Molecular-based Therapies
`for Renal Cell Carcinoma
`Amnon Zisman, MD, Allan J. Pantuck, MD,
`and Arie S. Belldegrun, MD, FACS
`
`Address
`Department of Urology, University of California School of
`Medicine, Le Conte Avenue, Room 66-118 CHS, Los Angeles,
`CA 90095-1738, USA.
`E-mail: tprintz@mednet.ucla.edu
`Current Urology Reports 2001, 2:55–61
`Current Science Inc. ISSN 1527–2737
`Copyright © 2001 Current Science Inc.
`
`The incidence of renal cell carcinoma (RCC) is rising
`steadily, but the ability to cure patients with metastatic
`RCC unfortunately remains limited. Emerging interest in
`gene therapy performance and safety is expressed by
`patients, medical institutes, and other agencies. It has
`become evident that better understanding of the genetic
`impairments and immune pathophysiology in RCC is
`essential for future improvement in patient care. Clinical
`trials now underway that are focusing on genetic and
`immune impairments will hopefully lead to future break-
`throughs in RCC therapy. This paper reviews available gene
`therapies and other related therapeutic approaches for
`RCC and lists some of the current clinical trials focused
`on molecular-based therapies.
`
`Introduction
`Renal cell carcinoma (RCC) is the most common tumor
`rising from the kidney. Approximately 30,000 cases
`per year are diagnosed in the United States, and incidence
`is increasing [1]. RCC is frequently resistant to radiation
`and chemotherapy, leaving surgical removal of the tumor
`the treatment of choice for localized renal cell cancer.
`However, patients with a disseminated disease have a poor
`prognosis, with an average survival of less than 1 year from
`the time of diagnosis [2]. Traditionally, nephrectomy is not
`usually performed when metastases are present, and the
`objective response rate to conventional chemotherapy is
`only 6% [3]. About 1% to 5% of patients with RCC have
`spontaneous regression, mainly of their pulmonary
`metastases [4]. The reason for this is unknown but is gener-
`ally attributed to self-immune mechanisms. With this in
`mind, methods are being developed to boost the human
`immune system to fight metastatic tumors (Fig. 1). These
`methods are generally termed immunotherapy.
`
`Immunotherapy
`The rationale for turning the immune system against tumor
`cells is based on the theory that the tumor cells survive
`through bypassing and inhibition of immune surveillance
`mechanisms (eg, by downregulation of cell surface antigens,
`such as the major histocompatibility complex [5] and by local
`hindrance of tumor-infiltrating immune competent cells).
`The first attempts to reinforce immunomodulation included
`direct systemic administration of nonspecific stimulants, such
`as the cytokines IL-2, interferon-a (IFN-a), IL-4, GM-CSF, and
`IL-12. Alternatively, adoptive (passive) immunotherapy was
`applied, in which self-lymphocytes were exposed ex vivo to
`the cytokines and then reinjected to the patient. The first
`attempts to apply adoptive immunotherapy used nonspecific
`LAK cells that were isolated from the patient’s peripheral
`blood, incubated in vitro with IL-2, and reinfused into the
`patient. A number of clinical trials demonstrated an average
`response rate of 23.5 % [6]. A more specific application is the
`combination of tumor-infiltrating leukocytes (TILs), which
`are isolated from the tumor tissue and are assumed to have
`immunologic memory to tumor antigens. On a cellular
`basis, TILs are 50 to 100 times more potent than LAK cells in
`mediating tumor regression. Few clinical studies have been
`undertaken with TILs. At the University of California, Los
`Angeles (UCLA), TILs are isolated from fresh nephrectomy
`specimens of RCC patients, expanded in vitro with IL-2 in the
`presence of tumor extract, and reinfused to the patient. The
`response rate obtained with this protocol was 33%, and the
`average response duration was 14 months, with a mean sur-
`vival of 22 months [7]. Recently, more tumor-specific
`approaches have been developed, targeting specific tumor
`markers in an attempt to limit the immune destruction to the
`malignant cells. These techniques are tailored to each patient.
`One promising example is the use of autologous dendritic
`cells (DCs) that were exposed ex vivo to specific tumor anti-
`gens. These DCs serve as antigen-presenting cells that present
`the tumor epitopes to T cells in situ, leading to anti-tumor
`immunity. This approach is currently being tested in a
`clinical trial at UCLA [8].
