The addition of recombinant vaccinia HER2/neu to oncolytic vaccinia-GMCSF given into the tumor microenvironment overcomes MDSC-mediated immune escape and systemic anergy.

Effective immunotherapeutic strategies require the ability to generate a systemic antigen-specific response capable of impacting both primary and metastatic disease. We have built on our oncolytic vaccinia a granulocyte-macrophage colony-stimulating factor (GM-CSF) strategy by adding recombinant tumor antigen to increase the response in the tumor microenvironment and systemically. In the present study, orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) was associated with increased Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) both systemically and in the tumor microenvironment. This MDSC population had inhibitory effects on the HER2/neu-specific Th1 immune response. VVneu and VVGMCSF are recombinant oncolytic vaccinia viruses that encode HER2/neu and GM-CSF, respectively. Naive FVB mice vaccinated with combined VVneu and VVGMCSF given systemically developed systemic HER2/neu-specific immunity. NBT1-bearing mice became anergic to systemic immunization with combined VVneu and VVGMCSF. Intratumoral VVGMCSF failed to result in systemic antitumor immunity until combined with intratumoral VVneu. Infection/transfection of the tumor microenvironment with combined VVGMCSF and VVneu resulted in development of systemic tumor-specific immunity, reduction in splenic and tumor MDSC and therapeutic efficacy against tumors. These studies demonstrate the enhanced efficacy of oncolytic vaccinia virus recombinants encoding combined tumor antigen and GM-CSF in modulating the microenvironment of MDSC-rich tumors.

Phase i study of intravesical vaccinia virus as a vector for gene therapy of bladder cancer.

PURPOSE: Vaccinia virus is a DNA poxvirus previously used as a vaccine to eradicate smallpox. The virus has a high efficiency of infection, replicates in the cytoplasm without chromosomal integration and can transport a large amount of recombinant DNA without losing infectivity. Therefore, it is an excellent choice as a vector for gene delivery in vivo. Large quantities of vaccinia have been injected into dermal, subcutaneous and peripheral lymph node melanoma metastases without significant side effects, and with efficient infection of the tumor cells and recombinant gene transfection. To determine if vaccinia, when given intravesically, can effectively infect bladder mucosa and tumor with acceptable toxicity, we performed a phase I trial of intravesical vaccinia in patients with muscle invasive transitional cell carcinoma before radical cystectomy. MATERIALS AND METHODS: After documenting immune competence and demonstration of a major reaction after revaccination, patients received 3 increasing doses of intravesical Dryvax vaccinia virus (Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania) that was provided by the Centers for Disease Control. Approximately 24 hours after the third dose, cystectomy was performed and the tissue was examined microscopically. RESULTS: There were 4 patients who were treated. The 3 patients who received the highest doses (100 x 106 plaque forming units) had significant mucosal and submucosal inflammatory infiltration by lymphocytes, eosinophils, and plasma cells into tumor and normal tissue. Dendritic cells were recruited to the site after exposure to the vaccinia. Significant mucosal edema and vascular ectasia were seen. Tumor and normal urothelial cells showed evidence of viral infection, including enlarged vacuolated cells with cytoplasmic inclusions. There were no clinical or laboratory manifestations of vaccinia related toxicity except mild dysuria. Of the 4 patients 3 survived and were free of disease at 4-year followup. CONCLUSIONS: Our study demonstrates that vaccinia virus can be administered safely into the bladder with recruitment of lymphocytes and induction of a brisk local inflammatory response. To our knowledge, this is the first report of direct delivery of live virus into the human bladder. The role of wild type vaccinia as immunotherapy for bladder cancer warrants further study. Furthermore, these data support the exploration of recombinant vaccinia as a putative gene therapy vector for intravesical infection and transfection of bladder tumor cells with cytokine or other genes, an approach that our group pioneered and most recently studied in patients with superficial melanoma.

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma.

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10(4)-2 x 10(7) plaque-forming units (PFU)/lesion; 10(4)-8 x 10(7) PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of > or =10(7) PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4+ and CD8+ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14-21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-beta-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. 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. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.

Tumor-induced interleukin-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non-tumor antigen present at the tumor site.

