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Title:  Methods of using BCL-2 for the therapeutic treatment and prevention of diseases

United States Patent:  6,514,761

Issued:  February 4, 2003

Inventors:  Reed; John C. (Carlsbad, CA)

Assignee:  The Burnham Institute (La Jolla, CA)

Appl. No.:  188066

Filed:  November 6, 1998


The invention provides a method of treating a disease or pathological condition resulting in apoptotic cell death. The method includes increasing the activity of Bcl-2 in cells affected by the disease or pathological condition. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and viral infections. Also provided is a method of prolonging the in vivo survival of transplanted cells for the treatment of a disease or pathological condition. The method includes increasing the activity of Bcl-2 in a population of cells and transplanting the population of cells having increased Bcl-2 activity into a subject. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and viral infections. A method to enhance the sensitivity of malignant cells to therapy is provided that includes decreasing the activity of Bcl-2 in the malignant cells. Methods to identify compounds that alter apoptotic cell death and to enhance monoclonal antibody production are also provided by the invention disclosed herein.


This invention is directed to general and effective methods to augment the treatment of diseases and pathological conditions. The methods are applicable to the treatment of cancer, neurodegenerative disorders, viral infections, autoimmune diseases and also to the modification of transplanted tissues and cells. The modified tissues and cells can be used, for example, in the treatment of neurological disorders, as well as diseases caused by hormonal and protein insufficiencies such as diabetes (insulin) and hemophilia (coagulation factors). The methods described herein also enable the development of novel pharmaceutics for the treatment and prevention of diseases and pathological conditions. Additionally, the methods of this invention can be further applied to the production of superior research and diagnostic reagents and compositions for use in essentially all disciplines of the basic and applied sciences.

The invention takes advantages of the ability of Bcl-2 to prevent the process of programmed cell death known as apoptosis. Targeting a physiological mechanism common to many diverse diseases is efficient and cost effective in that the specialized development of different drugs to each of the specific diseases is not required. Instead, a small number of therapeutic compounds or methods of treatment can be developed for the treatment of essentially all diseases that manifest their pathological condition through the aberrant regulation of apoptosis. In many cases, such compounds can be recombinant nucleic acids whose administration is by mode of gene therapy. Thus, reagents that either promote or impair apoptosis are used in the methods of the invention to retard disease-induced apoptotic cell death or to selectively enhance tumor cell killing by conventional chemotherapeutic drugs as well as to protect normal non-neoplastic cells from the toxicity of these drugs.

In one embodiment, gene transfer technology or "gene therapy" is used to create Bcl-2 recombinant DNA molecules and viral vectors to promote the in vivo survival of cells affected by a disease or pathological condition that results in apoptotic cell death. Bcl-2 recombinant DNA molecules and viral vectors are also used to genetically modify cells prior to transplantation to prolong their in vivo survival time and, where applicable, to simultaneously correct protein deficiencies. The use of Bcl-2 to immortalize or prolong the survival rate of targeted cells is advantageous in that it does not block cellular differentiation and is essentially non-tumorigenic when expressed in either primary or established cells compared to other oncogenes. Specific examples of such therapies include the production of recombinant viruses that direct Bcl-2 expression to specific types of neurons and the administration such viruses to patients having Alzheimer's or Parkinson's diseases via direct injection into the brain or spinal fluid; the intracranial implantation of Bcl-2 expressing fetal neuronal precursor cells for the treatment of Parkinson's disease; the mass expansion of human Bcl-2 expressing cells in vitro for prolonging their in vivo survival after transplantation and the use of recombinant vectors for directing Bcl-2 expression to specific cell types for the prevention of cell death induced by viral infections. Bcl-2-expressing cells for use in transplantations can be genetically modified, for example, to secrete various hormones and peptides such as neurotrophic factors in the setting of spinal cord injury, dopamine for the treatment of Parkinson's disease, enkephalins for pain control in terminally ill cancer patients, coagulation factors for patients with hemophilia and insulin producing islet cells for patients with diabetes.

In another embodiment, Bcl-2 gene transfer technology is used to "immortalize" human antibody-producing B-cells and thereby enhance the development of human monoclonal antibodies. The survival promoting function of Bcl-2 is also utilized to generate Bcl-2 transgenic mice for isolating B-cells for enhanced monoclonal antibody production. Such monoclonal antibodies are useful for diagnostic and therapeutic purposes.

