Patent Publication Number: US-2016222080-A1

Title: Multiple target t cell receptor

Description:
RELATED APPLICATIONS 
     This application claims priority to European Patent Application No. EP 09 160 679.8, filed May 19, 2009, the contents of which are incorporated by reference in the entirety for all purposes. 
     FIELD OF THE INVENTION 
     The present invention is directed to a functional T cell receptor (TCR) fusion protein (TFP) recognizing and binding to at least one MHC-presented epitope, and containing at least one amino acid sequence recognizing and binding an antigen. The present invention is further directed to an isolated nucleic acid molecule encoding the same, a T cell expressing said TFP, and a pharmaceutical composition for use in the treatment of diseases involving malignant cells expressing said tumor-associated antigen. 
     BACKGROUND OF THE INVENTION 
     TCR&#39;s are members of the immunoglobulin superfamily and usually consist of two subunits, namely the α- and β-subunits. These possess one N-terminal immunoglobulin (Ig)-variable (V) domain, one Ig-constant (C) domain, a transmembrane/cell membrane-spanning region, and a short cytoplasmic tail at the C-terminal end. The variable domains of both the TCR α-chain and β-chain have three hypervariable or complementarity determining regions (CDRs), whereas the variable region of the β-chain has an additional area of hypervariability (HV4) that does not normally contact the MHC-peptide complex and therefore is not considered a CDR. 
     CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the β-chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC. HV4 of the β-chain is not thought to participate in MHC-peptide complex recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains. 
     The affinity of TCR&#39;s for a specific antigen makes them valuable for several therapeutic approaches. For example, cancer patients, such as melanoma patients, can be effectively treated by using adoptive immunotherapy. That is to say, significant tumor regression can occur following adoptive transfer of T cells with anti-tumor specificity. However, patient-derived T cells may have sub-optimal activity. Therefore, there is current interest in using pre-characterized TCR genes to create designer lymphocytes for adoptive cell therapies. 
     The first clinical trials using adoptive transfer of TCR-transgenic T cells showed some clinical efficacy. However, the problem appears that therapies might fail due to so called antigen loss variants developed by cancer cells. Antigen- or MHC-loss variants are frequent mechanisms used by cancer cells that lead to their escape from detection and elimination by T cells. Whether it is due to active immune selection, as demonstrated by immunotherapy studies or due to genomic instability, required to maintain tumor outgrowth and the acquisition of its fully malignant phenotype, these escape mechanisms are very heterogeneous. They include mutations that lead to decreased or no MHC expression such as mutations in the MHC molecule heavy chains encoding genes or in those encoding for molecules involved in antigen processing and presentation including transporter associated with antigen presentation (TAP) and the light chain of MHC-I molecule; β2-microglobulin. 
     Altered level of expression of MHC can also be due to an effect mediated by suppressive cytokines such as interleukin 10. Recognition of tumor associated antigens can also be lost due to mutations or decreased expression level of these antigens. This was shown for melanoma antigens as well as for carcinomas and in an experimental mouse model and was associated with immune ignorance. 
     Another suggested mechanism for tumor escape from immune recognition is based on immunodominance. This mechanism favours sequential escape and selection of cancer cells expressing subdominant or cryptic antigen loss variants that can not be recognised by the mislead immune system. 
     The development of antigen loss variants represents a serious challenge for any successful immunotherapy approach, which is often based on targeting a single antigen or even an epitope (Leen et al. 2007). Yet, each single cancer cell expresses several antigens and 6-12 different MHC class I and II alleles. Studies of tumor-specific and -associated antigens have led to the isolation of many potential immunotherapy targets (Wang et al. 2006). With this myriad of targets, the earnest is on the ability of using these targets to kill tumor cells via the utilisation of their counter receptors. 
     Therapeutic interventions based on using monoclonal antibodies or the transfer of T cells or natural killer (NK) cells engineered with T cell receptor (TCR) or chimeric receptor genes represent a promising advance in immunotherapy (Leen et al. 2007). Several tumor-recognising antibodies and TCRs were recently isolated and used in experimental studies and in clinical trials and the list is continuously growing. Furthermore, some antibodies have already passed the scrutiny of regulatory bodies and are being currently used as a standard therapy. These include antibodies targeting angiogenesis, which is an essential process for solid tumor formation and survival. 
     Alternatively, targeting tumors with short peptide sequences derived from phage display or synthetic peptide library represents a promising approach in biotherapy (Arap et al. 1998). Among others, peptides specific for breast, prostate and colon carcinomas as well as those specific for neo-vasculatures were successfully isolated. 
