MXPA97003535A - Lymphocytes dirigi - Google Patents

Abstract

The present invention relates to cytotoxic T lymphocytes having a TcR consisting of alpha and heterologous polypeptides, whose heterologous polypeptides confer specificity restricted to MHC class I on the lymphocyte for the cells causing the disease. The lymphocytes can be monovalent, with a single species of TcR that confers specificity for a single class of target cells. Lymphocytes find application as vaccines and in adoptive immunotherapy, for example, cancer, AIDS or antiviral therapy

Description

DIRECTED LYMPHOCYTES T The present invention relates to targeted cytotoxic T lymphocytes and their uses, and in particular to targeted cytotoxic T lymphocytes that have T-cell antigen receptor a and β-hcrclear polypeptides that confer restricted specificity to MHC for T cells. white cells that cause disease. The immune system consists of a multitude of different molecules and cell types distributed throughout the body. In healthy individuals the immune system defends and protects against pathogens, parasites and invading cells that have been inserted or have become cancerous. The cellular basis for the various fundamental properties of the immune system (in which immunological memory, specificity and diversity of responses are included) is the lymphocyte. There are two d - lymphocyte classes: the B lymphocyte (or B cell), which is finally responsible for generating the humeral responses (mediidpr: for soluble antibodies) and the T lymphocyte (or T cell), which is finally the responsible for generating the responses mediated by the cells. T lymphocytes are further divided inter alip into cytotoxic T lymphocytes (which identify and destroy infected, aberrant or malignant white cells) and helper T lymphocytes (which stimulate, recruit and activate a wide range of cell types involved in preparing a immune response) . The T lymphocytes recognize the target cells via the T cell receptors (hereafter, the TcR). TcRs do not usually show antigen binding alone. In contrast, the TcRs recognize an antigen as part of a glycoprotein-cell surface complex encoded by the Class I or Class II genes of the major histocompatibility complex (hereinafter defined as the MHC Class I or MHC Class II). Cytotoxic T lymphocytes "usually recognize antigens in association with MHC Class I, whereas helper T lymphocytes usually recognize antigens in association with MHC class II.As a result, bl mro cell recognition by lymphocytes T is restricted by b 'mm cells capable of presenting antigen in association with particular host proteins (ie, -noléc -'- 1 ?? - MHC Class I or Class II) .The main function of the liní • t >T * can, therefore, be seen as the verification of 1 • = cellular surfaces in the body, so that aberrant, infected or malignant cells can -GP identified (or detected in the case of cells). cooperating) and finally eliminated.

The T cell receptor (TcR) The TcR is a protein complex, of a type that includes two polypeptide, disulfide, and crosslinked chains (a chains) that associate on the cell surface of the T lymphocyte with other molecules. The a chains of the TcR are sufficient to endow a T lymphocyte with both antigenic and MHC specificity (Class I or Ciase TI): therefore, only the a components of the TcR are necessary to completely define the double specificity (ie as MHC and as an antigen) of the T lymphocyte. The α chains contain constant and variable regions that are homologous to the V and C regions of the immunoglobulin, and the structural genes correspond to recurrent DNA arrangements such as the immunologlobin genes. Actually, the structural deterioration < IP immunoglobulins and TcRs are similar (about 1012 per individual). The genes encoding the a-chains of the TcR have been cloned and found their sequence (see, for example, Robertson (1985), Nature (317, 768-771). In addition, the ß chains of the TcR have been transferred from one T cell to another to generate a T cell clone of different specificity (see, for example, Dembic et al., 1906, Nature 320. pp. 232-238 ). The TcRs are distributed clonally: the TcRs of each T lymphocyte are specific only for an antigenic determinant (although there may be hundreds of thousands of TcRs, the same species in each cell). In this way, each T lymphocyte is usually monovalent with respect to .1 to TcR mediated binding. The β chains are associated with several other molecules, in which the proteins of the CD3 complex are included and are associated with the peptides zeta and eta. The TcR-CD3 complex transduces a signal from the TcR into the T lymphocyte when it recognizes the appropriate peptide-MHC complex, thereby contributing to the activation of the T cell.

Aberrant genetic expression, disease and spiking of the T cell The health of an individual depends on the closely controlled and coordinated expression of many different genes. The aberration or alteration of \ n gene expression, even if it is subtle, by Lauto, t-n < > 'j < -! -order to a plethora of different diseases, including: cancers, AIDS and various diseases. - > , -) .- i < autoimmune Many different factors can induce aberrant gene expression. For example, the expression of the endogenous genes or the structure of the proteins encoded by them can be altered by mutation. These mutations may be spontaneous, but are often induced by carcinogens present in the environment. Alternatively, the introduction of exogenous genetic material into cells of an individual (e.g., as a result of viral infection) can result in the destruction of gene expression patterns, the activation of normally silent genes and / or protein synthesis. strange (for example, viral). . One of the important examples of the consequences of induced aberrations in gene expression is the formation of tumors. Tumor formation characterizes most cancers, and tumors arise as a consequence of the structural alteration or overexpression of the endogenous proteins concurrent in the mutation of tumor and tumor suppressor genes in somatic cells. For example, in certain types of cancers? > -c ho tumor formation arises from the amplification and overexpression of the proto-oncogenes HER-2 / nou. With the exception of the onset of disease in the body, another phenotypic consequence of the aberrant genetic expression may be the production of neoantigens.

