WO2000063373A1 - A method of altering the properties of a membrane-associated protein by substitution of the transmembrane domain - Google Patents

Abstract

The invention relates to methods of altering the properties of membrane-associated proteins, by substituting the transmembrane region. In this way, for example, the level of expression of the protein can be altered as well as properties of the protein itself, such as the relative response of the membrane-associated protein to cell surface-associated antigen versus antigen in solution and the sensitivity of intracellular signalling mediated by the membrane-associated protein.

Description

A METHOD OF ALTERING THE PROPERTIES OF A MEMBRANE-ASSOCIATED PROTEIN BY SUBSTITUTION OF THE TRANSMEMBRANE DOMAIN

The present invention relates to methods of altering the properties of membrane- associated proteins.

5 A chimeric receptor is a protein that is derived by the insertion or substitution of a partial sequence from one protein into another protein, wherein the resultant protein has a defined specificity for ligand. On binding of ligand, a signal is generated intracellularly.

Chimeric receptors have been designed that target cells such as T-cells to other cells that 0 express antigenic ligands on their surface. These receptors facilitate the activation of receptor-bearing cells and can induce subsequent physiological effects that can have therapeutic benefits to a patient. Such compounds are clearly of considerable use in therapy of certain diseases.

In a chimeric receptor, ligand recognition is provided by an extracellular binding region 15 of the molecule, in the same way that a naturally-occurring membrane receptor binds to its target. Accordingly, when designing a chimeric receptor, a suitable extracellular binding region is chosen which binds specifically to its target ligand with high affinity.

Binding of ligand to the chimeric receptor triggers a series of intracellular events leading to activation of the receptor-bearing cell. This activation is effected by the 0 presence in the chimeric receptor molecule of an intracellular signalling domain. Activation of this domain may lead to a variety of biological effects caused by the cell, such as increased cellular proliferation, increased expression of cytokines with, for example, pro- or anti-inflammatory responses, stimulation of cytolytic activity, differentiation or other effector functions, antibody secretion, phagocytosis, tumour 5 infiltration and/or increased cellular adhesion.

Spacer domains have been used in chimeric receptor design to link the intracellular and extracellular domains to each other. Spacer domains have also been used to arrange the domains of the receptor in the desired conformation to optimise the binding or signalling potential of the receptor molecule. The inclusion of such domains in the receptor may also facilitate the initial cloning steps when the nucleic acid elements encoding each domain of the protein are assembled.

The mechanisms by which conventional chimeric receptor proteins convert an extracellular binding event of ligand to receptor into intracellular signalling are unclear. It is thought that these mechanisms involve clustering and association with endogenous cellular effector molecules. However, the relationship between the level of expression of a chimeric receptor protein and strength of intracellular signalling generated appears to vary between different systems. Therapeutic success in using chimeric proteins can depend on matching carefully the level of expression of the protein to the level of expression of the particular target ligand, so that the appropriate degree of receptor cross-linking, clustering and oligomerisation can be achieved for optimal signalling.

The transmembrane component of a chimeric receptor protein and of membrane- associated proteins in general typically serves to link the extracellular binding region of the protein to the intracellular cytoplasmic signalling region and thus to anchor the protein in the membrane of a cell. This component has conventionally been incorporated into the protein as part of the intracellular signalling domain, the spacer domain or the extracellular binding region with which it is associated in the naturally- occurring proteins from which these sequences are derived.

The level of expression of proteins is generally controlled at the level of transcription. Thus, depending upon the level of expression of chimeric receptor protein that is required in the cell, a strong or weak promoter system is used. However, it is not possible to define precisely the level of receptor expression using such a method, since the efficacy of all promoter systems tends to vary between different cell types and under different physiological conditions. Furthermore, the use of strong promoter systems can be physiologically disruptive when used in vivo.

There thus exists a great need in this area of technology for a suitable method of modulating the level of expression of membrane-associated proteins.

A further issue with previously-described chimeric receptor proteins is that these conventional proteins are susceptible to signalling not only in response to cell surface bound ligand, but also in response to the presence of soluble antigen. This is a particularly undesirable characteristic of chimeric receptors and limits the therapeutic potential of this approach (Eshhar, Z. (1997) Cancer Immunol. Immunother. 45: 131- 136). Many antigens that would otherwise make attractive targets for chimeric receptor- based therapy are unsuitable because they are either shed from the surface of cells or are secreted. They thus initiate systemic activation if they are bound by a chimeric receptor.

Specifically, many tumour-associated antigens, such as polymorphic epithelial mucin (PEM) and carcinoembryonic antigen (CEA), which are expressed on a wide range of solid tumours, are shed and are detectable in serum at significant levels. The therapeutic use of chimeric receptors with specificity for this type of antigen ligand would be severely compromised by the presence of a circulating antigen component. This would not only lead to reduced efficacy but could potentially cause systemic toxicity due to the inappropriate release of cytokines.

Accordingly, in order for the chimeric receptor approach to achieve its full therapeutic potential (in particular in cancer immunotherapy), a way must be found to allow such receptors to distinguish between soluble and cell surface bound antigen.

Summary of the invention

According to the present invention there is provided a method of altering the properties of a membrane-associated protein comprising substituting a transmembrane region or a membrane-anchoring region in the membrane-associated protein for a transmembrane region or a membrane-anchoring region that is not naturally part of said membrane- associated protein or for a synthetic transmembrane component.

The properties altered by the substitution can be any property resulting from the inherent nature of the transmembrane region component. For example, it has been discovered that simply by altering this component, significant changes in the level of expression of a protein may be effected in a host cell. This allows the level of membrane-associated protein to be precisely tailored, as required. For example, it may be advantageous for tumour cell killing for T cells to express high levels of receptors with CD3 pathway signalling and low levels of chimeric receptors with CD28 pathway signalling. This could be achieved by use of, for example, a CD28 transmembrane region component for the chimeric receptor with CD3 signalling capability and transmembrane region component derived from the T-cell receptor zeta or alpha chain for the receptor with CD28, signalling capability.

