MOLECULAR UROLOGY
`Volume 4, Number 2, 2000
`Mary Ann Liebert, Inc.
`
`Ex Vivo Gene Therapy Using Granulocyte-Macrophage
`Colony-Stimulating Factor-Transduced Tumor Vaccines
`
`KOJI KAWAI, M.D., Ph.D.,1 KENZABURO TANI, M.D., Ph.D.,2 SHIGETAKA ASANO, M.D., Ph.D.,2 and
`HIDEYUKI AKAZA, M.D., Ph.D.1
`
`ABSTRACT
`
`There is no standard effective therapy for metastatic renal-cell carcinoma (RCC) or prostate cancer. Both of
`these cancers may be immunogenic, so therapy targeted to a tumor-associated antigen may be effective. Trans-
`duction of the gene encoding granulocyte-macrophage colony-stimulating factor has shown promise in pre-
`clinical studies, and clinical trials are in their early stages. Both autologous cancer cells and partially HLA-
`matched allogenic cells are being studied. No dose-limiting side effects have been observed, and a few patients
`have had transient objective tumor regressions. Further trials with more frequent and, probably, longer im-
`munization schedules are needed to define efficacy.
`
`INTRODUCTION
`
`THERE IS NO STANDARD EFFECTIVE THERAPY for metastatic re-
`
`nal-cell carcinoma (RCC) or hormone-refractory prostate
`cancer. Like melanoma, RCC is thought to be an immunogenic
`cancer because of its relatively high response rate to cytokine
`therapy. While prostate cancer has been regarded as an unlikely
`target for immunotherapy, recent basic studies indicate that sev-
`eral putative tumor-associated antigens are expressed in prostate
`cancer tissue or cell lines.1 This finding suggests that im-
`munotherapy targeted for a tumor-associated antigen may pro-
`vide an effective treatment for prostate cancer.
`In cancer gene therapy, various approaches, including tumor
`suppressor genes, prodrug in combination with a suicide gene,
`and gene-modified immunotherapy are now being tested in clin-
`ical studies, as reviewed by other contributors to this issue. The
`gene-modified tumor vaccine strategy has been the most inten-
`sively studied. In Japan, a Phase I study of a granulocyte-
`macrophage colony-stimulating factor (GM-CSF) gene-trans-
`duced tumor vaccine for metastatic RCC has begun as the first
`cancer gene therapy under the direction of a multi-institutional
`group at the Institute of Medical Science, University of Tokyo.
`In this paper, we briefly review the ex vivo gene therapy ap-
`proaches and early clinical trials of the GM-CSF gene-trans-
`duced tumor vaccine for RCC and prostate cancer.
`
`PRECLINICAL STUDIES USING
`GM-CSF-TRANSDUCED TUMOR VACCINES
`
`In animal models, the inoculation of cells engineered to con-
`tain cytokine genes induces tumor-specific immune responses,
`defined as either protection of the animal against challenge with
`parental tumor cells or regression of established parental tu-
`mors.2 Among several kinds of cytokine genes, Dranoff and as-
`sociates3 demonstrated the efficacy of GM-CSF-transduced
`vaccines by comparing the activity of the transduced tumor cells
`with that of nontransduced cells in animal models of melanoma,
`prostate cancer, RCC, and others. Results in the Dunning rat
`model also support the use of GM-CSF-transfected tumor cells
`in prostate cancer treatment.4 In this model, GM-CSF was
`shown to induce more potent antitumor activity than other mol-
`ecules tested in protecting mice from subsequent lethal chal-
`lenge with live melanoma cells. Those findings suggest that
`GM-CSF is the most promising candidate for augmenting an-
`titumor immunity.
`The action of GM-CSF involves the local spread of the cy-
`tokine at a high concentration, which activates the function of
`the antigen-presenting cells (APCs), including macrophages
`and dendritic cells. The dendritic cell is known to be the most
`potent APC for helper T lymphocytes. Both CD41 and CD81
`T cells are activated, which results in the destruction of tumor
`
`1Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan.
`2Department of Hematology/Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
`
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`44
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`KAWAI ET AL.
`
`cells at metastatic sites. The role of the CD4 T cells has been
`largely attributed to induction of tumor-specific CD8 cytotoxic
`T lymphocytes (CTL) in the effector phase.3 In addition, recent
`analyses indicate that the GM-CSF tumor vaccine activates the
`CD4 T cell to simultaneous Th1 and Th2 responses.5 Those
`findings suggest that multiple lymphocyte effector mechanisms
`including B-cell immunity, result in the potent antitumor re-
`sponse observed in the animal studies of GM-CSF-transduced
`tumor vaccines.
