WO2001032843A9 - Enhanced immune recognition of pathogenic cells by icsbp expression - Google Patents

Abstract

The present invention relates to the use of ICSBP to enhance an individual's ability to eliminate cells that cause a condition in the individual, particularly by enhancing an individual's immune response to the cells (e.g., tumor cells, such as leukemia cells, including chronic myeloid leukemia cells, and solid tumor cells and cells infected with a pathogen). It further relates to genetically engineered or modified cells, such as genetically engineered somatic cells or tumor cells, which express ICSBP from exogenous DNA that encodes ICSBP or a precursor thereof or from endogenous DNA which has been manipulated, as described herein, in order to activate normally silent endogenous ICSBP-encoding DNA or to enhance the level of expression of endogenous DNA.

Description

ENHANCED IMMUNE RECOGNITION OF PATHOGENIC CELLS BY ICSBP EXPRESSION

BACKGROUND OF THE INVENTION

The immune system plays an important role in protecting individuals from, a wide variety of diseases and conditions, such as malignancies, infections and autoimmune conditions. Unfortunately, in many instances, an individual's immune system is not able to adequately protect the individual from the adverse effects of such conditions and other forms of treatment, such as drugs, radiation and surgery, are necessary as well. The ability to manipulate and, particularly enhance, an individual's immune response to a particular condition would be of great benefit.

SUMMARY OF THE INVENTION

The present invention relates to the use of ICSBP to enhance an individual's ability to eliminate cells that cause a condition in the individual, particularly by enhancing an individual's immune response to the cells (e.g., tumor cells, such as leukemia cells, including chronic myeloid leukemia cells, and solid tumor cells and cells infected with a pathogen). It further relates to genetically engineered or modified cells, such as genetically engineered somatic cells or tumor cells, which express ICSBP from exogenous DNA that encodes ICSBP or a precursor thereof or from endogenous DNA which has been manipulated, as described herein, in order to activate normally silent endogenous ICSBP-encoding DNA or to enhance the level of expression of endogenous DNA. The cells of the present invention are administered to an individual by an appropriate route, such as injection (e.g., intramuscular, subcutaneous, intraperitoneal) or infusion (e.g., intravenous), and in a therapeutically effective quantity or amount in order to enhance the individual's ability to eliminate cells that cause a condition in the individual and particularly to enhance the individual's immune response to the cells. In particular, the present invention is a method of enhancing an individual's immune response to target cells (e.g., tumor cells) as a result of expression of ICSBP in target cells. The method of the present invention, thus, provides a method of reducing (totally or partially) the occurrence of tumor cells (cancer) in an individual and a method of reversing (totally or partially) tumor cells that have been established in an individual. It, thus, provides a method of preventing or reducing the extent to which tumor cells (e.g., leukemia cells, solid tumor cells) already present in an individual and a method of reducing (totally or partially) the extent to which tumor cells already present in an individual spread or increase in number. It further provides a method of preventing or reducing the extent to which an infection occurs in an individual.

The modified tumor cells used in the method are the same type of tumor cells against which an enhanced immune response is desired or another type of cell (a tumor cell different from the type against which an immune response is desired or a nontumor cells) that expresses an oncogene(s) and/or other gene(s) that cause the cells to be the functional equivalent of the type of tumor cell against which the enhanced response is desired. The modified tumor cells express ICSBP from exogenous DNA (DNA that is introduced into the cells or ancestors thereof) or from endogenous DNA that encodes ICSBP or a precursor thereof and is silent (not expressed) in cells as obtained and is activated in the cells or ancestors thereof or is expressed in cells but whose expression is enhanced in the cells or in ancestors thereof through genetic engineering techniques. Thus, the term genetically engineered or modified cells includes the cells that are manipulated (e.g., to introduce exogenous DNA or activate or enhance expression of endogenous DNA; and progeny and derivatives thereof. The modified cells, such as modified tumor cells, that are used in the present method can be cells obtained from the individual into whom modified cells are introduced or can be obtained from another individual (of the same or of a different species).

In one embodiment of the method of the present invention, the modified tumor cells are introduced into an individual, in whom an enhanced immune response against tumor cells is desired, in whom no tumor cells are known to be present (e.g., prior to the occurrence of leukemia or a solid tumor) and in whom the modified tumor cells can be seen to act as a vaccine. The individual responds to the introduced modified tumor cells by mounting an immune response, which can be a systemic response or a local response. Typically, in the case of an individual with leukemia, the route of administration used will be one (e.g., infusion) that will result in production of a systemic immune response. Subsequent challenge of the individual by tumor cells (e.g., the presence of leukemia cells, such as chronic myeloid leukemia cells) results in an enhanced immune response by the individual (a response greater than would have occurred if modified tumor cells had not been introduced into the individual), with the result that the tumor cells which are present in the individual are prevented from becoming established or become established to less of an extent than would have occurred if the modified tumor cells had not been previously introduced into the individual. In another embodiment of the method, modified tumor cells are introduced into an individual in whom tumor cells are known to be present, with the result that the individual mounts an enhanced immune response to the tumor cells (an immune response greater than that which would have been mounted in the absence of introduction of the modified tumor cells) and some or all of the tumor cells in the individual are eliminated. As a result, the extent to which the tumor cells (e.g., leukemia, such as chronic myeloid leukemia or solid tumor cells) are present in the individual is less than it would have been if the individual had not been treated with the modified tumor cells. Thus, the present method also provides an approach to reduce the extent to which tumor cells in an individual increase.

In a specific embodiment of the present invention, leukemia cells, such as chronic myeloid leukemia cells, are modified to express ICSBP. For example, a vector comprising DNA or RNA encoding ICSBP is introduced into leukemia cells, such as CML cells, to produce genetically engineered or modified tumor cells (modified CML cells) in which ICSBP is expressed. A sufficient number of modified leukemia cells to produce an enhanced immune response to tumor cells is introduced into an individual, such as an individual at risk for leukemia, an individual who has been or is being treated with other forms of therapy (e.g., chemotherapy, radiation therapy) or an individual for whom other therapies have not been effective. The quantity of cells and the schedule of their administration (e.g., the frequency with which and the time period over which they are given) can be determined empirically and will vary depending on such considerations as, for example, the severity of an individual's condition, the individual's general health, age, size, and gender.

The present invention also provides an adjuvant for use in enhancing an individual's immune response to a substance or molecule (e.g., a tumor cell antigen or antigen of a pathogen) against which a stronger immune response (one greater than is evident in the absence of administration of ICSBP according to the present invention) is desired. In this embodiment, ICSBP-expressing cells of the present invention are given in conjunction with the substance or molecule against which the enhanced immune response is to be elicited. For example, genetically engineered cells co-expressing ICSBP and the substance or molecule are administered to an individual, in whom an enhanced immune response occurs due to the presence of ICSBP. Alternatively, ICSBP-encoding DNA or RNA can be present and expressed in one cell and the target antigen (the substance or molecule against which an enhanced immune response is to be elicited) can be present in or on the surface of a second cell. In this embodiment, it is necessary that the two (ICSBP and the target antigen) are administered sufficiently closely in time and by appropriate route(s) that they are encountered by (and, thus, stimulate) the individual's immune system sufficiently closely in time to have the desired effect of producing an enhanced immune response. Alternatively, ICSBP and the target antigen can be administered without a host.

Thus, the present invention provides a therapeutic agent against a wide variety of cells (tumor cells, cells infected with a pathogen), in that genetically engineered cells that express ICSBP and present an antigen(s) against which an immune response is elicited can be used as a vaccine (e.g., to protect recipients against future development of a tumor or infection) and/or as an agent to result in the reversal (partial or total) of an existing tumor or infection and/or to prevent or limit the extent to which the tumor or infection spreads or increases. It provides a valuable addition to existing therapies. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D show results demonstrating that ICSBP abrogates leukemogenesis in vivo by stimulating immune rejection of BCR/ABL-transformed BaF3 cells. Figure 1A is a graphic representation of proliferation of TonB210.1 cells expressing BCR/ABL(B/A), +/- ICSBP, +/- UN. Figure IB, 1C and ID show survival curves for mice.

