Review Article

Cancer Gene Therapy (2002) 9, 1013–1021. doi:10.1038/sj.cgt.7700538

Virotherapy clinical trials for regional disease: In situ immune modulation using recombinant poxvirus vectors

Michael J Mastrangelo1 and Edmund C Lattime2

  1. 1Department of Medicine, Division of Medical Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
  2. 2Department of Surgery, UMDNJ/Robert Wood Johnson Medical School and The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, USA

Correspondence: Dr Edmund C Lattime, The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901, USA. E-mail: edmund.lattime@umdnj.edu

Received 16 September 2002.

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Abstract

The ability of viruses to readily infect tumor cells both in vitro and in vivo has resulted in their study as antitumor agents through a variety of strategies. Replicating and conditionally replicating viruses and recombinant viruses encoding genes for toxins and/or prodrugs have been studied for their direct antitumor activity with promising results. However, to date, the lack of a targettable construct able to localize to all tumors following systemic administration has proven to be a major limitation in their use for metastatic disease. The ability of a variety of well-characterized viruses to serve as vectors for expression of tumor antigens and/or cytokines has also resulted in their study as immunotherapeutic agents. In this review, we discuss preclinical and clinical data that support the use of recombinant poxviruses as vectors for in situ tumor transfection with immune-enhancing cytokines and immune costimulatory antigens. We hypothesize that such an approach will ultimately lead to enhanced immune recognition of tumor and the development of an effective systemic antitumor immune response capable of eradicating primary and metastatic tumor foci.

Keywords:

vaccinia; immunotherapy; clinical trials; vaccine; melanoma; bladder cancer

As reviewed in the contributions to this volume, studies from a variety of laboratories, each with its own particular focus, have used viruses and viral vectors as antitumor agents (reviewed in Ref. [1]). These approaches range from direct tumor lysis by replicating or conditionally replicating viruses, to the use of viral vectors to carry toxins or prodrugs to tumor, to the use of viral vectors to, in one way or another, induce antitumor immunity as discussed in this review. Each approach has its own strengths and limitations and it is likely, as has been the long-held conclusion from traditional anticancer therapies such as surgery, chemotherapy, and radiation, that no single modality will be the “magic bullet,” but rather that combinations of modalities will likely be required for effective tumor eradication. Regardless of the strategy used, for an approach to be effective, it will be necessary to eliminate both identifiable tumor deposits as well as micrometastatic disease. For this reason, immunotherapy strategies, which hold out the promise of developing systemic antitumor effects, have held particular interest. To date, given the inability to target viral vectors administered systemically to all tumor, immunotherapy-based strategies continue to represent a major focus of clinical virotherapy trials.

The concept that the immune response may be manipulated so as to eliminate established neoplasms is an appealing one, which has been under study for decades. This concept stemmed from the early suggestion of Thomas2 in 1959 that the immune response might be useful in ridding the body of aberrant cells and was later refined into the immune surveillance hypothesis of Burnet3 in 1970, which, in its simplest form, hypothesized that the immune system would recognize incipient tumors as foreign and reject them and that only those tumors that evaded this surveillance mechanism would persist and grow. Early clinical support for the potential effectiveness of the immune system in eradicating tumors was based on the reports that a number of human tumors, especially melanoma and renal cell, spontaneously regress presumably by the development of an antitumor immune response.4,5 Based on these and other findings as well as a rapidly evolving understanding of basic immune regulation, studies by numerous investigators in preclinical and clinical settings have continued to focus on harnessing the immune response as a therapeutic for malignancy.

To date, investigators have primarily approached tumor immunotherapy in three ways (reviewed in Ref. [6]). Local therapy with immune-active adjuvants has been shown to be highly effective in the case of localized tumors of the skin and bladder.7,8,9 Tumor vaccines represent the most studied approach to therapy. In their earliest manifestations, these included the use of either whole tumor cells given unmodified or following modification with viral antigens or haptens (reviewed in Refs. [10,11]) followed by the use of tumor extracts and most recently defined protein antigens and peptides12 injected directly or used to pulse antigen-presenting cells, which were subsequently used as vaccines. Success in inducing regression of clinically evident disease using these vaccines has been quite limited. However, in the adjuvant setting where one would expect minimal residual disease, prolongation of disease-free survival has been reported using these first-generation approaches. More recently, gene-based strategies have come to the fore with plasmid- and viral vectors–encoding tumor antigens being used as vaccines and as vectors for delivering immune-enhancing cytokines and costimulatory molecules to tumor in situ.

