MXPA98007178A - Method of quick expansion modified for in vitro propagation from lymphocyte - Google Patents

Abstract

The present invention relates to a modified rapid expansion method (termed "low PBMC REM" or "Modified REM") to rapidly generate large numbers of T lymphocytes, including cytolytic and auxiliary T lymphocytes, without using large amounts of mononuclear cells of peripheral blood (PBMC) or lymphoblastoid cells transformed with EBV (LCL) characteristic of REM of high PBMC. Clonal expansions greater than 500 times can be obtained with a single stimulation cycle, in approximately 8-14 days.

Description

METHODS OF QUICK EXPANSION. MODIFIED FOR PROPAGATION N V TRO FROM LYMPHOCYTES T FIELD OF THE INVENTION This invention relates to improved methods of T lymphocyte cultures, including cytolytic lymphocytes specific for human antigen and helper T lymphocytes. The methods of the present invention result in a very rapid and efficient expansion of T cells which are useful, for example, in cellular immunotherapy.

ANI'lii '&DENTES OF THE TECHNIQUE T lymphocytes are formed in the bone marrow, migrate and mature in the thymus, then enter the peripheral blood and the lymphatic circulation. T lymphocytes can be phenotypically subdivided into several different types of cells including: helper T cells, suppressor T cells and cytotoxic T cells. T lymphocytes, unlike B lymphocytes, do not produce antibody molecules, but they express a heterodimeric cell surface receptor that can recognize fragments REF. 28224 antigenic protein peptides that bind to major histocompatibility complex (MHC) proteins expressed on the surfaces of target cells; see, for example, Abbas, A.K., Lichtman, A.H., and Pober, J.S., Cellular and Molecular Immunology. 1991, especially pages 15-16. The T lymphocytes that can be expanded according to the present invention are of particular interest in the context of cellular "i-immunotherapy". As used herein, "cellular immunotherapy" refers to any of a variety of techniques that involve the introduction of cells of the immune system, especially T-lymphocytes, into a patient for therapeutic benefit. Such techniques may include, by way of illustration, "immuno-restorative" techniques (involving, for example, the administration of T cells to a patient who has a compromised immune system), * "immunosuppression" techniques (involving, for example, , administering T cells to a patient in order to improve the ability of the patient's immune system to avoid or fight a cancer or a pathogen such as a viral or bacterial pathogen); and "immunomodulation" techniques (involving, for example, the administration of T cells to a patient in order to modulate the activity of other cells of the patient's immune system, such as a patient afflicted with an autoimmune condition). Cytotoxic T lymphocytes (CTL) are typically of the CD3 +, CD4-, CD8 + phenotype and lyse cells that show fragments of foreign antigens associated with MHC class I molecules on their cell surfaces. CTLs that are CD3 +, CD4 +, CD8- have also been identified. Target cells for CTL recognition include normal cells that express antigens after infection by viruses or other pathogens; and tumor cells that have undergone transformation and that express mutated proteins or overexpress normal proteins. Most "helper" T cells (helper) are CD3 +, CD4 +, CD8-. Auxiliary T cells recognize fragments of antigens present in association with MHC class II molecules, and function primarily to produce cytokines that amplify antigen-specific T and B cell responses and activate accessory common cells such as monocytes or macrophages. See, for example, Abbas, A.K., et al., Supra. The helper T cells also participate in, and / or increase, the cytolytic activities. In addition to conventional T helper cells and killer or cytolytic T cells (killer), they will also be useful in rapidly expanding other populations of T cells. For example, T cells that express the gamma / delta T cell receptor present in relatively small proportions in the population of human T cells, but it is suspected that viral and bacterial pathogens as well as tumor cells play a role in the viral network (see, for example, . Haas et al. 1993. Annu. Rev. Immunol. 11: 637). Another population of T cells of potential clinical importance is the population of CD1-restricted T cells. CDl is a molecule similar to MHC that shows limited polymorphism and, unlike classical MHC molecules which "present" antigenic peptides, CD molins bind lipoglisans and appear to be important in the recognition of microbial antigens (see, for example, PA Sieling et al., 1995. Science 269: 227; and E. M. Beckman et al. 1994. Nature 372: 691). Therefore, T lymphocytes are key components of the host immune response to viruses, bacterial pathogens and tumors. The importance of the proper functioning of T cells becomes very clear in individuals with congenital, acquired or iatrogenic T cell immunodeficiency conditions (eg SCID, BMT, AIDS, etc.), which can result in the development of a broad range of T cells. variety of infections or malignant cancers that endanger life. People with diseases that are related to a deficiency of immunologically competent T lymphocytes or people with conditions that can be improved by administering additional T lymphocytes can benefit from cellular immunotherapies, as mentioned above. T cells for use in such therapy can be derived from immunodeficient hosts, or from another source (preferably a compatible donor). This latter source, of course, is especially important in situations in which an immunodeficient host has an insufficient amount of T cells, or has T cells that are insufficiently effective. In any case, it is difficult to obtain sufficient quantities of T cells for effective administration; and therefore the target T cells must first grow in large quantities in vitro before their administration to a host. After experiencing such cellular immunotherapy, hosts who have previously shown, for example, inadequate or no responses to antigens expressed by pathogens or tumors, may express sufficient immune responses to become resistant or immune to the pathogen or tumor. It has been shown that adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models (reviewed in Greenberg, P.D., Advances in Immunology (1992)). For adoptive immunotherapy to be effective, antigen-specific T cells usually need to be isolated and expanded to large quantities by in vitro culture, and subsequent to such adoptive transfer, such cultured T cells must persist and function in vivo. For the treatment of some human diseases, the use in immunotherapy of antibody-specific T cells, cloned, with respect to the progeny of single cells, offers significant advantages because the specificity and function of these cells can be rigorously defined and evaluated. easily and precisely the effects of dose: response. Riddell et al., Were the first to adoptively adopt human T cell clones specific for antibody, to restore poor immunity in humans. Riddell, S.R. et al., "Restoration of Viral Im unity of Immunodeficient Humans by the Adoptive Transfer of T Cell Clones", Sciense 257: 238-240 (1992). In that study, Riddell et al. Used adoptive immunotherapy to restore poor immunity to cytomegalovirus in allogeneic bone marrow transplant recipients. Cytoplasmic CD8 cytotoxic T cell clones were isolated for cytomegalovirus from three bone marrow donors seropositive to CMV, propagated in vitro for 5 to 12 weeks to obtain a numerical expansion of effector T cells, and then intravenously administered to recipients of T cells. respective bone marrow transplant (BMT). BMT receptors were deficient in CMV-specific immunity due to the suppression of host T cell responses by chemoradiotherapy prior to transplantation and the delay in recovery of donor immunity commonly observed after bone marrow allogeneic transplantation (Reusser et al. al., Blood 78: 1373-1380, 1991). Riddell et al., Found no toxicity and the transferred T cell clones gave these immunodeficient hosts a rapid and persistent reconstitution of CD8 + CTL responses specific for cytomegalovirus. Riddell et al (J. Immunology, 146: 2795-2804, 1991) used the following procedure to isolate and culture CD8 + T cell clones specific for CMV: peripheral blood mononuclear cells (PBMC) derived from bone marrow donors were first cultured with autologous fibroblasts infected with cytomegalovirus , to activate specific CTL precursors for CMV. Then the cultured T cells were restimulated with fibroblasts infected with CMV and the cultures were supplemented with PBMC subjected to radiation ?. 2-5 U / ml interleukin-2 (IL-2) was added in appropriate culture medium on days 2 and 4 after restimulation to promote the expansion of CTL or CD8 + (Riddell et al., J. Immunol. 146: 2795-2804, 1991). To isolate T cell clones, CMV-specific CD8 + polyclonal T cells were plated at a limiting dilution (0.3-0.6 cells / well) in 96-well round-bottom wells with either CMV-infected fibroblasts as host cells of antigen (Riddell, J. Immunol .. 146: 2795-2804, 1991); or monoclonal antibody against CD3 to mimic the stimulus provided by antigen-presenting cells (Riddell, J. Imm. Methods, 128: 189-201, 1990). Subsequently, peripheral blood mononuclear cells (PBMC) subjected to irradiation were added? and lymphoblastoid cells transformed by EBV (LCL) to the microwells, as feeding cells. Positive wells for clonal T cell growth were evident in 10-14 days. The clonally derived cells were then propagated to large quantities initially in 48 or 24 well plates and subsequently in 12 well plates or 75 cm2 tissue culture flasks. T cell growth was promoted by restimulation every 7-10 days with autologous fibroblasts infected with CMV and feeder cells subjected to irradiation? which consists of PBMC and LCL, and the addition of 25-50 U / ml of IL-2 at 2 and 4 days after restimulation.

A major problem that exists in the studies described above, and in general with the culture of T cells of the prior art, is the ability to grow large amounts of human T cell clones specific for antigen in a timely manner. It is not known whether the slow growth of T cells in culture represents an inherent property of the cell cycle time for human lymphocytes or if it is due to the culture conditions used for example, with the culture method used in the immunotherapy study of adoptive type of CMV, it took three months to grow T cells to obtain the highest dose of cells under the study, which was 1 x 109 cells T / cm2. This greatly limits the adhesion of adoptive immunotherapy to human viral diseases and to cancer since the disease process can progress during the prolonged interval necessary to isolate and grow the specific T cells that are to be used in the therapy. Based on the extrapolation of studies in animal models (reviewed in Greenberg, PD, Advances in Immunology, 1992), it is predicted that in humans, doses of antigen-specific T cells in the range of 109-1010 cells may be required. to increase immune responses to obtain a therapeutic benefit.