Figure 2 is a graph of days versus cell number showing the growth kinetics of Ba-P210 and Ba-P210-ICSBP cells and that ICSBP expression in BCR/ABL transformed BaF3 cells does not alter their growth characteristics in vitro; Open symbols: Ba-P210; closed symbols: Ba-P210-ICSBP.

Figures 3A-3C are graphs of Days After Injection versus % Survival showing that Ba-P210-ICSBP cells fail to induce leukemia in immunocompetent mice, but remain leukemogenic in irradiated and immunodeficient mice. 10⁶ Ba-P210 (diamond) or Ba-P210-ICSBP cells (square) were injected intravenously into normal (open symbols) or 450 c-Gy irradiated (close symbols) Balb/c mice, 10 mice were used in each arm (Figure 3A); Nod/scid mice, 5-6 mice were used in each arm (Figure 3B); and Rag-1 deficient mice, 5 mice were used in each arm (Figure 3C).

Figures 4A-4B are graphs of Days After Injection versus % Survival showing that injection of ICSBP modified cells (Ba-P210-ICSBP) generates immunologic memory for parental leukemia cells (Ba-P210). Figure 4A shows survival of naive Balb/c mice (diamond) or Balb/c mice pre-immunized with 10⁶ Ba-P210-ICSBP cells (square) following intravenous injection with 10⁶ Ba-P210 cells. 10 mice were used in each arm. Figure 4B shows the results of Balb/c mice who received an intravenous injection of 7 x 10⁶ spleen cells from naive (circle) or Ba-P210-ICSBP immunized mice (square), along with 10⁶ Ba-P210 cells on the following day. 5 mice were used in each arm.

Figure 5 is a graph of Days After Injection versus % Survival showing that Ba-P210-ICSBP immunization rescues mice with pre-established leukemia. Survival of Balb/c mice injected with 10⁶ Ba-P210 cells, followed by 10⁶ Ba-P210- ICSBP cells injected on the same day or after a delay of 3, 7, or 14 days. Control mice (naive) received no Ba-P210-ICSBP cells. 5 mice were used in each arm. Figures 6A-6C are graphs showing immunization with Ba-P210-ICSBP cells activates CD8⁺ cytotoxic T lymphocytes, which exhibit specific cytolytic activity against BCR/ABL-associated cell surface antigens. Figure 6A shows cytolytic activity of a Ba-P210-ICSBP immunized spleen cells against different cells targets. 5,000 ⁵¹Cr-labeled target cells of the indicated types were used. Figure 6B shows a cytotoxicity competition assay. 5,000 ⁵¹Cr labeled Ba-P210 cells were used as target cells. 125,000 Ba-P210-ICSBP immunized spleen cells were used as effector cells (effector/target ratio of 25x). An excess of unlabeled BaF3 or Ba-P210 cells were added to the target/effector mixture (40x or 90x as indicated). CTL activity was measured as described in methods. Graph represents the percentage of reduction of cytolytic activity in the presence of unlabeled cells normalized against total cytolytic activity in the absence of unlabeled cells. Figure 6C shows cytolytic activities of CD87CD4⁺ positive cells or the corresponding CD8/CD4 depleted Ba-P210-ICSBP immunized spleen cells. 5,000 Ba-P210 cells were used as target and the effector/target cell ratio was 25x.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically engineered cells, also referred to as modified cells, in which Interferon Consensus Sequence Binding Protein (ICSBP) is expressed; uses of such cells in enhancing an individual's (e.g., a mammal's, such as a human's) response (e.g., immune response) to cells (e.g., malignant cells, cells infected with a pathogen) in the individual; methods of enhancing an individual's response, particularly an individual's immune response, to cells that cause a condition in an individual as a result of ICSBP expression in the individual; methods of enhancing the ability of an individual to eliminate cells that cause a condition in the individual; and methods of increasing ICSBP expression in target cells. The cells and methods of the present invention are useful in preventing the establishment of a condition (such as a malignancy or infection), lessening the extent to which the condition occurs, reversing the condition (totally or partially) and reducing the extent to which the condition progresses, once it occurs or is established. For example, the cells and methods of the present invention are useful in preventing the occurrence of tumors or malignancy (e.g., solid tumors, leukemias) in an individual; reducing or lessening the extent to which the tumor or malignancy occurs in an individual; treating an individual by reversing (partially or completely) the tumor or malignancy in the individual and reducing the extent to which the tumor or leukemia progresses in an individual. The cells and methods of the present invention are similarly useful in preventing infection, reducing or lessening the extent to which an infection occurs, reversing (lessening or curing) an infection and reducing the extent to which an infection, once established, progresses. Thus, the cells and methods of the present invention can be used prophylactically or to treat an existing condition. As used herein, the terms "therapeutic" and "therapeutically" include both prophylactic use and use to treat an existing condition. The cells of the present invention are also useful as an adjuvant in that they can be co-administered with another substance or molecule against which an immune response is desired. ICSBP enhances the immune response of the recipient of the co-administered ICSBP- expressing cells (or ICSBP itself) and the target antigen (substance or molecule against which an immune response is desired) is enhanced (greater than it would be in the absence of the administered ICSBP).

Genetically engineered or modified cells of the present invention can be any type of somatic cell (e.g., endothelial cells, epithelial cells, fibroblasts, hepatic cells, spleen cells and blood cells) or a tumor cell (e.g., a leukemia cell or a solid tumor cell). Cells of the present invention express ICSBP from exogenous DNA (DNA introduced into the cell or an ancestor of the cell) that encodes ICSBP or a precursor thereof; from endogenous DNA (DNA present in the cell or an ancestor of the cell) that is normally silent in the cell and has been activated (turned on) so that it is expressed in the cell, or from endogenous DNA that is normally expressed in the cell but whose expression has been enhanced or increased. Exogenous DNA encoding ICSBP can be from any source (e.g., any mammalian source, such as mouse, pig, monkey, human) and will typically be from the same type or species of animal as the cell into which it is introduced. For example, human DNA encoding ICSBP is typically used in producing genetically modified human ICSBP-expressing cells of the present invention, but ICSBP-encoding DNA from another animal can be used, provided that it has the desired effect of enhancing recognition of cells in an individual. Exogenous ICSBP-encoding DNA can be introduced into cells by a variety of art-recognized techniques, such as transfection, infection, chemical methods, microprojectile bombardment, transmembrane protein transduction and electroporation. A wide variety of expression vectors, such as retroviral vectors, adenoviral vectors, and plasmid vectors can be used for expressing ICSBP in modified cells of the present invention. Once introduced into the cell, exogenous DNA can be expressed episomally/extrachromosomally or as DNA integrated into host cell chromosomal DNA. In the method of the present invention, a therapeutically effective quantity or amount of genetically engineered or modified cells is administered to an individual in whom immune recognition of cells and elimination of cells is to be enhanced (increased relative to the extent of immune recognition and the extent of elimination of cells in the absence of administration of such cells). A therapeutically effective quantity or amount of cells of this invention is one sufficient to produce the desired enhanced effect. It may differ for different types of conditions (e.g., different types of tumors, different types of infections). It may also differ for different individuals, based on such factors as age, gender, size and health status. The appropriate dose for a particular individual with a particular condition to be addressed can be determined empirically or may be extrapolated, for example, from doses used in animal studies. The immune response which results can be systemic or local. One embodiment of the present invention is a method of enhancing the ability of an individual to eliminate cells that cause an undesirable condition in the individual, such as a malignancy or infection. In the method, ICSBP levels are increased in target cells, such as tumor biopsy cells, using methods described herein. The resulting genetically engineered tumor cells are re-introduced (e.g., re-injected) into the individual to stimulate an immune response, which results in elimination of the injected cells and tumor cells present in the individual (the resident tumor cells/tumor cells that need to be treated). In particular embodiments, the cells that are recognized are tumor cells, such as solid tumor cells and leukemia cells (e.g., chronic myeloid leukemia cells) or cells infected with a pathogen, such as a virus, a bacterium, a mycobacterium, a parasite, a protozoan or a fungus. The cells that express ICSBP can be of any type, provided that they produce sufficient ICSBP and the desired immunostimulatory effect (systemic or local), and will typically be the same type of cell as the cells for which enhanced recognition and elimination are desired (e.g., if a solid tumor of a particular type is the target, cells of the same type will be genetically engineered to express ICSBP).