In yet another embodiment, a cell-free system is described that faithfully reproduces characteristics of apoptotic cell death. The system is useful for the screening of compounds that alter the apoptotic process. Selected compounds that either promote or inhibit apoptosis can be used for therapeutic treatment of a variety of diseases including neurodegenerative diseases, cancer and virus infected cells.

The Bcl-2 gene was first discovered because of its involvement in lymphomas in humans. This gene has now been shown to prolong cell survival in culture when expressed at high levels in a variety of cell types such as lymphocytes, hemopoietic cells, fibroblasts and neurons. Bcl-2 is a 26 kilodalton (kDa) protein that is unique among cellular genes. It contains a stretch of 17 hydrophobic amino acids near its carboxy terminus that causes its post-translational insertion into intracellular membranes. Gene transfer studies have demonstrated that Bcl-2 promotes cell survival by blocking programmed cell death, or apoptosis.

Apoptosis can be actively triggered in cells by, for example, exposure to X-radiation, cytotoxic drugs, free-radicals and heat, or it can be unmasked by removal of critical peptide growth factors, steroid hormones, lymphokines or neurotrophins that constantly suppress programmed cell death in various tissues. Many of these processes are the terminal events involved in numerous disease states or the final events by which therapeutic treatments effect their results. Thus, to specifically target and alter apoptosis would provide a general treatment for a broad range of diseases and pathological conditions. It should be noted, however, that there exist Bcl-2-independent pathways for apoptosis. Thus, the new uses for Bcl-2 reported here constitute previously undocumented circumstances under which Bcl-2 gene transfer is revealed for the first time to exert protection against programmed cell death.

Bcl-2 is normally expressed in a variety of types of cells but particularly those that either exhibit a long lifespan such as some types of neurons, long-lived "memory" lymphocytes or cell having proliferative, self-renewing potential such as basal epithelial cells and hemopoietic progenitor cells in the bone marrow. It is likely that Bcl-2 represents the prototype of an entire family of structurally similar genes that are expressed in a tissue-specific manner and contribute to the regulation of cellular life span.

As used herein, the term "apoptosis" or "apoptotic cell death" refers to the physiological process known as programmed cell death. Apoptosis is unlike other forms of cell death that occur, for example, as the result of ischemia or necrosis because apoptosis is an active, ATP-requiring form of cell death that typically requires new RNA and protein synthesis. A hallmark of apoptosis is the activation of endogenous endonucleases that initially cleave the genomic DNA at its most accessible sites, i.e., between nucleosomes, producing a ladder of DNA bands representing integer multiples of the internucleosomal distance. This DNA degradation occurs early in the apoptotic process, before loss of plasma membrane integrity. Apoptotic cells also have a shrunken size and the process is not usually accompanied by inflammation since there is no spilling of cytoplasmic contents into the extracellular space. With apoptosis, much of the cell's content is autodigested. In vivo cell lysis never occurs because the apoptotic cells are usually phagocytosed by macrophages and related cells before loss of plasma membrane permeability. Consequently, there is no inflammatory reaction or subsequent scarring. Other morphological characteristics of apoptotic cells include nuclear fragmentation, development of vesicular bodies, "apoptotic bodies" and plasma membrane blebbing, all in the setting of intact mitochondria and lysosomes. Specific examples of apoptotic cell death as a natural programmed event include, for example, the loss of redundant neurons during fetal development and the destruction of potentially autoreactive T-cells during thymic education.

As used herein, the term "Bcl-2" refers to the protein originally discovered due to its inappropriate activation in lymphomas. Bcl-2 controls normal cell growth and differentiation by promoting cell survival. It has a molecular weight of about 26 kDa as determined by SDS-PAGE and is characterized by a hydrophobic stretch of about 17 amino acids near its carboxy terminus that functions in intracellular membrane attachment.

Bcl-2 has substantially the same amino acid sequence as that shown in GenBank accession M13994 and is encoded by a nucleotide sequence substantially similar to that shown in GenBank accession M13994. The definition of "Bcl-2" is intended to include other Bcl-2 family members such as those proteins that are found to exhibit the above functional characteristic or sequence homologies. Such members include, for example, homologs of Bcl-2 cloned from lower organisms such as rats, mice, chickens, flies and worms.

A specific example of a Bcl-2 family member is the protein encoded by the BHRF-1 gene in Epstein Barr virus. The BHRF-1 gene, which exhibits about 22% sequence identity and 47% sequence similarity with Bcl-2, is functionally equivalent to Bcl-2 in promoting cell survival (see, for example, FIG. 5).