However, the rapid expansion of antigen-specific human T cells in culture to obtain such large amounts of cells has proven to be a major obstacle. Therefore, with the exception of the study by Riddell et al., Supra (in which several months were required to grow a sufficient number of cells), no adoptive immunotherapy studies have been performed using antigen-specific T cell clones. . The problem of producing large numbers of cells for adoptive immunotherapy was identified in U.S. Patent No. 5,057,423. In this patent, a method for isolating pure large granular lymphocytes and a method for the expansion and conversion of these large granular lymphocytes into lymphokine-activated killer cells (LAK) is described. The methods are described in such a way that they provide high levels of expansion, that is, up to 100 times in a culture of 3-4 days. Although LAK cells lysed some types of tumor cells, they do not share with T cells restricted for MHC the properties of defined recognition antigens and do not provide immunological memory. In addition, the methods used to expand LAK cells, which are predominantly based on high concentrations of IL-2, do not efficiently expand human T cells specific for antigen (Riddell et al., Unpublished), * and those methods that return to T cells subject to programmed cell death (ie, apoptosis) by the extraction of IL-2 or subsequent stimulation via the T-cell receptor (see discussion of documents by Lenardo et al., and Boehme et al., infra) . Prior methods that are based on the use of lectins, such as concanavalin A or phytohaemagglutinin (see, for example, Van der Griend et al., Transplantation 38: 401-406 (1984), and Van de Griend et al., J. Immunol. Methods 66: 285-289 (1984)), has been even less satisfactory due to the use of such non-specific stimulatory lectins tend to induce several phenotypic changes in the stimulated cells which make them very different from the simulated T cells via the CD3 receiver. The inability to grow antigen-specific T cell clones in large quantities in part has been responsible for the limitation of adoptive immunotherapy studies for human diseases such as cancer (Rosenberg, New Engl. J. Med. 316: 1310-1321, 1986; Rosenberg, New Engl. J. Med. F 319: 1676-1680, 1988) and HIV infection (Ho M. et al., Blood 81: 2093-2101, 1993) for the evaluation of polyclonal populations of activated lymphocytes. with poorly defined antigen specificities. In such studies, polyclonal lymphocyte populations are isolated from the blood of the tumor filtrate and cultured at high concentrations of the T cell growth factor, IL-2. In general, these cells have shown little specificity, if any, restricted to MHC for the pathogen or tumor in the minority of patients who have experienced therapeutic benefit, it has been difficult to discern the effector mechanism involved. Typically, adoptive immunotherapy studies with nonspecific effector lymphocytes have administered approximately 2 x 1010 to 2 x 1011 cells to the patient. (See, for example, U.S. Patent No. 5,057,423 in column 1, lines 40-43). The development of efficient cell culture methods to rapidly grow T lymphocytes would be useful in diagnostic and therapeutic applications. In diagnostic applications, the ability to rapidly expand T cells from a patient can be used, for example, to quickly generate sufficient numbers of cells for use in tests to monitor the specificity, activity or other attributes of the patients' T lymphocytes. In addition, the ability to rapidly obtain cell doses of 109-1010 cells will greatly facilitate the applicability of specific adoptive immunotherapy for the treatment of human diseases. There are several establi methods already described for culturing cells for possible therapeutic use including methods for isolating and expanding T cell clones. Cell culture methods for anchor-dependent cells (i.e., those cells that require binding to a substrate for proliferation cell) are limited for the amount of surface area available in the culture vessels used (ie, multiple well plates, petri di and culture flasks). For anchor-dependent cells, the only way to increase the number of cells that grows is generally to use larger containers with increased surface area and / or use of more containers. However, hematopoietic cells such as T lymphocytes are independent of the anchor. They can survive and proliferate in response to appropriate growth factors in a culture suspension without binding to a substrate. Even with the ability to grow antigen-specific lymphocytes in a suspension culture, methods reported to date have not consistently produced a rapid numerical expansion of T cell clones. For example, in a T cell study conducted by Gillis and Atson, it has been found that T cells grown at low densities, ie, 5 × 10 3 to 1 × 10 4 cells / ml in the presence of T-cell growth factor, IL-2, proliferate rapidly over a period of seven days and finally reach a saturation density of 3-5 x 10 5 cells / ml. Gillis, S. and atson, J. "Interleukin-2 Dependent Culture of Cytolytic T Cell Lines". Immunological Rev., 54: 81-109 (1981). In addition, Gillis and Watson also found that once the cells reach this saturation level, the cells invariably die. Gillis et al. , id. Another study reported three different methods to establish mouse T lymphocytes in a long-term culture. Paul et al. Reported that the most widely used method is to grow T lymphocytes from donors immunized for several weeks or more in the presence of antigen and antigen-presenting cells (APC) to provide the requisite T-cell receptor signal and costimulatory signals , and with the addition of exogenous growth factors, before attempting to clone them, Paul, WE, et al., "Long-term growt and cloning of non-transformed lymphocytes", Nature, 294: 697-699, (1981). T cells specific for protein antigens are then cloned by limiting dilution with antigen and irradiated spleen cells as a source of APC. A second method involves growing T cells as colonies on soft agar as soon as possible after taking the cells from. an immunized donor. T cells are stimulated in an initial suspension culture with antigen and a source of APC, usually irradiated spleen cells. In this second approach it has been found that, after 3 days, the cells are distributed in the upper layer of a two-layer soft agar culture system. Colonies are taken on days 4 to 8 and then expanded into long-term cultures. The third approach involves selecting cells for their functional properties instead of their antigenic specificity and then making them creser with a series of different irradiated feeder cells and supernatants containing growth factor. Paul, W.E. et al., "Long-term growth and cloning of non-transformed lymphocytes", Nature, 294: 697-699, (1981). It is evident that with each of these methods, it is not possible to expand individual T cell clones from a single cell to 109-1010 cells in a timely manner. Therefore, despite the ability to clone T cells specific for antigens, and convincing evidence of the therapeutic efficacy of T cell clones in accepted animal models, the technical difficulty in culturing human T cells in large numbers has impeded clinical evaluation and application of cellular immunotherapeutic procedures. Another additional problem with cultured T cells is that they must remain capable of functioning in vivo in order to be useful in immunotherapeutic procedures. In particular, it has been observed that antigen-specific T cells which were grown long-term in culture at high concentrations of IL-2 can develop abnormalities in the cell cycle and lose the ability to return to a resting phase when remove IL-2. In contrast, the normal cell cycle consists of four successive phases: mitosis (or "M" phase) and the three phases which constitute the "interface" stage. During phase M, the cell undergoes nuclear division and cytokinesis. The interphase stage consists of the Gl phase in which the biosynthetic activities are resumed at high speed after mitosis; the S phase in which DNA synthesis occurs and the G2 phase continues until mitosis begins. Although they are in the Gl phase, some cells seem to stop progressing through the division cycle; and they are said to be in a "resting" or quiescent state (referred to as a "GO" state). Several environmental factors (such as the lack of serum growth factors or the confluence of cell cultures) can cause the cells to enter the quiescent state. Once the factor is restored, the cells resume their normal development throughout the cycle. However, cells that have grown in culture may be unable to enter the quiescent phase when the growth factor is removed, resulting in the death of these cells. This dependence on the growth factor is particularly relevant for cultured T cells. T lymphocytes that have been exposed for a prolonged period to elevated concentrations of IL-2 to promote cell growth will often die by a process called apoptosis if IL-2 is removed or if they are subsequently stimulated through the T cell receptor, that is, if they find specific antigens (see, for example, Lenardo MJ, Nature, 353: 858-861, 1991, Boehme SA and Lenardo MJ, Eur. J. Immunol. 23: 1552-1560, 1992). Therefore, the culture methods used to propagate LAK cells or TIL cells, and the above methods for culturing T cells which are predominantly based on high long-term concentrations of IL-2 to promote in vitro expansion, can return to many of the cells susceptible to apoptosis, and therefore limit or eliminate their used for cellular immunotherapy. It would also be advantageous in cell immunotherapy studies to use gene transfer methods to insert foreign DNA into T cells to provide a genetic marker, to facilitate the assessment of in vivo migration and the survival of transferred cells, or to confer functions that may improve the safety and efficacy of transferred T cells. One established method for stably transferring genes to mammalian cells is the use of amphotropic retroviral vectors (see, eg, Miller AD, Current Topics in Microbiology and Immunology, 158: 1-24, 1992). The stable integration of genes in the target cell using retrovirus vectors requires that the cell is actively cyclized, specifically, that these cells transit through the M phase of the cell cycle. Previous studies have introduced a marker gene into a small proportion of polyclonal T cells boosted to proliferate with high doses of IL-2, and these cells were reinfused in humans as a therapy against tumors and provided a means of monitoring cell survival in vivo. transferred (Rosenberg et al., New Engl. J. Med., 323: 570-578, 1990). However, for human T cells (whose cycle decreases when they grow with standard techniques), the transfer efficiency of stable genes is very low, in the range of 0.1-1% of T cells (Springett CM et al., J. Virology, 63: 3865-69, 1989). Culture methods which recruit the target T cell more efficiently in the S and G2-M phases of the cell cycle can increase the efficiency of gene modification using retrovirus-mediated gene transfer (Roe T. et al., EMBO J. 2: 2099-2108, 1993), and thus to improve the prospects for using genetically modified T cells in cellular immunotherapy or to use T cells to supply defective genes in diseases of genetic deficiency. The rapid expansion method described by S. Riddell et al., (In PCT publication WO 96/06929, published March 7, 1996), hereinafter referred to as "high PBCM REM" or "REM-hp" was developed to provide functional antigen-specific T cell clones for use in clinical adoptive immunotherapy protocols. The hp REM protocol was designed to provide maximal expansion of T cells in a limited amount of time without loss of function or specificity of T cells. Generally, the hp REM protocol involves the steps of adding an initial population of T lymphocytes to a medium of in vitro culture add a disproportionately large amount of peripheral blood mononuclear cells that do not divide to the culture medium ("PBMC") as feeder cells so that the resulting population of cells contains at least about 40 PBMC feeder cells (preferably at least about 200, more preferably at least about 400) per each T lymphocyte in the initial population that it is going to expand; and incubate the crop. In the preferred embodiments of the REM hp protocol, the T cells that are to be expanded are also exposed to a disproportionately large amount of lymphoblastoid cells transformed with EBV.

("LCL"), to a monoclonal antibody against CD3 (for example 0KT3) (to activate the T cells via the T cell antigen receptor), and with the T cell growth factor, interleukin-2 (IL-2). In the REM hp protocol, T cells are generally expanded using a vast excess of feeder cells consisting of peripheral blood mononuclear cells (PBMC) and possibly also lymphoblastoid cells transformed with EBV (EBV-LCL). The expanding T cells typically represent less than about 0.2% of the cells in the REM hp culture method. As described, the T cells can be activated through the T cell antigen receptor using a monoclonal antibody against CD3 (e.g., OKT3) and the proliferation of T cells can be induced using IL-2. It has been reported that such REM hp culture conditions result in a T cell expansion level 100 to 200 times higher than those reported by other researchers. However, for most uses, it would be preferable to avoid the use of a large excess of feeder cells (ie, PBMC and EBV-LCL) in the preparation of T cells intended for clinical use. For example, PBMCs are derived from human blood and may represent a potential source of adventitious agents (eg, human immunodeficiency virus, type 1 and 2, human T cell leukemia virus I, types 1 and 2, and hepatitis virus). , such as hepatitis B, C and G), and EBV-LCL may represent a potential source of Espein-Barr virus. In addition, the large-scale application of the REM hp protocol may require a large supply of human peripheral blood to provide adequate amounts of feeder cells. Therefore, it would be particularly advantageous to reduce the amount of such necessary feeder cells or to replace them completely. With these concerns in mind, the methods of the present invention (hereinafter referred to as "low PBMC REM" or "modified REM") are designed to obtain rapid in vitro expansion of T cells without using the vast excess of PBMC and / or EBV-LCL feeder cells that are the key feature in the REM hp protocol.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a method for quickly producing large amounts of T cells including human antigen-specific cytolytic T cells and helpers, isolated from an initial population of T cells, without using the vast excess of PBMC feeder cells and / or EBV-LCL which are the key feature of the REM hp protocol. Although the methods of the present invention are applicable to rapid expansion of T lymphocytes, generally, the rapid expansion method will be especially advantageous in situations in which a single clone of T cells must be expanded to provide a large population of T lymphocytes. Therefore, the present invention provides an especially important tool in the context of human adoptive immunotherapy, as exemplified in studies (using REM hp, described below) involving human bone marrow transplant recipients in the Fred Hutchinson Canser Research Center. The present invention also provides a method for improving the stable transfer efficiency of gels in T lymphocytes, as exemplified below. Accordingly, one object of the invention is to rapidly expand T lymphocytes in large quantities in vitro without using the vast excess of PBMC and / or EBV-LCL feeder cells which are the key feature of the REM hp protocol. Such rapidly expanded populations of T cells can be used, for example, for infusion in individuals for the purpose of conferring a specific immune response, as exemplified herein. The T cells that can be expanded using the present invention include any of the various T lymphocyte populations described herein (see, for example, the discussion above regarding CTL, helper T cells and other T lymphocytes, and the potential uses of T cells). such cells in immunotherapeutic techniques). Another objective of the invention is to use the method to grow T cells in a manner which facilitates the stable introduction of foreign genetic material which can be used to alter the function of T cells that are to be used in cellular immunotherapies, as describes before, or otherwise, resolving a defect or an inappropriate gene in the host. Several preferred embodiments of the present invention are described in the following list: 1. A method (hereinafter referred to as "REM low PBMC" or "modified REM") to rapidly expand an initial population of T lymphocytes in an in vitro culture medium. Vitro, which comprises the steps of: adding an initial population of T lymphocytes to an in vitro culture medium; adding to the culture medium a non-dividing mammalian cell line that expresses at least one T-cell stimulator component, wherein the cell line is not a line of lymphoblastoid cells transformed by EBV (LCL); and incubate the crop. REM cultures will generally be incubated under conditions of temperature and the like which are suitable for the growth of T lymphocytes. For the growth of human T lymphocytes, for example, the temperature will generally be about 25 ° Celsius, preferably at least about 30 ° C. , and most preferably approximately 37 °. The descriptions of an appropriate medium and other culture conditions are well known in the art, and are also exemplified herein. 2. A rapid expansion method according to the preceding clause, wherein the T-cell stimulator component is selected from the group consisting of a Fc receptor, an accessory cell of cell adhesion and a cytokine. 3. A method of rapid expansion according to any of the preceding paragraphs, wherein the T-cell stimulator component is selected from the group consisting of an Fc receptor, accessory cell adhesion molecules and a cytokine, and wherein the Initial population of T lymphocytes expands at least 200 times after an incubation period of less than about two weeks. 4. A rapid expansion method according to any of the preceding paragraphs, wherein the T-cell stimulator component is selected from the group consisting of a Fc receptor, accessory cell adhesion molecules and a cytokine, and wherein the initial population of T lymphocytes expands at least 500 times after an incubation period of less than about two weeks. 5. A rapid expansion method according to any of the preceding paragraphs, wherein the T-cell stimulator component is selected from the group consisting of a Fc receptor, accessory cell adhesion molecules and a cytokine, and wherein the Initial population of T lymphocytes expands at least 1000 times after an incubation period of less than about two weeks. 6. A method of rapid expansion according to any of the preceding paragraphs, further comprising the step of adding monoclonal antibody against CD3 to culture medium, wherein the concentration of monoclonal antibody against CD3 is at least about 1.0 ng / ml. Typically, a concentration of about 10 ng / ml is used, although much lower levels can be used, as illustrated below. 7. A method of rapid expansion according to any of the preceding paragraphs, further comprising the step of adding IL-2 to the culture medium, wherein the concentration of IL-2 is at least about 10 units / ml. Typically, a concentration of approximately 25 units / ml is used. Preferably, the incubation continues for at least about 9 days and wherein the step of adding IL-2 to the culture medium is repeated after each 3-5 day interval. Typically, IL-2 is added on day 0, again on day 5 or 6, and again on day 8 or 9. 8. A method of rapid expansion according to any of the preceding paragraphs, wherein the line of The mammalian cell comprises at least one type of cell that is present at a frequency at least twice that found in human peripheral blood mononuclear cells (human PBMC); preferably at least three times, at least ten times, or at least fifty times the frequency generally found in human PBMC. 9. A method of rapid expansion according to any of the preceding insiso, wherein the T-cell stimulator component is selected from the group consisting of an Fc receptor. and an accessory molecule of cell adhesion.