Another embodiment of the present invention is a method of stimulating, in a mammal (e.g., a human), an immune response to cells that cause a condition in the mammal which is to be treated. The method comprises administering to the mammal a therapeutically effective quantity of ICSBP-expressing cells, wherein the ICSBP-expressing cells are cells genetically engineered to express ICSBP (e.g., by introduction of exogenous DNA encoding ICSBP into the cells or ancestors thereof, by activating a normally silent ICSBP gene present in the cells or by enhancing or increasing expression of an ICSBP-encoding gene that is normally expressed in the cells). The immune response that is stimulated can be a systemic immune response or a local immune response. The nature of the immune response (systemic or local) may vary with the route of administration of the modified cells. For example, if cells are administered intravenously, a systemic immune response might occur, but if they are administered by injection at a particular site in the body, a local immune response might occur.

A further embodiment of the method of the present invention is a method of stimulating an immune response to tumor cells in a human. If tumor cells are the ICSBP-expressing cells, they can be viable tumor cells or tumor cells rendered proliferation incompetent, such as by treatment with irradiation or a chemotherapeutic agent, such as mitomycin C. One embodiment of the method comprises administering to the human (e.g., by injection or infusion) a therapeutically effective quantity of modified tumor cells which are proliferation incompetent and express ICSBP, are the same type of tumor cells as are present in the human (against which an immune response is to be stimulated) and elicit an immune response to the tumor cells in the human. In this embodiment, the response can be a systemic response or a local response. The tumor cells can be leukemia cells (e.g., chronic myeloid leukemia cells) or solid tumor cells.

Another embodiment of the present invention is a method of suppressing growth of tumor cells in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective quantity of modified tumor cells, wherein the tumor cells have been rendered proliferation incompetent by irradiation and express ICSBP from exogenous DNA. Alternatively, the cells can express ICSBP from endogenous DNA, as described herein. In either embodiment, the tumor cells present in the mammal and the modified replication-incompetent tumor cells are of the same type and the modified tumor cells elicit an immune response that results in suppression of growth of the tumor cells. In one embodiment, the immune response that is elicited is a systemic response. Alternatively, the response can be a local immune response. The tumor cells can be leukemia cells, such as chronic myeloid leukemia cells, or they can be solid tumor cells. In yet a further embodiment of the present invention, the method is one of enhancing the immune response of a mammal, such as a human, to leukemia cells (e.g., chronic myeloid leukemia cells), comprising administering to the mammal a therapeutically effective quantity of proliferation-incompetent leukemia cells that have been genetically engineered to express ICSBP (from exogenous DNA or endogenous DNA, as described herein), with the result that the proliferation-incompetent leukemia cells enhance the immune response of the individual to the leukemia cells. As a result, the individual is protected from the occurrence (establishment) of leukemia, such as chronic myeloid leukemia; develops the condition to a lesser extent than would occur in the absence of treatment by this method; or undergoes a lessening of an already existing leukemia (the leukemia is cured or is reduced in severity or the individual goes into remission).

The present invention also relates to a method of suppressing proliferation of cells in a mammal, such as a human, wherein proliferation of the cells in the mammal causes an undesirable condition. In the method, a therapeutically effective quantity of cells modified to express ICSBP (from exogenous DNA or endogenous DNA, as described herein) is administered to the mammal, in whom they elicit an immune response to the cells that cause the undesirable condition, such that proliferation of the cells that cause the undesirable condition is suppressed. In this embodiment, the cells that are modified to express ICSBP and the cells that cause the undesirable condition are of the same type. The undesirable condition can be, for example, cancer or an infection, such as a bacterial infection, a mycobacterial infection, a viral infection, a parasitic infection, or a protozoal infection, or any other condition in which cell proliferation is undesirable.

The present invention is also useful for enhancing immune recognition of cells, present in an individual, such as a human, that cause disease in the individual. In the method of enhancing immune recognition, cells, referred to as

ICSBP-expressing cells, that express ICSBP at a sufficient level to stimulate an immune response to the cells that cause the disease are introduced into the individual by an appropriate route (e.g., injection, infusion). As a result, an enhanced immune response to the cells that cause the disease (a response that is greater than would occur if ICSBP-expressing cells were not introduced into the individual) is stimulated in the individual, thereby enhancing immune recognition of the cells that cause disease in the individual. This embodiment of the invention is useful, for example, wherein the cells that cause disease in the individual are malignant cells (e.g., leukemia cells, such as chronic myeloid leukemia cells, or solid tumor cells) or cells infected with a pathogen (e.g., a virus, bacterium, mycobacterium, parasite, yeast, fungus or protozoan). As in other embodiments of the present invention, ICSBP can be expressed from exogenous DNA that encodes ICSBP or a precursor thereof or from endogenous DNA that has been activated or whose expression level has been enhanced. The present invention also relates to a method of increasing the immunostimulatory effect of a cell, comprising enhancing ICSBP expression in the cell, such as by introducing into the cell or an ancestor thereof exogenous DNA that encodes ICSBP and is expressed in the cell or by activating normally silent (unexpressed) ICSBP-encoding DNA present in the cell (activating endogenous ICSBP-encoding DNA) or enhancing expression of endogenous ICSBP-encoding DNA that is expressed in the cell. As described herein, the cell can be of any type, provided that it can be modified to express ICSBP. For example, it can be a nonmalignant somatic cell (e.g., an epithelial cell, an endothelial cell, a fibroblast, a hepatocyte) or it can be a malignant cell (e.g., a leukemia cell or a solid tumor cell). Tumor cells will typically be rendered replication-incompetent, such as by treatment with irradiation. Cells produced by this method, as well as cells produced by another method, that have an enhanced immunostimulatory effect when introduced into an individual (e.g., a human) are also the subject of this invention. Such modified or genetically engineered cells can be used alone, to increase or enhance an individual's immune response to subsequent challenges, or can be used as an adjuvant. In the former embodiment, the genetically-engineered cells can be administered to an individual whose immune response is in need of enhancement, such as an individual whose immune system is compromised (e.g., an individual undergoing chemotherapy or other drug therapy or an individual infected with HIV or other pathogen). In the latter instance, the ICSBP-expressing cells are administered to an individual in conjunction with another substance or molecule, such as a vaccine or protein against which an immune response is desired. It is not necessary that the genetically-engineered cells and the additional substance be administered simultaneously, provided that they are administered sufficiently closely that they are encountered by the individual's immune system essentially simultaneously.

A further method of the present invention is a method of enhancing the immune response to tumor cells in an individual comprising introducing into the individual cells that co-express an oncogene or tumor antigen that is expressed in the tumor cells and ICSBP, in sufficient quantity and by a route that results in an enhanced immune response to the tumor cells in the individual. The tumor cells can be leukemia cells or solid tumor cells. For example, CML is caused by a chromosomal translocation that result in production of a fusion protein, BCR/ABL. Peptides derived from normal proteins and BCR/ABL protein, including that from the junction region of BCR and ABL, are presented on the BCR/ABL + cell surface. Cells expressing the BCR/ABL fusion protein and ICSBP can be used in this embodiment. It is not necessary to both BCR/ABL and ICSBP to be expressed by the same vector or even in the same cells, provided that they are encountered sufficiently closely in time for the enhanced effect of ICSBP on immune recognition to occur.