     WO 01/62908 discloses a TCR fusion protein comprising a TCR recognition element and an immunoglobulin recognition element. However, there is no information contained regarding a TCR fusion protein comprising more than 2 functionalities and regarding the insertion sites suitable for introducing those functionalities in the TCR. 
     U.S. Pat. No. 6,534,633 B1 discloses a binding molecule including a sc-TCR and, covalently linked through a peptide linker, a single chain antibody. Furthermore, the fusion protein described therein is soluble, i.e. not membrane-bound. There is no information contained that a binding peptide should be inserted into the TCR and, further, no information regarding the insertion sites suitable for introducing the same into the TCR is provided. 
     WO 01/93913 shows soluble T cell receptor fusion proteins of bispecific nature. Also here, the TCR and the biologically active polypeptide are connected by a peptide linker. There is no information contained that a binding peptide can be inserted into the TCR and regarding the insertion sites suitable for introducing the same into the TCR. 
     Both experimental data and theoretical considerations suggest that the most effective approach of treating cancer would be through a vigorous immune response that can prevent the development of loss variants and enables the eradication of a large established tumor. This was most clearly established by the studies reported from H. Schreiber&#39;s lab, wherein successful T-cell immunotherapy was shown to operate at established tumors only when this tumor expressed a high level of the antigen, in a process that required recognition of the tumor stroma. Conversely, tumors established from the very same cell line but have a low level of the antigen expressed escaped the immune destruction and allowed the development of antigen-loss variant. 
     Therefore, new therapies are needed in order to address the above mentioned drawbacks of known therapeutic approaches. These new therapies should provide a vigorous immune response that can prevent the development of loss variants and should enable the eradication of a large established tumor. 
     SUMMARY OF THE INVENTION 
     Therefore, it is an object of the present invention to provide a new therapeutic approach which allows an improved therapy of several diseases, such as tumor related diseases, autoimmune, hereditary, or infectious diseases, immunodeficiency, allergy and which might find application in transplantation. 
     It is a further object of the present invention to provide a functional TCR fusion protein (TFP), which shows high affinity against tumor-associated antigens and MHC-presented epitopes. It is a still further object of the invention to provide pharmaceutical compositions for use in adoptive cell therapy which allow an effective treatment of the diseases and conditions outlined above. In particular, it is an object of the invention to provide a functional TFP, which can overcome the problems associated with tumor escape variants due to antigen loss or major histocompatibility complex protein loss and which may target neo-vasculature-specific antigens at the same time. 
     These objects are achieved by the subject-matter of the independent claims. Preferred embodiments are indicated in the dependent claims. 
     Herein, a new type of therapy is disclosed that will target simultaneously at least two different antigens and may further target angiogenesis as well. This is exemplified by targeted antigens such as HER-2, a cell surface molecule that is presented independently of MHC restriction and the MHC-presented epitopes gp100 209  and gp33 derived from the melanoma associated antigen gp100 or the model tumor antigen gp1 from the lymphocytic choriomeningitis virus, respectively. Angiogenesis may, for example, be targeted through the integrins αvβ3 and its homologue αvβ5 expressed on tumor neo-vasculatures (Arap et al. 1998). 
     The inventors surprisingly could show that a TCR fusion protein having at least two functionalities, i.e. one for a MHC-presented epitope, and one for an additional antigen, can be generated without affecting the original specificity for the MHC-complex. 
     Therefore, the TCR of the invention and the therapeutic approach disclosed herein enables multiple targeting of tumors via one genetic modification using one TCR. This is achieved by providing a TCR fusion protein which allows peptide guided and TCR guided attack of cells, in particular tumor cells, at the same time. Thus, the TCR fusion protein as disclosed herein will allow a remarkable improvement of therapies relying on adoptive T cell transfer. 
     DETAILED DESCRIPTION OF THE INVENTION 
     According to a first aspect, the present invention provides a functional TCR α and/or β chain fusion protein recognizing and binding to at least one MHC-presented epitope, and containing at least one amino acid (or peptide) sequence recognizing and binding an antigen. A TCR of the present invention may comprise only one of the α-chain or β-chain sequences as defined herein (in combination with a further α-chain or β-chain, respectively) or may comprise both chains. 
     In the context of the present invention, a “functional” T-cell receptor (TCR) α- and/or (3-chain fusion protein shall mean an α- and/or β-chain fusion protein that, although the chain includes a (foreign) amino acid sequence or peptide, maintains at least substantial biological activity in the fusion protein. In the case of the α- and/or β-chain of a TCR, this shall mean that both chains remain able to form a T-cell receptor (either with a non-modified α- and/or β-chain or with another fusion protein α- and/or β-chain) which exerts its biological function, in particular binding to the specific peptide-MHC complex of said TCR, and/or functional signal transduction upon peptide activation. 
