JP2003515321A - Adapter receptor - Google Patents

Abstract

  (57) [Summary] The present invention relates to chimeric receptor proteins, termed adapter receptors, which use adapter proteins as intracellular signaling components. By introducing a different adapter protein, optionally linked to other cytoplasmic signaling sequences, as the intracellular signaling domain of the adapter receptor, the level of intracellular signaling mediated by the adapter receptor can be regulated as desired. . Nucleic acids encoding adapter receptors and adapter receptor proteins suitable for use as a medicament are described.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to nucleic acids encoding adapter receptor proteins and their receptor proteins themselves, as well as methods of activating cells using such nucleic acids and proteins. The invention also includes the use of the nucleic acids, proteins and methods of the invention in the fields of medicine and research.

Throughout this application, various publications are referenced by their authors and publication years. Full citations for these publications are provided after the detailed description of the invention and the examples.

BACKGROUND OF THE INVENTION Studies in the area of immune cell signaling have yielded a considerable amount of information about signaling events that occur downstream of antigen-receptor association. Early work focused on enzymes that are stimulated in response to binding of the receptor itself and antigen (Review: We.
iss & Littman, 1994; DeFranco, 1997). More recent studies have explored how these early events are linked to secondary messenger signaling pathways, such as those activated by Ras, phospholipase Cγ and phosphoinositide 3-kinase. One class of proteins that play an important role in integrating these phenomena and regulating the signaling cascade are adapter molecules.

Adapter molecules lack enzymatic function but contain motifs and domains that enable them to participate in and mediate protein-protein interactions. The protein complex formed by these interactions acts as an intermediate, coupling activation of the receptor to downstream signaling cascades. Analysis of the roles of individual adapter molecules and their interactions has been the subject of intensive research (see Samelson, 1999; Peterson et al., 1998 and review of Rudd, 1999), but understanding the detailed mechanism of coupling. Is still poor.

In contrast, the components of the TCR complex that are required for early signaling events are well characterized. Some of these components have been used in the construction of chimeric receptor proteins as a tool to interpret the function of individual receptor subunits or domains (Kuwana et al., 1987; Romeo et al., 19).
92). More recently, chimeric receptors have been used for the regulation of cell activation levels (see, for example, published international patent specifications WO 97/23613 and WO 95/02686). Controls the biological effects of cell activation, such as increased cell proliferation, increased cytokine expression, stimulation of cytolytic activity, differentiation of other effector functions, antibody secretion, phagocytosis, increased tumor invasion and / or cell adhesion. The ability to do so has considerable therapeutic potential with chimeric receptors. While the chimeric receptors currently available can effectively activate cells, there is room for improvement in the efficiency of signaling to downstream members of the second messenger pathway.

[0006] It would be extremely useful if the level of intracellular signaling, and thus the activation level of effector cells, could be regulated to the required extent. It is highly desirable to be able to activate cells positively or negatively, ie to induce or inhibit their biological functions much more effectively than is currently possible.

The present invention fulfills these needs by providing cells with adapter receptor proteins that can regulate the level of signaling via the second messenger pathway. The totally unexpected finding that adapter proteins can play an active role in immune cell signaling pathways when removed from their natural environment results in highly efficient signaling. We were able to develop a possible adapter receptor. Such novel receptors use the adapter protein as the intracellular signaling domain of the chimeric receptor, eg in combination with an extracellular ligand binding domain and a transmembrane domain.

When the adapter receptor is expressed in effector cells, binding of the ligand to the extracellular binding domain results in signal transduction through the adapter protein component, which in turn induces cell activation. That is, signaling mediated by adapter proteins can be regulated in response to specific stimuli specified by the nature of the extracellular binding domain. In contrast, in their natural state, adapter proteins function in a ubiquitous manner, that is, they are recruited to assist cell signaling in response to stimulation of immune cells by any antigen.

Cellular activation is characterized by a number of biological responses including cytokine release and cell death, when the adapter protein is used as the intracellular domain of the receptor and signaling is mediated by the adapter protein. It is quite surprising that such a biological effect is observed. The present invention provides a number of adapter receptors that, when associated with extracellular ligands, can regulate biological responses either up or down with unpredictable greater efficiency than previously described chimeric receptors. To be able to build.

A first aspect of the invention provides an adapter receptor protein comprising an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain, wherein at least one intracellular signaling domain is present. And the extracellular ligand-binding domain is not a CD8 or MHC class I protein. The invention also includes a method of activating a cell comprising providing an adapter protein encoded by a nucleic acid of the invention described herein, as well as a cell having the adapter receptor of the invention.

[0011] Adapter proteins are defined herein as proteins that mediate protein-protein interactions and play a positive or negative regulatory role in immune cell signaling pathways and lack intrinsic enzyme activity.

The term “intrinsic enzymatic activity” of a protein or polypeptide domain means the ability of the protein or polypeptide domain to catalyze any enzymatic reaction, and thus the oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase activities. Includes.

The adapter proteins used in the present invention can be subclassified into two classes: pure cytoplasmic adapter proteins and a transmembrane domain as well as a suspected cytoplasmic domain. It is an adapter protein. Examples of cytoplasmic adapter proteins include SLAP, SLP-76.
, SKAP55, Grap, 3BP2, Grb-2, Nck, CRKL, Shc.
, And Cbl. The adapter protein Grap2 (Ellis et al., 2000), also known as GrbX, GrbLG, Grf40, Gads and GRID, is yet another example of a cytoplasmic adapter protein that can be used in the present invention. In contrast, LAT (also known as p36), TRIM and S
IT is an adapter protein that can be used in the present invention, which is presumed to have a transmembrane domain in addition to the cytoplasmic domain.

Any of the complete adapter proteins (including, for example, any of the adapter proteins described above) or, in the case of transmembrane adapter proteins, the cytoplasmic portion of any adapter protein is used as the intracellular signaling domain of the adapter receptor. be able to. It is particularly advantageous to include a cysteine residue in the membrane-proximal region of such a cytoplasmic portion. Such residues are often the site for palmitoylation and appear to be involved in membrane localization and function (Zhang et al., 1999). LAP, SLP-76, Grap, Grb2, T
It is preferred to use all of RIM, SIT or Cbl or cytoplasmic portions thereof in the present invention. Particularly preferred is the use of all or the cytoplasmic part of LAT, TRIM and SIT.

According to the invention, a further component of the adapter receptor is the transmembrane domain.
It may be formed from either natural or synthetic sources. If the source is natural, the domain is derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present invention includes α, β or ζ chain of T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16.
, CD22, CD33, CD37, CD64, CD80, CD86, CD134
, CD137 or CD154 (ie, including at least its transmembrane region (s)). Α, β or ζ chains of T-cell receptors, CD28,
CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, CD134 or CD15
Preferably, transmembrane regions from all or part of 4 are used in the present invention. Alternatively, the transmembrane domain is derived from all or part of the transmembrane domain of any adapter protein having such a domain, preferably LAT, SIT
Or derived from the transmembrane domain of TRIM. As another alternative, the transmembrane domain may be a synthetic domain, in which case it is composed largely of hydrophobic residues such as leucine and valine (eg published international patent specification WO 00/63).
374). Preferably, 3 of phenylalanine, tryptophan and valine
Alignments are found at each end of the synthetic transmembrane domain.