This invention further relates to a method of treating a mammal in whom tumor cells are present, comprising co-administering to the mammal (e.g., a human) at least one chemotherapeutic agent and modified tumor cells that express ICSBP from exogenous DNA or from silent endogenous DNA that is activated or endogenous DNA whose expression is enhanced, as described herein.

The invention also relates to an in vitro method of producing tumor-directed cytotoxic T-cell clones, in which T cells obtained from a mammal (e.g., a human), appropriate growth factors and antibodies sufficient to induce T cell activation and proliferation (e.g., IL-2) and target cells that express ICSBP against which cytotoxic T-cell clones are to be produced are combined, thereby producing a combination. The combination is maintained under conditions appropriate for T cell expansion, with the result that cytotoxic T-cell clones directed against the tumor cells are produced. The target cells can be tumor cells (leukemia cells, such as CML cells, or solid tumor cells) or cells infected with a pathogen.

Another subject of this invention is a modified tumor cell which is replication incompetent and expresses ICSBP encoded by exogenous DNA that encodes ICSBP or a precursor thereof or ICSBP encoded by an activated endogenous ICSBP-encoding gene or endogenous gene whose expression is enhanced. The modified tumor cells can be leukemia cells (e.g., chronic myeloid leukemia cells) or solid tumor cells. Genetically-engineered mammalian cells that express ICSBP from a normally silent, activated endogenous gene that encodes ICSBP are also the subject of the present invention.

The present invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1 Assessment of the Ability of ICSBP-Expressing BCR ABL- transformed BaF3 Cells to Induce Leukemias In Vivo The ability of ICSBP-expressing BCR/ABL-transformed BaF3 cells to induce leukemias in vivo was assayed. Following intravenous transfer, one million BCR/ABL-transformed BaF3 cells (BCR/ABL-BaF3 cells) form rapidly fatal leukemias in syngeneic Balb/C mice (Figure IB). Remarkably, BCR/ABL-BaF3 cells that co-express high levels of ICSBP (ICSBP-BCR/ABL-BaF3) are completely benign in syngeneic immunocompetent recipients, and surviving mice remain disease free for at least four months following challenge (the longest follow-up). In marked contrast, ICSBP-BCR/ABL-BaF3 cells kill immunodeficient mice, suggesting that ICSBP does not alter the survival of cells in vivo bur rather acts by rendering BCR/ABL-transformed cells susceptible to clearance by the immune system. This phenomenon can be demonstrated in syngeneic mice that are immunocompromised by prior irradiation (Figure IB); in NOD/SCID mice, which are deficient in T and B and most NK cell function (not shown); and in Rag-1 deficient mice, which lack T and B function but retain NK cell activity (Figure 1C). These results suggest that the mechanism of immune rejection is likely to be a T-cell mediated phenomenon. Moreover, once mice have survived challenge with the ICSBP-BCR/ABL-BaF3 cells, they survive a subsequent re-challenge with unmodified BCR/ABL-transformed cells (Figure ID). This observation rules out the possibility of an immune response to the ICSBP protein itself, begs the interesting question of what antigen accounts for the immune rejection, and raises the provocative possibility that ICSBP expression potentiates a specific immune response to BCR ABL. This novel immunostimulatory effect identified for ICSBP can be assessed using known methods.

EXAMPLE 2 Investigation of the Role of ICSBP in Antagonizing CML in a Mouse Model

Described in the following example are experiments carried out to investigate the role of ICSBP in antagonizing CML in a mouse model. A leukemic cell line was generated by overexpression of the CML-associated oncogene BCR/ABL. These cells caused rapidly aggressive lethal leukemias when injected into immunocompetent mice. However, when ICSBP was over-expressed in the same cell line, the mice were protected against lethal leukemia. Eventually, all leukemic cells were eliminated from the mice. The surviving mice failed to develop leukemia when re-challenged with the BCR/ABL-transformed leukemia cells that did not express ICSBP. Immunodeficient mice (either NOD/SCID strains or following irradiation ) were killed by the BCR/ABL-transformed cells even when ICSBP was over-expressed, arguing that the immune system is critical for eliminating the cells.

The BCR/ABL fusion protein induces human Chronic Myeloid Leukemia (CML). Interferon (IFN-α) prolongs the chronic phase of the disease and has become a mainstay of CML treatment. However, the precise mechanism by which IFN-α alters the natural history of CML is unclear. IFN-α signaling is mediated by Interferon Regulatory Factors (IRFs), a family of transcription factor proteins implicated in anti-proliferative and immunomodulatory effects. Mice deficient in the IFN Consensus Sequence Binding Protein (ICSBP) develop a CML-like myeloproliferative disease (Holtschke et al., Cell, 57:307 (1996)). To examine whether ICSBP acts as a tumor suppressor gene, whether ICSBP expression would antagonize the transformation of IL-3 dependent BaF3 cells by BCR/ABL was assessed. BaF3 cells expressing BCR/ABL (Ba-P210) proliferate in the absence of IL-3 and induce a rapidly fatal leukemia in syngeneic hosts. Overexpression of ICSBP in Ba-P210 (Ba-P210-ICSBP) had little demonstrable effect on proliferation, factor independence, or resistance to radiation-induced apoptosis in vitro. However, Ba-P210-ICSBP cells failed to induce leukemia in immunocompetent hosts, but remained leukemogenic in immunodeficient hosts. Furthermore, immunocompetent mice exposed to Ba-P210-ICSBP cells survived rechallenge with the parental Ba- P210 cells, suggesting that ICSBP expression in Ba-P210 cells induced potent immunity against Ba-P210 cells. Irradiated Ba-P210 cells failed to generate protective immunity, but irradiated Ba-P210-ICSBP cells were immunogenic and provided protective immunity, indicating a critical role for ICSBP expression. Relatively little is known about the function of ICSBP, and the mechanisms by which ICSBP alters the immune response to BCR ARL-transformed cells are presently being investigated. This includes utilizing cDNA arrays to identify cytokines and other immunomodulatory molecules whose expression is modified by ICSBP. Whether ICSBP expression will induce protective immunity against other leukemia cells and solid tumor cell lines is also being examined, in order to determine whether ICSBP gene transfer might be harnessed for the immunotherapy of malignancy. These results support a role for ICSBP in enhancing specific tumor antigen presentation to cytotoxic cells in vivo.

EXAMPLE 3 Expression of the Interferon Regulatory Transcription Factor ICBSP Induces Potent Immunity Against BCR/ABL-Induced Leukemia

Animals. Six to twelve week old female Balb/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). Rag-1 and NodJscid mice (Jackson Laboratory) were obtained at 6 weeks old and maintained in sterile conditions.

Cell lines, transfection, and infection. BaF3, a murine pro-B cell line (Palacios R. and Steinmetz M, Cell, 41:121-24 (1985)) was maintained in complete RPMI supplemented with 10% conditioned medium from WEHI-3B cells, as a source of EL-3. ICSBP and BCR/ABL constructs were introduced into the cells by electroporation using vectors pCXN2-ICSBP (kindly provided by Dr. Keiko Ozato, NTH) and MSCV-BCR ABL-PAC. Cells were selected in 2μg/ml puromycin for BCR/ABL and 2mg/ml G418 for ICSBP to achieve stable expression. Moloney murine sarcoma virus (M-MSV) transformed 3T3 cells and Lewis lung carcinoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI medium supplemented with 10% fetal bovine serum.

