Genetic engineering of TNF family protein-based vaccines for antitumor immunotherapy
The interaction between tumor cells and dendritic cells (DC) is a critical event for both initiation and regulation of specific antitumor immune responses. Based on the unique ability to present tumor antigen to and stimulate clonal expansion of naïve T cells, DC have been widely used to induce antitumor immunity in both preclinical animal models and human clinical trials. However, a growing body of clinical data and experimental evidence demonstrate that tumor exhibits a variety of inhibitory effects on the immune system and the DC system, in particular. It is well documented to date that tumor-derived factors influence DC generation, maturation, activity, and survival both in vitro and in vivo. Therefore, DC dysfunction in the tumor-bearing hosts could be largely responsible for the ability of tumor cells to escape from immune recognition or induce tumor specific tolerance. However, understanding the suppressive effect of tumor on the immune system, in particular on DC, and the protection of DC from tumor-induced dysfunction has not been studied. Therefore, the goals of this research project were (i) the understanding of DC immunobiology in the tumor microenvironment, revealing cellular and molecular mechanisms involved in tumor escape from immune recognition and elimination, (ii) the generation of novel antitumor vaccines which protect DC from tumor-mediated suppression, and (iii) the identification of primary mechanisms involved in vaccine-mediated antitumor immunity. The Tumor Necrosis Factor Ligand (TNFL) family is a group of cytokines, which through the interaction with corresponding receptors, regulate cell functions and activities. In this project we had examined three proteins which belong to the TNFL family: CD40L, RANKL, and 4-1BBL. Importantly, the receptors for these cytokines are found on activated DC. CD40L, RANKL and 4-1BBL were shown to influence DC activation and cytokine secretion. It also has been demonstrated that CD40L and 4-1BBL have a strong antitumor effect, whereas, the role of RANKL in antitumor immunity has not yet been examined. CD40L is a well studied cytokine, shown to play an important role in the development of both humoral and cell-mediated immunity. However, the mechanisms of action of all these three cytokines on immune effectors are not fully understood. Here, we have evaluated the ability of local adenoviral gene transfer of CD40L, RANKL and 4-1BBL to elicit an antitumor immune response to established tumors in mice. Adenoviruses encoding these genes (Ad-CD40L, Ad-RANKL and Ad-4-1BBL) were constructed and tested in murine MC38 colon and TS/A breast adenocarcinoma therapy models. Our results demonstrate that intratumoral administration of all three tested vectors resulted in a significant inhibition of MC38 and TS/A tumor growth when compared with control groups treated with either saline or control adenovirus. In addition, a single intratumoral injection of DC transduced with the adenoviral vectors also resulted in a significant inhibition of MC38 and TS/A tumor growth. Furthermore, treatment of TS/A tumors with DC transduced with Ad-CD40L induced a complete tumor rejection with the generation of a tumor-specific immune memory. Thus, these results demonstrate that DC genetically modified to express CD40L immunotherapy displays the strongest antitumor effect compare to Ad-CD40L or RANKL- and 4-1BBL-based therapeutic approaches. Next, we have observed that DC generated from tumor-bearing mice, in addition to expressing low levels of CD80 and CD86 and producing decreased amounts of IL-12, exhibit decreased expression of CD40 molecules. Moreover, we have detected that MC38 tumor suppressed CD40 expression on DC isolated from spleens of tumor bearers. These data suggest that tumors induce suppression of CD40 expression on DC, and, thus, result in dysfunction and inhibition of maturation of these cells. Subsequently, we have demonstrated that CD40L, in addition to generating a strong antitumor immunity, was able to protect DC from tumor-induced dysfunction. CD40L stimulates DC to express higher levels of co-stimulatory molecules, produce significantly higher levels of IL-12 protein, survive longer in cultures, efficiently stimulate T cells, induce high cytotoxic T lymphocyte activity, present tumor antigens to T cells more efficiently, and migrate to the lymphoid organs faster then control DC. We have shown that by up-regulating DC activity and function, CD40L rescues DC from tumor-induced suppression. In summary, our data demonstrate that CD40L-based immunotherapy is an effective approach for inducing antitumor immunity and rescuing DC from tumor-induced dysfunction. These results should guide the development of novel therapies for prevention of immunosuppression in cancer patients and design of novel effective immunotherapeutic strategies for cancer.
Advisor:Paul D. Robbins; Johnny Huard; William J. Federspiel; Daniel L. Farkas
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:02/02/2004