As used herein, the term "neoantigen" is Rropone to cover not only the new antigens arising from the mutant or foreign amino acid sequences, but also the "new" antigens that arise from the expression of normally silent natural genes, o The overexpression of natural genes that are expressed in a relatively weak manner The production of neoantigens can give rise to alterations in the antigens of the cell surface, these alterations can in turn induce an immune response. The use of Qncogenic products has been found in the serum of patients with tumor and cytotoxic T lymphocytes that recognize and eliminate tumor cells have been demonstrated in various model systems.The alterations in the antigens of the cellular supeí ficie able to activate a The response of cytotoxic cytokines can arise after the ini race process I or '-p prot mutant, foreign or overexpressed eins to produce fragments of peptides that can be exhibited as a cellular surface as part of a peptide or MH-Class I complex capable of being recognized by cytotoxic T lymphocytes. In this way, the aberrant cells? .'- > -: > be recognized and eliminated by T lymphocytes. In addition to alterations in cell surface antigens, protein overexpression, the production of foreign or mutant proteins may eventually result in the appearance of exogenous neoantigens (eg, particles or viral fragments). ). These can be recognized by the immunologlubins that bind to the menbrane on the surface of the lymphocytes B, can be internalized, processed and subsequently presented as antigens of the T helper cell as a complex with MHC Class II molecules on the surface of the B cell. A B cell that is presenting antigen in this way can be recognized by a T cell cooperating, via a specific interaction TcR-peptide-MHC Class II and can be stimulated to develop in the plasma cell secreting large amounts of soluble antibodies specific for an exogenous antigen. In this way, the helper T lymphocyte can stimulate an immune or humoral response and indirectly activate a wide range of immune cell types. There is a large amount of reports of tumor-specific neoantigens or tumor-associated ones. For example, overexpression of the ERBB2 receptor is associated with a large number of human breast and ovarian cancers and also gives rise to the presence of neoantigens derived from ERBB2 on the cell surface. In cocci cancer, the adenomatous polyposis coli (APC) gene usually undergoes early mutation. Although the mutation simply often results in the introduction of a nonsense codon (which terminates the translation resulting in the production of "a truncated APC protein"), in many cases the mutation is deficient giving rise to the production of new protein sequences and potentially unique antigens.Once processed intracellularly and bound to MHC proteins, these antigens can be present on the cell surface and be recognized by T lymphocytes. In many other cases it would be expected that the alleraion or structural overexpression of the endogenous proteins would give rise to alterations in the intracellular processing of these proteins and the presentation of neoantigens in association with MHC proteins on the cell surface, in spite of the immunogenic constellations CÍPS? the natural immune response prepared to combat c • i • ! - * - infected, aberrant or malignant is often inadequate. Although the reasons for this deficiency is not understood T-? - Completely, it is thought that neoantigens are usually treated by the immune system as tissue antigens and thus are tolerated in the same way. Ador i-, in general, neoantigens are only a minor source of peptides for the targeted processing of MHC-Class I, and, in this way, the processing and presentation of the neoantigen can be extremely inefficient. Finally, immunogenicity seems to be determined, in part, by the type of tissue in which the neoantigen is expressed, and thus it seems that tolerance may be greater (and / or less efficient processing and presentation) in a certain type of tissues. Various methods have been developed to generate more effective immune responses against aberrant, infected or malignant cell targets, for example, the use of antigens from malignant cells has been proposed as vaccines to induce cell-mediated, tumor-specific immunity. Recently adoptive immunotherapy was proposed as a mode of treatment against cancer. This mode of therapy is based on the transfer of immune cells with antitumor activity in patients with cancer. The immune cells that are used are derived from the patient with cannula, are cultured, and after the expansion is reintroduced into the tissue culture cells that are used in the adoptive imumnoterapi include killer cells. activated by lymphocyte, tumor infiltration lymphocytes, and in vitro sensitized lymphocytes derived from cytotoxic T lymphocytes The lymphosin-activated killer cells are cytolytic cells that react with a broad spectrum of target cells.These are not restricted MHC and 1 Lsan tumor cells. The therapies based on the use of these cells, therefore, suffer the disadvantage that the destruction of the tumor cells is accompanied by a significant damage in the normal tissue.The tumor infiltration lymphocytes are derived from tumor tissues. They are more potent than the lymphocyte-activated killer cells and are relatively specific for tumors of origin. In this way, problems arising from non-selective ablation of normal tissue associated with the use of murderous cells are avoided.; activated by lymphokine. However, the availability of these cells is severely restricted, being dependent on their natural presence in the tumor tissues and their expansion in vitro. In this way, therapies based on the use of these cells will be available only in a fraction of cases. Although each of the adoptive immunotherapies described above have been shown to be capable of mediating tumor regression to some extent, therapeutic responses have been observed only in some cases (and even then they occurred only in a fraction).