The third component of the adapter receptor is the extracellular ligand-binding domain. Introduction of such a domain confers on the adapter receptor the ability to display specificity for a specific ligand or class of ligands. By using the specificity of the extracellular ligand-binding domain to identify the exact ligand or class of ligands that can activate the receptor, it is possible to generate the desired cellular response within the cell in which it is expressed. Can be adjusted to.

The term “extracellular ligand-binding domain” as used herein refers to CD
With the exception of 8 and any MHC class I protein is meant any oligo or polypeptide capable of binding a ligand. Thus, antibody binding domains, antibody hypervariable loops or CDRs, receptor binding domains as well as other ligand binding domains are described by this term, examples of which will be apparent to those skilled in the art. Preferably, this domain is capable of interacting with cell surface molecules. Examples of proteins that are particularly useful in the invention in connection with binding to cell surface molecules include antibody variable domains (V _H or V _L ), T-cell receptor variable region domains (TCRα, TC).
Rβ, TCRγ, TCRδ) or CD11A, CD11B, CD11C, CD1
8, CD29, CD49A, CD49B, CD49D, CD49E, CD49F
, CD61, CD41 or CD51 chains are included. On the other hand, in some cases,
The use of whole domains or chains is advantageous and fragments can be used as appropriate.

Particularly useful binding moieties are derived from antibody binding domains and include Fab ′ fragments or especially single chain Fv fragments.

The choice of domain depends on the type and number of ligands that determine the surface of the target cell.
For example, the extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that act as ligands include markers associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells. In the latter case, specific examples of cell surface markers include bombesin receptors expressed on lung tumor cells, carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM) CD33, folate receptors, epithelial cells. There are adhesion molecules (EPCAM) and erb-B2. Other molecules of choice include cell surface adhesion molecules, inflammatory cells present in autoimmune diseases, and T-cell receptors or antigens that cause autoimmunity. The potential ligands listed above are by way of example, the list is not intended to be exclusive and further examples will be apparent to those of skill in the art. Is.

The adapter receptors of the present invention are bi- or multi-specifically designed, ie they contain two or more ligand binding domains and are capable of exhibiting specificity for more than one ligand. You can Such receptors include 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. Can be mobilized.

The inventors have surprisingly further enhanced the degree of cellular activation observed after binding of the ligand to the adapter receptor by the inclusion of an additional cytoplasmic signaling component as part of the receptor. I found that I could adjust it. That is, the second aspect of the present invention provides a nucleic acid encoding the adapter receptor described as the first aspect of the present invention, wherein the intracellular signaling domain contains at least one additional cytoplasmic signaling component. To do.

As used herein, the term “cytoplasmic signaling component” refers to the cytoplasmic sequence of the TCR and co-receptors (co) that work in concert to initiate signal transduction following antigen-receptor association.
-Receptors). These sequences have no intrinsic enzymatic activity.
The term also includes any other immune cell receptor having the same functional capacity, any signaling sequences derived from derivatives or variants of these sequences, as well as any synthetic sequences.

The signal generated by TCR alone is insufficient for full activation of T cells. Thus, the cytoplasmic signaling components of T cells can be subdivided into two classes: those that initiate antigen-dependent primary activation by the TCR (primary signaling sequences), and that work in an antigen-independent manner, Those that provide secondary or costimulatory signals (secondary signaling sequences). It should be understood that the primary signaling sequence can contain one or more signaling responsive primary signaling motifs. Thus, the cytoplasmic signaling component used in this aspect of the invention may include all (or one or more) primary (ie, one or more primary signaling motifs) and / or secondary signaling sequences.

The term “primary signaling motif” is defined as a sequence that transduces either a stimulatory or inhibitory signal that regulates the primary activation of the TCR complex. Examples of stimulatory primary signaling motif is a consensus _{sequence: Y-X 2 -L / I} -Xn
Any sequence that matches widely -Y-X ₂ -L / I, for example immunoreceptor tyrosine-based activation motif (ITAM) and the like. Motif acting inhibitory to the I / V-X-Y- X 2 -L broadly matching the defined as immunoreceptor tyrosine-based inhibitory motif consensus amino acid sequence (ITIMs) and the like (Burs
htyn et al., 1999). With the exception of FIG. 2 and some examples, where the canonical three letter code is used to describe amino acid sequences, the canonical one letter code is used throughout this application to describe both amino acid and nucleotide sequences. It

The stimulatory primary signaling motif for use in the present invention is the consensus amino acid sequence: may contain _{Y-X 2 -L / I-} Xn-Y-X 2 -L / I. In this formula,
X represents any amino acid, the subscript number indicates the number of residues present at this position in the motif, and the value n means any number greater than 0. The value n can be varied in the range 5-12, more preferably in the range 6-9. The term X ₂ or X _n can represent 2 or n amino acids (respectively) either homologous or heterologous.

When the value of n is 6-8, at least one of the additional primary signaling motifs in the adapter receptor is an immunoreceptor tyrosine-based activation motif (
ITAM), eg, TCRζ1, TCRζ2, TCRζ3 (ie, TCRζ
The first, second or third ITAMs of the chain), FcRγ, FcRβ, CD3γ, CD
3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66
It is preferable that all or part of d, or a variant thereof. Examples of primary signaling motifs derived from these molecules are shown in Table 1 below. Preferably the at least one additional primary signaling motif used in the present invention is TCRζ
1, TCRζ2, TCRζ3, FcRγ, FcRβ, CD3γ, CD3δ, CD
5, CD22, CD79a, CD79b or CD66d, or variants thereof in whole or in part.

[0027] Alternatively, at least one of the additional primary signaling motif is a motif unnatural Note _{sequence: Y-X 2 -L / I} -Xn-Y-X 2 -L / I The consensus amino acid sequence of Preferred examples of the value n such unnatural primary signaling motif is 6-8 is the SB 14 ^a or SB15 ^a, or a non-naturally occurring variants thereof as described in Table 1 herein. When the value n is 9 or more, SBX ^a , SBQ9 ^a , SB16 ^a or unnatural variants thereof can be used as additional motif (s), SB16 ^a being particularly preferred (Table 1 ).

[0028]

Primary signaling motifs, such as IT, that have the ability to inhibit cell activation
IMs can optionally be used as an additional component of the adapter receptor. Examples of ITIMs used in the present invention include FcγR (eg FcRγIIB),
ITIMs derived from CD22, EPOR, IL-2βR or IL-3βR
Is included.

