International Journal of Urology (2001) 8, S1–S4
`
`Conference Proceedings
`
`Gene and immune therapy for renal cell carcinoma
`
`ALLAN J PANTUCK, AMNON ZISMAN AND ARIE BELLDEGRUN
`Division of Urologic Oncology, Department of Urology, University of California School of
`Medicine, Los Angeles, California, USA
`
`Abstract
`
`Conventional therapy for metastatic renal cell carcinoma is associated with a poor response rate
`and few patients are long-term survivors. The occurrence of spontaneous regression and the pro-
`longed latency period between primary tumor removal and the appearance of metastases in some
`patients suggest the existence of important host immune responses to autologous tumor cells. With
`the advent of molecular gene transfer techniques and increased knowledge of the basic pathways
`of immune activation, the field of cancer immunotherapy has finally begun to develop novel
`and effective approaches for harnessing the immune system as a therapeutic agent. Current
`immunotherapy and gene therapy strategies, including methods of cytokine delivery and tumor-
`cell-based vaccines, are presented.
`
`Key words
`
`cytokine, gene therapy, immunotherapy, renal cell carcinoma, tumor vaccine.
`
`Introduction
`
`For at least 100 years, immunologists have proposed
`activating the immune system to specifically target and
`eradicate autologous tumor cells. The idea that tumor
`cells can be recognized as foreign to the host’s immune
`system is an essential component of tumor immunol-
`ogy. This concept of tumor cell recognition as foreign
`by their host was first postulated by Paul Ehrlich at the
`turn of the century. In 1943, Gross noted that when
`tumor cells were injected subcutaneously into synge-
`neic mice, the cells formed nodules that grew for a few
`days and then regressed.1 When tumor cells were re-
`injected into the mice, they failed to produce nodules
`or grow. This was interpreted to mean that the tumor
`cells did not grow because the mice had become
`immunologically resistant to the tumor, documenting
`the existence of tumor-associated antigens. In 1954,
`Billingham introduced the term ‘adoptive immunity’ to
`describe the acquisition of immunity as a result of the
`transference of immunologically competent cells rather
`
`Correspondence: Allan J Pantuck MD, University of
`California School of Medicine, Department of Urology,
`10833 Le Conte Avenue, Room 66-118 CHS, Los Angeles,
`USA. Email: apantuck@mednet.ucla.edu
`Presented at the 4th Urological Research Forum, 28–30
`January 2000, Okinawa, Japan.
`
`than of preformed antibody.2 In 1957, Prehn and Main
`demonstrated, further, that immunization of syngeneic
`mice with a given tumor protected the mice against a
`second challenge with the same tumor, but did not pro-
`tect them from other tumors.3 In 1959, Thomas sug-
`gested that the immune response might be able to rid
`the body of abnormal cells.4 His theories were later
`refined into the 1970 immune surveillance hypothesis
`of Burnet, which suggests that the immune system
`could recognize malignant cells as foreign and gener-
`ate a response against them and that only tumors capa-
`ble of evading the body’s surveillance would be able to
`grow.5 In 1972, Borberg successfully documented the
`ability of adoptively transferred immune cells to cause
`regression of established syngeneic tumors.6 Finally,
`the first successful clinical application of cellular ther-
`apy in humans was performed at the National Cancer
`Institute, Bethesda, USA, by Rosenberg in patients
`with metastatic melanoma and renal cell carcinoma.7
`Peripheral blood lymphocytes of patients were acti-
`vated ex vivo with IL-2 to generate lymphokine-
`activated killer cells (LAK) which, when reinfused into
`the patients, were capable of non-major histocompati-
`bility complex (MHC) restricted tumor lysis.
`With the advent of molecular gene transfer tech-
`niques and increased knowledge of the regulation of
`the immune response, effective methods for harnessing
`the immune system as a therapeutic agent are finally
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`S2 AJ Pantuck et al.
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`being realized. The existence of an immune response
`against a tumor is based on changes in the surface
`components of the tumor cell that do not occur in its
`non-malignant counterpart and that give rise to struc-
`tures that may be antigenic. Tumor-associated antigens
`on the surfaces of malignant cells may be unique to the
`cancerous cells and absent from their normal counter-
`part, or tumor antigens may be present on normal cells
`but become unmasked on the malignant cell or re-
`present products that are present during embryonic
`development but are absent in the normal adult
`tissue. Immune effector mechanisms capable of de-
`stroying tumor cells in vivo include humoral (antibody-
`mediated) and cell-mediated cytotoxicity.
`These recent achievements in basic science have not
`passed over the clinical realm of genitourinary oncol-
`ogy. The use of intravesical immunotherapy for super-
`ficial bladder tumors was the first immune-based
`therapy for bladder cancer and has become the gold
`standard. Developments in immunotherapy have re-
`sulted 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
`failed curative treatment. These achievements repre-
`sent only the beginning of new directions to be pur-
`sued. The future prospects of cancer therapy will be,
`without doubt, built upon the foundation of current
`investigative efforts in gene and immune therapy.