`Lately, preliminary data on two exciting new approaches
`have been reported showing impressive rates of regression
`of metastatic foci in patients with advanced RCC. These
`studies extend and improve the dendritic cell tumor vaccine
`approach by using an autologous tumor cell electrically
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`New Techniques, Molecular Gene Therapy
`
`CTL
`
`.........
`
`-cell
`medlailecl
`tlJI
`
`, ...
`
`/
`
`1d UC
`
`Figure 1. Schematic representation of potential molecular-based therapies for renal cell carcinoma. AP—antigen presenting;
`CTL—cytotoxic T cell; DC—dendritic cell; WT—wild type.
`
`fused with allogenic dendritic cells to form an RCC-DC
`hybrid cell [9••]. Similarly interesting reports on the use
`of nonmyeloablative allogeneic peripheral-blood stem-cell
`transplantation give hope for patients failing currently
`existing immunotherapies [10•].
`Cytokine-based immunotherapy has several advantages:
`1) it is easy to produce large quantities by recombinant DNA
`technology; 2) administration of this immunotherapy does
`not require complicated preparation procedures; and 3)
`FDA approval of a universal therapeutic agent, such as a
`cytokine, is more straightforward than a patient-dependent
`“tailor-made” treatment protocol. As a result, IL-2 has
`become an FDA-approved treatment for metastatic RCC in a
`relatively short period of time. Although interferon-based
`therapy generates similar overall response rates to IL-2 (15%
`to 20%) [6], high-dose IL-2 monotherapy causes a higher
`frequency of complete remission and prolonged response
`durations. Regardless of the specific immunotherapy regi-
`men used, patients included in immunotherapy clinical
`trials had better prognosis than patients treated by other
`clinical protocols in which no immunotherapy was given
`[11]. Some common protocols for IL-2 immunotherapy and
`their outcome are listed in Table 1 [12]. The overall outcome
`of high-dose intravenous recombinant interleukin (rIL-2)
`alone appears to be beneficial to each of the other regimens.
`However, this treatment is accompanied by very high toxic-
`ity and other adverse reactions [13]. Therefore, other strate-
`gies for therapy are continuously tested.
`
`Molecular-based Therapeutic Strategies
`Molecular-based therapeutic strategies have evolved beyond
`the previously described immunotherapeutic techniques to
`
`include a variety of genes and cell signaling pathways.
`Classification of these therapies, based on their strategic
`approach to eliminate tumor cells, is listed in Table 2.
`
`Immune-based gene therapy (cancer vaccines)
`This therapeutic approach aims to preserve the anti-tumor
`activity of cytokines while minimizing their systemic
`adverse effects. In this approach, cytokine genes or growth
`factor genes are introduced locally by various transfection
`and infection techniques, aiming at confined production
`of high levels of the gene products within the tumor vicin-
`ity. The gene products that are produced locally in high
`concentrations may directly alter neoplastic properties
`associated with invasion and metastasis. This approach
`carries significant advantage in that it is associated with
`lower toxicity than systemic administration of cytokines
`[14]. Animal studies using this technique have demon-
`strated prevention of tumor growth, decreased metastatic
`spread, and prolonged immunologic memory, resulting
`in rejection of subsequent tumor challenges. At UCLA we
`have transfected the genes for IL-2 and IFN-a into human
`RCC lines. When these cells were implanted subcutane-
`ously into nude mice, their cytokine secretion inhibited
`local tumor growth in a more effective manner than
`systemic administration of IL-2 and IFN-a [15]. In addi-
`tion to IL-2 and IFN-a, various other genes of immune
`system modulators, including GM-CSF [16] and B7
`(a costimulatory molecule needed for T cell activation
`[17]), were tested as possible tumor vaccines for RCC [18].
`One of the options used to confine the cytokines into the
`tumor site is by transfecting tumor infiltrating lymphocytes
`(TILs) with the cytokine genes [19]. The rationale is
`that TILs will home back to tumor deposits. In this way,
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`Molecular-based Therapies for Renal Cell Carcinoma • Zisman et al.