The term “secondary signaling sequence” is defined as a sequence that confers a secondary or costimulatory signaling ability on a molecule in T cells. Molecules containing such sequences include, for example, CD2, CD4, CD8, CD28, CD134 and CD154 (see Finney et al., 1998). Preferred secondary signaling sequences used in the present invention are sequences derived from CD28, CD134 and CD154, such as SB28 ^a : RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA, SB29 ^a : MIETYNQTSPRSAATGLPISMK and SB34 ^a : RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI. Further examples of secondary signaling sequences are SB28, SB29 and SB34.
As shown in FIG. In this case, a GS linker was introduced at each end of the sequence to facilitate cloning.

According to yet another aspect, activation of the cell is via an adapter receptor as described in any previous aspect of the invention, wherein the adapter receptor is not the primary signaling motif. It contains additional domains that are not secondary signaling sequences. This additional domain may be, for example, an enzymatic domain except hydrolases. This additional domain is preferably included as the cytoplasmic element of the receptor. If the additional domain represents an enzymatic activity, the transferase is more particularly protein tyrosine kinase activity, more particularly. Particularly preferred examples are members of the src and sky families of protein tyrosine kinases.

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

The cytoplasmic signaling and adapter components may be linked to each other or to the transmembrane domain in a random or specific order. Short oligo or polypeptide linkers, preferably 2-10 amino acids in length, may form bonds between the component parts of the receptor. Glycine-serine doublets provide a particularly suitable linker.

Positive and negative regulatory adapters for stimulatory and inhibitory primary signaling motifs,
And / or mixed and matched with different secondary signaling sequences, and / or domains that may be more enzymatic, provide multiple adapter receptors that can regulate cellular activation to different extents.

A spacer domain can be introduced between the extracellular ligand-binding domain and the transmembrane domain or between the cytoplasmic signaling component and the transmembrane domain. The term "spacer domain" as used herein generally refers to an oligo or polypeptide that has the function of linking a transmembrane domain to an extracellular ligand-binding domain, or a cytoplasmic signaling component in a polypeptide chain. The spacer domain is composed of up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids.

The spacer domain may be a whole or part of a naturally occurring molecule, eg CD8, C
All or part of the extracellular region of D4 or CD28; all or part of the antibody constant region;
All or part of the natural spacer component between the functional parts of the cytoplasmic signaling component,
For example, it can be derived from spacers between ITAMs. Alternatively, the spacer may be a synthetic sequence corresponding to the naturally occurring spacer sequence or a fully synthetic spacer sequence.

Spacer domains are such that they either minimize the structural association of adapter receptors and thus reduce the frequency of structural activation of the cell, or promote such association to promote structural activity of the cell. It can be designed to increase the level of commodification. Either possibility is achieved by artificially deleting, inserting, changing or otherwise modifying the amino acids and naturally occurring sequences in the transmembrane and / or spacer domains. These have side chain residues that can be covalently or non-covalently linked to amino acid side chains in other polypeptide chains. In general, specific examples of amino acids that can be expected to promote association include cysteine residues, charged amino acids or amino acids with internal glycosylated sites such as serine or threonine.

The adapter receptor can be designed such that the spacer and transmembrane component have a free thiol group, which provides the ability to multimerize, especially dimerize. Such multimer receptors, especially dimers, are preferred. A transmembrane and spacer domain derived from the CD28 component, the zeta chain of the native T cell receptor,
Adapter receptors with adapter transmembrane domains and / or antibody hinge sequences are particularly preferred.

Nucleic acid coding sequences used in the preparation of the nucleic acids of the invention are widely reported in the scientific literature,
It is also available in publicly available databases (eg Genebank, EMBL, etc.). Whether the DNA is commercially available, part of a cDNA library, or generated using standard molecular biology and / or chemical manipulations, as will be apparent to those skilled in the art. You can A particularly suitable technique is
It includes polymerase chain reaction (PCR), oligonucleotide-directed mutagenesis, oligonucleotide-specific synthesis techniques, enzymatic cleavage or filling of gap oligonucleotides. Such a technology is Sambrook & Fritsch
, 1989 and the examples contained herein.

The nucleic acid of the present invention is used with a carrier. The carrier can be a vector or other carrier suitable for introducing nucleic acids into target cells and / or target host cells ex vivo or in vivo. Examples of suitable vectors include viral vectors such as retrovirus, adenovirus, adeno-associated virus (AAV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) vectors. Non-viral vectors such as liposome vectors and International Patent Application Nos. WO96 / 10038, WO97 / 18185, WO97 / 25329, W
Vectors based on condensation reagents such as the cationic lipids described in O97 / 30170, WO97 / 31934 can also be used. Optionally, the vector further comprises promoters and regulatory sequences and / or replication functions from the virus, such as retroviral long terminal repeats (LTRs), AAV repeats, SV40, and human cytomegalosvirus (hCMV) promoters and / or enhancers,
Splicing and polyadenylation signals and EBV and BK virus replication functions are included. Tissue-specific regulatory sequences such as the TCR-α promoter, E
-The selectin promoter and the CD2 promoter and the locus regulatory region can be used. The carrier may be an antibody.

The present invention also includes cloning and expression vectors containing the nucleic acids of any of the above aspects of the invention. Appropriate transcriptional and translational regulatory sequences, such as enhancer elements, promoter / operator regions,
Termination stop sequences, mRNA stabilizing sequences, start and stop codons or ribosome binding sites are introduced in frame with the nucleic acid molecules of the invention as appropriate.

Vectors of the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses). Many expression systems suitable for expressing heterologous proteins are well known and reported in the art. The use of prokaryotic cells such as E. coli to express, for example, heterologous polypeptides and polypeptide fragments has been established (eg, Sambrook & Fritsch, 19).
89, Glover, 1995a). Similarly, eukaryotic expression systems have been developed and are commonly used to express heterologous proteins (eg Glover, 199).
5b and O'Reilly et al., 1993). In eukaryotic cells, apart from yeast, the vector of choice is a virus-based vector. Particularly suitable viral vectors include baculovirus, adenovirus and vaccinia virus based vectors.

Vectors containing the appropriate regulatory sequences (including promoter, termination, polyadenylation, and enhancer sequences, marker genes) may be selected from those described in the literature or for expression of the receptor of the invention, It can be easily constructed using standard molecular biology techniques. Such techniques and manipulation of nucleic acids,
Protocols for the production of nucleic acid constructs, mutagenesis, sequencing, DNA transformation and gene expression and protein analysis, for example, are described in detail in Ausubel et al., 1992 or Rees et al., 1993.