Immunoblotting analysis and Enzyme-linked immunosorbent assay (ELIS A). Cells were lysed in buffer containing 10% Glycerol, 150mM NaCl, 20 mM Tris (pH 7.4), 10 mM NaF, 1 mM ZnCU, 1 mM MgCl₂, and 1% NP-40. 40 μg total proteins were separated by SDS polyacrylamide gel electrophoresis (10%) and transferred to nitrocellulose membrane. Both polyclonal α-ICSBP (kindly provided by Dr. Keiko Ozato) and α-abl antibodies were used at 1 :5000 dilution. HRP conjugated Goat anti-rabbit IgG secondary antibody (Santa Cruz biotechnology, Santa Cruz, CA) and ECL was used to detect protein expression. Conditioned media from BaF3 and its derived cell lines were collected and concentrated with Centricon filters (YM-10; Millipore, Bedford, MA). Enzyme-linked immunosorbent assay (ELISA) for IL-2, IFN-γ, and GM-CSF were carried out using Quantikine immunoassay reagents according to manufacturer's protocols ® & D Systems, Minneapolis, MN).

CD8/CD4 fractionation. CD8+ or CD4+ cells were purified or depleted from single spleen cell suspensions using Dynalbeads conjugated with CD8 (Lyt2) or CD4 (L3T4) antibodies according to manufacturer protocol (Dynal Inc., Lake Success, NY). The purity of positively isolated cells and the efficiency of negative depletions were analyzed by flow cytometric analysis.

Flow cytometric analysis. 10⁶ cells were resuspended in PBS and incubated with Phycoerythrin (PE) conjugated antibodies against MHC class I, MHC class II, CD8, and CD4. For B7-1 and B7-2 detection, purified primary antibodies were used, followed by a biotinylated secondary antibody and PE-conjugated streptavidin staining. All antibodies were purchased from Pharmingen (San Diego. CA), and cells were analyzed on the Becton Dickenson FACSCalibur irnmunocytometry system (Becton Dickenson, San Jose, CA).

Mice injection and analysis. BaF3-derived cells were injected intravenously into mouse lateral tail vein. Mice were closely monitored and were sacrificed if moribund. Peripheral blood, spleens, and bone marrow samples were subsequently isolated from the mice for cell morphology analysis by staining centrifuged cells with the Hema 3 stain set (Biochemical Sciences, Inc., Swedesboro, M). Genomic DNA and mRNA were also isolated using DNA extraction reagent (Qiagen Inc., Valencia, CA) and RNA STAT-60 reagent (Tel-Test "B", Inc., Friendswood, TX).

Cell-mediated cytotoxicity and cold target inhibition assays. Fresh spleen cells were isolated from either naive or Ba-P210-ICSBP immunized mice two weeks after the immunization and were cultured in a complete K medium (RPMI 1640, 10% FCS, with 50μM β-mercaptoethanol, ImM non-essential AA, lOmM NaPyruvate; GibcoBRL, Rockville, MD). Irradiated Ba-P210-ICSBP cells (20,000-cGy) were co-cultured with the spleen cells for 6 days. On day 7, cytotoxic activities of stimulated cells were determined in a standard 4-hr ⁵¹Cr-release assay with the following modifications (Sykulev Y., et al, Immunity, 9:475-83, 1998.). 5,000 target cells were labeled with lOOμCi of sodium [⁵¹Cr] chromate and incubated for 1 hr before use. CTLs were added to achieve different effector/target ratio of 5/1, 25/1, 50/1, and 100/1. Specific lysis was determined as follows: percent specific release = 100 X (release by effector cells - spontaneous release)/(maximum release - spontaneous release). Spontaneous release was less than 20% of maximum release in all experiments. Competitive inhibition studies were carried out by measuring the specific lysis of ⁵¹Cr -labeled target cells by a fixed number of effector cells in the presence of various number of unlabeled tester cells. Unlabeled cells were added to the reaction wells at the same time as the ⁵¹Cr -loaded target cells.

Results

The expression of ICSBP did not alter BCR/ABL transformation fo BaF3 cells in vitro. The interleukin-3 dependent murine pro-B cell line BaF3 lacks endogenous ICSBP expression and is readily transformed by BCR/ABL, and therefore serves as a model to investigate the effect of exogenously expressed ICSBP on BCR ABL-induced leukemia. A Ba-P210 cell line was created by stably transfecting BaF3 cells with BCR/ABL vector. Subsequently, ICSBP was transfected into Ba-P210 cells to generate the Ba-P210-ICSBP cell line. Both cell lines expressed a high level of the BCR/ABL (P210) oncoprotein, while Ba-P210- ICSBP also expressed a high level of murine ICSBP. The expression level of

ICSBP in Ba-P210-ICSBP cells was comparable to the endogenous ICSBP level in the human lymphoid cell lines Namalwa, Daudi and Ramos.

The expression of BCR/ABL transforms BaF3 cells to IL-3 independence and resistance to γ-irradiation-induced apoptosis (Daley GQ and Baltimore D, Proc Natl Acad Sci USA, 55:9312-6 (1988)). The Ba-P210-ICSBP cell line remained growth factor independent and resistant to γ-irradiation in culture, indicating that the expression of ICSBP did not alter these in vitro transformation characteristics of BCR/ABL in BaF3 cells. The effect of ICSBP expression on cell proliferation was also examined. Two independent Ba-P210-ICSBP cell lines proliferated with the same growth kinetics as their parental cell line Ba-P210 (Figure 2). ICSBP expression did not exhibit an anti-proliferative effect on Ba-P210 cells.

ICSBP expression antagonized BCR/ABL induction of leukemia in vivo. When injected intravenously into healthy syngeneic Balb/c mice, Ba-P210 cells induced a rapidly progressive leukemia that killed the host in 3 weeks (Figure 3A). Spleens from injected animals were massively enlarged (10-20 fold above normal), and all mice had a pathologic burden of Ba-P210 cells in their peripheral blood (PB), spleen, and bone marrow (BM). In contrast, all mice receiving Ba-P210-ICSBP cells survived (maximal follow-up for 1 year; Figure 3A). Peripheral blood from mice injected with Ba-P210-ICSBP cells was monitored weekly and leukemia was not observed at any time even when up to 5 x 10⁷ Ba-P210-ICSBP cells were injected. The expression of ICSBP altered the leukemic behavior of BCR/ABL (P210) transformed BaF3 cells in a syngeneic host.

Intact host immunity was necessary for ICSBP-mediated protection. The failure of Ba-P210-ICSBP cells to induce a lethal leukemia in Balb/c mice could be due to a cell-intrinsic proliferation defect apparent only in vivo, or an indirect activation of host defense mechanisms. In order to see if impairment of host defenses altered tumorigenicity in vivo, 10⁶ Ba-P210 and Ba-P210-ICSBP cells were injected into Balb/c mice exposed to a sublethal dose of irradiation (450-cGy). Both mouse groups developed lethal leukemia and died with comparable disease latencies (Figure 3 A). This observation demonstrated that ICSBP expression in Ba-P210 cells did not alter the intrinsic tumorigenicity in vivo, and that the inability of Ba-P210- ICSBP cells to induce leukemia in healthy Balb/c mice was due to an inhibitory mechanism that could be destroyed by γ-irradiation. Because a major effect of γ- irradiation is compromise of host immunity, the importance of an intact immune system was investigated using immunodeficient mice. 10⁶ Ba-P210 and Ba-P210- ICSBP cells were injected into ^' iodlscid and Rag-1^"'^" mice. Nod/scid mice are severely deficient in T/B cells and NK cells (Shultz LD, et al, J. Immunol, 154: 1 SO- 91 (1995)), while Rag-1^"'^" mice have no T/B cell function, but maintain relatively normal function of NK cells (Mombaerts, P., et al, Cell, 68:869-11 (1992)). Similar to γ-irradiated Balb/c mice, the disease latencies induced by both Ba-P210 and Ba- P210-ICSBP cell lines were comparable in Nod/scid and Rag-1^"'^' mice (Figures 3B and 3C). This experiment indicates that the protective function of ICSBP was dependent on an intact immune system in the recipient mice. Furthermore, the result implicated T/B cells and not NK cells in the process.

