JP2003516127A - Polypeptides having an extended first signaling motif - Google Patents

Abstract

(57) Abstract: The present _{invention, YX 2 -L / IX n -YX} 2 -L / I, n is relates cytoplasmic signaling sequence including a new first signaling motifs comprising 9 or more common amino acid sequence is there. This novel motif is extremely effective at mediating immune cell signaling, especially when incorporated into the intracellular signaling domain of the chimeric receptor. Nucleic acids encoding the non-naturally stimulating first signaling motif and polypeptides containing the same are described, which are suitable for use in medicine.

Description

Detailed Description of the Invention

The present invention relates to novel cytoplasmic signaling molecules, nucleic acids encoding them, and polypeptides and nucleic acids thereof for use in medicine and research.

[0002] This specification cites various publications by author and year of publication. These publications are cited in the detailed description of the invention and examples below.

Research in the field of immune cell signaling has provided a significant amount of information on the signaling events that occur downstream of antigen-receptor binding. A considerable amount of research has focused on the receptors themselves and on the enzymes stimulated by antigen binding (Weis
s & Littman, 1994; DeFranco, 1997).

The individual components of the T cell receptor (TCR) complex have been well analyzed and often the function of receptor subunits or domains has been demonstrated by constituting chimeric receptor proteins. (Kuwana et al., 1987; Romeo et al., 1992)
. In particular, the cytoplasmic signaling domain and its role in TCR activation were identified using this method. However, only recently have the chimeric receptors been used as regulators of the process of cell activation (eg International patent application WO
See 97/23613 and WO 95/02686). Ability of chimeric receptors to regulate biological effects associated with cell activation, such as increased cell proliferation, increased cytokine expression, accelerated cytolytic activity, differentiation of other effect functions, antibody secretion, phagocytosis, tumor Increased infiltration and / or cell adhesion has considerable therapeutic potential.

While currently available chimeric receptors can effectively activate cells, the cytoplasmic signaling domain of chimeric receptors is derived from the extracellular ligand binding domain,
It signals downstream factors of the second messenger pathway, such as the src and syk-tyrosine kinase families, and there is room for improvement in terms of its effectiveness.

It would be advantageous if the cytoplasmic signaling domain could be improved or engineered to achieve the required level of effector cell activation. It would be of enormous benefit if cell activation could be performed more effectively than is currently possible and the induction or inhibition of any biological response could be controlled.

The present invention provides a nucleic acid encoding a novel cytoplasmic signaling sequence capable of effectively controlling the level of cell activation when expressed in effector cells,
It addresses these issues.

As used herein, the term “cytoplasmic signaling sequence” refers to the TCR and co-receptor cytoplasmic sequences that act in concert to initiate signal transduction following antigen-receptor binding. is there. The term also includes derivatives or variants of this sequence, as well as synthetic sequences, provided they have the same function. Signals generated by the TCR alone are not sufficient for full activation of T cells; secondary or supplemental stimulatory signals are required. Thus, cytoplasmic signaling components can be divided into two groups: those that initiate their initial activation in a TCR-dependent manner (first signaling sequence) and those that are secondary in an antigen-independent manner or Supplementary stimulation (second signal transduction sequence).

A first aspect of the invention provides a nucleic acid encoding a cytoplasmic signaling sequence comprising a first signaling motif having a common amino acid sequence of YX ₂ -L / IX _n -YX ₂ -L / I. In the above amino acid sequence, amino acid residues are represented by a standard one-letter code, X indicates any amino acid, subscript numbers indicate the number of residues at positions within the motif, and the value of n is 9 That is all. The term X ₂ or X _n is intended to indicate (respectively) 2 or n amino acids which may be the same or different. The value of n is preferably between 9 and 12, with a value of 9 being particularly preferred.

The term “first signaling motif” is defined as a sequence that transmits a stimulatory or inhibitory signal that regulates the first activation of the TCR complex. It will be appreciated that the first signaling sequence may include a single motif or more than one first signaling motif. The naturally occurring, stimulatory, first signaling motif is
The consensus sequence YX ₂ -L / IX _n -YX ₂ -L / I, where n is between 6 and 8, most typically
It is said to be 7. These motifs are also known as immunoreceptor tyrosine-containing activation motifs or ITAMs (Isakov, 1998). In contrast, the immunoreceptor tyrosine-containing inhibitory motif (ITIM) containing the consensus amino acid sequence I / VXYX ₂ -L (Brushtyn et al., 1999) is an example of an inhibitory first signaling motif. Except in some cases in Figure 2 and in the examples, where the standard three-letter code is used to describe the amino acid sequence, no matter what the amino acid or nucleotide sequence is described throughout this application. Note that we are using the standard one letter code.

The present invention provides novel first signaling motifs that can regulate different levels of first signaling and broaden the range of cell activation levels. The first activation is known to involve the src and syk families of protein tyrosine kinases binding to ITAM and subsequent phosphorylation of tyrosine residues within the motif. Thus, the novel first signaling motifs of the invention appear to mediate first signaling by possessing altered specificity and affinity for these kinases. This can be easily analyzed by an assay by Western blot using an anti-phosphotyrosine antibody (Sanchez-Garcia, et al., 1997). The assay can be used as the basis for a functional screen for the activity of the novel first signaling motif of the invention.

[0012]   The consensus sequence of the novel first signaling motif is that of the naturally occurring ITAM,
They differ in that the distance between the two tyrosine residues is increased by at least 1 amino acid.
(Ie, the new motif is a new consensus sequence Y-X with an n value of at least 9)₂-L /
I-X_n-Y-X₂-Same as L / I). These "extended" motifs are
At least the same, or better yet, able to signal
It The distance between tyrosine is decreased by one corresponding amino acid (that is, from n = 6 to n = 5)
This significantly reduces the signaling capacity of the first signaling motif, so
That is amazing. The first signaling motif used in the present invention may be
Is an odd array, Y-X₂-L / I-X_n-Y-X₂-L / I, n value is 9 or more.
However, desirable motifs are those with an n value of 9, and especially desirable motifs.
F is SBX^a(This has the amino acid sequence: RKNPQEGLYNELQKDKMAEDTYDALHMQA), SBQ
9^a(It has the amino acid sequence: GQNQLYNELQQQQQQQQQYDVLRRGRDPEM) and SB16 ^a (This has the amino acid sequence: GQDGLYQELNTRSRDEAAYSVLEGRKAR). That
Other examples are shown in Figure 4 as SBX, SBQ9 and SB16, which include cloning
Additional GS linkers are incorporated at each end of the sequence to facilitate.

