. CD28, the human CD28 transmembrane region, a stop codon and finishes with an EcoR I restriction site.

This PCR fragment was then digested with Spe I and EcoR I and substituted for the same fragment in our previously described cloning cassette system (Figure 2 of published International Patent application WO 97/23613) in order to clone it in-frame with the binding component.

Example 2: Cloning of adaptor molecules All whole adaptor molecules and portions thereof were cloned by PCR. The sequences of all oligonucleotides used in cloning adaptors in the examples described below, are given in Figure 3. For a number of adaptors the cloning process was carried out more than once, using different oligonucleotides. This provides DNA encoding adaptor molecules (or portions thereof) flanked by

different restriction endonuclease recognition sites and thus facilitates subsequent sub-cloning procedures. a) LAT (Weber et a/.. 1998). This adaptor was cloned from human leukocyte cDNA (Clontech). Residues 3 to 233 (comprising the transmembrane and cytosolic regions of LAT) were cloned on a Nar I to Mlu I fragment using oligos D3457 and F15163. The cytosolic region only (residues 28 to 233) was cloned on a Mlu I to Mlu I fragment using oligos F15162 and F15163 and on a Bcl I to BamH I fragment) using oligos F12315 and D3458. b) TRIM (Bruvns et al.. 1998). This adaptor was cloned from a human T cell library. Residues 9 to 186 (comprising the transmembrane and cytosolic regions of TRIM) were cloned on a Nar I to Mlu I fragment using oligos F15170 and F15164. The cytosolic region only (residues 29 to 186) was cloned on a Mlu I to Mlu I with oligos F15165 and F15164 and also on a Bgl II to BamH I with oligos F18148 and F16131. c) SIT (Marie-Cardine et al., 1999). This adaptor was also cloned from a human T cell library. Residues 42 to 196 (comprising the transmembrane and cytosolic regions of SIT) were cloned on a Nar I to Mlu I fragment using oligos F16130 and F15166. The cytosolic region only (residues 61 to 196), was cloned on a Mlu I to Mlu I using oligos F15167 and F15166. d) SLP-76 (Jackman et aL, 19aQ. This adaptor was also cloned from a human T cell library. Residues 2 to 533 (the cytosolic region of SLP-76) were cloned on a Mlu I to Mlu I fragment using oligos F15169 and F15168 and on a Bcl I to BamH I fragment using oligos F16128 and F12840. e) Grb-2 (Lowenstein et al. 1992). This adaptor was cloned from human leukocyte cDNA (Clontech). Residues 2 to 217 comprising the cytosolic region of Grb-2 were cloned on a Bcl I to BamH I fragment with oligos D8827 and D8828.

f) GRAP-2 (Qiu et al., 1998). This adaptor was also cloned from human leukocyte cDNA (Clontech). Residues 2 to 330 comprising the cytosolic region of GRAP-2 were cloned on a Bcl I to BamH I fragment using oligos D8825 and D8826.

These PCR products were then digested with the appropriate enzymes to produce suitable fragments for introduction into the cassette system (Example 1) in order to generate adaptor receptors (Example 4).

Example 3: The construction of sequence blocks (SBs) of primary and secondary signalling motifs Each sequence block was generated by annealing two oligos such that they had single-stranded overhangs forming half a Bcl I site at the 5'end and half a BamH I site at the 3'end. Oligos were annealed at a concentration of 1 pmole/l in a buffer consisting of 25mM NaCI, 12.5mM Tris-HCI, 2.5mM MgCI2 0.25mM DTE, pH7.5 by heating in a boiling water bath for 5 minutes and then allowing the bath to cool slowly to room temperature.

The predicted amino acid sequences of these examples of SBs are shown in Figure 4 and sequence of the oligonucleotides employed in their construction are given in Figure 3. All oligonulceotides were phosphorylated at their 5'end. a) SB1. This sequence is based on the first ITAM of human TCR and was constructed by annealing oligos A8816 and A8817. b) SB2. This sequence is based on the second ITAM of human TCR and was constructed by annealing oligos A8814 and A8815. c) SB3. This sequence is based on the third ITAM of human TCR and was constructed by annealing oligos A8812 and A8813. d) SB4. This sequence is based on the ITAM of the y chain of human FceR1 and was constructed by annealing oligos A8810 and A8811.

e) SB4*. This sequence was originally generated in error by mis-annealment of the above oligos but was subsequently made by annealing oligos A8810B and A8811B. f) SB5. This sequence is based on the ITAM of the D chain of human FceRt and was constructed by annealing oligos A9000 and A9001. g) SB6. This sequence is based on the ITAM of the y chain of human CD3 and was constructed by annealing oligos A9002 and A9003. h) SB7. This sequence is based on the ITAM of the 8 chain of human CD3 and was constructed by annealing oligos A9004 and A9005. i) SB8. This sequence is based on the ITAM of the e chain of human CD3 and was constructed by annealing oligos A9006 and A9007. j) SB9. This sequence is based on the ITAM of human CD5 and was constructed by annealing oligos A9008 and A9009. k) SB10. This sequence is based on the ITAM of human CD22 and was constructed by annealing oligos A9010 and A9011.