When one considers the choice of viral vectors for gene therapy, candidates usually include adenovirus and retrovirus families and, in the case of retrovirus, most recently, the HIV-derived lentiviral vectors (reviewed in Ref. [1]). Poxvirus vectors are often absent from the list of candidates for most studies. Indeed, whereas a number of characteristics of the lifecycle of poxvirus infection make them poor candidates for long-term expression, they have a number of properties that make them ideal for consideration in immunotherapy applications. Most notably, they are large viruses that are routinely shown to allow the insertion of multiple genes without compromising infectivity. In addition, the poxvirus lifecycle is limited to the cytoplasm, thus eliminating any concern for integration into the genome. Finally, they have a long history as effective and safe vaccines, first used successfully in the 1700s as a vaccine for smallpox.

While not traditionally thought of as viral or gene therapy, recombinant viral vaccines by virtue of their expression of foreign gene products in vivo meet the broad definition. Given the high level of immunogenicity of vaccinia and other poxvirus and the ease in generating recombinants noted above, poxvirus recombinants have been used extensively as vaccines for infectious organisms and more recently tumors. Preclinical studies using a variety of tumor transplants in wild-type and antigen-expressing transgenic mice have provided a strong basis for the use of poxvirus vaccines clinically (reviewed in Ref. [13]). Promising preclinical results have led to early stage clinical trials of vaccinia and nonreplicating poxviral vectors encoding defined tumor antigens such as CEA and PSA administered using a single viral vector or combinations of vectors in complex prime–boost strategies with and without genes encoding immune active cytokines and/or costimulatory molecules.14,15,16,17

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Cytokine gene transfer studies in antitumor immunity

The generation of a cell-mediated immune response to tumor or any antigen requires: (a) the presence of an antigen or target on the tumor; (b) the presence of a T-lymphocyte population with specificity for the particular antigen; and (c) a supportive cytokine milieu responsible for the recruitment of appropriate antigen-presenting cells and the modulation of the resultant response.6 Numerous studies both preclinical and clinical from a variety of investigators have shown that tumor-specific and/or tumor-associated antigens are present on tumors and that following in vivo and/or in vitro expansion, antigen-specific T-cell populations are demonstrable. Based on these and other findings, a major emphasis in current vaccine design has been placed on attempts to modulate responses by combining cytokine genes and/or genes encoding cell surface costimulatory molecules such as B7.1, LFA-3, and ICAM-1, which are expressed on professional antigen-presenting cell populations and not normally expressed on most tumors, together with genes encoding tumor antigen in single viral or DNA-based constructs. Towards this end, a number of laboratories have stably transfected murine and, more recently, human tumor with a variety of such genes for use as vaccines.18,19,20,21,22,23,24,25,26,27 In murine studies, such manipulation almost uniformly resulted in rejection of the transfected and coinjected nontransfected tumor. In some cases, mice were shown to generate a measurable systemic antitumor response based on rejection of subsequent challenge with the nontransfected tumor.18,19,28,29 In a limited number of cases, “vaccination” with such cells resulted in the elimination or reduced growth of preexisting tumor.22,23,29 Whereas these studies have been less than overwhelming in their effects on existing tumors, they do show that localized cytokine/lymphokine production can enhance the generation of tumor-specific immunity. More recently, this approach has been translated to clinical trials in renal carcinoma, prostate cancer, and melanoma.25,26,27,30,31,32 While, for the most part, there have been limited clinical antitumor responses, studies have shown positive immunologic findings.25,26,27,30,31,32

While the clinical trials to date using ex vivo transfected autologous tumor have demonstrated intriguing results, the requirement that autologous tumor, based on the need for proper antigen and MHC expression, be available, removed, transfected, cloned, and so forth, severely limits the number of suitable patients. A modified approach using cytokine-transfected HLA-matched allogeneic tumor or transfected autologous fibroblasts is currently under study at a number of centers, which may increase the numbers of potential patients.33,34,35 However, the labor-intensive nature of such approaches continues to be a limitation.

