Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumorassociated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virusmediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis. (Cancer Res 2005; 65(15): 6901-9)

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF)^secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Experimental Design: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a solubleVEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory Tcells were quantified by their CD4 + CD25hi and CD4 + FoxP3 + phenotype. Results: The present study established that GM-CSF^secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4 + and CD8 + tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory Tcells in the tumor, suggesting a novel mechanism ofVEGF-mediated immune suppression at the tumor site. Conclusion: GM-CSF^secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF)^secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1blockade, which has been shown to augment cellular immune responses. Experimental Design: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF^secret-ing tumor cell immunotherapy was administered s.c. and the anti^PD-1 antibody was administered i.p. Results:The studies reported here show that combining PD-1blockade with GM-CSF^secret-ing tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8 + T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specificT-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. Conclusion:The combination of PD-1 blockade with GM-CSF^secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti^PD-1antibody in subsequent immunotherapy cycles was required to reactivate theseT-cell responses.

Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer. [Cancer Res 2007;67(9):4399-407]