     As outlined above, the present functional TCR fusion protein has two basic functions: one is directed against an MHC-presented epitope, the other is a peptide-based or peptide-guided function in order to target an antigen. The peptide-guided function can in principle be achieved by introducing peptide sequences into the TCR and by targeting tumors with these peptide sequences. These peptides may be derived from phage display or synthetic peptide library (see supra, Arap et al. 1998; Scott 2001). Among others, peptides specific for breast, prostate and colon carcinomas as well as those specific for neo-vasculatures were already successfully isolated and may be used in the present invention (Samoylova et al. 2006). 
     In a preferred embodiment, the epitope is a tumor cell epitope and the antigen is a tumor cell antigen. Such a tumor cell epitope may be derived from a wide variety of tumor antigens such as antigens from tumors resulting from mutations, shared tumor specific antigens, differentiation antigens, and antigens overexpressed in tumors. Those antigens, for example may be derived from alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferaseAS fusion protein, HLA-A2 d , HLA-A11 d , hsp70-2, KIAAO205, MART2, ME1, MUM-1 f , MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1- or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-1, GAGE-1, 2, 8, Gage 3, 4, 5, 6, 7, GnTV f , HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-C2, mucin k , NA-88, NY-ESO-1/LAGE-2, SAGE, Sp17, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3, TRP2-INT2 g , XAGE-1b, CEA, gp100/Pmel17, Kallikrein 4, mammaglobin-A, Melan-A/MART-1, NY-BR-1, OA1, PSA, RAB38/NY-MEL-1, TRP-1/gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CPSF, cyclin D1, DKK1, ENAH (hMena), EP-CAM, EphA3, EZH2, FGF5, G250/MN/CAIX, HER-2/neu, IL13Ralpha2, intestinal carboxyl esterase, alpha fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-1, RGSS, RNF43, RU2AS, secernin 1, SOX10, STEAP1, surviving, Telomerase, VEGF, or WT1, just to name a few. 
     Further information in this regard my be derived from the data described in Nucleic Acids Research, 2006, Vol. 34, Database issue D607-D612; in tables 3a) and b) of Folkman et al.,  Angiogenesis: an organizing principle for drug discovery ?, Nature Reviews Drug Discovery, Vol. 6, April 2007; and in table 1 of Langenkamp et al.,  Microvascular endothelial cell heterogeneity: general concepts and pharmacological consequences for anti - angiogenic therapy of cancer , Cell Tissue Res (2009) 335:205-222. The aforementioned publications are incorporated herein by reference. 
     In a further embodiment, peptide sequences introduced into the TCR are providing a peptide-guided recognition and binding of an antigen which is a cell-surface antigen, a further MHC-presented epitope or an extracellular antigen, preferably a neo-vasculature specific antigen. 
     In a particularly preferred embodiment, the TFP of the present invention shows 3 different functionalties: the first is the function of the TCR α and/or β chain fusion protein to recognize and bind to at least one MHC-presented epitope; the second and third are based on amino acid sequences recognizing and binding an antigen, for example a cell-surface antigen and/or an extracellular antigen. It has never been shown before that a TFP having 3 functionalities can be expressed and will maintain all of these 3 functionalities without interfering with the TCR function as such. 
     It is referred to  FIG. 2  and  FIG. 8  providing examples of such a trifunctional TFP. As it can be seen from  FIG. 9 , it could be shown that these TFP&#39;s may be effectively expressed on cells (here Jurkat MA cells). 
     A preferred example of such a trifunctional TFP is one recognizing and binding to an MHC-presented tumor cell epitope; recognizing and binding a cell-surface antigen of a tumor cell and an extracellular tumor antigen, in particular a neo-vasculature specific antigen. This effectively provides an additional MHC independent activity of the TFP leading to the apoptosis of tumor cells and to an inhibition of angiogenesis at a time and thus helps to avoid the appearance of tumor escape mechanisms. 
     The cell surface antigen or the further MHC-presented epitope preferably is derived from HER2, CEA, PSMA, CD20 or one or more of the above mentioned antigens. The epitope is preferably selected from gp100 or gp1, or as well from one or more of the above mentioned antigens. 
     In a preferred embodiment, the amino acids (or peptides) recognizing and binding to an antigen are targeting a neo-vasculature specific antigen. Those antigens are valuable and promising targets in the attack on tumor cells. See, in this connection the above mentioned publications of Folkman et al.,  Angiogenesis: an organizing principle for drug discovery ?, Nature Reviews Drug Discovery, Vol. 6, April 2007, and Langenkamp et al.,  Microvascular endothelial cell heterogeneity: general concepts and pharmacological consequences for anti - angiogenic therapy of cancer , Cell Tissue Res (2009) 335:205-222. 
     Preferably, the neo-vasculature specific antigen is integrin αvβ3 or αvβ5. For a further selection of antigens, see also table 3a) and b) of Folkman et al. and table 1 of Langenkamp et al. 
     According to a further preferred embodiment, the TCR fusion protein of the invention is characterized in that 
     i) the one or more antigen binding amino acid (peptide) sequences are added to the N-terminus of the α and/or β chain,
 