`
`ONGOING CLINICAL STUDIES OF CYTOKINE
`GENE-TRANSDUCED VACCINES FOR RCC
`AND PROSTATE CANCER
`
`Table 1 shows the clinical trials of the gene-modified tumor
`vaccines designed for RCC and prostate cancer in the United
`States and Japan.6 The current approaches for RCC are gene-
`modified immunotherapy. Seven clinical trials are based on a
`tumor vaccine using ex-vivo gene-modified autologous cancer
`cells or partially HLA-matched allogeneic cancer cells. Four
`kinds of molecules—tumor necrosis factor, interleukin (IL)-2,
`GM-CSF, and B7.1 (CD80)—are being used to augment host
`immunity. An additional protocol is planned to use IL-4-trans-
`duced autologous fibroblasts in combination with untransduced
`autologous cells. Similarly, more than half of the gene thera-
`pies designed for prostate cancer are gene-modified im-
`munotherapy. The ex-vivo gene-modified tumor vaccines using
`GM-CSF or IL-2 are being tested in three protocols (Table 1).
`For the GM-CSF-transduced tumor vaccine, autologous and al-
`logeneic cancer cells are being studied in different protocols.
`To date, results from three clinical trials of the autologous GM-
`CSF tumor vaccine have been reported.7–9
`
`RESULTS OF CLINICAL STUDIES
`
`The vaccine dose and schedule, toxicity, and clinical re-
`sponse of individual trials are summarized in Table 2. The ca-
`
`pability of eliciting an antitumor immune response was exten-
`sively monitored using several methods, as listed in Table 3.
`In the RCC study, patients were randomized to treatment
`with escalating doses of autologous tumor vaccine with or with-
`out ex-vivo GM-CSF gene transfer. Cells were harvested from
`the patients’ primary tumors and transfected with a retroviral
`vector carrying the GM-CSF gene.7 The transduced and un-
`transduced tumor cells were then irradiated with a lethal dose
`(15 Gy) and administered intradermally or subcutaneously. No
`dose-limiting side effects were observed in 16 patients. One
`partial response was observed in a patient with multiple lung
`metastases who was treated with GM-CSF gene-transduced
`vaccine. The patient displayed the largest delayed-type hyper-
`sensitivity (DTH) conversion in this trial; however, progression
`of metastases was observed 4 months after the last vaccina-
`tion.10 Histologic evaluation of the vaccination site revealed ex-
`tensive infiltration of dendritic cells, macrophages, eosinophils,
`and T lymphocytes. In the DTH sites of patients treated with
`GM-CSF-transduced vaccine, the most characteristic finding
`was the recruitment of activated eosinophils. Eosinophil accu-
`mulation and degranulation were commonly observed in the
`postvaccination DTH response of melanoma and prostate can-
`cer patients also.
`In the melanoma study, not only DTH sites but also meta-
`static lesions resected after vaccination could be examined.8
`Diffuse infiltration of T lymphocytes and plasma cells was
`demonstrated in 11 of 16 patients in whom tissue could be ob-
`tained. In four patients, infiltration of eosinophils and lympho-
`cytes was associated with the targeted destruction of tumor vas-
`culature. Despite
`these promising histologically defined
`immune reactions, no objective clinical response was observed
`in the 22 patients.
`In the study of prostate cancer, the low yields of cells in the
`primary cultures of radical prostatectomy specimens limited the
`number of vaccination courses.9 Although no clinical response
`was noted in the eight patients, DTH site biopsies revealed in-
`filtration of CD45RO1 T cells and degranulating eosinophils
`consistent with the activation of Th1 and Th2 T-cell response.
`In addition to these in vivo responses, both T-cell and B-cell
`
`Gene
`
`RCC
`TNF
`IL-2
`IL-3
`
`IL-4
`
`GM-CSF
`B7.1
`(CD80)
`Prostate Ca
`GM-CSF
`IL-2
`GM-CSF
`
`TABLE 1. EX VIVO GENE THERAPY PROTOCOLS FOR RCC AND PROSTATE CANCER
`
`Phase
`
`Target
`
`Vehicle
`
`Research Site
`
`1
`I
`I
`
`I
`
`I
`I
`
`I/II
`I
`I/II
`
`Autologous RCC
`Autologous RCC
`Allogeneic RCC
`(partially HLA matched)
`Autologous fibroblasts
`(with untransduced RCC)
`Autologous RCC
`Autologous RCC
`
`Autologous CAP
`Autologous CAP
`Allogeneic CAP
`
`Retrovirus
`Retrovirus
`Retrovirus
`
`Retrovirus
`
`Retrovirus
`Adenovirus
`
`Retrovirus
`Lipid
`Retrovirus
`
`NIH
`NIH
`Memorial Sloan-Kettering
`
`U. Pittsburgh
`
`Johns Hopkins/U.Tokyo
`U. Southern California
`
`Johns Hopkins
`Duke
`Johns Hopkins/U California
`
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`EX VIVO GENE THERAPY USING GM-CSF
`
`45
`
`TABLE 2. SUMMARY OF PHASE I STUDENTS OF GM-CSF-TRANSDUCED TUMOR VACCINES
`
`Renal-cell carcinoma
`
`Melanoma
`
`Prostate cancer
`
`No. pts.
