Characterization of antitumor immunization to a defined melanoma antigen using genetically engineered murine dendritic cells

Abstract

A murine model of dendritic cell (DC)-based genetic immunization to a defined human melanoma antigen (Ag), MART-1/Melan-A (MART-1), was developed. The MART-1 gene was stably transfected into the nonimmunogenic mouse fibrosarcoma cell line NFSA that is syngeneic in C3Hf/Sem/Kam (C3H, H-2 k ) mice to generate the NFSA(MART1) cell line. In vivo protection from a lethal NFSA(MART1) tumor challenge could be generated by DCs transduced with a recombinant adenovirus (AdV) vector expressing MART-1 (AdVMART1). This model has the following characteristics: (a) immunological specificity and memory, (b) comparable protection for varying transduction multiplicities of infection, cell doses, and sites of DC inoculation but, interestingly, worse protection with increasing numbers of vaccinations, (c) the ability to treat small established tumors, (d) an absolute requirement for CD8 and CD4 T cells, (e) generation of MART-1-specific splenic cytotoxic T lymphocytes, and (f) up-regulation of both T helper type 1 and T helper type 2 cytokines. Genetically engineered DCs presenting defined tumor Ags represent an attractive method to generate effective immune responses.

Gene transduction efficiency and maturation status in mouse bone marrow-derived dendritic cells infected with conventional or RGD fiber-mutant adenovirus vectors

“…This method takes advantage of the natural properties of DCs as optimal immunological adjuvants and uses endogenous antigen gene expression to provide continuously antigenic epitopes for both MHC class Iand II-restricted expression. 7,8 Furthermore, gene transduction into DCs is an attractive technology for developing effective DC-based immunotherapy, because functional modification of DCs by cytokine genes or costimulatory molecule genes has already been demonstrated. [9][10][11][12][13] Since the efficiency of foreign gene transduction into DCs is very low, even when an adenovirus vector (Ad) is used, the establishment of a new vector system that can improve gene transduction into DCs is desirable.…”

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

“…The development of DC from murine bone marrow (BM) progenitor cells was performed as previously published [38,39,42,43]. BM cells were cultured overnight in RPMI 1640 (Life Technologies, Gaithersburg, Md) with 10% FCS, 1% penicillin, streptomycin, and amphotericin (Complete medium, CM) in a Petri dish.…”

Natural killer cells play a critical role in the immune response following immunization with melanoma-antigen-engineered dendritic cells

“…The construction and characterization of these vectors has been described previously. 35,38 The transgenes are driven by the human cytomegalovirus (CMV) early promoter/enhancer.…”

“…B16 cells used for tumor challenge were obtained from single-cell suspensions of progressively growing tumors in syngeneic mice to avoid the confounding effects of the presentation of media-and serum-derived epitopes. 34,35,38 Cell suspensions were washed extensively and injected into mice in a final volume of 0.2 ml of PBS/animal. Each treatment group typically contained five mice for in vivo tumor challenge studies, and one additional mouse per group to obtain splenocytes for in vitro studies (ELISPOT, 51 Cr release assays).…”

“…In this model, murine DC gene-modified to express MART-1 using a replication-incompetent adenoviral vector (Ad-VMART1/DC) protect syngeneic mice form a MART-1 positive B16 melanoma tumor challenge. This model has the following characteristics: AdVMART1/DC immunization in immunologically intact mice generates class I-restricted cytotoxic T-cell responses, 34,35 with a predominantly Th1 phenotype, 27,34-36 not as a result of cross-presentation, 34 in which multiple, shortly spaced immunizations results in decreased protection due to fasL/fas-dependent activation-induced cell death. 37 Cotransduction of MART-engineered DC with adenoviral vectors expressing cytokine genes IL2 and IL7 did not enhance protection, while co-transduction with an IL12 adenoviral vector had an adverse impact on protection.…”