Reviews
`
`Gene and Immune-based Theraples for Genitourinary
`Mallgnancles: Current Status and Future Prospects
`
`Amnon Zisman MD, Allan J. Pantuck MD and Arie 13elldegrun MD FACS
`
`Division of Urologic Oncology, Department of Urology, University of California, Los Angeles School of Medicine,
`Los Angeles, CA, USA.
`
`Key words: prostate cancer, renal cell carcinoma, transitional cell carcinoma, immunotherapy, gene therapy, suicide
`gene, tumor suppressor genes, antisense mRNA, immunomodulation
`
`IMAJ 2000,•2:33-42
`
`For Editorial see page 58
`
`In recent years we have witnessed improvements in many
`fields of cancer research and therapy, including enhance-
`ment of preventive medicine and early detection programs,
`improved surgical techniques, and major achievements in
`radio- and chemotherapy. However, the ability to cure the
`majority of cancer patients remains elusive. At the same
`time, it has become increasingly apparent that cells of the
`immune system play a key role in the recognition and
`elimination of neoplastic cells. Recently, therefore, new
`cancer therapies have been directed at modulating and
`exploiting components of the immune system in order to
`augment the immunogenicity of, and thus eradicate cancer
`cells. The science of vectorology has occupied itself with the
`task of designing and constructing new methods of
`delivering genes into tumor tissue with high efficiency.
`These methods, which are tumor or organ specific, are also
`capable of preventing systemic toxic effects.
`The above mentioned achievements in basic science have
`not bypassed the clinical realm of genitourinary oncology.
`The use of intravesical immunotherapy for superficial
`bladder tumors was the first immune-based therapy for
`bladder cancer and has become a gold standard. Develop-
`ments in immunotherapy have resulted in an improved
`outlook for patients presenting with advanced renal cell
`carcinoma as well as for those who develop both distant and
`local recurrences after curative treatment. These advances
`represent only the beginning of new directions to come.
`Clearly, the future prospects of cancer therapy will be built
`upon the foundation of current investigative efforts in gene
`and immune therapy.
`This article reviews the current role of immunotherapy
`and gene therapy in the treatment of metastatic and
`recurrent genitourinary neoplasms. First, we will discuss
`the fundamentals of immunotherapy and gene therapy. Next,
`we will review the applications of each of these therapeutic
`modalities individually with respect to prostate, bladder and
`renal cell carcinoma. We will then address future directions
`in gene and immunotherapy; and, finally, we will list the
`current clinical trials, focusing on gene and immune
`
`therapies. We contend that all the data on current cancer
`clinical trials should be available for the public to choose
`from [Tables 1 and 2].
`One of the fundamental dilemmas in gene therapy today
`involves the selection of genes to be used in clinical trials.
`Unlike many other genetic diseases, tumorigenesis is a
`multi-step pathway involving initiation, proliferation, loss of
`contact inhibition, invasion and ultimately metastasis of the
`cancer cell. Multiple genes involving cell cycle regulation,
`angiogenesis, immunoreactivity and cell adhesion are also
`involved. Since no single gene defect has been found to
`facilitate tumorigenesis for all cancer diseases, different
`mutation pathways may lead to the same end result.
`Consequently, the utilization of a single type of gene therapy
`may not be enough. Errors in gene regulation, transcription
`or translation can lead to morphologic and functional
`changes within the cell, whereafter the cancer phenotype
`may become apparent. There are several potential mecha-
`nisms by which gene therapy may achieve cancer control
`[Table 3].
`
`Cytoreductive Therapies
`Using this strategy, a gene (suicide gene) is injected into the
`tumor or specifically attached to it. After transfection, the
`gene that is expressed results in the production of a protein,
`usually an enzyme that is capable of converting an otherwise
`benign medication into a highly cytotoxic one. Obviously this
`will result in a high concentration of the cytotoxic agent in
`the tumor but without significant systemic concentrations.