Ba-P210-ICSBP immunization led to resistance against subsequent rechallenge with Ba-P210 cells. To assess whether the induced immunity was directed against ICSBP-specific antigens or antigens unrelated to ICSBP, whether mice that survived Ba-P210-ICSBP injection could survive subsequent challenge with parental Ba-P210 cells was tested. Thirty days after single dose of Ba-P 10- ICSBP immunization, mice were re-injected with 10⁶ Ba-P210 cells. All mice tested achieved long term leukemia- free survival (Figure 4A, maximal follow-up for 1 year). This result indicated that tumor rejection was not directed against ICSBP- induced antigens on the tumor cells. Immunized mice rechallenged with Ba-P210 cells 90 days after Ba-P210-ICSBP immunization also survived, demonstrating that the immunity induced by ICSBP expression in Ba-P210 cells was potent and long- lasting. Sublethal dose of P210 cells failed to provide protection against subsequent challenge, indicating that expression of ICSBP was critical to the development of protective immunity. A single dose of γ-irradiated Ba-P210-ICSBP cells (up to 10 ) also failed to induce immunity, suggesting that proliferating cells expressing ICSBP were required. ICSBP-induced immunity can be transferred to naive mice. The spleen is a primary immune organ in an adult mouse. To determine whether immunologic memory could be adoptively transferred from one animal to another, donor Baib/c mice were immunized with a single dose of 10⁶ Ba-P210-ICSBP cells. Four weeks after the immunization, spleens were removed from the donor mice, single ceil suspensions were made, and 6 x 10' mononuclear spleen cells were injected intravenously into non- irradiated recipient mice (representing about half the total cells from a single spleen). These mice were then challenged with 10⁶ Ba-P210 cells on the second day. At the time of spleen cell transfer, there was no residual Ba- P210-ICSBP cells detectable by genomic PCR reactions. All mice receiving ICSBP- immunized spleen cells were protected from Ba-P210 induced leukemia, while control mice receiving naive spleen cells died of leukemia with the expected latency (Figure 4B).

Ba-P210-ICSBP injection protected mice with pre-established leukemia. As demonstrated herein, ectopic expression of ICSBP in BCR ABL-transformed BaF3 cells induced potent cellular immunity. To test whether ICSBP-induced immunity could eradicate pre-existing disease, 10⁶ Ba-P210 cells were first injected into naive Balb/c mice to induce leukemia. A single dose of 10⁶ Ba-P210-ICSBP cells were injected simultaneously into the same hosts, or following a delay of 3, 7, or 14 days. Simultaneous injection of both cell lines allowed survival of all mice (Figure 5). More strikingly, even when Ba-P210-ICSBP cells were injected 3 to 7 days after the injection of the leukemic Ba-P210 cells, all mice survived. When leukemia was allowed to develop for 14 days, equivalent to 2/3 of the disease latency, all mice achieved prolonged survival and 20%) of the mice survived disease free. The results demonstrated that the anti-leukemic effect of the immunized cells could be initiated rapidly, and that ectopic ICSBP expression in leukemic cells was potent enough to eradicate established disease.

Cytotoxic T lymphocytes were induced by Ba-P210-ICSBP immunization. To determine whether mice immunized with Ba-P210-ICSBP cells generated Cytotoxic T Lymphocytes (CTL) against BCR ABL transformed BaF3 cells, spleens from naive or immunized Balb/c mice were isolated two weeks after immunization and tested in a cytotoxicity assay. Single cell suspensions were cultured for 6 days in the presence of γ-irradiated Ba-P210-ICSBP cells to stimulate specific CTL proliferation in vitro. A Cr⁵¹ release assay was performed on day 7. Spleen cells from immunized mice lysed both Ba-P210 and Ba-P210-ICSBP cells at comparable levels (maximum of 94.1%> & 84.9% respectively; Figure 6A). No cytolytic activity was observed when naive spleen cells were used as effector cells, or when Murine sarcoma virus transformed 3T3 (Balb/c derived) or LL2 Lewis Lung Carcinoma (C57BL/6 derived) cells were used as targets. This result not only demonstrated the generation of specific CTLs from in vivo immunization, but also confirmed that ICSBP-derived or ICSBP-induced antigens were unlikely to be the targets recognized by CTLs. Cytotoxicity was also observed when BaF3 cells were used as targets, though at a lower level, indicating that antigens on BaF3 cells were also targets of the immune response initiated by ICSBP expression.

To investigate whether there were specific CTLs directed against BCR/ABL- associated antigens, competition assays with unlabelled target cells were performed. Either BaF3 or Ba-P210 cells were added in excess as unlabelled cells to compete against ⁵¹Cr-labelled target Ba-P210 cells in cytotoxicity assays. The addition of excess unlabelled Ba-P210 cells was significantly more effective at inhibition of ICSBP-induced spleen cell cytotoxicity than a comparable number of unlabelled BaF3 cells (Figure 6B). This result confirmed that a proportion of spleen CTLs were directed against antigens associated with BCR/ABL expression, while other CTLs recognized antigens associated with the BaF3 cells.

CD8⁺ cells were the primary effector cells mediating ICSBP-induced immunity. To characterize which T-cell subtype mediated the cytotoxic effect that was observed, CD8⁺ or CD4⁺ cells from the immunized total spleen cell population were isolated by immunoaffmity fractionation. Both positively-selected CD8^" or CD4⁺ cells and the corresponding depleted cell populations were used for CTL assays. As determined by flow cytometry, positively selected cells were more than 85% pure and depletions for both CD4⁺ and CD8^÷ cells were more than 97%₀ efficient. Consistently, purified CD8⁺ cells exhibited ~80%> CTL activity, while CD8⁺ depletion resulted in more than 85% loss of CTL activity (Figure 6C). CD4^~ depletion had no effect on CTL activity, while purified CD4⁺ cells elicited only 20% activity. These results suggested that CD8^T cells were the primary cytotoxic effector cells in vitro and therefore likely to account for ICSBP-induced cellular immunity in vivo.

Discussion As shown herein, ectopic expression of ICSBP, a transcription factor regulated by interferon, induces rapid, potent, and long-lasting immune responses against BCR/ABL-transformed leukemic cells. ICSBP expression not only protects mice from developing leukemia, it also generates immunologic memory cells that provide long term immunity and protection from subsequent rechallenge. Adoptive transplantation of spleen cells from immunized mice transfers the immunity to naive recipients. In addition, a single dose vaccination with ICSBP-modified cells can eradicate pre-established leukemia. CD8⁺ cells appear to be the primary cytotoxic effector cells mediating SCSBP-induced protection. The mechanisms, by which ICSBP induces immunity, and the specific antigenic epitopes on BCR/ABL-transformed cells, remain to be defined. ICSBP can act as a negative regulator of MHC class I gene expression in cultured cells (Nelson N, et al, Mol Cell Biol, 13:588-99 (1993)), leading us to hypothesize that overexpression of ICSBP in our cell lines might down-regulate MHC expression and render cells susceptible to clearance by NK cells. Therefore, both MHC class I (H- 2D^d and H-2K^d) and class II (I-A^d and I-E^d) expression in the cell lines were examined by flow cytometry. BaF3 cells expressed relatively high levels of cell surface MHC Class I, which was increased modestly by expression of BCR/ABL. ICSBP overexpression did not alter the cell surface expression of MHC Class I. In contrast to MHC class I, MHC class II expression was not detected in any of our BaF3 cell lines. Since co-stimulatory signals enhance T-cell proliferation, and the co-stimulatory molecule B7 has been shown to induce cellular immunity against BCR/ABL-induced leukemia (Marulonis UA, et al, Blood, 85:2501-15 (1995)), its expression in the ICSBP modified cells was examined. BaF3 cells express low level of B7-1, and undetectable levels of B7-2, neither of which were affected by the expression of ICSBP. Therefore, the ICSBP-induced immune response against BCR/ABL transformed leukemic cells is unlikely to be mediated through altered MHC or B7 function.