ii) the one or more antigen binding amino acid sequences are inserted into a constant or a variable region of said α and/or β chain, and/or
 
iii) the one or more antigen binding amino acid sequences are replacing a number of amino acids in a constant or a variable region of said α and/or β chain.
 
     This selection of insertion sites of the amino acid sequence guarantees a proper function of the overall TCR, i.e. its biological function, in particular binding to the specific peptide-MHC complex of said TCR, and/or functional signal transduction upon peptide activation. 
     In a preferred embodiment, the one or more antigen binding amino acid (peptide) sequences are added to the N-terminus of the α and/or β chain, and/or are replacing a number of amino acids in the constant region of the β chain. More precisely, it is more preferred to replace the loop region of the constant region of the β chain with the respective antigen binding amino acid sequence. It is referred to  FIG. 8  showing examples of this approach: clones #1, 2, 3, 4 and 11 each carry an added antigen binding amino acid sequence at the N-terminus of the α chain and one further antigen binding amino acid sequence replacing parts of the loop region of the constant region of the β chain. This combination of one addition at the N terminus of the α chain and one replaced sequence in the β chain constant region (replacing parts of the β loop) is the most preferred embodiment of an insertion scheme for the trifunctional TFP of the present invention. The replaced region may be located at aa positions 242 to 255 of the β chain constant region, for example 244-253, 246 to 255, 244 to 253 or 242 to 251. 
     An alternative, but less preferred location for inserting antigen binding amino acid sequences is the α chain constant region, for example at aa positions 170-179 (there is no loop region present in the α chain constant region). 
     Generally, replacement of sequences in the constant regions is preferred, but an insertion the like is possible. 
     It is noted that the TFP of the present invention, in contrast to those of the prior art, does not require any peptide linker in order to link the individual components. 
     Preferably, said fusion protein comprises the one or more antigen binding amino acid sequences either spaced apart or directly in tandem, or the TCR is expressed as a single chain or soluble receptor. More preferred, the TFP of the present invention is expressed membrane-bound in order to achieve its full functionality. 
     In a further embodiment, the TCR α and/or (3 chain fusion protein of the invention may further comprise at least one epitope-tag, wherein said epitope-tag is selected from 
     i) an epitope-tag added to the N-terminus of the α and/or β chain,
 
ii) an epitope-tag inserted into a constant or variable region of said α and/or β chain, and
 
iii) an epitope-tag replacing a number of amino acids in a constant or variable region of said α and/or β chain.
 
