B7-1 Gene Research Articles

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Role of Podocyte B7-1 in Diabetic Nephropathy

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.

Phylogenetic Gaussian Process Model for the Inference of Functionally Important Regions in Protein Tertiary Structures

A critical question in biology is the identification of functionally important amino acid sites in proteins. Because functionally important sites are under stronger purifying selection, site-specific substitution rates tend to be lower than usual at these sites. A large number of phylogenetic models have been developed to estimate site-specific substitution rates in proteins and the extraordinarily low substitution rates have been used as evidence of function. Most of the existing tools, e.g. Rate4Site, assume that site-specific substitution rates are independent across sites. However, site-specific substitution rates may be strongly correlated in the protein tertiary structure, since functionally important sites tend to be clustered together to form functional patches. We have developed a new model, GP4Rate, which incorporates the Gaussian process model with the standard phylogenetic model to identify slowly evolved regions in protein tertiary structures. GP4Rate uses the Gaussian process to define a nonparametric prior distribution of site-specific substitution rates, which naturally captures the spatial correlation of substitution rates. Simulations suggest that GP4Rate can potentially estimate site-specific substitution rates with a much higher accuracy than Rate4Site and tends to report slowly evolved regions rather than individual sites. In addition, GP4Rate can estimate the strength of the spatial correlation of substitution rates from the data. By applying GP4Rate to a set of mammalian B7-1 genes, we found a highly conserved region which coincides with experimental evidence. GP4Rate may be a useful tool for the in silico prediction of functionally important regions in the proteins with known structures.

Recombinant adenoviral vector expressing human wild-type p53, GM-CSF, and B7-1 genes suppresses the growth of glioma in vivo

Malignant gliomas are the most common of primary brain tumors and have been proven incurable with conventional treatments. Evidence have shown that a recombinant adenoviral vector expressing human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes (BB-102) may have antitumor effects in vitro. In this study, we investigated the effects of BB-102-based vaccine on glioma in vivo. An animal model using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with human immune system was established. The mice were vaccinated with inactivated U251 glioma cells transduced with BB-102 or adenoviral vector expressing green fluorescence protein (Ad-GFP) as a control and followed by the challenge of live U251 glioma cells. Tumor growth and antitumor responses were measured. Data showed that mice vaccinated with BB-102 had significantly reduced local tumor growth compared to mice with Ad-GFP vaccination or the control group. Histopathological analysis displayed low tumor cell density and significant infiltration of human peripheral blood lymphocytes (HuPBLs) in the tumor tissues of mice transduced with BB-102. Immunohistochemical analysis showed that mutant p53 was not expressed in tumor tissues of mice with BB-102 vaccination, and the expression level of Ki67 was significantly lower in the tumor tissues of the BB-102 group than those in the Ad-GFP group or the control group. Further study demonstrated that mice with BB-102 vaccination had significantly increased total T cell numbers, total T cell proportion, CD4+ T cell proportion, and CD8+ T cell proportion in spleens, as well as higher value of IgG, IgA, and IgE in sera. These data suggest that the recombinant adenoviral vector expressing human wild-type p53, GM-CSF, and B7-1 genes could suppress glioma in NOD/SCID mice model and might be considered as a novel strategy for glioma therapy.

Messenger RNA expression of B7-1 and NPHS1 in urinary sediment could be useful to differentiate between minimal-change disease and focal segmental glomerulosclerosis in adult patients