Immunotherapy in the form of an allogeneic Graft-versus-Leukemia (GvL) effect of bone marrow transplantation has been exploited for decades in treating patients with Chronic Myeloid Leukemia (CML). Donor Lymphocyte Infusion has its major therapeutic benefit in CML patients (Sawyers, CL, N Engl JMed, 340:1330-40 (1999)). Since 95% of CML patients bear the abnormal chromosome translocation that generates BCR/ABL (P210), BCR/ABL peptides have been intensively studied for their ability to trigger tumor-specific immune reactivity. Indeed, peptides derived from the fusion sequences of BCR/ABL bind HLA molecules, demonstrating the potential for BCR/ABL-specific antigen presentation in vivo (Berke Z, et al, Leukemia, 14:419-26 (2000)). BCR/ABL peptide-sensitized dendritic cells generate cellular immune reactivity, indicating that leukemic cells can be targets for immune rejection (Mannering SI, et al, Blood, 90:290-1 (1997)). As shown herein, irradiated BCR/ABL-transformed leukemia cells do not induce immunity on their own, indicating that ICSBP expression potentiates some aspect of antigen presentation in the leukemic cells, or immune recognition and immune rejection by the host. The antigens that are responsible for the induction of immune reactivity against Ba-P210 cells remain to be defined. Cytotoxic T cells generated by Ba-P210-ICSBP immunization were more effectively competed by unlabelled Ba-P210 cells than by BaF3 cells, indicating the existence of specific CTLs against BCR/ABL-associated antigens on Ba-P210 cells. ICSBP-expression failed to induce immunity against BaF3 cell lines that become spontaneously transformed in culture. Therefore, the antigens on parental BaF3 cells are alone incapable of triggering an effective immune response. Antigens associated with BCR/ABL expression, either BCR/ABL derived peptides or other induced proteins, might serve as effective tumor-rejection antigens. Future efforts will be aimed at determining specific peptide epitopes from BCR/ABL that can bind to and be effectively presented in the context of the Balb/c class I molecules (H-2D^d, H-2K^d). ICSBP deficient mice are markedly impaired in their production of the T helper type 1 cytokines LL-12 and IFN-γ and have compromised Th-1 -driven immunity (Holtschke T, et al, Cell, 87: 307-17 (1996); Giese NA, et al, Exp Med, 186:1525-46 (1997)). Both JX-12 and IFN-γ are among the most potent antitumor cytokines. IL-12 modified tumor cells mediate CD8^"r T cell mediated cellular immunity and tumor rejection in animal models (Dunussi-Joannopoulos K, et al, Blood, 94:4263-13 (1999)). IFN-γ possesses both direct and anti-proliferative activity and immuno-modulating properties, including activation of macrophages and enhancement of T-cell-mediated immunity (Boehm U, et al, Annu Rev Immunol, 15: 749-95 (1997)). Recently, it was shown that overexpression of ICSBP stimulates IL-12 p40 promotor activity in macrophages (Wang LM, et al, J Immunol, 165:211-9 (2000)). Whether enhanced secretion of IL-12 and /or IFN-γ could be responsible for the antitumor activity of ICSBP-expressing cells in vivo was tested. Levels of IL-12, IFN-γ, and GM-CSF, another potent anti-tumor cytokine (Dranoff G, et al, Proc Natl Acad Sci USA, 90:3539-43 (1993)) were measured in conditioned culture medium of ICBSP-modified leukemia cells. None of these cytokines were detected by ELISA from Ba-P210 or Ba-P210-ICSBP cells. Moreover, altered mRNA expression for these cytokines were not detected by microarray expression profiling. Therefore, the mechanism by which ICSBP induces anti-leukemia immunity does not appear to be a direct consequence of increased secretion of these cytokines by ICSBP modified cells. Tumor rejection by the immune system is typically mediated by cytotoxic lymphocytes (T cells and NK cells) with specificity against tumor antigens. In contrast to syngeneic immunocompetent Balb/c mice, immunodeficient mice lacking normal T cell function (Rag-1 and Nod/scid) did not reject ICSBP-modified leukemia cells. Because NK cell function is largely intact in Rag-1 deficient mice, NK cells do not appear to play a major role in the elimination of ICSBP-modified cells. T cells appear indispensable for ICSBP-mediated protection, and the presence of cytotoxic CD8 T cells in immunized mice was confirmed by in vitro proliferation and CTL assays. In vitro stimulation with irradiated Ba-P210-ICSBP cells resulted in CTL expansion from immunized spleen cells but not naive spleen cells. High CTL activity was observed against both ICSBP modified cells (Ba-P210-ICSBP) and the parental leukemic cells (Ba-P210). Although both CD8" and CD4^T cells are capable of initiating tumor rejection in animal models, CD8^~ cells appear to be the primary cytolytic effector cells mediating leukemia rejection in the study. Purified CD8⁺ cells were capable of mediating CTL activity in vitro, and cytotoxicity was not affected by the deletion of CD4 cells. The primary role of CD4" T cells, specifically the Th-1 subset, is to enhance the induction and/or extend the persistence of CD8" CTL in vivo (Toes RE, et al, J Exp Med, 189:153-6 (1999)).

The molecular mechanisms controlling the maintenance of memory T cells against tumors are largely unknown. PCR analysis of spleen and bone marrow shows that immunized mice lack residual leukemic cells, indicating that persistent stimulation with leukemia-specific antigens is not required for maintenance of immuno logic memory in immunized mice.

Mycoplasma infection has been reported to alter the tumorigenicity of cell lines and the corresponding hose immune responses (Feng SH, et al, Mol Cell Biol,

19:1995-8002 (1999); Chambaud I, et al, Trends Microbiol, 7:493-9 (1999)). As shown herein, the BaF3 cell lines used were mycoplasma free by both PCR and antibody detection, ruling out the possible involvement of mycoplasma in the experimental system.

The data described herein provides evidence that ICSBP expression can also stimulate a cytotoxic T cell-mediated immune response against tumor cells, and represents the a demonstration that the expression of an intracellular transcriptional regulator rather than a cell surface protein can elicit a specific anti-leukemic response.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAJMSWhat is claimed is:

1. A method of stimulating an immune response to tumor cells in a human, comprising administering to the human a therapeutically effective quantity of modified tumor cells which are proliferation incompetent and express ICSBP from exogenous DNA encoding ICSBP, wherein the tumor cells present in the individual and the modified tumor cells are of the same type and the modified tumor cells elicit an immune response to the tumor cells present in the human.

2. The method of claim 1, wherein the immune response is a systemic response.

3. The method of claim 1, wherein the immune response is a local response.

4. The method of claim 1, wherein the tumor cells are leukemia cells.

5. The method of claim 4, wherein the leukemia cells are chronic myeloid leukemia cells.

6. The method of claim 1 , wherein the tumor cells are solid tumor cells.

7. A method of suppressing growth of tumor cells in a mammal, comprising administering to the mammal modified tumor cells, wherein the modified tumor cells have been rendered proliferation incompetent by irradiation and express ICSBP from exogenous DNA, wherein tumor cells present in the mammal and the modified tumor cells are of the same type and the modified tumor cells elicit an immune response that results in suppression of growth of the tumor cells.

8. The method of claim 7, wherein the immune response is a systemic response.

9. The method of claim 7, wherein the immune response is a local response.

10. The method of claim 7, wherein the mammal is a human.

11. The method of claim 7, wherein the tumor cells are leukemia cells.

12. The method of claim 11, wherein the leukemia cells are chronic myeloid leukemia cells.

13. The method of claim 7, wherein the tumor cells are solid tumor cells.

14. A method of enhancing the immune response of a mammal to leukemia cells, comprising administering to the mammal a therapeutically effective quantity of proliferation- incompetent leukemia cells that have been genetically engineered to express ICSBP, wherein the proliferation-incompetent leukemia cells enhance the immune response to the leukemia cells.

15. The method of claim 14, wherein the leukemia cells and the proliferation-incompetent leukemia cells are chronic myeloid leukemia cells.

