Dendritic cell-based vaccination: scientific background
Therapeutic dendritic cell-based vaccination of cancer patients represents one of the most promising non-toxic methods of treatment. The main goal of the vaccination is to stimulate the patient’s own immune system to combat cancer cells. DanDrit uses the patient’s own dendritic cells, taken from the blood and loaded with tumor antigens. The dendritic cells are then injected back into the cancer patient, where surface expressed antigenic peptides are recognized by T lymphocytes. T lymphocytes are stimulated by the dendritic cells to proliferate and differentiate into effecter cells that target and destroy tumor cells specifically. DanDrit’s therapeutic cancer vaccine is used either as separate treatment or complementary to other types of treatment such as surgery, chemotherapy and radiotherapy. Dendritic cells are considered to be the most efficient cells of the immune system, which are best suited to initiate an immune response against cancer cells.
The use of dendritic cells for anti-cancer immunotherapy became possible only after the development of methods to establish dendritic cell cultures from monocytes isolated from peripheral blood. The establishment of active, mature dendritic cells occurs in two steps. In the first step, two cytokines, GM-CSF (granulocyte macrophage colony stimulating factor) and IL-4, (interleukin 4) are added to monocyte cultures to transform the cells into immature dendritic cells. In the second step, maturation factors are added to stimulate further differentiation of the cells into active, mature dendritic cells. Immature dendritic cells are able to be efficiently loaded with exogenous antigens, but are unable to efficiently stimulate T lymphocytes. In contrast, mature dendritic cells can stimulate T lymphocytes but are not efficiently loaded with exogenous antigens. Therefore, tumor antigens are loaded onto dendritic cells at the immature stage.
A Dendritic cell (blue) interacting with a T-cell (gold).
An important aspect of anti-tumor immunotherapy is a selection of appropriate tumor-associated antigens for targeting. Tumor antigens may result from point mutations in genes that are expressed normally. These mutations usually affect the coding region of the gene and are unique to the tumor of an individual patient. Such antigens, which are strictly tumor-specific, may play an important role in the natural anti-tumor immune response of individual patients, but most of them cannot be easily used as immunotherapeutic targets because they are not shared by tumors from different patients. Tumor antigens may also arise by abnormal activation (expression) of normal genes in cancer cells specifically. These types of antigens are shared; they are expressed by many types of tumors in many different individuals. Shared tumor antigens are divided into three general classes. One class of shared tumor antigens, named differentiation antigens, is expressed in the normal tissue of origin of the malignancy. Antigens of this group are not tumor-specific and may not be strongly immuno-reactive. Furthermore, their use as targets for cancer immunotherapy may result in autoimmunity towards the corresponding normal tissue. A second class of shared tumor antigens is expressed in a wide variety of normal tissues and is over expressed in tumors. A low level of expression in normal tissues may permit immune activation that is tumor specific and does not result in autoimmunity. However, since these genes are expressed normally it may be difficult to induce an immune response against such antigens. The third class of shared tumor antigens corresponds to peptides encoded by "cancer-testis" genes that are expressed in many different types of tumors but not in normal tissues. The only normal cells in which significant expression of such genes has been detected are in reproductive cells. Since these cells are not accessible to the immune system such antigens can be considered as strictly tumor-specific. Vaccines that utilize cancer-testis antigens are likely to be effective for treating many different types of cancer. At DanDrit we are focusing primarily on the cancer-testis antigens in order to develop a vaccine that will be generally, and broadly, tumor specific.