Suitable host cells for high level in vitro expression of adapter receptor proteins include prokaryotic cells such as E. coli, eukaryotic yeasts such as Saccharomyces ce.
revisiae, Pichia sp., Schizosaccharomyces
Included are pombes, mammalian cell lines and insect cells. Alternatively, the adapter receptor is expressed in vivo, for example in insect larvae, plant cells or especially mammalian tissues.

Any available technique can be used to introduce the nucleic acid into the host cell.
Suitable techniques in eukaryotic cells include calcium phosphate transfection, DEAE-dextran, electroporation, particle bombardment, liposome-mediated transfection or retroviruses or other viruses such as vaccinia virus, or for insect cells. , Transduction using baculovirus can be mentioned. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation or transfection using bacteriophage. The nucleic acid may remain episomal within the cell, or it may be integrated into the cell's genome. If the latter is desired, sequences that facilitate recombination with the genome are included in the nucleic acid. After introduction of the nucleic acid into the host cell, the cell can be cultured under conditions where expression from the adapter receptor gene is increased or induced, as appropriate.

Accordingly, a further aspect of the invention provides a host cell containing a nucleic acid encoding an adapter receptor and a host cell expressing the adapter receptor protein.

According to yet another aspect, the nucleic acids of the invention can be used for either ex vivo or in vivo treatment of target cells and / or target host cells.

For ex vivo use, the effector cells from which the nucleic acid has been removed from the target cells are applied to methods well known in the art such as transfection, transduction (including viral transduction), biolistics ( biolisti
cs), protoplast fusion, calcium phosphate mediated DNA transformation, electroporation, cationic lipofection, or targeted liposomes. The effector cells are then reintroduced into the host using standard techniques. Examples of effector cells suitable for expressing the adapter receptor of the present invention include cells associated with the immune system, eg lymphocytes, eg cytotoxic T-lymphocytes,
Tumor infiltrating lymphocytes, neutrophils, basophils, or T-helper cells, dendritic cells, B
-Including cells, hematopoietic stem cells, macrophages, monocytes or NK cells. The use of cytotoxic T-lymphocytes is particularly preferred.

The nucleic acid according to this aspect of the invention is particularly suitable for in vivo administration. To achieve this, the DNA can be in the form of a targeted carrier system in which the carrier described above can direct the DNA to the desired effector cells. Examples of suitable targeted delivery systems include targeted naked DNA, targeted liposomes encapsulating and / or complexing DNA, targeted retroviral or adenoviral systems and targeted enriched DN.
A, for example protamine and polylysine enriched DNA is included.

Targeting systems are well known in the art and include, for example, cell surface antigens expressed in vivo on target cells such as CD8, CD16, CD4, CD3, selectin (
For example, the use of antibodies or fragments thereof against E-selectin), CD5, CD7, CD24, and activation antigens (eg CD69 and dlL-2R) is included. Also, other receptor-ligand interactions can be used to target. For example, the use of CD4 for targeting to HIVgp160-expressing target cells.

In general, the use of antibody-targeted DNA is preferred, especially antibody-targeted naked DNA.
, The use of antibody-targeted concentrated DNA is preferred, and the use of antibody-targeted liposomes is particularly preferred. The types of liposomes used include, for example, pH-sensitive liposomes, where a low pH-cleaving linker may be used to link the antibody to the liposome. The nucleic acids of the invention may also be directly targeted to the cytoplasm by using cationic liposomes which fuse to the cell membrane. The liposomes used in the present invention may also have hydrophobic molecules, such as polyethylene glycol polymers, attached to their surface to increase their circulating half-life. There are numerous examples in the art of groups suitable for attaching DNA to liposomes or other carriers, eg, International Patent Application No. WO 88/04924, WO 90/09782.
, WO91 / 05545, WO91 / 05546, WO93 / 19738, WO
See 94/20073 and WO94 / 22429. Antibodies or other targeting molecules can be linked to DNA, fused DNA or liposomes using conventional linking groups and reactive functional groups in antibodies and DNA or DNA containing materials such as thiols or amines.

Non-targeted carrier systems can also be used. Targeted expression of proteins is advantageous in these systems. This includes, for example, T cell-specific promoter systems such as the zeta promoter, the CD2 promoter and the locus control region, CD4, CD8, T
This is achieved using the CRα and TCRβ promoters, cytokine promoters such as the IL-2 promoter, and the perforin promoter.

The adapter receptor proteins of the invention or the nucleic acids encoding them are intended for application in methods of treating mammalian, especially human, patients. The adapter receptor proteins generated by the present invention are particularly useful in the treatment of many diseases and disorders. Such diseases or disorders include those listed under the general heading Infectious Diseases, such as HIV infection; inflammatory / autoimmune diseases such as asthma, eczema; congenital diseases such as cystic fibrosis, sickle type. Erythrocyte anemia; skin diseases such as psoriasis; neurological diseases such as multiple sclerosis; transplants such as organ transplant rejection, graft-versus-host disease; metabolic /
Idiopathic diseases such as diabetes; cancer are included.

For example, expression of an adapter receptor on the surface of a T cell can initiate activation of that cell by binding of the ligand binding domain to a ligand on the target cell. Subsequent release of inflammatory mediators stimulated by activation of the receptor's signaling function ensures destruction of target cells.

Accordingly, a further aspect of the invention comprises providing a cell with an adapter receptor protein as described in any one of the preceding aspects of the invention and binding a ligand to the extracellular domain of the adapter receptor. A method for activating cells is provided.

When the effector cells of the immune system are provided with the adapter receptor according to the invention, binding to the target activates the effector cells and the downstream effects of this activation also result in the destruction of the target cells. If the extracellular ligand binding domain of the adapter receptor exhibits specificity for surface markers on cells of the immune system, effector cells will be recruited to the site of disease. Therefore, expression of the adapter receptor in diseased cells ensures its destruction.

Expression of a multispecific adapter receptor protein, or two or more adapter receptors (with different ligand specificities), in a single host cell confers dual functionality on that receptor. For example, binding of the adapter receptor to its target not only activates the effector cells themselves, but also attracts other immune effectors to the disease site. Target cells are therefore destroyed by activation of the immune system.

A further aspect of the present invention provides a composition comprising a nucleic acid encoding an adapter receptor according to any one of the above aspects of the invention, or an adapter receptor protein together with a pharmaceutically acceptable excipient. To do.

Suitable excipients are well known to those skilled in the art, eg phosphate buffered saline (eg 0.01M phosphate, 0.138M NaCl, 0.0027M).
KCl, pH 7.4), optionally mineral salts such as hydrochlorides, hydrobromides, phosphates, sulfates; salts of organic acids such as acetates, propionates, malonates, benzoates, etc. It may comprise a liquid such as water, saline, glycerol or ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances may be present. For a complete discussion of pharmaceutically acceptable excipients, see Remingto
n's Pharmaceutical Sciences (Mack Pub
． Co. , NJ, 1991). The composition is preferably in a form suitable for parenteral administration, such as injection or infusion, such as single bolus injection or continuous infusion, or particle-mediated injection. When the composition is for injection or infusion, it is in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and contains a formulation agent such as a suspending agent, preservative, stabilizer and / or dispersant. Added. Alternatively, the composition can be in dry form for reconstitution with a suitable sterile liquid before use. For particle-mediated administration, the DNA can be coated onto particles such as microscopic gold particles.