When the novel cytoplasmic signaling motif of the present invention is used as the intracellular domain of the chimeric receptor, the magnitude of the signal transduced through the immune cell receptor is additionally determined by the cytoplasmic signaling motif and / or It is recognized that regulation can be achieved by incorporating sequences within the intracellular domain. Thus, in the second aspect of the present invention, a cytoplasmic signaling molecule consisting of the first signaling motif described in the first aspect of the present invention and a nucleic acid encoding at least one other first signaling motif provide.

The first signaling motif used in this aspect of the invention comprises the consensus amino acid sequence YX ₂ -L / IX _n -YX ₂ -L / I (one letter code). If n is 9, then S
BX ^a , SBQ9 ^a and SB16 ^a can be used as additional motifs. When the n value is between 6 and 8, at least one other first signaling motif is an immunoreceptor tyrosine-containing activation motif (ITAM), eg, derived from the TCR ζ chain, FcRγ, FcRβ, It is desirable to include at least a part of CD3γ, CD3δ, CD5, CD22, CD79a, CD79b or CD66d. Examples of first signaling motifs derived from these molecules are shown in Table 1 below. At least one other first signaling motif for use in this aspect of the invention is TCRζ1, TCRζ2, TCRζ3, FcRγ, FcR.
It is derived from all or part of β, CD3γ, CD3δ, CD5, CD22, CD79a, CD79b or CD66d.

Table 1. Origin and amino acid sequence of the first signaling motif particularly used in the present invention. The position of the common ITAM amino acid sequence is underlined. FIG. 4 also shows other examples of the first signaling motif used in the present invention (SB1, SB2, SB3, SB4, S
B4 ^* , SB5, SB6, SB7, SB8, SB9, SB10, SB11, SB12, SB13, SB14, SB15, SB16, SBX
And SBQ9), which correspond to the first signaling motif shown below with a GS linker incorporated at the end of each motif to facilitate cloning. Source 1st signal Amino acid sequence Transfer motif ^{TCRζ1 SB1 a GQNQL YNELNLGRREEYDVL DKRRGRDPEM TCRζ2 SB2} a RKNPQEGL YNELQKDKMAEAYSEI GMKGER TCRζ3 SB3 a RGKGHDGL YQGLSTATKDTYDAL HMQA FcRγ SB4 a YEKSDGV YTGLSTRNQETYETL KHEKP FcRβ SB5 a GNKBPEDRV YEELNIYSATYSEL EDPGEMSP CD3γ SB6 a KQTLLPNDQL YQPLKDREDDQYSHL QGNQLR CD3δ SB7 a ALLRNDQV YQPLRDRDDAQYSHL GGNWARNK CD3ε SB8 a QNKERPPPVPNPD YEPIRKGQRDLYSGL NQRRI CD5 SB9 ^{a HVDNE YSQPPRNSRLSAYPAL EGVLHRS CD22 SblO a PPRTCDDTVT} YSALHKRQVGDYENV IPDFPEDE CD79a SB11 a EYEDENL YEGLNLDDCSMYEDI SRGLQGTYQDV CD79b SB12 a KAGMEEDHT YEGLDIDQTATYEDI VTLRTGEV CD66d SB13 a PLPNPRTAASI YEELLKHDTNIYCRM DHKAEVA FcRγ SB4 * a YEKSDGV YTGLSTRNQETYDTL KHEKP non-natural SB14 ^a GQDGL YQELNTRSRDEYSVL EGRKAR non-natural SB15 ^a GQDGL YQELNTRSRDEAYSVL EGRKAR non Natural SB16 ^a GQDGL YQELNTRSRDEAAYSVL EGRKAR Non-natural SBX ^a RKNPQEGL YNELQKDKMAEDTYDAL HMQA Non-natural SBQ9 ^a GQNQL YNELQQQQQQQQQYDVL RRGRDPEM

Alternatively, the at least one additional first signaling motif is non-natural but identical to the amino acid sequence of the sequence YX ₂ -L / IX _6-8 -YX ₂ -L / I. "Non-natural ITAM" means a synthetic first signaling motif, the sequence of which is not found in nature. Preferred examples of such non-natural primary signaling motif is SB4 ^{* a,} SB14 ^a or SB15 ^a or a non-naturally occurring variants, described in Table 1 above.

The combination of desirable motif of this aspect of the invention, SBQ9 ^a, SBQX ^a or SB1,
6 ^a plurality; SBQ9 ^a, or combined with one another the SBX ^a or SB16 ^a, a first signaling motifs derived from TCRζ chain (i.e. SB1 ^a, SB2 ^a or SB3 ^a) when combined; or SB4 * combined with ^a. The combination SB16 ^a and SB2 ^a, SB4 * ^a and SB4 ^a, and SB
X ^a and SB3 ^a, it is particularly desirable.

In a third aspect of the invention there is provided a nucleic acid encoding a cytoplasmic signaling molecule comprising a first signaling motif of the first aspect of the invention and at least one second signaling sequence.

The term “second signaling sequence” means a sequence that conveys a second or costimulatory signaling capacity to T cells. Molecules containing such sequences are CD2, CD4, CD8, CD28, C
Such as D134 and CD154 (see Finney et al., 1998). Second signaling sequence desired to be used in the present invention are CD28, CD134 and CD154, for example, those derived SB28 ^a which has the amino acid sequence RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA, SB29 ^a which has the amino acid sequence MIETYNQTSPRSAATGLPISMK, SB34 ^a, from having the amino acid sequence RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI . Examples of other second signaling sequences are SB28, SB29 and SB34 in Figure 4.
, Which incorporates a GS linker at the end of each sequence to facilitate cloning.

The desirable combination of the first signaling motifs and secondary signaling sequence in this aspect of the invention includes combining SBQ9 ^a, the SBX ^a or SB16 ^a as SB28 ^a. The combination of SBQ9 ^a and SB28 ^a particularly desirable.

