Determining the immunologic response created toward peptide mimic and carbohydrate vaccines for the TF-antigen
Masters, April Rose
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Tumor antigens are selected for vaccine development based on characteristics such as their presence on tumor tissue, absence on normal tissue and the role which they play in tumor growth and development. The Thomsen-Friedenreich antigen (TF-Ag) is a tumor associated carbohydrate antigen that is an excellent target for anti-cancer vaccine development due to its wide spread prevalence on carcinomas, absence or inaccessibility on normal tissue and its role in the process of tumor cell adhesion and metastasis. In passive transfer studies as proof of principle for the potential efficacy of an immune response to TF-Ag, our monoclonal antibody to the TF-Ag, JAA-F11, has been shown to improve survival and decrease lung metastasis in a mouse metastatic breast cancer model. Making antibodies to the TF-Ag has shown not to be harmful to patients, due to the existence of naturally occurring low levels of antibodies to TF-Ag in human sera. Having higher levels of these naturally occurring antibodies to the TF-Ag has actually correlated with improved prognosis for cancer patients. Since the TF-Ag is a carbohydrate antigen it is difficult to generate long lasting protection from vaccination. Our lab has used various vaccine strategies to enhance the immune response created toward the TF-Ag. One vaccine design used in our laboratory involved the use of TF-Ag peptide mimics. Peptide mimics are protein sequences with similar shape as a carbohydrate, which fool the immune system into making a protein type response to a carbohydrate antigen like the TF-Ag. The TF-Ag peptide mimics and carbohydrate forms were used to create six vaccines; the constructs are 1-4) four different TF-Ag peptide mimics attached to the carrier protein bovine serum albumin (BSA), 5) TF-Ag linked to BSA and 6) asialoglycophorin (naturally occurring form of the TF-Ag). Each of these constructs was attached to tanned red blood cells as a carrier to increase the contact of the vaccine with certain immune cells involved in producing a lasting response located in spleen. Three groups of control animals were also vaccinated; one with PBS alone, one with BSA-tanned red blood cells and one with a control OVA peptide-BSA on the tanned red blood cells. Three additional mice were vaccinated intradermally with asialoglycophorin alone. This study was repeated with variations three times with tumor challenge and once without. These studies showed that one of the peptide mimic vaccines and the carbohydrate vaccines could produce protection in mice against breast cancer and prolong their survival compared to the controls. The other vaccine design consisted of two different TF-Ag-gold nanoparticle vaccine constructs with two associated controls. A stimulatory protein was also attached to the gold nanoparticle vaccine constructs to enhance the immune response made toward the TF-Ag along with another cancer antigen MUC4. Vaccination with one of these gold nanoparticle constructs improved the survival of the mice in a breast cancer tumor challenge. Although protection was seen using both vaccine designs the amount of antibody formed did not seem to be sufficient to cause the protective response. Thus these studies were undertaken to understand the mechanism of protection. Both humoral (antibody mediated) and cellular immunity were analyzed. The number of IL-2, IL-10 and INF-γ producing cells were measured by ELISPOT to assess the cellular response occurring along with in vitro cell mediated cytotoxicity studies. Humoral immunity was analyzed by antigen specific B cell ELISPOT, ELISA, complement dependent cytotoxicity and antibody dependent cell mediated cytotoxicity. No consistently significant differences were seen between the vaccinated animal groups and the corresponding controls for any of these parameters. Further analysis must be carried out to understand why protection occurred, but these results may indicate that measuring antibody levels in human tumor vaccine trials may not be indicative of the true response, and long term outcome of the patient groups should be compared.