`Active suicide genes enable not only destruction of the
`transfected cell, but the destruction of adjacent tumor cells
`(bystander effect). This means that not all tumor cells need
`to be directly transfected. Thus, one of the advantages of
`this approach is the need for less efficient transfection in
`comparison to other gene therapies. For example, the
`herpes simplex thymidine kinase gene (HSV-tk) is one of
`the most commonly used systems; another system uses
`cytosine deaminase [1]. Human thymidine kinase cannot
`phosphorylate certain pro-drugs like gancyclovir, while
`HSV-tk can phosphorylate gancyclovir to gancyclovir mono-
`phosphate. This is then converted to gancyclovir triphos-
`phate by cellular kinases. The resulting triphosphate acts as
`
`IMAJ - Vol 2 - January 2000
`
`Gene and Immune-based Therapies for G U Malignancies
`
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`Table 1. Clinical trials for prostate cancer using molecular-based approaches
`
`Study center
`Principal investigators
`Basic principle
`^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
`Anti-EGF Ab
`Belldegrun
`UCLA
`Liposomal IL-2
`Belldegrun
`UCLA
`Adenovirus P53 Belldegrun
`UCLA
`Intradermal vaccinia-PSA
`Chen
`Naval Medical Academy
`Autologous tumor cell vaccine + IFN/GM-CSF (phase II)
`Dillman
`Multicenter
`Intramuscular Vaccinia virus^MUC1-IL2
`Figlin
`UCLA
`Adenovirus gancyclovir/TK
`Hall
`Mount Sinai Hospital
`PSA vaccine-Vaccinia virus (phase I)
`Hamilton
`NMOB
`Adenovirus gancyclovir/TK
`Kadmon
`Baylor College of Medicine
`Intradermal vaccinia-PSA
`Kufe
`Dana Farber Cancer Inst.
`Adenovirus P53 Logothetis
`MD Anderson
`PSA vaccine-Vaccinia virus (phase I/II)
`Sanda
`University of Michigan
`Adenovirus gancyclovir/TK
`Scardino
`Baylor College of Medicine
`GM-CSF immunotherapy
`Simons
`John Hopkins
`MSKCC
`Autologous tumor cell vaccine + IFN/IL2
`Slovin
`Autologous tumor cell vaccine+ GM-CSF (phase I/II)
`Small
`UCSF
`PSA vaccine-Lipid envelope (phase II)
`Spitler (Chair, Ph)
`Multicenter
`Retrovirus antisense c-myc Steiner
`Vanderbildt
`^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
`Adopted with changes from *http:/cancernet.nci.nih.gov/ and from Rodrigez R. et al, Urologic applications of gene therapy.
`Urology 1999;54:401-6.
`MSKCC = Memorial Sloan-Kettering Cancer Center.
`
`Table 2. Clinical trials for renal cell cancer using molecular-based approaches
`^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
`Basic principle
`Principal investigators
`Study center
`^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
`HLA-B7 and IL-2
`Antonia
`University of South Florida
`Intra-tumoral injection of LEUVECTIN (phase II)
`Belldegrun
`UCLA
`Liposome HLA-B7/b 2 microglobulin
`Chang
`Multicenter
`Autologous tumor cell vaccine+IFN/GM-CSF (phase II)
`Dillman
`Multicenter
`Economou
`TIL+INF+IL2
`UCLA
`Liposome IL-2
`Figlin
`UCLA
`The HLA-B7 and IL-2 gene
`Figlin
`UCLA
`HLA-B7/beta-2 microglobulin
`Fox
`Chiles Research Institute
`MSKCC
`IL-2 (allogeneic)
`Gansbacher
`Multi-antigen loaded dendritic cell vaccine (adoptive
`immunotherapy — phase I)
`Gitlitz
`UCLA
`IL-4
`Lotze
`University of Pittsburgh
`Rosenberg
`TNFV-
`NIH
`IL-2
`Rosenberg
`NIH
`GM-CSF
`Simons
`Johns Hopkins
`^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
`Adopted with changes from *http://cancernet.nci.nih.gov and from Rodrigez R. et al, Urologic applications of gene therapy.