Podocyte proteins are involved in the pathogenesis of glomerular kidney disease (GKD). However, there is little information on messenger RNA (mRNA) expression patterns of B7-1 and NPHS1 in urinary sediment of patients with GKD. The objective of this study was to analyse the gene expression of B7-1 in urinary sediment and correlate it with the expression of podocyte-specific genes in patients with GKD. Adult patients with proliferative and non-proliferative GKD, proteinuria and stable renal function, were included. A group of healthy subjects was used to determine normal levels of urinary markers and to obtain reference RNA. Biochemical, clinical and experimental procedures included measurement of creatinine level and total urinary protein, renal biopsy, identification of urinary podocytes, gene expression analysis of B7-1, NPHS1, NPHS2 and SyNPO genes and urinary B7-1 protein analysis by enzyme-linked immunosorbent assay. Between June 2006 and November 2009, 69 patients with GKD (median age: 46 ± 15 years, 64% men) and 14 healthy subjects (median age: 34 ± 12 years, 43% men) were included. In both groups, urinary mRNA levels of B7-1 and NPHS1 were significantly higher in patients with GKD compared to healthy subjects (P = 0.050 and P = 0.008, respectively). Regarding GKD subtypes, patients with focal segmental glomerulosclerosis (FSGS), but not patients with minimal change disease (MCD), had a significantly higher mRNA expression of B7-1 and NPHS1 than healthy subjects (P = 0.012 and P = 0.030, respectively). Patients with MCD had a significantly lower NPHS1 mRNA expression than patients with FSGS (P = 0.012). The B7-1:NPHS1 urinary mRNA ratio was significantly higher in patients with MCD compared with patients with FSGS (P = 0.027). mRNA expression analysis of B7-1 and NPHS1 in urinary sediment may be useful to differentiate between different histologic subtypes of GKD, particularly between MCD and FSGS.

Triple expression of B7-1, B7-2 and 4-1BBL enhanced antitumor immune response against mouse H22 hepatocellular carcinoma

Costimulatory signals are essential for T-cell activation and hence play a very important role in antitumor immunity. B7 and 4-1BBL which belongs to tumor necrosis factor (TNF) family provide costimulatory interaction for T-cell activation and function. This study investigated the role of B7 and 4-1BBL in the amplification of tumor immunity by transduction of the B7-1, B7-2 and 4-1BBL into mouse hepatocellular carcinoma cell line H22. The tumorigenicity of H22 variants expressing either B7-1, B7-2 (H22/B7-1/B7-2) or 4-1BBL was compared with an H22 variant expressing B7-1, B7-2 and 4-1BBL (H22/B7-1/B7-2/4-1BBL). The study next investigated whether the combination of B7-1/B7-2 and 4-1BBL cell injection induced cytotoxic T lymphocyte (CTL) response and IL-2/IFN-γ secretion. The immune mechanisms underlying this combination treatment were then analyzed. Syngeneic BALB/c mice injected with H22/B7-1/B7-2/4-1BBL cells that expressed elevated levels of B7-1, B7-2 and 4-1BBL showed a tumor development frequency of 50% compared with 100% in mice injected with the H22 parental line, H22/neo, H22/B7-1/B7-2 and H22/4-1BBL. Mice inoculated with H22 tumor cells expressing B7-1, B7-2 and 4-1BBL developed a strong cytotoxic T lymphocyte response and long-term immunity against wild-type tumor, suggesting a synergistic effect between the B7 and 4-1BBL costimulatory pathways. Results showed that H22/B7-1/B7-2/4-1BBL tumor vaccines probably protect the infiltrating lymphocytes from apoptosis and induce NF-κB activation to improve T-cell-mediated antitumor response. In this study, the antitumor consequences of using B7-1, B7-2 and 4-1BBL gene transfer have demonstrated the therapeutic potential of gene therapy approach for hepatocellular carcinoma.

Adenovirus mediated transfer of p53, GM-CSF and B7-1 suppresses growth and enhances immunogenicity of glioma cells