The first signaling motifs can be combined with each other or with the second signaling sequence in any or specific order. Between the component motifs / sequences there are short oligo- or polypeptide linkages, preferably between 2 and 10 amino acids in length. The glycine diad is a particularly suitable linker.

The novel cytoplasmic signaling sequences and molecules of the invention can be used by themselves or as a component of large proteins such as chimeric receptors. They can be introduced or expressed in effector cells as individual protein molecules to act as cytoplasmic signaling sequences in place of immune cell receptors already expressed in cells. In this way they can increase the efficiency of signal transduction from the receptor. Alternatively, it can be introduced into cells to compete with existing src and syk-protein kinase binding sites and control the extent of cellular activity by acting as a protein kinase inhibitor.
Any one of a number of in vitro assays can be used to assess the efficacy of these novel cytoplasmic signaling molecules as protein kinase inhibitors, eg International Patent Application W098 / 11095. See.

However, it is believed that the cytoplasmic signaling sequences and molecules of the present invention are desirably used to mediate signal transduction when used as the intracellular domain of chimeric receptor proteins. The chimeric receptor further contains an extracellular ligand binding domain and a transmembrane domain.

By incorporating the extracellular ligand binding domain, the chimeric receptor is given the ability to exhibit specificity for a particular ligand or ligands. This specificity can be used to precisely define the ligand or ligands that can activate the receptor. In this way, of the selected group,
Alternatively, the receptor can be designed to bind individual ligands and activate the cells in which they are expressed.

When contact occurs between the ligand and the corresponding binding domain in the chimeric receptor,
Signal transduction occurs through the cytoplasmic signaling domain. Selection of motifs,
Alternatively, the combination of motifs and sequences with the cytoplasmic signaling domain, determines the magnitude of signal transduction and, consequently, the extent to which cells are activated.

Another aspect of the invention is an extracellular ligand binding domain, a transmembrane domain and
There is provided a nucleic acid encoding a chimeric receptor protein comprising a cytoplasmic signaling domain encoded by the nucleic acid described in any one of the aforementioned aspects of the present invention.

As used herein, the term “extracellular ligand binding domain” is defined as an oligo or polypeptide capable of binding a ligand. Thus, antibody binding domains, antibody hypervariable loops or CDRs, receptor binding domains and other ligand binding domains, examples of which will be apparent to those of skill in the art, are described in this term. It is desirable that this domain be able to interact with cell surface molecules. Examples of proteins involved in binding to cell surface molecules particularly useful in the present invention include antibody variable domain (V _H or V _L ), T cell receptor variable region domain (TCRα, TCRβ, TCRγ, T
CRδ), or CD8α, CD8b, CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B,
For example, a chain of CD49D, CD49E, CD49F, CD61, CD41, or CD51. In some cases it may be convenient to use the entire domain or chain, but fragments may be used where appropriate.

A particularly useful binding moiety is derived from the antibody binding domain and is derived from Fab ¹ and especially single chain.
For example, Fv fragment.

The choice of domain depends on the type and number of ligands defining the surface of the target cell. For example, the extracellular ligand binding domain can be chosen to recognize a ligand that serves as a cell surface marker for target cells associated with a particular pathology. Examples of cell surface markers that can serve as ligands are those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.
In the last 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, and epithelial cells. There are adhesion molecules (EPCAM) and erb-B2. Other selective ligands give rise to cell surface adhesion molecules, inflammatory cells present in autoimmune diseases, and autoimmunity
T cell receptor or antigen. The ligands that may be listed above are given as examples; the listing is not intended to be exclusive and other examples will be quite apparent to those of skill in the art.

Chimeric receptors can be bi- or multispecific designed, ie
One or more ligand binding domains may be included and thus rendered specific for one or more ligands. Such receptors may be cellular immune effector cells (eg, T cells, B cells, killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, or mast cells) or components of the complement cascade. Can be collected.

The other component of the chimeric receptor is the transmembrane domain. It can be derived from nature or obtained synthetically. If the source is natural, the domain can be derived from any membrane bound or transmembrane protein. The transmembrane region particularly useful in the present invention is α of T cell receptor (including at least the transmembrane region).
, Β, or ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,
It can be derived from CD37, CD64, CD80, CD86, CD134, CD137 or CD154. Transmembrane domain is α, β, or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, C
It is desirable to derive from all or part of D5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. It is particularly desirable to use the transmembrane domain derived from CD28. Alternatively, the transmembrane domain can be synthesized, in which case it will predominantly contain hydrophobic residues such as leucine and valine (see, eg, International Patent Application WO 00/63374). A phenylalanine, tryptophan and valine triplet will be found at each end of the synthetic transmembrane domain.

A spacer domain can be incorporated between the extracellular ligand binding domain and the transmembrane domain, or between the cytoplasmic signaling domain and the transmembrane domain. As used herein, the term "spacer domain" generally refers to an oligo or polypeptide that has the function of linking the transmembrane domain to the extracellular ligand binding domain or cytoplasmic signaling domain within the polypeptide chain.
The spacer domain can contain up to 300 amino acids, but is preferably 10 to 1
00 amino acids, most preferably 25 to 50 amino acids.

The spacer domain can be derived from all or part of a naturally occurring molecule, eg from all or part of the extracellular region of CD8, CD4 or CD28; all or part of the antibody constant region; cytoplasmic signaling. All or part of the natural spacer between the component functional parts, eg the spacer between ITAMs. Alternatively, the spacer may be an entirely synthetic spacer sequence.

The spacer domain should be designed to reduce the constitutive association of the chimeric receptor to reduce the occurrence of constitutive activation of cells, or the level of such association and constitutive activation in cells. You can The potential exists naturally with deletions, insertions, alterations or with side chain residues that may covalently or non-covalently interact with amino acids and amino acid side chains of other polypeptide chains. This can be achieved by modifying the sequence of the transmembrane and / or spacer domain.
Examples of amino acids that would normally be expected to promote association are cysteine residues, charged amino acids or amino acids such as serine or threonine within potential glycosylation sites, and the like.

The cytoplasmic signaling molecule of the present invention may optionally be linked to other intracellular components or transmembrane domains through a short oligo or polypeptide linker, preferably between 2 and 10 amino acids. The glycine-serine diad is a particularly suitable linker.