. A method of rapid expansion according to any of the preceding paragraphs, wherein the T-cell stimulator component is selected from the group consisting of accessory molecule of cell adhesion and a cytokine. 11. A method of rapid expansion according to any of the preceding paragraphs, wherein the T-cell simulator component is selected from the group consisting of an Fc-receptor. and a cytokine. 12. A rapid expansion method according to any of the preceding paragraphs, wherein the mammalian cell line expresses an accessory cell adhesion molecule. 13. A rapid expansion method according to any of the preceding paragraphs, wherein the accessory cell adhesion molecule is selected from the group consisting of MHC class II, MHC class I, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronictin, ligand to CD27, CD80, CD86 and haluronate. 14. A method of rapid expansion according to any of the preceding paragraphs, wherein the mammalian cell line expresses cytokine. Preferably, the cytokine is an interleukin. 15. A rapid expansion method according to any of the preceding paragraphs, wherein the T-cell stimulator component is a molecule that binds to CD21. 16. A method of rapid expansion according to any of the preceding paragraphs, wherein the cytokine is selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15. 17. A rapid expansion method according to any of the preceding paragraphs, further comprising the step of adding a soluble factor T cell stimulator to the culture medium. 18. A rapid expansion method according to any of the preceding paragraphs, wherein the soluble T cell stimulator factor is selected from the group consisting of a cytokine, an antibody specific for a T cell surface component and a specific antibody. for a component capable of binding to a T cell surface component. 19. A rapid expansion method according to any of the preceding paragraphs, wherein the soluble T-cell stimulator is a cytokine from the group consisting of IL- 1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15. 20. A method of rapid expansion according to any of the preceding paragraphs, wherein the soluble factor T cell stimulator is an antibody specific for a T cell surface component, and wherein the T cell surface component is selected of the group consisting of CD4, CD8, CDlla, CD2, CD5, CD49d, CD27, CD28 and CD44. 'twenty-one. A method of rapid expansion according to any of the preceding insiso, wherein the soluble factor T cell stimulator is an antibody specific for a component capable of binding to a surface component T, and wherein the cell surface component T is selected from the group consisting of CD4, CD8, CDlla, CD2, CD5, CD49d, CD27, CD28 and CD44. 22. A method of rapid expansion according to any of the preceding paragraphs, wherein the soluble factor T-cell stimulator is a molecule that binds to CD21. 23. A method of rapid expansion according to any of the preceding paragraphs, wherein the molecule that binds to CD21 is an antibody against CD21. 24. A rapid expansion method according to any of the preceding clauses, further comprising the step of adding to the culture a multiplicity of peripheral blood mononuclear cells (PBMC). Preferably, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads, more preferably at about 3300 rads.

. A method of rapid expansion according to any of the preceding paragraphs, wherein the ratio of the PBMC to the initial T cells to be expanded is less than about 40: 1. 26. A rapid expansion method according to any of the preceding paragraphs, wherein the ratio of PBMC to the initial T cells to be expanded is less than about 10: 1. 27. A method of rapid expansion according to any of the preceding paragraphs, wherein the ratio of PBMC to initial T cells that are to be expanded is less than about 3: 1. 28. A method of rapid expansion according to any of the preceding paragraphs, further comprising the step of adding to the culture a multiplicity of lymphoblastoid cells transformed with EBV (LCL). Preferably, the PBMCs are irradiated with gamma rays in the range of about 6000 to 10,000 rads, more preferably at about 8,000 rads. 29. A rapid expansion method according to any of the preceding paragraphs, wherein the LCL propionion with respect to initial T cells that are to be expanded is less than about 10: 1. 30. A rapid expansion method according to any of the preceding paragraphs, wherein the initial population of T lymphocytes comprises at least one cytotoxic T lymphocyte (CTL) antigen-specific, CD8 +. In the preferred embodiments of the present invention, the CTL is specific for an antigen present in a human tumor or encoded by a pathogen such as a virus or bacteria. 31. A method of rapid expansion according to any of the preceding paragraphs, wherein the initial population of T lymphocytes comprises at least one helper T lymphocyte specific for antigen, human CD4 +. 32. A method for genetically transducing a human T cell, comprising the steps of: adding an initial population of T lymphocytes to an in vitro culture medium, - adding to the culture medium a line of mammalian cells not transformed by EBV which express a T cell stimulator component; and incubate the culture; and add a vector to the culture medium. A vector refers to a unit of DNA or RNA in a form which is capable of being introduced into a target cell. Transduction is generally used to refer to the introduction of hexogen DNA or RNA into a target cell and includes the introduction of heterologous DNA or RNA sequences into the target cells by, for example, viral infection and electroporation. A currently preferred method of transducing T lymphocytes is to use retroviral vectors, as exemplified herein. 33. A method of genetic transduction according to item 32, wherein the vector is a retroviral vector containing a selectable marker that provides resistance to an inhibitory compound that inhibits T lymphocytes, and wherein the method further comprises * the steps of : continue the incubation of the culture for at least one day after the addition of the retroviral vector; and adding the inhibitor compound to the culture medium after continuing the incubation step. Preferably, the retroviral vector contains a positive and negative selectable marker. Preferred positive selectable markers are derived from genes selected from the group consisting of hph, neo and gpt, and preferred negative selectable markers are derived from genes that are selected from the group consisting of cytokine deaminase, HSV-1 TK, VZV TK, HPRT, APRT and gpt. Especially preferred labels are bifunctional selectable fusion genes in which a positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytokine deaminase or a gene for TK. 34. A method of genetic transduction according to any of paragraphs 32-33, which further comprises adding a multiplicity of human PBMCs. 35. A method of rapid expansion, according to any of paragraphs 32-34, wherein the ratio of the PBMC to the initial T cells is less than about 40: 1. 36. A method of genetic transduction according to any of paragraphs 32-35, which further comprises adding lymphoblastoid cells transformed with EBV, which are not in division (LCL). 37. A rapid expansion method, according to any of paragraphs 32-36, wherein the ratio of LCL to initial T cells is less than about 10: 1. 38. A method for generating a REM cell line capable of promoting rapid expansion of an initial population of T lymphocytes in vitro, comprising the steps of: suppressing one or more cell types from a population of human PBMCs to produce a population of deleted PBMCs in a cell type, use the suppressed PBMC population in a cell type, instead of the non-suppressed PBMCs, in a hp REM protocol to determine the contribution of the suppressed cell type to the activity provided by the Non-suppressed PBMCs, identify a T cell stimulatory activity provided by the type of suppressed cell, and transform a mammalian cell line with a gene that allows the expression of such T-cell stimulatory activity. 39. A method for generating a line of REM cells according to item 38, wherein the T-cell stimulator component is selected from the group consisting of a Fc receptor, an accessory cell of cell adhesion and a cytokine. 40. A line of REM cells capable of stimulating rapid expansion of an initial population of T lymphocytes in vitro, comprising a mammalian cell line generated according to a method according to paragraph 38 or preceding paragraph 39. 41. A line of REM cells according to item 40, wherein the cell line expresses an accessory cell adhesion molecule. 42. A REM cell line according to any of Claims 40-41, wherein the accessory cell adhesion molecule is selected from the group consisting of MHC class II, MHC class I, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, ligand to CD27, CD80, CD86 and hyaluronate. 43. A line of REM cells according to any of Claims 40-42, wherein the cell line expresses an Fc-1 receptor. 44. A line of REM cells according to any of Claims 40-43, wherein the cell line expresses at least one T-cell stimulatory cytokine. 45. A line of REM cells according to any of Claims 40-44, wherein the T cell-stimulating cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15. 46. A line of REM cells according to any of Claims 40-44, wherein the cell line expresses a molecule that binds to CD21.As used herein, a molecule that binds to CD21 may be a natural or synthetic conosida or determined molecule that binds to cell surface determinant CD21.M molecules known to bind to CD21 include antibodies against CD21, as well as molecules C3d, C3dg, iC3b and EBV gp350 / 220, and derivatives thereof 47. A culture medium capable of rapidly expanding an initial population of T lymphocytes in vitro, comprising a REM cell line according to any of the items 40-46. 48. A culture medium according to subsection 47 , which further comprises an exogenous cytokine 49. A culture medium according to any of the items 47-48, further comprising a multiplicity of exogenous cytokines, wherein the multiplicity comprises at least one interleukin. io of culture according to any of items 47-49, wherein, interleukin is selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15 . 51. A culture medium according to any one of items 47-50, further comprising a molecule that binds to CD21. As used herein, a molecule that binds to CD21 can be a known natural or synthetic molecule or that is determined to bind to the cell surface determinant CD21. Molecules known to bind to CD21 include antibodies against CD21, as well as molecules such as C3d, C3dg, iC3b and EBV gp350 / 220, and derivatives thereof which bind to CD21. 52. A culture medium according to item 51, wherein the molecule that binds CD21 is an antibody against CD21. 53. A culture medium according to any of items 49-52 further comprising a monoclonal antibody against CD3.

DESCRIPTION r > tgTAT.T.AT3A. OF THE PREFERRED MODALITIES AND APPLICATIONS OF THE INVENTION The invention described herein provides methods for rapidly expanding lymphocyte populations T, which include human cytotoxic T lymphocytes and helper T lymphocytes (helper), which may be particularly useful in the cellular immunotherapy of human diseases, without using a vast excess of PBMC and / or EBV-LCL feeder cells which are the key feature of the REM protocol hp. The T cells will be referred to as "T objective".

In general, the target T cells are added in small amounts to a culture vessel containing standard growth medium that has been supplemented with components that stimulate rapid expansion in vitro (REM) as described herein. Preferably, it is added Human recombinant IL-2 or other suitable preparation of IL-2 at low concentrations at 3-5 day intervals (typically on "day 0" (ie, at the start of the crop) or on "day 1" (the day after the start), and again on day 5 or 6, and again on day 8 or 9 ). REM protocols result in a rapid expansion of T cells, typically in the range of an expansion of 500 to 3000 times in the next 8 to 14 days. Therefore, such methods can provide expansion rates that are approximately 100 to 1000 times more efficient for each stimulation cycle compared to the previously described methods of culturing human T cells. In addition, REM protocols are applicable to the rapid expansion of any subpopulation of T cells including helper T cells and cytolytic T cells; and T-cell clones of many different antigenic specificities (e.g., to cytolytic T cells or CMV specific helper, HIV or other viral, bacterial or tumor derived antigens). In addition, REM protocols can be used for both small growth escapes (eg, to rapidly expand T cells from 104 to 107 cells); or for large-scale expansions (e.g., to rapidly expand T cells from 106 to more than 1010 cells); based on the size of the chosen culture vessel. Therefore, REM protocols make it possible to efficiently expand T-cell clones for use in adoptive immunotherapies by dramatically shortening the time necessary to make the numbers of cells necessary to restore, improve or modulate human immunity. In the study by Riddell et al., (Science r 257: 238-240, 1992), once the T cell clones were isolated, it was necessary to culture the clones for twelve weeks and accumulate multiple clones to obtain the cell dose Higher administered from 1 x 109 T cells specific for CMV CD8 + / m2 of body surface area. Using REM protocols, the expansion of individual T cell clones to more than 109 cells in less than three weeks can be carried out.

With respect to rapid expansion methods (ie, "REM" technology), the following abbreviations are used to differentiate the various REM protocols referred to herein. The basic Riddell protocol (as described above in the aforementioned Riddell patent application), which utilizes a disproportionately large amount of PBMC feeder cells (and preferably also EBV-LCL feeder cells) is referred to as "high REM PBMC" or simply "REM hp". In contrast, the methods of the present invention which do not utilize such large excesses of PBMC feeder cells (and preferably do not use EBV-LCL feeder cells) are referred to as "REM PBMC low" or "modified REM". Such methods are discussed in detail in the following. Unless indicated otherwise, the practice of the present invention will utilize conventional techniques of molecular biology, microbiology, cell biology, recombinant DNA and immunology which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: A LaboRatoy Manual. second edition (1989); Animal Cell Culture (R. Freshney, Ed., 1987); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M.P. Calos, efs, 1987); Handbook of Experimental Immiinoloav, (D.M. Weir and C.C.