`
`Immune and gene therapy for
`renal cell carcinoma
`
`Traditionally, there have been no other effective treat-
`ments for renal cell carcinoma (RCC) aside from
`surgery as RCC is radiation- and chemo-resistant.
`Metastatic RCC has a poor prognosis, with an average
`survival of only 6–12 months from the time of diagno-
`sis and with only a 6% objective response rate with
`conventional chemotherapy. Thus, additional therapy,
`mainly for patients with advanced RCC, is urgently
`needed. The first advance in this direction occurred
`when IL-2 was isolated and identified. The molecular
`cloning of IL-2 revolutionized the field of cancer
`immunotherapy and significantly altered the treatment
`of metastatic RCC.8 With the advent of recombinant
`DNA technology, the ability to produce large quantities
`of IL-2 has resulted in its widespread use. In a rela-
`tively short period of time, this agent has become
`a Federal Drug Administration approved treatment for
`metastatic RCC. Since then, other immunostimulatory
`cytokines have been identified and purified. To date,
`most studies investigating the use of cytokines are
`
`focusing on IFN-a, IL-2, combinations of these
`cytokines, or adoptive immunotherapy with tumor-
`infiltrating lymphocytes (TIL) or LAK cells. The role
`of other cytokines (IL-4, IL-7, IL-12 and GM-CSF) is
`currently under investigation.
`Impressive advances have occurred in the past two
`decades in the application of immunotherapy to treat
`RCC. At the University of California Los Angeles
`(UCLA), we have seen a progressive increase in
`response to treatment as therapy has evolved from
`systemic IFN-a administration (16%), to combination
`IFN + IL-2 (25%), to the current method of bulk TIL
`(33%) and CD8/TIL (40%). Patient characteristics that
`predict improved responsiveness to therapy have been
`identified and treatment protocols that decrease toxic-
`ity have been developed. The most encouraging results
`have been the improved rates of complete clinical
`response, most of which are durable and long-lasting.
`Further refinements in the treatment of renal cell
`disease with biologic and immunotherapeutic agents
`are still needed, yet there is no doubt that current
`immunotherapeutic protocols produce changes in the
`natural history of this disease and cause significant and
`lasting remissions in selected patients. Progress in
`understanding the genetic changes that are associated
`with the development of RCC has been made over sev-
`eral years. Thus, gene therapy for RCC has advanced
`further than in any other urological organ system.
`Intense efforts and progress have proceeded along sev-
`eral therapeutic paths.
`
`Immunotherapy
`
`It is well established that IL-2 has a beneficial activity
`in patients with advanced RCC; thus, a tumor vaccine
`seems appealing. By using a tumor vaccine, elevated
`cytokine concentrations can be achieved within the
`tumor causing an increase in MHC expression. By
`increasing the surface MHC expression, especially
`of HLA-Cw7, an immune response is anticipated
`once the TIL are able to recognize the MHC-restricted
`tumor-related peptides. By achieving locally high
`cytokine concentrations, the systemic toxicity that
`limits
`the efficacy of
`immunotherapy should be
`avoided.
`Initial studies with tumor vaccines in animal models
`have shown that the transfer of cytokine genes to
`tumor cells is feasible and can induce host antitumor
`effects.9 IL-2-transfected RCC cells inhibit the growth
`of parental tumor cells in rats.9 It has also been shown
`that the production of IL-2 is more intense after intra-
`tumor injection than after systemic administration of
`the transfecting agent. A synergistic antitumoral effect
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`was reported for retroviral transduction of the IL-2
`gene conjugated with systemic administration of IFN-
`a. This suggests that a synergistic, immunogenic effect
`can be obtained by using both local gene therapy and
`systemic, immune stimulation. IL-4, GM-CSF, HLA-
`137 and IFN-a gene transfection are other immune
`system modulators that may have a role in future
`tumor vaccines for RCC.10
`Recent phase I trials using tumor vaccines have
`been initiated in humans with metastatic RCC. Patients
`were given irradiated autologous tumor cells trans-
`fected in vitro with a retroviral vector carrying the
`GM-CSF gene. No significant toxicity was reported.
`One out of 16 patients had a partial response.
`Additionally, studies using genetically modified den-
`dritic cells and studies using the injection of cytokines
`into the tumor have been performed at UCLA, by
`using the HLA-B7 and IL-2 genes carried in a liposo-
`mal vector. In addition to these, at least three other
`tumor vaccine programs have been initiated using
`either intratumoral HLA-B7 or IL-2 gene transfection
`to enhance the immunogenicity of the tumor. At this
`time, although tumor vaccine-based gene therapy
`appears to be safe, its efficacy in metastatic RCC has
`yet to be proved.
`
`Gene therapy for RCC S3
`
`product in an attempt to reverse the cancer phenotype.