Chimeric receptors can be designed so that the spacer and transmembrane components have free thiol groups, and thus provide multimeric receptors, particularly dimeric receptors. Such multimeric receptors, especially dimers, are desirable. Receptors with transmembrane and spacer domains derived from the CD28 component, the zeta chain of native T cell receptors, and / or antibody hinge sequences are particularly desirable.

It is contemplated that the component motifs and novel cytoplasmic signaling molecules of the invention may be linked in such a way as to achieve a desired level of activation (or even inhibition) of multiple second messenger cascades. . When the cytoplasmic signaling motif / molecule of the present invention is used as the intracellular domain of the chimeric receptor, activation / activation
Inhibition can be initiated by a unimolecular binding event in the extracellular ligand binding domain. The cytoplasmic signaling motif, or a combination of motif and sequence, can be on separate polypeptide chains or can be contiguous on the same polypeptide chain. The idea that a single polypeptide chain or multiple polypeptide chains supply the components can be applied to the chimeric receptors of the invention as well.

The invention thus also provides novel cytoplasmic signaling proteins and chimeric receptor proteins encoded by the nucleic acids described in any of the above aspects of the invention.

The nucleic acid encoding the sequence for the novel first signaling motif used in the present invention can be easily derived from the specified amino acid sequence. Other nucleic acid sequences are widely reported in the scientific literature and are available in public databases. The DNA is available commercially, is part of a cDNA library, or can be generated using standard molecular biology and / or chemical methods, as will be apparent to those skilled in the art. . Particularly suitable techniques are polymerase chain reaction (PCR), oligonucleotide directed mutagenesis, oligonucleotide directed synthesis techniques, enzymatic cleavage or insertion of gap oligonucleotides, and the like. The technology is Sambr
ook & Fritsch, 1989 and is included in the examples below.

The nucleic acid of the present invention can be used with a carrier. The carrier may be a vector or other carrier suitable for introducing the nucleic acid into a target cell and / or target host cell ex vivo or in vivo. Examples of suitable carriers are retroviruses,
Adenovirus, adeno-associated virus (AAV), Epstein-Barr virus (EB
V) and herpes simplex virus (HSV). Non-viral vectors can also be used, examples of which are liposomal vectors and vectors based on condensing agents such as cationic lipids, International Patent Application No. W096 / 10038,
W097 / 18185, W097 / 25329, W097 / 30170 and W097 / 31934. If appropriate, the vector further comprises promoters and regulatory sequences and / or viral replication functions such as retrovirus long terminal repeats (LTRs), AAV repeats, SV40.
And a human cytomegalovirus (hCMV) promoter and / or enhancer, splicing and polyadenylation signals and EBV and BK virus replication functions. TCR-α promoter, E-selectin promoter and CD2
Tissue-specific regulatory sequences such as promoters and locus regulatory regions may also be used. The carrier may be an antibody.

The present invention also includes cloning and expression vectors that include a nucleic acid as described in any of the above aspects of the invention. The expression vector is linked to the nucleic acid molecule of the invention in the appropriate reading frame to provide suitable transcriptional and translational regulatory sequences, such as enhancer elements, promoter-operator regions, termination and termination sequences, mRNA stabilizing sequences,
It incorporates start and stop codons or ribosome binding sites.

Furthermore, it does not include an originally effective signal peptide in the protein sequence, which is convenient for secreting a recombinant cytoplasmic signaling protein from a host. Thus, other components of such vectors are secretion signals and processing sequences.

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 documented. For example, it has been established to use prokaryotic cells such as E. coli to express heterologous polypeptides and polypeptide fragments (see, eg, Sambrook & Fritsch, 1989, Glover, 1995a). Similarly,
Eukaryotic expression systems are well developed and widely used for heterologous protein expression (see, eg, Glover, 1995b and O'Reilly et al., 1993). In eukaryotic cells, apart from yeast, the vector of choice is virus-based. Particularly suitable viral vectors are those based on baculovirus, adenovirus and vaccinia virus.

Vectors containing the relevant regulatory sequences (including promoters, terminations, polyadenylation, and enhancer sequences, marker genes) can be selected from those described in the literature or can be prepared using standard molecular biology techniques. And can be readily constructed to express the proteins of the invention. Such techniques and procedures for handling nucleic acids, such as preparation of nucleic acid constructs, mutations, sequencing, DNA transformation and gene expression, and protein analysis are described in Ausubel et al., 1992 or Rees et al., 19
93 for more details.

Suitable host cells for high level in vitro expression of cytoplasmic signaling molecules or chimeric receptors include prokaryotic cells such as E. coli, eukaryotic yeast such as eg
Saccharomyces cerevisiae, Pichia species, Schizosaccharomyces pombe, mammalian cell lines and insect cells. Alternatively, the recombinant protein or chimeric receptor can be expressed in vivo, eg, in insect larvae or plant cells, or more desirably in mammalian tissue.

Nucleic acid can be introduced into host cells by any suitable technique. In eukaryotic cells, these techniques include calcium phosphate gene transfer, DEAE-dextran, electroporation, particle bombardment, liposome-mediated gene transfer or genes using other viruses such as retroviruses, adenoviruses or vaccinia. And baculovirus for insect cells. The nucleic acid can remain episomal in the cell or can be integrated into the cell's genome. If the latter is desired, a sequence that facilitates recombination with the genome is included in the nucleic acid. After introducing the nucleic acid into the host cell, the cell can be cultured under conditions that enhance or induce expression of the recombinant cytoplasmic signaling molecule or chimeric receptor protein.

In yet another aspect of the invention, the cytoplasmic signaling protein of the invention and / or
Alternatively, a host cell is provided that comprises a nucleic acid that encodes a chimeric receptor protein and that host cell expresses the protein.

In other embodiments, the nucleic acids of the invention are used for ex vivo or in vivo therapy.

For ex vivo use, the nucleic acid is introduced into the effector cells removed from the target host by methods well known to those skilled in the art, such as gene transfer, transfection (including viral transfection), It can be introduced by biolistics, protoplast fusion, calcium phosphate mediated DNA transformation, electroporation, cationic lipid transfer, or directional liposomes. Examples of effector cells suitable for expression of the chimeric receptors of the present invention include cells related to the immune system, such as lymphocytes, such as cytotoxic T lymphocytes, tumor infiltrating lymphocytes, neutrophils, neutrophils, Baseball,
Alternatively, they are T helper cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes or NK cells. It is particularly desirable to use cytotoxic T lymphocytes.