It is understood that limited modifications to the protein can be made without destroying the biological function of Bcl-2 and that only a portion of the entire primary structure may be required in order to effect activity. For example, minor modifications of the Bcl-2 protein or nucleotide sequence which do not destroy its activity are included within the definition of Bcl-2. Moreover, fragments of Bcl-2 which retain at least one function of the entire protein are included within the definition. It is understood that various modifications of primary amino acid or nucleotide sequence may result in proteins having substantially equivalent or enhanced function as compared to the sequences set forth in GenBank accession number M133994. These modifications may be deliberate, as through site-directed mutagenesis, or accidental, such as through mutation in hosts which are Bcl-2 producers. All of these modifications are included as long as Bcl-2 biological function is retained. Furthermore, various molecules, such as other proteins, carbohydrates, or lipids, can be attached to Bcl-2. Such modifications are included within the definition of Bcl-2.

The invention provides a method of treating a disease or pathological condition resulting in apoptotic cell death. The method includes increasing the activity of Bcl-2 in cells affected by the disease or pathological condition. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and virus-infected cells.

Alzheimer's disease, the most common neurodegenerative disorder, is estimated to affect four million Americans and represents a major economic burden to families and society. No treatment can stop or even slow the progression of this disorder. Amyloid .beta.-protein (ABP) has been identified as a possible causative agent of this disease. Addition of ABP, or of specific peptide fragments from this protein, to cultured neurons and neuronal cell lines results in cell death. Expression of Bcl-2 in these cultured cells by gene transfer can reduce neuronal cell killing by ABP. These results indicate that apoptosis contributes to neuronal cell death in Alzheimer's disease.

Parkinson's disease is a progressive and ultimately fatal neurodegenerative disorder characterized by loss of the pigmented dopaminergic neurons of the substantia nigra. The symptoms of Parkinson's disease can often be managed initially by administration of L-DOPA, the immediate precursor of dopamine. However, reduced efficacy of L-DOPA treatment often occurs possibly because metabolism of the drug prevents effective delivery to the CNS. Programmed cell death has also been implicated to play an important role in this neurodegenerative disorder inasmuch as withdrawal of neurotrophic factors from neurons leads to cell death through a mechanism consistent with apoptosis. Moreover, the absence of inflammatory cells or scar formation in the brains of patients with Parkinson's disease indicates that striatal neuron death can occur through apoptosis as opposed, for example, to necrosis.

In addition to neurodegenerative disorders, apoptosis has been indicated to result in cell death from glutamate-induced neurotoxicity arising from conditions such as stroke and amyotrophic lateral sclerosis (ALS; "Lou Gehrig's disease"). Glutamate-induced toxicity occurs when glutamate is released from dying neurons in the brain at times of acute injury. Glutamate released by dying neurons in turn binds to specific receptors for glutamate on adjacent healthy neurons, triggering signals that set-off a complex series of biochemical events leading to apoptotic cell death.

Diseases and pathological conditions such as those described above and those that will be described below can be treated by increasing the activity of Bcl-2 in the cells affected by the disease or pathological condition. Increasing Bcl-2 activity in these affected cells will inhibit the apoptotic death of such cells and therefore reduce or prevent progression of the disease or pathological condition.

The activity of Bcl-2 can be increased by a variety of means, including, for example, increasing the Bcl-2 synthesis rate or decreasing the Bcl-2 degradation rate or modulating the ability of Bcl-2 to interact with other proteins that control the apoptosis process. Increasing the synthesis rate of Bcl-2 will result in elevated protein accumulation and thereby increase Bcl-2 activity within the cell.

An elevated synthesis rate can be achieved, for example, by using recombinant expression vectors and gene transfer technology to express a Bcl-2-encoding nucleic acid. Such methods are well known in the art and are described below with reference to recombinant viral vectors. Other vectors compatible with the appropriate targeted cell can accomplish the same goal and, therefore, can be substituted for recombinant viral vectors in the methods described herein. For example, recombinant adenoviruses having general or tissue-specific promoters can be used to drive Bcl-2 cDNA expression and to deliver Bcl-2 expression constructs into a variety of types of tissues and cells, including non-mitotic cells such as neurons in the substantia nigra of the brain (the region affected in Parkinson's disease) (La Salle et al., Science 259:988-990 (1993), which is incorporated herein by reference).