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In situ cytokine gene transfer to enhance antitumor immunity

Given the limitations and the lack of clear successes resulting from the approaches outlined above, we developed a strategy of directly inserting the desired cytokine gene or gene-encoding costimulatory antigens into the tumor utilizing vaccinia virus recombinants. Injection of the virus intralesionally or intravesically in the case of bladder cancer would result in the infection of the tumor cells and, subsequently, the secretion of biologically active cytokines and/or the cell surface expression of costimulatory molecules. Supported by the preclinical tumor transfection studies described above, it is our hypothesis that production of proimmune cytokines locally at the tumor site in this way would enhance the generation of systemic tumor-specific immunity and resultant tumor destruction. We chose vaccinia virus vectors for our initial studies for a number of reasons. As noted above, vaccinia replicates solely in the cytoplasm, infects a variety of cell types with high efficiency, has the capacity to express several encoded genes in the same vector, and has been used in countless millions of individuals to eradicate smallpox.

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Tumor transfection by vaccinia recombinants

The overall hypothesis behind our studies is that by modulating the immune milieu at the local tumor site, and thus recruiting antigen-presenting and effector cell populations, it will be possible to engender a systemic tumor-specific immune response. The result would be to eliminate both localized and disseminated tumor. We pursued both preclinical and clinical studies to determine the feasibility of the use of recombinant vaccinia as a vector for in situ transfection with the result that highly supportive data have been generated, which enhances our enthusiasm for the approach.

Prior to developing recombinant vaccinia for our clinical studies, it was necessary to demonstrate that vaccinia virus recombinants were capable of transfecting murine and human tumor cells. A panel of cell lines including the murine melanoma B16, bladder tumors MBT2 and MB49,36,37 as well as human melanoma lines produced from our patients,38,39 bladder (T24), and prostate carcinoma (LNCAP, PC3) cell lines40 were examined for their ability to be infected/transfected with vaccinia recombinants. Cell lines were exposed in vitro to vaccinia virus recombinants encoding the genes for influenza hemagglutinin and nuclear protein antigens, which allow us to stain for productively infected/transfected cells in vitro. Without exception, all cell lines tested were highly susceptible to infection/transfection at a multiplicity of infection (MOI) of 10:1 PFU:cell.37,41

To determine if recombinant vaccinia were able to infect/transfect tumor in vivo, vaccinia recombinants containing reporter constructs (HA, NP, or the lacZ gene) were injected intralesionally into murine B16 melanoma lesions or instilled through urethral catheters into the bladders of C57BL/6 mice bearing the MB49 tumor,36,37,42 with results demonstrating significant infection/transfection in all systems tested. Given the immunogenicity of vaccinia and the possibility that immunity to the virus would prevent infection/transfection following in vivo administration, we examined tumor infection/transfection in vaccinia immune recipients and demonstrated significant activity in vaccinia-immune mice37,41 following intralesional or intravesical administration. Thus, systemic immunity to vaccinia, which would be expected to be present in adult patients and following initial vaccinia treatments, does not prevent in vivo tumor infection/transfection.

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Cytokine gene delivery using vaccinia recombinants

To determine if vaccinia recombinants could be used to transfect tumors with resultant cytokine production, we established a panel of vaccinia recombinants expressing murine IL-4, IL-5, IFNδ, and GMCSF.42,43 At the time at which these were being produced, a report by Ramshaw et al demonstrated cytokine production by such recombinants. They subsequently demonstrated that the resultant production of cytokines at the viral infection/immunization site had profound effects on the resultant antiviral immunity and viral clearance. Cytokines such as IL-2 and IL-12 demonstrated enhanced viral clearance and immunity, IFN-γ and TNF showed significant direct antiviral activity, whereas IL-4 significantly inhibited viral clearance and immunity.44 Our ELISA and functional cytokine analyses demonstrated that the vaccinia-recombinant–infected tumor cells produce significant levels of cytokine protein43 and, using vaccinia-specific primers designed in our laboratory which allow the elucidation of encoded cytokine mRNA in vivo, demonstrated prolonged cytokine gene expression in vivo following intralesional injection

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Intralesional vaccinia vector in patients with melanoma

