MXPA96004221A - Method for improving the immunotherapeutic activity of immune cells by exhaustion / positive selection of subconjunds of cells, and method to reduce tumor volume in v - Google Patents

Abstract

The present invention relates to the use of either a subpopulation of CD8 + -free cells stimulated, or of a subpopulation of CD4-stimulated T cells, for the manufacture of a medicament for treating a mammal that has tumors, in a method that it comprises optionally administering to said mammal an immunosuppressant, and then administering said said cellular subpopulation to said mammal, followed by the administration of IL-2 liposome

Description

METHOD FOR IMPROVING THE IMMUNOTHERAPEUTIC ACTIVITY OF IMMUNE CELLS BY EXHAUSTION / POSITIVE SELECTION OF CELL SUCCESSIONS. AND METHOD TO REDUCE LIVE TUMOR VOLUME Field of the Invention This invention relates to improving the immunotherapeutic activity of immune cells. Specifically, this invention relates to the stimulation of antitumor activity of immune cells after depletion or positive selection of specific cells or subsets of T lymphocyte cells, and the use of depleted or positively selected stimulated cells or subsets of T lymphocytes to reduce the volume of tumors in mammals, and to immunize mammals against tumors. This invention also relates to the use of depleted or positively selected stimulated cells or subsets of T lymphocyte cells to increase the production of colony forming units.

BACKGROUND OF THE INVENTION Peripheral blood mononuclear lymphocytes (PBL) can be stimulated to develop lytic activity against new tumor cells, as well as against various targets resistant to natural killer (NK), such as Daudi and HL60, after a relatively short-term culture (3-5 days) in the presence of interleukin-2 (IL-2). See, for example, M. Lotze et al., Cancer Res. , 41, 4420 (1981); C. Grimm et al., J. Exp. Med., 155, 1823 (1982); and E. A. Grimm et al., "The Limphokine-Activated Killer Cell Phenomenon: In Vi tro and In Vivo Studies", in INTERLEUKINS, LYMPHOKINES AND CYTOKINES, S. Cohen and J. Oppenheim, eds., Academic Press; New York, p. 739 (1983). This function has been called lymphokine-activated annihilating activity (LAK). Initial reports suggested that cell precursors with lymphocyte-killing annihilating activity did not express the T cell receptor as determined by anti-CD3 binding. See E.A. Grimm et al., J. Exp. Med. , 157, 884 (1983). More recent reports have shown that the effector cells obtained from the short-term culture with interleukin 2 (2-5 days) are a CD3 population "of cells expressing the natural annihilator markers CD16 and / or CD56.The CD3 + cells of these cultures have low lytic activity against targets resistant to natural killers, thus, the CD3 population ", with markers of natural annihilators CD16 and / or CD 56, is responsible for the great majority of the annihilating activity activated by lymphokine in cultures of mononuclear leukocytes of the peripheral blood. That is, CD3 cells "appear to be the classic effector cells of natural annihilators, see, for example, JR Ortaldo et al, J. Exp. Med., 164, 1193 (1986); S. Ferrini et al., J. Im unol., 138, 1297 (1987), K. Itoh et al., J. Immunol., 136 3910 (1986), and JH Phillips et al., J. Exp. Med., 164, 814 (1986). reported reversible induction of natural killer activity in cloned cytotoxic lymphocytes in response to interleukin 2 and interferon (IFN), see CG Brooks, NATURE, 305, 155 (1983). Large numbers of cells with lymphokine-activated annihilating activity using relatively long-term cultures (10-30 days) of peripheral blood mononuclear leukocytes stimulated with the monoclonal antibody (MoAb) OKT3 anti-CD3, in combination with interleukin 2 (cells CD3-LAK or T-AK cells.) See AC Ochoa and colab speakers, J. Ipununol. , 138, 2728 (1987). Effector cells in these long-term cultures interleukin 2 and OKT3 include CD3 + T cells, CD3"cells, as well as a population of CD3 + that is CD4 and CD8 negative, and expresses the α-chains of the T cell receptor. , the effector cells in short-term cultures t interleukin 2 and OKT3 (2-5 days) are predominantly CD3 cells ". Numerous studies have shown that it seems that very little lymphokine-activated annihilating activity is mediated by the CD4 + or CD8 + T cells classically described. further, CD4 + or CD8 + cells isolated from cultures of mixed peripheral blood mononuclear leukocyte populations, which are activated with an antibody to a lymphocyte surface receptor, such as the anti-CD3 monoclonal antibody 0KT3, and are continuously cultured with interleukin 2 , do not develop significant levels of activity of natural annihilators or annihilating activity activated by lymphokine, as determined immediately after the isolation of the total population. See, for example, A.C. Ochoa et al., Cancer Res. , 49, 963 (1989). For example, in an effector-to-target ratio of approximately 30: 1, ie, a ratio of the number of T cells capable of mediating cytotoxicity against the number of targets of tumor cell lines, the cytotoxicity of the CD4 + subsets or CD8 + is no greater than about 15 to 20 percent. . It has been noted that when mononuclear leukocytes from peripheral blood are stimulated in a culture of mixed lymphocytes (MLC), CD4 + cells are minimally cytotoxic. Furthermore, when the population of CD4 + is stimulated in the culture of mixed lymphocytes in the absence of other T cells, they develop a greater cytolytic activity. See, E.L. Reinherz et al., Proc. Na ti. Acad. Sci. USA, 76, 4061 (1979). However, this cytotoxicity is antigen-specific, and does not involve tumor killing activity. It has also recently been shown that CD8 + CDllb + cells can develop lymphokine-activated annihilating activity. In this specific situation, CD8 + cells were isolated from the mononuclear leukocyte population of the peripheral blood before the start of culture in the presence of interleukin-2 alone. However, this method did not involve stimulation of the anti-CD3 monoclonal antibody; This method involved separating the T cells with red blood cells from sheep, which in themselves can produce a stimulating signal through the CD2 receptor. Thus, natural killer cells expressing the CD2 receptor can also be activated. See, U. Dianzani et al., Eur. J. Im unol. , 19, 1937 (1989). It has been shown that CD4 + and CD8 + cells cultured in the presence of interleukin-2 alone express the lytic machinery, but lynphokine-killing annihilation activity was not demonstrated and cell growth was not reported. See, M.J. Smyth et al., J. Exp. Med., 171, 1269 (1990). Finally, it has been shown that lymphocytes that infiltrate tumors (TIL), which seem to be effective in the treatment of solid tumors, are mainly CD8 +.

See, for example, S. Shu et al., J. Immunol. , 139, 295 (1987); and A. Belldegrun et al., Cancer Res. , 48, 206 (1988). The identification of the cells that can mediate cytotoxicity, for example the lymphokine-killing annihilating activity, is important for an understanding of the interactions of the immune system as well as for the potential development of effective methods of immunotherapy. One of the limitations of current lymphokine-activated annihilator therapies for the treatment of tumors is that lymphokine-activated annihilating cells appear to be transported via the reticuloendothelial system which, in some cases, limits the accessibility of lymphocyte-activated annihilating cells. to certain tumors. See, for example, A.A. Maghazachi et al., J. Immunol. , 141, 4039 (1988). On the other hand, T cells circulate through the lymphatic system and provide greater accessibility to most tumors. While most natural killer and lymphokine-mediated killing activity in cultures stimulated with interleukin-2 alone or interleukin-2 + anti-CD3 seem not to be mediated by CD4 + or CD8 + cells, what has been necessary is to determine whether these cells T, under the right conditions, could develop high cytotoxicity, for example specific or non-specific lytic activity. Thus, what is needed is a method for the stimulation of immune cells to produce high cytotoxicity, preferably non-specific high lytic activity, for example of natural annihilators or lymphokine-activated annihilator, in T cells, which may provide antitumor therapeutic efficacy . Patients undergoing bone marrow transplantation find a period of severe immunodeficiency after high-dose chemotherapy and / or total body radiation (TBI) since the patient's bone marrow is replaced with a small amount of healthy bone marrow cells that proliferate in the body until enough bone marrow cells have been generated to reach a repopulation of the peripheral blood by red blood cells, platelets and white blood cells of the immune system. A typical bone marrow graft can take 20 to 30 days to regenerate (graft) sufficient amounts of bone marrow cells. During this period, the patient's immune system is virtually nonfunctional, and the patient must be closely monitored to prevent the aggravation of the disease. The proliferation of these bone marrow cells can be induced with factors such as G-CSF, GM-CSF, IL-3 and the like. However, all these factors have to be administered at high doses that can be toxic to the patient. Thus, there is a need to provide a method that is capable of stimulating the proliferation of bone marrow cells to increase the number of bone marrow cells produced and thereby decrease the time needed to regenerate a sufficient number of bone marrow cells. There is also a need to develop a method to reduce the volume of tumors in mammals, and to generate long-term immunity against tumors by means of which a population of activated immune cells has the high. The aforementioned cytotoxicity, for example, of natural killer or lymphokine-activated annihilating activity, can be administered to the mammal to reduce tumor volume. There is also a need to develop a method of transferable immunity whereby the immune cells of an immunized mammal are administered to another mammal of the same species, thereby conferring immunity to the tumor to the other mammal.