It is also possible to use carriers which do not induce the production of antibodies harmful to the individual to whom the composition is administered and which can be administered without inconvenient toxicity. Suitable carriers are usually large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid,
Polyglycolic acid, amino acid polymers, amino acid copolymers and inactive virus particles. The pharmaceutical composition may also include a preservative to prolong the shelf life on storage.

When the composition is suitable for oral administration, the formulation contains, in addition to the active ingredient, additives such as starch (eg potato, corn or wheat starch, cellulose), starch derivatives such as microcrystalline cellulose. , Silica, various sugars such as lactose, magnesium carbonate and / or calcium phosphate can be included. Formulations suitable for oral administration should be well tolerated by the patient's digestive system. For this purpose, it is desirable to include a mucus forming agent and a resin. It is also desirable to improve tolerability by formulating the composition in capsules that are insoluble in gastric juices. In addition, it is also preferable to include the composition in a controlled release formulation.

According to yet another aspect of the invention, a nucleic acid encoding an adapter receptor, or an adapter protein, or a pharmaceutical composition of the pharmaceutical composition described herein above for the treatment or prophylaxis of a human or animal. Provide use in manufacturing.

Various aspects and embodiments of the invention are illustrated in further detail below by examples.
It should be understood that modifications of detail can be made without departing from the scope of the invention.

[0064]     (Example)   Example 1: Construction of cloning vector, pHMF393

In order to facilitate the construction of adapter receptors with various binding, extracellular spacer, transmembrane and signaling components, pBluescript SK + (St
The cloning cassette system was devised in the ratagene). This is a modification of our cassette system described in published international patent specification WO 97/13613.

This new cassette system is shown in FIG. The binding component has a 5 '(relative to the coding direction) NotI and HindIII restriction sites and a 3' (again toward the coding direction) SpeI restriction site. The extracellular spacer is flanked by a SpeI site (thus encoding Thr, Ser at the 5'end) as well as a NarI site (thus encoding Gly, Ala at the 3'site). The transmembrane component has a NarI site at its 5'end (thus encoding Gly, Ala) and an MluI at its 3'end (thus encoding Thr, Arg) and a BamHI site at its 3'end (thus Gly, Ser). Code). The signaling component can be cloned in frame at the BamHI site. This Bam
Following the HI site is a stop codon for transcription termination, downstream of which also an EcoRI site is placed to facilitate subsequent rescue of the overall construction.

In order to generate the cassette, the following oligos, namely S0146, A60
The 200 bp fragment was assembled by PCR using 81, A6082 and A6083 (FIG. 3). The nucleotide and amino acid sequences of this fragment are shown in Figure 2. It begins at the SpeI site and begins with the extracellular spacer h. It consists of CD28, the human CD28 transmembrane region, a stop codon, and ends with an EcoRI restriction site. This PCR fragment was then digested with SpeI and EcoRI to clone it in-frame with the binding component, in order to clone it in the cloning cassette system previously described by the inventors (see published international patent application WO97 / 23613, FIG. ) Was replaced.

Example 2 Cloning of Adapter Molecules All whole adapter molecules and their parts were cloned by PCR. The sequences of all oligonucleotides used to clone the adapters in the examples described below are shown in FIG. For many adapters, the cloning process was performed more than once with different oligonucleotides. This provides DNA encoding adapter molecules (or parts thereof) flanked by different restriction endonuclease recognition sites, thus facilitating subsequent subcloning operations.

A) LAT (Weber et al., 1998). This adapter is a human leukocyte cDNA
Cloned from A (Clontech). Residues 3 to 233 (including the transmembrane and cytosolic regions of LAT) were labeled with oligos D3457 and F15163 to N
It was cloned on the arI to MluI fragment. Only the cytosolic region (residues 28-233) was digested with oligos F15162 and F15163, MluI-Ml.
B on the uI fragment and with oligos F12315 and D3458
It was cloned on the clI to BamHI fragment.

B) TRIM (Bruyns et al., 1998). This adapter was cloned from a human T cell library. Residues 9-186 (including the transmembrane and cytosolic regions of TRIM) use NarI-M with oligos F15170 and F15164.
It was cloned on the Iul fragment. Cytosolic region only (residues 29-18
6) is on oligo MluI to MluI using oligos F15165 and F15164,
And BglII to BamHI with oligos F18148 and F16131
Cloned above.

C) SIT (Marie-Cardine et al., 1999). This adapter was also cloned from a human T cell library. Residues 42-196 (SIT
Containing the transmembrane and cytosolic regions of C. elegans) was cloned onto the NarI to MluI fragment using oligos F16130 and F15166. Only the cytosolic region (residues 61-196) was Mlu with oligos F15167 and F15166.
I-MluI was cloned.

D) SLP-76 (Jackman et al., 1995). This adapter is a human T
It was also cloned from a cell library. Residues 2 to 533 (cytosolic region of SLP-76) are MluI to Ml using oligos F15169 and F15168.
Bcl on uI fragment and with F16128 and F12840
It was cloned onto the I-BamHI fragment.

E) Grb-2 (Lowenstein et al., 1992). This adapter was cloned from human leukocyte cDNA (Clontech). Residues 2 to 217 containing the cytosolic region of Grb-2 were Bcl with oligos D8827 and D8828.
It was cloned onto the I-BamHI fragment.

F) GRAP-2 (Qiu et al., 1998). This adapter is also human leukocyte cd
It was cloned from NA (Clontech). Residues 2 to 330 containing the cytosolic region of GRAP-2 were converted to BclI to B using oligos D8825 and D8826.
It was cloned on the amHI fragment.

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

Example 3 Construction of Sequence Blocks (SB) of Primary and Secondary Signaling Motif Each sequence block is such that they are half of the BclI site at the 5'end and B at the 3'end.
It was generated by annealing two oligos so that they have a single-stranded overhang that forms half of the amHI site. Oligo is 25 mM NaC
1, 12.5 mM Tris-HCl, 2.5 mM MgCl ₂ , 0.25 mM
Annealing was performed for 5 minutes in a boiling water bath at a concentration of 1 pmole / μL in a buffer composed of DTE, pH 7.5, and then the water bath was allowed to cool slowly to room temperature.

The predicted amino acid sequences for these examples of SB are shown in FIG. 4 and the sequences of the oligonucleotides used in their construction are shown in FIG. All oligonucleotides
Phosphorylated at their 5'ends.

A) SB1. This sequence is based on the first ITAM of human TCR zeta, oligo A8
It was constructed by annealing 816 and A8817.