Appropriate choice of the transmembrane region may also be used to match the level of expression of the chimeric receptor to the level of expression of the particular target ligand for the receptor, so that the optimal degree of receptor cross-linking, clustering, oligomerisation and association with endogenous molecules can be achieved, so optimising the activation of the signalling cascade within the cell in which the membrane protein is expressed. In this respect, specific transmembrane regions may be chosen to optimise cross-linking of the protein with other components of the membrane.

Thus, according to a particular aspect of the invention there is provided a method of altering the level of expression of a membrane-associated protein comprising substituting a transmembrane or membrane anchoring region in the membrane- associated protein for a transmembrane region that is not naturally part of said membrane associated protein, or for a synthetic transmembrane component.

Another property, which may be altered in the method of the invention, is the sensitivity of a membrane-associated protein for antigen. Thus, in a further aspect the method of the invention may be used to alter the sensitivity of a membrane-associated protein for antigen, by incorporating in the protein a transmembrane region that is not naturally part of the membrane-associated protein or by incorporating a synthetic transmembrane region. The sensitivity of intracellular signalling, stimulated by binding of antigen to an extracellular ligand binding domain of the protein may also be modified by appropriate choice of transmembrane region.

Similarly a further property, which may be altered by the method of the invention, is the relative response of a membrane-associated protein to cell surface associated antigen versus antigen in solution. By "relative response" is meant the ratio of cell surface- associated antigen to soluble antigen that is bound by a membrane-associated protein. It has been found that the specific inclusion in a chimeric receptor molecule of transmembrane components that are not naturally fused to the other components of the receptor molecule has an effect in modulating the response function of the chimeric receptor to types of antigen. For the avoidance of doubt, the term "not naturally part" as used herein is intended to mean that the transmembrane region does not exist in association with the adjoining domains to which it is attached in nature.

As used herein, the term "transmembrane region" is defined as a predominantly hydrophobic sequence of amino acids that is predicted to span the cell membrane. The transmembrane region may be composed of one or more transmembrane domains. A transmembrane domain may in general be any oligo- or polypeptide which when folded under physiological conditions is of sufficient length to span the membrane of a cell. This domain should be of between 15 and 35 amino acids in length, preferably between 20-31 amino acids, enabling the domain to span a typical cell membrane, which is of the order of between 2 and 6nm in width.

The extremities of a transmembrane domain may be defined by helix -breaker residues that disrupt the structure, for example, proline. Charged residues may also define the ends of the transmembrane region, since these residues are energetically unstable in the hydrophobic environment of the membrane.

A transmembrane domain may be derived from any naturally occurring transmembrane region. In this case the majority of the membrane-associated protein, in which the transmembrane region is to be introduced, will be derived from a different protein to that from which the transmembrane domain is derived. Of particular interest in this aspect of the invention are transmembrane regions and/or domains derived from all or part of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD137, or CD 154. A transmembrane domain from a cytokine receptor may also be used (for example an interleukin receptor, a TNF receptor, or an interferon receptor). It may also be derived from a colony stimulating factor receptor such as GMCSF receptor. Further examples will be clear to those of skill in the art.

Alternatively, a transmembrane domain may be synthetic. Suitable synthetic transmembrane domains will comprise predominantly hydrophobic amino acids such as leucine and valine. Preferably, the synthetic transmembrane component comprises a sequence of amino acid residues of which at least 50%, more preferably at least 80%, are hydrophobic amino acid residues. In this respect, suitable amino acid residues include the hydrophobic residues Ala, Leu, Val, He, Pro, Phe or Met.

The synthetic transmembrane region may also be designed so as to possess an alpha helical structure, by constructing the component from one or more alpha helix- promoting amino acid residues such as Ala, Asn, Cys, Gin, His, Leu, Met, Phe, Tip, Tyr or Val. Preferably, the hydrophobic alpha helix-promoting residues Ala, Met, Phe, Trp or Val are used. The hydrophobic alpha helix-promoting residues Phe, Trp or Val are preferred.

It has also been found preferable to include at each end of the transmembrane component the triplet phenylalanine, tryptophan, valine (FWN).

Examples of synthetic transmembrane regions are given in accompanying Figure 4. The regions of choice are TM20, TM24, TM27, TM29 and TM31. As is clear from this Figure, the preferred synthetic sequences are flanked by "FWN" triplets, whilst the membrane spanning section comprises a string of leucine residues. This sequence is predicted to form an helical structure. Other protein structures capable of spanning a membrane (such as β sheet structures) may also be used.

As used herein, the term " membrane anchoring region" is defined as a sequence that facilitates the attachment of a molecule to a cell membrane.

The degree to which the level of expression of the protein has been altered by replacing the transmembrane region in accordance with the invention can be assessed by a number of methods, as will be clear to the person of skill in the art. Particularly suitable methods are fluorescence-activated cell sorting (FACS) or Western blotting, using an antibody specific for the protein of interest and measurement of cytokine release in response to cell bound and/or soluble antigen.

The term "membrane- associated protein" as used herein is intended to mean any protein that contains one or more transmembrane domains. The protein may be, for example, a chimeric receptor comprising an extracellular ligand binding domain and at least one transmembrane domain optionally linked to one or more intracellular signalling domains as described hereinafter. Alternatively, the membrane-associated protein may consist solely of one or more transmembrane domains linked to one or more intracellular domains as described below.

For membrane associated proteins with more than one transmembrane domain, such as proteins of the tetraspan family, substitutions may be made of one or more transmembrane domains.

An extracellular ligand binding domain may be present in the membrane-associated protein to define the required specificity of the protein for antigen. As used herein, the term "extracellular ligand binding domain" is intended to refer to any oligo- or polypeptide that is capable of binding to a ligand. Accordingly, this term is intended to include any binding domain of any molecule with affinity for ligand. The term thus includes antibody binding domains, antibody hypervariable loops and CDR domains, receptor binding domains and other ligand binding domains, examples of which will be readily apparent to those of skill in the art.