Alternatively, recombinant adeno-associated viruses can be used for this purpose, with the added advantage that the recombinant virus can stably integrate into the chromatin of even quiescent non-proliferating. cells such as neurons of the central and peripheral nervous systems (Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988), which is incorporated herein by reference). Receptor-mediated DNA delivery approaches also can be used to deliver Bcl-2 expression plasmids into cells in a tissue-specific fashion using a tissue-specific ligand or antibody non-covalently complexed with DNA via bridging molecules (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987), both of which are incorporated herein by reference). Direct injection of DNA (mammalian expression plasmids of various types) or of DNA encapsulated in cationic liposomes also can be used for stable gene transfer to non-dividing and dividing cells in vivo (Ulmer et al., Science 259:1745-1748 (1993), which is incorporated herein by reference). In addition, DNA transfer by the particle bombardment method can be used to transfer DNA into a variety of tissues (Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991), which is incorporated herein by reference).

Moreover, recombinant expression vectors encoding Bcl-2 can also contain additional non-Bcl-2-encoding nucleic acids that are useful for the therapeutic treatment of a disease or pathological condition. For example, Bcl-2-encoding vectors can be constructed to encode enzymes used in the synthesis of dopamine when treating Parkinson's disease, for example, with the idea of simultaneously providing genes for enhancement of cell survival (Bcl-2) and cell function (dopamine-.beta.-hydroxylase). Other examples are Bcl-2 plus recombinant DNA sequences engineered to allow for nerve growth factor secretion for sustaining the survival of the cholinergic neurons that are typically lost in Alzheimer's disease (Rosenberg et al., Science 242:1575-1578 (1988), which is incorporated herein by reference), Bcl-2 plus insulin for the treatment of diabetes or Bcl-2 plus encephalin for treatment of intractable pain. Whether other non-Bcl-2-encoding nucleic acids are also contained within a vector will depend on the disease and the therapeutic need. One skilled in the art will be able to determine such a need.

Viruses are very specialized infectious agents that have evolved in many cases to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes this natural specificity, in turn, to specifically target predetermined cell types and, thereby, introduce a recombinant gene engineered into the viral genome into the infected cell. The vector to be used in the methods of the invention will depend on desired cell type to be targeted. For example, if neurodegenerative diseases are to be treated by increasing the Bcl-2 activity of neuronal cells affected by the disease, then a vector specific for cells of the neuronal cell linage could be used. Such viral vectors include, for example, Herpes simplex virus-based vectors (Battleman et al., J. Neurosci. 13:941-951 (1993), which is incorporated herein by reference). Similarly, if a disease or pathological condition of the hematopoietic system is to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, should be used. Such viral vectors include, for example, HIV-based vectors (Carroll et al., J. Cell. Biochem. 17E:241 (1993), which is incorporated herein by reference).

Moreover, such vectors can additionally be modified with specific receptors or ligands to modify or alter target specificity through receptor mediated events. These modification procedures can be performed, for example, using recombinant DNA techniques or synthetic chemistry procedures. Specific examples of viral vectors and their specificity include, for example, Herpes simplex virus for neuronal cell lineages, HIV for T lymphocytes, hepatitis virus for liver cells and Adenovirus for lung and other tissues. In cases where viral infections cannot be made tissue-specific, it may be possible to make viral gene expression specific for only the desired type of cell through the use of tissue-specific promoters and enhancers (Dai et al., Proc. Natl. Acad. Sci. USA 89:10892-10895 (1992), which is incorporated herein by reference).

Viral vectors commonly used for in vivo targeting and therapy procedures are retroviral vectors or DNA-based vectors. Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. However, oncogenic transformation properties of these viruses are destroyed. Once the viral proteins are synthesized, the host cell packages the RNA into new viral particles, which can undergo further rounds of infection. The viral genome is also engineered to encode and express the desired recombinant gene.

In the case of non-infectious viral vectors, the helper virus genome is usually mutated to destroy the viral packaging signal, which is required to encapsulate the RNA into viral particles, but retains the structural genes required to package the co-introduced recombinant virus containing a gene or genes of interest. Without such a signal, any particles that are formed will not contain a genome and, therefore, cannot proceed through subsequent rounds of infection.

The methods for constructing and using such viral vectors are known in the art and are reviewed, for example, in Miller and Rosman, Biotechniques 7:980-990 (1992), which is incorporated herein by reference. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art.

Bcl-2-encoding viral vectors can be administered in several ways to obtain expression and, therefore, increased activity of Bcl-2 in the cells affected by the disease or pathological condition. If viral vectors, for example, are used, the procedure can take advantage of their target specificity and the vectors need not be administered locally at the diseased site. However, local administration can provide a quicker, more effective treatment. Administration can also be by intravenous or subcutaneous injection into the subject. Injection of the viral vectors into the spinal fluid can also be used as a mode of administration, especially in the case of neurodegenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection.

