Laboratory of Immunotherapy
RNDr. Michal Šmahel, Ph.D.
Phone: +420 325 873 921, +420 325 873 947, +420 325 873 941
The Laboratory of Immunotherapy was launched in 2015 by a group of researchers who came from the Institute of Hematology and Blood Transfusion to join the programme Cellular Biology and Virology in BIOCEV, a newly established research centre located at Vestec, Central Bohemia (research group Immunization against Tumours Caused by Human Viruses). The Laboratory focuses on the immunotherapy of virus-induced tumours tested experimentally on murine models of tumours in which human papillomaviruses are known to play a role.
The laboratory has been involved in the development of anti-tumour DNA vaccines to be administered biolistically using a gene gun. The immunization targets not only the tumour cells themselves but also the tumour stromal cells that may support tumour growth, in particular macrophages. Our attention is also drawn to antigens of tumour stem cells known to be resistant to various cancer therapies and thus to be the major cause of disease relapse and metastasis.
The efficiency of DNA vaccines can be enhanced by various modifications of immunizing genes to construct fusion (chimeric) genes. The first principle used in the construction of fusion genes is altering the cellular localization of proteins to target the immunogen to the MHC class I or class II epitope presentation pathway. This is achieved by using lysosome-associated membrane protein 1 (LAMP-1) localization sequences which enable the delivery of fusion proteins into the endoplasmic reticulum and subsequently to the endosomes and lysosomes. Next, to enhance the immunogenicity of DNA vaccines, a helper epitope is added to activate CD4+ T helper cells (Th). To this aim, the universal epitope from the tetanus toxin (TT), p30 (TT947-967), was used and more recently, the in silico designed PADRE epitope has been preferred as it has proven superior efficacy in our model system. As our DNA vaccines are often targeted against proteins with oncogenic potential, transformation properties of the immunogens are reduced by mutagenesis to enhance the safety of DNA vaccines.
If the H-2b epitopes of the antigens against which the immune responses are induced are not known, they can be predicted in silico. The reactivity of the candidate epitopes is tested by ELISPOT assays. The proven epitopes enable examination of immune reactions in vitro and the results serve as a basis for the optimization of immunotherapeutic protocols.
DNA vaccines containing foreign antigens, such as papillomavirus oncoproteins E6 and E7, can reduce tumour growth while the immune responses resulting from the immunization against self-antigens often have only low anti-tumour activity. Therefore, the effect of DNA vaccines is enhanced by inhibiting the immunosuppressive mechanisms contributing to tumour growth. One of the possible strategies is the depletion of regulatory T (Treg) cells by the anti-CD25 antibody recognizing the interleukin 2 (IL-2) receptor. Another strategy is to combine the DNA vaccine with antibodies that block immune checkpoints CTLA-4 or PD-1. These receptors are expressed on different subpopulations of T cells and regulate their activity at various stages of activation. Their blockade prevents the natural suppression of immune responses induced e.g. by a vaccine or partial elimination of tumour cells by other therapeutic modalities. Anti-CTLA-4 and anti-PD-1 antibodies have already proven their potential in clinical trials and are being introduced into routine clinical practice.
The major anti-tumour effect of the antibodies blocking the CTLA-4 and PD-1 receptors consists in the stimulation of the activity of the cytotoxic T cells that recognize tumour cells presenting on their surface epitopes bound to MHC class I (MHC-I) molecules. However, tumours often evade the immune cells because of the genetic instability of cancer cells and selection of their variants with reduced expression of MHC-I molecules. Therefore, in the development of our murine model, we focused on deriving oncogenic cell clones with reduced expression of MHC-I molecules. Such a reduction can be either reversible or irreversible. In the reversible modification induced by epigenetic regulations, the cell surface expression of MHC-I molecules can be restored by induction by e.g. interferon gamma (IFN-g). Irreversible modifications result from gene mutations which may be reflected in diverse phenotypic alterations. The complete loss of the cell surface expression of MHC-I molecules can result from e.g. defective production of β2-microglobulin. The tumour cell clones with reversibly reduced MHC-I expression were previously obtained from murine tumours where they had been naturally selected after DNA immunization against the E7 oncoprotein. Recently, the cell clones have been prepared in which the gene for β2-microglobulin is inactivated using the CRISPR-Cas9 system and consequently, irreversible loss of cell surface MHC-I expression is achieved.
Tumour cells with reduced MHC-I expression are not susceptible to cytotoxic T cells, but they often exhibit higher susceptibility to NK cells and can also be killed by other cells of innate immunity such as macrophages. Therefore, we attempt to develop combined immunotherapeutic protocols aimed at the activation of diverse cells of adaptive and innate immunity to be effective against tumour cells with different levels of MHC-I expression. Apart from the above mentioned protocols, we use the activation of immune cells by toll-like receptor (TLR) agonists and the next step will be the blockade of inhibitory Tim-3, which is expressed not only on activated T cells but also on macrophages and dendritic cells. One of the aims of the combined immunotherapy is to convert pro-tumour M2 type macrophages to anti-tumour M1 type macrophages with potential inhibitory activity against the growth of tumours induced by cells with reduced MHC-I expression.
The tumour microenvironment is a complex system of various types of cells involved, through different mechanisms, either in the tumorigenesis or in the anti-tumour activity. One of our goals is to characterize these mechanisms and to study their modifiability in response to different immunotherapeutic challenges. To this aim, we isolate cells from tumours and perform their phenotyping. Furthermore, we isolate RNA from tumours and examine the expression of genes the products of which modify immune responses.