`Urology 1999;54:401-6.
`
`a false base inhibiting DNA polymerase and DNA synthesis,
`ultimately leading to cell death. The bystander effect seen
`with this method is not clearly understood. Proposed
`explanations for this effect include the distribution of
`gancyclovir triphosphate via gap junctions into neighboring
`cells, a local inflammatory response due to direct injection
`into prostatic tissue, or a systemic immune response. The
`bystander effect and requirement for only transient and
`modest trans-gene expression has made suicide gene
`therapy very appealing and has contributed to its early
`approval for human trials. Other pro-drug systems include
`the cytosine deaminase gene and the Escherichea coli
`xanthine-guanine phosphoribosyltransferase. Cytosine de-
`aminase is a bacterial gene that converts 5-fluorocytosine
`into the anti-metabolite 5-fluorouracil, which causes cell
`
`death by inhibiting the host cell DNA synthesis. E. coli
`xanthine-guanine phosphoribosyltransferase phosphorylates
`6-thioxanthine to its cytotoxic monophosphate.
`
`Corrective Gene Therapy
`Many different genetic alterations have been recognized in
`urologic tumors. Most of them are distinguished by either
`the overexpression of an oncogene or the inactivation of a
`tumor-suppressor gene such as the p53 gene. In diseases
`like cystic fibrosis where the disorder is caused by a single
`gene defect, gene replacement is particularly attractive.
`Unfortunately, these approaches have not proven successful
`in cancer patients. This may be due to the fact that there is
`no single oncogene or tumor-suppressor gene defect that
`can be definitely implicated in the formation of all tumors.
`
`34 A. Zisman et al.
`
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`Table 3. Potential mechanisms by which molecular-based
`approaches may attain cancer control
`
`•
`
`Cytoredactive therapies
`• Suicide gene (e.g., thimidine kinase gene therapy followed by
`gancyclovir administration).
`• Drug activation of suicide genes.
`• Oncolytic viruses (e.g., adenovirus that replicates in p53-deficient
`cells).
`Toxic gene therapy (e.g., diphtheria toxin that induces necrosis and
`apoptosis)
`Corrective gene therapy
`• Correction of defective tumor suppressor genes by insertion of wild
`type genes (e.g., p53, p16, p27).
`• Growth factor modulation using antisense mRNA techniques (e.g.,
`antisense bcl-2 and antisense TGF().
`Immanotherapy/cancer (genetic) vaccine elicitingimmane response
`Transfection of tumor cells with cytokines or growth factor genes.
`•
`Secretion of the gene product (IL-2, GM-CSF (G-VAX), IL-12) for
`immune activation and effect on primary or systemic immunity. Lower
`toxicity than with direct injection of cytokines.
`• Adoptive immunotherapy: expose cytotoxic lymphocytes or dendritic
`cells to cancer-specific antigens and instigate an immune response by
`returning cells to patient (e.g., PSMA).
`Immanomodalation by administering cytokines directly (e.g., IL-4,
`GM-CSF and co-stimulation with 13-7).
`
`Thus, the simultaneous targeting of multiple gene defects
`will probably be a more appealing strategy in the future.
`
`Immunotherapy
`It has been hypothesized that tumor cells escape surveil-
`lance and destruction by the immune system through down-
`regulation of cell surface antigens, such as the major
`histocompatibility complex [2]. Tumor vaccine approaches
`involve the use of cytokine genes (interleukin-2, IL-6, tumor
`necrosis factor-, interferon gamma, granulocyte-macrophage
`colony-stimulating factor, IL-4, IL-12) transfected into tumor
`cells in vitro [3]. Cytokines originating in the transfected
`cells induce expression of cell surface proteins such as HLA
`class I and II that then augment their immunogenicity. Cells
`
`Reviews
`
`are thereafter irradiated to eliminate proliferative capability
`and are administered back to the patient as a vaccine in an
`attempt to generate an immune response against the
`remaining tumor burden. Efficacy of this approach depends
`on achieving a high level of cytokine expression in the tumor
`tissue and on the availability of tumor tissue for the process.