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`Table 1. Protocols tested in interleukin-2 immunotherapy for renal cell carcinoma
`
`Regimen
`
`Response rate, %
`
`Complete
`response rate, %
`
`Median response
`duration, mo
`
`High-dose rIL-2 alone
`High-dose rIL-2/rIFN-a
`Outpatient subcutaneous rIL-2/rIFN-a
`Outpatient rIL-2/rIFN-a, plus 5-FU/rIFN-a
`
`17
`11
`17
`16
`
`7
`0
`4
`4
`
`53
`7
`12
`9
`
`Table 2. Classification of molecular-based therapies for renal cell carcinoma
`
`Immunotherapy
`Cytokine administration (eg, IL-2, interferon, IL-4, GM-CSF, IL-12)
`Adoptive immunotherapy: ex vivo activation of autologous immune effector cells using recombinant cytokines followed
`by reinfusion to the patient
`Antigen-presenting cells therapy alone or in combination with cytokines (dendritic cell vaccines)
`Immune-based gene therapy (cancer vaccines)
`Cells transfected with cytokine genes or growth factors genes (IL-2, GM-CSF [G-VAX], IL-12) are introduced to the tum
`or site and locally release the transfected gene product, inducing immune activation with lower systemic toxicity than
`with direct injection of cytokines. The transfected cells can be autologous tumor cells or TILs.
`Viruses as gene carriers (adeno viruses, retro viruses, pox, and vaccinia)
`Direct transfection with DNA/genes
`Cytoreductive therapies
`Suicide genes (eg, thymidine kinase gene therapy followed by gancyclovir administration)
`Drug-activated suicide genes
`Oncolytic viruses (eg, adenovirus replicating in p53 deficient cells only)
`Toxic gene therapy (necrosis and apoptosis induction by diphteria toxin)
`Corrective gene therapy
`Introduction of active wild type suppressor genes (eg, p53, VHL gene, p16, p27) into tumor cells with defective or
`inactive tumor suppressor genes
`Tumor growth and survival factor modulation by antisense mRNA molecules (eg, antisense bcl-2 and antisense TGF-b)
`
`secretion of the relevant gene product may be localized
`and continuous, leading to augmented killing. A phase I
`clinical trial using irradiated RCC cells transfected ex vivo
`with human GM-CSF gene was performed to test the safety
`and the induction of immune responses in patients with
`metastatic RCC [20]. No significant toxicity or autoim-
`mune responses were reported, and one of 16 patients had
`a partial response. Furthermore, recent studies using modi-
`fied dendritic cells [21] and studies directly introducing the
`cytokines into the tumor have been performed at UCLA as
`well as in other centers. At this time, tumor vaccine-based
`gene therapy appears to be safe, but its efficacy in meta-
`static RCC has yet to be proven.
`
`Cytoreductive therapy
`This therapeutic line shares the concept of introducing
`specific genes into the tumor site. However, in cyto-
`reductive therapy, a suicidal gene is introduced into the
`tumor. In most cases, the gene encodes for an enzyme
`capable of converting an otherwise benign medication
`(pro-drug) into a highly cytotoxic one. Administration
`of the pro-drug produces a high concentration of the cyto-
`toxic metabolite in the tumor area, but not in normal
`tissues that did not encounter the gene, resulting in
`reduced systemic toxicity. The most commonly used
`system in this group is herpes simplex thymidine kinase
`
`gene (HSV-tk). HSV-tk phosphorylates ganciclovir to ganci-
`clovir monophosphate, which is converted to ganciclovir
`triphosphate by cellular kinases. The resulting triphos-
`phate acts as a false base, inhibiting DNA polymerase and
`DNA synthesis, leading to cell death. Other examples of
`cytoreductive strategies include virus systems that target
`only p53 deficient cells and the diphtheria toxin gene,
`whose product induces necrosis and apoptosis.
`
`Corrective gene therapy
`This therapeutic approach attempts to correct a specific
`genetic alteration that was found to be present in a certain
`malignancy, such as overexpression of an oncogene or
`inactivation of a tumor suppressor gene (eg, p53 gene) by a
`mutation. The logic for corrective gene therapy is that the
`rectification should restore normal growth control. Correc-
`tive gene therapy uses the same vectors as immunotherapy
`and cytoreductive therapy to introduce the relevant wild
`type gene into the tumor cells. Alternatively, corrective
`gene therapy can be achieved by introducing the comple-
`mentary stretch of an mRNA, whose further translation to
`an active protein is undesired. This complementary stretch,
`called “antisense mRNA” binds to the sense-mRNA and
`blocks the translation of its protein. This was experimented
`with oncogenes like bcl-2 and growth factor genes like
`transforming growth factor (TGF) b [22].