Objectives: Malignant gliomas are good targets for gene therapy because they have been proven incurable with conventional treatments. However, malignant gliomas are genetically and physiologically highly heterogeneous, and current gene therapy interventions have been designed to target only a few variations of this kind of disease. Hence, we developed a combined gene therapy approach using a recombinant adenovirus carrying human wild-type p53 (WT-p53), granulocyte–macrophage colony-stimulating factor (GM-CSF) and B7-1 genes (designated BB-102) to combat the disease.Methods: Human malignant glioma cells U251 and U87 were transduced with BB-102. Expression of WT-p53, GM-CSF and B7-1 genes were determined by Western blot, enzyme linked immunosorbent assay and flow cytometric analysis, respectively. Growth rates were determined by serial cell counts. Apoptosis was detected by flow cytometric analysis. Proliferation of autologous peripheral blood lymphocytes (PBLs) and cytotoxicity against primary glioma cells were assessed by cell proliferation and cytotoxicity assay kits, respectively.Results: By the transduction of BB-102, high expression levels of the three exogenesis genes were detected in glioma cells. Cell growth was inhibited and apoptosis was induced. Significant proliferation of autologous PBLs and specific cytotoxicity against primary glioma cells were also induced by the infection of BB-102 in vitro, with the effect being more evident than that of Ad-p53.Conclusion: These results suggest that glioma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a feasible and effective immunotherapeutic approach in glioma treatments.

Metronomic Chemotherapy Enhances Antitumor Effects of Cancer Vaccine by Depleting Regulatory T Lymphocytes and Inhibiting Tumor Angiogenesis

Although cancer vaccines are emerging as innovative methods for cancer treatment, these alone have limited potential for treating measurable tumor burden. Thus, the importance of identifying anticancer strategies with greater potency is necessary. The chimeric DNA vaccine CTGF/E7 (connective tissue growth factor linked to the tumor antigen human papillomavirus 16 E7) generates potent E7-specific immunity and antitumor effects. We tested immune-modulating doses of chemotherapy in combination with the CTGF/E7 DNA vaccine to treat existing tumors in mice. Metronomic low doses of paclitaxel, not the maximal tolerable dose, are synergistic with the antigen-specific DNA vaccine. Paclitaxel, given in metronomic sequence with the CTGF/E7 DNA vaccine enhanced the vaccine's potential to delay tumor growth and decreased metastatic tumors in vivo better than the CTGF/E7 DNA vaccine alone. The two possible mechanisms of metronomic paclitaxel chemotherapy are the depletion of regulatory T cells and the inhibition of tumor angiogenesis rather than direct cancer cell cytolytic effects. Results indicate that combination treatment of metronomic chemotherapy and antigen-specific DNA vaccine can induce more potent antigen-specific immune responses and antitumor effects. This provides an immunologic basis for further testing in cancer patients.

Effects of wt-p53, BT-1 and CM-CSF genes cotransfection on proliferation and immunogenicity of SH-SY5Y cell lines

Objective To observe the effeet of cotransduction of wt-p53,B7-1 and GM-CSF genes mediated by recombinant adenovirus on cell growth, apoptosis and immunogenicity of neuroblastoma cell line SH-SY5Y, Methods We transfected SH-SYSY cells separately with recombinant adenovirus, recombinant adenovirus mediating human wild type p53(wt-p53) gene and BB-102.We separately named them with SH/Ad,SH/p53 and SH/BB-102 group.The protein expression of p53, B7-1 and GM-CSF were measured by Western blot, FCM and enzyme linked immunosorbent assay. Cell number was counted and growth curves were drawed. Cell apoptosis was tested by FCM. Lymphocyte proliferation and cytokine secretory was evaluated by mixed lymphocyte culturing. Results After transection with BB- 102, p53, B7-1, and GM-CSF, high-efficiency expression of target genes were found. Growth of the cells was inhibited, apoptosis was induced in the SH/p53 and SH/BB-102 groups. Proliferation of lymphocytes was stimulated and cytokine increased visorously in the SH/BB-102 group. Condusions After transfection with BB-102, growth of the SH-SY5Y cells was inhibited,apoptosis was induced,and immunogenicity was greatly enhanced. Key words: Neuroblastoma; Adenoviruses,Human; Genes,p53; Antigens,CD80; Granulocyte-macrophage colony-stimulating factor

Gene Therapy for Human Nasopharyngeal Carcinoma by Adenovirus–Mediated Transfer of Human p53, GM-CSF, and B7-1 Genes in a Mouse Xenograft Tumor Model

Incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions. Prevention of tumor recurrences and metastases is a crucial approach to improve therapeutic outcome in NPC patients. In this study, we investigated the effects of the cotransfer of the tumor suppressor gene, p53, in combination with the immunostimulatory genes, GM-CSF and B7-1, on tumor regression and subsequent tumor recurrence. We constructed a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes (Ad-p53/GM-CSF/B7-1), which mediated high-level expression of these three genes in NPC CNE-1 cells. Ad-p53/GM-CSF/B7-1 infection inhibited the growth of CNE-1 cells and induced tumor-specific cytotoxic T-lymphocytes (CTLs) in vitro. In CNE-1 xenograft tumor models in huPBL-nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, an intratumoral injection of Ad-p53/GM-CSF/B7-1 resulted in a reduced tumor burden, compared to normal saline (NS) and Ad-p53 controls. Tumors in the Ad-p53/GM-CSF/B7-1 group displayed diffuse necrosis and infiltration of human T-cells. Further, the tumor occurrence of CNE-1 cell rechallenge largely decreased after the primary tumor was intratumorally injected with Ad-p53/GM-CSF/B7-1 in the HuPBL-NOD/SCID mice model. Only 2 of 8 (25%) animals in the Ad-p53/GM-CSF/B7-1 group had developed measurable tumors, which demonstrated extensive necrosis and much more human T-cell infiltration, compared to 5 of 7 (71%) in the NS and Ad-p53 groups. Therefore, the adenovirus-mediated introduction of p53, GM-CSF, and B7-1 genes could improve local control and prevent the recurrence or metastases of NPC tumors, which suggests a potential therapeutic value in NPC treatment.

Treatment of Intracerebral Glioblastomas with G422 Tumour Cell Vaccine in a Mouse Model

The aim of this study was to develop a tumour vaccine with the ability to induce and expand higher affinity cytotoxic T lymphocytes and stimulate an effective antitumour immune response. The hypothesis tested was that G422 glioblastoma cells modified with B7-1 and interferon (IFN)-gamma genes could serve as a tumour vaccine. It was found that therapeutic subcutaneous immunizations with this tumour vaccine significantly induced a cytotoxic T-cell response and prolonged the survival of female Kuming mice with intracerebral G422 tumour isografts. The data collectively suggested that G422 glioblastoma cells genetically modified with B7-1 and IFN-gamma genes could serve as a tumour vaccine.

A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes

Oncolytic adenoviruses, also called conditionally replicating adenoviruses (CRADs), have been widely applied in cancer gene therapy. However, the construction of CRADs is still time-consuming. In this study, we attempted to establish a simplified method of generating CRADs based on AdEasy system. A novel plasmid pTE-TPE-GM was constructed, containing sequentially positioned promoter of telomerase reverse transcriptase (TERTp), coding sequence of E1A gene, promoter of E1B gene, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, internal ribosome entry site sequence and coding sequence of E1B55K gene. The CRAD-generating system reported here include three plasmids: pTE-TPE-GM, pShuttle-CMV and AdEasy-1, one Escherichia coli strain BJ5183, and the packaging cell line 293. Using this system, an oncolytic adenovirus carrying B7-1 (CD80) and GM-CSF genes was successfully constructed and designated as Ad-CD80-TPE-GM. The expression of GM-CSF increased more than 9000 times in tumor cell lines infected by Ad-CD80-TPE-GM at a multiplicity of infection (MOI) of 5, compared with the cells infected by replication-defective control virus. Similarly, the expression of CD80 also increased 9-140 times. Ad-CD80-TPE-GM selectively replicates in TERT-positive tumor cells, and the progeny viruses can reach up to 375 infection units (IU) per cell. In vitro study showed that the Ad-CD80-TPE-GM induced an obvious oncolytic effect at MOI of 0.1, and killed about 80% TERT-positive tumor cells within 7 days at an MOI of 1. The antitumor effect of this vector was also investigated in Hep2 xenograft model of nude mice, and the tumor inhibition rate reached 74% at day 30 after the administration with a total dose of 1 x 10(9) IU Ad-CD80-TPE-GM. Intratumoral injection of Ad-CD80-TPE-GM slightly induced neutralizing antibody against the oncolytic adenovirus in nude mice, which might contribute to the virus clearance in vivo. In conclusion, we successfully constructed an oncolytic CRAD carrying GM-CSF and CD80 gene. More importantly, this system can be modified to generate novel transcriptionally regulated CRADs with different tissue-specific promoters or transgenes.