Interleukin (IL)-10 is a potent immunosuppressive cytokine that has been found to be present at the tumor site in a wide variety of human cancers, including transitional cell carcinoma of the bladder. Using a murine bladder tumor (MB49), which we show to express the male transplantation antigen (HY), we tested the hypothesis that IL-10 at the tumor site can block the generation of a tumor-specific type 1 immune response. We show that, despite its expression of HY, MB49 fails to prime for an HY-specific type 1 (IFN-gamma) response in normal female mice. Although MB49 does not constitutively produce IL-10, our data support a model whereby MB49 induces infiltrating cells to produce IL-10. This feature rendered the IL-10 knockout (KO) mouse, whose infiltrating cells are incapable of IL-10 production, a suitable model in which to study MB49 in the absence of IL-10. When injected into IL-10 KO mice, MB49 does prime for an HY-specific, type 1 immune response. Furthermore, IL-10 KO mice show prolonged survival and an increased capacity to reject tumors as compared with normal mice. We also tested the ability of tumor-induced IL-10 to inhibit immunization to a non-tumor antigen present at the tumor site. When vaccinia virus encoding beta-galactosidase (beta-gal) is injected into the tumors of normal mice, no beta-gal-specific IFN-gamma response is mounted. However, when this same viral construct is injected into the tumors of IL-10 KO mice, it produces a strong beta-gal-specific, IFN-gamma response. These studies demonstrate that tumor-induced IL-10 can block the generation of a tumor-specific type 1 immune response as well as subvert attempts to elicit a type 1 immune response to a non-tumor antigen at the tumor site.

Phase I study of R24 in patients with metastatic melanoma including evaluation of immunologic parameters.

R24 is a mouse IgG3 monoclonal antibody with specificity for the disialoganglioside GD3. Most human melanomas have substantial surface GD3; in addition, a significant proportion of T lymphocytes display surface GD3. In a phase I study, we have investigated the toxicity and effect on selected immunological parameters of three dose levels of R24 given intravenously daily for five days (10 mg/m2/d, 30 mg/m2/d and 50 mg/m2/d) to patients with advanced melanoma. R24 administration neither consistently diminished nor augmented expression of delayed type hypersensitivity (DTH) skin reactions to anergy panel antigens or to a contact allergen dinitrofluorobenzene. R24 was infrequently found on tumor cells, or on lymphocytes from DTH biopsies, despite measurable serum levels of R24. The 30 mg/m2/d dose of R24 produced a statistically significant drop in peripheral blood lymphocytes on treatment Day 5. Likewise, on Day 5 there was a modest but statistically significant decrement in the proportion of circulating cells which were R24+. While there was one mixed response, there were no complete or partial tumor regressions in the R24 treated patients; there was no evident clinical benefit from the R24 therapy. The toxicity of the R24 at the higher dose levels can be very substantial. One patient, on the highest dose level, died on the 4th day of R24 treatment; in the absence of a plausible alternative explanation, a relationship of the death to the administered R24 must be considered. A precipitous drop in serum albumin coincident with R24 administration was found in all cases; this effect has not been previously reported with R24.

Neutralizing anti-IL-10 antibody upregulates the induction and elicitation of contact hypersensitivity.

Various cytokines have been shown to modulate the acquisition and expression of delayed-type hypersensitivity. In a mouse model, we tested the notion that neutralization of interleukin-10 (IL-10), a cytokine that inhibits T cell-mediated reactions, would upregulate delayed-type hypersensitivity. We used two different monoclonal antibodies with specificity for murine IL-10 and used allergic contact dermatitis as a prototypical example of delayed-type hypersensitivity. When anti-IL-10 antibody was given at the time of sensitization to a contact allergen, there was a substantial increase in the induced contact hypersensitivity (CHS). In other experiments, the challenge reactions to contact allergen in routinely sensitized mice were increased when anti-IL-10 antibody was given at the time of challenge. Primary irritant reactions to croton oil were increased but only if anti-IL-10 antibody was given at the time of challenge and not when it was given a week previously. It appears that anti-IL-10 antibody can potentiate CHS reactivity by inactivating otherwise downregulating endogenous IL-10.

Interleukin 10 production by human melanoma.

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.

Development of secondary structure, growth characteristics and cytogenetic analysis of human transitional cell carcinoma xenografts in scid/scid mice.

PURPOSE: The growth of human bladder transitional cell carcinoma (TCC) in scid/scid mice was examined. MATERIALS AND METHODS: Cystocopically obtained TCC biopsies were implanted in scid/scid mice, and successful xenografts were compared with original tumors for growth and genetic characteristics. RESULTS: Low grade papillary tumors formed fluid-filled pseudobladders lined with malignant urothelium and papillary fronds containing fibrovascular cores recruited from the murine host. High grade xenografts grew without these secondary structures. When compared with the patient tumors, xenograft growth fractures, as measured by proliferating cell nuclear antigen, p53 expression and ploidy, were similar in each. CONCLUSIONS: The scid/scid xenografts maintain phenotype and architecture. This model may be useful for studying factors determining tumor grade, angiogenesis and tissue organization.

