Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5’s wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17–71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17–71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17–71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins.
Osteosarcoma is one among the most common neoplasms in dogs. Current treatments show limited efficacy and fail to prevent metastasis. Conditionally replicative adenoviruses (CRAd) replicate exclusively in targeted tumor cells and release new virus particles to infect additional cells. We proposed that OC-CAVE1 (CAV2 with the E1A promoter replaced with the osteocalcin promotor) may also enhance existing immunity against tumors by overcoming immune tolerance via exposure of new epitopes and cytokine signaling. Eleven client-owned dogs with spontaneously occurring osteosarcomas were enrolled in a pilot study. All dogs were injected with OC-CAVE1 following amputation of the affected limb or limb-sparing surgery. Dogs were monitored for viremia and viral shedding. There was minimal virus shedding in urine and feces by the 6th day and no virus was present in blood after 4 weeks. CAV-2 antibody-titers increased in all of the patients, post-CRAd injection. Immunological assays were performed to monitor 1) humoral response against tumors, 2) levels of circulatory CD11c + cells, 3) levels of regulatory T cells, and 4) cytotoxic activity of tumor specific T cells against autologous tumor cells between pre-CRAd administration and 4 weeks post-CRAd administration samples. Administration of the CRAd OC-CAVE1 resulted in alteration of some immune response parameters but did not appear to result in increased survival duration. However, 2 dogs in the study achieved survival times in excess of 1 year. Weak replication of OC-CAVE1 in metastatic cells and delay of chemotherapy following CRAd treatment may contribute to the lack of immune response and improvement in survival time of the clinical patients.
S206 high titer (1,000vp/cell) in an in vitro assay. To look at the efficacy of the O-Ad and CART combination, we performed a killing assay. O-Ad clearly enhanced killing by CART cells in luciferase based assay. We also used xCELLigence real time cell analyzer (RTCA) for kinetic analysis of killing. In combination with O-Ad, more rapid killing kinetics by CART cells were observed especially in lower E:T ratio. To look at the impact of the combination in vivo, subcutaneous ASPC1-CBG-GFP in NSG mice were treated with CART alone or in combination with intra-tumoral injection of O-Ad. O-Ad showed significant synergy with CART in luciferase based photon assay. And higher number of T cells was observed in spleen in the O-Ad armed with IL2 combination group. Conclusions: These results suggest that combination therapy of O-Ad armed with cytokine(s) and CART cells is effective against solid tumors by enhancing T cell activity.
Background: Osteosarcoma is one among the most common neoplasms in dogs. Current treatments include leg-amputation or limb-sparing techniques along with chemotherapy that show limited efficacy and fail to prevent metastasis. Conditionally replicative adenoviruses (CRAd) are gene therapy tools that replicate exclusively in targeted tumor cells, causing a cytopathic effect and in turn release of new virus particles to infect additional cells. We proposed that OC-CAVE1 (CAV2 with the E1A promoter replaced with the osteocalcin promotor) may also enhance existing immunity against tumors by overcoming immune tolerance via exposure of new epitopes and cytokine signaling. A previous study indicated administration of OC-CAVE1 was safe and showed specific replication in osteosarcoma cells. Results: We enrolled eleven client-owned dogs with spontaneously occurring osteosarcomas in a pilot clinical-trial. All dogs were injected with OC-CAVE1 following amputation of the affected limb or limb-sparing surgery. Dogs were monitored for viremia and viral shedding. There was minimal virus shedding in urine and feces by the 6th day and no virus was present in blood after 4 weeks. CAV-2 antibody-titers increased in all of the patients, post-CRAd injection. OC-CAVE1 injections did not result in a statistically significant increase in life span, although 2 dogs in the study achieved survival times in excess of 1 year. Immunological assays were performed to monitor 1) humoral response against tumors, 2) levels of circulatory CD11c+ cells, 3) levels of regulatory T cells, and 4) cytotoxic activity of tumor specific T cells against autologous tumor cells between pre-CRAd administration and 4 weeks post-CRAd administration samples. Western blots indicated an increased humoral immune response against pre-existing antigens in some dogs, but flow cytometry did not support this apparent increase. Cytotoxic T-cell activity did not increase in a consistent pattern. Levels of circulatory regulatory T-cells did decrease statistically in all patients. Conclusions: Administration of the CRAd OC-CAVE1 resulted in alteration of some immune response parameters but did not appear to result in increased survival duration. Weak replication of OC-CAVE1 in metastatic cells and delay of chemotherapy following CRAd treatment may contribute to the lack of improvement in survival time of the clinical patients.
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