Preferably, the extracellular ligand binding domain is capable of interacting with a cell surface molecule. For example, this domain may be chosen to recognise a cell surface marker ligand expressed on target cells associated with a disease state such as viral, bacterial and parasitic infection, auto-immune disease, inflammation and cancer.

Examples of markers for cancer cells are the bombesin receptor expressed on lung tumour cells, CEA, PEM, CD33, Folate receptor, epithelial cell adhesion molecule (EPCAM) and erb-B2. Other molecules of choice are cell surface adhesion molecules, inflammatory cells present in auto-immune disease and T-cell receptors or antigens that give rise to autoimmunity. Further examples will be readily apparent to those of skill in the art.

In one aspect of the invention, the extracellular ligand binding domain may be chosen such that it interacts with one or more other extracellular ligand binding domains of other receptors. This aspect of chimeric receptor design is described in detail in co- pending co-owned patent application GB9809658.9 (Biological Products), the content of which is incorporated by reference herein in its entirety. Membrane-associated proteins produced according to the method of the invention may provide multiply-associated domains that are capable of recognising a cell surface marker ligand expressed on a target cell. In this respect, particularly useful extracellular ligand binding domains include parts of receptors associated with binding two cell surface-associated molecules and especially include an antibody variable domain (V_H or V_L), a T-cell receptor variable region domain (TCRα, TCRβ, TCRγ, TCRδ) or a CD8α, CD8β, CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B, CD49C, CD49D, CD49E, CD49F, CD61, CD41 or CD51 chain. Of course, fragments of these domains or chains may be used where appropriate.

More than one extracellular ligand binding domain may be incorporated into the membrane-associated protein. Proteins which feature more than one extracellular ligand binding domain may, for example, recruit cellular immune effector cells such as T-cells, B-cells, NK-cells, macrophages, neutrophils, eosinophils, basophils or mast cells or components of the complement cascade. A particularly suitable combination of ligand specificities is anti-CD3 with anti-CD28, to specifically recruit and stimulate T-cells.

As will be clear to the skilled artisan, these combinations of extracellular ligand binding domains can be on separate polypeptide chains or may be in series on a single polypeptide chain.

It may also be desired for the extracellular ligand binding domains to be able to interact co-operatively with each other to form a ligand binding site. Particular examples include a V_H domain paired with a V_L domain, two or more TCRα, TCRβ, TCRγ and/or TCRδ domains, a CD8α or CD8β homo or heterodimer, CD 18 paired with one or more of CD1 la, b, or c, CD29 paired with one or more of CD49a, b, c, d, e or f and CD61 paired with CD41c and/or CD51. In this aspect of the invention, in binding to ligand, each extracellular ligand binding domain forms part of a ligand binding site and in doing so establishes a close spatial proximity of the chains which constitute the chimeric receptor.

In embodiments of the invention that involve co-operative interaction of chimeric receptor molecules, the transmembrane region component will desirably be chosen or designed so as to minimise its constitutive association with any other domain in the chimeric receptor molecule. Ideally, in these embodiments the transmembrane region component will be designed to allow association of the receptor polypeptide chains only when ligand is bound by one or more of the extracellular domains. This preferable feature reduces undesirable random signal generation by ensuring that the intracellular signalling domains only interact when ligand is bound by the extracellular domain.

Where the membrane-associated protein produced according to the invention contains an extracellular ligand binding domain linked to one or more transmembrane domains, but lacks any intracellular signalling domain, such a protein may function as a recruitment receptor. Examples of recruitment receptors and their use is described in co- pending International patent application entitled "Synthetic Transmembrane Components" (Reference PA448; P021405WO) filed by Celltech Therapeutics Limited on even date herewith.

The membrane-associated proteins produced according to the invention may incorporate an intracellular signalling domain. As used herein, the term "intracellular signalling domain" is intended to mean any oligopeptide or polypeptide that can participate in the transduction of a signal which results in the direct or indirect activation of one or more intracellular messenger systems. Suitable intracellular messenger systems include, for example, kinase pathways such as those involving tyrosine kinase, protein kinase C or MAP kinase; G-protein or phospholipase-mediated pathways; calcium-mediated pathways; and pathways involving synthesis of a cytokine such as an interleukin e.g. LL-2, including NFAT and cAMP mediated pathways.

The intracellular signalling domain may be a naturally-occurring polypeptide signalling sequence or may be synthetic. Examples of suitable naturally-occurring sequences include sequences derived from: the T cell receptor, such as all or part of the zeta, eta or epsilon chain; CD28; CD4; CD8; the gamma chain of an Fc receptor or signalling components from a cytokine receptor, such as the interleukin, TNF or interferon receptors; a colony stimulating factor receptor e.g. GMCSF, tyrosine kinase e.g. ZAP- 70, fyn, lck, Itk and syk; and binding domains thereof; an adhesion molecule e.g. LFA-1 and LFA-2; B29; MB-1; CD3 delta; CD3 gamma; CD5; or CD2.

Suitable synthetic intracellular signalling domains may contain peptide sequences that are similar to or are derived from any natural domain or portion thereof. As the skilled artisan will appreciate, amino acid mutations, deletions, insertions or substitutions may be made from natural sequences in order to modify the precise properties of the domains, in accordance with what is required for the membrane-associated protein. Examples of suitable synthetic signalling domains are given in co-pending International patent application entitled "Synthetic signalling molecules", (Ref. PA451; P021408WO) filed by Celltech Therapeutics Limited on even date herewith.

These signalling domains may be combined so as to allow the activation of a number of secondary messenger cascades through a single binding event. As will be clear to the skilled artisan, combinations of intracellular signalling domains can be on separate polypeptide chains or may be in series on a single polypeptide chain.

Between the extracellular ligand binding domain and transmembrane region, or between the intracellular ligand binding domain and the transmembrane region, there may be incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link the transmembrane domain to either of the extracellular ligand binding domains or intracellular signalling domains in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 2 to 100 amino acids and most preferably 25 to 50 amino acids.