As a prelude to studying the effects of intralesional recombinant vaccinia in human melanoma, we obtained an IND from the FDA to inject the Wyeth strain of vaccinia (the vaccine used in the US for smallpox immunization and our nonrecombinant parent) intralesionally in patients with recurrent superficial melanoma.39,45 Five patients with histologically documented, surgically incurable melanoma with at least one dermal, subcutaneous, or lymph node metastasis that was evaluable for local response and accessible for injection were enrolled. All patients were immunocompetent as demonstrated by one or more positive cutaneous delayed-type hypersensitivity reactions to recall microbial antigens or to dinitrofluorobenzene after sensitization. Following the demonstration of systemic immunity to vaccinia through an intradermal administration of vaccine to the patients, increasing doses of vaccinia were injected intratumorally. Patients were treated twice weekly with increasing doses of intralesionally injected virus. The number of treatments and total dose varied from a low of four treatments for a total of 8×106 PFU to a high of 19 treatments for a total of 12.85×107 PFU. Four of five patients developed high titers of anti-vaccinia antibody within 14–21 days. (For a detailed description of treatment and response of each patient see, Ref. [46].) Antitumor activity varied among the patients, with one patient exhibiting no regression of treated nor untreated lesions, three patients had partial but brief (<1 month) regression of treated lesions with the onset of progression coinciding with the failure to maintain erythema and induration with repeated injections of virus. The fifth patient sustained clinically complete remission of a large exophytic mass that was treated repeatedly as well as a smaller untreated lesion in close proximity. The size of the lesion in this latter patient allowed serial biopsies to be taken to assess viral gene function over time. In this patient, 106 PFU was injected into four sites in a 3-cm superficial melanoma lesion. The lesion was biopsied at 6 hours and 4 days following administration. The biopsies were processed as frozen sections and stained using the monoclonal antibody TW2-3, which is specific for an early viral protein product of the EL3 gene present at sites of viral replication.39 To determine if increasing immunity to vaccinia induced by multiple treatments would block productive infection/transfection, additional biopsies were similarly analyzed over the course of therapy in this patient who received 19 biweekly injections of as high as 107 PFU of virus (total cumulative dose of 12.85×107 PFU). Whereas the duration of expression was diminished with increasing immunity as measured by antiviral antibody titer, productive infection was seen throughout the treatment course.39,45 It should be noted that minimal systemic side effects were seen in the trial. These findings demonstrate, as did our murine studies above, that vaccinia recombinants are able to infect/transfect tumor in vivo following intralesional injection even in the face of systemic immunity to the virus. We concluded from these studies that systemic immunity to the virus acts to protect the patients from toxicity while not preventing local gene expression. Our demonstration of sustained infection in virus-immune individuals strongly supports our approach in demonstrating that infection/transfection using cytokine gene–encoding vaccinia should result in cytokine production for a prolonged period.

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Intralesional vaccinia–GMCSF recombinant in patients with melanoma

Having been satisfied that the vaccinia vector met our requirements of safety and efficacy, we carried out a Phase I trial of intralesional vaccinia–GMCSF in patients with therapy-refractory recurrent melanoma. The recombinant virus was produced in our laboratories using the Wyeth vaccine strain of vaccinia obtained from the CDC, which was also used in our vector alone trial above. The full-length cDNA for human GMCSF was obtained from the American Type Culture Collection (Manassas, VA) (pCSF-1, no. 39754) and cloned behind the pSC65 Synthetic Early/Late Promoter provided by Dr Bernard Moss (NIAID). The β-gal gene was included as a reporter gene cloned behind the p7.5 Early/Late vaccinia promoter. (For a complete description of the viral construct and production, see Ref. [47].) Clinical grade virus was produced under GMP conditions, allowing us to obtain an IND from the FDA (BB-IND-6486). All patients were required to have accessible dermal and/or subcutaneous disease with a number also having visceral disease. Following the demonstration of immune competence — important given the replicative nature of the vector — patients receive twice weekly intralesional injections of the recombinant with dose escalation within each patient. Patients were initially treated for a 4-week induction period (eight treatments). Those patients who continued to manifest clinical indications of local infectivity were maintained on treatment until maximal benefit or tumor progression. Table 1 summarizes the results seen in the first seven patients who are described in detail in Ref. [47]. At the highest doses, patients received 2×107 PFU per lesion with the injection of multiple lesions resulting in as high as 8×107 per session. For comparison, vaccinia was used as a smallpox immunization at a scarification dose of 2.5×105. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease-free. One patient with only dermal metastases confined to the scalp achieved a complete remission.