LO of the treated patients). It has been proposed (Moritz et al. (1994), PNAS, 91, pp. 4318-4322) to improve the efficacy of lymphocyte-mediated tumor therapy by in vitro manipulation of the recognition specificity of cytotoxic T lymphocytes to equip them with a specificity defined for the tumor cell. Moritz et al. addressed this problem by avoiding specificity based on the TcR (and therefore, the restricted MHC) to create a "synthetic pseudo-receptor" consisting of a single-chain antibody (to confer link specificity) to bind it (via a pivot region) ) to a z-chain of the TcR complex. It was found that the cytotoxic T cells carrying the synthetic pseudo-receptor exhibit MHC-independent recognition with a specificity conferred by the antibody component. However, the use of a completely synthetic pseudo-receptor seems to give rise to a transduction of inefficient L and a reduction in the activity of the Lul * s T. Furthermore, given the recognition specificity or the T lymphocytes. directed cytotoxic agents described by M < * *! -et al, it is independent of the MHC, the efficiency of the exploration on the cell surface by lymphocytes is likely to deteriorate. Finally, the synthetic pseudo-receptor itself may manifest undesirable immune responses. An objective of the present invention is to provide targeted T lymphocytes as an alternative for use, for example, in adoptive immunotherapy, which does not express the completely synthetic pseudo receptors, but instead is directed via the TcRs which are essentially similar in their structure to the normal TcRs. The normal signal transduction pathway, therefore, is conserved and the risks of immune responses minimized. In addition, the targeted T cells of the invention are restricted to the MHC, so that they can efficiently expel the cell surfaces of the aberrant antigens. Accordingly, the present invention provides a targeted cytotoxic T-lymphocyte having a TcR containing heterologous αβ polypeptides, whose heterologous polypeptides confer restricted specificity to "Class I MU1" on the lymphocyte for the target cells causing the disease. in the present, the term "polypeptide" TcR ß hetérologos "is proposed to determine the c ms n and ß of the TcR that are not normally expressed on the "Go T." In one embodiment, the heterologous TcR polypeptides ":" chimeric or synthetic molecules derived from the expression of recombinant or completely synthetic DNA. In other embodiments, the heterologous TcR polypeptides are derived, directly or indirectly (for example, via a TcR gene library), from another T cell. The other T cell can be derived from any source, as long as it expresses the components of the TcR of the required specificity. For example, the other T cell can be from the same species (or even from the same individual), or from different species. It can also be a T cell hybridoma. The specificity of the target cells causing disease need not be absolute. For the purposes of the invention, it is sufficient if the specificity is such that any ablation of the concomitant autoimmune type of normal or non-diseased tissue can be tolerated by the patient. For example, in circumstances where the cell? causes of the disease are all members of a particular tissue which is dispensable, it is sufficient if the targeted T lymphocyte is effectively specific for ra < - «- < -• tissue. This could be the case where the bl inc * cells causing the disease have malignant tumor cells - in this case it is sufficient if the cytotoxic T cells are targeted with sufficient specificity to remove both normal and ! Caneco: ^ (while other tissues are unaffected or removed to a lesser and tolerable degree). Another example is melanoma - although some melanocyte-specific antigens also appear in certain cells of the retina, the brain and the inner ear, these latter cells appear to be less sensitive to immunotherapy based on the specific antigenicity of the melanocyte. Another situation where absolute specificity may not be necessary arises when the counterpart of the normal tissue of a particular tumor expresses MHC class I at extremely low levels, thereby protecting it from attack by cytotoxic T cells. The term "disease-causing cell" is used herein in a broad sense to determine not only the cells that are directly involved with the disease (e.g., tumor cells or autoimmune cells) but also the cells associated with the progress of the disease or that help to promote or maintain the disease state (for example, cells infected by viruses). Therefore, <; > ! term covers any cell that is aberrant or harmful to the health of the individual in any way. The cytotoxic T lymphocytes targeted by the invention are preferably recombinant, being transduced, for example, by a viral vector. The targeted cytotoxic T lymphocyte of the invention of preference is monovalent with respect to target cells, with a single species of TcR conferring specificity restricted to MHC class I for a single * target cell class. These monovalent lymphocytes can be generated, for example, by replacing (rather than complementing) the genes of the α chain and β residents (or endogenous) with heterologous α and β chains that confer the required specificity. The monovalent lymphocytes of the invention can also be generated by down-regulation or blocking the expression of endogenous TcR using any of the wide range of available techniques. For example, targeted insertion mutagenesis can be used to break the endogenous TcR genes. The use of monovalent lymphocytes avoids problems that arise from the formation of dimeros a and ß mal acomplad s (and therefore inactive and / or nonselective). The formation of these inactive heterodimers reduces the elective density of the TcR cell surface which can damage * avoid recognition or lysis of the target cell. By ensuring the monovalence of the lymphocytes of the invention, the cell surface density of the TcRs is optimized. However, in some circumstances it may be preferable or convenient to provide polyvalent cytotoxic T lymphocytes having two or more different TcR species which together confer restricted specificity to MHC class I for two or more classes of target cells. These polypeptide lymphocytes can be generated, for example, by complementing (rather than substituting) the α and β chains residing with one or more pairs of heterologous α and β chains that confer the necessary specificity. The polyvalent, targeted, cytotoxic T lymphocytes of the invention can find particular application in cases where it is necessary to direct two or more different classes of target cells; In the case of the targeted cytotoxic T lymphocyte of the invention is polyvalent, the heterologous TcR a and β polypeptides are preferably provided as a single fusion polypeptide. This avoids the formation of dimples and ß badly coupled (and therefore, inactive and / or non-selective), thus avoiding the problems associated with the reduction of the effective density of the TOR on the cell surface as described above. . r. Preferably, the target cells comprise the tumor cells, immune cells that contribute to an autoimmune response and / or cells infected with pat? 