`Second-generation tumor vaccines utilize retroviruses,
`lipid-packaged segments or naked DNA-encoding cytokine
`genes. These constructs are injected into the tumor.
`Transfection takes place in vivo and a chronic cytokine
`production results [4].
`
`Molecular-based Approaches to
`Urologic Malignancy
`Bringing prostate cancer into the framework of
`molecular-based therapy
`The strategies for different gene and immune therapies in
`prostate cancer are illustrated in Figure 1. The expectation
`of a breakthrough in gene therapy for prostate cancer is
`based on the fact that cells in the prostate gland express
`tissue-specific molecules (prostate-specific antigen and
`prostate-specific membrane antigen). With the discovery
`and sequencing of additional genes encoding specific
`prostate antigens — such as the PSA enhancer promoter
`[5], specific oncogens, cell surface receptors and tumor
`suppressor genes [7] such as p53, Rb, p2l, PML, BRCAl, c-
`myc, Bcl-2 and transforming growth factor- b [6] — the
`potential for prostate-specific targeting at the genetic level
`becomes apparent as the number of possible genetic targets
`increase. Phase I studies using PSA promoter-driven genes
`are now underway. The expression of these genes has been
`shown to be both androgen-responsive and -specific for
`prostate tissue [7]. These studies have combined the PSA
`gene enhancer or promoter with suicide genes like the HSV-
`tk gene or cytosine deaminase gene, or tumor suppressor
`genes. Studies utilizing intratumoral injection of adenovirus
`vector carrying the suicide gene HSV-tk alone have already
`been conducted and have shown it to
`be effective in the Dunning prostate
`cancer rat tumor model and nontoxic
`for both cancer patients and care-
`givers.
`Cytoreductive therapy^ Gene re-
`placement and antisense strategies
`require highly efficient transfection of
`prostate cancer cells in order to be
`effective. Thus, suicide gene ap-
`proaches that do not require as high
`levels of transfection seem appealing.
`Advances in prostate-specific suicide
`genes have been considerably pro-
`moted by the identification and clon-
`ing of the PSA promoter and
`enhancer. By using these sequences
`
`Figure 1. The strategies for different gene and immune-based therapies in prostate cancer.
`PBL - peripheral blood lymphocytes, VEGF - vascular endothelial growth factor, FGF -
`fibroblast growth factor
`
`PSA = prostate-specific antigen
`
`IMAJ • Vol 2 • January 2000
`
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`to drive suicide gene expression, fairly high levels of
`prostate tissue specificity can be achieved while incurring
`minimal risk to non-prostatic tissues.
`Direct intratumoral injections of suicide genes without
`the aid of the PSA promoter have also been tested and were
`found to be target-specific and effective. Intratumoral
`injection of the HSV-tk suicide gene in a Dunning rat
`prostate cancer model has shown a growth inhibition of
`subcutaneously implanted tumors with little systemic
`toxicity when compared to control animals [8]. The intra-
`tumoral injection route is used to minimize systemic effects,
`but investigators have recently demonstrated that the
`intratumoral injection of the HSV-tk gene also induces
`systemic anti-tumor effects. For example, one study showed
`growth inhibition of pulmonary metastasis in a mouse model
`of prostate cancer [1]. Moreover, when attenuated repli-
`cation-deficient adenovirus carrying the HSV-tk gene was
`injected into the tumor in 18 patients, lower dose levels
`yielded no toxicity or clinical responses while higher doses
`showed 20-25% response rates.
`The concept of suicide genes has led to new possibilities
`utilizing previously tested regulatory genes for prostate
`cancer to increase the effect of tumor cell death. Gene
`complexes that utilize promoter sequences requiring
`mutated p53 for activity are being created. The suicide
`gene is placed under the control of a promoter. When the
`abnormal p53 binds to it the suicide gene is expressed,
`leading to tumor cell death [9].