     Preferred is a functional T-cell receptor (TCR) α- and/or β-chain fusion protein according to the present invention, wherein said epitope-tag is selected from, but not limited to, CD20 or HER2/neu tags, or other conventional tags such as a myc-tag, FLAG-tag, T7-tag, HA (hemagglutinin)-tag, His-tag, S-tag, GST-tag, or GFP-tag. myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc-tag can preferably be used because high quality reagents are available to be used for its detection. Epitope tags can of course have one or more additional functions, beyond recognition by an antibody. The sequences of these tags are described in the literature and well known to the person of skill in art. 
     In the functional T-cell receptor (TCR) α- and/or β-chain fusion protein according to the present invention, said fusion protein may be for example selected from two myc-tag sequences that are attached to the N-terminus of an α-TCR-chain and/or 10 amino acids of a protruding loop region in the β-chain constant domain being exchanged for the sequence of two myc-tags. 
     In an embodiment of the present invention, the inventors inserted an amino acid sequence that corresponds to a part of the myc protein (myc-tag) at several reasonable sites into the structure of a T cell receptor and transduced this modified receptor into T cells. By introducing a tag into the TCR structure, it is possible to deplete the modified cells by administering the tag-specific antibody to the patient. 
     Those functional TCR fusion proteins may be used in a method for selecting a host cell population expressing a fusion protein selected from the group consisting of a fusion protein comprising a) at least one epitope-providing amino acid sequence (epitope-tag), and b) the amino acid sequence of an α- and/or β-chain of a TCR as defined above, wherein said epitope-tag is selected from an epitope-tag added to the N-terminus of said α- and/or β-chain or added into the α- and/or β-chain sequence, but outside the CDR3 region, an epitope-tag inserted into a constant region of said α- and/or β-chain, and an epitope-tag replacing a number of amino acids in a constant region of said α- and/or β-chain; and a TCR comprising at least one fusion protein as above on the surface of the host cell; comprising contacting host cells in a sample with a binding agent that immunologically binds to the epitope-tag, and selection of said host cells based on said binding. 
     In the TCR fusion protein, the amino acid sequence recognizing and binding an antigen has preferably a length of between 5-20 amino acids, more preferably between 6 and 12 amino acids. The amino acid sequence recognizing and binding an antigen is preferably selected from SEQ ID NO: 1 and/or 2 corresponding to MARSGL and CDCRGDCFC. 
     In a second aspect, the present invention provides an isolated nucleic acid coding for the TCR fusion protein as defined herein. 
     The term “isolated nucleic acid” as used herein with reference to nucleic acids refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the cell from which it is derived. For example, a nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, a nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e. g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e. g., a retrovirus, or adenovirus). In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence. 
     Furthermore, the term “isolated nucleic acid” as used herein also includes artificially produced DNA or RNA sequences, such as those sequences generated by DNA synthesis based on in silico information. 
     The nucleic acids of the invention can comprise natural nucleotides, modified nucleotides, analogues of nucleotides, or mixtures of the foregoing as long as they are capable of causing the expression of a polypeptide in vitro, and preferably, in a T cell. The nucleic acids of the invention are preferably RNA, and more preferably DNA, but may also take the form of PNA, CNA, mRNA or mixtures thereof. 
     In a third aspect, the invention is directed to a vector, preferably a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, adenoviral vector or particle and/or vector to be used in gene therapy, which comprises the above defined nucleic acid molecule. 
     In the context of the present invention, a “vector” shall mean a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. A vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid. 
     A fourth aspect provides a host cell transfected with a vector or a nucleic acid as defined above, or which has been infected or transduced with a particle as described above. 
     The host cell preferably is a T-cell, a natural killer cell, a monocyte, a natural killer T-cell, a monocyte-precursor cell, a hematopoietic stem cell or a non-pluripotent stem cell. More preferably, it is a peripheral blood lymphocyte (PBL) which has been transfected or transduced with the above vector. The step of cloning the T cell receptor (TCR) of the isolated T cells and/or expressing the TCR transgenes in PBMC can be done according to established methods. 
     In a still further preferred embodiment, the host cell expresses a fusion protein as disclosed herein on its surface. 
     A fifth aspect of the present invention provides a method for producing a fusion protein according to the invention, comprising a chemical synthesis or genetic engineering of said peptide. Those methods for chemically synthesizing proteins and peptides are described in the literature, and well known to the person of skill. 
     Peptides (at least those containing peptide linkages between amino acid residues) may be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433-3436, and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is achieved by using 20% piperidine in N, N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4′-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N, N-dicyclohexyl-carbodiimide/lhydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. 
     Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. 
     Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (usually) reverse-phase high performance liquid chromatography. 
     Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis. 
     Furthermore, the fusion protein may be prepared by using genetic engineering: such a method, as an example, may comprise expressing a nucleic acid molecule encoding a fusion protein according to the present invention in a host cell of the invention, and purifying said fusion protein or said TCR from said host cell. 
     One skilled in the art will understand that there are myriad ways to express a recombinant protein such that it can subsequently be purified. In general, an expression vector carrying the nucleic acid sequence that encodes the desired fusion protein will be transformed into a microorganism for expression. Such microorganisms can be prokaryotic or eukaryotic. A eukaryotic expression system will be preferred where the protein of interest requires eukaryote-specific post-translational modifications such as glycosylation. Also, protein can be expressed using a viral (e.g., vaccinia) based expression system. 
     Protein can also be expressed in animal cell tissue culture, and such a system will be appropriate where animal-specific protein modifications are desirable or required in the recombinant protein. Such expression is particularly appropriate where native assembly and export of the inventive fusion protein is desirable, since the activity of TCRs is influenced by native dimerization (folding and assembly) and presentation on the cell. 
     In a sixth aspect, the present invention provides a pharmaceutical composition which comprises a fusion protein, a nucleic acid molecule, a vector according or a host cell as defined hereinabove, together with a pharmaceutically acceptable carrier or excipient. 
     Those active components of the present invention are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art. 
     The term “pharmaceutically acceptable” defines a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application. 
     The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects. 
     Techniques for the formulation or preparation and application/medication of active components of the present invention are published in “Remington&#39;s Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. An appropriate application is a parenteral application, for example intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. The intravenous injection is the preferred treatment of a patient. 
     According to a preferred embodiment, the pharmaceutical composition is an infusion or an injection. 
     An injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g., an expanded T-cell population (for example autologous or allogenic to the patient to be treated) expressing a TCR. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the fusion proteins of this disclosure are conventional; appropriate formulations are well known to those of ordinary skill in the art. 
     According to a further aspect, the present invention is directed to a method of treating a patient in need of adoptive cell therapy, said method comprising administering to said patient a pharmaceutical composition as defined above. The disease to be treated preferably is a tumor, autoimmune, hereditary, or infectious disease, immunodeficiency, allergy and/or the composition will be designed for use in transplantation. 
     The present invention now will be illustrated by the enclosed Figures and the Examples. The following examples further illustrate the invention but, of course, should not be construed as limiting its scope. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1 : Jurkat MA T cells untransduced or transduced with the wilde-type (WT) or with the myc-taged gp 100 (DAN) TCRs were unstimulated or stimulated with a CD3 (OKT-3) antibody as a positive control, or with an anti myc-tag (9E10) antibody. Response was measured using the luciferin conversion assay and measured in relative light unit. 
         FIG. 2 : Schematic representation of the TCR (left) and the TFPhv (right) with the HER-2 specific peptide (MARSGL) and the neo-vasculature integrins αvβ3 and αvβ5 specific peptide (RGD4C) are depicted at the N-termini of the TCR molecule. 
         FIG. 3 : Overview over two cell types (Jurkat MA and B3Z) expressing peptide-guided TCR&#39;s (bifunctional). The insertion positions and myc-binding are indicated in the tables. 
         FIG. 4 : Monoclonal anti-Myc antibody can activate Jurkat MA cells. Jurkat MA T cells were activated with anti-CD3 or anti-Myc-tag antibody. The activation was measured in relative light unit (RLU), indicating IL-2 secretion, and compared to wild type (wt). 
         FIG. 5 : Gp100-pulsed T2 can activate Jurkat MA. Jurkat MA T cells were cultivated with T2 cells loaded with the peptide specific for the TCR (gp100) or with an irrelevant peptide (Marti). The activation were measured in relative light unit (RLU), indicating IL-2 secretion, and compared to wild type (wt). 
         FIG. 6 : Monoclonal anti-Myc antibody can activate B3Z cells. B3Z cells were activated with anti-CD3 or anti-Myc-tag antibody. The activation were measured by IL-2 secretion, and compared to wild type (wt). 
         FIG. 7 : Gp33-pulsed RMAS can activate B3Z. B3Z cells were cultivated with RMAS cells loaded with the peptide specific for the TCR (gp33) or with an irrelevant peptide (IV Tag). The activation were measured by IL-2 secretion, and compared to wild type (wt). 
         FIG. 8 : Insertion of Her-2 specific (eg.: MARSGL) or neovasculature specific (RGD) peptides into these selected positions. 
         FIG. 9 : Peptide-guided gp100 TCR (Mig 1, 2, 3, 4 and 11) can be expressed on Jurkat MA cells. 
     