Blackwell, Eds.); Current Protocols in Molecular Biology (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Siedman, J. A. Smith and K. Struhl, eds., 1987); Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Oligonucleotide Synthesis (M. J. Gait, Ed., 1984), * and in the series Methods in Enzymology (Academic Press, Inc.). All patents, patent applications and publications mentioned herein, both in the foregoing and in the following, are incorporated herein by reference. As an aid in the understanding of this invention, the following is a list of some abbreviations commonly used therein: CTL cytotoxic T lymphocytes APC antigen presenting cells CMV cytomegalovirus HIV human immunodeficiency virus EBV Epstein Barr virus hIL-2 interleukin 2 human MHC comp principal principle of histocompatibility PBMC peripheral blood mononuclear cells EBV-LCL line of lymphoblastoid cells transformed with EBV (sometimes simply abbreviated as "LCL") PBS solution buffered with phosphate "REM method of rapid expansion REM hp REM PBMC high REM lp REM PBMC low or "modified" As used herein, a "cytosine" refers to any of a variety of intercellular signaling molecules (of which the best known are involved in the regulation of mammalian somatic cells). Several families of cytokines have been characterized, both growth promoters and growth inhibitors in their effects and include, for example: interleukin (such as IL-la, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 (P40), IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15); CSF-like cytokines such as GM-CSF, G-CSF, M-CFS, LIF, EPO, TPO, ("thrombopoietin"), TNF-a and TNF-β), * interferons (such as IFN-a, IFN-β , IFN-?); cytokines of the TGF-β family (such as TGF-β1, TGF-β2, TGF-β3, inhibin A, inhibin B, activin A, activin B); Growth factors, such as EGF, VEGF, SCF, ("pluripotent cell factor" or "steel factor"), TGF-a, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS , IGF-I, IGF-II, NGF-ß), * intercrine cytokines type a (such as IL-8, GRO / MGSA, PF-4, PBP / CATP / ßTG, IP-10, MIP-2, KC , 9E3); and β-type intercrine cytokines (such as MCAF, ACT-2 / PAT 744 / G26, LD-78 / PAT, 464, RANTES, G26, 1309, JE, TCA3, MIP-la, β, CRG-2); and chemotactic factors (such as NAP-1, MCP-l, MlP-la, MlP-lβ, MIP-2, SISβ, SISd, SIS6, PF-4, PBP,? IP-10, MGSA). Many other cytokines are also known to those familiar in the art. The sources, characteristics, targets and effector activities of these cytokines have been described and, for many of the cytokines, the DNA sequences encoding the molecules are also known; see, for example, R. Callard & A. Gearing, The Cytokine Facts Book (Academic Press, 1994), and the particular publications reviewed and / or mentioned therein, which are incorporated herein by reference in their entirety. As referenced in catalogs such as The Cytokine Facts Book. many of the DNA sequences and / or proteins encoding such cytokines are also generally available from sequence databases such as GENBANK (DNA); and / or SWISSPROT (protein). Typically, the cloned DNA encoding such cytokines is already available as plasmids, although it is also possible to synthesize polynucleotides that code for cytokines based on published sequence information. Polynucleotides that code for cytokines can also be obtained using the polymerase chain reaction (PCR) methodology as described in the art. See, for example Mullís & Faloona, Met. Enzymology 155: 355 (1987). The detection, purification and characterization of cytokines, including assays to identify effective new cytokines before a given cell type, has also been described in numerous publications, as well as the references referred to therein. See, for example, Lymphokines and Interferons, 1987; and DeMaeyer, E., et al., "Interferons and Other Regulatory Cytokines," (John Wiley &Sons 1988). As used herein, a mammalian "cell line" refers to a population of mammalian cells (preferably human cells) that have undergone repeated in vitro propagation, unlike "primary cells" taken from such an individual as a human. Generally, a mammalian cell line will have been propagated in vitro for at least about 10 generations, more typically at least about 40 generations, more typically at least about 100 generations. More preferably, the mammalian cell line can be propagated and maintained for a long term (i.e., at least several months in vitro, preferably at least one year). Such cell lines include, but are not limited to "clonal" lines (in which all of the cells in the population are derived from a single ancestral cell). Inversely, a population of mixed peripheral blood such as PBMC does not constitute a mammalian cell line. A mammalian cell line for use in the present invention, however, may contain a type of cell found in peripheral blood but in that case, the cell type will generally be present at a much higher frequency than that normally found in cells. mononuclear cells of human peripheral blood (at least twice the frequency usually found in human peripheral blood mononuclear cells); preferably at least five times, at least ten times, at least twenty times or at least fifty times the frequency generally found in human peripheral blood mononuclear cells). A particular "cell type" can be, for example, one of the cell types typically found in peripheral blood (such as B lymphocytes, monocytes, cytotoxic T lymphocytes, helper T lymphocytes, granulocytes, eosinophils or NK cells), * or a type of cell that is not normally found in peripheral blood (such as fibroblasts, endothelial cells, etc.), * or a more specific subpopulation of such cell types (eg, a subpopulation that is relatively homogeneous with respect to specificity to antigen or expression of a particular receptor). Therefore, a cell line can be relatively homogeneous with respect to attributes such as antigen specificity of the cell surface receptors / ligand, as discussed in more detail below. By way of illustration, a receptor-specific monocyte line refers to an in vitro population of cells, in which most of the cells are monocytes that possess a particular cell surface receptor (sural cell line may have been obtained, for example, by transforming a population of monocytes with genes that express the particular receptor). Again, by way of illustration, a line of antigen-specific CTL cells refers to a population of cells in vitro in which most of the cells are cytotoxic T lymphocytes specific for a particular antigen such as a viral, bacterial or antigen. tumoral (cell line which may have been obtained, for example, by exposing a population of T cells to repeated stimulation with a particular antigen and subsequently enriching for antigen-specific CTL). Preferably, such a cell line for use with the present invention will become non-divisible before use in the modified culture (eg, by irradiation). However, alternatively, a cell line that is divisible (preferably at a rate similar to or smaller than the expanding T cells) can be alternatively used, but which can be subsequently deleted by virtue of having a negative selectable marker (for example, example, a suicide gene that can be used to inhibit or destroy cells that present the gene, or a cell surface marker that can be used to isolate and / or eliminate cells that present the marker). In the latter case, the cell line may be allowed to expand to some extent in the REM culture before being selected negatively. Preferably, the mammalian cell lines to be used with the present invention are relatively homogeneous lines (ie, at least 50% of the cells are of a particular cell type, more preferably at least 70%, so less 90%, at least 95% or at least 99% of the cells are of a particular cell type). However, it should be noted that the T cells that are to be expanded by exposure to such a cell line must also be exposed to additional cell lines (at the same time or in sequence). Thus, by way of illustration, a modified REM culture (containing a population of T lymphocytes to be expanded) can be exposed to a mammalian cell line or to several such lines. For modified REM, the T cells to be expanded will be exposed to at least one of such mammalian cell lines and / or to a mixture of non-cellular factors (including, for example, cytokines, antibodies, soluble ligands, etc. ), as discussed in the present. The T cells that are to be propagated in culture (ie, the "target" T cells) can be obtained from the subject to be treated. Alternatively, the T cells can be obtained from a source other than the subject to be treated, in which case the receptor and the transferred cells are preferably immunologically compatible (or the receptor in some other way becomes immunotolerant to the transferred cells). Typically, target T cells are derived from tissue, bone marrow, fetal tissue or peripheral blood. Preferably, the cells are derived from peripheral blood. If T cells are derived from tissues, suspensions of cells alone can be prepared using a suitable medium or diluent. Mononuclear cells containing the T lymphocytes of a heterogeneous population can be isolated, according to any of the methods well known in the art. As illustrative examples, Ficoll-Hypaque gradient centrifugation, fluorescence activated cell sorting (FAC), washing (panning technique) on plates coated with monoclonal antibody, and / or magnetic separation techniques (separately) can be used. or in combination) to obtain purified populations of cells for expansion according to the present invention. Antigen-specific T cells can be isolated by standard culture techniques known in the art involving initial activation of antigen-specific T cell precursors by stimulation with antigen-presenting cells and, for a clonal population, by limiting dilution cultures using techniques known in the art, such as those described in Riddell and Greenber (J. Immunol.Meth., 128: 189-201, 1990); and Riddell et al., (J. Immunol., 146: 2795-2804, 1991). See also, the examples later. T-cell clones isolated in microwells in limiting dilution cultures have typically expanded from a single cell to 2 × 10 4 to 5 × 10 5 cells after 14 days. For expansion, T cells can be placed in an appropriate culture medium in plastic culture vessels with T cell stimulating components as described herein. The initial rapid expansion phase is generally carried out in a culture vessel, the size of which depends on the number of target cells, and which typically can be a 25 cm2 flask. The size of the culture vessel used for subsequent cycles of T cell expansion depends on the initial number of T cells and the number of cells needed. The typical initial cell numbers for different sized culture vessels are as follows: 5 x 104 to 2 x 105 - flask approximately 25 cm2; 2 x 105 to 5 x 105 - flask of approximately 75 cm2; 5 x 105 to 1 x 106 - flask of approximately 225 cm2; and 1 x 106 to 2 x 106 - rotating bottle. The approximate initial volume of medium used with each flask is: 25 cm2 - 20-30 ml; 75 cm2 - 60-90 ml; 225 cm2 - 100-200 ml; rotating bottle -500 ml. For expansions even on a larger scale, a variety of culture media can be used including, for example, spinner flasks, cell culture bags and bioreactors (such as hollow fiber bioreactors). As used herein, "feeder cells" are accessory cells that provide costimulatory functions in conjunction with the activation of T cell receptor (which can be obtained by ligation of the T cell receptor complex with monoclonal antibody against CD3). PBMC feeder cells for use in REM can be obtained by techniques known in the art, for example by leucaphoresis, which is a standard medical procedure with minimal risks (see, for example, Weaver et al., Blood 82: 1981-1984 , 1993); and these feeder cells can be stored by cryopreservation in liquid nitrogen until their use. LCL can be generated from B cells of peripheral blood by transformation with EBV, for example, EBV strain B95-8, using standard methods (see, for example, Crossland et al., J. Immunol, 146: 4414- 20, 1991), or by spontaneous growth in the presence of cyclosporin A. Such LCL cells will grow rapidly and indefinitely in culture. Before adding any feeder cells to the culture vessel (either PBMC or cells derived from a cell line as described herein), such feeder cells are preferably prevented from experiencing mitosis. Techniques to avoid mitosis are well known in the art and include, for example, irradiation. For example, any PBMC can be irradiated with gamma rays in the range of about 3000 to 4000 rads (preferably, the PBMCs are irradiated at about 3600 rads); any LC can be irradiated with gamma rays in the range of about 6000-12000 rads (preferably, LCLs are irradiated at approximately 10,000 rads); and any cell derived from other cell lines can also be irradiated with gamma rays in the range of about 6000-12000 rads. As discussed above, negative selectable feeder cells can also be used. Since the antigen specificity of the T cell clone is generally defined before expanding into the cell in the culture system, autologous or allogeneic feeder cells can be used to support the growth of T cells. The availability to use allogeneic feeder cells It is important in situations in which the patient is infected with a virus that is present in PBMC, for example, HIV, which could, therefore, contaminate the T cell cultures. Under such circumstances, they can be used in the method of culture the use of allogeneic feeder cells derived from an individual who is examined and who is considered a suitable blood donor by the criteria of the American Red Cross. The T cell receptor activation signal (usually provided by antigen and antigen-presenting cells) can be obtained by the addition of monoclonal antibodies against CD3 to the culture system. The most commonly used monoclonal antibody against CD3 is "0KT3", which is commercially available from Ortho Pharmaceuticals in a formulation suitable for clinical use. The use of MAB against CD3 ("aCD3") instead of an antigen as a means to bind the T cell receptor eliminates the need to have a source of antigen presenting cells, which, for T cells specific for viruses, would require maintain large numbers of adequate autologous cells and infect these cells in vitro with high titers of virus. A concentration of monoclonal antibody against CD3 of at least about 0.5 ng / ml, preferably at least about 1 ng / ml, more preferably at least about 2 ng / ml, promotes rapid expansion of T cells so that You can obtain an extension of 500 to 3000 times within approximately 10 to 13 days of growth. Typically, a concentration of about 10 ng / ml of monoclonal antibody against CD3 is used. Of course, as an alternative to the monoclonal antibody against CD3, the T cell receptors can be activated and the cells stimulated by the addition of antigen-presenting cells, as described in Riddell et al., J. Immunol. 1476: 2795-2904, 1991. Suitable antigen presenting cells include, for example, virus infected cells, tumor cells and cells pulsed with the relevant peptide antigen. The culture medium for use in the methods of the invention can be any of the commercially available media, preferably one containing: RPMI, 25 mM HEPES, 25 μM 2-mercaptoethanol, 4 mM L-glutamine and 11% human AB serum . Fetal bovine serum can be substituted by human AB serum. Preferably, after the addition of any feeder cells, the monoclonal antibody against CD3 and culture medium is added to the target cells, and the mixture is allowed to incubate at 37 ° C in a humidified atmosphere, with 5% C02. under standard cell culture conditions which are well known in the art Typically, such conditions may include ventilation, and addition of C02 if necessary (eg, 5% C02 in a humidified incubator). is supplemented with interleukin-2 (IL-2) Typically, recombinant human IL-2 is used, although a functional equivalent thereof can also be used Preferably, IL-2 is added on day 1, and it becomes To add at intervals of 3-5 days, therefore, IL-2 is usually added on day 1, on day 5 or 6, and again on day 8 or 9. Expansion can be improved by using a At least IL-2 concentration s about 5 U / ml, more preferably at least about 10 U / ml. Generally, a concentration of approximately 25 U / ml can be used. As described in Riddell et al., Supra, antigen-specific T cells are expanded using REM and retain their antigen-specific functionality. For example, 4 different clones of CD8 cytotoxic T cells more specific for HIV require their ability to kill infected cells with viruses that express the relevant antigen (ie, HIV), and do not acquire nonspecific cytolytic activities against target cells infected with virus or transformed, not relevant. Similarly, four different clones of CD8 + cytotoxic T cells specific for CMV retain their ability to kill cells infected with CMV and do not acquire non-specific cytolytic activities against target cells infected with viruses and relevant or transformed. These characteristics are also applicable to CD4 + helper T cells. Therefore, CD4 + cells propagated using REM retain the ability to proliferate in response to appropriate viral antigens and appropriate antigen presenting cells (APCs). In addition, antigen-specific T cells cultured under REM are also able to enter an adjacent, non-dividing phase of the cell cycle; and are able to remain viable for at least four weeks in vitro. Therefore, aliquots of T cells can be removed from the cultures at the end of a stimulation cycle (usually on day 12-14) and can be placed in a culture vessel with an approximately equal amount of irradiated PBMC (without mAb). against CD3, antigen or IL-2). The addition of irradiated PBMC as feeder cells during storage of expanded populations improves the ability of T cells to enter a resting phase and remain viable. Preferably, the ratio of PBMC feeder cells to resting T cells during storage is at least about 2: 1. Without the addition of PBMC feeder cells, the viability of the T cells generally decreases significantly (typically at levels of about 10% or less). As described in Riddell et al., Supra, the T cells expanded by REM assume a small round morphology and 60-95% remain viable by trypan blue dye exclusion test even after 28 days in culture. The T cells propagated by REM hp also enter a resting phase when IL-2 is no longer administered; and do not experience programmed cell death (i.e., apoptosis) upon restimulation via a receptor for antigen-specific T cells. Upon restimulation (for example with mAb against CD3 or antigen), the T cells again acquire a response layer to IL-2, and can enter the S and G2 phases of the cell cycle and increase the number of cells. It is considered that such characteristics are important for the in vivo survival of the cells and for the effectiveness of cellular immunotherapy. In contrast, certain methods described previously for the propagation of T cells have been reported to cause apoptotic cell death in a proportion of cells after cytokine suppression or restimulation of T cell receptor (see, for example, Boehme SA and Lenardo MJ, Eur. J. Immunol. 23: 1552-1560, 1992). There are numerous different causes in which the introduction of functional genes into T cells that are to be used in immunotherapy may be desirable. For example, the introduced gene or genes can improve the efficacy of the therapy by promoting the viability and / or function of transferred T cells; or can provide a genetic marker to allow selection and / or evaluation of in vivo survival or migration; or they may incorporate functions that enhance the safety of immunotherapy, for example, by returning to cells susceptible to negative selection in vivo, as described by Lupton S.D. et al. , Mol. and Cell Biol .. 11: 6 (1991); and Riddell et al., Human Gene Theraphy 3: 319-338 (1992); see also publications WO / 9208796 and WO / 9428143 by Lupton et al. , which describe the use of selectable bifunctional fusion genes derived from a dominant positive selectable marker with a negative selectable marker.