Summary of the Invention It is an object of the present invention to develop a method for improving the immunotherapeutic activity of a population of immune cells by stimulating immune cells to produce high cytotoxicity., preferably high non-specific lytic activity, for example of natural annihilators or lymphokine-activated annihilator, in T cells, which can provide therapeutic antitumor efficacy. It is also an object of the invention to provide a method for treating a mammal having tumors using a population of immune cells that has improved immunotherapeutic activity against these tumors. It is a further object of the present invention to provide a method for reducing tumor volume in mammals, and for generating long-term immunity against tumors. It is also an object of the invention to provide a method for transferable immunity whereby the immune cells of an immunized mammal are administered to a second mammal, and the second mammal also develops a tumor immunity. In accordance with these and other objects of the present invention, there is provided a method for enhancing the immunotherapeutic activity of immune cells by separating at least a subset of cells that mutually inhibit each other within a population of immune cells. By separating at least a subset of cells that mutually inhibit each other within a population of immune cells, the population of remaining immune cells or the subset of separate cells are able to more fully express their immune function when stimulated. The stimulation of immune cells to improve their immunotherapeutic activity can be carried out mainly in one of four methods, the first two being "depletion methods" and the other two being "positive selection methods". The first method includes: separating at least a subset of cells, or subpopulation, that is capable of down-regulating the immunotherapeutic activity, eg, cytotoxicity, of a population of immune cells, from that population of immune cells to form a "population of depleted immune cells"; and culturing the population of depleted immune cells in the presence of an antibody to the lymphocyte surface receptor, optionally in the presence of interleukin 2, to form a "population of stimulated depleted immune cells". In addition, the population of stimulated depleted immune cells can optionally be further cultured in the presence of interleukin 2. Preferably, this method reduces or eliminates a regulatory mechanism of the immune cell population, which allows the remaining cells to more fully express their immune function. . The immunotherapeutic activity of the remaining immune cell population, represented by the measurement of the ability of the immune cell population to reduce tumor volume, is increased when compared to a population of non-depleted immune cells treated in a similar manner. . A second "depletion method" that enhances immunotherapeutic activity in accordance with an objective of the present invention, includes: (i) first culturing a population of immune cells to form a "population of cultured immune cells", (ii) separating a subset of cells or subpopulation, which is capable of developing immunotherapeutic activity, eg, cytotoxicity to form a "population of stimulated depleted immune cells"; and optionally (iii) separately culturing, i.e., subculturing, the population of depleted immune cells stimulated in a second medium in the presence of interleukin 2. Preferably, by using this method and separating a subpopulation that is capable of down-regulating the immunotherapeutic activity of the cell population, the immunotherapeutic activity, represented by the ability to reduce the volume of the tumors, of this population of stimulated depleted immune cells is improved when compared to a population of non-depleted cells treated in a similar manner. A third method ("positive selection method") for improving immunotherapeutic activity in accordance with an objective of the present invention includes: 'separating and positively selecting at least a subset of cells, or subpopulation, which is capable of up-regulating the immunotherapeutic activity, or develop the immunotherapeutic activity, of a population of immune cells, from that population of immune cells to form a "subset of immune cells"; and then culturing the subset of immune cells, eg, CD4 + or CD8 + cells, in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin 2, to form a "subset of stimulated immune cells". In addition, the population of stimulated depleted immune cells can optionally be further cultured in the presence of interleukin 2. The immunotherapeutic activity of the subset of stimulated immune cells, represented by a measure of the ability of the population of immune cells to reduce tumor volume, it is increased in comparison with a population of similarly treated non-depleted immune cells. An additional method of positive selection for enhancing immunotherapeutic activity according to the present invention, includes: (i) first - culturing a population of immune cells to form a "population of cultured immune cells", (ii) separating, for example, a subset of cells, or subpopulation, that is capable of developing immunotherapeutic activity, for example, cytotoxicity to form a "subset of stimulated immune cells"; and optionally (iii) separately culturing, i.e., subculturing, the population of the subset of immune cells stimulated in a second medium in the presence of interleukin 2. Preferably, by using this method and separating a subpopulation of cells that is capable of regulating down the immunotherapeutic activity of the cell population, immunotherapeutic activity, represented by the ability to reduce the volume of tumors, of this subset of immune cells is improved when compared to a population of non-depleted cells treated in a similar manner. In addition, the separated cells are preferably CD4 + or CD8 + lymphocytes, or subsets of each of these populations. According to the methods described above, the population of depleted immune cells or conveniently, subsets of immune cells are preferably cultured in a first medium in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin 2. Also of According to the methods described above, the whole population of immune cells is cultured in a first medium in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin 2. More preferably, the cells (population of immune cells or population of depleted cells) are cultured in interleukin 2 and in an antibody to a lymphocyte surface receptor for only the first 48 hours. Therefore, any culture either from the population of stimulated depleted immune cells or the subset of stimulated immune cells occurs preferably in the presence of interleukin 2 without any additional amount of antibody to the lymphocyte surface receptor in order to conserve and culture. the cells in vi tro. If the population of stimulated immune cells or the subsets of stimulated immune cells, ie "stimulated cells", are administered to a mammal shortly after the initial culture, no additional culture is necessary in the presence of interleukin 2. However, it is typically administering interleukin 2 to the mammal together with the stimulated cells as a method to generate lymphokine-activated annihilating activity in vivo. In addition, the population of depleted immune cells, or conveniently, subset of immune cells, can be stimulated by an antibody to a lymphocyte surface receptor for the first 48 hours, and then cultured with interleukin 2.; however, additional culture with interleukin 2 is not necessary if the stimulated depleted immune cells are administered to a mammal shortly after culturing. The population of depleted immune cells stimulated or the subset of stimulated immune cells can also be optionally grown in vitro in the presence of interleukin 2 to generate immune cells or subsets of immune cells that exhibit improved immunotherapeutic activity. Subsequent in vitro culture serves not only to proliferate and maintain the cell population, but also to return cytotoxic in vivo to the stimulated cells as well. The population of stimulated depleted immune cells or the subset of stimulated immune cells can also be injected in vivo after interleukin 2 is administered. Although no attempt is made to be limited by any theory, it is believed that by culturing the population of immune cells or the subset of immune cells in the presence of an antibody to a lymphocyte surface receptor "primes" the cells to up-regulate the production of the interleukin 2 receptor sites in the cells. Thus, when interleukin 2 is administered either in vivo together with the stimulated cells, or in vi tro, the stimulated cells are stimulated more significantly by accepting more interleukin 2. These stimulated cells, when bound to interleukin 2, are then able to use tumors because interleukin 2 can generate a cascade signaling reaction inside the cells to generate lymphokine-activated annihilating activity. Immune cell populations stimulated according to any of the above methods are useful in the following methods to treat mammals with tumors, immunize mammals against tumors and transfer immunity from one mammal to another mammal. According to a further objective of the present invention there is provided a method for treating a mammal having tumors with immune cells having improved immunotherapeutic activity, prepared by the procedures outlined above for preparing a population of stimulated depleted immune cells or a subset of stimulated immune cells; and then culturing the population of depleted immune cells, or the subpopulation of cells in the presence of an antibody to the lymphocyte surface receptor, optionally in the presence of interleukin 2. The population of stimulated depleted immune cells or the subset of immune cells stimulated then they may optionally be further cultured in the presence of interleukin 2, although additional culture is not necessary. This method therefore includes (i) enhancing the immunotherapeutic activity of a population of immune cells as just described; (ii) administering the population of immune cells to a mammal having tumors and optionally treating it previously with an immunosuppressant; and (iii) administering interleukin 2, preferably liposomal interleukin 2, to the mammal in addition to the population of immune cells of step (ii). According to another object of the present invention, there is provided a method for immunizing a mammal against a tumor by (i) enhancing the immunotherapeutic activity of a population of immune cells according to any of the procedures outlined above; (ii) administering the population of stimulated depleted immune cells or the subset of immune cells stimulated to a mammal having tumors, wherein the mammals are optionally pretreated with an immunosuppressant; and (iii) administering interleukin 2, preferably interleukin 2 liposomal, to the mammal in addition to the population of stimulated depleted cells or the subset of immune cells stimulated from step (ii). According to yet another object of the present invention, there is provided a method for transferring immunity from a mammal to another mammal by (i) extracting and separating splenocytes from the spleen of a mammal immunized against tumors, whereby the mammal is immunized against tumors by: (i) improving the immunotherapeutic activity of a population of immune cells according to any of the procedures outlined above; (ii) administering the population of stimulated depleted immune cells or the subset of immune cells stimulated to a mammal, optionally pretreated with an immunosuppressant; and (iii) administering interleukin 2, preferably liposomal interleukin 2, to the mammal in addition to the population of stimulated depleted cells or the subset of immune stimulated immune cells of step (ii) to produce an immunity in the mammal against tumors. The splenocytes of this treated mammal that has developed the aforementioned immunity are then extracted using conventional techniques and administered to a second mammal, optionally with interleukin 2, preferably liposomal interleukin 2. The second mammal is also pre-treated optionally with an immunosuppressant before the administration of the splenocytes. According to this method, the second mammal that receives the splenocytes also develops an immunity against the tumors. According to another object of the present invention, there is provided a method for stimulating the proliferation of bone marrow cells comprising the steps of first improving the immunotherapeutic activity of a population of immune cells according to any of the methods outlined above. The population of stimulated depleted immune cells or the subset of stimulated immune cells can be further cultured in vitro with interleukin 2 for in vitro efficacy. These stimulated cells are then incubated in the presence of bone marrow, optionally in the presence of additional cytokines including granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, Kit Ligand (KL), erythropoietin (Epo) or interleukin 2 for increase by this the number of bone marrow cells. Bone marrow cells cultured in vi tro, then can be administered to a mammal to stimulate the proliferation of additional bone marrow cells. Alternatively, the stimulated cells can be administered directly to a mammal having a population of bone marrow cells or compromised to generate bone marrow cells in vivo.

Brief Description of the Drawings FIGURE 1 illustrates the lymphokine-mediated killing activity (percent cytotoxicity) of a mononuclear leukocyte population of CD8 + depleted peripheral blood, mononuclear leukocyte population of CD4 + depleted peripheral blood, and mononuclear leukocytes of the peripheral blood not separated, stimulated with the anti-CD3 monoclonal antibody OKT3 and continuously cultured with interleukin 2. FIGURE 2 illustrates the lymphokine-activated annihilating activity (percent cytotoxicity) of: (a) a population of mononuclear leukocytes of peripheral bleeding depleted of CD4 + stimulated with the monoclonal antibody anti-CD3 OKT3 and continuously cultured with interleukin 2; (b) CD8 + cells isolated from the mononuclear leukocytes of the cultured peripheral blood depleted of CD4 +, and (c) the population of remaining CD4CD8 cells isolated from peripheral blood mononuclear leukocytes depleted of CD4 +.

FIGURE 3 illustrates the lymphokine-activated annihilating activity (percent cytotoxicity) of: (a) a population of mononuclear leukocytes from the depleted peripheral blood of CD8 + stimulated with the anti-CD3 monoclonal antibody 0KT3 and continuously cultured with interleukin 2; (b) CD4 + cells isolated from cultured peripheral blood mononuclear leukocytes depleted of CD8 +; and (c) the remaining CD4CD8 cell population isolated from CD8 + depleted peripheral blood mononuclear leukocytes FIGURE 4 illustrates lymphokine-activated annihilating activity (percent cytotoxicity) of: (a) a population of mononuclear leukocytes of peripheral blood not depleted, that is, not separated or total, stimulated with monoclonal antibody OKT3 anti-CD3 and continuously cultured with interleukin 2; (b) CD4 + cells isolated from cultured peripheral blood mononuclear leukocytes not depleted, * (c) CD8 + cells isolated from peripheral blood mononuclear leukocytes cultured not depleted: (d) CD4"1 cells isolated from a population of cultured peripheral blood mononuclear leukocytes depleted of CD8 +; and (e) CD8 + cells isolated from a cultured population of peripheral blood mononuclear leukocytes depleted of CD4 +.

FIGURES 5A and 5B illustrate lymphokine-activated annihilating activity (percent cytotoxicity) of CD4 + and CD8 + cells, respectively isolated from cultured non-depleted peripheral blood mononuclear leukocyte populations (0KT3 + interleukin 2) and subsequently cultured with interleukin 2 alone These data were obtained after five days of culturing the mononuclear leukocytes of the non-depleted peripheral blood, "sorting day", and after ten and thirteen days of culturing each subset of cells in interleukin 2. FIGURE 6 illustrates the capacity of stimulated CD4 + cells to reduce tumor volume in vivo when stimulated only in the presence of anti-CD3 for less than 48 hours. FIGURE 7 illustrates the ability of non-depleted CD4 + cells stimulated to substantially eradicate a tumor in vivo when stimulated in the presence of anti-CD3 and a relatively minor amount of interleukin 2, and then administered to a mammal initially and then administered by a second mammal. time.

