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Documents and Publications  >  report  >  PhD SCIENCE REPORT ISABEL P...




I have been registered as a PhD student at the Karolinska Institute on the 31th of May, 2007 and expect to finish my PhD in June 2011.

Project 1 - Preparation and immunomontioring of a clinical trial in patients with advanced melanoma ? collaboration with Carl Figdor
Patients will receive a DC vaccination regimen (3 vaccinations at weekly intervals) followed by 3 adoptive transfers of autologous in vitro expanded T cells. This novel approach combines in vivo priming by the DC vaccine with passive immunotherapy by lymphocyte infusion.
Patients will undergo surgical removal of a metastatic lesion and leukapheresis at the beginning of the trial to provide sources for tumor antigens as well as monocytes and T cells, which will be isolated from the leukapheresis product by elutriation. Patients will undergo lymphodepletion prior to vaccination and prior to adoptive transfer of autologous T cells. The DC vaccine will consist of monocyte derived DC loaded with tumor lysate generated from the excised lesion. The T cell transfer will consist of T cells expanded by co-culture with tumor lysate loaded DC.
The study protocol has been reviewed by the local ethical committee and is now ready to be submitted to Läkemedelsverket, the Swedish regulatory authority for clinical trials. I am involved in the planning of the protocol and in the design of immune-monitoring experiments, including setup of the necessary techniques (Elispot, multi-parametric Flow Cytometry, Elisa) in the lab. Upon approval by swedish authorities we plan to include, treat and evaluate 10 patients during a period of one to two years.

Project 2 – Characterization of myeloid derived suppressor cells patients with cancer
Accumulation of myeloid derived suppressor cells (MDSC) with the ability to suppress T cells has been observed in serveral types of cancer, including melanoma.
Using blood from melanoma patients I am investigating phenotypic markers that can be used to further characterize MDSC, focusing on molecules that are related to their suppressive function. As it is believed that recruitment from the bone marrow by tumor derived factors leads to increased presence of immature myeloid cells in the blood, I am testing substances that that could induce maturation and thereby turn immature suppressor cells into mature cells capable of efficient antigen presentation.
So far I have collected blood from >15 melanoma patients and compared the MDSC phenotype and function with healthy donor derived cells. There are some candidate suppressive mechanisms that will be studied in more detail
Using some model substances I am testing a group of drugs for its ability to induce MDSC maturation. I am also interested in studying the effect of MDSC on other immune cells.
Most of these experiments rely on multiparametric flow cytometry to phenotypically characterize the MDSC, study suppression of T cells proliferation and cytokine production as well intracellular signalling molecules. In the future we plan to run a gene-microarray or potentially a phospoproteomcis approach to discover further important molecules.
Recently a new clinical collaboration was established that will allow us to collect fresh melanoma tumors. This will allow us to study MDSC phenotype and function also in the tumor environment.

b) Ovarian cancer
Currently there is no knowledge about the relevance of MDSC in ovarian cancer patients. However, it is known that both Tregs and suppressive macrophages are increased in this maligancy and have been suggested to correlate with worse prognosis. I am collecting blood and ascitic fluid of ovarian cancer patients to study the presence of immature myeloid populations as compared to healthy donor blood. I have identified two candidate populations that will be tested for the ability to suppress T cells. I also aim to characterize these cells phenotypically and will study potential suppressive mechanisms. Collection of material for this project is ongoing.

Project 3 - Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas
Our lab has a long standing interest in optimizing lymphocytes to achieve improved tumor-reactivity.
In a collaboration with Dr. Charo at the Max Delbrück Center for Molecular Medicine in Berlin, Germany human primary NK cells were engineered to recognize Her2-expressing target cells in addition to their natural activity. For this project I have isolated and provided material from patients with ovarian cancer. During a visit to Dr. Charos lab A. Kruschinski and I have isolated and transduced healthy donor and patient NK cells and tested their activity against autologous and allogeneic tumor cells isolated from ascitic fluid of several ovarian cancer patients. We found that NK cells transduced with a chimeric Her2-specific receptor could recognize freshly isolated tumor cells as well as tumor cell lines. Recognition was dependent on the level of Her2 expression of the target cells and Her2 negative target cells or control-transduced NK cells yielded negative results. This collaboration has resulted in a publication in PNAS.

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