Various infection techniques have been developed which use recombinant infectious virus particles for gene delivery. This represents a currently preferred approach for the transduction of T lymphocytes of the present invention. Viral vectors which have been used in this manner include viral vectors derived from simian virus 40 (SV40) (see, for example, Karlsson et al., Proc. Nati. Acad. Sci.

USA 84 82: 158, 1985); adenovirus (see, for example, Karlsson et al., EMBO J. 5: 2377, 1986); adeno-associated virus (AAV) (see, for example, B.J. Carter, Current Opinion in Biotechnology 1992, 3_: 533-539); and retroviruses (see, for example, Coffin, 1985, pp. 17-71 in Weiss et al. (eds.), RNA Tumor Viruses, 2nd ed., Vol. 2, Cold Spring Harbor Labor, New York). Therefore, gene transfer and expression methods are numerous, but essentially function to introduce and express genetic material in mammalian cells. Many previous techniques have been used to transduce hematopoietic or lymphoid cells, which include transfection with calcium phosphate (see, for example Berman et al., supra, 1984); protoplast fusion (see, for example, Deans et al., supra, 1984); electroporation (see, for example Cann et al., Oncogene 3: 123, 1988); and infection are resovinant adenovirus (see, for example, Karlsson et al., supra; Reuther et al., Mol. Cell. Biol. 6: 123, 1986); adeno-associated virus (see, for example, LaFace et al., supra); and retroviral vectors (see, for example, Overell et al., Oncogene 4: 1425, 1989). Primary T lymphocytes have been successfully transduced by electroporation (see, * for example, Cann et al., Supra, 1988), and by retroviral infection (see, for example, Nishihara et al., Cancer Res. 48: 4730, 1988 Kasid et al., Supra, 199.0; and Riddell, S. et al., Human Gene Therapy 3: 319-338, 1992). Retroviral vectors provide a highly efficient method for gene transfer in eukaryotic cells. In addition, retroviral integration takes place in a controlled manner and results in a stable integration of one or some copies of the new genetic information per cell. Retroviruses are a class of viruses which replicate using RNA-encoded DNA polymerase, or reverse transcriptase, to replicate a viral RNA genome and provide a double-stranded DNA intermediate which is incorporated into the Chromosomal DNA from a bird or mammalian host cell. Most of the retroviral vectors are derived from mouse retroviruses. Adaptive retroviruses for use according to the present invention, however, can be derived from any cellular source of birds or mammals. Preferably, these retroviruses are amphotropic, which means they are capable of infecting host cells of several species, including humans. A characteristic feature of the retroviral genomes (and retroviral vectors used as described herein) is the long-terminal retroviral repeat sequence, or LTR, which is an untranslated region of approximately 600 base pairs found in slightly variant forms. at the 5 'and 3' ends of the retroviral genome. When incorporated into the DNA as a provirus, the retroviral LTR includes a short repeat sequence at each end and signals for initiation of transcription by RNA polymerase II and 3 'cleavage and polyadenylation of RNA transcripts. The LTR contains all other cis-action sequences necessary for viral replication. A "provirus" refers to a reverse transcript of a retrovirus DNA which is stably integrated into chromosomal DNA in a suitable host cell, or a cloned copy thereof, or a cloned copy of non-integrated intermediate forms of retroviral DNA. Direct transcription of the provirus and assembling in infectious virus occurs in the presence of an appropriate helper virus or in a cell line containing appropriate sequences that allow the formation of capsid without coincident production of a contaminating helper virus. Mann et al. (Cell 33: 153, 1983) describes the development of cell lines (for example,? 2) which can be used to produce free accumulators of auxiliaries, of recombinant retroviruses. These cell lines contain integrated retroviral genomes which lack in cis sequences for encapsidation, but which provide the entire gene product necessary in trans to produce intact virions. Transcribed mutated provirus RNA itself can not be packaged, but these cells can form RNA capsids transcribed from a recombinant retrovirus introduced into the same cell. The resulting virus particles are infectious, although replication-defective, which renders them useful vectors which are incapable of producing infectious virus after introduction into a cell that lacks complementary genetic information that permits encapsidation. Encapsidation in a cell line harboring the trans-acting elements encoding an ecotropic viral envelope (e.g. "2") provides an ecotropic progeny virus (limited host range). Alternatively, assembly in a cell line containing amphotropic packaging genes (eg PA317, ATCC CRL 9078; Miller and Buttimore, Mol. Cell. Biol. 6: 2895, 1986) provides amphotropic progeny virus (broad host range). Such packaged cell lines provide the necessary retroviral ga, pol and env proteins in trans. This strategy results in the production of retroviral particles which are highly infectious for mammalian cells, but at the same time they are incapable of further replication after they have been integrated into the genome of the target cell. The product of the env gene is responsible for the binding of the retrovirus to viral receptors on the surface of the target cell and therefore determines the host range of the retrovirus. PA317 cells produce retroviral particles with an amphotropic envelope protein which can transduce cells of human and other species origin. Other packing cell lines produce particles with ecotropic envelope proteins, which are capable of transducing only mouse and rat cells. Numerous retroviral vector constructs have been used successfully to express many foreign genes (see, for example, Coffin, in Weiss et al. (Eds.), RNA Tumor Viruses, 2nd, vol.2 (Cold Spring Harbor Laboratory, New York , 1985, pp. 17-71) Retroviral vectors with inserted sequences are generally functional, and some sequences that are consistently inhibitory for retroviral infection have been identified.Functional polyadenylation motifs (repeated sequences) inhibit retroviral replication by blocking the retroviral synthesis of retroviral RNA, and there is a upper size limit of approximately 11 kb of sequence which can be packed into retroviral particles (Coffin, supra, 1985), however, the presence of multiple internal promoters, which initially was considered problematic ( Coffin, supra et al, 1985) was found to be well tolerated in several retroviral constructs (Overell et al., Mol.Cell. Biol. 8: 1803, 1983). Retroviral vectors have been used as genetic labels by several groups to follow the development of mouse hematopoietic pluripotent cells which have been transduced in vitro with retrovirus vectors and transplanted into recipient mice (Williams et al., Nature 310: 476, 1984; Dick et al., Cell 42: 71, 1985; Keller et al., Nature 318: 149, 1985). These studies have shown that infected hematopoietic cells reconstitute the hematopoietic and lymphoid tissue of the recipient animals and that the cells show a potential for normal development in vivo. The labeled cells can be visualized using any of several molecular biological techniques which can demonstrate the presence of retroviral vestibular sequelae, most notably Southern analysis and PCR (polymerase chain reaction). The ability to genetically label cells using retroviral vectors is also useful in clinical systems in which the technique for following autologous cell grafts can be used. This approach has already been used to monitor TIL (tumor infiltrating lymphocytes) in patients given TIL therapy for terminal cancer treatment by Rosenberg et al. (N. Engl. J. Med. 323: 570, 1990). Transduction of these cells with the marker gene is not associated with cell dysfunction in vitro (Kasid et al., Proc. Nati, Acad. Sci. USA 87: 473, 1990). Many gene products have been expressed in retroviral vectors. This can be carried out by placing the sequences to be expressed under the transcriptional control of the promoter incorporated in the retroviral LTR, or by placing them under the control of a heterologous promoter inserted between the LTRs. This latter strategy provides a way of co-expressing a dominant selectable marker gene in the vector, and thus allowing the selection of cells which express specific sequences of the vector. It has been contemplated that overexpression of a stimulatory factor (e.g., a lymphokine or a cytokine) may be toxic to the treated individual. Therefore, it is within the scope of the invention to include gene segments that cause the T cells of the invention to be susceptible to negative selection in vivo.

By "negative selection" is meant that the cells subjected to infusion can be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype can result from the insertion of a gene that confers sensitivity to a administered agent, e.g., a compound. The selectable negative genes known in the art include, for example, the following: the gene for thymidine kinase of Herpes virus if plex type I (HSV-1 TK) (Wigler e_t al-, Cell 11: 223, 1997) which confers sensitivity to ganciclovir; the gene for hypoxanthine cellular phospho-ribosyltransferase (HPRT) the gene for adenine phosphoribosyltransferase (APRT), and the bacterial cytokine deaminase (Mullen e_t al-, Proc. Nati, Acad. Sci. USA, 89:33 (1992)). In addition, it is useful to include in the T cells a positive marker that allows the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker can be a gene which, when introduced into the host cell, expressed a dominant phenotype allowing a positive selection of the cells presenting the gene. Genes of this type are known in the art and include, for example, the gene for hygromycin-B phosphotransferase (hph) which confers resistance to hygromycin B, the gene for aminoglycoside phosphotransferase (neo or aph) of Tn5, which codes for resistance to the antibiotic G418, the gene for dihydrofolate reductase (DHFR), the adenosine deaminase (ADA) gene and the gene for multiple drug resistance (MDR). Preferably, the positive selectable marker and the negative selectable element are linked such that the loss of the negative selectable element is necessarily also accompanied by the loss of the positive selectable marker. Even more preferably, the positive and negative selectable markers are fused so that the loss of one inevitably leads to the loss of the other. An example of a fused polynucleotide that provides as an expression product a polypeptide that confers both desired selection characteristics, positive and negative, described above, is the fusion gene of hygromycin phosphotransferase thymidine kinase (HyTK). Expression of this gene provides a polypeptide that confers hygromycin B resistance for positive selection in vitro, and sensitivity to ganciclovir for negative selection in vivo. See Lupton, S .., stal., Mol. and Cell. Biology ll: 3374-3378, 1991). In addition, in the preferred embodiments, the polynucleotides of the invention encoding chimeric or recombinant receptors are in retroviral vectors containing the fused gene, particularly those that confer resistance to hygromycin B for positive selection in vitro, and sensitivity to ganciclovir for negative selection. in vivo, for example, the retroviral vector HyTK described in Lupton, SD et al. (1991) supra. See also publications PCT / US91 / 08442 and PCT / US94 / 05601 by S.D. Lupton describing the use of selectable fusion genes derived from the fusion of dominant positive selectable markers with selectable negative markers. Preferred selectable markers are derived from genes selected from the group consisting of hph, neo and gpt, and preferred negative selectable markers derived from genes that are selected from the group consisting of cytokine deaminase, HSV-1 TK, VZV TK, HPRT , APRT and gpt. Especially preferred labels are bifunctional selectable fusion genes wherein the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from cytokine deaminase or a TK gene. Various methods can be used to transduce T lymphocytes, as is well known in the art. Typically, retroviral transductions can be carried out as follows: on day 1 after stimulation using REM as described herein, it can be provided to the cells with 20-30 units / ml of IL-2; on day 1, 2 or 3, half of the medium can be replaced with retroviral supernatant prepared according to standard methods and the cultures are supplemented with 5 μg / ml of polybrene and 20-30 units / ml of IL-2; on day 4, the cells are washed and placed in fresh culture medium supplemented with 20-30 units / ml of IL-2; on day 5, exposure to retroviruses can be repeated; on day 6, the cells can be placed in selective medium (containing, for example, an antibiotic corresponding to an antibiotic resistance gene provided in the retroviral vector) supplemented with 30 units / ml of IL-2; on day 13, viable cells can be separated from dead cells using Ficoll-Hypaque density gradient separation, and then viable cells can be subcloned using REM. Using antigen-specific CTL (55E1, a CD8 + clonal line specific for EBV) and a retroviral vector (LAPSN, Clowes et al., 1994, J. Clin.