Detailed Description of the Invention As used herein, the phrase "depletion method" denotes a method for improving the immunotherapeutic activity of a population of immune cells using a "population of depleted immune cells". A population of depleted immune cells is the portion of the population of immune cells that remain after at least one subset of cells, or subpopulation, that is layer. From down-regulating the immunotherapeutic activity of that population of immune cells, has been separated and removed from it. The resulting depleted immune cell population, when stimulated, it is called in the present "population of stimulated immune cells". Throughout this description, the phrase "positive selection method" denotes a method for highlighting the immunotherapeutic activity of a population of immune cells using a "subset of immune cells". A subset of immune cells is the portion of the population of immune cells that has been removed and removed, or removed, from the population of immune cells. This subset of immune cells, when stimulated, is referred to herein as a "subset of stimulated immune cells." As used herein, lymphokine-mediated killing activity is defined as the ability of lymphocytes to use tumor cells, and to a lesser degree, normal cells. This activity in lymphocytes is typically stimulated by lymphokines, such as interleukin 2. In the examples in vi tro herein, lymphokine-killing activity refers to the ability to use the tumor target resistant to natural human annihilators. , HL60. The activity of the natural annihilators is defined as the ability to use tumor cells, but not the normal cells, which does not result from the previous stimulation. In the examples in vi tro used herein, the activity of the natural annihilators refers to the ability to use the K562 human tumor line. Since similar results were obtained with the two tumor lines, only the results of the lynchin-activated annihilator with HL60 are shown. In the in vivo examples used herein, the annihilating activity activated by lymphokine and natural annihilators refers to the ability to reduce tumor volume. The population of immune cells can include all immune cells that are part of an immune system, such as T cells, B cells, natural killer cells, and macrophages. Any of these cells, or a combination of these cells, can be depleted by the method of the present invention, with an improved immunotherapeutic effect resulting, for example, antitumor. As used herein, the "population of immune cells" may be, as is preferable, a total population, ie, not separated or not depleted, as obtained from a whole blood sample.; however, the "immune cell population" can be any portion of a total population that contains a subset of cells or subpopulation that down-regulates the immunotherapeutic activity of the larger population, or is capable of developing immunotherapeutic activity itself. As used herein "cultivar" indicates the process by which the cells are placed in a tissue culture medium comprising nutrients to support the life of the cells, and other additives, such as interleukin 2 growth factor. process can take place in any container or appliance. The process may involve several steps of cultivating and subculturing. Typically, the cells are initially cultured and expanded, that is, they increase in size and number. These expanded cells are then counted and divided into groups, or subcultures, for further culture and expansion. The expanded cells of these subcultures are then divided into additional subcultures, for further expansion and cultivation. Each of the culture or subculture steps typically takes approximately 48 hours, using new tissue culture medium in each culture. As used herein, "immunotherapeutic activity" refers to any of a variety of immune cell immune responses. This includes a cytotoxic or antitumor effect. As used herein, "cytotoxicity" includes specific lytic activity, and the non-specific lytic activity of lymphokine-activated annihilating cells (LAK) and natural killer (NK) cells in vi tro. As used herein, "antitumor activity" includes specific lytic activity, and the non-specific lytic activity of lymphokine-activated annihilating cells (LAK) and natural killer (NK) cells both in vi tro and in vivo. In general, the methods of the present invention are preferably directed to enhance the antitumor activity of immune cells, preferably T lymphocytes. Preferably, the method of improving the immunotherapeutic activity of a population of immune cells involves first depleting a subpopulation of lymphocytes. T, for example, populations of peripheral blood mononuclear leukocytes, before starting the culture with an antibody to a lymphocyte surface receptor, optionally with interleukin 2. Initial culture with only one antibody to a lymphocyte surface receptor, and optionally with a relatively minor amount of interleukin 2, it is conveniently used in the present invention. As used herein, the phrase "relatively minor amount of interleukin 2" denotes a stabilizing amount of interleukin 2, ie, an amount sufficient to sustain the culture at approximately its initial cell density. Conveniently, interleukin 2 is present in an amount of less than about 30 percent of the amount of interleukin 2 typically used in culture procedures. When immune cells are optionally cultured in the presence of interleukin 2, this typically means that if interleukin 2 is used, it is used in a "relatively minor amount". For example, if approximately 1,000 interleukin 2 units are used typically to culture a population of specific immune cells, then less than about 300 units would be used in the present invention if interleukin 2 culture is optional, and interleukin 2 is used. Conveniently, interleukin 2 is not used in the initial culture process with the antibody to a lymphocyte surface receptor. In addition, subsets of immune cells are usually conveniently CD4 + or CD8 + lymphocytes, or more specific subsets of each of these populations. As a result of the removal or depletion of specific cell subsets that inhibit antitumor activity, the remaining immune cellsi.e., the population of depleted immune cells and the subsets of immune cells, preferably develop enhanced immunotherapeutic activity as represented by an ability to reduce the volume of tumors in vivo. The cytotoxic activity of the natural killer cells cultured in some situations may be increased above the natural killer cells not cultured by the methods of the present invention. Thus, the population of stimulated depleted immune cells and subsets of stimulated immune cells preferably develop improved immunotherapeutic activity as represented by an ability to reduce tumor volume, ie, antitumor activity, when compared to a population of non-depleted immune cells treated in a similar manner. In order to effect this enhanced immunotherapeutic activity, eg, improved antitumor activity, the initial culture processes described above either of the immune cell populations or the depleted immune cell populations preferably occur over a period of about two days. The cultivation process may be in a period of at least about ten days, but a period of less than 48 hours is more convenient, and more conveniently, less than about 24 hours. According to one embodiment of the invention for improving in vitro cytotoxicity, the culturing process involves: stimulating a depleted population of immune cells or a subset of immune cells with an antibody to a lymphocyte surface receptor during the first 48 hours of culture in a first medium which also optionally contains a relatively minor amount of interleukin 2; remove the population of stimulated depleted immune cells or the subset of stimulated immune cells from the first medium; and optionally subculturing the population of stimulated depleted immune cells or the subset of immune cells stimulated in a second medium containing interleukin 2 without any additional amount of an antibody to a lymphocyte surface receptor. However, further subculturing of the population of stimulated depleted immune cells or subsets of stimulated immune cells is not required to improve immunotherapeutic activity in vivo since the cells are typically administered to a mammal shortly after stimulation. The antibody to a lymphocyte surface receptor can be any variety of monoclonal antibodies against a surface antigen receptor complex. Conveniently, the antibody for a lymphocyte surface receptor is an anti-CD3 antibody, ie, an antibody against the CD3 antigen receptor complex, such as 0KT3. Other useful antibodies include a monoclonal antibody (MoAb) anti-CD2, anti-CD4, anti-CD5, anti-CD28, and anti-CDII, and so on. The antibodies can be used alone or in various combinations with other antibodies. For example, anti-CD3 can be used in combination with anti-CD2, anti-CD4, anti-CD5, anti-CD28, or anti-CDII, for effective results. Anti-CD3 or anti-CD2 can each be used individually as the antibody in the cultures. The anti-CD3 monoclonal antibody can be, but is not limited to, 0KT3, T32, Leu-4, SPV-T3C, RIV9, 64.1, et cetera. More preferably, the anti-CD3 monoclonal antibody is 0KT3, which is available from Ortho, a division of Johnson & Johnson. An additional method useful for improving the immunotherapeutic activity of a population of immune cells comprises (i) first culturing a population of immune cells to form a population of cultured immune cells; (ii) separating a subset of cells, or subpopulation, that is capable of developing immunotherapeutic activity, eg, cytotoxicity to form a population of depleted immune cells or a subset of stimulated immune cells; and optionally (iii) culturing separately, i.e., subculturing, the population of depleted immune cells or a subset of immune cells stimulated in a second medium in the presence of interleukin 2. Preferably, g this method, the immunotherapeutic activity, represented by the ability to reduce tumor volume, this subpopulation is improved compared to a population of non-depleted cells treated in a similar manner. In addition, the separated cells are preferably CD4 + or CD8 + lymphocytes, or subsets of each of these populations. To effect the enhanced immunotherapeutic activity, for example, to increase the antitumor activity, of subpopulations of immune cells prepared according to the methods described above, the initial culture process preferably involves the use of interleukin 2 and an antibody to a cell surface receptor. lymphocyte to produce what are known as annihilating cells activated by CD3 lymphokine or activated killer T cells (T-AK). The process of initial culture of immune cell populations, total, ie not separated or not depleted, may occur in a period of at least about three days, and possibly at least about five days. Alternatively, the initial culture process may involve stimulation by an antibody to a lymphocyte surface receptor and then culturing it with interleukin 2; however, it may not be necessary to culture it with interleukin 2. In any of the above scenarios, the initial culture may be presented over a period of less than about 48 hours, and more conveniently less than about 24 hours. The optional subsequent subculture process in interleukin 2 of each subset of cells preferably occurs in a period of at least about three days, and more preferably at least about ten days. To decrease tumor volume in a mammal that has tumors, the immunotherapeutic activity of a population of immune cells is first improved according to any of the methods described above. This population of stimulated depleted immune cells or subset of stimulated immune cells is then administered to a mammal that has tumors. Optionally, the mammal is pretreated with an immunosuppressant that preferably is also chemotherapeutic. Although not intended to be limited by any theory, the use of an immunosuppressant may serve to suppress the activity of other immune cells thereby allowing the stimulated cells to function more effectively after administration to the mammal, or the immunosuppressant may serve to decrease the volume Total tumor Typically, immunosuppressants such as doxorubicin or cyclophosphamide (Cytoxan) are used, although those skilled in the art readily recognize that other immunosuppressants can be used in accordance with the present invention. The population of stimulated depleted immune cells or the subset of stimulated immune cells is conveniently added with interleukin 2 liposomal. More conveniently, stimulated immune cells are administered intravenously while interleukin-2 liposomal is administered intraperitoneally. Usually, interleukin-2 liposomal is added periodically after initial administration of the population of stimulated immune-boosted cells or the subset of stimulated immune cells. . In addition, the liposomal interleukin 2 and the population of stimulated depleted immune cells or the subsets of stimulated immune cells can be administered to the mammal a second time after the initial treatment. Preferably, the second treatment is carried out about one week after the initial treatment, although a second treatment at any time after about 4 days is effective to substantially eradicate the tumor. As used herein, the phrase "substantial eradication of the tumor" denotes a reduction in tumor volume to a point at which the tumor is completely destroyed or is so small that it is not easily recognized by conventional methods. Mammals that have been treated according to the procedures outlined above are immunized against recurrent tumors. Mammals that are treatable in accordance with the present invention include, but are not limited to, mice, rats, farm animals and pets, primates and humans, and so on. Conveniently, mammals that have received an initial treatment and a second treatment of liposomal interleukin 2 and population of stimulated depleted immune cells or subsets of stimulated immune cells that have improved immunotherapeutic activity, resist and substantially eradicate tumors that are subsequently introduced. These mammals are therefore immunized against the original tumor and tumors similar to this one. The aforementioned immunity can also be transferred to other mammals. Particularly, splenocytes extracted from a mammal that has been treated with interleukin 2 liposomal and a population of stimulated depleted immune cells or a subset of stimulated immune cells that have improved immunotherapeutic activity are useful for immunizing other mammals. Those skilled in the art are capable of extracting splenocytes from the spleen of a mammal using conventional methods. In addition, those skilled in the art are capable of separating several populations of immune cells such as T and B cells from the separated splenocytes. Conveniently, splenocytes are removed from the spleen of a mouse that has been immunized according to the aforementioned procedures. These cells are then administered to a second mouse. This second mouse then develops an immune response to it or similar tumors when it is attacked. For example, the splenocytes of a mouse that has developed antitumor activity by virtue of the immunization procedures mentioned above with respect to, for example the MC-38 tumor, can be administered to a second mouse. If this second mouse is attacked with the MC-38 tumor, the mouse will be able to reduce the size of, and substantially eradicate the tumor. The present invention therefore utilizes a transferable immunity protocol for transferring immunity from a mammal to another mammal with respect to tumors. Interleukin 2 used in culturing depleted cell populations or non-depleted populations is typically free interleukin 2. However, interleukin 2 used when the populations of depleted cells stimulated to a mammal are administered, is conveniently liposomal interleukin 2. Interleukin 2 liposomal is used because it provides a longer, more controlled, and substantial release of interleukin 2, thereby mitigating the toxicity of interleukin 2 and reducing inconvenient side effects from its use. Mammals that undergo bone marrow grafting have a population of bone marrow cells compromised for a period of about 15 to about 25 days. Throughout this description, the term "population of compromised bone marrow cells" denotes a population of incompletely or depleted bone marrow cells compared to the normal bone marrow cell population of mammals. For example, a population of compromised bone marrow cells may include a population of bone marrow cells that is insufficient to achieve homeostasis in the mammalian immune system. The present invention therefore further conveniently provides a method for stimulating the proliferation of bone marrow cells to generate bone marrow cells more easily in a population of compromised bone marrow cells. According to this method, the immunotherapeutic activity of a population of immune cells is improved according to one of the four procedures outlined above. Preferably, the immunotherapeutic activity is enhanced by culturing positively selected CD4 + cells in the presence of anti- CD3 and interleukin 2 for a period of at least 24 hours. More preferably, CD4 + cells are cultured in the presence of anti-CD3 and interleukin-2 during the first 24 hours, and then further cultured in the presence of interleukin-2, and optionally anti-CD3 for at least an additional 48 hours. The population of resultant stimulated depleted immune cells or the subset of stimulated immune cells is capable of stimulating the proliferation of bone marrow cells defined, for example, by an increase in the number of colony forming units. Alternative methods of stimulating the production of bone marrow cells according to the present invention can be carried out. First, bone marrow cells from a mammal can be extracted and purified using techniques well known in the art, and then incubated with the population of stimulated depleted immune cells or the subset of immune cells stimulated for a period of time sufficient to generate cells. of bone marrow. Preferably, the cells are incubated for a period of about 10 to about 20 days and more preferably, about 14 days at about 37 ° C. In addition, cytokines such as GM-CSF, IL-3, KL and Epo can be added to the bone marrow during culture. Kit Ligand (KL) is a soluble factor, such as interleukin 2, which is known in the art, and is being useful in the stimulation of O initial growth and progression of progeny cells during and bone marrow grafting. Obtaining and using KL within the confines of the present invention is within the routine skill of those skilled in the art after reading the present specification. Although it is not intended to be linked to any theory, it is believed that the addition of one or more of the cytokines described above (GM-CSF, IL-3, KL, and the like) to the bone marrow provides a synergistic proliferative effect. The bone marrow cells produced according to this method can be infused in a mammal having a population of committed bone marrow cells using known techniques, particularly, by intravenous injection. Bone marrow cells can also be injected at the same time as the population of stimulated depleted immune cells or the subset of immune cells stimulated during a bone marrow transplant so that the stimulated cells stimulate the proliferation of additional bone marrow and cells. Alternatively, the population of stimulated depleted immune cells or the subset of stimulated immune cells can be administered to a mammal having a population of committed bone marrow cells using known techniques, for example intravenous injection. Optionally, the mammal is pretreated with an immunosuppressant such as cytoxan, which preferably is also chemotherapeutic. Advantageously the population of stimulated depleted immune cells or the subset of stimulated immune cells is periodically administered to the mammal, for example, every four or five days. More advantageously, the population of stimulated depleted immune cells or the subset of stimulated immune cells is administered with an additional cytokine such as interleukin 2, IL-3, GM-CSF, KL, Epo, and the like. The administration of the population of stimulated depleted immune cells or the subset of stimulated immune cells serves to stimulate the proliferation of additional bone marrow cells in a population of compromised bone marrow cells of the treated mammal to generate bone marrow cells more rapidly in comparison with the proliferation of a population of compromised bone marrow cells.