`Another suicide gene system utilizes the human inducible
`heat shock protein 70 promoter sequence as a regulatory
`component. Thus, suicide gene expression is induced by a
`temperature elevation. A recent study using both cytosine
`deaminase and HSV-tk suicide genes under the control of
`this system employed a pro-drug. After appropriate heat
`induction and pro-drug administration, targeted PC-3
`prostate cancer cell line growth was inhibited significantly.
`Additional suicide gene candidates for prostate cancer
`therapy have recently been identified and characterized.
`These include diphtheria toxin, which has significant p53-
`independent cytotoxic effects on the LnCaP cell line; and
`cerulenin, a fatty acid synthesis inhibitor that has demon-
`strated apoptotic effects on the TSU-Prl cell line. This gene
`system holds promise for future therapies.
`
`Corrective gene therapy.Alternatively, correction of
`aberrant gene expression, such as through the replacement
`of malfunctioning tumor suppressor genes, is another route
`for gene therapy in prostate cancer.
`
`• p53: Abnormal expression of the p53 has been
`implicated in a variety of tumor systems. The p53
`protein manifests control on cellular proliferation by
`blocking the binding of DNA polymerase to the DNA
`strand [10] if sufficient damage has been incurred. This
`results in the arrest of cell proliferation at the G1
`checkpoint of the cell cycle. If the p53 gene product is
`absent, cell proliferation will continue in the face of
`
`•
`
`severe DNA damage, resulting in increased genetic
`instability and possible tumorigenic effects. Approxi-
`mately 60% of prostate cancer cell lines have mutations
`in the p53 gene [11]. Abnormal p53 expression has been
`found in more aggressive tumors, and appears to be an
`independent predictor of cancer recurrence after radical
`prostatectomy. The transfection of normal wild type p53
`gene into p53-deficient prostate cancer cell line resulted
`in decreased tumorigenicity and decreased proliferation
`after being injected back into nude mice. Unfortunately,
`80% of sporadic prostate cancers do not have an
`identifiable defect in the p53 protein, but many other
`oncogenes and tumor suppressor genes have been
`identified as potential targets for gene therapy, including
`c-ras, H-ras, c-myc, TGF- b and bcl-2 [12].
`p21: This gene is thought to play a similar role as p53 by
`inhibiting DNA replication when severely damaged. The
`p2l gene encodes for a protein that functions as an
`inducer of a cyclin-dependent kinase inhibitor, which may
`also play a role in DNA replication and repair [13]. Thus,
`lack of the wild type p2l protein results in perpetuation of
`the cell cycle in the presence of DNA damage. Recent
`studies comparing the transfection effects of p53-
`deficient prostate cancer cell lines with wild type
`adenovirus-mediated p53 and p2l genes, both in vitro
`and in vivo, have shown greater growth suppression with
`p2l than p53 gene replacement. This has led to the
`assumption that certain subsets of p53-deficient prostate
`tumors may be more responsive to p2l-directed gene
`replacement therapy than conventional therapy with p53.
`• PML gene (progressive multifocal leukoencephal-
`opathy). Aberrant expression of this gene has been
`observed in various human cancers, including leukemia,
`breast cancer and prostate cancer. This gene encodes a
`protein that is involved in the suppression of growth and
`transformation of cells. Direct intratumoral injection of
`this vector into nude mice decreased tumor growth by
`more than 60%. These results suggest a promising role
`for the PML gene in future prostate cancer gene therapy.
`• BRCA1: The breast cancer susceptibility gene
`(BRCA1), a tumor suppressor gene, has been implicated
`in hereditary prostate cancer. The BRCA1 gene is
`involved in transcriptional regulation, but its exact role
`is still poorly understood. In vitro experiments using wild
`type and mutant BRCA1 transfection into a low BRCA1-
`expressing prostate cancer cell line (DU145) have
`demonstrated increased tumor doubling time, increased
`susceptibility to drug-induced apoptosis, reduced capa-
`bility to repair single strand DNA breaks, and alteration
`in regulatory genes such as p2l and Bcl-2. Phase I clinical
`trials using wild type BRCA1 incorporated into retro-
`virus vectors have shown encouraging results, and
`further studies are currently underway.