Figure 1 demonstrates the complete resolution of dermal metastases seen in patient 3 of the study following treatment and that tumor eradication was accompanied by recruitment of large numbers of CD3+ T cells (both CD4 and CD8 phenotype, not shown) into injected lesions. Figure 2 demonstrates the regression of a noninjected lesion outside of the lymphatic drainage area from the injected site (below the knee on the back of the calf) associated with significant CD8+ T-cell infiltration. It was the regression of uninjected lesions in four of seven patients that we have taken as evidence of the induction of tumor-specific immunity. Laboratory studies demonstrated that patients developed high levels of immunity to both vaccinia and the included β-galactosidase gene product.47 We also confirmed that, in the face of maximal antibody titers, we continued to be successful at achieving recombinant gene expression as measured both as vaccinia-encoded GMCSF (V-GMCSF) (RT-PCR using primers designed to specifically identify viral encoded GMCSF) and viral thymidine kinase (V-TK) gene mRNA expression (Fig 3).

Figure 1.
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Resolution of dermal metastases following intralesional injection of recombinant vaccinia–GMCSF. Patient 3, a 32-year-old female with extensive dermal metastases of the left thigh before treatment (A), on day 81 (B), and on day 600, 150 days following cessation of treatment (C). Regression was accompanied by gross (D) and histologic (E) evidence of inflammation including significant T-cell (CD3+) involvement (F) (reprinted with permission from Cancer Gene Ther 1999;6:409–422).

Full figure and legend (678K)

Figure 2.
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Regression of uninjected lesions is accompanied by T-cell infiltration. A representative uninjected distant regressing lesion prior to (A) and following patient treatment (B) demonstrated T-cell (CD8) infiltration (C) (reprinted with permission from Cancer Gene Ther 1999;6:409–422).

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Figure 3.
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Expression of vaccinia-encoded genes following intralesional injection. RT-PCR of mRNA from melanoma biopsies for vaccinia thymidine kinase (V-TK); vaccinia-encoded human GMCSF (V-GMCSF); human GMCSF (GMCSF); and biopsies from injected (lanes 1–3) and an uninjected lesions. Lane 1: Biopsy 18 hours following the last of a series of multiple injections; lanes 2 and 3: biopsies 18 hours following a single injection; lane 4: uninjected lesion. All biopsies were taken from patient 3 at week 31 (reprinted with permission from J Clin Invest 2000;105:1031).

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Intravesical vaccinia in patients with bladder cancer

As a first step to our planned expansion of this strategy to the localized treatment of bladder cancer (our preclinical data demonstrated significant infection/transfection of the orthotopically growing murine bladder tumor MB49 following intravesical administration of recombinant vaccinia37), we have completed a Phase I study of intravesical vaccinia vector in patients with advanced transitional cell carcinoma. As with our Phase I study of vaccinia vector alone in melanoma,39 we used the vaccinia vector in a dose escalation study, with each patient receiving three intravesical doses over a 2-week period. Given safety concerns, this study focused on patients with invasive transitional cell carcinoma scheduled for cystectomy with the cystectomy scheduled for the day following the third dose. Table 2 summarizes patient characteristics, doses employed, and toxicity. As noted in our prior clinical trials, patients developed high titers of antivaccinia antibody, although maximal titers were measured after cystectomy given the shortened course of therapy (not shown). Also as noted above, treatment was associated with a significant recruitment of activated T lymphocytes. Figure 4 demonstrates recruitment of activated CD3+ T lymphocytes as well as dendritic cells that we feel will enhance prospects for the induction of immunity to tumor.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Recruitment of activated T lymphocytes and dendritic cells to the bladder wall following intravesical vaccinia instillation. Immunohistochemical staining of bladder from patient 2. Pretreatment biopsies (A,C,E) and posttreatment cystectomy sections (B,D,F) were stained for CD3 (A,B), CD45RO (C,D), and Factor XIIIa (dendritic cells) (E,F) (reprinted with permission from J Clin Invest 2000;105:1031).

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Completed and ongoing clinical trials of localized immune gene transfer

Subsequent to our preclinical and clinical studies outlined above, there have been a number of clinical trials carried out or in the planning stage that utilize the above strategy. Table 3 outlines studies using vaccinia as well as related replicating39,47,48,49,50,51,52 and nonreplicating poxvirus vectors encoding cytokines such as GMCSF47 and IL-249,50 with and without cell surface immune costimulatory molecules such as B7.1 alone or in combination with LFA-3 and ICAM-1 (TRICOM)51,52 and shown above in preclinical studies to enhance the interaction of antigen-presenting cells with antigen-specific T lymphocytes used in a variety of tumors. Whereas antitumor responses have been varied in the studies completed to date, demonstration of significant tumor transfection following repeated injections where neutralizing antibodies are found is uniformly seen supporting the use of poxvirus as vectors for sustained production of immune-enhancing cytokines and cell surface molecules in vivo.


