Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-γ when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type β1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-γ. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr51 release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-β1 or anti-V-β2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-β can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type β1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8+ cytolytic T cells.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC50) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC50 values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC50 in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared in vivo in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral γ34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 γ34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a γ34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the γ34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Targeting p53 as a general tumor antigen.

A major barrier to the design of immunotherapeutics and vaccines for cancer is the idiosyncratic nature of many tumor antigens and the possibility that T cells may be tolerant of broadly distributed antigens. We have devised an experimental strategy that exploits species differences in protein sequences to circumvent tolerance of high-affinity T cells. HLA transgenic mice were used to obtain cytotoxic T lymphocytes specific for peptides from the human p53 tumor-suppressor molecule presented in association with HLA-A2.1. Although such p53-specific cytotoxic T cells did not recognize nontransformed human cells, they were able to lyse a wide variety of human tumor cells lines, thus confirming the existence of broadly distributed determinants that may serve as targets for immunotherapy.

Selective tumor kill of cerebral glioma by photodynamic therapy using a boronated porphyrin photosensitizer.

The prognosis for patients with the high-grade cerebral glioma glioblastoma multiforme is poor. The median survival for primary tumors is < 12 months, with most recurring at the site of the original tumor, indicating that a more aggressive local therapy is required to eradicate the unresectable "nests" of tumor cells invading into adjacent brain. Two adjuvant therapies with the potential to destroy these cells are porphyrin-sensitized photodynamic therapy (PDT) and boron-sensitized boron neutron capture therapy (BNCT). The ability of a boronated porphyrin, 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX tetrakiscarborane carboxylate ester (BOPP), to act as a photosensitizing agent was investigated in vitro with the C6 rat glioma cell line and in vivo with C6 cells grown as an intracerebral tumor after implantation into Wistar rats. These studies determined the doses of BOPP and light required to achieve maximal cell kill in vitro and selective tumor kill in vivo. The data show that BOPP is more dose effective in vivo by a factor of 10 than the current clinically used photosensitizer hematoporphyrin derivative and suggest that BOPP may have potential as a dual PDT/BNCT sensitizer.

The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro.

Herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) viral-directed enzyme prodrug gene therapy causes potent, tumor-selective cytotoxicity in animal models in which HSV-tk gene transduction is limited to a minority of tumor cells. The passage of toxic molecules from HSV-tk+ cells to neighboring HSV-tk- cells during GCV therapy is one mechanism that may account for this "bystander" cytotoxicity. To investigate whether gap junction-mediated intercellular coupling could mediate this bystander effect, we used a flow cytometry assay to quantitate the extent of heterocellular coupling between HSV-tk+ murine fibroblasts and both rodent and human tumor cell lines. Bystander tumor cytotoxicity during GCV treatment in a coculture assay was highly correlated (P < 0.001) with the extent of gap junction-mediated coupling. These findings show that gap junction-mediated intercellular coupling contributes to the in vitro bystander effect during HSV-tk/GCV therapy and that retroviral transduction of tumor cells is not required for bystander cytotoxicity.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.