`
`TGF-b =transforming growth factor-beta
`
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`Other prospective tumor suppressor genes that are
`currently being investigated for possible roles in prostate
`cancer and future gene therapy include the retinoblastoma
`gene, CDKN2, STEAP, KA^1, GSTP1 and various cellular
`adhesion molecule genes. These genes have all been
`implicated as having roles in different prostate cancer cell
`lines.
`
`• Antisense gene therapy is an arm of corrective gene
`therapy [Table 3]. Antisense molecules use complimen-
`tary mRNA segments and oligonucleotides that bind to
`and inactivate target genes by inhibiting transcription or
`translation. This strategy has been proposed as a method
`to disarm the cancer cell by counteracting the over-
`expression of a particular oncogene. The antisense
`molecule can be delivered directly, or inside a vector
`as part of a gene consolidate, which will then transcribe
`antisense molecules in the presence of a promoter.
`c-myc is an oncogene whose mechanism of enhanced
`expression has yet to be explained. Its expression has
`been found in various prostate cancer cell lines [14].
`Transduction of the LnCaP, PC3 and DU145 prostate
`cancer cell lines with antisense c-myc mRNA revealed
`dose-dependent reduction in DNA synthesis and cell
`viability [15]. Nude mice with established DU145
`xenografts showed a significant decrease in tumor size
`and histological aggressiveness after intratumoral in-
`jection of antisense c-myc mRNA. This has led to Phase I
`clinical trials currently in progress.
`
`•
`
`Further proposed targets for antisense therapy include the
`Bcl-2 and TGF-b genes, both of which are overexpressed in
`certain aggressive prostate cancers.
`
`• Bcl-2: The Bcl-2 protein inhibits cell apoptosis and has
`been implicated in the development of androgen-
`resistant prostate cancer cells. It is believed that Bcl-2,
`by blocking apoptosis, is responsible for the poor
`response to anti-neoplastic drugs and radiation therapy
`seen in tumors. In vitro studies using ribozymes to
`cleave the Bcl-2 RNA have demonstrated efficacy, and
`thus may be applicable for gene therapy.
`• TGF-b
`: High levels of TGF-b enable prostate cancer
`cells to adhere to bone marrow stroma and, furthermore,
`to suppress local immune system responses against their
`tumoral antigens [16]. Recent experiments have also
`demonstrated the restoration of TGF-b growth inhibition
`function on prostate cancer cell lines via the over-
`expression of TGF-b 1 type II receptors. Current experi-
`ments have also shown that TGF- b has a proliferative
`effect on the highly aggressive prostate cancer cell line,
`TSU-Pr1 [17]. Clearly the role of TGF- b in prostate
`cancer is a complex one. Work is currently underway to
`further delineate the function of this interesting sub-
`stance in prostate cancer.
`
`Numerous other genes are also being studied for their
`apoptotic effect in tumor cells in the hope of inducing cell
`
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`death pathways by means of gene therapy. Possible targets
`being studied include Bax, Bcl-X-S, Bcl-X-L, E2F, DP,
`cytokines IL-1, tumor necrosis factor- α and viral proteins
`E1A and E1B.