An alternate mode of administration of Bcl-2 encoding vectors can be by direct inoculation locally at the site of the disease or pathological condition. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve Bcl-2 expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement needed with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and expression elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genomes, plasmids, phagemids and the like. Transfection vehicles such as liposomes can be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known to one skilled within the art.

The Bcl-2 encoding nucleic acid used in the methods of the invention is known in the art and available from GenBank under the locus identification "HUMBCL2A" as accession number M133994. The nucleotide sequence available from the above database is all that is necessary for one skilled in the art to obtain a Bcl-2 cDNA for use in the disclosed methods. Moreover, since the Bcl-2 cDNA has been published in Tsujimoto and Croce, Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986), which is incorporated herein by reference, it is also readily available to those skilled in the art.

Using the Bcl-2 sequence described in GenBank accession number M1344, one skilled in the art can clone the Bcl-2 nucleotide sequence using conventional library screening methods. Oligonucleotide probes useful for screening can be synthesized using known methods in the art such as phosphoramidite chemistry. Other method such as the polymerase chain reaction (PCR) also can be used to rapidly and efficiently clone Bcl-2-encoding nucleic acids. Using PCR, a DNA segment of up to approximately 6,000 base pairs in length can be amplified from a single gene copy.

Briefly, PCR involves incubating a denatured DNA sample with two oligonucleotide primers that direct the DNA polymerase-dependent synthesis of complementary strands. Multiple cycles of synthesis are performed, wherein each cycle affords an approximate doubling of the amount of target sequence. Each cycle is controlled by varying the temperature to permit denaturation of the DNA strands, annealing the primers and synthesis of new DNA strands. Use of a thermostable DNA polymerase eliminates the necessity of adding new enzyme for each cycle and permits fully automated DNA amplification. Twenty-five amplification cycles increase the amount of target sequence by approximately 106 -fold. The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 all of which are incorporated herein by reference.

The invention also provides a method of treating a disease or pathological condition resulting in apoptotic cell death by increasing the activity of Bcl-2 wherein the disease or pathological condition is mediated by viral infection. The methods described above for the treatment of neurological diseases and pathological conditions can also be applied to various other disease states such as virus-infected cells. Many viral infections, such as HIV, culminate in cell death through apoptosis.

Apoptosis can be prevented or retarded by expressing a Bcl-2 encoding nucleic acid or functional equivalent thereof in viral-infected cells (see, for example, FIG. 3). Elevated levels of Bcl-2 inhibit the programmed cell death induced by an infecting virus and result in prolonged survival of the infected cells. Bcl-2-containing viral vectors that appropriately target the infected cells can be used to specifically introduce and increase the Bcl-2 activity within the infected cells. Such vectors also can contain non-Bcl-2-encoding nucleic acids that are useful for treating the virus-infected cells.

The invention provides a method of prolonging the in vivo survival of transplanted cells for the treatment of a disease or pathological condition. The method includes increasing the activity of Bcl-2 in a population of cells and transplanting the population of cells having increased Bcl-2 activity into a subject. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and virus-infected cells.

The transplantation of genetically modified cells that secrete proteins, hormones or neurotransmitters, for example, can be used to treat the above diseases as well as many chronic, metabolic and inherited disorders such as diabetes and hemophilia. Employing the methods described herein, the in vivo survival of such diseased cells can be improved by Bcl-2 gene transfer. An advantage of treating cells using Bcl-2 is that Bcl-2 is not oncogenic in most cells and, therefore, can be used to "immortalize" cells that would be responsive to normal growth control mechanisms in vivo.

Cell transplantation is now being explored for the treatment of certain diseases, notably Parkinson's disease. For example, potential therapies in animal models of Parkinson's disease have included cell transplantation of genetically modified fibroblasts, which produce L-DOPA in the vicinity of the substantia nigra. Although the results of these experiments have been encouraging, the survival time of the transplanted cells is limited and, therefore, results in only a temporary and minor improvement of the condition.

Transplantation of fetal brain cells, which contain precursors of the dopaminergic neurons, has also been examined as a potential treatment for Parkinson's disease. In animal models and in patients with this disease, fetal brain cell transplantations have resulted in the temporary reduction of motor abnormalities. Furthermore, it appears that the implanted fetal dopaminergic neurons form synapses with surrounding host neurons. However, the transplantation of fetal brain cells is again limited due, for example, to the limited survival time of the implanted neuronal precursors.

