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EP1226245A2 - Recepteurs-adaptateurs - Google Patents

Recepteurs-adaptateurs

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Publication number
EP1226245A2
EP1226245A2 EP00973001A EP00973001A EP1226245A2 EP 1226245 A2 EP1226245 A2 EP 1226245A2 EP 00973001 A EP00973001 A EP 00973001A EP 00973001 A EP00973001 A EP 00973001A EP 1226245 A2 EP1226245 A2 EP 1226245A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
adaptor
acid according
signalling
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00973001A
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German (de)
English (en)
Inventor
Helene Margaret Finney
Alastair David Griffiths Lawson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Celltech R&D Ltd
Original Assignee
Celltech R&D Ltd
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Filing date
Publication date
Application filed by Celltech R&D Ltd filed Critical Celltech R&D Ltd
Publication of EP1226245A2 publication Critical patent/EP1226245A2/fr
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to nucleic acids encoding adaptor receptor proteins, as well as the receptor proteins themselves and a method of activating cells using such nucleic acids and proteins.
  • the invention also encompasses the use of the nucleic acids, proteins and method of the invention in the fields of medicine and research.
  • 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.
  • the components of the TCR complex which are required for the initial signalling events, are well characterised.
  • chimeric receptor proteins 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.
  • 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 intraceUular signalling domain of a chimeric receptor, in combination with, for example, an extracellular ligand binding domain and a transmembrane domain.
  • 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.
  • 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 intraceUular 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 intraceUular signalling domain, wherein the intraceUular 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 includes 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.
  • cytoplasmic adaptor proteins include SLAP, SLP-76, SKAP55, Grap, 3BP2, Grb-2, Nek, CRKL, She, and Cbl.
  • the adaptor protein Grap2 which is also known as GrbX, GrbLG, Grf40, Gads and GRID (Ellis et al., 2000), is yet another example of a cytoplasmic adaptor protein that may be used in the invention.
  • 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 intraceUular 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.
  • 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 ⁇ , ⁇ or ⁇ chain of the T-cell receptor, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.
  • transmembrane regions derived from all or part of the ⁇ , ⁇ or ⁇ 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • extracellular ligand-binding domain 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 (V or VL), T-cell receptor variable region domains (TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ ) 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 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.
  • cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.
  • 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.
  • 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.
  • NK natural killer
  • 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.
  • cytoplasmic signalling component 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.
  • T cell cytoplasmic signalling components can be subdivided 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).
  • primary signalling sequence may contain one or more primary signalling motif responsible for signal transduction.
  • 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.
  • 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.
  • stimulatory primary signalling motifs include any sequence that broadly conforms to the consensus sequence Y- X 2 - -X n -Y-X 2 -L/I such as for example immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • 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 l/V-X-Y-X 2 - 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 nucle
  • Stimulatory primary signalling motifs may contain the consensus amino acid sequence Y-X 2 -IJI-X n -Y-X 2 -L/I.
  • X represents any amino acid
  • a subscripted number indicates the number of residues present at that position within the motif
  • 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 X 2 or X ⁇ can represent 2 or n amino acids (respectively) which may either be the same or different.
  • n lies between 6 and 8
  • 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 TCR ⁇ l , TCR ⁇ 2, TCR ⁇ 3 (i.e the first, second or third, ITAMs of the TCR ⁇ chain), FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b or CD66d, or variants thereof.
  • ITAM immunoreceptor tyrosine based activation motif
  • At least one additional primary signalling motif for use in the invention will be derived from all or part of TCR ⁇ l , TCR ⁇ 2, TCR ⁇ 3, FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b or CD66d, or a variant thereof.
  • 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-X 2 -L/I-X n -Y-X2-L/I.
  • Preferred examples of such non-natural primary signalling motifs with a value of n between 6 and 8 will be SB14 a or SB15 a as described herein in Table 1 , or non-natural variants thereof. Where the value of n is 9 or greater, SBX a , SBQ9 a , SB16 a or non-natural variants thereof, may be employed as the additional motif(s), with SB16 a being especially preferred (Table 1 ).