Gene therapy for human cancer: an essay for clinicians.

In the last decade our understanding of the processes that govern growth and differentiation has become quite sophisticated. A variety of tumor suppressor genes and more than 100 oncogenes have been identified. The roles of developmental genes in shaping the expression of neoplasia and of defective housekeeping genes in allowing mutations to persist and be transcribed have been appreciated. These advances have revolutionized our ability to diagnose and to formulate prognoses for patients with cancer. However, successful gene therapy for cancer has been elusive. This review highlights the current approaches to gene therapy for cancer and their scientific bases. The requirement that the therapy repair or destroy every cancer cell seems an insurmountable hurdle. Environmental manipulation through systemic administration of exogenous antisense may circumvent this problem in cases where it is appropriate. The more practical application of the technology of genetic engineering to facilitate cancer chemotherapy and immunotherapy is also reviewed. Particularly encouraging are the preclinical and clinical results of in vivo, in situ gene transfer. It remains to be determined if this local approach impacts favorably on survival.

In situ cytokine gene transfection using vaccinia virus vectors.

Studies by a number of investigators have focused on inducing tumor-specific immunity as a therapeutic approach to cancer. Successful immunotherapeutic strategies have involved localized treatment with immune-active adjuvants, systemic administration using cytokines such as interleukin-2, and the use of whole tumor cells or tumor cell fragments as vaccines. With an increasing understanding of the requirements for the development of an immune response, immunotherapeutic strategies have focused on providing mechanistic requirements, such as tumor or accessory antigen expression and cytokine-based "immune help." Recent preclinical studies have shown that ex vivo cytokine gene transfection of tumor cells and their use as vaccines result in the enhanced development of antitumor immunity and in some cases can be used to successfully treat pre-existing tumors. Studies from our laboratory have explored the use of vaccinia virus recombinants to directly transfect tumor cells in situ with cytokine genes as a strategy for enhancing the development of antitumor immunity. We have demonstrated that vaccinia virus recombinants are highly efficient in transfecting a wide range of murine and human tumors in vitro and can be used with similar effects in in vivo murine models. In addition, we have found that vaccinia virus productively infects human melanoma cells following intratumoral injection in patients with accessible lesions. In situ transfection is highly efficient, and therapy with increasing doses of virus is safe with only minor side effects. The results of our studies support the use of cytokine-encoding recombinant vaccinia virus vectors for in situ transfection in patients with cancer.

Expression of cytokine mRNA in human melanoma tissues.

We have reported that patients with metastatic melanoma treated with an autologous, dinitrophenol-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with tumor cell destruction. We tested biopsy specimens of eight subcutaneous metastases that had developed inflammation following vaccine treatment for expression of mRNA for interferon gamma (IFN gamma), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF alpha), and IL-10. Post-vaccine, inflamed biopsies contained mRNA for IFN gamma (5/8), IL-4 (4/8) or both (3/8), and for TNF alpha (4/7). In contrast, IFN gamma mRNA was detected in only 1/17 and TNF alpha mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL-10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ the reverse transcriptase/polymerase chain reaction confirmed that melanoma cells were the major source. These findings may provide a new parameter by which to measure the effects of cancer immunotherapy.

De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

Induction of TH1- and TH2-associated cytokine mRNA in mouse bladder following intravesical growth of the murine bladder tumor MB49 and BCG immunotherapy.

Productive immunity to murine and human parasites is associated with the development of a type I T cell response (interferon-gamma-producing) while type II responses (interleukin-4-producing) suppress the development of delayed-type hypersensitivity (DTH) and the elimination of the parasite. To determine if a similar regulatory pathway might exist in tumor systems and may be effected by immunotherapeutic manipulation, we have studied the localized cytokine response to the murine bladder tumor MB49 growing intravesically in syngeneic mice. Intravesical growth of MB49 results in the host-derived expression of mRNA for both interleukin-4 (IL-4) (TH2) and interferon gamma (IFN gamma) (TH1), as well as tumor necrosis factor alpha (TNF alpha) expression of indeterminate origin. Intravesical instillation of bacillus Calmette-Guérin (BCG), highly effective in eliminating bladder tumors clinically and in experimental systems, results in IFN gamma and TNF alpha mRNa production in the bladder wall, but no IL-4. Following BCG treatment of intravesical MB49, the number bladders expressing IL-4 mRNA decreases, while IFN gamma and TNF alpha expression remains constant. These results are consistent with the mechanism of action of BCG involving the generation of an enhanced TH1 immune milieu in the bladder wall, which may contribute to the generation of productive tumor-specific immunity.