I) SB11. This sequence is based on the ITAM of human CD79a and was constructed by annealing oligos A9012 and A9013. m) SB12. This sequence is based on the ITAM of human CD79b and was constructed by annealing oligos A9014 and A9015. n) SB13. This sequence is based on the ITAM of human CD66d and was constructed by annealing oligos A9016 and A9017.

o) SB14. This sequence is synthetic and was constructed by annealing oligos D5258 and D5259. p) SB15. This sequence is synthetic and was constructed by annealing oligos F6392 and F6394. q) SB16. This sequence is synthetic and was constructed by annealing oligos F6393 and F6395. r) SB28. This sequence is based on the secondary signalling (costimulation) sequence of human CD28 and was constructed by annealing oligos A9018 and A9019. s) SB29. This sequence is based on the secondary signalling (costimulation) sequence of human CD154 and was constructed by annealing oligos A9020 and A9021. t) SB34. This sequence is based on the secondary signalling (costimulation) sequence of human CD134 and was constructed by annealing oligos F1340A and F1340B.

Example 4: The construction of adaptor receptors The cloning of adaptor molecules on the various fragments described, along with primary and secondary signalling motifs on Bel I to BamH I fragments, allows the construction of a multiplicity of adaptor receptors. Thus, the cytoplamsic signalling regions of these receptors comprise an adaptor molecule either on its own or in combination with any number of any additional adaptor molecules and/or primary signalling motifs and/or secondary signalling sequences.

Adaptor molecules with a natural transmembrane region may be cloned on a Nar I to BamH I fragment into the cassette described in Example 1, thus replacing the transmembrane region. This fragment may be cloned upstream of other primary signalling motifs or secondary signalling sequences or adaptor

molecules, alternatively other primary signalling motifs, secondary signalling sequences, or adaptor molecules may subsequently be cloned downstream.

Adaptor molecules may be cloned on a Mlu I to Mlu I fragment into the cassette described in Example 1, downstream of the transmembrane region. This fragment may be cloned upstream of other signalling motifs or adaptor molecules. Subsequently, other primary signalling motifs, secondary signalling sequences or adaptor molecules may be cloned downstream, by insertion at the BamH I site in the vector.

Adaptor molecules may be cloned on a Bcl I or Bgl II to BamH I fragment into the BamH I site of the cassette described in Example 1, downstream of the transmembrane region. Cloning in the correct orientation allows the subsequent cloning of other primary signalling motifs, secondary signalling sequences or adaptor molecules downstream. Alternatively/in addition, such a fragment may be cloned downstream of other primary signalling motifs, secondary signalling sequences or adaptor molecules that have already been cloned into the cassette.

Primary signalling motifs and secondary signalling sequences (encoded on SBs, see Figure 4) on Bcl I to BamH I fragments may be cloned into the BamH I site of the cassette described in Example 1, downstream of the transmembrane region. Cloning in the correct orientation allows the subsequent cloning of other signalling motifs/sequences or adaptor molecules downstream. Alternatively/in addition such a fragment may be cloned downstream other primary signalling motifs, secondary signalling sequences or adaptor molecules that have already been cloned into the cassette.

This cassette also facilitates exchange of binding components on a Not I/Hind III to Spe I fragment; exchange of extracellular spacers on a Spe I to Nar I fragment and exchange of transmembrane regions on a Nar I to Mlu I fragment. Thus adaptor receptors with different binding, extracellular spacer, transmembrane and signalling components can be easily assembled.

Example 5: Analysis of Adaptor receptors a) Construction of expression plasmids. The adaptor receptor constructs were subcloned from pBluescript KS+ into the expression vector pEE6hCMV. ne (Cockett, et al., 1991) on a Hind III to EcoR I restriction fragment. The empty expression vector (i. e. lacks in chimeric receptor genes) is used as a negative control. b) Transfection into Jurkat E6.1 cells. To generate stable cell lines, the expression plasmids were linearised and transfected into Jurkat E6.1 cells (ECACC) by electroporation using a BioRad Gene Pulser : cells (-2. 5 X106) were mixed with DNA (10Rg) and pulsed twice at 1kV, 3RF (0. 4cm electrode gap cuvette) in 1 ml PBS. Cells were left to recover overnight in non-selective media before being selected and cultured in media supplemented with the antibiotic G418 (Sigma) at 1.5mg/ml. After approximately four weeks cells were ready for analysis.