Spacer domains may be 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, including the hinge region; all or part of natural spacer components between the functional parts of intracellular signalling molecules, for example spacers between ITAMS (immunoglobulin tyrosine based activation motifs) 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.

In one aspect of the invention, spacer domains may be incorporated into a membrane- associated protein that do not associate with one another. This aspect of receptor design is intended to minimise constitutive association of the membrane-associated proteins and so prevent constitutive activation of the molecules. Of course, the opposite effect may also be achieved if constitutive activation is desired. 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 sidechain 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.

According to a further aspect of the invention there is also provided a membrane- associated protein comprising an intracellular signalling domain and a transmembrane region, wherein said transmembrane region is not naturally fused to said intracellular signalling domain.

The transmembrane region of the membrane-associated proteins produced according to, or featuring as the above aspects of the invention may be a natural transmembrane domain derived from a naturally-occurring membrane associated protein, or may be synthetic. Suitable candidate transmembrane domains, both natural and synthetic, are discussed in some detail above, as are suitable extracellular ligand binding domains, intracellular signalling domains and spacer domains. Preferably the extracellular ligand binding domain, spacer domain, transmembrane region and intracellular signalling domains of the membrane-associated proteins of the invention are derived from or are based on mammalian, most preferably human, sequences.

According to a still further aspect of the invention, there is provided a nucleic acid molecule encoding a membrane-associated protein according to any one of the above- described aspects of the invention. Preferably, the nucleic acid molecule comprises DNA.

Nucleic acid coding sequences for use in the invention are widely reported in the scientific literature and are also available in public databases. 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 oligonucleotides. Such techniques are described by Maniatis et al in Molecular Cloning, Cold Spring Harbor Laboratory, New York 1989 and in the Examples contained herein. The DNA of this aspect of the invention may be used with a carrier. The carrier may be a vector or other carrier suitable for introduction of the DNA ex-vivo or in-vivo into target cells 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 vectors may also be used, such as liposomal vectors and vectors based on DNA condensing agents such as the cationic lipids described in International patent applications nos. WO96/10038, WO97/18185, WO97/25329, WO97/30170 and WO97/31934. Where appropriate, 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. Tissue-specific regulatory sequences such as the TCR-α promoter, E-selectin promoter and the CD2 promoter and locus control region may also be used. The carrier may be an antibody.

Each DNA molecule coding for a polypeptide chain of the chimeric receptor may be incorporated into a different carrier as described above. Preferably however, the DNA is incorporated into the same carrier. For this the DNA may be located for example on separate plasmids or may be advantageously part of a single plasmid additionally containing one or more promoter and/or regulatory sequences and/or replication functions as described above. The invention extends to a plasmid comprising DNA coding for a chimeric receptor according to the invention. Particularly useful plasmids of this type include the modified pBluescript SK+ (Stratagene) plasmid described in International patent application no. WO97/23613 and in the Examples contained herein.

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

Additionally, in the absence of a naturally-effective signal peptide in the protein sequence, it may be convenient to cause the recombinant protein to be secreted from certain hosts. Accordingly, further components of such vectors may include nucleic acid sequences encoding secretion signalling and processing sequences.

Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses). Many such vectors and expression systems are well known and documented in the art. Particularly suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus-based vectors.

The expression of heterologous polypeptides and polypeptide fragments in prokaryotic cells such as E. coli is well established in the art; see for example Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or DNA cloning: a practical approach, Volume II: Expression systems, edited by D.M. Glover (IRL Press, 1995). Expression in eukaryotic cells, including plant cells, in culture is also an option available to those skilled in the art for the production of heterologous proteins; see for example O'Reilly et al., (1994) Baculovirus expression vectors - a laboratory manual (Oxford University Press) or DNA cloning: a practical approach, Volume IV: Mammalian systems, edited by D.M. Glover (IRL Press, 1995) .

Suitable vectors can be chosen or constructed for expression of the membrane- associated proteins of the invention, containing the appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. bacteriophage, or phagemid, as appropriate. For further details see Molecular Cloning: a Laboratory Manual. Many known techniques and protocols for manipulation of nucleic acid, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., (John Wiley & Sons, 1992) or Protein Engineering: A practical approach (edited by A.R. Rees et al., IRL Press 1993). For example, in eukaryotic cells, the vectors of choice are virus-based.

A further aspect of the present invention provides a host cell containing a nucleic acid encoding a membrane-associated protein of the above-described aspects of the invention. A still further aspect provides a method comprising introducing such nucleic acid into a host cell or organism.

Suitable hosts include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities. Mammalian cell lines grown in vitro are also suitable, particularly when using virus-driven expression systems such as the baculovirus expression system which involves the use of insect cells as hosts. Compounds may also be expressed in vivo, for example in insect larvae, mammalian tissues or plant cells.

Introduction of nucleic acid may employ any available technique. In eukaryotic cells, suitable techniques may include calcium phosphate transfection, DΕAΕ-Dextran, electroporation, liposome-mediated transfection or transduction using retrovirus 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. Bacterial cells will be of particular use in the methods described above in which the aim of the method is to increase levels of expression of membrane-associated protein. For example, one of the above-described aspects involves the alteration of the level of expression of a membrane-associated protein by substituting a membrane-anchoring region in the membrane-associated protein for a transmembrane region that is not naturally part of said membrane- associated protein or for a synthetic transmembrane component. High expression levels are generally more easy to achieve in prokaryotic cells than in eukaryotic cells. Furthermore, these systems are simpler and may more easily be manipulated. However, the system of choice will depend upon the particular membrane-associated protein of interest. Other factors will also be relevant, for example, the degree to which it is important for the fidelity of post-translational modification to be retained.

Introduction of the nucleic acid may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.