'Joi *. "(And in particular virus-infected cells.) In a particularly preferred embodiment, the cells white include HIV-infected lymphocytes Other viral infections include hepatitis (eg, hepatitis B, C and NANB) and virus-induced Burkitt's lymphoma • The heterologous TcR and aβ polypeptides can be chimeric, and can contain, for example, a variable domain of in unologlobina or fragment of it.These chimeric TcRs exploit the close structural similarities between the constant and variable regions of the α and β chains of the TcR and the V and C regions of the immunoglobulin. these chimeric TcR and ß-polypeptides may be particularly convenient when an immunoglobulin having the required specificity is available or It easily obtains (for example, by developing antibodies to known or predicted T cell antigens). In another aspect, the invention also contemplates a method for producing the targeted cytotoxic T lymphocyte of the invention, the method consists of the steps of: (a) a vector containing DNA is provided (eg, AI * KJ derived from a lymphocyte T csitotoxic of a donor) which encodes TcR and ß-specific polypeptides for the target cells causing the disease, and (b) transfection of a cytotoxic T lymphocyte receptor with ^ 1 vector from step (a) to produce a T lymphocyte Recombinant cytotoxic with DNA encoding the TcR and ß-specific polypeptides for the target cells causing the disease, whereby the cytotoxic T lymphocyte of step (b) expresses a DNA encoding the TcR and ß polypeptides to endow them with the lymphocyte? with restricted specificity MHC / class I and therefore direct them to target cells. The vector of step (a) can be providedby cloning, assembling or synthesizing (for example, by synthesizing oligonucleotides in its solid phase) the DNA encoding the TcR a and β polypeptides specific for the target cells causing the disease. Preferably, the DNA encoding the TcR and ß polypeptides are cloned by: (a) obtaining a sample of T lymphocytes from the donor, for example, from a blood bank, a blood sample or tumor biopsy (d) ) enrichment of the donor T lymphocyte sample for cytotoxic T lymphocytes -which have specificity to the target cells causing the disease, for example, through the prolifer to:? o- * specifically induced and / or expansion clonal espodio - (c) the extraction of chromosomal DNA from the cytotoxic T lymphocytes of the donor; (d) the isolap-i ent 'of the DNA encoding the TcR or y 3 polypeptides, for example, by the amplification of a primer-specific PCR. The vector can be introduced into the cytotoxic T lymphocyte receptor using any sule method. Many of the different methods are known to those skilled in the art, in which transfection by electroporation, protoplast fusion or viral transfection (eg, retroviral) is included. The invention also relates to another aspect for a vector for use in the method of the invention, which vector 'consists of DNA encoding the TcR a and β polypeptides specific for a disease-causing cell. The vector DNA preferably can be operably linked to an expression element or elements to provide expression of the TcR polypeptides. Any of a wide variety of expression elements can be used and the element or elements can have any form, as long as they can (at least according to some circumstances) be elaborated to direct or control the expression of the rn | -which are coupled operably. The elements or expression elements can, for example, be "transcription and / or translation elements, and include promoters, ribosome binding sites, sites j or p? and regulators that include activator and repressor sites (operator). By way of example only, the expression elements for use in the invention can be selected from those naturally associated with the TcR / and / or β peptide genes. It is convenient that the vector of the invention is a viral vector, for example, based on the simian virus 40, the adenovirus (human adenovirus), retrovirus and papillomavirus. In addition, the vector may contain: (a) a selectable positive marker, the marker, for example, is selected from neomycin phosphotransferase, hyrocholine phosphostransferase, xanthinguanin phosphoribociltransferase, thymidine kinase from Herpes simplex virus type 1, adenine phosphoribociltransferase and hypoxanthine phosphoriboyltransferase and / or (b) a selectable negative label, the label, for example, is selected from thymidine kinase of Herpes simplex virus type 1, adenine phosphoribociltransferase, hygromycin phosphotransferase and hypoxanthine phosphoribociltransfei asa. The use of a positive marker selectable by the selection and / or identification of transfected lymphocytes, while the presence of a selectable negative ooR allows subsequent elimination of transfected T cells either in vivo or in vivo. in vitro (for example, if undesirable side effects arise.) The methods of preparing the plasmid to produce Constructs that allow the transcription and translation of, for example, the α and β units in mammalian cells, and in particular, in engineered cytotoxic T lymphocytes, are well known to those skilled in the art. A particularly advantageous vector expresses both the α and β chains under the control of a single promoter. Particularly preferred are promoters specific for cellular expression of T cells and / or T cell precursors / progenitors, and constructs can be separated by a poliovirus derived from the internal ribosomal entry site (IRES). The IRES allows two separate genetic elements to be expressed under the control of the same promoter / speaker element placed at the 5 'end of the sequence. The vector can advantageously be a retroviral vector. The retroviral transduction of mammalian cells is very efficient and can be used to transduce human cells with genes such as the α and β chains of a selected T cell receptor. The retroviral vector preferably has a specific elimination 1 i i-, 3 '. During transduction with the vector of the cell to be engineered, and reverse transcription of the vector to form the proviral AI * N for subsequent incorporation into the host genome, the activity of the promoter / enhancer element LTR 5 will be lost. ' This will allow expression to be placed under the control of selected promoters, thus allowing the specific expression of cytotoxic T cells (Yu SF et al 1986 PNAS USA 83 3191). However, if the restrictions on the size of the vector are too limited in this retroviral vector, alternative vectors can also be considered as can be the adenoviral vectors. The targeted cytotoxic T lymphocytes and the vectors of the invention find application in various forms of therapy, and in particular in adoptive immunotherapy. Lymphocytes and vectors are particularly useful in adoptive immunotherapy which consists of the steps of: (a) eliminating the cytotoxic T lymphocytes of a patient and optionally the selective expansion of these into tissue, (b) the transection of the cytotoxic T lymphocytes removed in step (a) with the vector of the invention to produce cytotoxic T lymphocytes irigida, and (c) the reintroduction of cytotoxic T lymphocytes directed from step (b) in the patient. In the method described above, the T lymphocytes that are used in adoptive immunotherapy are autologous. This has particular advantages because it ensures that the T lymphocytes have the appropriate stimulation and adhesion factors, necessary for the efficient recognition of target cells after reintroduction into the patient. The therapy can be applied to patients with cancer, patients with AIDS, individuals suffering from an autoimmune disease or immunosuppressed individuals (for example, individuals carrying a transplanted organ) who suffer an opportunistic infection. Targeted cytotoxic T lymphocytes and vectors of the invention also find application as vaccines, for example, for prophylactic use in individuals at risk of disease. Examples of these high-risk individuals include individuals predisposed by genetic or environmental factors to diseases (eg, cancer, AIDS or hepatitis). For example, T lymphocytes', -. The present invention can be used to provide immunity against AIDS of the type described by Rowland-Jones et al. (1995), Nature Medicine, Vol. 1 (1), pages, < -'-. Now the invention will be described in more detail by means of specific examples of proposed protocols which are believed to be practicable (with or without modification). These examples are not proposed as limiting in any way.