The nucleic acids according to this aspect of the invention are particularly suitable for in vivo administration. To achieve this, the carrier may take the form of a directional carrier system in which the DNA directs the effector cells of interest. Suitable directional administration systems include directional naked DNA, directional liposome-encapsulated and / or complex DNA, directional retroviral systems and directional condensed DNA such as protamine and polylysine condensed DNA.

Directed systems are well known, eg cell surface antigens expressed in vivo on the surface of target cells, eg CD8, CD16, CD4, CD3, selecting (eg E-selectin), CD5, CD7. , CD24, and activation antigens (eg, CD69 and IL-2R), or fragments thereof. Alternatively, other receptor-ligand interactions can be used for tropism, eg, CD4 against target HIV gp160-expressing target cells.

In general, it is desirable to use antibody-target DNA, especially antibody-target naked DNA.
, Antibody-target condensed DNA and especially antibody-target liposomes. Types of liposomes that can be used include, for example, pH sensitive liposomes, when a linker that is cleaved at low pH is used to link the antibody to the liposome. The nucleic acid of the present invention can also be directed directly into the cytoplasm by using a cationic liposome that fuses with the cell membrane. The liposome used in the present invention is a hydrophilic molecule,
It can also have, for example, a polyethylene glycol polymer and can be attached to its surface to prolong its circulation half-life. There are numerous examples in this field of groups suitable for attaching DNA to liposomes or other carriers; eg, International Patent Application Nos. W088 / 04924, W090 / 09782, W091 / 05545, W091 / 05546, W093 / 19738. , W094 / 20073
And W094 / 22429). Antibodies or other targeting molecules are linked to DNA, condensed DNA or liposomes using conventional linking groups and reactive functional groups of antibodies, such as thiols or amines, and reactive functional groups of DNA or DNA-containing materials. To do.

Non-directional carrier systems can also be used. In this system, directed expression of proteins is advantageous. This includes, for example, T cell-specific promoter systems such as the zeta promoter, CD2 promoter and locus regulatory regions, CD4, C
This can be achieved by using the D8, TCRα and TCRβ promoters, cytokine promoters such as the IL2 promoter, and the perforin promoter.

The cytoplasmic signaling protein and / or chimeric receptor protein of the present invention, or the nucleic acid encoding the same is intended to be applied to a therapeutic method for mammals, particularly humans and patients. The signaling and chimeric receptor proteins produced by the present invention are particularly useful for treating many diseases or disorders. Such diseases or abnormalities include those described under the general title of infectious diseases, such as HIV.
Infection; inflammatory disease / autoimmunity such as asthma, eczema; congenital disease such as cystic fibrosis, sickle cell anemia; skin disease such as psoriasis; neurological disease such as multiple sclerosis; transplantation such as Organ transplant rejection, graft-versus-host disease; metabolic / idiopathic disease,
For example, diabetes; cancer.

For example, expression of the chimeric receptor on the surface of T cells can cause the ligand-binding domain to bind to a ligand on the target cell and initiate activation of that cell. When inflammatory mediators are reliably released by the activation of receptor signaling function,
Destruction of target cells is ensured.

When the chimeric receptors of the present invention are expressed on effector cells of the immune system, binding to the target activates the effector cells: a downstream effect of this activation results in destruction of the target cells. If the extracellular ligand binding domain of the chimeric receptor shows selectivity for surface markers on immune cells, effector cells can be recruited to the disease site. Therefore, expressing the chimeric receptor in diseased cells would ensure its destruction.

Expression of a multispecific chimeric receptor protein or a plurality of chimeric receptors (different ligand specificities) in one host cell can give the receptor two functions. For example, the binding of the chimeric receptor to its target can not only activate the effector cells themselves, but also attract other immune effector cells to the site of the disease. Target cells can thus be destroyed by activation of the immune system.

In another aspect of the present invention, the cytoplasmic signaling protein of the present invention and / or
Alternatively, there is provided a composition comprising a chimeric receptor protein or a nucleic acid encoding the protein together with a pharmaceutically acceptable additive.

Suitable additives are well known to the person skilled in the art, for example phosphate buffered saline (eg 0.01M phosphate, 0.138M NaCl, 0.0027M KCl, pH7.4), any Water with added mineral salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., saline, liquids such as glycol or ethanol; and acetates, propionates,
It contains organic acid salts such as malonate, benzoate and the like. Auxiliary agents such as wetting or emulsifying agents, and pH buffering agents can also be added. For a thorough examination of pharmaceutically acceptable excipients, see Remington's Pharmaceutical Sciences (
Mack Pub. Co., NJ 1991). The composition is preferably in a form suitable for parenteral administration, eg, by injection or infusion, such as single injection or continuous infusion or microparticle-mediated injection. When the composition is for injection or infusion, it can be in the form of suspension, solution or emulsion in oil or aqueous solvent, and also as a suspending agent, preservative, stabilizer and / or dispersion. Formulation auxiliaries such as agents may also be included. Alternatively, the composition can be in dried form, reconstituted with a suitable sterile liquid immediately before use. For microparticle-mediated administration, DNA is coated on the surface of particles such as microscopic gold particles.

Carriers which do not induce harmful antibody production in the individuals to whom the composition is administered and which do not give rise to adverse toxicity upon administration can be used. Suitable carriers are typically large,
Macromolecules that are metabolized slowly, such as proteins, polysaccharides, polyactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The pharmaceutical composition may also contain a preservative to extend its shelf life.

When the composition is suitable for oral administration, the formulation may contain, in addition to the active ingredient, starch (eg potato, corn or wheat starch, cellulose), starch derivatives such as microcrystalline cellulose, silica, lactose. Various sugars, additives such as magnesium carbonate and / or calcium phosphate, can be included. Formulations suitable for oral administration are desired so that they do not burden the digestive system of the patient. Finally, it is desired to include a mucus forming agent and a resin. It is also desirable to improve the tolerability by formulating the composition in a capsule that does not dissolve in gastric juice. Additionally, it may be desirable to formulate the composition for controlled release.

