Xiao-n Li

Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.

BackgroundIn view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity.MethodsWe describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5′-untranslated region (5′-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA.ResultsBoth in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity.ConclusionsOur study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

Trogocytosis of MHC-I/Peptide Complexes Derived from Tumors and Infected Cells Enhances Dendritic Cell Cross-Priming and Promotes Adaptive T Cell Responses

The transporter associated with antigen processing (TAP) and the major histocompatibility complex class I (MHC-I), two important components of the MHC-I antigen presentation pathway, are often deficient in tumor cells. The restoration of their expression has been shown to restore the antigenicity and immunogenicity of tumor cells. However, it is unclear whether TAP and MHC-I expression in tumor cells can affect the induction phase of the T cell response. To address this issue, we expressed viral antigens in tumors that are either deficient or proficient in TAP and MHC-I expression. The relative efficiency of direct immunization or immunization through cross-presentation in promoting adaptive T cell responses was compared. The results demonstrated that stimulation of animals with TAP and MHC-I proficient tumor cells generated antigen specific T cells with greater killing activities than those of TAP and MHC-I deficient tumor cells. This discrepancy was traced to differences in the ability of dendritic cells (DCs) to access and sample different antigen reservoirs in TAP and MHC-I proficient versus deficient cells and thereby stimulate adaptive immune responses through the process of cross-presentation. In addition, our data suggest that the increased activity of T cells is caused by the enhanced DC uptake and utilization of MHC-I/peptide complexes from the proficient cells as an additional source of processed antigen. Furthermore, we demonstrate that immune-escape and metastasis are promoted in the absence of this DC ‘arming’ mechanism. Physiologically, this novel form of DC antigen sampling resembles trogocytosis, and acts to enhance T cell priming and increase the efficacy of adaptive immune responses against tumors and infectious pathogens.

Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Priming of immune responses against transporter associated with antigen processing (TAP)‐deficient tumours: tumour direct priming

We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP‐negative CMT.64, a major histocompatibility complex class I (MHC‐I) down‐regulated mouse lung carcinoma cell line, enhanced T‐cell immunity against TAP‐deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co‐expression of TAP1 with B7.1 or H‐2Kb genes, and (2) which T‐cell priming mechanism (tumour direct priming or dendritic cell cross‐priming) plays the major role in inducing an immune response against TAP‐deficient tumours. We introduced the B7.1 or H‐2Kb gene into TAP1‐expressing CMT.64 cells and determined which gene co‐expressed with TAP1 was able to provide greater protective immunity against TAP‐deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co‐expressing but not H‐2Kb and TAP1 co‐expressing CMT.64 cells dramatically augments T‐cell‐mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T‐cell‐mediated immunity against TAP‐deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross‐priming as they show that T cells generated by tumour cell‐lysate‐loaded dendritic cells recognized TAP‐deficient tumour cells much less than TAP‐proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co‐expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP‐deficient tumour cells.

Establishment of a Mammary Carcinoma Cell Line from Syrian Hamsters Treated with N-Methyl-N-Nitrosourea

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.

Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP−) or TAP1-transfected (TAP1+) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP− and TAP1+ B7.1 expressing CMT.64 cells depending on the doses of γ-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1+ cells rejected TAP− tumors, only high dose immunization with B7.1-expressing TAP− cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP− tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of γ-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP− cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.

In vivo Survivors of Transformed Mouse Ovarian Surface Epithelial Cells Display Diverse Phenotypes for Gene Expression and Tumorigenicity

Ovarian cancer is the fifth most common cause of cancer death in women. Due to a lack of appropriate animal models, studies involving tumorigenicity, tumor progression and immune response at the molecular level are limited. We isolated many clones derived from thesurvivors of a transformed mouse ovarian epithelial cell line IG-10 in immune- competent mice and found that the clones displayed diverse phenotypes. Most clones were deficient in components of the MHC-I antigen presentation pathway. Soft-agarose colony assays showed different growth rates among clones. However, this did not completely correlate with each clone’s in vivo tumorigenicity regarding growth, tumor mass and ascites formation, suggesting the possibility that the clones may display contrasting intrinsic gene expression. We therefore performed two types of arrays to evaluate gene expression at transcriptional and translational levels. The results showed differences in expression of COL4α5, NOS-2, and SOCS-1 genes at the transcriptional level, MIP-2 gene at the protein level and CCL5, CXCL-10, IL-1α genes at both transcriptional and protein levels between low and high tumorigenic clones. Thus, our animal cell model together with the identified genes may provide a useful tool to study ovarian cancer immune response, tumorigenicity and tumor-host cell interactions in the tumor microenvironment.