For ex vivo use, the nucleic acid of this aspect of the invention may be introduced into effector cells removed from the target host using methods well known in the art e.g. transfection, transduction, biolistics, protoplast fusion, calcium phosphate-precipitated 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 expression of the chimeric receptors of the invention include cells associated with the immune system such as lymphocytes e.g. cytotoxic T- lymphocytes, tumour infiltrating lymphocytes, natural killer cells, neutrophils, basophils or T-helper cells, dendritic cells, B-cells, haematopoietic stem cells, macrophages, monocytes or natural killer (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 the DNA to a desired effector cell. Examples of suitable targeted delivery systems include targeted naked DNA, targeted liposomes encapsulating and/or complexed with the DNA, targeted retroviral 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; CD 16; CD4; CD3; selectins e.g. E-selectin; CD5; CD7; CD34; and activation antigens e.g. CD69 and LL-2R. Alternatively, other receptor-ligand interactions can be used for targeting e.g. CD4 to target H±V_gp160-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 cleaved at low pH may be used to link the antibody to the liposome. Cationic liposomes that fuse with the cell membrane and deliver the recombinant chimeric receptor DNA according to this aspect of the invention directly into the cytoplasm may also be used. Liposomes for use in the invention may also have hydrophilic molecules, for example, 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 to liposomes or other carriers; see for example International patent applications nos. WO88/04924, WO90/09782, WO91/05545, WO91/05546, WO93/19738, WO94/20073 and WO94/22429. The antibody or other targeting molecule may be linked to the DNA, condensed DNA or liposome using conventional readily available linking groups and reactive functional groups in the antibody, e.g. thiols or amines, and in the DNA or DNA-containing materials.

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

The DNA according to this aspect of the invention may be used ex vivo and in a further aspect of the invention provides effector cells that have been transfected with DNA according to this aspect of the invention. The effector cells may be any of those described above which are suitable for ex vivo use and are preferably T-cells, most preferably cytotoxic T-cells.

According to a further aspect of the invention there is provided a composition comprising a membrane-associated protein according to the above-described aspects of the invention or a nucleic acid molecule coding therefor, 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 (0.01M phosphate salts, 0J38M NaCl, 0.0027M KCl, 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., NJ. 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 emulsion 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 to ensure a long 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 (for example potato, maize or wheat starch or cellulose), starch derivatives such as microcrystalline cellulose, silica, various sugars such as lactose, magnesium carbonate and/or calcium phosphate. It is desirable that, if the formulation is for oral administration it will be well tolerated by the patient's digestive system. To this end, it may be desirable to include in the formulation 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. It may also be preferable to include the composition in a controlled release formulation.

The membrane-associated proteins of the invention and nucleic acids coding therefor may be of use in medicine. According to a further aspect of the invention there is provided a method of treatment of a human or animal subject, the method comprising administering to the subject an effective amount of: a membrane-associated protein according to the above-described aspects of the invention; a DNA delivery system as described above; or transfected effector cells, in a therapeutically-effective amount. The exact amount of active composition to be used will depend on the age and condition of the patient, the nature of the disease or disorder and the route of administration, but may be determined using conventional means, for example by extrapolation of data derived from animal experiments. In particular, for ex vivo use the number of transfected effector cells required may be established by ex vivo transfection and reintroduction into an animal model of a range of effector cell numbers.

Similarly the quantity of DNA required for in vivo use may be established in animals using a range of DNA concentrations.

The present invention may be 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. rheumatoid arthritis, osteoarthritis, inflammatory bowel disease; cancer; allergic/atopic diseases 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.

According to a yet further aspect, the present invention provides for the use of a membrane-associated protein according to the above-referenced aspects of the invention, a nucleic acid encoding such a protein or a pharmaceutical composition comprising either or both of these agents in therapy.

According to a still further aspect of the invention there is provided the use of membrane-associated protein according to the above-referenced aspects of the invention or a nucleic acid encoding therefor, in the manufacture of a medicament for the treatment or prevention of a disease in a mammal, preferably a human.

Transgenic animals transformed so as to express or overexpress in the germ line one or more membrane-associated proteins as described herein form a still further aspect of the invention, along with methods for their production. Many techniques now exist to introduce transgenes into the embryo or germ line of an organism, such as for example, illustrated in Watson et al., (1994) Recombinant DNA (2nd edition), Scientific American Books. Preferred host animals are rodents. Various aspects and embodiments of the present invention will now be described 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.

All documents mentioned in the text are incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Cloning cassette for chimeric receptor construction.

Figure 2: Sequence of signalling component cassette.

Figure 3: Oligonucleotide sequences for chimeric receptor construction.

Figure 4: Sequence of synthetic transmembrane components.

Figure 5: Effect of transmembrane component on the expression level of recruitment receptors in Jurkat cells.

Figure 6: Effect of transmembrane component on the expression level of recruitment receptors in Cos cells.

Figure 7: Expression in Cos cells of recruitment receptors with different synthetic transmembrane components.

Figure 8: Chimeric receptor expression in Jurkat cells and IL-2 production in response to solid phase antigen.

Figure 9: Chimeric receptor expression in Jurkat cells and IL-2 production in response to solid phase and cell surface antigen.

Figure 10: Chimeric receptor expression in Jurkat cells and IL-2 production in response to solid phase and cell surface antigen EXAMPLES

Example 1: Construction of cloning cassette system

To facilitate construction of chimeric 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 International Patent Specification No. WO97/23613.

This new cassette system is shown in Figure 1. The binding component has 5' Not I and Hind III restriction sites and a 3' Spe I restriction site. The extracellular spacer has a 5' Spe I site (Thr, Ser) and a 3' Nar I site (Gly, Ala). The transmembrane component has a 5' Nar I site (Gly, Ala) and 3' Mlu I (Thr, Arg) and BamHI sites (Gly, Ser). The signalling component has a 5' BamHI site and a 3' EcoRI site. In between this BamHI and EcoRI site is a stop codon for receptors without a signalling component.