`
`Immunotherapy: Prostate cancer may escape immune
`response partly due to the lack of MHC class I expression,
`resulting in poor antigen presentation. Gene therapies have
`been constructed to overcome this lack of immunogenicity
`and loss of MHC-I expression by transfecting cytokine
`genes directly into prostate cancer cells. Because some
`segments of the PSA molecule are immunogenic and
`stimulate T and B cell immune responses [18,19], efforts
`have been made to transfect dendritic cells with PSM or
`PSA genes. The transfected dendritic cells will present the
`prostate antigens as foreign, thus stimulating an immune
`system-mediated, anti-tumor response [20]. Clinical trials
`are now underway in which patients are vaccinated against
`the PSA protein, with the hope that a systemic immune
`response against prostate cancer will develop. Local
`production of cytokines in the prostate can generate an
`immune response. The Dunning rat R3327-MatLyLu cell
`line was transfected with the IL-2 gene and then injected
`back to a site remote from the primary tumor. This
`procedure cured the rats and also protected them from
`future tumor cell challenges [20]. Similar work done with
`the GM-CSF gene formed the basis for current clinical trials
`using the GM-CSF gene in prostate cancer [25]. The long-
`term role that tumor vaccine therapies will play in the
`treatment of prostate cancer is yet to be determined.
`Summary
`Recent demonstrations that prostate tumors are immuno-
`genic, coupled with new developments in gene delivery
`technology, and better understanding of prostate tissue-
`specific regulation of gene expression, have enhanced the
`prospects of gene therapy as a feasible approach to the
`treatment of advanced prostate cancer. Currently, phase I
`dose-escalation and safety-evaluation clinical trials are
`underway. These studies involve the use of PSA
`promoter-driven suicide genes, IL-2 gene transfection, PSA
`and GM-CSF tumor vaccines, and p53 and anti c-myc gene
`replacement approaches. The biological efficacy of these
`approaches should become clear in the next few years. Some
`of these clinical trials are listed in Table 1.
`From intravesical instillation of Bacillus
`calmette-guerin to molecular-based therapy for
`patients with transitional cell carcinoma
`Although superficial papillary tumors are easily resected by
`trans-urethral resection of bladder tumor, there is a high
`recurrence rate after surgery. Risk factors for recurrence
`include the presence of multiple lesions, high grade tumors,
`and previous recurrences. Likewise, the presence of TCC in
`
`MHC = major histocompatibility complex
`PSM =prostate-specific membrane
`GM-CSF = granulocyte macrophage colony-stimulating factor
`TCC = transitional cell carcinoma
`
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`situ forecasts a worse outcome. Prevention of invasion and
`recurrence by superficial tumors and the amelioration of
`TCC in situ are the goals in the treatment of bladder cancer,
`thereby reducing the morbidity and mortality associated
`with advanced disease.
`Intravesical instillation of Bacillus calmette-guerin myco-
`bacteria strain is one of the earliest and most effective
`immunotherapies known. BCG plays an important role in
`the treatment of noninvasive bladder cancer and is
`recommended for prophylaxis against tumor recurrence
`after successful trans-urethral resection of bladder tumors.
`Its mechanism of action is currently unknown, although
`IVC-BCG is believed to induce anti-tumor effects through
`immune effector agents. IVC-BCG may initiate a tumor-
`specific immune response involving T cell-dependent
`agents, including T helper cells, macrophages, IL-2, IFN,
`and tumor necrosis factor. However, The degree of the local
`inflammatory/immune response correlates directly with the
`likelihood of a patient remaining disease free. Although IVC-
`BCG is generally well tolerated by 90-95% of patients,
`complications occasionally do occur and mortality is rarely
`reported [22]. Indeed, 2-5% of patients develop severe,
`potentially life-threatening complications that necessitate
`intensive treatment. Despite the impressive success of IVC-
`BCG treatment, an average of 20% of patients treated with
`combined trans-urethral and IVC-BCG fail to respond to
`therapy and will either develop recurrent tumors or
`progress to invasive disease [23]. For these patients,
`treatment options are limited. In the search for therapies
`as effective as IVC-BCG but with less toxicity, other
`immunotherapeutic agents have been investigated:
`
`Cytoreductive therapy: Transfection with the HSV-tk
`suicide gene and administration of gancyclovir resulted in a
`tenfold decrease in tumor size in a murine model. One
`limitation to this approach is that the penetration of
`intravesical instillation of the transfecting agent is limited
`to the superficial cell layers.