Invest. 93: 644 (which allows the monitoring of alkaline phosphatase expression by flow cytometry)), high transduction frequencies can be obtained when cells are exposed to the vector on day 1, 2 or 3 after the onset of REM. As described above, the T cells prepared according to the invention can be used to restore, improve and / or modulate immunity in recipient individuals. By "immunity" is meant a loss of one or more physical symptoms associated with a response to infection by a pathogen, or a tumor, to which the response of the lymphocytes is directed. The amount of cells usually administered is in the range present in normal individuals with immunity to the pathogen. Therefore, CD8 + CD4- cells are usually administered by infusion, with each infusion in a range of at least 10 6 to 10 10 cells / m2, preferably in the range of at least 10 7 to 10 9 cells / m2. The clones can be administered by simple infusion or by multiple infusions over a time interval. However, since different individuals are expected to vary in their responsiveness, the type and amount of cells delivered by infusion, as well as the number of infusions and the time interval over which the multiple infusions are administered, is determined by the doctor who attends, and can be determined by usual examination. The generation of sufficient levels of T lymphocytes (including cytotoxic T lymphocytes and / or helper T lymphocytes) can be easily achieved using the rapid expansion method of the present invention, as exemplified herein.

It has also been observed that expanded T cells using REM show very high levels of transduction using vectors such as retroviral vectors which will be of great use in the context of cellular immunotherapy and gene therapy using lymphocytes. The examples in Riddell et al., Supra, exemplify the basic REM protocol (ie, REM hp) and also help illustrate the general applications of REM technology for the preparation and use of expanded T cell populations and, in this respect, , the exemplification techniques and the principles that can also be applied in the context of modified REM. The examples below illustrate "exemplary modifications of the REM technology according to the present invention (ie, modified REM) to allow a reduction or elimination of the PBMC and / or EBV-LCL feeder cells that are characteristic of the REM hp protocol. All the examples provided below are provided as an additional guide for whoever habitually brings the technique into practice, and are not considered as limiting the invention in any way.

EXAMPLES Use 1 Contribution of the Recipients Fc? of Monocyte in the Rapid Expansion The PBMC feeder cells, which are used in large excess to activate REM hp, are a heterogeneous population of cells that include B lymphocytes, T lymphocytes, monosites, macrophages and granulocytes and natural killer cells ("NK"). One of the activities considered to be supplied by PBMC in the REM hp protocol is to provide Fc? which can bind to the Fe portion of the IgG antibody molecules. In particular, it is considered that the cell activation. T in the protocol REM hp can be mediated by the binding of Fc receptors.

("Fc? R") on monocytes within the PBMC population to the Fe portion of antibody against CD3 (for example OKT3), which can thus be "presented" by monocytes to T cells within the population that it's going to expand. After such activation, it is considered that T cells are capable of initiating an "autocrine" growth stimulator cycle in which activated T cells secrete growth-promoting cytokines and also increase the expression of cell surface receptors for such cytokines. By supplying Fc receivers, or by effectively presenting in some other way antibody against CD3, it is considered that this works in the initiation and promotion of T cell expansion. The confirmation and quantification of the contribution of the Fc receptors. of monocytes in the REM protocol hp can be carried out by removing monocytes from the PBMC feeder cells. Peripheral blood mononuclear cells can be obtained from any of a variety of sources, as described above. For the following effects, layers of the yellow coat (derived from healthy human donors) were obtained from a Red Cross blood bank. The PBMC were isolated using a Ficoll gradient, washed and stored in cell culture medium (at 4o Celsius) using standard techniques, as mentioned above. A variety of techniques can be used to separate various cell types from a mixed population such as PBMCs. By way of illustration, the deletion of the monocyte / macrophage population was performed using chromatography on Sephadex G-10 (see, for example, section 3.6 in "Current Protocols in Immunology" (Wiley Interscience, 1992)). Monocyte suppression was monitored by flow cytometry following staining with monoclonal antibody against CD14 conjugated to FITC (available from, for example, PharmaGen). The expression of CD14 before suppression is approximately 7.4% of the total cells. After the deletion, it is approximately 1.5%. In order to determine the impact of suppressing monocytes on the ability of PBMCs to promote rapid expansion, a standard REM hp protocol was used and PBMC without monocytes were compared with PBMC without suppression. For purposes of illustration, a CTL line (designated "27EB") prepared by procedures analogous to those described above was prepared. Briefly, PBMC were obtained from an individual blood sample and cultured in EBV-LCL derived from the same individual. After two weeks, the CD8 + CTLs were isolated by "washing" (panning) with a flask coated with antibodies against CD8 (for example, the CELLector flask "AIS-CD8 + from Applied Immune Sciences.) For all of these illustrative examples, the PBMC were subjected to gamma irradiation at 3600 rads (using a Cs -137 source) and the EBV-LCL was They irradiated to 10,000 rads.

The cultures were generally maintained as described above for REM hp except that 10% fetal bovine serum was used instead of human serum; and IL-2 was used at 25 units / ml, and was usually added first on "day 0" (as opposed to day 1 after the start of culture), and then at intervals of 3-5 days (generally, at day 5, and then again on day 8), as described otherwise in the foregoing for the REM protocol hp. Generally, 0KT3 was used at approximately 10 ng / ml. The cells were typically collected and quantified after 14 days of culture. For this example, cultures were established with 5 x 104 CTL ("27EB", as described above), 5 x 106 irradiated EBV-LCL (ie, an excess of 100: 1 with respect to CTL) (prepared and irradiated as described above), 10 ng / ml of OKT3, 25 U / ml of IL-2 and 2.5 x 107 of irradiated PBMC (ie, an excess of 500: 1 with respect to CTL) (either deleted in terms of monocytes or non-suppressed, prepared and irradiated as described above). Typical control crops may include, for example, crops without any added CTL. After 14 days of culture, the cells are harvested and quantified. In the case of the standard protocol hp REM using the PBMC without deletion, approximately 4.86 x 107 T cells were recovered, representing an expansion of approximately 882 times. As shown in Table 1, when PBMC suppressed with monocytes was used, only recovered approximately 2.7 x 107 T cells, which indicates that the rate of expansion has decreased by approximately 55% with respect to the control rate.

TABLE 1 The above results provide further indication that monocytes within the PBMC population apparently contribute significantly to the ability of PBMCs to carry out the rapid expansion of T cells. In the following example the ability to provide Fc activity will be determined. ? R or its equivalent from a source other than PBMC as a means to reduce the dependence of REM to a large excess of PBMC feeder cells. 2 Replacement of receiver activity Fc? of monocytes in modified REM As discussed before, the Fc? found in monocytes are believed to be responsible for a significant portion of the stimulatory activity provided by the PBMC in the presence of antibodies such as antibody against CD3 (for example 0KT3). Having identified a stimulator component supplied by the heterogeneous PBMC population, it is possible to reduce the dependence on the PBMC itself by providing this activity (or its equivalent) from another source. Preferably, for use in modified REM as described herein, the identified stimulatory activity will be provided by a mammalian cell line or a non-cellular additive to the REM culture. Illustrative examples of both options are provided below. to. Use of mammalian cell lines in modified REM Cell lines expressing one or more T-cell stimulatory activities identified by PBMCs (or LCL) can be used effectively to reduce reliance on REM on such PBMC (or LCL) feeder cells. When such a cell line is to be incorporated into the protocol, it is preferable, as described above, that the cell line is not a potential source of adventitious agents such as viruses. Accordingly, the supplementary cell line used preferably is not a cell line transformed by EBV (such as EBV-LCL). Furthermore, for the rapid expansion of human T cells, it is generally preferable to use a cell line derived from a higher mammal, especially a primate, more preferably a human. In the literature, mammalian cell lines expressing Fc? Receptors have been described. that can be obtained from various sources. For example, it has been shown that numerous human tumor lines express receptors Fc-? (see, for example, R.J. Looney et al., J. Immunol. 136: 1641 (1986) (describing K562, an erythroleukemia cell line), * S.J. Collins et al. 1977. Nature 270: 347 (1997) (describing HL60, a promyelocytic cell line); C. Lozzio and B. Lozzio, Blood 45: 321 (1975) (describing U937, a cell line of histiocytic lymphoma); and GR. Crabtree et al., Cancer Res. 38: 4268 (1979). The cell lines that express Fc? They can also be easily prepared using standard molecular biology techniques. By way of illustration, lines of cells positive for Fc? R can be obtained by immortalizing cells that already express receptors in Fc? using any of a variety of well-known techniques to transform mammalian cells. Alternatively, an existing cell line such as a human cell line can be genetically modified to express Fc receptors. by introducing genes that code for Fc receptors? in the cells. Therefore, a cell line of choice, such as the line of human cells that already express a stimulatory component such as a cytokine or an accessory cell adhesion molecule (both of which are discussed below) can be further modified by gene introduction. that code for an Fc? R. By way of illustration, human monocytes apparently express two different types of Fc receptor. ("Fc? RI" and "Fc? RII") which differ in their affinity for the binding of IgG antibody (see, for example, Ravetch and Kinet Annu, Review of Immunol., 9: 457-492, 1991). In particular, Fc? RI is generally a high affinity receptor (Ka = 108-109) whereas Fc? RII is generally a lower affinity receptor (Ka = 107). The genes for both Fc? RI and Fc? RII have been identified and cloned. Previous studies have shown that fibroblasts expressing the FcγRII receptor after gene transfer can effectively re-establish antibody-dependent proliferation against CD3 from T cell cultures without monocytes (see, for example, Peltz et al., J. Immunol 141: 1891 (1988)). Therefore, a genetically modified cell line to express Fc? R could be capable of delivering a significant portion of the stimulatory activity provided by PBMCs in the REM hp protocol. The use of such a cell line, together with other components such as cytokines or accessory adhesion molecules as described below would therefore allow a decrease in the amount of PBMC necessary for rapid expansion. b. Use of non-cellular additives for modified REM In addition to providing stimulatory components for T cells by means of a cell line, as described above, it will be possible to provide various components (or their functional equivalents) as non-cellular additives to a modified REM culture medium. Therefore, as an alternative to the activity for FcγR that PBMC monocytes apparently provide, it will be possible to provide a substitute or structural equivalent for FcγR activity. For example, an alternative means to obtain "presentation" of antibodies such as antibodies against CD3 to T cells, is to conjugate such antibodies to spheres (such as Sephadex spheres or magnetic spheres). In order to determine the ability of such conjugated antibodies to spheres to substitute soluble antibodies (which are probably presented via Fc? R), we carried out experiments to determine whether the magnetic spheres conjugated with antibody against CD3 can effectively replace the soluble monoclonal antibodies against CD3 in the REM protocol. The magnetic spheres conjugated with antibody against CD3 ("BioMag anti-CD3") were obtained from Perceptive Diagnostics. The particles used were approximately 1 μm in size and had covalently bound monoclonal antibodies against CD3 loaded to approximately 20 μg antibody / l x 107 spheres. A range of spheres was chosen to approximate the number of antigen-presenting cells ("APCs") that are present within the PBMC population used in REM hp. To quantify the real impact on REM hp, T-cell expansion cultures containing 5 x 10 4 CTL, 5 x 10 6 irradiated EBV-LCL, 2.5 x 10 7 irradiated allogeneic PBMC and 25 U / ml of IL-2 were established, essentially as described in Example 1. they added either 10 ng / ml of soluble antibody against CD3 (OKT3) or various amounts of spheres conjugated with antibody against CD3 as the T cell activation reagent. Cell cultures were expanded and T cells were counted, essentially as described for example 1. The results are shown in table 2. Although the expansion of T cells using spheres conjugated with antibody against CD3 is somewhat lower than with soluble OKT3 (in the range of approximately 80%) the results suggest that the spheres coated with antibody are capable of inducing substantial levels of activation / proliferation of T cells within a modified REM protocol. A more detailed quantification of the relative role of presentation with antibody against CD3 can be easily obtained compared to other activities potentially provided by APCs within the PBMC population by determining the expansion rates that can be obtained with spheres with antibody. against CD3 using cultures without APC, either in the presence or absence of various cytokines or other soluble stimulatory factors (which are described in greater detail later).

TABLE 2 Comparisons of relative efficiency can easily be obtained by providing various substitution components of PBMC, as described herein, by standard titration analyzes in which the various components are again aggregated at varying concentrations to a REM culture limited by PBMC (ie, a culture in which PBMCs are included at a suboptimal level). By way of illustration, the experiments described below determined the impact of adding various combinations of exogenous cytokines to suboptimized REM hp cultures in which the PBMC population has been reduced to half or a quarter of an optimal start level. Analogous assays can easily be performed for other components such as Fc [reg] R expressing cells with accessory adhesion components, spheres conjugated with antibody against CD3 and / or other soluble stimulatory factors (such as monoclonal antibodies directed to the surface components of T cells) as described in more detail in the following.