To generate this cassette, a 200bp fragment was PCR assembled using oligos:- S0146, A6081, A6082 and A6083 (Figure 3). This fragment 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 EcoRI site (see Figure 2). This PCR fragment was then restricted with Spel and EcoRI and substituted for the same fragment in our previously described cloning cassette system to join the binding component (International Patent WO97/23613; Figure 2).

Example 2: Construction of chimeric receptors with different transmembrane components

a) P67scFv/h.CD28/CD28Tm/FcRΥ chimeric receptor

This construct was generated from the cassette described above and forms the basis for chimeric receptor constructs (b) to (f). The FcRγ intracellular component was PCR cloned with oligos A9515 and A9516 (Figure 3) from human Leukocyte cDNA (Clontech) and cloned into the BamHI site of the described cassette (Figure 1).

The binding component, P67 single chain Fv (scFv) with specificity for CD33 and CD33 on HL60 cells, consists of a human antibody leader sequence and the variable component of the light chain of the engineered human antibody linked via a (Gly4Ser)5 linker to the variable component of the heavy chain of the engineered human antibody. This binding component is described in WO 97/23613. The extracellular spacer component h.CD28, consists of residues 234 to 243 of human IgGl hinge and residues 118 to 134 of human CD28. The transmembrane component consists of residues 135 to 161 of human CD28 (A. Aruffo & B. Seed 1987 PNAS 84 8573-8577). The intracellular component consists of residues 27 to 68 of the γ chain of human FcεRl (Kuster et al (1989) J. Biol. Chem. 255, 6448-6452).

b P67scFv/h.CD28/Tm20 FcRγ chimeric receptor

This chimeric receptor is the same as in 2 (a) above, except that the transmembrane component consists of 20 synthetic amino acid residues (Figure 4). This transmembrane component was constructed by annealing oligos B6471 and B6472 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 2(a) on a Narl to BamHI fragment.

c) P67scFv/h.CD28/Tm24/FcRγ chimeric receptor

This chimeric receptor is the same as in 2 (a) above except that the transmembrane component consists of 24 synthetic amino acid residues (Figure 4). This transmembrane component was constructed by annealing oligos B6469 and B6470 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 2(a) on a Narl to BamHI fragment.

d) P67scFv/h.CD28/Tm27/FcRγ chimeric receptor

This chimeric receptor is the same as in 2 (a) above except that the transmembrane component consists of 27 synthetic amino acid residues (Figure 4). This transmembrane component was constructed by annealing oligos B6467 and B6468 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 2(a) on a Narl to BamHI fragment. e) P67scFv/h.CD28/Tm29/FcRγ chimeric receptor

This chimeric receptor is the same as in 2 (a) above except that the transmembrane component consists of 29 synthetic amino acid residues (Figure 4). This transmembrane component was constructed by annealing oligos B6465 and B6466 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 2(a) on a Narl to BamHI fragment.

f) P67scFv/h.CD28/Tm31/FcRγ chimeric receptor

This chimeric receptor is the same as in 2 (a) above except that the transmembrane component consists of 31 synthetic amino acid residues (Figure 4). This transmembrane component was constructed by annealing oligos B6463 and B6464 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 2(a) on a Narl to BamHI fragment.

g P67scFv/h.CD28/CD28Tm/TCR recruitment receptor

This construct was generated from the cassette described above and forms the basis for chimeric receptor constructs 2(h) to 2(k). The TCR ζ intracellular component was PCR cloned with oligos C3208 and C3209 (Figure 3) from human Leukocyte cDNA (Clonetech). This PCR fragment was restricted with Mlu I and EcoR I and substituted for this fragment in the described cassette (Figure 1).

The binding component, P67 single chain Fv (scFv) consists of a human antibody leader sequence and the variable component of the light chain of the engineered human antibody linked via a (Gly4Ser)5 linker to the variable component of the heavy chain of the engineered human antibody. This binding component is described in WO 97/23613. The extracellular spacer component h.CD28, consists of residues 234 to 243 of human IgGl hinge and residues 118 to 134 of human CD28. The transmembrane component consists of residues 135 to 161 of human CD28 (A. Aruffo & B. Seed 1987 PNAS 84 8573-8577). The intracellular component consists of residues 31 to 142 of human TCR ζ chain (Weissman et al : PNAS 85,9709-9713,1988. Moingeon et al : Eur. J. Immunol. 20,1741-1745- 1990.)

h) P67scFv/h.CD28/Tm24/TCRC chimeric receptor

This chimeric receptor is the same as 2 (g) except that the transmembrane component consists of 24 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (c) for the same fragment in chimeric receptor 2 (g).

i P67scFv/h.CD28/Tm27/TCRC chimeric receptor

This chimeric receptor is the same as in 2 (g) above except that the transmembrane component consists of 27 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (d) for the same fragment in chimeric receptor 2 (g).

i) P67scFv/h.CD28/Tm29/TCR chimeric receptor

This chimeric receptor is the same as in 2 (g) above except that the transmembrane component consists of 29 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (e) for the same fragment in chimeric receptor 2 (g).

k) P67scFv/h.CD28/Tm31/TCRζ chimeric receptor

This chimeric receptor is the same as in 2 (g) above, except that the transmembrane component consists of 31 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (f) for the same fragment in chimeric receptor 2 (g).

1) P67scFv/h.CD28/CD45Tm/Zeta-CD28 chimeric receptor

The Zeta-CD28 fusion signalling component was PCR cloned from our P67scFv/h.28/Zeta-CD28 construct (WO 97/23613 page 23) with oligos A9514 and B4006. This PCR fragment was then substituted for the BamHI to EcoRI fragment in the P67scFv/h.CD28/CD45Tm.stop construct described in Examples (Figure 1). m) P67scFv/h.CD28/CD28TmZeta-CD28 chimeric receptor

The Zeta-CD28 fusion signalling component was PCR cloned from our P67scFv/hJ8/Zeta-CD28 construct (WO 97/23613 page 23) with oligos A9514 and B4006. This PCR fragment was then substituted for the BamHI to EcoRI fragment in the P67scFv/h.CD28/CD28Tm.stop construct described in Examples (Figure 1).

