The role of FAPa-expressing stromal cells in therapeutic vaccination in a mouse model of pancreatic ductal adenocarcinoma. (360G-Wellcome-085748_Z_08_A)

Mesothelin as a potential target for immunotherapy- project aim 1 The fir.;t objective of this project is defining an antigen that could be used in the vaccine. Several TAA have already been described to be uniquely expressed in human pancreatic cancer and not in the normal tissue. On~ of the most promising candidates is mesothelin - GPI anchored differentiation antigen that was found in all mesotheliomas an~ pancreatic aden9carcinomas (22), and approximately 70% of ovarian cancer.; and 50% of lung adenocarcinomas [23]. Additionally, mesothelin expression was restricted to pancreatic adenocarcinomas, whereas adjacent normal pancreas did not stain for mesothelin. Mesothelin as an antigen has already been used in allogeneic granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumour vaccines to treat patients with surgically resected adenocarcinoma of the pancreas (24). 3 out of 14 patients developed post-vaccination delayed-type hypersensitivity (OTH) response to autologous tumour, which was associated with prolonged survival. To find whether mesothelin is also expressed in KPC mouse model we isolated RNA from lung, pancreas from healthy mice and POA from tumour-bearing animals. Our preliminary qPCR data suggest that 3 out of 5 tumour.; that were examined showed high mesothelin RNA level (Fig. 3). In order to verify this result on protein level tumour sections will be stained with anti-mesothelin antibody. However, as there are few antibodies (Ab) directed against mouse mesothelin, it is required to screen commercially available Ab and validate then on western blot. Preliminarily, cancer cell lines isolated from PDA tumours can be used for those tests. Cancer/Testis antigens as potential targets for immunotherapy- project aim la (contingency plan) Mesothelin is not the only potential PDA-associated antigen. Cancer/testis antigens (CTA) are immunogenic molecules expressed in normal tissues, but restricted to placenta, testis, fetal ovary, germ cells and placenta, as well as in a wide variety of tumours. There are approximately 150 CT As identified to date (www.cta.lncc.br) strictly regulated by epigenetic mechanisms. Vaccines containing the CT antigens- MAGEA and NY-ES0-1/CTAGIB- are utilized in patients with melanoma as well as lung and ovary cancers. Generation of viral vectors for immunization- Vaccinia-project aim 2 In this project, to mount an effective anti-tumour T cell response, a vaccine strategy utilizing ~ poxvirus as TAA delivery vehicles in combination with T -cell costimulatory molecules will be introduced. The attenuated strain of Modified Vaccinia virus Ankara (MV A) is widely used. as a safe non-replicating recombinant vaccine vector in humans and other mammals. Among the first TAA successfully used for immunization with VV are carcinoembrionic antigen (25), prostate-specific antigen [26] and many melanoma-associated antigens. Generation of viral vectors for immunization-gamma-herpesvirus- project aim 2 Herpesviruses persist latently in the infected host indefinitely effectively evading the immune response. Sporadic reactivations lead to release of infectious virions and facilitate spreading to new host. As y-herpesvirus (?HV) reactivation is only observed in immunosuppressive conditions, it suggests that immune system is able to control the infection. It was demonstrated that more potent antiviral response mediated by antigen-specific COS+ T cells was found in persistent ?HV infection [27)[28). Moreover, during latent infection with ?HV -68 increased turnover rate of CD8+ T cells indicates that these cells are restimulated with antigen. Using recombinant methodology sequence of the antigen of interest will be inserted in place of ovalbumin in both vectors. Infection of LL20VA-bearing mice with ?HV -OVA maintained high level of SIINFEKL-specific CD8+ T cells (data unpublished), which controlled tumour growth when F APa ablation was performed. Assessment of immune response to vaccines project aim 3 Before performing therapeutic vaccination on tumour-bearing mice it is necessary to evaluate the effect of immunization. Cellular response will be assessed by performing killing assay or by studying the profile of major cytokines (IL-2, IL-4, IL-10 and IFN-y). In order to evaluate CDS+ T cell killing antigen-expressing cell line with matching MHC will be used and chromium release will be monitored to detect lysis. EVALUATION OF TUMOUR GROWTH- PROJECT AIM 4 For the purpose of measuring tumour growth, tumour volume will be quantified by 30 ultrasonography [ 17]. 30 ultrasound provides a new method for the objective quantitative assessment of stromal echogenicity and vascularity as well as blood flow within the tumour. Moreover, the biggest advantage of this technique is its non-invasiveness, which enables assessment of the effects of therapy without sacrificing animals. This will be done in collaboration with Tuveson's lab.