Example 1: TCR cloning The cloning of T cell receptors has historically depended on the formation of the cDNA libraries from the T cell carrying the receptor of interest. This means that for each T-cell receptor a new cDNA library had to be made from the appropriate clonal cell line, which is a process that requires time. However, between the genes of the two subunits of the T cell receptor, a and ß, there are regions of sequence identity. These are known as constant regions and are found in all subunit and β genes. The sequences for these regions can be found in the published data of the receptor subunits of the cTone T cell, and, by comparing this, it is possible to identify the constant regions of the identity of the sequences. Although these regions are called constant regions, the analysis has shown that there is some variation between constant regions that have so far been cloned. However, small regions of consistent sequences can be identified within a receiving subunit. Using these regions it is possible to clone the transcripts of the a and β subunit to the full length using a method known as the inverse polymerase chain reaction (PCR) described by Ochman et al (Genetics (1988) 120: 621-623). This method depends on the ability of the cDNA, derived from the mRNA, to be circularized and using the PCR primers designated against the known sequence of the transcription, to amplify the entire cDNA by lengthening the chain through an uncharacterized region of the Circularized DNA However, the products resulting from the PCR do not give the sequence of interest in the correct orientation and, therefore, it is "necessary to clone the PCR products for sequencing." By using the sequence data it is possible to visualize the sequence In the correct orientation and design the normal PCR primers to amplify the full-length cDNA in the correct orientation, these cDNA sequences can then be cloned into expression vectors for further manipulation. Inverse primers have been designated against a constant region, the same methodology / primers can be used for different T cell clones. This significantly reduces the cell number requirements and increases the speed and efficiency of TcR cloning. The method can be summarized as follows (see Figures 1 to 3): 1) The total RNA is isolated from the T cell clone of interest used All the Trizol reagent. 2) The cDNA is produced from the total RNA by reverse transcription using an Oligo dT as the initiator for transcription. 3) The synthesis of the second strand is achieved by DNA polymerase 1 in the presence of RNase H to create notches and holes in the hybrid mRNA strand, which provides 3'-0H start sites for DNA synthesis. 4) The circularization is performed with the T4DNA ligase in a concentration of diluted DNA that favors the formation of monomer circles. 5) The linear ADNc is then removed by treatment with exonuclease III. 6) Utilizing the resulting closed circular cDNA, the transcripts of interest are amplified by r r. reverse. The reverse primers are designed in such a way as to initially replicate the cDNA in opposite directions (as shown in Fig. 1) giving rise to the linear products. These products are then replicated as expected with the normal i- PCR methodology. 7) The PCR products are separated on an agarose gel and some of the larger bands are reopened. of the gel and purified. Then the purified products are used in another PCR reaction. However, as a control, normal PCR primers designed to amplify a short length of the constant region of one of the T cell receptor subunits are used. As the fragments that were obtained from the inverse PCR will contain constant regions , if they are really transcripts of the T cell receptor subunit, the normal PCR that uses these primers will amplify short fragments which can be visualized on an agarose gel. Fragments that do not give normal PCR products when amplified using the appropriate normal primers do not represent interest and are discarded. ) The remaining fragments can be cloned into the TA vector (commercially available from Invitrogen) which is a plasmid vector that depends on the 3 'adenylation sites created during each round of replication by the Taq polymerase., to incorporate the PCR product. The competent bacteria are then transformed with the resulting plasmids and the transformants selected for ampicillin resistance. Plasmids with insertions have a <; n 'lac Z broken and therefore appear white when plated on nutrient agar containing X-gal. ) Confirmatory digestions are carried out on plasmid DNA minipreparations from the selected ones. The selected samples are then expanded and large-scale plasmid preparations are made for automated sequencing. 10) Once the sequence data have been obtained it is possible to distinguish the correct orientation of the transcripts by identifying the initial ATG codon (see Figure 2 for more detailed explanation). Using these data it is then possible to design the PCR primers to amplify the entire transcript from the preparation of the original T cell cDNA line. This can then be cloned into a variety of vectors, including vectors. of eukaryotic expression. It is possible to use this methodology for an unlimited number of subunits of the T-cell receptor, and when there are available T cells, without the need to design new initiare- (exception for the amplification of transcription). complete at the end of the procedure). In addition, the complete procedure can be done in a matter of days, contrary to the weeks in the case of the generated < '- •) of the cDNA library.