`Normal (wild-type) VHL gene was transfected into
`RCC cell lines lacking the normal expression of the
`gene. The wild-type VHL gene was attached to a con-
`stitutively activated cytomegalovirus promoter and put
`into a liposome vehicle. Transfection of the wild-type
`VHL gene had no effects on the transfected cell line
`growth in vitro, but the expression of the wild-type
`VHL gene resulted in growth suppression of other
`RCC cell lines. This study showed that the suppression
`of cell growth was specific to RCC cell lines, which
`implied that the VHL protein is important in control-
`ling the proliferation of kidney cells. Thus, gene
`replacement therapy using the wild-type VHL gene
`may have a role in treating patients with RCC,
`although the safety and efficacy of this treatment is yet
`to be defined.
`In vitro attempts to replace 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 meta-
`static lung lesions.13 The use of the p53 wild-type gene
`by intratumoral injection may prove to be efficacious
`in the future.
`
`Corrective gene therapy
`
`Cyto-reductive therapy
`
`From studies of familial RCC in patients with the von
`Hippel–Lindau syndrome, the molecular basis for
`tumorigenesis of the kidney is becoming clearer; loss
`of chromosome 3p in many sporadic and familial
`renal cell cancers has been noted,11 with restriction
`fragment length polymorphisms (RFLP). The von
`Hippel–Lindau (VHL) gene was identified at 3p25.5 of
`chromosome 3.11 It has been hypothesized that the
`VHL protein functions as a cell-cycle regulator, con-
`trolling cellular proliferation by restricting gene tran-
`scription, translation or repair. However, only 45–60%
`of all patients with sporadic RCC have a detectable
`mutation in the VHL gene. Furthermore, the pheno-
`typic expression of the VHL gene defect varies, with
`loss of the VHL gene product not always resulting in
`RCC.12 Thus, the defect in the VHL gene is probably
`influenced by many other yet to be defined epigenetic
`phenomena. Moreover, aberrations at chromosome 5,
`7, 14 and at the Y-chromosome have also been associ-
`ated with RCC. These factors may be able to act
`independently from the VHL locus, resulting in the
`development of RCC.
`Despite the limitations of the VHL gene as a target
`for gene therapy, initial studies have been performed in
`an attempt to replace the defective tumor suppressor
`
`A tumor marker has been recently identified for RCC.
`This new tumor antigen has been named G250. The
`function of this protein is unclear. High levels of G250
`antigen can be detected in up to 90% of all kidney can-
`cer cells, with normal renal parenchyma showing no
`detectable G250 antigen. This antigen has been used as
`a target for monoclonal antibody immunohistochemi-
`cal staining for diagnostic purposes and has been also
`used in radionuclide scans to localize tumor sites.14
`Because this antigen is found in a high proportion of
`RCC cases, it has the potential to be a target for gene
`therapy. Initial studies looking at cytokine-stimulated
`human RCC xenografts showed that the antitumor
`activity of the immune system could be enhanced by
`the administration of antibodies to G250. For now, it is
`unknown whether the G250 protein itself can stimulate
`an immune response. Furthermore, because expression
`of this gene product is also seen in normal bile ducts
`and normal gastric mucosa, the safety of an anti-G250
`treatment needs to be further tested.
`Thus, at this time, there is no perfect target for all
`patients who have RCC, although restoration of the
`wild-type VHL gene product and the development of
`targeted therapy against the G250 protein do hold sig-
`nificant promise for future trials.
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`S4 AJ Pantuck et al.
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`Conclusion
`
`Although surgical management continues to be an
`effective
`treatment
`for organ-confined neoplastic
`disease, the treatment options for patients with dis-
`seminated cancer are limited. Immunotherapy has
`demonstrated significant success in the management of
`advanced RCC. Recent exciting research has demon-
`strated that prostate cancer may also be susceptible to
`immuno-therapeutic protocols, finally offering a glim-
`mer of hope to physicians treating this very prevalent
`and morbid disease. Molecular-based therapy has
`many potential applications
`in
`the
`treatment of
`advanced genitourinary cancers. The reinsertion of
`inactivated tumor suppressor genes, the inactivation
`of oncogenes, the insertion of immunomodulatory
`genes and the insertion of suicide genes have all been
`used to treat genito-urinary malignancies, in vitro and
`in animal models. Progress is being made in better
`understanding the genetic and cellular mechanisms
`that underlie tumorigenesis. Human clinical trials are
`already in phase I testing in some tumor systems,
`including RCC,
`transitional cell carcinoma and
`prostate cancer. However, limitations still have to be
`overcome. Safe and effective gene vectors will be
`needed to carry the therapeutic gene to the host cell.
`Treatments need to be tailored so that the desired
`effects occur only in the tumor cells. In conclusion,
`molecular-based therapy is appealing because of its
`ability to treat cancer at the level of the gene defect
`that causes the malignant phenotype, and it offers
`novel and exciting approaches for the treatment and
`ultimate eradication of cancer.
`
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