Construction and identification of recombinant adenovirus co-expressing NK4 and B7-1 genes

目的 构建能同时表达B7-1和HGF/NK4基因的重组腺病毒载体,同时制备相应的复制缺陷型重组腺病毒,检测其在喉癌细胞中的表达.方法 构建以串联方式携带人B7.1和HGF/NK4基凶的重组穿梭载体pAdtrack-NK4-B7-1.Pme I线性化后与腺病毒载体pAdEasy-1共转化大肠杆菌BJ5183,通过同源重组得到重组腺病毒载体pAd-NK4-B7-1.将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒,应用病毒悬液感染人喉癌细胞株.用RT-PCR检测感染细胞中B7.1和NK4基因mRNA表达.结果 酶切鉴定证实阳性pAdTrack-NK4-B7-1重组穿梭载体含有目的基因B7-1和HGF/NK4,同源重组后制备的病毒载体DNA经酶切鉴定获得了阳性重组腺病毒载体,该载体能有效转染HEK293细胞并在细胞内成功包装,转染3 d后可以观察到绿色荧光蛋白(GFP)表达并逐渐增多、增强.制备的Ad-NK4-B7在体外能有效感染Hep-2细胞,感染后可维持6 d高水平的B7-1和NK4基因的表达,整体表达可持续2周.结论 成功构建了同时表达B7-1和NK4基因的重组腺病毒载体并制备出重组腺病毒颗粒,该病毒能有效地感染喉癌细胞并表达高水平的NK4和B7-1基因,为进一步研究B7-1和NK4基凶的功能及应用B7-1和NK4进行喉癌的联合基因治疗提供了依据和素材。

Induction of Immunological Antitumor Effects by Adenovirus-Mediated Gene Transfer of B7-1 in a Murine Squamous Cell Carcinoma Cell Line

To evaluate the antitumor immune effects of B7-1 gene expression mediated by adenoviral vectors against squamous cell carcinoma. Transfection of the costimulatory molecule B7-1 gene into certain murine tumors increases antitumor immunity and suppresses tumor growth. In vitro and in vivo study. A murine squamous cell carcinoma cell line, KLN205, was infected with adenoviral vectors carrying either B7-1 (AdB7) or LacZ (AdCL) genes. Infected cells were injected subcutaneously into the flanks of DBA/2 mice. The growth of tumors infected with adenviral vectors was measured. AdB7-infected cells grew significantly slower than AdCL-infected cells in vivo, while there was no significant difference in the growth rates between the 2 groups in vitro. Moreover, significant growth suppression of rechallenged noninfected parental cells was observed in the mice immunized with AdB7-infected cells but not in those immunized with AdCL-infected cells. These results suggest that the B7-1 gene has therapeutic potential for immunotherapy against head and neck squamous cell carcinoma.