In yet another aspect of the present invention, a novel cytoplasmic signaling protein or chimeric receptor protein, a nucleic acid encoding the protein, or a pharmaceutical composition is used for treating or preventing a human or animal disease. It can be used when manufacturing.

Various aspects and embodiments of the invention are described in further detail by giving examples. It will be appreciated that partial modifications may be made without departing from the spirit of the invention.

(Example) Example 1. Construction of Cloning Vector, pHMF393 To facilitate the construction of chimeric receptors with different linkages, extracellular spacers, transmembrane and signal transduction components, the cloning cassette system was constructed in pBluescrip.
It was incorporated into t SK + (Stratagene). This is a modification of our cassette system described in International Patent W097 / 23613.

This new cassette system is shown in FIG. The binding component has 5 '(to the coding direction) Not I and Hind III restriction sites and 3' (to the coding direction) Spe I restriction site. The extracellular spacer consists of Spe I site (thus encoding Thr and Ser at the 5'end) and Nar I site (hence Gly at the 3'end,
Adjacent to Ala code). The transmembrane component has a Nar I site at its 5'end (thus encoding Gly, Ala) and Mlu I at its 3'end (thus encoding Thr, Arg).
And adjacent to the BamHI site (hence the code Gly, Ser). The signaling component can be cloned in frame into the BamHI site.
Following this BamH I site is a stop codon for transcription termination and an EcoR I site downstream of this to facilitate removal of the entire construct.

To generate the cassette, the following oligos: S0146, A6081, A6082 and A6083 (
A 200 bp fragment was constructed by PCR using (Fig. 3). The nucleotide and amino acid sequence of this fragment is shown in Figure 2. It begins at the Spel site and is composed of the extracellular spacer h.CD28, the human CD28 transmembrane region, the stop codon, EcoR I
End with a restricted site. This PCR fragment was then digested with Spe I and EcoR I,
The same fragment of the cloning cassette system described above (FIG. 2 of WO 97/23613) was replaced for cloning in frame with the binding component.

Example 2. Construction of sequence blocks for the first and second signaling motifs Each sequence block (SB) forms a single-stranded overhang, such as half of the 5'end Bcl I site and half of the 3'end BamH I site. Generated by annealing two oligos. Oligos are buffered in 1 pmole / μl buffer (25 mM NaCl, 12.5 mM Tris-
HCl, 2.5mM MgCl ₂ , 0.25mM DTE, pH 7.5) after heating for 5 minutes in a boiling water bath,
Annealing was performed by gradually cooling to room temperature.

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

A) SB1: This sequence is based on the first ITAM of human TCRζ, oligos A8816 and A881
Built by 7 annealing.

B) SB2: This sequence is based on the second ITAM of human TCRζ, oligos A8814 and A881.
Constructed by 5 annealing.

C) SB3: This sequence is based on the third ITAM of human TCRζ, oligos A8812 and A881.
Constructed by 3 annealing.

D) SB4: This sequence is based on the ITAM of the human FcεR1γ chain and contains oligos A8810 and A88.
Constructed with 11 anneals.

E) SB4 ^* : This sequence was initially generated by error due to misannealing of the above oligos, but was later produced by annealing oligos A8810B and A8811B.

F) SB5: This sequence is based on the ITAM of the human FcεR1β chain and contains oligos A9000 and A90
Built by 01 Annealing.

G) SB6: This sequence is based on the ITAM of the human CD3γ chain and contains oligos A9002 and A9003
Was constructed by annealing.

H) SB7: This sequence is based on the ITAM of the human CD3 delta chain and contains oligos A9004 and A9005
Was constructed by annealing.

I) SB8: This sequence is based on the ITAM of the human CD3ε chain and contains oligos A9006 and A9007
Was constructed by annealing.

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

K) SB10: This sequence is based on the ITAM of human CD22 and was constructed by annealing oligos A9010 and A9011.

L) SB11: This sequence is based on the ITAM of human CD79a and was constructed by annealing oligos A9012 and A9013.

M) SB12: This sequence is based on the ITAM of human CD79b and was constructed by annealing oligos A9014 and A9015.

N) SB13: This sequence is based on the ITAM of human CD66d and was constructed by annealing oligos A9016 and A9017.

O) SB14: This sequence was synthetic and was constructed by annealing oligos D5258 and D5259.

P) SB15: This sequence was synthetic and was constructed by annealing oligos F6392 and F6394.

Q) SB16: This sequence was synthetic and was constructed by annealing oligos F6393 and F6395.

R) SB28: This sequence is based on the second (costimulatory) sequence of human CD28, oligo A90
Constructed by annealing 18 and A9019.

S) SB29: This sequence is based on the second (costimulatory) sequence of human CD154, oligo A9
Constructed by annealing 020 and A9021.

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

U) SBQ5: This sequence was synthetic and was constructed by annealing oligos D7609 and D7610.

V) SBQ7: This sequence was synthetic and was constructed by annealing oligos D7613 and D7614.

W) SBQ9: This sequence was synthetic and was constructed by annealing oligos D7617 and D7618.

X) SBX: This sequence resulted from recombination between SB2 and SB3 during cloning.

Example 3. Construction of receptors with different signaling components. The first signaling motif and the second signaling motif (in the form of SB, see Fig. 4) on the Bcl I to BamH I fragment were located downstream of the transmembrane region of the BamH I site of the cassette described in Example 1. Can be cloned. Cloning in the correct orientation allows subsequent downstream insertion of other signaling motifs / sequences.

This cassette facilitates the exchange of the binding component on Not I or Hind III for the Spe I fragment, as well as the exchange of the extracellular spacer on Spe I for the Nar I fragment and on Nar I. It also facilitates the exchange of certain transmembrane regions for Mlu I fragments. Thus, chimeric receptors with different linkages, extracellular spacers, transmembranes and signaling subdivisions can be easily assembled.

Example 4. Analysis of chimeric receptor a) Construction of expression plasmid Chimeric receptor construction from pBluescript KS + to Hind III ~
Expression vector on EcoR I restriction fragment pEE6hCMV.ne (Cockett, et al., 199
It was cloned into 1). The empty expression vector (ie the vector lacking the chimeric receptor gene) was used as a negative control.