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Conclusions

In summary, we have discussed an approach to immunologically based gene therapy logically designed from the requirements to generate a productive cellular immune response. As outlined in this review, numerous strategies have been hypothesized and tested in both preclinical and clinical settings with this goal as an endpoint.

It is our hypothesis that in situ tumor transfection with cytokine genes will provide a logical extension of the vaccine strategies that have been previously studied. By incorporating genes selected based on their known contribution to the generation of systemic immune responses, we anticipate the ability to optimize the generation of an antitumor response. In addition to this logical in vivo vaccine design, this methodology will allow the generation of a single reagent in a bottle that will be of use in any tumor type provided that it is accessible to injection. This will preclude the need to have sufficient autologous tumor for harvest and subsequent vaccine production and will overcome the significant limitation of the in vitro transfectants for tumor transfection and selection in the laboratory. As noted above, the use of the patients' own tumor as a source of antigens in our system optimizes the generation of a T-cell response and has significant advantages over allogeneic vaccine strategies that rely on shared antigens restricted by common MHC antigens.

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References

  1. Lattime EC, Gerson SL. Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation 2nd ed San Diego: Academic Press 2002 534
  2. Thomas L. In: Lawrence HS, ed Cellular and Humoral Aspects of the Hypersensitive States New York: Hoeber-Harper 1959 529–532
  3. Burnet FM. The concept of immunological surveillance Prog Exp Tumor Res 1970 13: 1 | PubMed | ISI | ChemPort |
  4. Bodurtha AJ, Berkelhammer J, Kim YH, Laucius JF, Mastrangelo MJ. A clinical, histologic, and immunologic study of a case of metastatic malignant melanoma undergoing spontaneous remission Cancer 1976 37: 735–742 | PubMed | ISI | ChemPort |
  5. Spontaneous Remission: An Annotated Bibliography Sausalito, CA: Institute of Noetic Sciences 1993 1–710
  6. Ostrand-Rosenberg S, Clements VK, Dissanayake S, Gilbert M, Pulaski BA, Qi L. Immunologic targets for the gene therapy of cancer In: Lattime EC, Gerson SL, eds Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation 2nd ed San Diego: Academic Press 2002 128–144
  7. Bornstein RS, Mastrangelo MJ, Sulit H et al. Immunotherapy of melanoma with intralesional BCG Natl Cancer Inst Monogr 1973 39: 213–220
  8. Laucius JF, Bodurtha AJ, Mastrangelo MJ, Creech RH. Bacillus Calmette–Guerin in the treatment of neoplastic disease J Reticuloendothel Soc 1974 16: 347–373 | PubMed | ChemPort |
  9. Lamm DL, Thor DE, Harris SC, Reyna JA, Stogdill VD, Radwin HM. Bacillus Calmette–Guerin immunotherapy of superficial bladder cancer J Urol 1980 124: 38–42
  10. Mastrangelo MJ, Maguire HCJ, Lattime EC, Berd D. Whole cell vaccines In: DaVita VT, Hellman S, Rosenberg SA, eds Biological Therapy of Cancer 2nd ed Philadelphia: Lippincott 1995 648–658
  11. Mastrangelo MJ, Sato T, Lattime EC, Maguire HC Jr, Berd D. Cellular vaccine therapies for cancer In: Foon KA, Muss HB, eds Biological and Hormonal Therapies of Cancer Boston: Kluwer Academic Publishing 1998 35–50
  12. Hu X, Chakraborty NG, Sporn JR, Kurtzman SH, Ergin MT, Mukherji B. Enhancement of cytolytic T lymphocyte precursor frequency in melanoma patients following immunization with MAGE-1 peptide loaded antigen presenting cell–based vaccine Cancer Res 1996 56: 2479–2483 | PubMed | ISI | ChemPort |
  13. Schlom J, Tsang K-Y, Kantor J et al. Strategies in the development of recombinant vaccines for colon cancer Semin Oncol 1999 26: 672–682 | PubMed |
  14. Marshall JL, Hoyer RJ, Toomey MA et al. Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses J Clin Oncol 2000 18: 3964–3973 | PubMed | ISI | ChemPort |