Example 3: Construction of recruitment receptors with different transmembrane components

a) P67scFv/h.CD28/CD28Tm.stop recruitment receptor

This construct was generated as described for the cloning cassette and forms the basis for subsequent recruitment receptor constructs (Figure 1 and 2).

The binding component, P67 single chain Fv (scFv) consists of a human antibody leader sequence and the variable component of the light chain of the engineered human antibody linked via a (Gly4Ser)5 linker to the variable component of the heavy chain of the engineered human antibody. This binding component is described in WO 97/23613. The extracellular spacer component h.CD28, consists of residues 234 to 243 of human IgGl hinge and residues 118 to 134 of human CD28. The transmembrane component consists of residues 135 to 161 of human CD28 (A. Aruffo & B. Seed 1987 PNAS 84 8573-8577). This is followed by an in frame stop codon.

b) P67scFv/h.CD28/Tm20.stop recruitment receptor

This recruitment receptor is the same as in 3 (a) above except that the transmembrane component consists of 20 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (b) for the same fragment in recruitment receptor 3 (a).

c) P67scFv/h.CD28/Tm24.stop recruitment receptor

This recruitment receptor is the same as in 3 (a) above except that the transmembrane component consists of 24 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (c) for the same fragment in recruitment receptor 3 (a). d) P67scFv/h.CD28/Tm27.stop recruitment receptor

This recruitment receptor is the same as in 3 (a) above except that the transmembrane component consists of 27 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (d) for the same fragment in recruitment receptor 3 (a).

e) P67scFv/h.CD28/Tm29.stop recruitment receptor

This recruitment receptor is the same as in 3 (a) above except that the transmembrane component consists of 29 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (e) for the same fragment in recruitment receptor 3 (a).

f) P67scFv/h.CD28/Tm31.stop recruitment receptor

This recruitment receptor is the same as in 3 (a) above except that the transmembrane component consists of 31 synthetic amino acid residues (Figure 4). This construct was generated by substituting a Spel to Mlu I fragment from chimeric receptor 2 (f) for the same fragment in recruitment receptor 3 (a).

g) P67scFv/h.CD28/TCRαTm.stop chimeric receptor

This chimeric receptor is the same as 3(a) above, except that the transmembrane component consists of residues 132 to 152 of human TCRα (P. Del Porto et al, 1995 PNAS 92, 12105-12109). This transmembrane component was constructed by annealing oligos A8350 and A8351 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 3(a) on a Narl to BamHI fragment.

h) P67scFv/h.CD28/TCRCTm.stop chimeric receptor

This chimeric receptor is the same as 3 (a) above, except that the transmembrane component consists of residues 132 to 152 of human TCRζ (A.M. Weissman et al 1988

PNAS 85 9709-9713). This transmembrane component was constructed by annealing oligos A6248 and A6249 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct 3(a) on a Narl to BamHI fragment.

i) P67scFv/h.CD28/CD3εTm.stop chimeric receptor

This chimeric receptor is the same as 3 (a) above, except that the transmembrane component consists of residues 105 to x 130 human CD3ε (D.P. Gold et al 1986 Nature 321 431-434). This transmembrane component was constructed by annealing oligos A8352 and A8353 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct (a) on a Narl to BamHI fragment.

i) P67scFv/h.CD28/CD45Tm.stop chimeric receptor

This chimeric receptor is the same as 3 (a) above, except that the transmembrane component consists of residues 553 to 574 of human CD45 (M. Streuli et al 1987 J. Exp. Med. 166 1548-1566). This transmembrane component was constructed by annealing oligos A8354 and A8355 (Figure 3) which are designed so that a 5' overhang forms a Narl site and a 3' overhang forms a BamHI site. These annealed oligos were then substituted for the CD28 transmembrane in construct (a) on a Narl to BamHI fragment.

Example 4: Analysis of receptors

a) Construction of expression plasmids

The chimeric receptor constructs were subcloned from pBluescript KS+ into the expression vector pEEόhCMV.ne (C.R. Bebbington (1991), Methods 2, 136-145) on a Hindlll to EcoRI restriction fragment. The expression vector with no chimeric receptor genes is used as a negative control in subsequent experiments.

b) Stable transfection into Jurkat E6.1 cells

The expression plasmids were linearised and transfected into Jurkat E6.1 cells (ECACC) by electroporation using a Bio-rad Gene Pulser. lOμg of DNA per 2.5 X10^ cells were given two pulses of 1000V, 3μF in 1ml 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.

c Transient transfection into Cos-1 cells

Sub-confluent 6 well plates were transfected by coating with 15μg of expression plasmid DNA complexed with DEAE/DEXTRAN at a final concentration of 0.4 mg/ml for 3 hours at 37°C/8% CO2 and then shocking with 10% DMSO. Cells were analysed three days later.

d) FACS analysis of surface expression

For both Jurkat and Cos-1 cells approximately 5X10-5 _ceu_{s were} stained with 1 μg/ml FITC labelled antigen, CD33. Fluorescence was analysed by a FACScan cytometer (Becton Dickinson).

e) IL-2 production analysis of function

2X10⁵ cells were incubated at 37°C/8% CO2 for 20 hours in 96 well plates with soluble CD33 at 5 μg/ml or HL60 target cells at an effector : target ratio of 1: 1 or in 96 well plates (Nunc Immunol) pre-coated with soluble CD33 at 5 μg/ml. Cell supernatants were then harvested and assayed for human IL-2 (R & D Systems Quantikine kit).

Example 5: Results

The choice of transmembrane domain has been found to have a profound effect on the level of expression of receptors, with use of the transmembrane domain from CD28 in particular conferring a high level of transient and stable expression of recruitment receptors in COS (Figure 6) and Jurkat (Figure 5) cells respectively. Use of the transmembrane domains from the alpha or zeta chain of the T cell receptor led to low level expression. An intermediate level of expression was observed when the transmembrane domain from CD3 epsilon or CD45 was incorporated into the design.