Example 2: Construction of cytotoxic T cells directed to the melanoma MAGE-1 antigen. Cytotoxic T lymphocytes that recognize the MZ2-E product of the MAGE-1 gene (see for example, Chen et al. (1994), PNAS 91, pp 1004-10Ó8; Van den Eynde et al. (1989), Int. J Cancer, 44, pp 634-640, van der Bruggen et al. (1991), Science, 254, pp. 1643-1647; Travesari et al. (1992), J. Exp. Med. 176, pp. 1453- 1457) were collected and their DNA extracted. From the extracted DNA, a complementary DNA library was constructed using a gtll lambda vector.

The . DNA sequences (cDNAs) coding for the o and 3 chains were then isolated using constant region hybridization probes for the o- and 3-chain described in, for example, Dembic et al. (1985) Nature 311 pp. 271-273 and Snodgrass et al. (1985) Nature 315, pp. 2 < 2-233. The procedure used was similar to that described in Dembic et al. (1986) Nature, 320, pp. 232-1 'M !.

The genes for the chains o and 3 were then cloned into a cosmid vector in the same transcription orientation, together with the n-oir-icin-resistant gene as a positive selection marker. The appropriate expression elements were functionally coupled with the coding sequences o and 3 to ensure efficient expression. The cosmid vector containing the o and 3 chains was then transferred by protoplast fusion into a suitable cytolytic receptor T-cell hybridoma to produce a recombinant T-cell hybridoma having specificity for the MAGE-1 gene product. Those skilled in the art will understand that the above-described method can be easily adapted for the recovery and transfer of other TcRs, and can be applied with suitable modifications to any population of cytotoxic T-cell receptors. Those skilled in the art will understand that the above-described method can be easily adapted for the recovery and transfer of other T? Rs, and can be applied, with appropriate modifications to any population of cytotoxic T-cell receptors.

Example 3: Generation of human T lymphocytes treated with genetic engineering to express cytotoxic activity against cells expressing the influenza virus (Fluj.) Mononuclear, complete cells of human erythrocyte blood (PHMC), derived from a blood donor H A-A2 H class T were prepared and cultured in vtro in standard medium in the presence of Flu peptides known to occur in a restricted HLA-A2 mode.Antong this, antigen presenting cells were formed.Alternatively, antigen presenting cells can formed by addition of peptide to a population of dendritic cells isolated from a restricted HLA-2 donor After 24 hours of incubation with the peptide, the PBMCs were added to CD4 / CD8 cells derived from a human donor recently immunized against influenza, also of HLA-2 MHC Class I restricted idiotype The population of CD4 / CD8 cells derived from an immunized donor were plated in a limiting solution (from 100 to 1 cell) in microtitre plates and left in the presence of antigen presenting cells for 10-14 days with. suitable medium changes. Then, the plates were selected to extend the colonies of T lymphocytes, which presented activated CD4 / CD8 cells capable of recognizing the cells that present antigen and being cytotoxic to them. These extended colonies of activated T-lymphocytes, capable of recognizing influenza antigen when presented by cells in a restricted class I MHC mode, were then cloned and used to prepare complete sequences of the α and β chain of the recipient. T cells, as described in the example. Alternatively other methods of producing and isolating cDNA a and β known to those skilled in the art may be employed.

Once isolated, the? DNc of the α and β chains were then used to engineered the T cells derived from an HLA-A2 donor. MHC class I, not immune. This conferred the engineered T cells the ability to recognize influenza-infected cells in a restricted class I mode and mediate cytotoxicity as determined by the chromium release assay.