  15. Horig H, Lee DS, Conkright W et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule Cancer Immunol Immunother 2000 49: 504–514 | Article | PubMed | ISI | ChemPort |
  16. Eder JP, Kantoff PW, Roper K et al. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer Clin Cancer Res 2000 6: 1632–1638 | PubMed | ISI | ChemPort |
  17. Sanda MG, Smith DC, Charles LG et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer Urology 1999 53: 260–266 | Article | PubMed | ISI | ChemPort |
  18. Fearon ER, Pardoll DM, Itaya T et al. Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response Cell 1990 60: 397–403 | Article | PubMed | ISI | ChemPort |
  19. Asher AL, Mulé JJ, Kasid A et al. Murine tumor cells transduced with the gene for tumor necrosis factor-α: evidence for paracrine immune effects of tumor necrosis factor against tumors J Immunol 1991 146: 3227–3234 | PubMed | ISI | ChemPort |
  20. Watanabe Y, Kuribayashi K, Miyatake J et al. Exogenous expression of mouse interferon-gamma cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity Proc Natl Acad Sci USA 1989 86: 9456–9460 | Article | PubMed | ChemPort |
  21. Tepper RI, Pattengale PK, Leder P. Murine interleukin-4 displays potent anti-tumor activity in vivo Cell 1989 57: 503–512 | Article | PubMed | ISI | ChemPort |
  22. Connor J, Bannerji R, Saito S, Heston W, Fair W, Gilboa E. Regression of bladder tumors in mice treated with interleukin 2 gene–modified tumor cells J Exp Med 1993 177: 1127–1134 | Article | PubMed | ChemPort |
  23. Saito S, Bannerji R, Gansbacher B et al. Immunotherapy of bladder cancer with cytokine gene–modified tumor vaccines Cancer Res 1994 54: 3516–3520 | PubMed |
  24. Dranoff G, Jaffee E, Lazenby A. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte–macrophage colony stimulating factor stimulates potent, specific, and long lasting anti-tumor immunity Proc Natl Acad Sci USA 1993 90: 3539–3543 | Article | PubMed | ChemPort |
  25. Simons JW, Mikhak B, Chang JF et al. Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte–macrophage colony-stimulating factor using ex vivo gene transfer Cancer Res 1999 59: 5160–5168 | PubMed | ISI | ChemPort |
  26. Simons JW, Jaffee EM, Weber CE et al. Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte–macrophage colony-stimulating factor gene transfer Cancer Res 1997 57: 1537–1546 | PubMed | ISI | ChemPort |
  27. Nelson WG, Simons JW, Mikhak B et al. Cancer cells engineered to secrete granulocyte–macrophage colony- stimulating factor using ex vivo gene transfer as vaccines for the treatment of genitourinary malignancies Cancer Chemother Pharmacol 2000 46: S67–S72 | PubMed | ChemPort |
  28. Perussia B, Chan SH, D'Andrea A et al. Natural killer cell stimulatory factor or interleukin-12 has differential effects on the proliferation of TCRαβ+, TCRτδ+ T lymphocytes and NK cells J Immunol 1992 149: 3495–3502 | PubMed | ISI | ChemPort |
  29. Golumbek PT, Lazenby AJ, Levitsky HI et al. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4 Science 1991 254: 713–716 | Article | PubMed | ISI | ChemPort |
  30. Kusumoto M, Umeda S, Ikubo A et al. Phase 1 clinical trial of irradiated autologous melanoma cells adenovirally transduced with human GM-CSF gene Cancer Immunol Immunother 2001 50: 373–381 | Article | PubMed | ISI | ChemPort |
  31. Chang AE, Li Q, Bishop DK, Normolle DP, Redman BD, Nickoloff BJ. Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte–macrophage colony-stimulating factor Hum Gene Ther 2000 11: 839–850 | Article | PubMed | ISI | ChemPort |
  32. Soiffer R, Lynch T, Mihm M et al. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma Proc Natl Acad Sci USA 1998 95: 13141–13146 | Article | PubMed | ChemPort |
  33. Tahara H, Zeh HJ, Storkus WJ et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in-vivo Cancer Res 1994 54: 182–189 | PubMed | ISI | ChemPort |