Exhaustion of cell subpopulations Depletion of at least a subset of cells, or subpopulation, such as CD4 +, or CD8 + cells, or specific subsets of any of these cell populations, which is able to down-regulate (prevent effector cells from developing the immunotherapeutic machinery, eg, cytolytic) the immunotherapeutic activity of a population of immune cells, of the total population of immune cells results in the development of improved immunotherapeutic activity, as represented by an improved antitumor effect. The cells can be depleted from natural total immune cells or from a population of immune complete cells that has been stimulated. Specifically, the depletion of a T-cell subpopulation, which inhibits the antitumor activity of a population of immune complete cells, from the population of immune cells results in the development of high levels of lymphokine-killing annihilating activity in "depleted" immune cells. " remaining. Conveniently, depletion of a T cell subpopulation that inhibits the antitumor activity of a total immune cell population from the population of total immune cells, and then stimulation of the depleted cell subpopulation results in the development of High levels of lymphokine-triggered killing activity in the population of stimulated depleted immune cells. This effect occurs in response to culturing the population of depleted immune cells (or whole cells to be depleted), in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin-2 (IL-2). ). Preferably, this is presented in response to the initial stimulation with an antibody to a lymphocyte surface receptor and continuous culture, or subculture, in the presence of interleukin 2 for "in vitro efficacy." Those skilled in the art will recognize, however, that for In vivo efficacy, additional culture in the presence of interleukin 2 is not necessary since the population of stimulated depleted cells will be administered directly to the mammal, together with interleukin 2. For in vitro efficacy, cells are preferably cultured in a first tissue culture medium, the lymphocyte surface receptor antibody, optionally with interleukin 2. After this, the cells are cultured, or subcultured, in a second tissue culture medium with interleukin 2 but are no additional amount of an antibody For a lymphocyte surface receptor, additional cultures, or subcultures, may also be present in the presence of interleukin. ucina 2. Alternatively, the cells can be stimulated with an antibody to a lymphocyte surface receptor alone, and then optionally cultured with interleukin 2 without subsequent culture with interleukin 2. Although preferably no lymphocyte surface receptor antibody is added to any of the subcultures after the first 48 hours, it may be present in subsequent subcultures if the cultured cells of the first culture are not removed before the addition of a second culture medium containing interleukin-2 without any surface receptor antibody. Any protocol, however, for culturing immune cells in which immune cells are in the presence of interleukin-2 and an antibody to a lymphocyte surface receptor at any time and for any period of time in the total course of the culture process as well it is within the scope of the present invention. In addition, any protocol in which immune cells are stimulated with the antibody alone in the tissue culture medium without any further culture with interleukin 2 is within the scope of the present invention. In the present invention, to maintain in vitro viability, cells (population of depleted immune cells) are preferably cultured with interleukin 2 for at least about 2 days, more preferably during at least ten days. Similar results have been obtained from cells cultured in the presence of interleukin 2 for as much as 30 days, with subculture occurring approximately every 48 hours. As mentioned above, cells are preferably stimulated with an antibody to a lymphocyte surface receptor during the first 48 hours of culture, and preferably, only during the first 24 hours. However, for in vivo efficacy the depleted immune cells are typically cultured in the presence of an antibody to a lymphocyte surface receptor for less than about 48 hours, preferably less than about 24 hours. If interleukin 2 is added during the initial culture period, only a relatively minor amount of interleukin 2 is added. Conveniently, less than about 100 units / milliliter of interleukin 2 is added during this initial culture period. After the initial culture period, if the population of stimulated depleted immune cells is to be maintained in vi tro, the presence of additional interleukin 2 is advantageous to maintain and possibly grow the population of stimulated depleted cells or the subpopulation of stimulated cells. After the initial culture period, if the population of depleted immune cells stimulated in a mammal is administered in vivo, then additional culture with interleukin 2 is not required. Interleukin-2 (IL-2) is a T cell growth factor commercially available. It can be interleukin 2 that occurs naturally or can be recombinant interleukin 2. It is believed that other lymphokines may also be used in the present invention to provide the cells activated by lymphokine. These include IL-1, IL-4, IL-6, interferons, and so on. It is envisioned that they can be used alone, in succession, or in combination with interleukin 2 in the culture medium. The liposomal interleukin-2 that is preferably administered in vivo is the commercially available T cell growth factor, only that encapsulated in a liposome. Immune cells, preferably T lymphocytes, and peripheral blood mononuclear lymphocytes more preferably can be depleted of specific T cell subsets by any method. Depletion can be carried out either before the initial culture with an antibody to a lymphocyte surface receptor and, optionally interleukin 2 or after the initial culture of the whole cells. Preferably mononuclear leukocytes from peripheral blood are depleted of specific subsets by negative depletion using magnetic beads. Typically, this involves the labeling of peripheral blood mononuclear leukocytes with an antibody to the lymphocyte surface receptor for T cells that are to be removed from the total population. This mixture of labeled and unlabeled cells is then mixed with magnetic beads coated with goat anti-mouse IgG. A complex of granules and labeled T cells is formed, ie, those complex cells with the surface receptor antibody. The granule / labeled T cell complexes are then separated from the mixture using a magnetic separator. In this way, a specific subset of T cells, or portion thereof, of the mixture of mononuclear leukocytes from the peripheral blood can be removed. The subset of specific immune cells removed can be any that down regulates the immunotherapeutic activity, preferably the cytotoxic activity, of the total population of immune cells. Removed subsets may include: CD4 +, or any of its subsets such as 2H4 or 4B4; CD8 +, or any of its subsets; natural killer cells, or any of their subsets; macrophages; B cells; and similar. Preferably, the subsets of immune cells removed are subsets of T cells, and more preferably are CD4 + or CD8 + cells. In general, a typical sample of mononuclear leukocytes from the peripheral blood of a human whole blood sample contains approximately 20 to 30 percent of CD8 cells and approximately 30 to 50 percent of CD4 + cells. To increase the immunotherapeutic activity, eg, antitumor activity, of a population of immune cells according to the present invention, cells that inhibit or down-regulate the immunotherapeutic activity of the population need only be removed until an increase in immunotherapeutic activity in the remaining cell population. Preferably, in order to increase the immunotherapeutic activity of the immune cells according to the present invention, the number of CD4 + or CD8 + cells is reduced in the depleted immune cell populations by at least about 75 percent, more preferably by. at least approximately 90 percent. More preferably, however, populations of "substantially completely depleted" immune cells, eg, peripheral blood mononuclear leukocyte populations, contain less than about 5 percent of the subset of cells removed. For example, a "population of immune cells substantially completely depleted of CD4 +" contains less than about 5 percent of CD4 + cells. Thus, the method of the present invention includes preferably separating at least about 75 percent, and more preferably at least about 90 percent of the CD4 + or CD8 + cells from peripheral blood mononuclear leukocytes to increase immunotherapeutic activity, by example, antitumor activity, of the remaining depleted immune cell population. The increased immunotherapeutic activity of immune cell populations is determined in vivo by the ability of immune cells to reduce or substantially eradicate a tumor target. However, antitumor activity can be determined in vi tro by comparing the level of radioactivity released in the tissue culture medium of the effector / target combination for the level of radioactivity in the culture medium released from the target alone. Thus, the immunotherapeutic activity of immune cells can be demonstrated by an increase in the percentage cytotoxicity of effector cells in human tumor cells in vi tro, however, efficiency in mammals in vivo is preferred.