Example 4: Generation of human T lymphocytes treated by genetic engineering to express cytotoxicity activity against cells expressing a member of the oricidal family. Complete mononuclear cells of human peripheral blood (PMBC), derived from a blood donor HLA-A2 class I were prepared and cultured in vitro in a standard medium in the presence of peptides of the Harvey oncogene ra - or Kirsten Ras or N-ras known by be presented in a reframed HLA-? 2 mode. In this way they formed cells that present antigen. Alternatively, the cells exhibiting antigens can be formed by addition of the peptide (s) to a pcblaci < of dendritic cells isolated from a restricted LA-P ^ 1 donor. After 24 hours of incubation with the PBMC peptide they were added to CD4 / CD / 8 cells derived from a series of human donors, also of the restricted HLA-A2 MHC class I idiotype. The CD4 / CD8 cell populations derived from the donors were plated in dilution. limiting (from 100 to 1 cell) in microtiter plates and left in the presence of the presenter cells for 10-14 days with adequate changes of the medium. The plates were then selected to extend the colonies of T lymphocytes which presented activated CD4 / CD8 cells capable of recognizing the antigen presenting cells and of being cytotoxic to them. These extended cells of activated T lymphocytes, capable of recognizing the RAS oncogene antigen when presented by the cells in a restricted class I MHC mode, were then cloned and used to prepare the sequences of the complete s and β! Chains. T cell receptor, as described in the above. Once isolated, the cDNA of the α- and β-chains related to the knowledge of the peptides shrinking • •. Harvey ras and Kirsten ras or N-ras were then used to engineer the T cells derived from the donor. HLA-A2 MHC non-immune class I. This conferred the ability to recognize the activation of the ras oncogene in cells in class I restricted mode and provide cytotoxicity mediated to these cells when determined by the test of Chromium release.

Example 5- Generation of human T-lymphocytes engineered to express cytotoxic activity against cells expressing HIV peptpet. Complete mononuclear cells of human peripheral blood (PBMC), derived from a blood donor HLA-B35 MHCclass I were prepared and cultured in vitro in a standard medium in the presence of HIV peptides known to be presented in a restricted HLA-B35 mode, thus forming antigen presenting cells. Alternatively, antigen-presenting cells can be formed by adding the peptide to a population of dendritic cells isolated from a donor HLA-B35 restricted. After 24 hours of incubation with the peptide, ', - PBMC were added to the human CD4 / CD8 derived from the donor) with potential immunity to HIV, also from idio1 < 'pn HLA-B35 class I MHC restricted. The cell population CD4 / CD8 derived from a potential immune donor sr. plaquearon in limiting dilution (from 100 to a cell in microtiter plates and left in the presence or presenter cells for 10-14 days with the appropriate changes of the medium.) The plates were selected to extend the colonies of the T lymphocytes which presented cells Activated CD4 / CD8 capable of recognizing antigenic host cells and those that were cytotoxic to these.Extended colonies of activated T lymphocytes capable of recognizing HIV antigen processed with HLA-B35, when presented by the cells in a restricted MHC class I, the complete sequences of the α and β chain of the T cell receptor were then cloned and prepared, as described above, or by alternative methods of production and α and β cDNA isolation known to those skilled in the art. Once annealed, the cDNA of the α and β chains was then used for genetic engineering treatment in T cells derived from a HLA-B35 class I MHf donor. This conferred T cells treated with genetic immunity, recognition of HIV-infected cells in a restricted class I MHC mode. The average cytotoxicity by cells treated with engineering was demonstrated by the chromium release assay.

Claims (17)

1. A targeted cytotoxic T lymphocyte having a TcR consisting of polypeptides a and β-heterológos, whose heterologous polypeptides confer specificity restricted to MHC class I on the lymphocyte for the target cells of the disease, where the T lymphocyte is monovalent, has a single a species of TcR that confers restricted specificity MHC class I for a single class of target cells.

2. Targeted cytotoxic T lymphocyte, in accordance with claim 1, which is recombinant.

3. Targeted cytotoxic T lymphocyte, of identity with reinvidication 2, which is transduced by a viral vector.

4. The targeted cytotoxic T lymphocyte according to any of the preceding claims, wherein the target cells comprise 1 > 'tumor cells, immune cells that I contribute' * to u- > i autoimmune response and / or cells infected with a pathogen.

5. The targeted cytotoxic T lymphocyte according to claim 4, wherein the target cells comprise the human melanoma cells and the heterologous peptides a and β confer specificity for: (a) the tumor antigen MAGE-1, (b) the tumor antigen MAGE-3, (c) tumor antigen MART 1 / Aa, (d) tumor antigen gplOO and / or (e) tyrosine tumor antigen.

6. The targeted cytotoxic T lymphocyte according to claim 4, wherein the pathogenic agent is a virus.

7. The targeted cytotoxic T lymphocyte, in accordance with reinvication 6, wherein the target cells comprise the lymphocytes infected with HIV.

8. The targeted cytotoxic T lymphocyte, in accordance with the above claims, wherein the heterologous TcR a and β polypeptides are provided with a single fusion polypeptide.