  34. Lotze MT, Rubin JT, Carty S et al. Gene therapy of cancer: a pilot study of IL-4-gene–modified fibroblasts admixed with autologous tumor to elicit an immune response Hum Gene Ther 1994 5: 41–55 | PubMed | ISI | ChemPort |
  35. Kang WK, Park C, Yoon HL et al. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study Hum Gene Ther 2001 12: 671–684 | Article | PubMed | ISI | ChemPort |
  36. Lee SS, Eisenlohr LC, McCue PA, Mastrangelo MJ, Lattime EC. Intravesical gene therapy: vaccinia virus recombinants transfect murine bladder tumors and urothelium Proc Am Assoc Cancer Res 1993 34: 337
  37. Lee SS, Eisenlohr LC, McCue PA, Mastrangelo MJ, Lattime EC. Intravesical gene therapy: in-vivo gene transfer using vaccinia vectors Cancer Res 1994 54: 3325–3328 | PubMed | ISI | ChemPort |
  38. Lattime EC, Maguire HCJ, McCue PA et al. Infection of human melanoma cells by intratumoral vaccinia J Invest Dermatol 1994 102: 568
  39. Mastrangelo MJ, Maguire HC Jr, McCue PA. A pilot study demonstrating the feasibility of using intratumoral vaccinia injections as a vector for gene transfer Vaccine Res 1995 4: 55–69
  40. Gomella LG, Mastrangelo MJ, Eisenlohr LC, McCue PA, Lee SS, Lattime EC. Localized gene therapy for prostate cancer: strategies for intraprostatic cytokine gene transfection using vaccinia virus vectors J Urol 1995 153: 308A
  41. Lattime E, Eisenlohr L, Gomella L, Mastrangelo M. The use of vaccinia virus vectors for immunotherapy via in-situ tumor transfection In: Lattime E, Gerson S, eds Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation San Diego: Academic Press 1999 125–137
  42. Lee SS, Eisenlohr LC, McCue PA, Mastrangelo MJ, Fink E, Lattime EC. In-vivo gene therapy of murine tumors using recombinant vaccinia virus encoding GM-CSF Proc Am Assoc Cancer Res 1995 36: 248
  43. Lee SS, Eisenlohr LC, McCue PA, Mastrangelo MJ, Lattime EC. Vaccinia virus vector mediated cytokine gene transfer for in vivo tumor immunotherapy Proc Am Assoc Cancer Res 1994 35: 514
  44. Ramshaw IA, Ramsay AJ, Karupiah G, Rolph MS, Mahalingam S, Ruby JC. Cytokines and immunity to viral infections Immunol Rev 1997 159: 119–135 | Article | PubMed | ISI | ChemPort |
  45. Lattime EC, Maguire HCJ, McCue PA et al. Gene therapy using vaccinia vectors: repeated intratumoral injections result in tumor infection in the presence of anti-vaccinia immunity Proc Am Soc Clin Oncol 1994 13: 397
  46. Ostrand-Rosenberg S, Pulaski BA, Armstrong TD, Clements VK. Immunotherapy of established tumor with MHC class II and B7.1 cell–based tumor vaccines Adv Exp Med Biol 1998 451: 259–264 | PubMed | ChemPort |
  47. Mastrangelo MJ, Maguire HC Jr, Eisenlohr LC. Intratumoral recombinant GM-CSF–encoding virus as gene therapy in patients with cutaneous melanoma Cancer Gene Ther 1999 6: 409–422 | Article | PubMed | ISI | ChemPort |
  48. Gomella LG, Mastrangelo MJ, McCue PA, Maguire HC, Mulholland SG, Lattime EC. Phase I study of intravesical vaccinia virus as a vector for gene therapy of bladder cancer J Urol 2001 166: 1291–1295 | Article | PubMed | ISI | ChemPort |
  49. Mukherjee S, Haenel T, Himbeck R et al. Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation Cancer Gene Ther 2000 7: 663–670 | Article | PubMed | ISI | ChemPort |
  50. Robinson BW, Mukherjee SA, Davidson A et al. Cytokine gene therapy or infusion as treatment for solid human cancer J Immunother 1998 21: 211–217 | Article | PubMed | ISI | ChemPort |
  51. Kaufman HL, Conkright W, Divito J Jr et al. A phase I trial of intra lesional RV-B7.1 vaccine in the treatment of malignant melanoma Hum Gene Ther 2000 11: 1065–1082
  52. Kaufman HL, DeRaffele G, Divito J et al. A phase I trial of intralesional rV-Tricom vaccine in the treatment of malignant melanoma Hum Gene Ther 2001 12: 1459–1480 | PubMed | ChemPort |
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Acknowledgements

Supported by ACS Grants IM-742 and EDT-78842; USPHS Grants CA-42908, CA-55322, CA-69253, CA-74543; and the Nat Pincus Trust.