B) SB2. This sequence is based on the second ITAM of human TCRζ and is based on oligo A8.
It was constructed by annealing 814 and A8815.

C) SB3. This sequence is based on the third ITAM of human TCR zeta, oligo A8
It was constructed by annealing 812 and A8813.

D) SB4. This sequence is based on ITAM of the human FcεR1 γ chain and is based on oligo A
It was constructed by annealing 8810 and A8811.

E) SB4 ^* . This sequence was initially generated with an error due to oligo misannealing above, but was subsequently constructed by annealing oligos A8810B and A8811B.

F) SB5. This sequence is based on the ITAM of the β chain of human FcεR1 and is based on oligo A
It was constructed by annealing 9000 and A9001.

G) SB6. This sequence is based on the ITAM of human CD3 γ chain and is based on oligo A90.
02 and A9003 were annealed and constructed.

H) SB7. This sequence is based on the ITAM of the human CD3 delta chain and is based on oligo A90.
It was constructed by annealing 04 and A9005.

I) SB8. This sequence is based on the ITAM of human CD3 ε chain and is based on oligo A90.
It was constructed by annealing 06 and A9007.

J) SB9. This sequence was constructed by annealing oligos A9008 and A9009 based on human CD5 ITAM.

K) SB10. This sequence is based on the human CD22 ITAM, oligo A90
It was constructed by annealing 10 and A9011.

L) SB11. This sequence is based on the ITAM of human CD79a and is based on oligo A9
It was constructed by annealing 012 and A9013.

M) SB12. This sequence is based on the human CD79b ITAM and is based on oligo A90.
It was constructed by annealing 14 and A9015.

N) SB13. This sequence is based on ITAM of human CD66d and is based on oligo A90.
It was constructed by annealing 16 and A9017.

O) SB14. This sequence is a synthetic sequence and includes oligos D5258 and D5259.
Built by annealing.

P) SB15. This sequence is a synthetic sequence and contains oligos F6392 and F6394.
Built by annealing.

Q) SB16. This sequence is a synthetic sequence and includes oligos F6333 and F6395.
Built by annealing.

R) SB28. This sequence is based on the human CD28 secondary signaling (costimulatory) sequence and was constructed by annealing oligos A9018 and A9019.

S) SB29. This sequence is based on the secondary signaling (costimulatory) sequence of human CD154 and was constructed by annealing oligos A9020 and A9021.

T) SB34. This sequence is based on the secondary signaling (costimulatory) sequence of human CD134 and was constructed by annealing oligos F1340A and F1340B.

Example 4: Construction of Adapter Receptor Adapter molecules on the various fragments described were labeled with BclI to Bam.
Cloning with primary and secondary signaling motifs on the HI fragment allows the construction of multiplicity of adapter receptors. Thus, the cytoplasmic regions of these receptors include adapter molecules either by themselves or in combination with any number of additional adapter molecules and / or primary signaling motifs and / or secondary signaling sequences.

The adapter molecule can be cloned into the cassette described in Example 1 on the NarI to BamHI fragment along with the native transmembrane region. That is, the transmembrane region can be replaced. This fragment may be cloned upstream of another primary signaling motif or secondary signaling sequence or adapter molecule, or another primary signaling motif, secondary signaling sequence or adapter molecule may then be cloned downstream.

The adapter molecule can be cloned on the MluI to MluI fragment in the cassette described in Example 1 downstream of the transmembrane region. This fragment may be cloned upstream of other signaling motifs or adapter molecules. It may then be cloned downstream by inserting another primary signaling motif, secondary signaling sequence or adapter molecule at the BamHI site in the vector.

The adapter molecule is BclI or BglII to BamH of the cassette described in Example 1.
It can be cloned on the I fragment downstream of the transmembrane region. Cloning in the correct orientation allows subsequent downstream cloning of other signaling motifs, secondary signaling sequences or adapter molecules. Alternatively and / or such fragments can be cloned downstream of other primary signaling motifs, secondary signaling sequences or adapter molecules already cloned into the cassette.

The primary signaling motifs and secondary signaling sequences on the BclI to BamHI fragment (encoded on SB, see FIG. 4) should be cloned downstream of the BamHI site, the transmembrane region of the cassette described in Example 1. You can Cloning in the correct orientation allows downstream subsequent cloning of other signaling motifs / sequences or adapter molecules. Alternatively and / or such fragments can be cloned downstream of other primary signaling motifs, secondary signaling sequences or adapter molecules already cloned into the cassette.

This cassette exchanges the binding component on NotI / HindIII for a SpeI fragment; the extracellular spacer on SpeI for a NarI fragment,
And also facilitates the exchange of the transmembrane region on NarI for a MluI fragment. Therefore, adapter receptors with different binding, extracellular spacers, transmembrane and signaling components can be easily assembled.

Example 5: Analysis of Adapter Receptors a) Construction of expression plasmids: Adapter Receptor Constructs were prepared using pBluescript
pt KS + to HindIII to EcoRI restriction fragment expression vector pEE6hCMV. subcloned into ne (Cockett et al., 1991). The empty expression vector (ie, lacking the chimeric receptor gene) was used as a negative control.

B) Transfection of Jurkat E6.1 cells: The expression plasmid was linearized and BioRad Gene Pu to generate a stable cell line.
Transfected into Jurkat E6.1 cells (ECACC) by electroporation with lser: cells (~ 2.5x10 ⁶ ) were DN
Mixed with A (10 μg) and pulsed twice with 1 kV, 3 μF (0.4 cm electrode gap cuvette) in 1 mL PBS. Cells were left overnight in non-selective medium to allow recovery, then selected and antibiotic G418 (Sigma) 1.5 mg / m 2.
The cells were cultured in a medium supplemented with L. After about 4 weeks the cells were ready for analysis.

For transient expression in Jurkat cells, DuoFect (Quantu)
m Biotechnologies Inc. ) Was used to transfect the expression plasmid according to the manufacturer's instructions.

C) Surface expression analysis: FACS: Approximately 5 × 10 ⁵ Jurkat cells at 1 μg /
Stained with mL of FITC-labeled antigen or antibody specific for the binding component of the adapter receptor. FITC-labeled CD33 antigen was used for analysis of receptors by the P67scFV binding component. Fluorescence is FACScan cytometer (Becton
Dickinson).

D) Functional analysis: IL-2 production: 2 × 10 ⁵ cells in 96 well plates at 37 ° C., 8% CO ₂ for 20 hours, target cells to effector: target ratio 1:
Incubated at 1. To further provide antibody stimulation, either primary stimulation with OKT3 or secondary stimulation with anti-CD28 (Caltag) was used, these antibodies were added to a final concentration of 2-5 μg / mL. Cell supernatants were then harvested and assayed for human IL-2 (R & D Systems Qu).
antikine kit).