Eg mp o 3 Contribution of B cells in the rapid expansion With the purpose of. To quantify the contribution of B lymphocytes to the stimulus provided by PBMC, we examined the relative layering of PBMC-deleted PBMC populations to support REM. Isolation of cells such as B cells (or other cells referred to herein) can be conveniently obtained using antibodies directed to a cell surface marker known to be present on the cells to be suppressed. A variety of such markers are well known, including several "CD" or "differentiation group" markers; and antibodies can easily be obtained for many such markers. In addition, for many such markers, spheres conjugated with the antibodies are readily available and can greatly facilitate cell separation. By way of illustration, CD19 is a well-known cell surface marker for B lymphocytes. Magnetic spheres were obtained which have been conjugated with antibodies against CD19 from Dynal, and were used to suppress a B cell PBMC population, following standard procedures as It is described by the manufacturer. Deletion was evaluated by fluorescence activated cell sorting ("FACS") after CD19 staining. It is estimated that the PBMC population contains approximately 12% B cells before suppression, and less than 1% B cells after suppression. To test the impact of cell suppression B on REM hp, T cell expansion cultures containing 5 x 104 CTL, 5 x 10s irradiated EBV-LCL, 2.5 x 10 7 irradiated allogeneic PBMC were established (suppressed or without their B-cell pressure) 10 ng / ml of OKT3 and 25 U / ml of IL-2; and after 14 days, the T cells were harvested and quantified as described above. The results shown in Table 3 suggest that B cells also contribute to the stimulating activity provided by PBMC.

TABLE 3 The decreased levels of T cell expansion in this and in previous experiments suggests a role for monocytes and B cells as antigen-presenting cells ("APC") in the REM hp protocol. (The inability to inhibit the expansion of T cells to levels observed in Example 1 up to the levels observed in this experiment may be the result of differences in cell suppression by the various methods used and / or the presence of small amounts of APC not detected by the assays used It should also be noted that monocyte suppression, measured in example 1, only reduces the monocyte population from about 7.5% to about 1.5%). There are many different well known techniques that can be used to suppress various cell types from a PBMC population and which can therefore be used to provide additional confirmation and quantification of the results described herein. Thus, for example, nylon wool can be used to remove both monocytes and B cells (as well as any fibroblasts) from the PBMC population. The following example describes the substitution of various APC activities in modified REM.

Example 4 Substitution of various APC activities in modified REM The results obtained in the experiments of B cell suppression and monocyte suppression, described above, indicate that putative APCs in the PBMC population seem to contribute to the stimulus provided by the PBMC.

As described in Examples 1-2, it is expected that the role of FcγR activity in the presentation of antibody against CD3 constitutes some portion of the activity provided by putative APCs. Such Fc? R activity can be supplied by another source (other than PBMC), for example a cell line expressing Fc? R or spheres conjugated with antibody against CD3, as also described above. It is considered that APC within the PBMC population also contributes with accessory adhesion molecules and stimulatory cytokines that can be expressed to further enhance activation / proliferation processes. (In addition, as described below, T lymphocytes within the PBMC population are also expected to produce stimulatory cytokines as a result of activation via the antibody against CD3). The roles of such accessory adhesion molecules and cytokines are described in greater detail in the examples below.

Example 5 Contribution of cytokines in REM Although antibody against CD3 (for example OKT3) is used to activate and induce the proliferation of T cell clones for their expansion in vitro, the PBMC population of food subjected to irradiation? it also contains a substantial population of T lymphocytes that are considered to be activatable by the antibody against CD3. Although such irradiated feeder cells are incapable of dividing, it is considered that their activation via antibody to CD3 results in the secretion of multiple cytokines which can provide additional lymphoproliferative signals. For example, in addition to IL-2, it is believed that activation of T cells by antibody against CD3 results in the secretion of other stimulatory cytokines, including IL-1 a and β, IL-6, IL-8, GM-CSF, INF-a and TNF-a and β (du Moulin et al., 1994. Cytotechnology 15: 365). It is considered that the sesssion of one or more of these cytokines can contribute substantially to the proliferative stimulation provided by PBMCs within the REM hp protocol. Of the numerous additional cytokines that have been characterized, several of these are known to stimulate the growth of T cells, including, for example, IL-7 and IL-15. Others can easily examine their ability to improve T-cell proliferation and their relative ability to reduce REM dependence on large amounts of PBMC, as described herein. By way of illustration, we analyze the ability of numerous cytokines supplied exogenously to reconstitute the expansion of T cells in REM cultures in which the amount of PBMC feeder cells has been reduced to suboptimal levels, in order to quantify the potential role of such cytokines in promoting REM. Following procedures essentially analogous to those described above, cultures were established containing 5 x 104 CTL, 5 x 106 EBV-LCL, 10 ng / ml of OKT3 and 25 U / ml of IL-2, either with 2.5 x 10 7 PBMC irradiated (100% control), 1.25 x 107 irradiated PBMC (50%) or 6.12 x 106 irradiated PBMC (25%). It is considered that numerous cytokines can act synergistically with IL-2 to promote T cell proliferation. In this illustrative experiment, the following exogenous cytokines were added to the cultures, either alone or in various combinations, as described: IL-1 (40 U / ml), IL-4 (200 U / ml), IL-6 (500 U / ml) and IL-12 (20 U / ml). The results, shown in Table 4, confirm that such cytosines can substantially improve the expansion of T cells when populations of PBMC are reduced to suboptimal levels. It is not unexpected that the expansion levels do not return to those observed with the optimal amount of PBMC feeders, because the PBMC population is considered to provide additional stimulatory activities as described herein. The data suggest that replacement of IL-4 with IL-2 in a cytokine mixture can further enhance proliferation. The properties, sources and sequences of DNA and proteins of many such cytokines are described in reference books of cytokines such as "The Cytokine Facts Book" by R. Callard et al., Supra. To take a single example for purposes of illustration, it is known that IL-12 is a heterodimeric cytokine comprising two peptide chains (p35 and p40) that induces the production of IFNα. by T lymphocytes and co-stimulate the proliferation of lymphocytes in peripheral blood. IL-12 also stimulates the proliferation and differentiation of THL lymphocytes, and is known to be produced by B cells, monocytes / macrophages and B lymphoblastoid cells. The complete amino acid sequences of both p35 and p40 chains are known and are available in Genbank (access numbers provided in Callard). The receptor for IL-12 (id) has also been characterized. Cytokines and additional mixtures thereof can be easily tested in an analogous manner, * and a comparison of stimulatory mixtures can be made using even lower levels of PBMC.

TABLE 4 Additional evidence is obtained that the soluble components of the supernatant of feeder cells can provide a stimulus effect for low REM PBMC by reducing the PBMC population to sub-optimal levels and using a supernatant of REM to provide soluble stimulatory signals. Briefly, a standard protocol of REM hp was carried out as described above, using a CTL clone specific for antigen and performing a 48 hour REM expansion with PBMC (500: 1), EBV-LCL (100: 1), antibody against CD3 (10 ng / ml) and recombinant human IL-2 (25 units / ml). After 48 hours, the cells were harvested and the supernatant ("REM supernatant") was examined as a source of soluble stimulatory factors in a REM expansion in which the PBMCs were reduced to suboptimal levels (ie, 1/2, 1 / 4 or 1/8 of the optimum or "SOP"). The results, as shown in Table 5, confirm that such soluble factors can provide an effective stimulatory signal in the context of low REM PBMC. In particular, a large proportion of the reduction in proliferation levels observed when PBMC is reduced can be resolved by using REM supernatant instead of standard medium. Furthermore, the more PBMC is reduced (ie, 1/8 of the optimal), the greater is the effect observed when the REM supernatant is used (an average expansion of 1022 times using the REM supernatant versus a 359-fold expansion, without the same ). Such supernatants and / or their components such as individual cytokines or "mixtures" thereof can be used in this manner to reduce the need to carry out REM with a large excess of feeder cells such as PBMCs).

TABLE 5 Ejepplo 6 Replacing the modified REM cytokine activity As described above, a large number of cytokines have been described and are widely available, including numerous cytokines that are known to stimulate T lymphocytes. It will be apparent to those familiar with the art that such cytokines can be easily tested (whether known or not). or not previously stimulating T cells) in order to determine their ability to increase rapid expansion using methods such as the above. In addition, for any of the rapid expansion techniques described herein, the resulting expanded T cells can be monitored for the maintenance of various desired characteristics, using methods such as those illustrated above for REM hp. Cytokines that are to be used in modified REM can be introduced into the target T cells in any of several ways, as illustrated in the following. Thus, for example, the medium can be added one or more cytokines, as exemplified above. Alternatively, or additionally, cytosines can also be supplied are cells that secrete the cytokines in the REM medium. Thus, by way of illustration, a mammalian cell line which is known to secrete a particular cytokine or combination of cytokines can be used. Alternatively, a mammalian cell line that no longer secretes a particular cytokine (or secretes it at suboptimal levels) can be easily modified by introducing a gene encoding the desired cytokine. As is well known, the gene can be placed under the control of any of a variety of promoters (as alternatives to its original promoter), so that the expression of the cytokine can be controlled to maximize its effectiveness. The somatic sequencing of a large number of cytokines is known and coding DNA is often available. Many such sequences are published in nucleic acid and / or protein databases (such as GenEMBL, GENBANK or Swissprot); see, for example, the Cytokine Facts Book, R.E. Callard et al., Academic Press, 1994). In addition, as described above, such additional mammalian cell lines can be modified to provide several T-cell stimulatory activities at one time.

Example 7 Role of. the accessory adhesion molecules in the rapid expansion As discussed above, APCs such as monocytes and B cells also provide other T cell costimulatory signals which serve to enhance T cell activation / proliferation. Therefore, although T cell activation involves specific recognition of peptides antigenics bound to MHC on the surface of APCs (which interact with the T cell / CD3 receptor complex), many receptor-ligand interactions not antigen-specific between APCs and T cells can further enhance the activation / proliferation of T cells. In particular, APCs that express ligands for a variety of receptors on T cells, and that appear to activate / proliferate T cells are results of a combination of signals derived through the T cell receptor and other molecules Signaling. Many such receptor-ligand interactions have already been identified and, for many of these, it has been reported that the inhibition of receptor: ligand interactions inhibits T cell proliferation and cytokine secretion. By way of illustration, numerous receptor: ligand pairs that are considered likely to play a role in the activation / proliferation of T cells are included in Table 6 below.

TABLE 6 Although it has been reported that many of these molecules function in adhesion (improving cell: cell and / or cell: substrate interactions), it has also been shown that many provide costimulatory signals to T cells in a way that improves intracellular calcium and activation of Pl and PKC (see, for example Geppert et al., 1990, Immunol. Reviews 117: 5-66). The interactions of such accessory adhesion molecules as described above have been shown to positively enhance the activation of resting T lymphocytes. It has been shown that antibodies which bind to these accessory molecules, under specific conditions, provide signals for T cell activation (see, for example, the references mentioned below). In addition, it has been shown that the addition of purifissated ICAS-1 and LFA-3 ligand ligands (ligands for CDlla and CD2, respectively) to purified T cells that have been stimulated with monoclonal antibody against CD3, provides costimulatory signals for activation and T cell proliferation (see, for example, Semnani et al., 1994. J. Exp. Med. 180: 2125). Therefore, bodies directed against CD4 and CD8 are capable of inhibiting the activation of T cells (see, for example, I. Bank and L. Chess, 1985. J. Exp. Med. 162: 1194; GA van Seventer, 1986. Eur. J. Immunol., 16: 1363) or synergize with mAb against CD3 to induce T cell proliferation (see, for example, F. Emmrich et al., 1986. PNAS 83: 8298, T. Owens et al., 1987. PNAS 84: 9209: K. Saizawa et al., 1987. Nature 328: 260). As is well known to those of ordinary skill in the art, a collection of antibodies raised against a particular antigen is expected to contain antibodies that bind to a variety of different sites on the antigen. Numerous studies have shown that antibodies to other accessory adhesion molecules are capable of increasing the stimulation / proliferation of T cells. By way of illustration, see, for example, J.A. Ledbetter et al. 1985. J. Immunol 135: 2331 (antibodies directed to CD5 and CD28 that increase the proliferation of T cells induced by antibody against CD3); P.J. Martin et al., 1986. J. Immunol. 136: 3282 (antibodies to CD28 that increase the proliferation of T cells induced by antibodies against CD3); R. Galandrini et al. 1993. J. Immunol. 150: 4225, and Y. Shimizu. 1989. J. Immunol 143: 2457 (antibodies directed against CD44 that increase the proliferation of T cells induced by antibodies against CD3); S.C. Meur et al. 1984. Cell 36: 897 (antibodies directed against the Til.2 and Til.3 epitopes of CD2 that stimulate T cell proliferation); R. van Lier. 1987. J. Immunol. 139: 1589 (antibodies directed against CD27 that increase the proliferation of T cells induced by antibody to CD3); Bossy et al. 1995. Eur. J. Immunol. 25: 459 (antibodies to CD50 (ICAM-3) that increase the proliferation of T cells induced by antibodies against CD3), * M.C. Wacholtz et al. 1989. J. Exp. Med. 170: 431 (antibodies directed to LFA-1 that increase antibody-induced proliferation against CD3 when the two antibodies cross-link on the surface of T cells); G.A. van Seventer et. to the. 1990. J. Immunol. 144: 4579 (purified ICAM-1, immobilized in plastic with mAb against CD3 that co-stimulates the proliferation of T cells via the molecule LFA-1); Y. Shimizu et al. 1990. J. Immunol. 145: 59 (purified fibronectin on plastic with mAb against CD3 that co-stimulates the proliferation of T cells, and antibodies for VLA4 and VLA5 that inhibit this activity indicating the role of VLA4 and VLA5 as co-stimulatory receptors of T cells); N.K. Damle et al. 1992. J. Immunol. 148: 1985 (Soluble ICAM-1, B7-1, LFA-3 and VCAM that increase the proliferation of T cells induced by antibodies against CD3). The quantification of the relative contribution of such accessory adhesion factors within the REM protocol can be easily carried out using suppression techniques and titration experiments in hp REM assays with limited PBMC analogous to those illustrated above for the combinations of the various cytokines .