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`New Techniques, Molecular Gene Therapy
`
`Tumorigenetic Pathways
`of Renal Cell Carcinoma:
`Mechanisms and Therapeutic Implications
`Chromosome 3 and von Hippel-Lindau gene
`Genetic studies of families with hereditary RCC, in
`the presence or absence of von Hippel-Lindau (VHL)
`syndrome, contributed to our current view of the tumori-
`genetic pathways of RCC, possibly leading to the design
`of future gene-targeted therapies. These studies identified
`a loss of chromosome 3p in many sporadic and familial
`RCC patients [23] and localized the VHL gene on chro-
`mosome 3p25-26 [23,24]. The wild type (WT) VHL gene
`product, pVHL, is a 213 amino acid polypeptide that
`forms multiprotein complexes with elongin B, elongin C,
`and Cul-2 and negatively regulates hypoxia-inducible
`mRNAs, such as the mRNA encoding vascular endothelial
`growth factor (VEGF). pVHL is suspected to play a role in
`ubiquitination given the similarity of elongin C and Cul-
`2 with Skp1 and Cdc53, respectively. Furthermore, pVHL
`can interact with fibronectin and is required for the
`assembly of a fibronectin matrix. Thus, pVHL is a tumor
`suppressor protein that regulates angiogenesis, extracellu-
`lar matrix formation, and the cell cycle [25]. More than
`half of the patients with sporadic RCC have a detectable
`mutation in one allele of the VHL gene [26], and allelic
`loss in the other allele is seen in up to 98% of tumors
`[27,28]. Gross karyotypic changes of chromosome 3,
`such as chromosomal 3p loss or monosomy, were also
`observed in some patients [29,30]. Moreover, subtle
`molecular changes in the form of hypermethylation of
`the DNA in regulatory areas of the VHL gene, which were
`found in up to 20% of sporadic RCC [31,32], caused tran-
`scriptional arrest. Additional gene loci on chromosome
`3p [33,34] and aberrations in chromosomes 5, 7, 14, and
`Y [35] were also associated with RCC. These loci may be
`able to act independently from the VHL locus, resulting
`in the development of RCC. Moreover, it was recently
`shown that the loss of VHL gene is associated with
`increased expression of the inhibitory cytokines TGFa
`and b1 [36•]. This ties together the genetic and immuno-
`logic alterations seen with RCC.
`Therefore, the VHL gene and gene products may serve
`as potential targets for corrective gene therapy. Initial
`studies have been performed to replace the defective tumor
`suppressor product by the WT gene in an attempt to reverse
`the cancer phenotype. The WT VHL gene was transfected
`into RCC cell lines that lacked the normal expression of the
`gene, attached to a constitutively activated cytomegalovirus
`promoter via a liposome vehicle [37••]. Transfection of the
`WT VHL gene resulted in growth suppression of the RCC
`cell line. In contrast to cultures of mutant VHL RCC cells,
`which formed very compact and cohesive spheroids, the
`WT VHL transfectants were loosely arranged and formed a
`network of tubular and trabecular structures within the
`spheroids [38]. The morphologic changes of the WT VHL
`spheroids were associated with the deposition of fibronec-
`
`tin in the extracellular space, a feature that was absent in the
`mutant and inactivated VHL gene-expressing spheroids.
`The results suggest that the VHL gene may be involved in
`the maintenance of the epithelial phenotype of renal tubu-
`lar cells, ie, it may act as a differentiation/morphogenetic
`factor. Moreover, this effect in tumor cells appears to be
`highly dependent on multicellular growth conditions that
`mimic the basic nature of solid tumors, such as RCC [38].
