Prostate cancer (PC) is the most common visceral cancer amongst men in Australia and elsewhere in the world. The American Cancer Society estimated that in 2006, PC alone would account for about 33% of newly diagnosed cancer in men, and be responsible for 9% of cancer related deaths in men. In men with advanced, metastatic PC, hormone therapy is widely accepted as the treatment of choice and produces good initial responses in most patients. However, many patients will relapse and become resistant to further hormone manipulation. Currently used treatment modalities for these patients are intended to palliate symptoms and therefore improve quality of life; the long term survival benefit of currently used management strategies is marginal at best. The limited treatment modalities with survival benefit for patients with advanced PC results in the need for development of novel therapies. Enhancement of the natural anti-tumour defences of the immune system to recognise and destroy tumour cells appears as a favourable alternative to prior therapies. Use of autologous dendritic cells (DC) as stimulators of an anti-tumour response has shown promise in several phase I, II and III trials. The Mater Medical Research Institute has developed a novel system for the isolation of blood DC (BDC). This system utilizes an antibody selection system based on a human/mouse chimeric CMRF-56 (huCMRF-56) monoclonal antibody (mAb) which has been engineered from the prototype murine IgG1 CMRF-56 mAb. Pre-clinical studies have demonstrated that the huCMRF-56 mAb isolates a CMRF-56+ cell population comparable to that obtained with the murine IgG1 CMRF-56 mAb with a sufficient CMRF-56+ BDC purity and yield to warrant its use as a BDC based immunotherapy. The objective of this research project was to validate the use of the huCMRF-56 mAb isolated BDC preparations in PC immunotherapy. This was achieved by; i) determining the optimal concentration of the huCMRF-56 mAb at which to perform isolations in order to obtain a CMRF-56+ cellular preparation with purity, yield and cellular composition, in terms of DC subsets, B cells, monocytes and contaminating cells, comparable to that obtained with the murine mAb, which has shown efficacy in in vitro studies; ii) Demonstrating that CMRF-56+ preparations obtained from PC patients using the huCMRF-56 BDC mAb are comparable to those from healthy donors (HD) and iii) demonstrating functional capacity of both freshly isolated and cryopreserved CMRF-56+ BDC isolated preparations to induce anti-tumour cytotoxic T lymphocyte (CTL) responses in both HD and PC patients. These studies utilised the appropriate immunostaining techniques and flow cytometry to determine the CMRF-56+ cell yield, CMRF-56+ BDC purity and viability of the preparation. To determine functional capacity CMRF-56+ preparations were isolated from HD and PC patients, and loaded with the prostate tumour associated antigens (TAA) and control antigen peptides: prostate specific antigen-3 (PSA-3), prostatic acid phosphatase- 5 (PAP-5), prostatic specific membrane antigen-2 (PSMA-2), flu matrix protein (FMP) and/or melanoma antigen recognised by T cells (MART). Co-culture experiments were set up with autologous peripheral blood mononuclear cells (PBMC) and induction of CTL responses were assessed using pentamer staining and ELISPOT assay. The working concentration of the huCMRF-56 mAb of 1.28mg/ml was determined to immunoselect a cellular preparation with maximal BDC purity, BDC yield and total viable cell number. Phenotyping studies of this preparation demonstrated it to be predominately comprised of antigen presenting cells (APC) and enriched for BDC with several BDC subsets immunoselected. CMRF-56+ preparations immunoselected from HD and PC donors were similar and comparable to previously described preparations immunoselected using the original murine CMRF-56 mAb. From antigen loaded CMRF-56+ preparations, specific major histocompatibility complex (MHC) class 1 restricted CD8 lymphocyte responses were generated against prostate TAAs in 2 of 5 HD and 1of 3 PC donors and against FMP in 1 of 3 PC and 5 of 5 HD. Cryopreserved antigen loaded CMRF-56+ BDC preparations from HD generated antigen specific FMP or MART-1 CTL responses in 3 of 4 HD, however anti-prostate TAA CTL responses were not observed. The humanised CMRF-56 mAb immunoselects a cellular preparation enriched in BDC and capable of effective uptake and presentation of antigen. The CMRF-56+ BDC enriched preparation, loaded with PC TAA is capable of inducing specific anti tumour responses in vitro. While further preclinical studies are required, the preparation, loaded with PC TAA shows promise as a potential immunotherapy for PC.
| Identifer | oai:union.ndltd.org:ADTP/279377 |
| Creators | Robert Coleman |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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