Example 8 Substitution of accessory adhesion molecule activity in modified REM In a manner analogous to the modifications described above, and perhaps in combination with such modifications, the REM protocol can thus be modified to include a characterized cell line that expresses high levels of these receptor ligands (obtained by, modification of the gene of a cell line of choice or by identification of established cell lines that already express such molecules). It is also possible to use antibodies directed against accessory molecules that are known to induce signal transduction and / or which use purified accessory ligand molecules as a means to substitute the corresponding activity provided by the PBMC feeder cells, thereby allowing a reduction in the amount of the PBMC necessary to activate REM.

Example 9_ Substitution of additional stimulatory activities provided by EBV-LCL Although EBV-LCL does not appear to be sufficient to obtain maximal expansion of T cells, they are capable of increasing expansion in the REM hp protocol. The analysis of EBV-LCL indicated that express adhesion molecules such as LFA-1, ICAM-1 and LFA-3, as well as Fc? R. In addition, EBV-LCL secrete IL-1 (Liu et al., Cell.Immunol., 108: 64-75, 1987) IL-2 (Kobayashi et al., 1989. J. Exp. Med. 170: 827), both which are also secreted by the APC. As described above, it is considered that such components can be easily supplied by other sources - thereby reducing the need for large numbers of PBMC and / or EBV-LCL feeder cells characteristic of REM hp.

Example 10 Use of antibody against CD21 in modified REM CD21 is an absorbing molecule expressed in mature B lymphocytes and, at low levels, in T lymphocytes.

We examined the ability of a molecule to bind CD21 to provide a stimulatory signal in the context of REM modified. In a first set of experiments, we used antibody against plate bound CD21 to examine the ability to provide a stimulator signal in modified REM in which the EBV-LCL feeder population is completely eliminated. Two different CTL clones specific for antigen ("R7" which is specific for haloantigen, and "11E2" which is specific for EBV) were tested in a modified REM procedure, in which the EBV-LCL were eliminated, but they were maintained the other components, as described above (PBMC at 500: 1, IL-2 at 25 units / ml). Antibodies against CD21 were available from commercial sources. We used an antibody against CD21 available from Pharmingen. The antibody against CD3 was also used, and bound to plates, together with the CD21 antibody. The cultures were expanded for two weeks in a standard REM cycle, essentially as described above. The data, as shown in table 7, show that the inclusion of antibody against CD21 results in a large increase in the proliferation times obtainable without the use of EBV-LCL (up to 650% of the control and 408% of the control for R7 and 11E2, respectively). A second set of experiments, performed using soluble antibody against CD21, provided additional confirmation data. In particular, a range of antibody concentrations against CD21 in REM was used as above, except that the antibodies against CD21 and against CD3 were supplied as soluble antibodies (the antibody against CD21 at concentrations ranging from 0 ng / ml to 1.75. ng / ml; the antibody against CD3 at 10 ng / ml). As shown in Table 8, the removal of all EBV-LCL feeder cells from the cultures results in a substantial reduction in the average proliferation times (up to 10% of the control and 14% of the control for R7 and 11E2, respectively) . The addition of even smaller amounts of antibody against CD21 to the culture medium results in a large increase in proliferation rates (at 72% of the control and 57% of the control for R7 and 11E2, respectively). Although the antibody against CD21 provides a convenient method for improving the stimulatory signal, it is also possible to stimulate CD21 in other ways. For example, in addition to the antibody to CD21, other molecules that can be used to bind to CD21 include C3d, C3dg, iC3b and gp350 / 220 or EBV (see, for example, W. Timens et al., Pages 516-518 in " Leucocite Typing V. White Cell Differentiation Antigens, "Schlossman, SF, et al (eds.), Oxford University Press, Oxford, 1995). Furthermore, as described above, although such T-cell stimulating components can be provided as soluble factors in the modified REM medium, they can also be provided by a cell line included in the medium (for example a cell line that secretes or presents a molecule to join CD21). TABLE 7 TABLE 8 It is noted that in relation to this date, the best method conosido by the applicant to implement the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (53)

1. A method for rapidly expanding an initial population of T lymphocytes in a culture medium in vitro, characterized in that it comprises the steps of: adding an initial population of T lymphocytes to an in vitro culture medium, - adding a line of culture to the culture medium. non-dividing mammalian cells that express at least one T-cell stimulator component, wherein the cell line is not a line of lymphoblastoid cells transformed by EBV (LCL); and incubate the crop.

2. A rapid expansion method according to claim 1, characterized in that the T cell stimulator component is selected from the group consisting of an Fc receptor, an accessory cell of cell adhesion and a cytokine.

3. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is selected from the group consisting of a Fc receptor, accessory cell adhesion molecules and a cytokine, and wherein the initial population of T lymphocytes expands at least 200 times after an incubation period of less than about two weeks.

4. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is selected from the group consisting of a Fc receptor, accessory cell adhesion molecules and a cytokine, and wherein the initial population of T lymphocytes expands at least 500 times after an incubation period of less than about two weeks.

5. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is selected from the group consisting of a Fc receptor, accessory cell adhesion molecules and a cytokine, wherein the initial population of T lymphocytes expands at least 1000 times after an incubation period of less than about two weeks ..

6. A rapid expansion method according to claim 1, characterized in that it further comprises the step of adding monoclonal antibody against CD3 to the culture medium, wherein the concentration of monoclonal antibody against CD3 is at least about 1.0 ng / ml.

7. The method of rapid expansion according to claim 1, characterized in that it further comprises the step of adding IL-2 to the culture medium, wherein the concentration of IL-2 is at least about 10 units / ml.

A rapid expansion method according to claim 1, characterized in that the mammalian cell line comprises at least one type of cell that is present at a frequency at least three times that found in peripheral blood mononuclear cells human (human PBMC).

9. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is selected from the group consisting of an Fc receptor. and an accessory molecule of cell adhesion.

10. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is selected from the group consisting of accessory molecule of cell adhesion and a cytokine.

11. A rapid expansion method according to claim 1, characterized in that the T cell simulator component is selected from the group consisting of an Fc receptor. and a cytokine.

12. A rapid expansion method according to claim 1, characterized in that the mammalian cell line expresses an accessory cell adhesion molecule.

13. A rapid expansion method according to claim 12, characterized in that the accessory cell adhesion molecule is selected from the group consisting of MHC class II, MHC class I, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, ligand for CD27, CD80, CD86 and haluronate.

14. A rapid expansion method according to claim 1, characterized in that the mammalian cell line expresses a cytokine.

15. A rapid expansion method according to claim 1, characterized in that the T-cell stimulator component is a molecule that binds to CD21.

16. A rapid expansion method according to claim 14, characterized in that the cytokine is selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL -fifteen.

17. A rapid expansion method according to claim 1, characterized in that it further comprises the step of adding a soluble factor T cell stimulator to the culture medium.

18. A rapid expansion method according to claim 17, characterized in that the soluble factor T cell stimulator is selected from the group consisting of a cytokine, an antibody specific for a cell surface component • T and an antibody specific for a component capable of binding to a T cell surface component.

19. A rapid expansion method according to claim 17, characterized in that the soluble factor T cell stimulator is a cytokine selected from the group consisting of IL-1. , IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.

20. A rapid expansion method according to claim 17, characterized in that the soluble factor T cell stimulator is an antibody specific for a T cell surface component, and wherein the T cell surface component is selected from the group which consists of CD4, CD8, CDlla, CD2, CD5, CD49d, CD27, CD28 and CD44.

21. A rapid expansion method according to claim 17, characterized in that the soluble factor T cell stimulator is an antibody specific for a component capable of binding to a surface component of T cells, wherein the cell surface component T is selected from the group consisting of CD4, CD8, CDlla, CD2, CD5, CD49d, CD27, CD28 and CD44.

22. A rapid expansion method according to claim 17, characterized in that the soluble factor T-cell stimulator is a molecule that binds to CD21.

23. A rapid expansion method according to claim 22, characterized in that the molecule that binds CD21 is an antibody against CD21.

24. A method of rapid expansion according to claim 1, characterized in that it also comprises the step of adding a multiplicity of peripheral blood mononuclear cells (PBMC) to the culture.

25. A rapid expansion method according to claim 24, characterized in that the ratio of the PBMC to the initial T cells to be expanded is less than about 40: 1.

26. A method of rapid expansion according to claim 24, characterized in that the proportion of PBMC with respect to the initial T cells to be expanded is less than about 10: 1.

27. A rapid expansion method according to claim 24, characterized in that the ratio of PBMC to initial T cells that are to be expanded is less than about 3: 1.

28. A method of rapid expansion according to claim 1, characterized in that it further comprises the step of adding to the culture a multiplicity of lymphoblastoid cells transformed with EBV (LCL).

29. A rapid expansion method according to claim 28, characterized in that the ratio of LCL to initial T cells that are to be expanded is less than about 10: 1.

30. A rapid expansion method according to claim 1, characterized in that the initial population of T lymphocytes comprises at least one antigen-specific cytotoxic T lymphocyte, CD8 + (CTL).

31. A rapid expansion method according to claim 1, characterized in that the initial population of T lymphocytes comprises at least one antigen-specific T lymphocyte, human CD4 +.

32. A method for genetically transducing a human T cell, characterized in that it comprises the steps of: adding an initial population of T lymphocytes to an in vitro culture medium; adding to the culture medium a line of mammalian cells not transformed by EBV that express a T-cell stimulator component; and incubate the culture; and add a vector to the culture medium.

33. A method of genetic transduction according to claim 32, characterized in that the vector is a retroviral vector containing a selectable marker that provides resistance to an inhibitory compound that inhibits T lymphocytes, and wherein the method further comprises the steps of : continue incubation of the culture for at least one day after the addition of the retroviral vector; and adding the inhibitor compound to the culture medium after continuing the incubation step.

34. A method of genetic transduction according to claim 32, characterized in that it further comprises adding a multiplicity of human PBMC.

35. A rapid expansion method, according to claim 34, characterized in that the proportion of the PBMC with respect to the initial T cells is less than about 40: 1.

36. A method of genetic transduction according to claim 32, characterized in that it further comprises adding lymphoblastoid cells transformed with EBV, which are not in division (LCL).

37. A method of genetic transduction, according to claim 35, characterized in that the ratio of LCL to initial T cells is less than about 10: 1.

38. A method for generating a REM cell line capable of promoting a rapid expansion of an initial population of T lymphocytes in vitro, characterized in that it comprises the steps of: suppressing one or more cell types from a population of human PBMCs to produce a population of PBMC deleted in a cell type, use the suppressed PBMC population in a cell type, instead of the non-suppressed PBMCs, in a hp REM protocol to determine the contribution of the suppressed cell type to the activity provided by the non-suppressed PBMCs, identify a T-cell stimulatory activity provided by the type of suppressed cell, and transform a mammalian cell line with a gene that allows the expression of such T-cell stimulatory activity.

39. A method to generate a REM cell line according to claim 38, characterized in that the T-cell stimulator component is selected from the rupo consisting of an Fc receptor ?, an accessory molecule of cell adhesion and a cytokine.

40. A REM cell line capable of stimulating rapid expansion of an initial population of lymphocytes T in vitro, characterized in that it comprises a line of mammalian cells generated in accordance with a method of claim 38.

41. A line of REM cells according to claim 40, characterized in that the cell line expresses an accessory molecule of cell adhesion.

42. A line of REM cells according to claim 41, characterized in that the accessory cell adhesion molecule is selected from the group consisting of MHC class II, MHC class I, ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, binding to CD27, CD80, CD86 and hyaluronate.

43. A line of REM cells according to claim 40, characterized in that the cell line expresses an Fc-1 receptor.

44. A line of REM cells according to claim 40, characterized in that the cell line expresses at least one T-cell stimulatory cytokine.

A REM cell line according to claim 44, characterized in that the cytokine stimulant T cells are selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15.

46. A line of REM cells according to claim 40, characterized in that the cell line expresses a molecule that binds to CD21.

47. A culture medium capable of rapidly expanding an initial population of T lymphocytes in vitro, characterized in that it comprises a line of REM cells according to claim 40.

48. A culture medium according to claim 47, characterized in that it comprises in addition an exogenous cytokine.

49. A culture medium according to claim 47, characterized in that it also comprises a multiplicity of exogenous cytokines in which the multiplicity comprises at least one interleukin.

50. A culture medium according to claim 49, characterized in that the interleukin is selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15.

51. A culture medium according to claim 47, characterized in that it also comprises a molecule that binds to CD21.

52. A culture medium according to claim 51, characterized in that the molecule that binds to CD21 is an antibody against CD21.

53. A culture medium according to claim 49, characterized in that it also comprises a monoclonal antibody against CD3.