Human tumor cell lines useful for determining in vitro efficacy can be any variety of commercially available cell lines, including leukemia cells and new tumor targets, preferably, leukemia cells. However, conveniently, the antitumor activity of stimulated depleted immune cell populations or subpopulations of stimulated cells is determined in vivo by a capacity of the cells to substantially decrease or eradicate tumor volume. As can be seen from FIGURE 1, the amount of cytolytic activity of CD4 + depleted or CD8 + depleted cultures is greater than that of non-separated, ie total or non-depleted, peripheral blood mononuclear leukocytes independently of the effector versus target ratio. The annihilating activity activated by lymphokine was measured at several time points between 10 and 30 days of culture, with similar results regardless of the day of the test. Results are obtained with populations depleted of CD4 + and depleted of CD8 + in cultures of approximately three to five days. However, longer culture periods, that is, in the order of less than 48 hours, are capable of producing similar results. These observations could be explained in various ways. For example, the improved lymphokine-killing annihilator activity could be the result of relative enrichment in CD3"CD16 + and / or CD56 + natural killer cells, or CD8 + CD4 + CD4 + CD4 + CD4 + cells, both of which have demonstrated Previously, they mediate lymphokine-mediated killing activity, however, it has been determined that improved LAK, as well as the activity of natural killers, is preferably the result of the activation of cell subpopulations ie CD4 + or CD8 + T cells This was determined by labeling the cells of mononuclear leukocyte populations of peripheral blood depleted with anti-CD8 monoclonal antibody (in CD4 + depleted cultures) or anti-CD4 monoclonal antibody. (in CD8 + depleted cultures), and positively classifying them using fluorescence activated cell sorter (FACS). When cells from the depleted CD4 + cultures are separated into CD8 + and CD4"CD8" populations, and tested for lymphokine-activated annihilating activity and natural killer cells immediately after sorting, both CD8 + and CD4"CD8 cells "they mediate significant levels of annihilating activity activated by lymphokine and of natural annihilators. As shown in FIGURE 2, "CD8" CD4 cells demonstrate approximately 65 percent cytotoxicity at an effector to target ratio of approximately 30: 1. CD8 + cells demonstrate approximately 40 percent cytotoxicity at an effector to target ratio of approximately 30: 1. Similar results are observed when populations depleted of CD8 + are labeled with anti-CD4 monoclonal antibody and classified into CD4 + and CD4"CD8" populations (Figure 3). Additional phenotyping of CD4CD8 cells "in both types of cultures showing that they are mainly CD16 + Leul9 + or CD3 + CD4" CD8 \ However, in comparison, when cultured with interleukin 2 an antibody to a lymphocyte surface receptor in a population not depleted of peripheral blood mononuclear leukocytes, isolated CD4 + or CD8 + cells do not necessarily develop these significant levels of natural killer activity or lymphokine-triggered annihilator. as can be seen from the results shown in FIGURE 4, at an effector-to-target ratio of approximately 10: 1, the cytotoxicity of the CD4 + or CD8 + cell subsets is not greater than about 15 to 20 percent. Results similar to those shown in FIGURE 4 were obtained at various times between 10 and 30 days of cultivating peripheral blood mononuclear leukocytes not depleted with IL_2 and the anti-CD3 monoclonal antibody OKT3.

CD4 + and CD8 + cells were isolated by negative exhaustion, however, it is also possible to isolate them by positive selection of FACS. The control cultures of CD4 + depleted or CD8 + depleted cells were classified to obtain CD8 + and CD4 + cells, respectively. As shown in FIGURE 4, the CD8 + and CD4 + cells of the depleted cultures developed significant levels of lymphokine-triggered killing activity, i.e., at least about 40 percent cytotoxic activity at an effector to target ratio of approximately 30. :1. Thus, although CD4 + and CD8 + cells do not show significant killing activity triggered by lymphokine or of significant natural killers when tested immediately after isolation of CD3-LAK cultures, CD4 + and CD8 + cells can develop high lymphokine-killing annihilating activity if one of these subsets the mononuclear leukocyte population of the peripheral blood is depleted from the population prior to culture initiation and then stimulated to form a subpopulation of stimulated cells. Conveniently, populations of cells stimulated according to the depletion method described above are used. Although no attempt is made to be limited in any way, these results suggest that the development of lymphocyte-killing activity by T-cell subpopulations is inhibited in peripheral blood mononuclear leukocyte cultures. It is believed that this inhibitory effect is the result of T cells, and possibly other immune cells such as macrophages or B cells, which prevent the development of lymphokine-triggered killing activity of the other subsets of T cells. Furthermore, it is believed that Inhibitory T cells generally exert their effect only if they occur throughout the culture period.

Positive selection of cell subsets The subpopulations, such as CD4 + and CD8 + cells, of specific subsets of cells from these populations, separated from CD3-LAK cells, ie, T-AK cells, show negligible activity. These T-AK cells are typically cultured in the presence of an antibody to a lymphocyte surface receptor and, optionally, interleukin-2 for about five days. However, these subsets of cells can be removed from T-AK cells and then cultured in the presence of an antibody to a lymphocyte surface receptor and, optionally, interleukin 2. It has been determined that if CD4 + and CD8 + cells are cultured sequentially separately in the presence of interleukin-2 alone, each individual population rapidly develops in vi tro a population of stimulated depleted immune cells or a subset of immune cells stimulated with lymphokine-activated annihilating activity. The initial culture of undifferentiated peripheral blood mononuclear leukocyte populations is preferably present in a period of at least about three days, and more preferably at least about five days. The initial culture of positively selected cells from a population of unseparated peripheral blood mononuclear leukocytes that has been or is not first cultured for less than 48 hours, preferably less than 24 hours, is also within the scope of the present invention. The subsequent culturing process in interleukin 2 of each cell subset of preference is presented in a period of at least about three days, and more preferably at least about ten days. The subsequent cultivation process can be carried out up to approximately 30 days. However, for in vivo applications, subsequent culture in the presence of interleukin 2 for extended periods of time is not necessary; instead, subpopulations of cells separated from the stimulated peripheral blood mononuclear leukocyte populations can be administered directly to a mammal or administered after subsequent culture with interleukin 2.

Subsequent culture of the separated CD4 + and CD8 + cell populations is done in the presence of preferably about 100-1000 units / milliliter of interleukin 2. Initial culture in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of an amount Relatively minor interleukin 2, typically is made in the presence of less than about 100 units / milliliter of interleukin 2. After testing for lymphokine-activated annihilating activity at various times throughout the culture process of the subsets of stimulated immune cells, both populations develop rapidly and maintain high levels of activity of natural annihilators and annihilator activated by lymphokine. Specific immune cells, preferably T lymphocytes, can be separated from a non-separate, ie total, population of immune cells, preferably peripheral blood mononuclear leukocytes by any method. Preferably, the subpopulations of specific cells are separated from the total populations by positive selection using monoclonal antibodies labeled by fluorescence. Typically, this involves adding a monoclonal antibody conjugated to fluorescein isothiocyanate or a monoclonal antibody conjugated to phycoerythrin for a population of cultured immune cells, incubating the cells with the conjugate for 30 minutes at 4 ° C, washing the cells, and sorting or selecting the cells labeled using a fluorescence activated cell sorter. To positively select CD8 + cells, the monoclonal antibody 0KT8 can be used, and to select positively CD4 + cells, the monoclonal antibody 0KT4, both available from the Ortho Division of Johnson & Johnson. In addition, subpopulations of murine CD4 + and CD8 + cells can be separated from T cells by using the counterpart of the murine monoclonal antibody, ie, LY2.2 to separate CD4 +. As can be seen in FIGURE 5, both the subsets of CD4 + and CD8 + cells show improved antitumor activity in vi tro after ten days of culturing them in interleukin 2. Although not shown, the enhanced activity is seen in vitro only After three days of cultivation in interleukin 2. Thus, using this methodology both CD4 + and CD8 + T cells can develop high levels of lymphokine-activated annihilating activity. Although this is by no means limiting, the results of the exhaustion and positive selection experiments suggest that mutual inhibition involves some progressive regulatory interaction between CD4 + and CD8 + cells that is abolished effectively once they are separated, and that they do not it is due to an event in the initial activation process. That is, the observation that CD4 + or CD8 + cells can rapidly acquire lymphokine-activated annihilating activity once they are isolated from peripheral blood mononuclear leukocyte cultures suggests that the absence of expression of lymphokine-triggered killing activity by these Cells in the non-separated populations is not the result of an irreversible process. Instead, inhibition requires continued interactions by reciprocal subsets of T cells. Mixed experiments designed to test this hypothesis demonstrate that the maintenance of a suppressive effect requires the continued interaction of metabolically active viable T cell subsets. The inhibition could be mediated through direct contact of the cell or via soluble factors. Regulatory networks have been described in which both subsets of T cells must be present in order to obtain suppression of function [N.K. Damle et al., J. Exp. Med., 158, 159 (1983)]. These data suggest that there are inhibitory signals that prevent the development of lymphocyte-mediated killing activity by CD4 + or CD8 + cells in populations of peripheral blood mononuclear leukocytes not separated, that is, total or not exhausted. That is, there is a negative regulation of the function of the T cells in the mononuclear leukocyte populations of the peripheral blood that seems to be mediated by the same T cells. There are several soluble factors that could be involved to regulate the development of the annihilating activity activated by lymphokine by T cells, including interleukin-4 (IL-4) and transforming growth factor β (TGF- / 3). Of course it is not necessary that the two subsets of T cells are regulated by the same factor. IL-4 has been shown to inhibit both growth and development of effector functions by cellsLAK, although those effects appear to be mainly in natural killer cells instead of T cells. See, for example, M.B. Idmer et al., J ". Exp. Med., 166, 1477 (1987); H. Spits et al., J. Immunol. , 141 ,. 29 (1988); A. Nagler et al., J. I unol. , 141, 2349 (1988); Y. Kawakami et al., J ". Exp. Med., 168, 2183 (1988). IL-4 is mainly made up of CD4 + cells, which suggest that this could play a role in regulating the development of lymphokine-triggered killing activity in CD8 + cells; however, there is no attempt to be limited in any way. See D.B. Lewis et al., Proc. Nati Acad. Sci. USA, 85, 9743 (1988). TGF-jS has also been shown to inhibit both natural killer activity and lymphokine-activated annihilator. See, for example, A. Kasid et al., J. Immunol. , 141, 690 (1988); and J.J. Mulé et al., Cancer Immuno 1. Immunother. , 26, 95 (1988). In addition, this inhibition in some cases has been shown to be based on the balance between the levels of interleukin 2 and TGF-β. See, for example, J.H. Kehrl et al., J. Exp. Med., 163, 1037 (1986). The addition of TGF-j (TGF-β), which is available in R & D Systems, Minneapolis, MN,, to depleted CD4 + or CD8 + populations depleted after the start of the culture process results in the inhibition of lytic function of depleted populations. Specifically, the addition of TGF-β at concentrations ranging from 0.1 to 30 ng / milliliter after the start of the culture, and during each of the subculture steps, demonstrates a dose-dependent decrease in lytic activity, to say, the lymphokine-activated killing activity of T cells. This effect is reversible after the removal of TGF-β from the culture medium. TGF-β is produced by CD4 + and CD8 + cells under certain culture conditions. Since both subsets of T cells produce TGF-β, it is possible that only when both CD4 + and CD8 + cells are present that the level of TGF-β produced reaches the point where it has an inhibitory effect in the presence of high levels of interleukin 2. After depletion of one of the subsets of T cells, TGF-β levels would be too low to have an inhibitory effect on T cells; however, this is not intended to be a limitation in any way. Observations that CD4 + or CD8 + cells can, under appropriate conditions, develop lymphokine-activated annihilating activity and that the generation of that lymphokine-killing annihilating activity is regulated by the presence of the reciprocal T-cell subset, has implications for immunotherapy protocols adoptive. For example, under certain conditions it is preferable to have T-cell-mediated lymphocyte-killing activity. Thus, the present invention includes using T cells with lymphokine-activated annihilating activity for therapy in humans. Stimulated depleted cell populations or subpopulations of stimulated cells can be used to immunize humans against tumors and to transfer immunity from one human to another. These cells may possibly achieve complementary or different antitumor effects that have been observed in conventional protocols. The following examples are presented as representative of the specific and preferred embodiments of the present invention. These examples are not to be construed as limiting the scope of the invention in any way.