`In addition, it was recently shown that loss of VHL gene was
`associated with increased expression of TGF-a and TGF-b1,
`and that transfection of RCC cell lines with WT VHL sub-
`stantially decreased TGF-a and TGF-b1 mRNA and protein
`by shortening their mRNA half-life [36,39]. Thus, gene
`replacement therapy using the WT VHL gene may have a
`role in treating patients with RCC, although the safety and
`efficacy of this treatment is yet to be defined.
`
`P53 mutation
`p53 was named Molecule of the Year in 1993 by the editors
`of Science [40], and the “guardian of the genome” by many
`researchers. The human p53 tumor suppressor gene is
`located on chromosome 17p13.1 and encodes a nuclear
`transcription factor that has been implicated in normal
`development and cellular responses to stress. The WT protein
`plays a major role in DNA transcription, cell growth and pro-
`liferation, and a number of metabolic processes. It
`suppresses abnormal cell proliferation and is involved in
`programmed cell death, or apoptosis in damaged cells
`[41,42]. The mechanisms of growth arrest and apoptosis are
`both direct, intracellular effects moderated by p53-respon-
`sive genes and bystander, intercellular mechanisms involving
`secretion of growth inhibitory factors [43]. Mutations in the
`p53 gene lead to loss of the tumor suppressor capabilities
`and to cancer promotion. A large number of human cancers
`contain p53 mutations, including cancers of the breast, cer-
`vix, colon, lung, liver, prostate, bladder, and skin. In RCC,
`p53 is associated with the aggressive sarco-matoid variant
`(79%) [44]. The loss of p53 function in these tumors is asso-
`ciated with more aggressiveness, higher frequency of
`metastasis, and resistance to anti-cancer therapy.
`The WT p53 suppresses progression through the cell cycle
`in response to DNA damage, thereby allowing DNA repair to
`occur before replicating the genome. Therefore, p53 prevents
`the transmission of damaged genetic information from one
`cell generation to the next and initiates apoptosis if the
`damage to the cell is severe. The carboxy-terminus of human
`p53 plays an important role in controlling the specific DNA
`binding function. WT p53 is found in a latent form, which
`does not bind to DNA. The specific DNA binding activity was
`shown to be activated by various pathways: phosphorylation,
`antibody specific for the carboxy-terminus of the protein,
`small peptides that could mimic the carboxy-terminus of the
`p53, short single-stranded DNA, deletion of the last
`30 amino acids, and the interaction with a cellular protein
`[45–47]. Thus, all naturally occurring mutations in p53
`directly or indirectly affect the interaction of p53 with DNA,
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`Table 3. Current clinical trials using molecular based therapies for advanced renal cell carcinoma
`
`Basic principle
`
`Principal investigator
`
`Intratumoral injection of LEUVECTIN (phase II)
`TIL+INF+IL-2
`Liposome IL-2
`HLA-B7 and IL-2 gene
`Multi-antigen loaded dendritic cell vaccine
`(Adoptive immunotherapy - phase I)
`HLA-B7 and IL-2
`IL-4
`TNF- a
`IL-2
`Liposome HLA-B7/b-2 microglobulin
`Autologous tumor cell vaccine+IFN/GM-CSF (phase II)
`IL-2 (allogeneic)
`GM-CSF
`HLA-B7/b-2 microglobulin
`
`Belldegrun
`Belldegrun
`Figlin
`Figlin
`Gitlitz
`
`Antonia
`Lotze
`Rosenberg
`Rosenberg
`Chang
`Dillman
`Gansbacher
`Simons
`Fox
`
`Site
`
`UCLA
`UCLA
`UCLA
`UCLA
`UCLA
`
`University of South Florida
`University of Pittsburgh
`NIH
`NIH
`multicenter
`multicenter
`MSKC
`Johns Hopkins
`Chiles Research Institute
`
`Adapted with changes from the National Cancer Institute web site: www.cancernet.mci.nih.gov and from Rodriguez et al. [58].
`MSKC—Memorial Sloan-Kettering Cancer Center; NIH—National Institutes of Health; UCLA—University of California, Los Angeles.
`
`demonstrating that sequence-specific DNA binding is central
`to the normal functioning of p53 as a tumor suppressor.