B7-1 and 4-1BB ligand expression on a myeloma cell line makes it possible to expand autologous tumor-specific cytotoxic T cells in vitro

The aim of this study was to confer an antigen-presenting cell (APC) ability on multiple myeloma cell lines (HMCLs) using B7-1 and/or 4-1BBL gene transfer. HMCLs were retrovirally transduced with B7-1 and/or 4-1BBL cDNAs. Allogeneic or autologous T cells were stimulated by coculture with B7-1- and/or 4-1BBL-transduced HMCLs in the presence of interleukin-2. T cell clones were obtained by limiting dilution. T-cell activation was assessed by interferon-gamma Elispot assays and cytotoxicity by (51)Cr release assays. Neither primary multiple myeloma cells (MMCs) nor HMCLs expressed B7-1 or 4-1BBL, and these molecules could not be induced by CD40 triggering. HMCLs failed to stimulate allogeneic or autologous T cells. Transduction of HMCLs with B7-1 and/or 4-1BBL retroviruses induced a high expression of B7-1 and 4-1BBL molecules and a strong T-cell activation ability. Long-term cultured CD8(+) T-cell lines could be obtained by stimulation with the autologous B7-1/4-1BBL XG-19 HMCL. These cytotoxic T lymphocytes (CTL) efficiently killed the autologous parental XG-19 HMCL as well as autologous primary MMCs and allogeneic HMCLs. They did not kill autologous CD34 cells and autologous EBV cell line or natural killer target K562 cells. Cloned CTL could recognize allogeneic HMCLs, demonstrating that a shared anti-MMC repertoire was expanded. Transduction with B7-1 and 4-1BBL retroviruses turned HMCLs into efficient APCs. It permitted the long-term expansion of autologous anti-tumor CTL with a shared anti-MMC repertoire, for one HMCL. These data suggest developing an immunotherapy using modified tumor cells in patients with multiple myeloma.

Concurrent delivery of GM-CSF and B7-1 using an oncolytic adenovirus elicits potent antitumor effect

Oncolytic adenoviral vectors are currently being developed as biologic anticancer agents. Coupling the lytic function of an oncolytic adenovirus (Ad) with its ability as a transgene delivery system represents a powerful extension of this methodology. A clear advantage is the amplification of a therapeutic gene, as replicating vectors would be able to infect and deliver the gene of interest to neighboring cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the most potent stimulators of a specific and long-lasting antitumor immunity and its important role in the maturation of antigen-presenting cells to induce T-cell activation has been well documented. Similarly, the B7 family has also been shown to play an integral role in mediating an antitumor response. Most tumor cells, however, lack the expression of these costimulatory molecules on their surface, thus escaping immune system recognition. To increase the antitumor effect of an oncolytic Ad, we have generated an E1B 55 kDa-deleted oncolytic adenoviral vector, YKL-GB, that expresses both GM-CSF and B7-1. The therapeutic efficacy of YKL-GB Ad was evaluated in immunocompetent mice bearing murine melanoma B16-F10 tumors. Significant inhibition of tumor growth was seen in mice treated with YKL-GB compared to those treated with the analogous vector, YKL-1. Moreover, YKL-GB oncolytic Ad demonstrated enhanced antitumor activity and higher incidences of tumor regression compared to a replication-incompetent Ad, dl-GB, which coexpresses GM-CSF and B7-1. Localized GM-CSF and B7-1 gene transfer also conferred long-lasting immunity against a tumor re-challenge. To establish that the observed antitumor effect is associated with the generation of a tumor-specific immune response, we carried out interferon-gamma enzyme-linked immune spot assay. We observed that YKL-GB induced significantly higher immune cell activation than YKL-1. Furthermore, immunohistochemical studies demonstrated robust dendritic cells and CD4(+)/CD8(+) T-cell infiltration in these mice compared to the YKL-1-treated groups. In agreement with these results, splenocytes from tumor-bearing mice treated with YKL-GB expressed high levels of the costimulatory and activation molecules. These findings demonstrate the effectiveness of enhancing the immune response against tumors with an oncolytic Ad expressing both GM-CSF and B7-1 and provide a potential therapeutic strategy for the management of neoplasia.