B) Transfer to Jurkat E6.1 cells To generate stable cell lines, the expression plasmid was linearized and transferred to Jurkat E6.1 cells (ECACC) by electroporation using the BioRad Gene Pulser. did. Cells (-2.5 x 10 < ⁶ >) were mixed with DNA (10 [mu] g) and pulsed twice in 1 ml PBS at 1 kV, 3 [mu] F (0.4 cm electrode gap cuvette). The cells were allowed to recover overnight in non-selective medium and then cultured in medium supplemented with the antibiotic G418 (Sigma) at a concentration of 1.5 mg / ml. After about 4 weeks the cells were submitted for analysis.

For transient expression in Jurkat E6.1 cells, DuoFect (Quar
ntum Biotechnologies Inc.) was used to transfer the expression plasmids.

C) Analysis of surface expression: FACS approximately 5 × 10 ⁵ Jurkat cells were stained with 1 μg / ml FITC-labeled antigen or an antibody against the binding component of the chimeric receptor. FITC-labeled CD33 antigen was used for analysis of receptors with P67scFV binding components. Fluorescence is FACScan cy
It was analyzed with a tometer (Becton Dickinson).

D) Analysis of function: 2 × 10 ⁵ cells of IL-2 production were cultured at 37 ° C. in 8% CO 2 for 20 hours on a 96-well plate with target cells and an effector: target ratio of 1: 1. Collect the culture supernatant,
Human IL-2 (R & D Systems Quantikine kit) was assayed.

When P67scFv was used as an example of the extracellular binding domain, the target cells used were HL60 cells—a human cell line expressing the native antigen, CD33, N.EE6—a mouse transfected with a control expression vector. It was myeloma (NSO). This cell was used as a negative control target cell line. N.CD33-mouse myeloma (NSO) that has been transfected with an expression vector that facilitates expression of the antigen CD33 on the cell surface.

Example 5. Results In response to antigen challenge (by HL-60 or N.CD33 cells as shown),
The specific production of IL-2 by Jurkat cells expressing the chimeric receptor is used as an indicator of signal transduction ability and the degree of cell activation in all experiments described below.

A) Comparison of Signal Transduction Ability of Chimeric Receptors Having Different First Signal Transduction Motif (FIG. 5) Antigen Challenge to Jurkat Cells Expressing Chimera Receptors Having First Signal Transduction Motif with Different Tyrosine Distances To do this, N.CD33 cells were used. The results (shown in Figure 5) indicate that chimeric receptors containing the first signaling motif with a long (9 amino acid) spacer between the YXXL motifs, such as SB16, are more atypical than SB14. It is shown to be superior to that of the distance (7 amino acids).

[0103] b) signaling by the chimeric receptor with an extended first signaling motifs and the additional first signaling motifs (Figure 6) Jurkat cells expressing chimeric receptors with extended first signaling motif antigen challenge N.CD33 cells were used to induce. The results shown in Figure 6 indicate that IL-2 production by cells expressing the novel chimeric receptor is extremely efficient. In this particular example, the first signaling motifs SB3 ^a is included upstream of the extended motif SBX ^a, to generate a high-efficiency receptor P67scFV / h.CcD28 / CD28tm / SB3.SBX.

[0104] c) extending the first signaling motifs and signal transduction by chimeric receptor having a second signaling sequence (Figure 7) new first signaling motifs, SBQ5 ^a, SBQ7 ^a and SBQ9 ^a, and the second signaling Jurkat cells expressing chimeric receptors consisting of SEQ SB28 ^a derived challenged with N.CD33 cells. All chimeric receptors were able to transduce a signal, but the average native length ((9 amino acids) and short (5 amino acids) of the spacer motif between YXXL of the first signaling motif were 7 amino acids) was more effective than the spacer.

[0105] (Cited documents) Ausubel, F. W. & Kingston R. E. (1992) Short protocols in Molecular Biology. 2nd edition. John Wiley & Sons Ltd. Burshtyn, D. N., Lam, A. S., Weston, M., Gupta, N., Warmerdam, P. A. & Long, E. O. (1999) Journal of Immunology 162: 897-902. Cockett, M. I., Bebbington, C. R. & Yarranton, G. T. (1991) Nucleic Acids Research 19: 319-325. DeFranco A. L. (1997) Current Opinion in Immunology 9: 296-308. Finney, H.M., Lawson, A. D. G., Bebbington, C. R. & Weir, A. C. N. (1998) Journal of Immunology 161: 2791-2797. Glover, D. M. 1995a. DNA cloning: a practical approach, Volume II: Expression systems. IRL press. Glover, D. M. 1995b. DNA cloning: a practical approach, Volume IV: Mammalian systems. IRL press. Kuwana, Y., Asakura, Y., Utsunomiya, N., Nakanishi, M., Arat, Y., Itoh, S., Nagese, F. & Kurosawa, Y. (1987) Biochemical and Biophysical Research Communications 149: 960-968. O'Reilly, D. R., & Miller, L. K. (1993). Baculovirus expression vectors- A laboratory manual. Oxford University Press. Rees, A. R (1993) Protein Engineering: a practical approach. IRL press Romeo, C., Kolanus, W., Amiot, M. & Seed, B. (1992) Cold Spring Harbor Symposia on Quantitative Biology. Volume LVII pp 117-125. Sambrook, J., & Fritsch, E. (1989) Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Press, N. Y. Sancez-Garcia, F. J., Aller, W. W. & McCormack, W. T. (1997) Immunology 91: 81-87. Weiss, A. & Littman, D. R. (1994) Cell 76: 26-274.

[Brief description of drawings]

FIG. 1: Schematic representation of the cloning cassette used to construct the chimeric receptor with novel signaling components.

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

[Figure 3]   Oligonucleotide sequence used to construct the chimeric receptor.

FIG. 4 Amino acid sequences of the first and second signaling motifs used to construct the chimeric receptor.

FIG. 5: Antigen-specific stimulation of chimeric receptors with the first signaling motif with different tyrosine distances.

FIG. 6: Antigen-specific stimulation of chimeric receptors having an extended first signaling motif with an additional first signaling motif.