  • FIG. 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.
  • TCR ⁇ 3 SB3 a RGKGHDGLYQGLSTATKDTYDALHMQA
  • FcR ⁇ SB5 a GNKBPEDRVYEELNIYSATYSELEDPGEMSP
  • CD3 ⁇ SB6 a KQTLLPNDQLYQPLKDREDDQYSHLQGNQLR
  • CD3 ⁇ SB8 a QNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI
  • CD5 SB9 a HVDNEYSQPPRNSRLSAYPALEGVLHRS
  • CD22 SB10 a PPRTCDDTVTYSALHKRQVGDYENVIPDFPEDE
  • CD66d SB13 a PLPNPRTAASIYEELLKHDTNIYCRMDHKAEVA
  • Non- SB15 a GQDGLYQELNTRSRDEAYSVLEGRKAR natural
  • Non- SB16 a GQDGLYQELNTRSRDEAAYSVLEGRKAR natural
  • Non- SBX a RKNPQEGLYNELQKDKMAEDTYDALHMQA natural
  • Non- SBQ9 a GQNQLYNELQQQQQQQQYDVLRRGRDPEM natural
  • ITIMs 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.
  • ITIMs for use in the present invention include those derived from Fc ⁇ R (e.g. Fc ⁇ RIIB), CD22, EPOR, IL-2 ⁇ R or IL-3 ⁇ R.
  • 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, SB28 a RLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FA, SB29 a MIETYNQTSPRSAATGLPISMK and SB34 a 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.
  • 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.
  • 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.
  • 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, potentially enzymatic, domains thus provides a multiplicity of adaptor receptors, each capable of regulating cellular activation to a different degree.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • oligonucleotide-directed mutagenesis oligonucleotide-directed synthesis techniques
  • 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.
  • 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 WO96/10038, W097/18185, W097/25329, WO97/30170 and W097/31934.
  • the vector may additionally 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.
  • 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.
  • TTRs retrovirus long terminal repeats
  • AAV repeats SV40 and human cytomegalovirus (hCMV) promoters and/or enhancers
  • splicing and polyadenylation signals and EBV and BK virus replication functions.
  • TCR- ⁇ promoter such as the TCR- ⁇ promoter, E-selectin promoter and the CD2 promoter and locus control region
  • 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.
  • prokaryotic cells such as Escherichia coli to express heterologous polypeptides and polypeptide fragments is well established (see for example, Sambrook & Fritsch, 1989, Glover, 1995a).
  • 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).
  • the vectors of choice are virus-based.
  • viral vectors include baculovirus-, adenovirus-, and vaccinia virus-based vectors.
  • Vectors containing the relevant regulatory sequences 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 ai, 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.
  • adaptor receptors may be expressed in vivo, for example in insect larvae, plant cells, or in particular in mammalian tissues.
  • nucleic acid may employ any available technique.
  • 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.
  • 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.
  • the cells may be cultured under conditions to enhance or induce expression from the adaptor receptor gene as appropriate.
  • further aspects of the invention provide host cells containing a nucleic acid encoding an adaptor receptor, and host cells expressing an adaptor receptor protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable targeted delivery systems include targeted naked DNA, targeted liposomes encapsulating and/or complexed with the DNA, targeted retroviral or adenoviral systems and targeted condensed DNA such as protamine and polylysine-condensed DNA.
  • Targeting systems 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 dlL-2R.
  • CD8 e.g. CD8
  • CD16 e.g. CD16
  • CD4 e.g. CD5
  • CD7 e.g. CD24
  • activation antigens e.g. CD69 an dlL-2R.
  • other receptor-ligand interactions can be used for targeting e.g. CD4 to target HIV gp 160-expressing target cells.
  • 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.
  • 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.
  • 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 TCR ⁇ and TCR ⁇ promoters, cytokine promoters, such as the IL2 promoter, and the perform promoter.
  • T cell specific promoter systems such as the zeta promoter, CD2 promoter and locus control region, CD4, CD8 TCR ⁇ and TCR ⁇ promoters, cytokine promoters, such as the IL2 promoter, and the perform promoter.
  • adaptor receptor proteins of the present invention 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.
  • 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.
  • infectious diseases e.g. HIV infection
  • inflammatory disease/autoimmunity e.g. asthma, eczema
  • congenital e.g. cystic fibrosis sickle cell anaemia
  • dermatologic e.
  • 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.
  • 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.
  • an effector cell of the immune system 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.
  • 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 NaCl, 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.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates and the like
  • 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.
  • 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.
  • parenteral administration e.g. by injection or infusion