    
    
     EXAMPLES 
     The P14 TCR, which recognises the gp33 epitope from the lymphocytic choriomeningitis virus, was studied for identifying neutral sites for the introduction of peptide sequences. Similarly, the gp100 TCR, which recognises the human melanoma antigen derived epitope gp100 209  was studied for identifying such sites. This was achieved by introducing a myc-tag sequence (table 1) into 11 different sites of the TCR molecule, five of which were used in both TCRs resulting in a total number of 16 different TFPs. T cells transduced with any of these 16 constructs expressed the TFP at a level similar to that of the wild type P14 or gp100 TCRs and maintained their specificity to the corresponding tetramer and to stimulation by the specific peptide-pulsed target. 
     Testing TFP for the Ability of Responding to De Novo Stimulation: 
     The inventors have recently used CD3 or myc-tag specific antibodies to investigate the possibility of stimulating the transduced T cells. They found that 9 of the 16 TFPs located in 3 different positions of the TFPs can be triggered by the myc-tag specific antibody to produce similar amount of IL-2 to that produced through the control stimulation by the CD3 antibody ( FIG. 1 ). These data indicate that a targeting-peptide introduced to the TCR can be used to trigger the TCR similarly to the triggering of this molecule by the corresponding MHC-peptide complex and provides 3 potential sites for the introduction of targeting peptides. 
     Molecular Cloning of TFP Encompassing TCR with Three Specificities: 
     Peptide sequences specific for the tumor-associated antigen HER-2 and/or tumor vasculature (based on the RGD4C sequence) indicated in table 1 are cloned into the identified optimal sites of the TCR sequences. This is achieved by introducing the corresponding nucleotide sequences into the TCR chains using overlapping PCR primers. Unique restriction sites with silent substitutions are introduced to enable a potential further cloning of alternative targeting peptides. Constructs identities are confirmed by restriction mapping and sequencing. The resulting TFPs recognise HER-2, neovasculature and gp33-expressing H2Kb target cells or gp100 209  HLA-A2-expressing target cells. Three different types of TFPs with specificities to TCR epitope and HER-2 (TFPh), TCR epitope and angiogenesis (TFPv) or TCR epitope, HER-2 and angiogenesis (TFPhv) are constructed ( FIG. 2 ). 
     Establishing a Tumor Cell Line with Two Different Model Antigens with the Expression Level of One being Controllable: 
     A tumor cell line is constructed in which the level of HER-2 will be constant, while the level of gp33 is controlled by an inducible cre recombination system based on the MC57G-gp33 cell line provided by Dr. H. Schreiber, Chicago. In this system the gp33 eptitope is expressed at low but detectable levels due to inhibition by a floxable sequence containing the HLA-A2 and puromycin. Following cre recombination, which is induced by tamoxifen administration, this inhibitory sequence is floxed, which enables a higher expression level for the gp33 epitope. The uninduced tumor cell line is designated MC57G-gp33 low , and the induced cell line is designated MC57G-gp33 high . In this model a control cell line MC57G-neo, which expresses the inducible cre construct is used as a gp33 negative cell line. 
     HER-2 is introduced to the uninduced MC57G-gp33 or to MC57G-neo by retroviral transduction using the MIG-R1 HER-2 construct (provided by Dr. Helga Bernhard, GSF, Munich). The resulting tumor cell lines are designated MC57G-neo-HER-2, MC57G-gp33 low -HER-2, and MC57G-gp33 high -HER-2. 
     T Cell Transduction 
     Human T cells are derived from peripheral blood lymphocytes, while mouse T cells are derived from splenocytes of OT-I-Rag−/− TCR transgenic mice. 
     T cells are transduced with the different TFPs by spinocculation. Cryopreserved peripheral blood mononuclear cells are collected from healthy donor blood donation by centrifugation on Ficoll-Hypaque gradients. Peripheral blood mononuclear cells are stimulated with 10 ng/mL CD3 (ORTHOCLONE OKT3) and 600 IU IL-2/mL. The retroviral production is done in 293T cells that are cotransfected with the TFP encoding plasmid or GFP as a mock control and the pCL-10A1 plasmid encoding gag, pol and env using lipofection. Virus supernatant is collected and used to transduce primary activated human T cells three days after the activation. Enrichment of mouse T cells for retroviral transduction is achieved by isolating splenocytes from OT-I mice, followed by red blood cell lyses by ammonium chloride solution and stimulation with the OT-I epitope (SIINFEKL), anti CD3 and anti CD28 antibodies. The ecotropic virus supernatant is produced by transiently transfecting plasmid-DNA encoding the appropriate TFP or GFP as a mock control using lipofection into the ecotropic packaging cell line Plat-E. T cells are transduced on two subsequent days and the transduction efficacy will be analysed by FACS. 
     Measuring TFP Expression and Binding Capacity to Three Independent Antigens 
     TFPs expression is confirmed using specific antibodies, tetramers and by using recombinant proteins followed by specific antibody staining. This is achieved using commercially available recombinant HER-2 or αvβ3 (which binds to the RGD4C peptide) proteins and antibodies specific to these proteins. Cells expressing the TFPs or control TCRs are incubated with the recombinant protein for 30 minutes after which the residual non-binding protein is washed away. Fluorochrome-conjugated antibodies specific for either of the recombinant proteins are then added to the cells and incubated for further 30 min followed by washing and fixing with 4% paraformaldehyde solution. Binding is then detected by FACS. Specificity is confirmed by adding a competing soluble synthetic peptide. 
     In Vitro Analysis of the Functional Activity of TFP 
     TFPs function is confirmed using cytokine release assay with the transduced cells as effectors and target cell lines expressing any or all of the target antigens. HUVECS is used as target for the vasculature targeting as well as tumor cell lines expressing αvβ3 such as MDAMB435 breast carcinoma. To evaluate HER-2 specificity of the TFP, C1R/A2 and its transfectant C1R/A2HER2, which expresses HER-2 are used as well as carcinoma cell lines expressing HER-2. Functional specificity is confirmed by adding a competing soluble synthetic peptide. TFPs specificity for the gp33 and gp100 209  epitopes are confirmed by using peptide-pulsed RMA-S and T2 cells, respectively, and by using MC57-neo and MC57-gp33 expressing tumor cell lines and 624-28 (HLA-A2 negative and gp100 positive) and 624-38 (HLA-A2 and gp100 positive) melanoma cell lines. 
     Adoptive Therapy with TFP-Engineered T Cells Targeting Antigen-Loss Variants 
     The rational for this study is to answer the question of whether it is possible to provide an immunotherapeutic strategy that prevents “immune escape” of tumor cells expressing low amount of a targeted antigen. An MHC-restricted epitope, a cell surface-expressed proto-oncogene and tumor vasculature are targeted simultaneously. 
     MC57G tumor cell lines are used to compare the therapeutic potential of P14 TCR versus the TFPs based on P14 with the added specificity to HER-2 and neo-vasculature. Six different populations of T cells are used in the adoptive transfer:
         1. OT-I   2. P14 T cells from P14 TCR transgenic mice   3. P14 transduced OT-I   4. P14 TFP transduced OT-I with specificities to H2Kb-gp33 and HER-2 (TFPh)   5. P14 TFP transduced OT-I with specificities to H2Kb-gp33 and angiogenesis (TFPv)   6. P14 TFP transduced OT-I with specificities to H2Kb-gp33, HER-2 and angiogenesis (TFPhv)       