FIG. 7: Antigen-specific stimulation of chimeric receptors containing a first signaling motif with a different tyrosine distance together with a second signaling sequence.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/06 A61P 17/00 11/06 17/06 17/00 25/00 17/06 29/00 25 / 00 31/18 29/00 35/00 31/18 37/06 35/00 C07K 14/47 37/06 14/705 C07K 14/47 19/00 14/705 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 15/00 ZNAA 1/21 5/00 A 5/10 A61K 37/02 (81) Designated country 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, Z W), 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, J P, 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, ZW F terms (reference) 4B024 AA01 AA11 BA63 BA80 CA02 DA02 EA04 GA14 4B065 AA26X AA72X AA77X AA80X AA90X AA90Y AA93X AB01 AC14 BA03 CA24 CA44 4C084 AA02 AA03 AA07 AA13 BA01 BA02 BA19 CA53 NA14 ZA022 ZA552 ZA592 ZA892 ZB082 ZB112 ZB262 ZB332 ZC352 ZC552 4C087 AA01 AA02 BC83 CA12 NA14 ZA02 ZA55 ZA59 ZA89 ZB08 ZB11 ZB26 C55 FA41 Z41

Claims (34)

[Claims] 1. A common amino acid sequence of YX ₂ -L / IX _n -YX ₂ -L / I, in which amino acid residues are represented by a standard one-letter code, X represents any amino acid, and a subscript The number indicates the number of residues at that position in the motif, n is 9 or more, a nucleic acid encoding a cytoplasmic signaling sequence consisting of the first signaling motif. 2. The nucleic acid according to claim 1, wherein the value of n is between 9 and 12. 3. The nucleic acid according to claim 1, wherein the value of n is 9. 4. (designated SBX ^a) first signaling motif amino acid sequence RKNPQEGLYNELQKDKMAEDTYDALHMQA, GQNQLYNELQQQQQQQQQYDVLRRGRDPEM (SBQ9 ^a named), or GQDGLYQELNTRSRDEAAYSVLEGRKAR claim 3, wherein the nucleic acid having a (SB16 ^a named). 5. A nucleic acid encoding a cytoplasmic signaling molecule comprising the first signaling motif according to any one of claims 1 to 4 and at least one other first signaling motif. 6. The nucleic acid of claim 5, wherein the at least one other first signaling motif comprises the common amino acid sequence: YX ₂ -L / IX _n -YX ₂ -L / I. 7. The nucleic acid of claim 6, wherein the value of n in at least one other first signaling motif is 9. 8. At least one other first signaling motif is SBQ9 ^a ,
The nucleic acid according to claim 7, which is selected from SBX ^a and SB16 ^a . 9. The nucleic acid according to claim 6, wherein the value of n in at least one other first signaling motif is between 6 and 8. 10. At least one other first signaling motif is TCRζ.
Chain, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66
10. The nucleic acid of claim 9, comprising at least a portion of an immunoreceptor tyrosine-containing activation motif derived from d. 11. At least one additional first signaling motif is SB4 ^{* a.}
, SB14 ^a, SB15 ^a a is 9. The nucleic acid according. 12. The nucleic acid of claim 10, wherein the cytoplasmic signaling molecule comprises SBX ^a and SB3 ^a . 13. cytoplasmic signaling molecules SB16 ^a, SB4 ^{* a,} SB2 nucleic acid of claim 11, further comprising ^a and SB4 ^a. 14. A nucleic acid encoding a cytoplasmic signaling molecule comprising the first signaling motif according to any one of claims 1 to 4 and at least one second signaling sequence. 15. The nucleic acid of claim 14, wherein at least one second signaling sequence is derived from CD28, CD134 or CD154. 16. At least one second signal transduction sequence RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA, MIETYNQTSPRSAATGLPISMK, or RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHS 16. The nucleic acid according to claim 15, which is 17. First signaling motif SBQ9 ^a, a SBX ^a or SB16 ^a, and the nucleic acid of claim 14, wherein at least one second signaling sequence ArueruerueichiesudiwaiMNMTPRRPGPTRKHYQPYAPPRDFA. 18. A first signaling motif SBQ9 ^a, and the nucleic acid of claim 17, wherein at least one second signaling sequence ArueruerueichiesudiwaiMNMTPRRPGPTRKHYQPYAPPRDFA. 19. A chimeric receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a cytoplasmic signaling domain encoded by the nucleic acid of any one of claims 1-18. Nucleic acid. 20. The nucleic acid of claim 19, wherein the extracellular ligand binding domain comprises an antibody binding domain or fragment thereof. 21. The extracellular ligand-binding domain has a Fab ^' fragment or sc.
20. The nucleic acid according to claim 19, which is Fv. 22. The nucleic acid according to claim 21, wherein the extracellular ligand binding domain is scFv. 23. The transmembrane domain comprises a T cell receptor α, β or ζ chain, CD28, CD
3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86
23. The nucleic acid according to any one of claims 19 to 22, which is derived from CD134, CD137 or CD154. 24. The nucleic acid of claim 23, wherein the transmembrane domain is derived from CD28. 25. The nucleic acid according to any one of claims 19 to 22, wherein the transmembrane domain is synthesized. A vector comprising the nucleic acid according to any one of claims 1 to 25. 27. A cytoplasmic signaling protein encoded by the nucleic acid according to any one of claims 1-18. 28. A chimeric receptor protein encoded by the nucleic acid of any one of claims 19-25. 29. The nucleic acid according to any one of claims 1 to 25 or the vector according to claim 26, for use in therapy. 30. The cytoplasmic signaling protein of claim 27 or the chimeric receptor protein of claim 28, for use in therapy. 31. The cytoplasmic signaling protein of claim 27, or the chimeric receptor protein of claim 28, with a pharmaceutically acceptable additive.
Alternatively, a composition comprising the nucleic acid according to any one of claims 1 to 25. 32. A nucleic acid, a cytoplasmic signaling protein, or a chimeric receptor protein according to any one of claims 1-31, in the manufacture of a medicament for the treatment or prevention of human or animal disease. Use of the composition. 33. The nucleic acid according to any one of claims 1 to 25 or claim 26.
A host cell containing the described vector. 34. A host cell that expresses the cytoplasmic signaling protein of claim 27 or the chimeric receptor protein of claim 28.