For transient expression in Jurkat cells, the expression plasmids were transfected using DuoFect (Quantum Biotechnologies Inc.) according to the supplier's instructions. c) Analysis of surface expression: FACS. Approximately 5X105 Jurkat cells were stained with 1Rg/ml FITC labelled antigen or antibody specific for the binding component of the adaptor receptor. For analysis of receptors with a P67scFV binding component, FITC-labelled CD33 antigen was employed.

Fluorescence was analysed by a FACScan cytometer (Becton Dickinson). d) Analysis of function: IL-2 production 2X105 cells were incubated at 37°C with 8% C02 for 20 hours in 96 well plates with target cells at an effector: target ratio of 1: 1. When further stimulation was provided by antibodies, either primary stimulation with OKT3 or secondary stimulation with anti-CD28 (Caltag) was used; these antibodies were added to give a final concentration of 2 to 5 Rg/ml.

Cell supernatants were then harvested and assayed for human IL-2 (R & D Systems Quantikine kit).

In the case of the binding component being P67scFV, the target cells used were: HL60 cells-a human cell line naturally expressing the antigen, CD33.

N. EE6-a mouse myeloma (NSO) transfected with a control expression vector.

These cells are used as a negative control target cell line.

N. CD33-a mouse myeloma (NSO) transfected with an expression vector facilitating the expression of antigen CD33 on the cell surface.

Example 6: Results The specific production of IL-2 by Jurkat cells expressing adaptor receptors, in response to antigen challenge (either by HL-60 or N. CD33 cells as indicated), is used as a measure of the degree of cellular activation in all of the experiments described below. a) Antigen specific stimulation of an adaptor receptor (Figure 5). HL60 cells were used to provide an antigen challenge to Jurkat cells expressing the adaptor receptor P67scFV/h. CD28/LATtm/LAT, (comprising the P67 scFv binding component, h. CD28 spacer, LAT transmembrane and signalling components).

The results in Figure 5 demonstrate that cells expressing adaptor receptors are capable of producing IL-2 as a specific response to this challenge. b) Provision of additional, primary, signaling to an adaptor receptor using an OKT3 antibody (Figure 6). HL60 cells were used to provide an antigen challenge to Jurkat cells expressing the adaptor receptor P67scFV/h. CD28/LATtm/LAT. Figure 6 shows that the additional provision of primary signalling, using the antibody OKT3, to cells expressing adaptor receptors results in increased IL-2 production. This implies that the addition of a primary signalling motif is advantageous and would result in enhanced cellular activation.

c) Provision of additional, primary, siqnallinq to an adaptor receptor using a primary signaling motif (Figure 7). Jurkat cells expressing the adaptor receptors P67scFV/h. CD28/LATtm/LAT and P67scFV/h. CD28/LATtm/LAT. SB14 were challenge with HL60 cells. The results, illustrated in Figure 7, show that IL-2 production by cells expressing adaptor receptors can be enhanced significantly by the inclusion of a primary signalling motif within the receptor. In this particular instance the primary signalling motif SB14a has been included in a position 3'to the adaptor molecule to generate the receptor P67scFV/h. CD28/LATtm/LAT. SB14. d) Further exemplification of the provision of additional primary signallinq capabilitv to an adaptor receptor by the inclusion of a primary signalling motif (Figure 8). The data presented in Figure 8 provides further evidence that IL-2 production in Jurkat cells expressing adaptor receptors can be improved by the inclusion of a primary signalling motif within the adaptor receptor. In this particular experiment, the antigen challenge is provided by N. CD33 cells. This again demonstrates that that IL-2 is produced in response to a specific stimulus i. e. the CD33 antigen and proves that it is not the result of non-specific stimulation by a human cell-line. e) Provision of additional primary siqnallinq capacity to an adaptor receptor already havinq a primarv sianalling motif using the OKT3 antibody (Figure 9).

N. CD33 cells were used to provide an antigen challenge to Jurkat cells expressing the adaptor receptor P67scFV/h. 28/LATtm/LAT. SB14. The results shown in Figure 9 demonstrate that IL-2 production can be increased even more by providing further primary signalling using the antibody, OKT3. This suggests that the addition of a further (i. e. more than one additional) primary signalling motif would be advantageous. f) Provision of additional, secondary, signalling to adaptor receptors using an anti-CD28 antibody (Figure 10). N. CD33 cells were used to provide an antigen challenge to Jurkat cells expressing the adaptor receptors P67scFV/h. 28/LATtm/LAT and P67scFV/h. 28/LATtm/LAT. SB14. The results in