16. A method of suppressing proliferation of cells in a mammal, wherein proliferation of the cells in the mammal causes an undesirable condition in the mammal, comprising administering to the mammal a therapeutically effective quantity of cells modified to express ICSBP, wherein the cells modified to express ICSBP elicit an immune response to the cells that cause the undesirable condition such that proliferation of the cells that cause the undesirable condition is suppressed and the cells modified to express ICSBP and the cells that cause the undesirable condition are of the same type.

17. The method of claim 16, wherein the undesirable condition is selected from the group consisting of: cancer, bacterial infection, mycobacterial infection, viral infection, parasitic infection, protozoal infection, and autoimmune disease and rejection of transplanted tissue.

18. A method of enhancing immune recognition of cells, present in an individual, that cause disease in the individual, comprising introducing into the individual cells, referred to as ICSBP-expressing cells, that express ICSBP at a sufficient level to stimulate an immune response to the cells that cause disease in the individual that is greater than the immune response to the cells that cause disease in the individual that occurs if ICSBP-expressing cells are not introduced into the individual, thereby enhancing immune recognition of the cells that cause disease in the individual.

19. The method of claim 18, wherein the cells that cause disease in the individual are selected from the group consisting of: malignant cells and cells infected with a pathogen.

20. The method of claim 19, wherein the malignant cells are leukemia cells or solid tumor cells.

21. The method of claim 19, wherein the pathogen is selected from the group consisting of: a virus, a bacterium, a mycobacterium, a parasite, a yeast and a protozoan.

22. A method of increasing the immuno stimulatory effect of a cell, comprising enhancing ICSBP expression in the cell.

23. The method of claim 22. wherein expression is enhanced in the cell by introducing into the cell or an ancestor of the cell exogenous DNA that encodes ICSBP and expresses ICSBP in the cell or by increasing expression of endogenous DNA that encodes ICSBP.

24. The method of claim 22, wherein the cell, as obtained, does not express ICSBP.

25. The method of claim 23, wherein the cell, as obtained, expresses ICSBP.

26. The method of claim 23, wherein the cell, is a malignant cell.

27. The method of claim 26, wherein the malignant cell is a leukemia cell or a solid tumor cell.

28. The method of claim 27, wherein the leukemia cells are chronic myeloid leukemia cells.

29. A method of enhancing the immune response of an individual to tumor cells present in the individual, comprising administering to the individual modified tumor cells, which have been made proliferation-incompetent by irradiation, wherein the modified tumor cells are the same type of tumor cells as the tumor cells present in the individual and express ICSBP encoded by exogenous

DNA, wherein the modified tumor cells are introduced into the individual in sufficient quantity and by an appropriate route to enhance the immune response of the individual to tumor cells present in the individual.

30. The method of claim 29, wherein the tumor cells are leukemia cells.

31. The method of claim 30, wherein the leukemia cells are chronic myeloid leukemia cells.

32. The method of claim 31 , wherein the modified tumor cells are introduced into the individual by injection or infusion.

33. A method of enhancing the immune response to tumor cells in an individual, comprising introducing into the individual cells that co-express an oncogene that is expressed in the tumor cells and ICSBP, in sufficient quantity and by a route that results in an enhanced immune response to the tumor cells in the individual.

34. The method of claim 33, wherein the tumor cells are leukemia cells.

35. The method of claim 34, wherein the leukemia cells are chronic myeloid leukemia cells.

36. The method of claim 34, wherein the cells are introduced into the individual by injection or infusion.

37. A tumor cell, referred to as a modified tumor cell, which is replication incompetent and expresses ICSBP encoded by exogenous DNA.

38. The modified tumor cell of claim 37, which is a leukemia cell.

39. The modified tumor cell of claim 38, which is a chronic myeloid leukemia cell.

40. A replication incompetent chronic myeloid leukemia cell that expresses ICSBP encoded by exogenous DNA.

41. A method of treating a mammal in whom tumor cells are present, comprising co-administering to the mammal at least one chemotherapeutic agent and modified tumor cells which express ICSBP from exogenous DNA.

42. The method of claim 41, wherein the mammal is a human.

43. An in vitro method of producing tumor-directed cytotoxic T-cell clones, comprising combining T cells obtained from a mammal, appropriate growth factors and target cells that express ICSBP and against which cytotoxic T-cell clones are to be produced, thereby producing a combination, maintaining the combination under conditions appropriate for T cell activation and proliferation, thereby producing cytotoxic T-cells clones directed against the target cells.

44. The method of claim 43, wherein the target cell is a tumor cell or a cell infected with a pathogen.

45. A method ofproducing a mammalian cell that expresses ICSBP comprising activating a gene that encodes ICSBP and is a silent gene that is not normally expressed in the mammalian cell.

46. The mammalian cell of claim 45 selected from the group consisting of: a fibroblast, an endothelial cell, an epithelial cell, a hepatocyte, and a blood cell.

47. A genetically engineered mammalian cell that expresses ICSBP from a normally silent, activated endogenous gene.

48. A method of enhancing the ability of an individual to eliminate cells that cause a condition in the individual, comprising increasing ICSBP levels in the individual to a level which results in elimination of the cells to a greater extent than would occur if ICSBP levels were not increased in the individual, thereby enhancing the ability of the individual to eliminate the cells.

49. The method of claim 48, wherein the enhanced ability of the individual to eliminate the cells that cause a condition results from an immunostimuiatory effect of increased ICSBP levels in the individual and the cells are destroyed or inactivated by the immune system of the individual.

50. The method of claim 49, wherein the cells that cause a condition are selected from the group consisting of: a cancer cell or a cell infected with a pathogen.

51. The method of claim 50, wherein the cancer cells are leukemia cells or solid tumor cells.

52. The method of claim 51 , wherein the leukemia cells are chronic myeloid leukemia cells.

53. The method of claim 50, wherein the pathogen is selected from the group consisting of: a virus, a bacterium, a parasite, a mycobacterium, and a fungus.

54. The method of claim 48, comprising increasing ICSBP levels in the individual by introducing into the individual ICSBP-expressing cells, wherein ICSBP is expressed from exogenous DNA encoding ICSBP.

55. The method of claim 54, wherein the ICSBP-expressing cells are the same type of cells as the cells that cause the condition to be treated.

56. The method of claim 55, wherein the cells that cause the condition to be treated are cancer cells or cells infected with a pathogen.

57. The method of claim 56, wherein the cancer cells are leukemia cells or solid tumor cells.

58. The method of claim 57, wherein the leukemia cells are chronic myeloid leukemia cells.

59. The method of claim 56, wherein the pathogen is selected from the group consisting of: a virus, a bacterium, a parasite, a mycobacterium and a fungus.

60. A method of stimulating, in a mammal, an immune response to cells that cause a condition in the mammal which is to be treated, comprising administering to the mammal a therapeutically effective quantity of ICSBP-expressing cells, wherein the ICSBP-expressing cells are cells genetically engineered to express ICSBP.

61. The method of claim 60, wherein the ICSBP-expressing cells and the cells that cause the condition which is to be treated are the same type.

62. The method of claim 61, wherein the immune response is a systemic response.

63. The method of claim 60, wherein the immune response is a local response.

64. The method of claim 60, wherein the condition to be treated is the presence of tumor cells in the mammal.

65. The method of claim 61, wherein the cells are tumor cells and the ICSBP-expressing tumor cells are proliferation-incompetent tumor cells.

66. The method of claim 63, wherein the tumor cells are leukemia cells or solid tumor cells.

67. The method of claim 66, wherein the leukemia cells are chronic myeloid leukemia cells.

68. The method of claim 60, wherein the mammal is a human.

69. The method of claim 61, wherein the mammal is a human.

70. The method of claim 64, wherein the mammal is a human.

71. The method of claim 65, wherein the mammal is a human.

72. The method of claim 66, wherein the mammal is a human.