In the specific case of Parkinson's disease, intervention by increasing the activity of Bcl-2 can improve the in vitro and in vivo survival of fetal and adult dopaminergic neurons, their precursors and dopamine-secreting fibroblasts and, thus, can provide a more effective treatment of this disease. Likewise, improved in vivo survival of essentially any cell type to be transplanted will improve the treatment of that disease. For example, neuronal cells or their precursors can be used for the treatment of other neurodegenerative diseases such as Alzheimer's disease and glutamate-induced neuronal cell death by enhancing the in vivo survival of cells using Bcl-2.

Specific examples of cell types other than neuronal cells include hepatocytes for the treatment of liver failure, .beta. cells for the treatment of insulin-dependent diabetes and skin cells for the treatment of burns. Additionally, Bcl-2 expression can be used to enhance survival of transplanted cells for cosmetic treatments. One example of such a cosmetic purpose is for the treatment of alopecia, the medical term for baldness. Moreover, viral vectors can be employed using the methods that target Bcl-2 expression to the cells of the hair follicle or Bcl-2 transfer vehicles can be applied topically to the scalp, resulting in a novel genetic treatment for hair loss.

Cells to be transplanted for the treatment of a particular disease can be genetically modified in vitro so as to increase the activity of Bcl-2. Such methods are known within the art and are essentially the same as those described above, except that Bcl-2 expression is first achieved within the cells in vitro. Bcl-2 expressing vectors can be constructed using recombinant DNA techniques and can utilize, for example, viral vectors, viral genomes, plasmids, phagemids and the like (see, for example, FIG. 1). Such vectors can also encode one or more non-Bcl-2 nucleotide sequences to facilitate the therapeutic function of the cells once they are transplanted.

Bcl-2-encoding vectors are introduced into recipient cells using transfection methods known to one skilled in the art. Such methods include, for example, infection using viral vectors, lipofection, electroporation, particle bombardment and transfection. Detailed procedures for these methods can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and the references cited therein, which are incorporated herein by reference.

Following transfection, cells having increased levels of Bcl-2 activity are selected for use in transplantation treatment. The screening procedure will depend on the method by which the Bcl-2 activity is increased. For example, if increased activity is accomplished through elevated Bcl-2 protein levels, then a quantitative assay that determines the accumulated Bcl-2 protein level can be used. Such assays include, for example, immunoblot analysis, immunoprecipitation and ELISA. Such methods are known to one skilled in the art and can be found in Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, 1989) or in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988), both of which are incorporated herein by reference. Functional assays also can be employed such as the inhibition of apoptotic DNA degradation or nuclear disintegration, such as disclosed herein or known in the art (see Example VII).

Similar to the use of Bcl-2 for prolonged in vivo survival of transplanted cells, the expansion of mammalian cells in culture for subsequent transplantation or the expansion of cells for industrial scale production of proteins, metabolites or other clinically useful factors can benefit from the enhanced survival due to Bcl-2. Among the limitations that plague these types of procedures are the dependence of many cell types on adherence to a solid surface. Moreover, many types of cells die through apoptosis when their required growth factors run in short supply or when toxic by-products accumulate in cultures of cells grown at high densities. Such problems can be overcome by increasing the levels of Bcl-2 within the cultured cells.

For example, increased levels of Bcl-2 can allow anchorage-dependent cells to survive and grow in the absence of attachment to a solid surface. Bcl-2 overexpression can also allow cells to grow to-higher densities when compared to cells expressing low or normal Bcl-2 levels. Moreover, a novel form of the Bcl-2 protein, Bcl2/P59S, is substantially more active than the wild-type protein in allowing higher density growth and preventing cell death due to the depletion of growth and survival factors in tissue culture medium (FIG. 2). All of these properties of Bcl-2 can be utilized for mass expansion of cells in culture.

The invention also provides a method of prolonging the in vivo survival of transplanted cells for the treatment of a disease or pathological condition by increasing the Bcl-2 activity of immune cells. The immune system consists of a variety of cell types that protect the body from infectious organisms and continually monitor the body for the appearance of abnormal cells such as cancer cells. Some immune cells have the capacity to bind to and kill other cell types, particularly tumor cells and virus-infected cells. One outcome of the killer cell response, besides target cell death, is the activation of apoptosis within the killer cells, themselves. The physiological role of-this self-induced suicide may be a negative feedback mechanism for controlling an immune response. However, this regulatory mechanism of immune cell death prevents immune responses from being of sufficient duration and intensity to effectively eliminate malignant or virus-infected cells. Tumor-specific and virus-specific immune cells can also die from lack of sufficient growth factors such as interleukin-2 (Il-2) in vivo. This process can be arrested or prevented by Bcl-2 gene transfer. Thus, the augmentation of immune cell survival due to increased Bcl-2 expression can result in more effective treatment of cancer and virus-induced diseases.