`Therefore, p53 gene and gene products may serve
`as potential targets for corrective gene therapy. In vitro
`attempts to correct the p53 gene in RCC cell lines using
`liposome-p53 gene complexes have resulted in decreased
`growth of tumor cells in culture. Transfection of the p53
`gene into a mouse xenograft model resulted in a decrease in
`the number of metastatic lung lesions [48]. A phase I dose
`escalation study of a single intratumoral injection of a
`replication-defective adenoviral expression vector encoding
`WT p53 was carried out in patients with incurable non-
`small cell lung cancer. All patients enrolled had p53 protein
`overexpression as a marker of mutant p53 status in pretreat-
`ment tumor biopsies. No clinically significant toxicity
`was observed; however, successful transfer of WT
`p53 was achieved only with higher vector doses [49]. In a
`recent phase I study, patients with non-small cell lung
`cancer received repeated intratumoral injections of Ad-p53.
`Repeated injections appear to be equally tolerated, resulted
`in transgene expression of WT p53, and seemed to mediate
`antitumor activity in a subset of patients [50]. The use of
`the p53 WT gene therapy by intratumoral injection may
`prove effective for additional tumors.
`
`G250
`G250 is a tumor marker recently introduced for RCC. This
`protein is detected on most primary and metastatic RCCs
`but is absent from kidney and other normal tissues,
`with the exception of gastric mucosal cells and cells of the
`larger bile ducts [50,52]. G250 is a transmembrane protein
`identical to the tumor-associated antigen MN/CA IX that
`was previously identified in cervical carcinoma. It is a
`carbonic anhydrase IX tumor antigen and is expressed only
`when a mutation occurs either within the coding region of
`
`the elonging binding domain of the VII gene, or when a 5’
`non-sense or frameshift mutation occurs [53]. However, its
`unique expression on RCC tissues makes it a potential can-
`didate for both tumor diagnosis and therapy [54,55]. Vari-
`ous potential therapeutic approaches can be considered,
`including targeting the tumors by specific anti-G250 anti-
`bodies or educating T cells to specific killing by presenting
`them with G250 epitopes using dendritic cell tumor vac-
`cines [56•]. Further studies are currently ongoing to eluci-
`date its potential role. At UCLA we fused the DNA
`sequences of the G250 renal cell carcinoma-associated
`antigen together with granulocyte-macrophage colony
`stimulating factor (GM-CSF) and cloned a novel G250-
`GM-CSF fusion protein [57]. The fusion protein retains
`its GM-CSF biologic activity and together with IL-4 is
`capable of inducing dendritic cell (DC) differentiation.
`The activated DCs induce specific killing activity by cyto-
`toxic T cells against allogeneic tumors cultured from meta-
`static RCC patients. This new strategy opens multiple
`therapeutic possibilities and may obviate fresh tumor spec-
`imen for the generation of anti-tumor vaccines. It may be
`possible that patients’ DCs will be activated with the fusion
`protein and, together with systemic IL-2 administration,
`will activate tumor-infiltrating lymphocytes specifically
`against G250 on RCC cells. A list of clinical trials using
`molecular-based therapies is presented (Table 3).
`
`Conclusions
`In the past 20 years, impressive advances in the application
`of immunotherapy to treating renal cell carcinoma were
`witnessed. At UCLA we have seen a progressive increase
`in responses to treatment as therapy has evolved, from
`systemic IFNa administration (16%), to combination
`IFN+IL-2 (25%), to the current method of bulk TILs (33%)
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`and CD8-/TILs (40%). Patient characteristics that predict
`improved responsiveness to therapy have been identified,
`and treatment protocols that decrease toxicity have been
`developed. The most encouraging results have been the
`improved rates of complete clinical response, most of
`which are durable and long-lasting.
`There is no doubt that current immunotherapeutic
`protocols produce changes in the natural history of this
`disease and cause significant and lasting remissions in
`select patients. Moreover, the place of surgery in advanced
`RCC is no longer anecdotal. With the advantages of tumor-
`infiltrating leukocyte technology, nephrectomy is essential
`for the preparation of tumor-infiltrating leukocytes.
`
`References and Recommended Reading
`Papers of particular interest, published recently, have been
`highlighted as:
`•
`Of importance
`•• Of major importance
`
`6.
`
`7.
`
`2.
`
`3.
`
`4.
`
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`An excellent description of a milestone in the evolution of
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`An excellent description of a milestone in the evolution of modern
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