It should be understood that many variations and modifications can be made by remaining within the spirit and scope of the invention.

Example 1 Isolation and culture of cells with LAK activity in vi tro Peripheral blood lymphocytes (PBL) were isolated from heparinized venous blood (human whole blood) by centrifugation over Ficoll-Hypaque according to the method of A. Boyum, Scand. J. of Clin. Lab. Invest. , 99, 77 (1968), which is incorporated herein by reference. Isolated mononuclear cells were washed three times with phosphate buffered saline (PBS, pH 7.4) (GIBCO Laboratories, Grand Island, NY) and counted, enriched CD4 + and CD8 + cultures were obtained by negative depletion using magnetic beads (obtained in Baxter Healthcare Corporation, Deerfield, IL; also available in Advanced Magnetics, Massachusetts; or Dynal Corp., Norway). Briefly, peripheral blood mononuclear leukocytes were labeled by incubation with one of the monoclonal antibodies 0KT4 or OKT8 (Ortho, Raritan, NJ) for 30 minutes on ice. Cells were washed twice with cold phosphate buffered saline and mixed with goat anti-mouse IgG-coated magnetic beads (obtained from Baxter Healthcare, also available from Dynal Corp., Norway) at a granule: cell ratio. 10: 1 The granule / cell mixture was incubated for 30 minutes at 4 ° C while rotating at 5-6 revolutions per minute. At the end of the incubation, the granule / cell suspension was diluted twice with cold phosphate buffered saline. Using a magnetic separator (Baxter Healthcare Corporation, Deerfield, IL), the granules were allowed to gather against the side of the test tube for five minutes. The supernatant containing the unbound cells was then collected and transferred to a new tube. This process was repeated three times to completely remove the granules and the cells bound to the granules. Cells that remained in suspension (PBL depleted of CD4 + or depleted of CD8 +) were washed and counted. This resulted in populations of peripheral blood mononuclear leukocytes depleted with less than about 5 percent contamination by the subset of T cells removed. The cells remained essentially unchanged during the subsequent culture. Peripheral blood lymphocytes were cultured (5 x 106 cells) in 25 cm2 flasks (Corning, Corning, NY) in tissue culture medium (TCM). The TCM consisted of Rosewell Park Memorial Institute (RPMI) 1640 medium (available from GIBCO, Grand Island, NY) supplemented with 25 mM of [N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid)] Hepes, 2 M L-glutamine, 100 units / milliliter of penicillin, 100 μg / milliliter streptomycin (the penicillin / streptomycin mixture available from GIBCO, Grand Island, NY), and 5 percent combined heat-inactivated human serum. The cultures were supplemented with 1000 units / milliliter of highly purified recombinant human interleukin 2 from E. coli (Hoffman-LaRoche, Nutley, NJ). [See, A. Wang et al., Science, 224, 1431 (1984); and S.A. Rosenberg et al., Science, 223, 1412 (1984), which are incorporated herein by reference]. The cultures were supplemented with 10 ng / milliliter of the anti-CD3 monoclonal antibody OKT3 (Ortho Division, Johnson &Johnson, Raritan, NJ). 0KT3 was present in the culture during the first 48 hours. After that, OKT3 was diluted due to the addition of new TCM and interleukin 2. No more OKT3 was added during the culture process. The cultures were incubated at 37 ° C in a humidified atmosphere of 5 percent C02. After the first 48 hours of culture, the cells were counted and subcultured at "0.5 x 106 cells / milliliter in TCM containing interleukin 2. Subsequently, the cells were counted and subcultured every 48 hours in fresh TCM with interleukin 2 at a concentration of 0.5 x 106 cells / milliliter. EXAMPLE 2 Cell Classification by Immunofluorescence Mononuclear leukocytes from the peripheral blood, depleted CD4 + peripheral blood mononuclear leukocytes, CD8 + peripheral blood mononuclear leukocytes depleted in 0KT3 + interleukin 2 were cultured as shown in FIG. previously described, at various times during the culture period, the cells were classified in a fluorescence activated cell sorter (FACS) .The populations were labeled with fluorescein isothiocyanate conjugated antibody (FITC) or phycoerythrin conjugate (PE); and OKT8 (Orto). Depleted populations of CD4 + were labeled with OKT8, and quenched populations depleted of CD8 + with OKT4. The cells were incubated for 30 minutes at 4 ° C and then washed twice with cold phosphate buffered saline containing 2 percent fetal bovine serum. The cells were classified in a FACS IV (Becton Dickinson, Mountain View, CA). The sorted cells were centrifuged and an aliquot was stained again to test the purity of the populations. All the positively classified populations used to determine lymphokine-killing annihilating activity were more than 97 percent positive for the desired surface marker.

EXAMPLE 3 Cell-mediated lympholysis (CML) In Vitro CML tests were performed as described in S.L. Wee et al., Hum. I unol. 3, 45 (1981), which is incorporated herein by reference. The human tumor lines K562 (chronic myelogenous leukemia, obtained from the American Tissue Type Culture Collection (ATTCC)) and HL60 (promyelocytic leukemia, ATTCC) were maintained in culture in RPMI 1640 with 10 percent fetal bovine serum (GIBCO, Grand Island, NY). The cells were subcultured at 0.5 x 106 / milliliter in fresh medium twice a week. Cells of the HL60 line were not used by peripheral blood mononuclear leukocytes not stimulated and therefore considered resistant to natural annihilators. The annihilating activity activated by lymphokine was measured as cytolytic against HL60 targets resistant to natural annihilators. The activity of the natural annihilators was measured as a cytolytic activity against the K562 targets. Targets of tumor cell lines were labeled with 250-270 μCi of Na51Cr04 (5000 μCi / milliliter, New England Nuclear, Boston, MA) for one hour at 37 ° C. These cells were washed three times in TCM, resuspended in culture medium that did not contain interleukin 2, counted and aliquots of 500 targets / well were removed in a 96-well V-bottom tray (Costar, Cambridge, MA ) in which peripheral blood lymphocytes or cultured populations were previously aliquoted as fixed concentrations as described above in Example 1. The target cell effector ratios ranged from 30: 1 to 1: 1. The trays were centrifuged at 65 g for five minutes and incubated in 5 percent CO2 at 37 ° C for four hours, after which lOOμl of average from each well was harvested in a flask bottle with 2.5 milliliters of fluid. scintillation (Biofluor, New England Nuclear, Boston, MA). 'The radioactivity was counted in a liquid scintillation counter (LKB, Turku, Finland). The percentage of cytotoxicity was determined by the following equation (cpm = counts per minute): (average experimental cpm) - (cpm mean spontaneous release) x 100 (cpm mean maximum release) - (cpm mean spontaneous release) where "average release spontaneous "is defined as the amount of 5iCr released from target cells alone (background); "maximum release" is the total of 51 Cr in the targets after lysis with a detergent such as Triton X-100; and "experimental media" is the 5 | Cr released in wells with objectives and effectors. Representative samples of the results of all the in vi tro experiments are exhibited in FIGURES 1-5. Each data point in each FIGURE represents an analysis of three separate samples analyzed after the same growing period, that is, between 10 and 30 days, and the same period of contact between the effectors and the objectives, that is, approximately four hours . Example 4 Immunophenotyping Cells (1 x 106) were washed 3 times with HBSS after which they were incubated at 4 ° C with 20 μl of the corresponding monoclonal antibody (0KT4 or 0KT8 from Ortho, Rareton, New Jersey). They were washed again 3 times with Hanks Balanced Saline Solution (HBSS) including 2 percent fetal bovine serum and resuspending in 0.2 percent paraformaldehyde. Two color fluorescence measurements were performed in a Coulter Profile (Coulter Cytometry, Hialeah, FL) or a FACS IV (Becton Dickinson, Mountain View, CA). Example 5: Mixed experiments CD4 + or CD8 + cells isolated from peripheral blood cultures of non-separated mononuclear blood cells were added.

T-AK on day 5, in a 1: 1 ratio in populations depleted of CD4 +, or depleted of CD8 + respectively, on day 0 of autologous cultures. Some of the cultures received radiated cells. The lithic function was tested four days later as a percentage of cytotoxicity. The results as shown below in Table 1 demonstrated that the addition of non-viable radiated cells did not prevent the development of lytic activity. However, the addition of non-irradiated, ie, metabolically active, CD4 + or CD8 + cells, completely suppressed the development of lymphokine-killing annihilating activity by depleted CD4 + or CD8 + depleted populations, respectively. Table 1 CD4 + and CD8 + cells of PBL cultures inhibit the development of lytic function by depleted CD4 + and depleted CD8 + cultures.

% Cytotoxicity 30: 1 10: 1 3: 1 CD4 + depleted 38 26 15 CD4 + depleted + CD4 + de PBL -4 - 1 1 CD4 + depleted + CD4 + b irrad. 42 26 11 CD8 + depleted 25 14 - 9 CD8 + depleted + CD8 + de PBL -1 2 -4 CD8 + depleted + CD8 + b irrad. 46 30 15 a CD4 + and CD8 + cells were positively classified as mononuclear leukocytes from the peripheral blood stimulated with 0KT3 + interleukin 2 and cultured for 5 days. The cells were added in a ratio of 1: 1 to autologous cultures which have been depleted of CD4 + or CD8 + cells, respectively. The lytic function was tested in the HL60 cell line. b The isolated CD4 + and CD8 + cells received 250 rads.