     Rag−/− OT-I mice are challenged subcutaneously on contra-lateral sites with either of the following 4 tumor pairs: 
     A. MC57-neo and MC57-gp33 low    
     B. MC57-neo and MC57G-neo-HER-2 
     C. MC57G-neo-HER-2 and MC57G-gp33 low -HER-2 
     D. MC57G-neo and MC57G-gp33 high    
     Once the tumors reach 1 cm mean tumor diameter, each of the 4 groups receive intravenous injection with 10e6 activated CD8+ positive P14, OT-I, P14 transduced OT-I, TFP transduced OT-I T cells or TFP transduced OT-I T cells and the RGD4C synthetic peptide, which are used to block the recognition of the RGD4C sequence of the TFP. Efficient tumor destruction would require targeting tumor stroma either directly through RGD4C specificity of the TFP or through targeting stroma cells cross presenting the gp33 epitope. The expected recognition spectrum of these different effectors is outlined in table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Amino acid sequences of peptides used  
               
               
                 in the TFPs. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Length 
               
               
                 Peptide 
                 Target 
                 Sequence 
                 aa 
               
               
                   
               
               
                 myc-tag 
                 myc  
                 EQKLISEEDL  
                 10 
               
               
                   
                 antibody 
                 (SEQ ID NO: 3) 
                   
               
               
                   
               
               
                 2xmyc-tag 
                 myc  
                 EQKLISEEDLEQKLISEEDL 
                 20 
               
               
                   
                 antibody 
                 (SEQ ID NO: 4) 
                   
               
               
                   
               
               
                 MARS 
                 HER-2 
                 MARSGL  
                  6 
               
               
                   
                   
                 (SEQ ID NO: 1) 
                   
               
               
                   
               
               
                 RGD4C 
                 av  
                 CDCRGDCFC  
                  9 
               
               
                   
                 Integrins 
                 (SEQ ID NO: 2) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 The predicted recognition pattern of transduced/transferred T cells. 
               
            
           
           
               
               
            
               
                   
                 Transferred T cells and recognition 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 TFPhv- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 OT-I + 
               
               
                   
                   
                   
                 P14- 
                 TFPh- 
                 TFPv- 
                 TFPhv- 
                 RGD4C 
               
               
                 Target 
                 OT-I 
                 P14 
                 OT-I 
                 OT-I 
                 OT-I 
                 OT-I 
                 peptide 
               
               
                   
               
               
                 H2Kb- 
                 No 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
               
               
                 gp33 
               
               
                 HER-2 
                 No 
                 No 
                 No 
                 Yes 
                 No 
                 Yes 
                 Yes 
               
               
                 Neo- 
                 No 
                 No 
                 No 
                 No 
                 Yes 
                 Yes 
                 No 
               
               
                 vascu- 
               
               
                 lature 
               
               
                   
               
            
           
         
       
     
     PUBLICATIONS 
     
         
         Arap, W., R. Pasqualini and E. Ruoslahti (1998). “Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model.”  Science  279(5349): 377-80. 
         Folkman, J. (2007). “Angiogenesis: an organizing principle for drug discovery?”  Nat Rev Drug Discov  6(4): 273-86. 
         Leen, A. M., C. M. Rooney and A. E. Foster (2007). “Improving T cell therapy for cancer.”  Annu Rev Immunol  25: 243-65. 
         Samoylova, T. I., N. E. Morrison, L. P. Globa and N. R. Cox (2006). “Peptide phage display: opportunities for development of personalized anti-cancer strategies.”  Anticancer Agents Med Chem  6(1): 9-17. 
         Wang, X., H. Zhao, Q. Xu, W. Jin, C. Liu, H. Zhang, Z. Huang, X. Zhang, Y. Zhang, D. Xin, A. J. Simpson, L. J. Old, Y. Na, Y. Zhao and W. Chen (2006). “HPtaa database-potential target genes for clinical diagnosis and immunotherapy of human carcinoma.”  Nucleic Acids Res  34(Database issue): D607-12. 
         Becker, C., H. Pohla, B. Frankenberger, T. Schuler, M. Assenmacher, D. J. Schendel and T. Blankenstein (2001). “Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay.”  Nat Med  7(10): 1159-62. 
         Charo, J., S. E. Finkelstein, N. Grewal, N. P. Restifo, P. F. Robbins and S. A. Rosenberg (2005). “Bcl-2 overexpression enhances tumor-specific T-cell survival.”  Cancer Res  65(5): 2001-8. 
         Gladow, M., W. Uckert and T. Blankenstein (2004). “Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors.”  Eur J Immunol  34(7): 1882-91. 
         Kieback E, Charo J, Sommermeyer D, Blankenstein T, Uckert W (2008). A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer. Proc Natl Acad Sci USA. 105(2):623-8. 
         Kruschinski A, Moosmann A, Poschke I, Norell H, Chmielewski M, Seliger B, Kiessling R, Blankenstein T, Abken H and J Charo (2008). Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas. Proc Natl Acad Sci USA. 105(45):17481-6. 
         Sommermeyer, D., J. Neudorfer, M. Weinhold, M. Leisegang, B. Engels, E. Noessner, M. H. Heemskerk, J. Charo, D. J. Schendel, T. Blankenstein, H. Bernhard and W. Uckert (2006). “Designer T cells by T cell receptor replacement.”  Eur J Immunol  36(11): 3052-9.