850. Concurrent Delivery of GM-CSF and B7-1 Using an Oncolytic Adenovirus Elicits Potent Anti- Tumor Effect on the Growth Established B16-F10 Tumor Model

Coupling the lytic function of an oncolytic adenovirus with its ability as a transgene delivery system represents a powerful extension of this methodology. A clear advantage is the amplification of a therapeutic gene, as replicating vectors would be able to infect and deliver the gene of interest to neighboring cells. To increase the anti-tumor effect of an oncolytic adenovirus, we have generated an E1B 55kDa deleted oncolytic adenoviral vector, YKL-GB that expresses both GM-CSF and B7-1. The therapeutic efficacy of YKL-GB adenovirus was evaluated in immunocompetent mice bearing murine melanoma B16-F10 tumors. Significant inhibition of tumor growth was seen in mice treated with YKL-GB compared to those treated with the analogous vector, YKL-1. Moreover, YKL-GB oncolytic adenovirus demonstrated enhanced anti-tumor activity and higher incidences of tumor regression compared to a replication-incompetent adenovirus, dl-GB, which co-expresses GM-CSF and B7-1. Localized GM-CSF and B7-1 gene transfer also conferred long-lasting immunity against a tumor re-challenge. To establish that the observed anti- tumor effect is associated with the generation of a tumor-specific immune response, we examined the cytolytic activity using an IFN-γ ELISpot assay. We observed that YKL-GB induced significantly higher T cell-mediated anti-tumor activity than YKL-1. Furthermore, immunohistochemical studies demonstrated robust DCs and CD4+/ CD8+ T-cell infiltration in these mice compared to the YKL-1-treated groups. In agreement with these results, splenocytes from tumor-bearing mice treated with YKL-GB expressed high levels of the co-stimulatory and activation molecules. These findings demonstrate the effectiveness of enhancing the immune response against tumors with an oncolytic adenovirus expressing both GM-CSF and B7-1 and provide a potential therapeutic strategy for the management of neoplasia.

Adenoviral-mediated transfer of human wild-type p53, GM-CSF and B7-1 genes results in growth suppression and autologous anti-tumor cytotoxicity of multiple myeloma cells in vitro.

Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.

Versatile Prostate Cancer Treatment with Inducible Caspase and Interleukin-12

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12-containing apoptotic, but not necrotic TRAMP-C2 cell-based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of gamma-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1- and IL-12-containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1- and IL-12-containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.

702. Concurrent Delivery of GM-CSF and B7-1 Using an Oncolytic Adenovirus Elicits Potent Anti-Tumor Effect on the Growth Established B16-F10 Tumor Model

Oncolytic adenoviral vectors are currently being developed as biological anti-cancer therapeutic agents. Coupling the lytic function of oncolytic adenovirus with virus-mediated transgene delivery represents a powerful extension of this methodolgy. To increase the potential anti-tumor activity of oncolytic adenovirus, we constructed an E1B 55kDa deleted oncolytic adenoviral vector, YKL-GB, which expresses both GM-CSF and B7-1. The therapeutic efficacy of YKL-GB adenovirus was evaluated in immunocompetent mice bearing murine melanoma B16-F10 tumors. Significant inhibition of tumor growth was seen in mice treated with YKL-GB compared to those treated with an analogous vector, YKL-1, that lacks GM-CSF and B7-1. Moreover, YKL-GB oncolytic adenovirus demonstrated enhanced anti-tumor activity and a higher incidence of complete tumor regression compared to a replication-incompetent adenovirus, dl-GB, which co-expresses the GM-CSF and B7-1. Localized GM-CSF and B7-1 gene transfer also conferred long-lasting immunity against tumor rechallenge. To establish that anti-tumor effect is associated with the generation of tumor-specific immune response, we examined the cytolytic activity by IFN-|[gamma]| ELISpot assay. We observed that YKL-GB induced significantly higher T cell-mediated antitumor activity than YKL-1. Furthermore, immunohistochemical studies demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 groups. In agreement with these results, splenocytes from tumor-bearing mice treated with YKL-GB expressed high levels of costimulatory and activation molecules. In conclusion, the findings of this study demonstrate the effectiveness of augmenting the immune response against tumors with oncolytic adenovirus expressing both GM-CSF and B7-1, which provide a potential therapeutic strategy for the management of neoplasia.