Figure 10 show that IL-2 production by cells expressing adaptor receptors can be increased further by the additional provision of secondary signalling using an anti-CD28 antibody. This suggests that the addition of secondary signalling sequence would be advantageous in the presence or absence of a primary signalling motif. g) Comparison of the signallinci capability of different LAT adaptor receptors (Figure 11). HL60 cells were used to provide an antigen challenge to Jurkat cells transiently expressing different LAT adaptor receptors. The results in Figure 11 show that LAT adaptor receptors with different additional signalling motifs/sequences (primary and/or secondary), are capable of modulating signalling to different degrees. h) Comparison of the signalling capability of different SIT adaptor receptors (Figure 12). HL60 cells were used to provide an antigen challenge to Jurkat cells transiently expressing SIT adaptor receptors with different additional signalling motifs/sequences (primary and/or secondary). The results are shown in Figure 12. SIT may be providing negative signalling through the ITIM (intracellular tyrosine inhibitory motif) reported in its cytosolic region. i) Comparison of the signalling capability of different TRIM adaptor receptors (Figure 13). HL60 cells were used to provide an antigen challenge to Jurkat cells transiently expressing different TRIM adaptor receptors. The results in Figure 13 show that TRIM adaptor receptors with different additional signalling motifs and/or sequences (primary and/or secondary), are capable of modulating signalling to different degrees.

REFERENCES CITED Ausubel, F. W. & Kingston R. E. (1992) Short protocols in Molecular Biology.

2nd edition. John Wiley & Sons Ltd.

Bruyns, E., Marie-Cardine, A., Kirchgessner, H., Sagolla, K., Shevchenko, A., Mann, M., Autschbach, F., Bensussan, A., Meuer, S. Shraven, B. (1998) Journal of Experimental Medicine 188: 561-575.

Burshtyn, D. N., Lam, A. S., Weston, M., Gupta, N., Warmerdam, P. A. & Long, E. O. (1999) Journal of Immunology 162: 897-902.

Cockett, M. I., Bebbington, C. R. & Yarranton, G. T. (1991) Nucleic Acids Research 19: 319-325.

DeFranco A. L. (1997) Current Opinion in Immunology 9: 296-308.

Ellis. J. H., Ashman, C., Burden, M. N., Kilpatrick, K. E., Morse, M. A. & Hamblin, P. A. (2000) Journal of Immunology 164: 5805-5814.

Finney, H. M., Lawson, A. D. G., Bebbington, C. R. & Weir, A. C. N. (1998) Joumal of Immunology 161: 2791-2797.

Glover, D. M. 1995a. DNA cloning : a practical approach, Volume Expression systems. IRL press.

Glover, D. M. 1995b. DNA cloning : a practical approach, Volume IV : Mammalian systems. IRL press.

Jackman, J. K., Motto, D. G., Sun, Q., Tanemoto, M., Turck C. W., Peltz, G. A., Koretzky, G. A. & Findell, P. R. (1995) Journal of Biological Chemistry 270 : 7029-32 Kuwana, Y., Asakura, Y., Utsunomiya, N., Nakanishi, M., Arat, Y., Itoh, S., Nagese, F. & Kurosawa, Y. (1987) Biochemical and Biophysical Research Communications 149: 960-968.

Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D. & Schlessinger, J. (1992) Ce//70 : 431- 442.

Marie-Cardine, A., Kirchgessner, H., Bruyns, E., Shevchenko, A., Mann, M., Autschbach, F., Ratnofsky, S., Meuer, S. & Schraven, B. (1999) Journal of Experimental Medicine 189: 1181-1194.

O'Reilly, D. R., & Miller, L. K. (1993). Baculovirus expression vectors-A laboratory manual. Oxford University Press.

Qiu, M., Hua, S., Agrawal, M., Li, G., Cai, J., Chan, E., Zhou, H., Luo, Y. & Liu, M. (1998) Biochemical and Biophysical Research Communications 253: 443-447.

Peterson, E. J., Clements, J. L., Fang, N. & Koretzky, G. A. (1998) Current Opinion in Immunology 10: 337-344.

Rees, A. R (1993) Protein Engineering : a practical approach. IRL press Romeo, C., Kolanus, W., Amiot, M. & Seed, B. (1992) Cold Spring Harbour Symposia on Quantitative Biology. Volume LVII pp 117-125.

Rudd, C. E. (1998) Current Biology. 8 (22): R805-8.

Sambrook, J., & Fritsch, E. (1989) Molecular cloning : a laboratory manual.

2nd edition. Cold Spring Harbour Press, N. Y.

Samelson, L. E. (1999) Progress in Biophysics & Molecular Biology 71: 393- 403.

Weber, J. R., Orstavik, S., Torgersen, K. M., Danbolt, N. C., Berg, S. F., Ryan, J. C., Tasken, K., Imboden, J. B. & Vaage, J. T. (1998). Journal of Experimental Medicine 187: 1157-1161.

Weiss, A. & Littman, D. R. (1994) Ce//76 : 26-274.

Zhang, W., Trible, R. P. & Samelson, L. E. (1999) Immunity 9: 239-246.