`Initial studies showed the safety and efficacy of a
`genetically modified BCG strain that also expresses the
`gene for IL-2. Even more promising results were reported
`in an animal model using the strategy of transfection with
`IL-2 plus B7, which is a potent T cell stimulator. The vector
`for the delivery of the transfecting agent is the rate-limiting
`factor. A recent report of a phase I study describes the
`intravesical administration of vaccinia virus to patients
`before radical cystectomy, with no apparent toxicity [24].
`Adenovirus vectors appear to be promising as intravesical
`gene delivery vehicles with easy access and safety profile.
`Because they are not given systemically, pre-existing
`humoral blocking antibodies against adenovirus should not
`decrease the efficacy of treatment. Adenovirus vectors may
`be able to provide tumor-specific gene transfection because
`they seem to preferentially infect bladder tumor cells
`
`instead of normal mucosa [25]. Moreover, the administration
`of the naked IL-2 gene alone protected by a lipid envelope
`(liposomal IL-2) was reported to be very efficacious and a
`high concentration of post-transfection urinary IL-2 was
`demonstrated.
`
`Corrective gene therapy: Gene therapy for TCC is
`appealing because the bladder urothelium is readily
`accessible for intravesical instillation of the genetic agent.
`The effect may be monitored with cystoscopy as well as
`cytology and other available tests (BTA, NMP-22 and CK-
`20). To date, the tumor suppressor genes Rb, c-myc, C-CAM
`1, Bdx-1 (cell apoptosis regulator) and p53 have all been
`identified as potential targets for gene replacement therapy
`in TCC.
`
`•
`
`• Rh gene: Epidemiological evidence shows an increased
`incidence of TCC in retinoblastoma families [26]. More-
`over, retinoblastoma gene activation has been observed
`in patients with sporadic bladder cancer. Rb-deficient
`TCC cell lines have been established. When these cell
`lines were transfected with the wild type Rb, a decreased
`tumorigenicity and slowed cellular proliferation were
`recorded both in vitro and in vivo. Phase I and Phase II
`clinical trials have already been initiated in humans, and
`some studies have shown that selected bladder cancer
`cell lines transfected by the wild type Rb gene still
`maintain some malignant features [26]. It was suggested
`that mutations in the Rb gene represent late events in
`carcinogenesis, leading to increased aggressiveness.
`c-myc: Expression of this gene has been associated with
`tumor progression and chemotherapy resistance. Since
`the c-myc gene product confers resistance to cisplatin-
`induced DNA injury, cell replication continues despite
`DNA damage, leading to a severe genomic instability.
`Gene therapy against c-myc has been tried by adding c-
`myc antisense oligonucleotides to cell cultures containing
`human bladder tumor cell lines that are known to
`express c-myc and are resistant to cisplatin-based
`chemotherapy. The result was a significant decline in
`the translation of c-myc sense mRNA with an enhanced
`cytotoxic effect when used in combination with cisplatin
`[27]. The combination of c-myc antisense gene therapy
`and chemotherapy may significantly improve the out-
`come of patients with advanced TCC.
`• p53: Changes in p53 are believed to be one of the first
`steps in bladder tumorigenesis. Initial studies using a
`wild type p53 transfected into murine and human TCC
`cell lines and animal models are promising.
`
`At this time a few clinical trials are applying gene therapy to
`human subjects with bladder cancer, and most of them are
`using adenoviral vectors. The genes tested are the Rb gene
`at the University of California in San Francisco, and the p53
`gene at the University of California in Los Angeles and the
`
`IVC-BCG = intravesical instillation of Bacillus calmette-guerin
`IFN = interferon
`
`CAM = cell adhesion molecule
`
`38 A. Zisman et al.
`
`IMAJ • Vol 2 • January 2000
`
`NOVARTIS EXHIBIT 2099
`Breckenridge v. Novartis, IPR 2017-01592
`Page 6 of 10
`
`

`

`M.D. Anderson Cancer Institute [28]. The combination of
`the ability to directly examine and inject tumors intravesi-
`cally, together with the accum