`
`Pretreatment
`Dose and schedule
`
`GM-CSF production
`(ng/106 cells/24h)
`DTH conversion
`Objective tumor
`response
`Toxicities
`
`Enrolled: 31
`Treated: 18
`(-)
`Level 1: 4 3 106 cells
`(N 5 3) q 28d 3 3
`Level 2: 4 3 107 cells
`(N 5 4) q 28d 3 3
`Nontransduced cells
`(N 5 11)
`
`42–149
`
`Enrolled: 33
`Treated: 26
`(-)
`Cell dose fixed
`at 1 3 107
`Level 1: q 28d 3 3
`(N 5 4)
`Level 2: q 14d 3 6
`(N 5 7)
`Level 3: q 7d 3 12
`(N 5 15)
`84–965
`
`Enrolled: 23
`Treated: 8
`Radical prostatectomy
`Level 1: 1 3 107 cells
`(N 5 5) q 7d 3 6
`Level 2: 5 3 107 cells
`(N 5 3) q7 d 3 6
`
`143–1403
`
`All at level 2 (2–10 cm)
`1 PR
`
`All evaluated (5–10 cm)
`None
`
`All evaluated (0.5–1.0 cm)
`None
`
`Local:
`erythema, swelling
`pruritus
`Systemic:
`constipation (1 case)
`pruritus (1 case)
`
`Local:
`erythema, swelling
`pruritus
`Systemic:
`fatigue (grade 1)
`occasionally
`
`Local:
`erythema, swelling,
`pruritus
`Systemic:
`fever , grade 2 (2 cases)
`chills (3 cases)
`
`antitumor responses were monitored by in vitro tests. Specific
`CTL activity against autologous tumor was detected in tumor-
`infiltrating lymphocytes from residual melanoma metastases.8
`Ellem and associates11 reported an increase in the frequency of
`CTL precursors by the limiting dilution assay in a melanoma
`patient who was treated with a GM-CSF gene-transduced tu-
`mor vaccine in another clinical study. The immune response
`was associated with a transient partial antitumor effect. How-
`ever, neither a clinical effect nor an immune response was de-
`tectable 2 months after the last vaccination.
`For detecting the B-cell antitumor response, immunoblotting
`analyses using autologous tumor cell lysates and sera were per-
`formed in the melanoma and prostate cancer studies.8,9 An in-
`crease in antibody titers was observed in seven melanoma and
`three prostate cancer patients. In prostate cancer, the induced
`immunoglobulin recognized the 150-kDa polypeptide com-
`monly expressed by LNCaP and PC-3 cell lines, as well as by
`normal prostate epithelium and a number of other human can-
`cer cell lines.
`
`CONCLUSION
`
`Although the side effects of gene-modified immunotherapy
`appear to be mild, the clinical efficacies shown in the clinical
`studies are limited. Because the primary purpose of a Phase I
`study is to test safety, the clinical efficacy of the GM-CSF-
`transduced tumor vaccine has not been fully examined as yet.
`Further clinical studies with more frequent and probably longer
`periods of vaccination are needed to define the efficacy. For the
`long-term treatment schedule, however, the efficiency of the
`propagating autologous tumor cells in culture is the major fac-
`
`tor limiting the quantity of vaccine. In prostate cancer trials,
`sufficient cells to provide a higher dose were recovered from
`surgical specimens in only three of the seven patients. The use
`of allogeneic tumor cells, if they can be sources of tumor anti-
`gens, will permit long-term vaccinations. Some preclinical stud-
`ies suggest that HLA matching may be less critical in the ap-
`plication of tumor vaccines than previously thought.12 The
`benefits of long-term treatment with a nontransduced allogeneic
`tumor vaccine were reported from clinical studies for
`melanoma.13 On the basis of experiences, a Phase II clinical
`trial of irradiated GM-CSF-secreting LNCaP and PC-3 vac-
`cines, using doses higher than are possible with an autologous
`tumor vaccine, is currently under way in the United States.
`There is hope that further understanding of clinical efficacy as
`well as the immunologic response induced by the GM-CSF-
`transduced tumor vaccine will be gained from the ongoing and
`future clinical trials.
`
`TABLE 3. IMMUNE RESPONSES EVALUATED IN CLINICAL
`TRIALS OF GM-CSF-TRANSDUCED TUMOR VACCINE
`
`In vivo
`Delayed-type hypersensitivity
`Vaccine site (histology)
`DTH site (histology)
`Metastases (histology)
`Hematology findings
`In vitro
`T-cell immunity
`Cytotoxic activity of tumor-infiltrating lymphocytes
`B-cell immunity
`Antitumor activity of postimmunization serum
`
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`46
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`Address reprint requests to:
`Koji Kawai, M.D.
`Department of Urology
`Institute of Clinical Medicine
`University of Tsukuba
`1-1-1 Tennodai Tsukuba-city
`Ibaraki 305, Japan
`
`E-mail: rkawa@md.tsukuba.ac.jp
`
`NOVARTIS EXHIBIT 2013
`Breckenridge v. Novartis, IPR 2017-01592
`Page 4 of 4
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