P67scFv/h.28/zeta-CD28, a chimeric receptor containing the transmembrane component from the zeta chain of the T cell receptor, is expressed in Jurkat at relatively low level (see Figure 5). Use of the zeta chain transmembrane component is here shown to lead to a low expression of recruitment receptor.

Use of a transmembrane component derived from CD28 in place of the zeta chain transmembrane component in the chimeric receptor P67scFv/hJ8/zeta-CD28 led to increased expression in Jurkat, as predicted from Figure 5. Substitution of the zeta chain transmembrane component with a transmembrane component derived from CD45 led to an intermediate level of expression of this chimeric receptor in Jurkat.

The surface expression of chimeric receptors in Jurkat was found to closely match the ability to signal from solid phase antigen, with a correlation coefficient between expression level and IL-2 production of 0.89. Constructs with signalling sequences from both zeta and CD28 in series and which incorporated the CD28 transmembrane domain mediated lOx more IL-2 production than constructs featuring an identical signalling region but with the zeta chain transmembrane domain instead (Figure 8).

Substitution of a non-naturally occurring transmembrane component for the CD28 transmembrane domain still enabled expression of chimeric receptors in Jurkat (Figure 9) and recruitment molecules in Cos-1 cells (Figure 7). Use of the transmembrane component TM27 conferred expression of chimeric receptors in Jurkat equivalent to that seen with the naturally occurring CD28 transmembrane domain. TM31 led to relatively low expression of the chimeric receptor in Jurkat; intermediate levels of expression were seen with TM20, TM24 and TM29.

Choice of synthetic transmembrane component was found to influence the response of chimeric receptors to antigen. The FcRγ signalling component in the chimeric receptor described, when coupled to a CD28 transmembrane domain, demonstrated a marked preference for soluble rather than cell surface-expressed antigen. Substitution of just the transmembrane domain for either TM24 or TM31 led to a dramatic change in antigen preference, with a greater than five fold increase in IL-2 production from cell surface-expressed antigen and an up to three fold decrease in IL-2 production from soluble antigen. The net effect of this was to convert a three fold deficit of IL-2 production from cell surface-expressed antigen with respect to soluble antigen into a five fold excess. In constructs featuring synthetic transmembrane components linked to the zeta signalling component a similar antigen preference was observed. In this case substitution of the CD28 transmembrane region for either TM24 or TM29 led to an increase in IL-2 production in response to cell surface expressed antigen compared to soluble antigen. With TM24 the net effect was to convert a greater than two fold deficit of IL-2 production from cell surface-expressed antigen with respect to soluble antigen into a two fold excess (Figure 10).

Summary

Substitution of naturally-occurring transmembrane domains for synthetic sequences has shown the ability to alter function by reversing the preference of chimeric receptors to signal in response to soluble antigen over cell surface-expressed antigen.

Careful selection and design of the transmembrane domain of proteins will allow expression of the protein to be adjusted. From the data presented here, use of, for example, the CD28 transmembrane domain may be appropriate where a high level of expression is desirable, while use of, for example, the alpha or zeta chain from the T cell receptor in the design would be applicable where low level expression is sought. In addition, where more than one protein is expressed, the relative amounts of each can be modulated by the choice of the transmembrane component.

Claims

1. A method of altering the properties of a membrane-associated protein comprising substituting a transmembrane region or a membrane-anchoring region in the membrane-associated protein for a transmembrane region or a membrane-anchoring region that is not naturally part of said membrane-associated protein or for a synthetic transmembrane component.

2. A method of altering the level of expression of a membrane-associated protein comprising substituting a transmembrane or a membrane-anchoring region in the membrane-associated protein for a transmembrane region that is not naturally part of said membrane-associated protein or for a synthetic transmembrane component.

3. A method according to claim 1, wherein said properties comprise the relative response of the membrane-associated protein to cell surface-associated antigen versus antigen in solution.

4. A method according to claim 1, wherein said properties comprise the sensitivity of intracellular signalling mediated by the membrane-associated protein.

5. A method according to any one of claims 1-4 wherein said membrane-associated protein is a chimeric receptor.

6. A method according to any one of the preceding claims wherein said membrane- associated protein is a recruitment receptor.

7. A method according to any one of the preceding claims wherein the transmembrane region that is substituted into the membrane-associated protein is derived from all or part of a naturally-occurring membrane-associated protein.

8. A method according to claim 7 wherein said substituted transmembrane region is derived from all or part of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,

CD80, CD86, CD 137 or CD 154.

9. A method according to claim 8, wherein said transmembrane region is derived from all or part of CD28.

10. A method according to any one of claims 1-6, wherein said synthetic transmembrane component comprises a sequence of amino acid residues of which at least 50% are hydrophobic amino acid residues.

11. A method according to claim 10, wherein said synthetic transmembrane component comprises a sequence of amino acid residues of which at least 80% are hydrophobic amino acid residues.

12. A method according to claim 10 or claim 11, wherein said hydrophobic amino acid residues are Ala, Leu, Val, lie, Pro, Phe or Met.

13. A method according to claim 10 or claim 11, wherein said synthetic transmembrane component comprises a sequence of alpha helix-promoting amino acid residues.

14. A method according to claim 13 wherein said sequence of alpha helix-promoting amino acid residues comprises one or more of the residues Ala, Asn, Cys, Gin, His, Leu, Met, Phe, Trp, Tyr or Val.

15. A method according to claim 14, wherein said sequence of alpha helix-promoting amino acid residues comprises one or more of the hydrophobic alpha helix- promoting residues Ala, Met, Phe, Trp or Val.

16. A method according to claim 15, wherein said sequence of alpha helix-promoting amino acid residues comprises the hydrophobic alpha helix-promoting residues Phe,

Trp or Val.

17. A method according to any one of claims 1-6 or 10-16 wherein said synthetic transmembrane region comprises one of the components TM20, TM24, TM27, TM29 or TM31 as set out in Figure 4.