Example 6 Improve the inunotherapeutic activity of cell subpopulations in vivo using anti-CD3 alone The animal model used in the following experiments uses a tumor known as murine carcinoma MC-38 or MCA-38. The methods for using this animal model are well documented and described in, for example Lafreniere et al., MC-38 Adenocarcinoma Tumor Infil trating Lymphocytes. . . , J. SURG. ONCOL. 43: 8-12 (1990); Johnkoski et al., Hepatic Metastasis Al ters the Immune Function of Murine Liver Nonparenchymal Cells, ARCH. SURG. 127: 1325-29 (1992); Lafreniere and collaborators, Adoptive I munotherapy of Murine Hepatic Metastaees. . . , J. IMMUNOL., 135: 4273-80 (1985); and Pope and collaborators, Ant tumor Effi cacy of Lymphokine -Activated Killer Cells. . . , CANCER RES. 46: 4973-78 (1986). The descriptions of these documents are incorporated by reference herein in full. T cells were purified from splenocytes that were removed from the spleen of a C57BL / 6 mouse according to conventional procedures. See, Lafreniere (1985), supra. The purified T cells were divided into two groups, and the first group of T cells were not depleted. The second group of T cells was contacted with anti-CD8 (LY2.2) and passed over a cell purification column (R & D Systems of Minneapolis) to produce enriched CD4 + cells. The first and second groups of T cells (ie, non-separated and subpopulation of CD4 + cells, respectively) were cultured with murine anti-CD3 (145-2C11) for a period of about 24 hours. Then the cells were harvested, washed and counted according to the procedures delineated in the present specification. C5BL / 6 mice were injected with MCA-38 as described above, and had tumors ranging from about 140 to about 220 mm 3. These mice were initially injected with 150 mg / kg / mouse of Cytoxan, an immunosuppressant and a chemotherapeutic agent, before immunotherapy with cultured cells depleted and not depleted. Approximately 20 million stimulated and unextracted CD4 + cells cultured per mouse were administered intravenously together with an intraperitoneal injection of approximately 50,000 liposomal interleukin 2 units. The volume of the tumors was measured after a period of 2, 5 and 8 days. The results are illustrated in FIGURE 6. A group of mice that had tumors in the range of 140 to approximately 220 mm3 did not receive treatment (represented by the black box in Figure 6) another group received Cytoxan injections only (represented by the empty triangle in FIGURE 6). A third group of mice received Cytoxan and liposomal interleukin 2 (represented by the empty box in Figure 6), while a fourth group of mice received an intravenous injection of cultured, non-exhausted cells, and an intraperitoneal injection of interleukin 2 (represented by the black circle in FIGURE 6). The fifth group of mice represented the group of the invention, and received an intravenous injection of cultured depleted cells and an intraperitoneal injection of liposomal interleukin 2 (represented by the black triangle in FIGURE 6). As can be seen from FIGURE 6, the subpopulation of stimulated cells cultured only in the presence of an antibody to a lymphocyte surface receptor for less than 48 hours, showed improved antitumor immunotherapeutic activity compared to unseparated cells treated in a similar manner. The person skilled in the art would reasonably expect that the subpopulation of immune cells stimulated in the presence of an antibody to a lymphocyte surface receptor for less than 48 hours would provide a similar result in humans.

Example 7 Enhance the immunotherapeutic activity of cell subpopulations in vivo using anti-CD3 and interleukin 2 CD4 + cells and whole T cells were prepared according to the procedures delineated in Example 6 above. Whole T cells and CD4 + cells were then stimulated in the presence of a murine anti-CD3 (145-2C11) and approximately 100 units / milliliter of free interleukin-2 for a period of about 24 hours in a murine lymphocyte culture medium. at a concentration of approximately 1.5 million cells per milliliter. The cells were then harvested, washed and counted according to the procedures delineated in the present specification. CC57BL / 6 mice were injected with MCA-38 as described above approximately 7 days before treatment with cytoxan, and had tumors ranging from about 140 to about 220 mm 3. These mice were initially injected with 150 mg / kg / mouse of Cytoxan, an immunosuppressant and a chemotherapeutic agent, approximately four days prior to immunotherapy with subsets of immune cells and cultured non-depleted cells. Stimulated CD4 + cells and non-depleted T cells were intravenously administered to C57BL / 6 mice affected with the MCA-38 tumor together with an intrapritoneal injection of approximately 50,000 units of interleukin-2 liposomal. Intraperitoneal injections of liposomal interleukin 2 were repeated for approximately 4 days. After seven days after treatment with the subset of stimulated immune cells and cultured whole cells, the mice that received the subsets of stimulated CD4 + cells were again injected intravenously with stimulated CD4 + cells and with an intraperitoneal injection of interleukin 2 liposomal. The volume of tumors was periodically measured over a period of approximately 45 days. The results are illustrated in FIGURE 7. A group of mice having tumors in the range of 140 to about 220 mm 3 (represented by the empty rhombus in Figure 7), another group received Cytoxan injections only (represented by the empty circle in Figure 7). A third group of mice received Cytoxan and liposomal interleukin 2 (represented by the empty triangle in Figure 7), while a fourth group of mice received an intravenous injection of cultured unexploited cells and cytoxan, and an intraperitoneal injection of liposomal interleukin 2 (represented by the empty box in Figure 7). The fifth group of mice represented the group of the invention, and received an intravenous injection of cytoxan before an intravenous injection of subpopulation of CD4 + cells and an intraperitoneal injection of interleukin-2 liposomal (represented by the black box in Figure 7). As can be seen in Figure 7, the subpopulation of stimulated cells cultured in the presence of an antibody to a lymphocyte surface receptor and a relatively minor amount of interleukin 2 for less than 48 hours, showed improved antitumor immunotherapeutic activity compared to cells not separated treated in a way to assimilate. Undoubtedly, administration of subpopulations of CD4 + cells to the mice initially and on day 7 together with cytoxan and interleukin 2 liposomal resulted in a substantial eradication of the MCA-38 tumor after approximately 14 days. One skilled in the art would expect that a subset of human stimulated immune cells grown in the presence of an antibody to a lymphocyte surface receptor and a relatively lower amount of interleukin 2 for less than 48 hours would provide a similar result in humans.

Example 8 Long-term immunity of mammals treated with subpopulations of cells having improved immunotherapeutic activity in vivo Eight C57BL / 6 mice received the initial and second administration of stimulated CD4 +, cytoxan and liposomal interleukin 2 were challenged again with the same MCA tumor -38 (1 x 106 cells administered subcutaneously) in the right hind flank 82 days after the initial treatment. Control C57BL / 6 mice that did not receive treatment were also challenged again with the same MCA-38 tumor. All control mice died by tumor while all eight treated mice survived all and had no tumor after 45 days. Four of these eight mice were challenged again with a different tumor (melanoma B16 1 x 106 cells administered subcutaneously) and died in two weeks of tumor. The remaining four treated mice as well as a group of control mice were challenged again with the same MCA-38 tumor after 180 days of initial treatment. Again, all the control mice died of tumor, but the treated mice did not develop any tumor after 30 days. This example clearly illustrates the long-term immunity of subpopulations of stimulated CD4 + cells when administered with cytoxan intravenously together with an intraperitoneal injection of interleukin-2 liposomal. Therefore, the mice to which the stimulated cells mentioned above were administered developed an immunity to the tumors and substantially eradicated the tumors even before being threatened with the tumors three times in a period of 180 days.

Example 9 Transferable immunity of mammals treated with subpopulations of cells having improved immunotherapeutic activity in vivo The four remaining C57BL / 6 mice that survived the tumor challenges described in Example 8 above were sacrificed. Splenocytes were extracted from the sacrificed mice using the conventional techniques outlined above. Approximately 5 x 10 7 cells of splenocytes from the treated mice were injected into mice without C57BL / 6 treatment. These treated mice as well as the control mice were threatened with MCA-38 tumor (1 x 105 cells subcutaneously). All control mice died of tumor in 30 days while mice treated according to the present invention exhibited no evidence of tumor. These results indicate the effect of transferable immunity in mammals of the subpopulations of CD4 + cells stimulated and administered according to the present invention.

Example 10 Increasing the number of bone marrow cells Bone marrow cells were obtained from a donor by conventional methods and separated on Ficoll-Hypaque and frozen. Cells from the same human donor (P.L; M42389) were also obtained by leukophoresis, separated on Ficoll-Hypaque and frozen. These cells were subsequently thawed and separated using human T cells and columns of human CD4 + subsets (R &D Systems Human Select) according to the game instructions. The subsets of separate CD4 + cells were then activated with 10 ng / milliliter of human anti-CD3 and placed in a culture with 100 units / milliliter of interleukin-2 for 24 hours to produce subsets of stimulated CD4 + cells. These subsets of cells were then placed in colonies tests, according to conventional procedures, in various combinations with new bone marrow cells, separated by Ficoll (P.L., M42961) and incubated for 14 days at 37 ° C. Bone marrow (BM) and CD4 + cells stimulated in a ratio of 1: 1 (105: 105, BM: CD4 +) were plated when used in combination or plated at a concentration of 10 5 cells / plate when plaqueed alone. Several combinations of BM and CD4 + cells and conditioned medium (CM) were incubated together with cytokines such as interleukin 2, GM-CSF, IL-3, KL, Epo for 14 days at 37 ° C. HE . They counted the colonies after 14 days of incubation where 50 or more cells constitute a colony. Three trials were performed for each combination. The results are shown in Table 2 below.

Table 2 These results illustrated above indicate that stimulated CD4 + cells produce several cytokines that when combined with bone marrow cells are able to stimulate the growth of bone marrow cells. Even without the addition of cytokines, stimulated CD4 + cells, when contacted with bone marrow and interleukin 2 cells stimulated the production of significantly more bone marrow cells than bone marrow or CD4 + cells alone. The addition of KL to the bone marrow cells provided a synergistic proliferative effect since, it is believed, KL helps stimulate the early growth and progression of bone marrow progenitor cells.

EXAMPLE 11 Increase in the number of bone marrow cells after 72 hours of activation The same procedures were carried out as above, only that the separated CD4 + were activated for 24 hours and for 72 hours to produce two separate groups of subsets of CD4 + cells stimulated. These two groups were then incubated with bone marrow and various combinations of exogenous cytokines for 14 days at 37 ° C. In addition, the Ligand Kit cytokine was added to the combination BM + CD4 + + interleukin 2 used in Example 10. The results are shown in Table 3 below.

Table 3 The results illustrated in Table 3 above indicate that three additional days of culture of the stimulated CD4 + cells stimulated the production of bone marrow cells further without the addition of exogenous cytokines. However, the use of CD4 + cells, interleukin 2 and KL provide significantly enhanced stimulation of the production of bone marrow cells. The additional activation period of 72 hours compared to that of 24 hours indicates that the amount of cytokine production by the stimulated cells was increased further, thereby increasing the total number of bone marrow cells produced. The invention has been described with reference to several specific and preferred embodiments. However, it should be understood that many variations and modifications can be made as long as they remain in the spirit and scope of the invention. The relevant portions of the references cited herein are incorporated by reference.