When the binding component was P67scFV, the target cells used were as follows: HL60 cells-a human cell line that naturally expresses the antigen CD33. Mouse myeloma transfected with EE6-control expression vector (
NS0) These cells are used as negative control target cell lines. N. CD33-mouse myeloma (NS0) transfected with an expression vector that facilitates expression of the antigen CD33 on the cell surface

Example 6: Results IL with Jurkat cells expressing adapter receptor in response to antigen challenge (either with HL-60 or N.CD33 as indicated).
The specific production of -2 is used as an indicator of the degree of cell activation in all experiments described below.

A) Antigen-specific stimulation of adapter receptor (FIG. 5): Adapter receptor P67s
cFV / h. CD28 / LATtm / LAT (P67 scFV binding component, h.
HL60 cells were used to provide the antigen challenge to Jurkat cells expressing the CD28 spacer (including the LAT transmembrane and signaling components). The results in Figure 5 demonstrate that cells expressing the adapter receptor can produce IL-2 as a specific response to this challenge.

B) Providing additional primary signaling to the adapter receptor using the OKT3 antibody (FIG. 6): Adapter receptor P67scFV / h. CD28 / LATt
To provide an antigenic challenge to Jurkat cells expressing m / LAT,
HL60 cells were used. FIG. 6 shows antibody O to cells expressing adapter receptor.
It is shown that provision of additional primary signaling with KT3 results in increased production of IL-2. This means that the addition of primary signaling motifs is advantageous and leads to increased activation of cells.

C) Providing additional primary signaling to the adapter receptor using the primary signaling motif (FIG. 7): Adapter receptor P67scFV / h. CD2
8 / LATtm / LAT and P67scFV / h. CD28 / LATtm / LA
T. Jurkat cells expressing SB14 were challenged with HL60 cells. The results illustrated in Figure 7 show that IL-2 production by cells expressing the adapter receptor can be significantly increased by inclusion of a primary signaling motif within the receptor. In this particular example, the primary signaling motif S
B14 ^a is, receptor P67scFV / h. CD28 / LATtm / LAT. SB
To generate 14, it was included at the 3'position relative to the adapter molecule.

D) Further exemplification of the provision of additional primary signaling capacity to the adapter receptor by inclusion of a primary signaling motif (FIG. 8): The data shown in FIG. 8 shows that IL-in Jurkat cells expressing adapter receptor. It provides further evidence that the production of 2 can be improved by the inclusion of a primary signaling motif within the adapter receptor. In this particular experiment, the antigen challenge was N. Provided by CD33 cells. Again, in response to a specific stimulus, the CD33 antigen, IL
-2 is produced, further demonstrating that it is not the result of non-specific stimulation by human cell lines.

E) Using the OKT3 antibody, providing additional primary signaling capacity for adapter receptors already having the primary signaling motif (FIG. 9): adapter receptor P67scFV / h. 28 / LATtm / LAT. To provide antigenic challenge to Jurkat cells expressing SB14, N. CD33 cells were used. The results shown in FIG. 9 demonstrate that the production of IL-2 can be increased even more by providing additional primary signaling with the antibody OKT3. This suggests that additional additions of primary signaling motifs (ie additions of 2 or more) are advantageous.

F) Use of anti-CD28 antibody to provide additional secondary signaling to the adapter receptor (FIG. 10): Adapter receptor P67scFV / h. 28 / LAT
tm / LAT and P67scFV / h. 28 / LATtm / LAT. To provide antigenic challenge to Jurkat cells expressing SB14, N. CD33 cells were used. The results shown in FIG. 10 show that I by cells expressing adapter receptor
It has been shown that L-2 production can be further increased by the additional provision of secondary signaling with anti-CD28 antibodies. This suggests that the addition of secondary signaling sequences is advantageous in the presence or absence of primary signaling motifs.

G) Comparison of Signaling Ability of Different LAT Adapter Receptors (FIG. 11): HL60 Cells Used to Provide Antigen Challenge to Jurkat Cells Transiently Expressing Different LAT Adapter Receptors . The results in Figure 11 show that LAT adapter receptors with different additional signaling motifs / sequences (primary and / or secondary) can regulate signaling to varying degrees.

H) Comparison of signaling abilities of different SIT adapter receptors (FIG. 12): SIT with different additional signaling motifs / sequences (primary and / or secondary)
HL60 cells were used to provide an antigen challenge to Jurkat cells transiently expressing the adapter receptor. Results are shown in FIG. SIT, as reported in its cytosolic region, is ITIM (intracellular tyrosine inhibitory motif)
Negative signaling may be provided via

I) Comparison of signaling capacity of various TRIM adapter receptors (FIG. 13):
HL60 cells were used to provide an antigen challenge to Jurkat cells transiently expressing various TRIM adapter receptors. The results in Figure 13 show that TRIM adapter receptors with different additional signaling motifs and / or sequences (primary and / or secondary) can regulate signaling to varying degrees.

References Ausubel FW & Kingston R. et al. E. (1992) Short protocols in Molecular Biology
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GA, Koretzky, GA & Findell, PR (1995) Journal Biological Chemistry
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M., Autschbach, F., Ratnofsky, S., Meuer, S. & Schraven, B. (1999) Jour
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[Brief description of drawings]

FIG. 1: Cloning cassette for the construction of adapter receptors with multiple signaling components.

FIG. 2 is a schematic diagram of the h.2 used in the construction of the cloning cassette described in FIG. Nucleotide and amino acid sequences of the CD28 extracellular spacer and human CD28 transmembrane region.

[Figure 3]   Oligonucleotide sequences used to construct the adapter receptor.

FIG. 4 Amino acid sequences of primary and secondary signaling sequences used in the construction of adapter receptors.

FIG. 5: Adapter receptor, p67scFv / h. Antigen-specific stimulation of CD28 / LATtm / LAT.

FIG. 6: Adapter receptor p67scFv / h. CD28 / L
Providing primary signaling for ATtm / LAT.

FIG. 7: Adapter receptor p67s upon addition of the primary signaling motif SB14 ^a
cFv / h. Addition of primary signaling to CD28 / LATtm / LAT.

FIG. 8: Adapter receptor p67s upon addition of the primary signaling motif SB14 ^a
cFv / h. Addition of primary signaling to CD28 / LATtm / LAT.

FIG. 9. Adapter receptor p67scFv / h. CD
28 / LATtm / LAT. Addition of further primary signaling to the SB 14.

FIG. 10. Adapter receptor p67scFv / h.h. Stimulated with anti-CD28 antibody. CD
28 / LATtm / LAT. Providing secondary (costimulatory) signaling to SB14.

FIG. 11: LA with various additional primary signaling motifs and secondary signaling sequences
Analysis of transient transfections in Jurkat of the T adapter receptor.