  • it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents.
  • the composition may be in dry form, for reconstitution before use with an appropriate sterile liquid.
  • 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.
  • 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.
  • a formulation suitable for oral administration be well tolerated by the patient's digestive system.
  • 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 SB14 a .
  • Figure 8 Provision of primary signalling to the adaptor receptor p67scFv/h.CD28/LATtm/LAT by addition of a primary signalling motif SB14 a .
  • 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 1 1 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.
  • FIG. 13 Transient transfection analysis in Jurkat of TRIM adaptor receptors with various additional primary signalling motifs and secondary signalling sequences.
  • Example 1 Construction of the cloning vector, pHMF393
  • 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.
  • a 200bp fragment was assembled 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.
  • LAT (Weber et al., 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 Bel I to BamH I fragment) using oligos F12315 and D3458.
  • TRIM (Bruyns 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.
  • 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.
  • SLP-76 (Jackman et al., 1995). 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 Bel I to BamH I fragment using oligos F16128 and F12840.
  • 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 Bel 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 Bel I to BamH I fragment using oligos D8825 and D8826.
  • Each sequence block was generated by annealing two oligos such that they had single-stranded overhangs forming half a Bel 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 NaCl, 12.5mM Tris-HCI, 2.5mM MgCI 2 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.
  • SB1 This sequence is based on the first ITAM of human TCR ⁇ and was constructed by annealing oligos A8816 and A8817.
  • SB2 This sequence is based on the second ITAM of human TCR ⁇ and was constructed by annealing oligos A8814 and A8815.
  • SB4 This sequence is based on the ITAM of the ⁇ chain of human Fc ⁇ R1 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 A8811 B.
  • SB8 This sequence is based on the ITAM of the ⁇ chain of human CD3 and was constructed by annealing oligos A9006 and A9007.
  • n) SB13 This sequence is based on the ITAM of human CD66d and was constructed by annealing oligos A9016 and A9017.
  • SB14 This sequence is synthetic and was constructed by annealing oligos D5258 and D5259.
  • r) SB28 This sequence is based on the secondary signalling (costimulation) sequence of human CD28 and was constructed by annealing oligos A9018 and A9019.
  • 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.
  • 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 Bel 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.
  • 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.
  • 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.
  • ECACC electroporation using a BioRad Gene Pulser
  • cells (-2.5 X10 6 ) were mixed with DNA (10 ⁇ g) and pulsed twice at 1kV, 3 ⁇ F (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.
  • the expression plasmids were transfeeted using DuoFect (Quantum Biotechnologies Inc.) according to the supplier's instructions.
  • FITC-labelled CD33 antigen was employed for analysis of receptors with a P67scFV binding component. Fluorescence was analysed by a FACScan cytometer (Becton Dickinson).
  • IL-2 production 2X10 5 cells were incubated at 37°C with 8% C0 2 for 20 hours in 96 well plates with target cells at an effector:target ratio of 1 :1.
  • additional 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 ⁇ g/ml.
  • Cell supernatants were then harvested and assayed for human IL-2 (R & D Systems Quantikine kit).
  • the target cells used were: HL60 cells - a human cell line naturally expressing the antigen, CD33.
  • N.EE6 - a mouse myeloma (NSO) transfeeted with a control expression vector. These cells are used as a negative control target cell line.
  • N.CD33 - a mouse myeloma (NSO) transfeeted with an expression vector facilitating the expression of antigen CD33 on the cell surface.
  • IL-2 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.
  • Figure 8 Further exemplification of the provision of additional primary signalling capability to an adaptor receptor by the inclusion of a primary signalling motif.
  • 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.
  • 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.

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Abstract

L'invention concerne des protéines réceptrices chimériques, des récepteurs-adaptateurs, utilisant des protéines adaptatrices en tant que composants de marquage intracellulaire. Le niveau de marquage intracellulaire diffusé par l'adaptateur-récepteur peut être ajusté comme on le souhaite, en y incorporant diverses protéines adaptatrices et en y associant éventuellement d'autres séquences de marquage cytoplasmique, comme domaine de marquage intracellulaire d'un adaptateur-récepteur. L'invention concerne également des acides nucléiques codant des récepteurs-adaptateurs, ainsi que des protéines récepteurs-adaptateurs adéquates pour un usage médical.
EP00973001A 1999-11-01 2000-11-01 Recepteurs-adaptateurs Withdrawn EP1226245A2 (fr)

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