Claims (30)

1. A method for improving the ability to develop cytotoxic activity of immune cells comprising a method selected from the group consisting of method 1: (a) culturing a population of immune cells in the presence of an antibody to a lymphocyte surface receptor, optionally in presence of interleukin 2 to form a population of cultured immune cells; (b) separating at least one subpopulation of cells, which is capable of developing immunotherapeutic activity, from the population of cultured immune cells to form a population of stimulated depleted immune cells; and (c) optionally subculturing the population of depleted immune cells stimulated in a second medium containing interleukin-2; wherein the subpopulation of depleted immune cells exhibits improved ability to develop cytotoxic activity compared to a population of immune cells not depleted treated in a similar manner, and method 2: (a) separating and selecting at least one subpopulation of cells from a population of immune cells to form a subset of immune cells, wherein the subpopulation of cells removed is capable of down-regulating the immunotherapeutic activity of the immune cell population, (b) culturing the subset of immune cells in the presence of an antibody for a lymphocyte surface receptor, optionally in the presence of interleukin 2 for a sufficient time to form a subset of stimulated immune cells, and (c) optionally subculturing the eubset of stimulated immune cells in a second medium containing interleukin 2; wherein the subpopulation of stimulated depleted immune cells display u An improved ability to develop cytotoxic activity compared to a population of non-depleted immune cells treated in a similar manner.

The method of claim 1, wherein the step of separating cells comprises separating CD4 + lymphocytes from the population of immune cells to form a population of CD4 + depleted immune cells and a subpopulation of CD4 + cells.

The method of claim 2, wherein the antibody for a lymphocyte surface receptor is an anti-CD3 monoclonal antibody, and the cytotoxic activity is lymphokine-activated annihilating activity.

4. The method of claim 2, wherein the step of separating cells comprises separating at least about 75 percent of the subpopulation of cells from immune cells.

5. A method for improving the lymphokine-activated killing activity of immune cells comprising: (a) separating CD4 + lymphocytes from a population of immune cells to form a subset of immune CD4 + cells; (b) culturing the subset of immune CD4 + cells in the presence of an anti-CD3 monoclonal antibody, optionally in the presence of interleukin-2 for less than 48 hours from a subset of stimulated immune CD4 + cells; and (c) subculturing the subset of immune CD4 + cells stimulated in the presence of interleukin 2 wherein the subset of stimulated immune CD4 + cells exhibits improved ability to develop lymphokine-killing annihilating activity compared to a population of non-depleted immune cells treated in a similar manner .

6. A method for treating a mammal having tumors comprising the steps of: (a) enhancing the ability to develop cytotoxic activity of a population of immune cells according to the method of claim 1, (b) administering optionally to the mammal an immunosuppressant before steps (c) and (d); (c) administering to the mammal the population of stimulated depleted immune cells or the subset of stimulated immune cells prepared in step (a); and (d) administering the liposomal interleukin 2 mammal.

The method of claim 6, wherein the step of separating cells comprises separating CD4 + lymphocytes from the population of immune cells to form a population of depleted immune CD4 + cells and a subset of CD4 + cells.

8. The method of claim 7, wherein the antibody for a lymphocyte surface receptor is the anti-CD3 monoclonal antibody, and the cytotoxic activity is lymphokine-activated annihilating activity.

The method of claim 8, wherein the immunosuppressant is Cytoxan.

The method of claim 6, wherein step (a) comprises separating a subset of CD4 + cells from the population of immune cells, and culturing the subset of CD4 + cells in the presence of anti-CD3 and a relatively minor amount of interleukin 2 for less than 48 hours to provide a subset of stimulated CD4 + immune cells.

The method of claim 6, wherein the relatively minor amount of interleukin 2 is less than about 100 units / milliliter.

12. A method for treating a mammal having tumors comprising the steps of: (a) separating CD4 + lymphocytes from a population of T cells to form a subpopulation of removed CD4 + cells; (b) culturing the subpopulation of CD4 + cells removed in the presence of a? nti-CD3 monoclonal antibody and approximately 100 units / milliliter of interleukin-2 for a sufficient time to form a subpopulation of stimulated CD4 + cells; wherein the subpopulation of stimulated CD4 + cells exhibits increased ability to develop cytotoxic activity compared to a population of non-depleted immune cells treated in a similar manner; (c) administering to the mammal Cytoxan before steps (d) and (e); (d) administering to the mammal the subpopulation of stimulated CD4 + cells prepared in step (b); and (e) administering the mammal liposomal interleukin 2.

13. A method of immunizing a mammal against tumors comprising the steps of: (a) enhancing the ability to develop cytotoxic activity of a population of immune cells according to the method of claim 1, (b) optionally administering to the mammal a immunosuppressant before steps (c) and (d); (c) administering to the mammal the population of stimulated depleted immune cells or the subset of stimulated immune cells prepared in step (a); and (d) administering the liposomal interleukin 2 mammal.

The method of claim 13, wherein the step of separating cells comprises separating CD4 + lymphocytes from the population of immune cells to form a population of depleted immune CD4 + cells and a subset of CD4 + cells.

The method of claim 14, wherein the antibody for a lymphocyte surface receptor is the anti-CD3 monoclonal antibody, and the cytotoxic activity is lymphokine-activated annihilating activity.

16. The method of claim 15, wherein the immunosuppressant is Cytoxan.

The method of claim 13, wherein step (a) comprises separating a subset of CD4 + cells from the population of immune cells, and culturing the subpopulation of CD4 + cells in the presence of anti-CD3 and a relatively minor amount of interleukin. 2 for less than 48 hours to provide a subset of stimulated CD4 immune cells.

18. The method of claim 15, wherein the relatively minor amount of interleukin 2 is less than about 100 units / milliliter.

19. A method for immunizing a mammal against tumors comprising the steps of: (a) separating CD4 + lymphocytes from a population of T cells to form a subset of immune CD4 + cells; (b) culturing the subset of immune CD4 + cells in the presence of an anti-CD3 monoclonal antibody and approximately 100 units / milliliter of interleukin-2 for less than 48 hours, and then subculturing in the presence of interleukin-2 to form a subset of immune cells. CD4 + stimulated; wherein the subset of stimulated CD4 + cells exhibits increased ability to develop lymphokine-activated annihilating activity compared to a population of non-depleted immune cells treated in a similar manner; (c) administering to the mammal Cytoxan before steps (d) and (e); (d) administering to the mammal the subset of stimulated CD4 + immune cells prepared in step (b); (e) administering the mammal liposomal interleukin 2; (f) repeat steps (c), (d) and (e) after 7 days.

20. A method for transferring immunity of a mammal to a second mammal comprising the steps of first immunizing a mammal by: (a) enhancing the ability to develop cytotoxic activity of a population of immune cells according to the method of claim 1, (b) optionally administering to the mammal an immunosuppressant before steps (c) and (d); (c) administering to the mammal the population of stimulated depleted immune cells or the subset of stimulated immune cells prepared in step (a); and (d) administering the liposomal interleukin 2 mammal. (e) repeat steps (b), (c) and (d) after a period of more than approximately 5 days; and (f) extracting splenocytes from the mammal treated according to steps (a) to (e); and (g) administering the splenocytes from step (g) to a second mammal.

The method of claim 20, wherein the step of separating cells comprises separating CD4 + lymphocytes from the population of immune cells to form a population of depleted immune CD4 + cells and a subset of CD4 + immune cells.

22. The method of claim 21, wherein the antibody for a lymphocyte surface receptor is the anti-CD3 monoclonal antibody, and the cytotoxic activity is lymphokine-activated annihilating activity. 2. 3 .

The method of claim 22, wherein the immunosuppressant is Cytoxan.

The method of claim 20, wherein step (a) comprises separating a subset of CD4 + cells from the population of immune cells, and culturing the eubpopulation of CD4 + cells in the presence of-anti-CD3 and a relatively smaller amount of interleukin 2 for less than 48 hours to provide a subset of stimulated CD4 + immune cells.

25. The method of claim 20, wherein the relatively minor amount of interleukin 2 is less than about 100 units / milliliter.

26. A method for transferring the immunity of a mammal to a second mammal comprising the first birds to immunize a mammal by: (a) separating CD4 + lymphocytes from a population of T cells to form a subset of immune CD4 + cells; (b) culturing the subset of immune CD4 + cells in preemption of an anti-CD3 monoclonal antibody and about 100 units / milliliter of -interleukin 2 for less than 48 hours, and then subculturing in the presence of interleukin 2 to form a subset of immune cells CD4 + stimulated; wherein the subset of stimulated CD4 'cells exhibits increased ability to develop lymphokine-triggered killing activity compared to a population of non-depleted immune cells treated in a similar manner; (c) administering to the mammal Cytoxan before paeos (d) and (e); (d) administering to the mammal the subset of stimulated CD4 + immune cells prepared in step (b); (e) administering the mammal liposomal interleukin 2; (f) repeat steps (c), (d) and (e) after 7 days. (g) extracting splenocytes from the mammal treated in accordance with steps (a) through (f); and (h) administering the splenocytes from step (g) to a second mammal.

27. A method for stimulating the proliferation of bone marrow cells comprising the steps of: (a) enhancing the immunotherapeutic activity of a population of immune cells according to one of methods 1 or 2; and (b) incubating the population of immune cells of step (a) with bone marrow and interleukin 2, wherein method 1 comprises: (a) culturing a population of immune cells in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin 2 to form a population of cultured immune cells; (b) separating at least one cell subpopulation, which is capable of de-coiling immunotherapeutic activity, from the cultured cell population and immune to form a population of stimulated depleted immune cells; and (c) optionally eubcultivating the population of diseased immune cells and stimulated in a second medium containing interleukin-2; wherein the subpopulation of depleted immune cells exhibits improved ability to develop immunotherapeutic activity compared to a population of non-depleted immune cells treated in a similar manner; and method 2 comprises: (a) separating and selecting at least one subpopulation of cells from a population of immune cells to form a subset of immune cells; wherein the removed cell subpopulation is capable of down-regulating the immunotherapeutic activity of the immune cell population; (b) culturing the eubset of immune cells in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of interleukin 2 for a long time to form an eubset of stimulated immune cells; and (c) optionally subculturing the subset of immune cells stimulated in a second medium containing interleukin 2; wherein the subpopulation of stimulated depleted immune cells exhibits an enhanced ability to develop immunotherapeutic activity compared to a population of immune cells not depleted treated in an animilar manner.

28. The method of claim 27, wherein the population of immune cells from the pae (a) is eubset of stimulated CD4 + immune cells, and the immunotherapeutic activity of the eubset of CD4 + immune cells is enhanced by the stimulation in preemption of anti-CD4 +. CD3 and interleukin 2 over a period of approximately 24 hours.

29. The method according to claim 27, wherein the stimulation is carried out for a period of about 72 hours.

30. The method according to claim 29, wherein Kit Ligand is added during the incubation step (b).