MelCancerVac® – polyvalent vaccine targeting shared tumor-specific antigens
The main product of DanDrit Biotech A/S is MelCancerVac® that uses a patient’s own dendritic cells loaded with tumor antigens from the lysate of a specifically selected melanoma cell line. The melanoma cell line expresses a broad spectrum of cancer/testis antigens (in particular, the antigens of the MAGE family) and has low levels of expression of the melanocyte differentiation antigens. The original cell line used for the selection was isolated from a patient with a long-lasting disease-free period after the removal of a metastatic tumor, reflecting the natural high immunogenicity of these particular melanoma cells. The melanoma cell line has been tested for safety and was found to be free of infectious agents. The melanoma cell lysate is combined with the patient’s own dendritic cells generated by the method described above. The method of dendritic cell generation was initially optimized in order to increase the capacity of immature dendritic cells to load the exogenous antigens and present them for recognition by T lymphocytes. The method was also optimized to increase the efficiency of transformation of monocytes into dendritic cells and permits us to produce up to five portions of vaccine using only 200 ml of patient’s blood. In pre-clinical studies performed in vitro, dendritic cells loaded with melanoma cell lysate were able to stimulate the generation of cytotoxic T lymphocytes with broad anti-tumor specificity. This demonstrated that DanDrit’s dendritic cell system is capable of inducing an immune response against shared tumor-restricted antigens expressed in many types of solid tumors. The extent of tumor cell death was correlated with the number of expressed antigens of the MAGE family, the main group of the cancer/testis antigens. These data indicate that the lysate of our melanoma cell lines could be used for treatment of many kinds of solid tumors where cancer/testis antigens are expressed in significant amounts.
Safety of vaccination
In general, dendritic cell-based vaccination demonstrates no life-threatening side effects, as is also described in many published clinical studies. The only side effects are flu-like symptoms with fever (up to 39-40 deg. C), chill, and headache in some patients. The occurrence of these effects did not require additional treatment or prolonged hospitalization. Some patients develop vitiligo when melanocyte differentiation antigens are used as targets in immunotherapy. However, this is does not occur with MelCancerVac® due to a lack of differentiation antigens in the melanoma cell lysate.
MelCancerVac® is produced according to the principles of Good Manufacturing Practice (GMP) in facilities approved by the Danish Medicine Agency for the production of medicines from patient blood in aseptic conditions. No products of animal origin are used during vaccine preparation. Quality control is performed for each individual batch of the vaccine as well as for the lysate used in the loading of dendritic cells. Analysis
RT-PCR analysis of the tumor antigen expression profile
The technology developed at DanDrit requires that a thorough analysis is performed for each batch of melanoma cells expanded for producing lysate. The method used to analyze tumor antigen expression is Reverse Transcriptase – Quantitative Polymerase Chain Reaction (RT-QPCR). RT-QPCR detects the messenger RNAs that code for genes in cells. Briefly, when a gene is active it is expressed –the DNA sequence of the gene is transcribed into messenger RNA and the messenger RNA is then translated into polypeptide or protein product. The protein products are the antigens that are recognized by the immune system. In RT-QPCR the messenger RNAs are amplified in a quantitative PCR reaction, thus enabling a direct measure of gene expression. The above mentioned properties of the melanoma cell lysate are confirmed by RT-QPCR: that a broad spectrum of tumor-specific antigens is present and that the expression of differentiation antigens is kept low, in comparison to a selected standard cell line.
Comparison of tumor antigen expression in MelCancerVac® with two patient biopsies by RT-QPCR (TA = tumor antigen).
The analysis of tumor antigen expression may also be applied to the patient’s tumor. A small sample of tumor (biopsy) can be analyzed to reveal which tumor antigens are expressed in the particular tumor. Profiling of tumor antigen expression in patient tumor samples can be used to improve cancer immunotherapy outcomes by ensuring that treated patients have tumors that actually express tumor antigens and by targeting patients with immunotherapy that is specific to the individual tumor. The ability to examine the expression of many tumor antigen genes simultaneously provides a powerful tool to monitor these essential targets of immunotherapy. For many tumor antigens the immunogenic peptide sequences recognized on the cell surface are already known. This enables researchers to determine whether a patient has responded by inducing an immune response towards specific peptide antigens known to be present in both MelCancerVac® and the patient tumor.