Apoptotic death of immune cells can be inhibited by isolating these cells or their precursors and modifying them to express elevated levels of Bcl-2. The methods for modifying these cells are essentially the same as those described above. The transplantation of the Bcl-2-expressing cells into a subject suspected of having a cancer or a viral infection will ensure a more prolonged and active immune response against the condition. As an alternative to extracorporeal treatment, tissue-specific gene transfer and expression technology can be used to specifically increase Bcl-2 gene expression in the killer cells in vivo.

Other non-Bcl-2 genes that augment the cell survival function of Bcl-2 or enhance the killer activity of immune cells can be introduced into cells in combination with Bcl-2. A specific example of introducing a second gene that enhances the effector activity of immune cells is the coexpression of the Lck protein tyrosine kinase. When constitutively overexpressed, for example, by mutation of the regulatory tyrosine residue at amino acid position 505 to a phenylalanine (Y505F), Lck confers T cell effector functions on the Y505F-expressing cell in the absence of IL-2. Abrogation of the IL-2 requirement is clinically advantageous because side effects due to treatment with an immunostimulatory drug such as IL-2 can be avoided. Nucleic acids encoding proteins other than Lck-can be coexpressed to enhance effector functions of immune cells. Such nucleic acids include, for example, Lyn, Hck, Fyn, Yes, Atk, Fgr and Blk.

Raf-1, which encodes a serine-threonine protein kinase, is an example of a non-Bcl-2 gene that can be administered in conjunction with Bcl-2 to enhance the action of Bcl-2 (see Example V and FIG. 6). Infection of cells with DNA sequences encoding a mutant version of the Raf-1 kinase having constitutive, non-inducible kinase activity acts synergistically with Bcl-2 to prolong cell survival by blocking apoptotic cell death (see, for example, FIG. 6A). Thus, coexpression of non-Bcl-2 genes to augment function of Bcl-2 or to enhance other desired functions provides a means of preventing or limiting virus infections and malignant cell growth.

The invention also provides a method for enhancing the sensitivity of malignant or virus-infected cells to therapy by decreasing the activity of Bcl-2 in the malignant or virus-infected cells. Decreased activity can be accomplished by expressing an alternative form of Bcl-2 capable of forming a bound complex with Bcl-2, wherein the bound Bcl-2 is inactive, or with other proteins that interact with Bcl-2, thus inhibiting the normal function of Bcl-2.

Many types of malignant cells become resistant or refractory to treatment due to high endogenous levels of Bcl-2. These malignant cells having high levels of Bcl-2 include prostate, colorectal and nasopharyngeal cancers and lymphomas, leukemias and heuroblastoma. Similarly, virus-infected cells can be intrinsically resistant to treatment because of endogenous Bcl-2 expression. In contrast to the previously described methods for increasing the Bcl-2 activity, such diseases can be effectively treated by utilizing the opposite approach, i.e., inhibiting Bcl-2 activity. Suppression of Bcl-2 function can be employed alone or in combination with conventional therapies to provide a more effective means of decreasing the resistance of malignant or virus-infected cells to killing by chemotherapeutic drugs and irradiation.

Bcl-2 expression can be inhibited, for example, by targeting and/or expression vectors that produce the alternative form of the Bcl-2 protein, Bcl-2.beta.. When expressed in conjunction with a normal Bcl-2 gene, Bcl-2.beta. binds to the cellular proteins with which Bcl-2 normally interacts and prevents Bcl-2 function, probably through a competition mechanism. The inhibition of a wild-type function through the coexpression of a variant form of a gene product is known in the art as a dominant negative mutation (FIG. 7; see, also, Kolch et al., Nature 349:426-428 (1991), which is incorporated herein by reference.

Bcl-2.beta. arises through an alternative splicing mechanism and lacks the hydrophobic stretch of amino acids found in the normal Bcl-2 protein, the hydrophobic region being necessary for membrane insertion of Bcl-2 and its function as a blocker of apoptosis. Other examples of mutant Bcl-2 are proteins that have been genetically engineered to contain deletions within the region of amino acids 85-219 of the 239 amino acid Bcl-2 protein. In malignant cells where Bcl-2 function has been markedly reduced by dominant negative mutation, enhanced sensitivity to killing by a wide variety of chemotherapeutic drugs such as methotrexate, Adriamycin, Ara-C and dexamethasone can be observed.