FIG. 12 SI with various additional primary signaling motifs and secondary signaling sequences.
Analysis of transient transfections in Jurkat of the T adapter receptor.

FIG. 13: TR with various additional primary signaling motifs and secondary signaling sequences
Analysis of transient transfection in Jurkat of IM adapter receptor.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] December 27, 2001 (2001.12.27)

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 3

[Correction method] Change

[Contents of correction]

[Figure 3]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/00 A61P 17/00 3/10 17/04 7/04 17/06 11/06 19/04 17 / 00 25/00 17/04 25/28 17/06 29/00 19/04 31/00 25/00 31/18 25/28 35/00 29/00 37/06 31/00 43/00 111 31 / 18 C07K 14/705 35/00 C12N 1/15 37/06 1/19 43/00 111 1/21 C07K 14/705 15/00 ZNAA C12N 1/15 5/00 A 1/19 A61K 37/02 1 / 21 37/52 5/10 37/48 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH , GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ , VN, YU, ZA, ZWF F terms (reference) 4B024 AA01 AA11 BA63 CA04 CA12 DA03 HA11 HA17 4 B065 AA94X AA94Y AB01 CA24 CA44 CA46 4C084 AA02 BA01 BA08 BA19 CA18 CA53 DA00 DA45 DC50 NA14 ZA15 ZA55 ZA59 ZA89 ZA96 ZB08 ZB11 ZB26 ZB31 ZC21 ZC35 4H045 AA10 AA20 AA30 BA20 CA40EA50 DA50

Claims (37)

[Claims] 1. A nucleic acid encoding an adapter receptor protein comprising an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises at least one cytoplasmic portion of the adapter protein. The above nucleic acid, wherein the nucleic acid comprises and the extracellular ligand binding domain is not a CD8 or MHC class I protein. 2. At least one adapter protein is LAT, SLP-
The nucleic acid according to claim 1, which is selected from 76, Grap, Grb2, TRIM, SIT or Cbl. 3. The nucleic acid according to claim 2, wherein the adapter protein is selected from LAT, TRIM or SIT. 4. The transmembrane domain is an α, β or ζ chain of the T-cell receptor, C
D28, CD3ε, DC45, CD4, CD5, CD8, CD9, CD16, C
The nucleic acid according to any one of claims 1 to 3, which is derived from D22, CD33, CD37, CD64, CD80, CD86 or CD154. 5. The nucleic acid according to claim 1, wherein the transmembrane domain is derived from an adapter protein. 6. The nucleic acid according to claim 5, wherein the adapter protein is LAT, SIT or TRIM. 7. The nucleic acid according to any one of claims 1 to 3, wherein the transmembrane domain is a synthetic domain. 8. The nucleic acid according to any one of claims 1 to 7, wherein the extracellular ligand-binding domain comprises an antibody binding domain or a fragment thereof. 9. The nucleic acid according to claim 8, wherein the extracellular ligand-binding domain is a Fab ′ fragment or scFv. 10. The nucleic acid according to any one of the preceding claims, wherein the adapter receptor comprises at least two extracellular ligand binding domains. 11. The nucleic acid of claim 10, wherein each ligand binding domain exhibits specificity for a different ligand. 12. The nucleic acid according to any one of the preceding claims, wherein the intracellular signaling domain comprises at least one cytoplasmic part of an adapter protein and at least one further cytoplasmic signaling component. 13. The nucleic acid of claim 12, wherein at least one additional cytoplasmic signaling component is a primary signaling motif. 14. The primary signaling motif comprises a consensus amino acid sequence: YX ₂ -L / I-Xn-YX ₂ -L / I (wherein, the amino acid residue is represented by a one-letter code, and X represents any amino acid. , The subscript number represents the number of residues present at that position in the motif, the number n being 5-12). 15. The nucleic acid according to claim 14, wherein the number n is 6-8. 16. The nucleic acid of claim 15, wherein the primary signaling motif is a naturally occurring immunoreceptor tyrosine-based activation motif (ITAM). 17. The primary signaling motif is TCRζ chain, FcRζ, Fc
Rβ, CD3γ, CD3δ, CD5, CD22, CD79a, CD79b or C
The nucleic acid according to claim 16, which is derived from D66d or is a mutant thereof. 18. The nucleic acid of claim 15, wherein the primary signaling motif is a non-natural motif. 19. The primary signaling motif is:       GQDGLYQELNTRSRDEYSVLEGRKAR or       GQDGLYQELNTRSRDEAYSVLEGRKAR 19. The nucleic acid of claim 18, which is 20. The nucleic acid according to claim 14, wherein the number n is 9 or more. 21. The primary signaling motif is:       RKNPQEGLYNELQKDKMAEDTYDALHMQA,       GQNQLYNELQQQQQQQQQYDVLRRGRDPEM or       GQDGLYQELNTRSRDEAAYSVLEGRKAR 21. The nucleic acid of claim 20, which is 22. The nucleic acid of claim 13, wherein the primary signaling motif is an immunoreceptor tyrosine-based inhibitory motif (ITIM). 23. The primary signaling motif is FcγR, CD22, EPO
23. The nucleic acid of claim 22, which is derived from R, IL-2βR, or IL-3βR. 24. The nucleic acid of claim 12, wherein the at least one additional cytoplasmic signaling component is a secondary signaling sequence. 25. The secondary signaling sequence is CD28, CD134 or CD1.
25. The nucleic acid of claim 24, which is derived from 54. 26. The secondary signaling sequence is       RLLHSDYMNMTPRRPGPTRKHYQPYAPPRD,       MIETYNQTSPRSAATGLPISMK or       RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 26. The nucleic acid of claim 25, which is 27. The nucleic acid according to any one of the preceding claims, wherein the adapter receptor comprises an additional domain representing an intrinsic enzymatic activity, provided that the intrinsic enzymatic activity is not a hydrolase activity. 28. The nucleic acid of claim 27, wherein the additional domain forms the cytoplasmic domain of the adapter receptor. 29. The nucleic acid of claim 28, wherein the additional domain represents transferase activity. 30. The nucleic acid of claim 29, wherein the additional domain exhibits protein tyrosine kinase activity. 31. A vector comprising the nucleic acid according to any one of the preceding claims. 32. An adapter receptor protein encoded by the nucleic acid of any one of the preceding claims. 33. A nucleic acid according to any one of claims 1 to 33 or an adapter receptor protein according to claim 33 for use in therapy. 34. A composition comprising a nucleic acid according to any one of the preceding claims, or a vector or adapter receptor protein, together with a pharmaceutically acceptable excipient. 35. Use of the nucleic acid or adapter receptor protein or composition according to any one of the preceding claims for the manufacture of a medicament for the treatment or prevention of human or animal disease. 36. A host cell containing the nucleic acid according to any one of claims 1 to 30 or the vector according to claim 31. 37. A host cell containing the adapter receptor protein of claim 33.