9. The targeted cytotoxic T lymphocyte, in accordance with any of the above claims, wherein the heterologous TcR a and β polypeptides are chimeric.

10. The targeted cytotoxic T lymphocyte, in accordance with reinvication 9, wherein the TcR polypeptides contain a variable domain of the inraunologlobin or fragment thereof.

11. A method for producing a targeted cytotoxic T lymphocyte, in accordance with any of the above claims, the method consists of the steps of: (a) a vector consisting of nucleic acid (e.g., DNA derived from a lymphocyte) is provided Cytotoxic T of a donor) that encodes the TcR and ß polypeptides for the target cells causing the disease, and (b) Transfecting a cytotoxic T lymphocyte receptor with the vector of step (a) to produce a recombinant cytotoxic T lymphocyte having DNA encoding the TcR a and β polypeptides specific for the target cells causing the disease. By means of which the cytotoxic T-lymphocyte of step (b) expresses the DNA encoding the TcR a and β polypeptides to endow the lymphocyte with specificity restricted to the MHC class I and thereby direct it to the target cells.

12. The method according to claim 11, wherein the vector of step (a) is obtained by cloning, assembling or synthesizing (for example, by synthesizing solid-phase oligonucleotides) from DNA encoding the TcR polypeptides. a and ß specific for Ll cells ?? causing the disease.

13. The method according to claim 1, no. 12, wherein the DNA encoding the polypeptides ^ a and ß is cloned by means of: (a) a sample of T lymphocytes is obtained from a donor, for example, from a blood bank, blood sample or .. tumor biopsy; (b) enrichment of the T lymphocyte sample for cytotoxic T lymphocytes having specificity for the target cells causing the disease, for example, by specifically induced proliferation and / or specific clonal expansion. (c) extraction of the chromosomal DNA from the donor, cytotoxic T lymphocytes; (d) the isolation of the DNA encoding the a and β polypeptides, for example, the amplification of the PCR specific for the initiator. The method according to any one of claims 11 to 13, wherein the vector is transfected into a recipient cytotoxic T lymphocyte by means of electroporation, protoplast fusion or viral transfection (e.g., retroviral). 15. A targeted cytotoxic T lymphocyte that can be produced by the method according to any of claims 11 to

14. 16. A vector for use in the method according to any of claims 11 to 11 comprises DNA encoding the TcR and ß-specific polypeptides for a disease-causing cell, the "P" J, for example, operably linked to an expression element or elements, the expression element or elements are selected from, for example , transcription and / or translation elements, promoters, ribosome binding sites, enhancers, regulatory sites (eg, activators and repressors (operator) and expression elements naturally associated with TcR and / or β peptide genes. The vector, according to claim 16, which is a viral vector, is based, for example, on a simian virus 40, adenovirus (human adenovirus), retrovirus and papillomavirus. according to claim no.16 or la. claim 17 further comprises: (a) A positive selectable marker, the marker, for example, is selected from neomycin phosphotransferase, hygromycin phosphostransferase, xanthinguanin phosforiboyltransferase, thymidine kinase from Herpes simplex virus type 1, adenine phosphoribociltransferase or hypoxanthine phosphoriboyltransferase and / or (b) a selectable-negative label, the label, for example, is selected from Herpes urine virus type 1 thymidine kinase, adenine phosphoribociltransferase, hygromycin phosphotransferase and hypoxanthine phosphoriboyltransferase. 19. A targeted cytotoxic T lymphocyte, in accordance with any of the claims of! t 1 heterologous a and β, whose heterologous polypeptides confer restricted specificity to MHC class 1 on the lymphocyte for target cells, for use in therapy. 20. The use of (a) the? directed cytotoxic T lymphocyte as defined in claim 19, or (b) the vector according to any of claims 16 to 18, for the preparation of a medicament for use in adoptive immunotherapy. 21. The use, in accordance with claim no. 20, wherein immunotherapy consists of the steps of: (a) Cytotoxic T lymphocytes are obtained from a patient, (b) The transfection of the cytotoxic T lymphocytes obtained in step (a) is done with the vector according to any of claims 16 to 18 to produce targeted cytotoxic T lymphocytes. (c) The reintroduction of the cytotoxic T lymphocytes directed from step (b) in the patient. 22. The use according to claim no.21 further comprises the step of: the selective expansion in tissue culture of the cytotoxic T cells obtained in step (a) prior to steps (b) and (c) ). 23. The use, according to any of the claims from 20 to 22, where the patient e? a patient with cancer, a patient with AIDS, an indid! i-suffering from an autoimmune disease or an immunosuppressed individual (eg, an individual carrying a transplanted organ) who suffers an opportunistic infection. 24. The use of: (a) the targeted cytotoxic T lymphocyte, as defined in claim 19, or (b) the vector according to any of claims 16 to 18, for the preparation of a vaccine for use in immunotherapy or prophylaxis, for example, for use in the treatment or prophylaxis of SIE'A. 25. A vaccine containing the targeted cytotoxic T lymphocyte according to any one of claims 1 to 10 or 15, for example, also contains a pharmaceutically acceptable excipient.