Genetic Engineering of T Lymphocytes for Cancer Immunotherapy Optimisation of Gene Transfer Thesis for M. Sc. degree in laboratory science within the programme of Biomedical Laboratory Sciences June 2006 Camilla Lindqvist Supervisor: Angelica Loskog, Ph.D. Division of Clinical Immunology, Uppsala University Abstract T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer. Keywords Chimeric receptor, tumour, retroviral transduction, plasmid transfection, retroviral enhancers Sammanfattning Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen. 2 Abbreviations APC antigen presenting cell CTL cytotoxic T lymphocyte FACS fluorescent-activated cell sorting FBS foetal bovine serum GFP green fluorescent protein ITAM immunoreceptor tyrosine-based activation motif LTR long terminal repeat Luc Luciferase MHC major histocompatibility complex PBMC peripheral blood mononuclear cell PBS phosphate buffered saline PEST penicillin-streptomycin PLL poly-L-lysine Polybrene 1,5-dimethyl-1,5diazaundecamethylene polymethobromide RN retronectin TCR T cell receptor Introduction Cancer is one of today’s most prevalent diseases with almost 50 000 cases being diagnosed in Sweden every year 1. It is the second most common cause of death (after ischemic heart disease) in developed countries, accounting for about 23% of all mortality2. The risk of developing cancer increases by age and for individuals over 75 the risk is almost 30% (statistics for Sweden)1. There are about 200 different types of cancers, where prostate cancer and breast cancer is the most common in men and women, respectively3. Cancer arises from the uncontrolled proliferation of transformed endogenous cells. The growth of malignant tumours is largely dependent on the tumour cells’ proliferative capacity and the ability of these cells to invade surrounding tissues and metastasise to distant sites. Each neoplastic cell has an alteration in its genome that is responsible for its abnormal growth. The four main genetic mechanisms that are believed to transform cells into neoplasms are expression of oncogenes (stimulates growth), loss of activity of tumour suppressor genes or anti-oncogenes (inhibit cell growth), over-expression of genes whose products prevent normal cell death and/or loss of activity of genes that under normal circumstances repair damaged DNA. Cells normally need to have more than one of these genetical errors to be transformed into cancer cells. Many tumour cells express antigens on their surface that triggers the immune system. They can either be tumour specific, i.e. they are only expressed on the cancer cells, or are tumour associated, i.e. the antigens are found on other cells than the tumour, but are over-expressed by the cancer cells. These antigens enable the immune system to recognize the tumour while ignoring adjacent normal cells. The principal mechanism of tumour immunity is killing of tumour cells by CD8+ cytotoxic T lymphocytes (CTLs). CTLs recognize antigens presented to them by the class I major histocompatibility complex (MHC) molecule on antigen presenting cells (APCs) such as dentritic cells (DCs). However, the DCs need to be activated in order to stimulate the CTLs. Upon DC activation they express MHC I together with co stimulatory molecules. In absence of co stimulation, the CTLs become anergic or inactive. Since the tumour cells may down regulate MHC I or have other defects in the antigen 3 presentation pathway, they may be poor targets for CTLs. Except down regulation of antigen presentation they may secrete growth factors for T regulatory cells and/or secrete inhibitory proteins. T regulatory cells and inhibitory proteins can directly suppress the antitumour reactivity of CTLs. To defeat cancer, all the malignant tumour cells need to be eliminated. There are curative treatments for some of the different cancer types, providing the disease is detected early. The relative five-year-survival in Sweden are approximately 60%3. Surgery is the most commonly used therapy against solid tumours. It can be used as a single therapy or in combination with radiation therapy and/or chemotherapy. Most patients that are cured from cancer disease are so by this method. Even though surgery treats many patients, it is an invasive procedure that always involves a risk. Furthermore, healthy tissues surrounding the tumour usually need to be removed as well. Radiation therapy uses a method were X-rays creates free radicals in the irradiated tissue. This damages the DNA of cells and hence their ability to replicate. Tumour cells have a less efficient repair system than healthy cells, which often results in the death of the tumour, while healthy cells may survive. Even though this therapy seems to be efficient for killing cancer cells, it requires high specificity. Furthermore, cancer therapy gives severe side effects, some of which are alopecia (hair loss), skin reactions and nausea (early side effects). Among some of the later side effects are secondary malignancies and impaired fertility or sterility at worst. Chemotherapy functions by disturbing the cell replication by targeting the DNA and/or transport systems within the cell. It is most efficient on rapidly dividing cells, which is well concordant with cancer cells. Unfortunately, many of the bodies own cells are also rapidly dividing, e.g. blood cells, hair and epithelia, and the chemotherapeutic drugs cannot distinguish between healthy cells and cancer cells. This results in side effects such as alopecia, nausea, anaemia, thrombocytopenia and also an increased sensitivity for infections due to the decrease of leukocytes. One of the biggest problems with the current therapies against cancer is that they are not specific enough. Both radiation therapy and chemotherapy affects healthy cells as well as cancer cells, which leads to the many side effects that can be seen within these therapies. An alternative approach for cancer therapy is to re-educate the body’s own immune system to defeat cancer cells. Immunotherapy may decrease the side effects due to its higher specificity. Several strategies have been evaluated within this field and there are several novel therapies that are currently being clinically explored. Among these are e.g. adoptive T cell therapy where a biopsy from the patient’s tumour are excised and the tumour infiltrating lymphocytes (TILs) are extracted. Cells with high avidity to the tumour are selected and expanded. For the tumour specific cells to be effective, the patient need to undergo immunosupression before being re-infused with high numbers of the tumour specific cells4. The immunosupression creates a space in the lymphocyte population that allows the transferred cells to proliferate4. Furthermore, it has been shown that there are a subpopulation of CD4+ cells, CD4+ CD25+ (and according to more recent findings FOXp3+ 5) that are the so called T regulatory cells and these are believed to protect the body against self reactive immune responses6. The adoptive transfer of expanded autologous tumour reactive T lymphocytes has been successful in the regression of e.g. metastatic melanoma. To enhance these effects the T regulatory cells need to be depleted prior therapy. This can be done by a general immunosupression drug, but as a side effect of this the patients developed different autoimmune diseases7. For this treatment to be effective, the patient also needs to receive high doses of IL-2, the principal growth factor for T cells, which is known to be toxic in high doses8. Furthermore, T regulatory cells also 4 thrive upon IL-2 administration9. Instead of finding means to deplete T regulatory cells the cytotoxic T lymphocytes can be genetically engineered to resist the negative actions of the T regulatory cells. The first signal for T lymphocyte activation is provided to the cell by antigen recognition by the T cell receptor (TCR) binding to the MHC molecule. The second signal is from binding of costimulatory molecules which function together with the TCR to activate the T cell.