In addition to inhibiting Bcl-2 activity to enhance the sensitivity of malignant or virus infected cells to therapy, other non-Bcl-2 gene products involved in the progression of the diseased state can be inhibited to increase the efficacy of treatment. For example, high levels of Raf-1 kinase activity are associated with radioresistance of tumors. Gene transfer manipulations that cause an increase of decrease of Raf-1 kinase activity can increase or decrease, respectively, the sensitivity of tumor cells to killing by chemotherapeutic drugs. Moreover, elevations in Raf-1 activity in the presence of increased Bcl-2, activity indicate that the combined use of reagents designed to interfere with Raf-1 and Bcl-2 can act synergistically to render tumor cells more sensitive to killing by conventional chemotherapeutic drugs and irradiation (see Example V).

As an alternative to gene therapy and transfer approaches, chemical compounds that alter the activity of Bcl-2 can be used. The same rationale as described above for treating diseases or pathological conditions can be applied to these applications, except that the specific compounds that alter the Bcl-2 activity are substituted in place of recombinant methods. Thus, all the therapies described previously using Bcl-2 gene transfer are equally applicable to the use of Bcl-2 specific compounds.

Novel Bcl-2 specific compounds can be obtained, for example, through rational design or random drug-screening methods. All that is required is a method to accurately identify active compounds. Active compounds include both those that increase Bcl-2 activity as well as those that decrease its activity. Thus, the determination of an activity will depend on the desired outcome.

The invention provides a method to identify compounds that alter the process of apoptosis (see Example VII). This method includes treating an apoptotic cell extract with one or more compounds and selecting the compound that alters the apoptotic process in the cell extract. Thus, the method allows the identification of active Bcl-2 specific compounds. The method consists of a cell free extract that faithfully reproduces the apoptotic process. Briefly, when Xenopus egg extracts are mixed with sperm chromatin, the chromatin is assembled into a nucleus that is surrounded by a nuclear enveloped. These cell free nuclei undergo degeneration spontaneously with time or inducibly in the presence of particular drugs. The nuclear degeneration process is indicative of the process that occurs in cells dying by apoptosis. Addition of Bcl-2 protein to the extracts prevents nuclear breakdown. Active Bcl-2 specific compounds can be identified by substituting the compound for Bcl-2 in the cell free extract. An advantage of the cell free extract method is that it can be automated by monitoring, for example, the transport of radiolabeled or fluorescent-tagged peptides into nuclei; this transport process is prevented by nuclear breakdown.

The invention also provides a method to enhance monoclonal antibody production by prolonging the in vitro survival of hybridoma precursor cells by increasing the activity of Bcl-2 in the precursor cells. Similar to the methods described above, Bcl-2 can enhance the survival, for example, of antibody producing cells. Such enhanced survival can increase the efficiency of monoclonal antibody production by allowing the generation of a greater number of successful fusions.

Precursor hybridoma cells, such as myeloma fusion partners and antibody-producing B cells, can be modified using methods described herein to elevate the expression of Bcl-2. Bcl-2 expression vectors can be introduced in vitro into the myeloma cells using the disclosed methods. Since the antibody-producing B cells are isolated from an immunized animal, increasing Bcl-2 expression in these cells can be accomplished by immunizing transgenic animals expressing a Bcl-2 encoding transgene. B cells taken from the spleen of such immunized animals will have enhanced survival characteristics compared to B cells from normal animals. In addition, hybridomas prepared using B cells obtained from transgenic mice expressing a human bcl-2 gene produced a higher frequency of antigen-positive clones than did B cells obtained from normal mice, thereby increasing the likelihood of obtaining a hybridoma cell line expressing a desirable monoclonal antibody. Thus, the invention also provides transgenic mice expressing Bcl-2 as the transgene. The B lymphocytes of such mice are useful for obtaining more efficient monoclonal antibody production. Moreover, increased Bcl-2 activity can also be used to immortalize human B cells for the production of human monoclonal antibodies.

Claim 1 of 19 Claims

What is claimed is:

1. A method to prolong the in vitro survival of primary, explanted tumor cells, comprising introducing into the primary, explanted tumor cells a nucleic acid molecule encoding a polypeptide selected from the group consisting of human Bcl-2, a species homolog of human Bcl-2 and BHRF-1 and expressing said polypeptide, whereby expression of said polypeptide prolongs the in vitro survival of said cells.


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