Periodic Reporting for period 1 - AngioCAR (Chimeric antigen receptor (CAR) T-cell therapy against solid tumors by targeting the vasculature)
Période du rapport: 2020-06-01 au 2022-05-31
In the last decades, breast cancer rates have been increasing worldwide which can be related to higher exposure to risk factors, delayed childbearing, lower pregnancy rate, longer life expectancy, and obesity. Although in breast cancer treatment several promising therapeutic approaches are being introduced and investigated, unresponsiveness and/or resistance to distinct pharmacotherapies has been reported. In addition, not all breast cancer patients respond to traditional approaches, such as resection surgery, chemo and /or radiation therapy.
In the past decade immunotherapy has revolutionized the field of cancer treatment. Cancer immunotherapy can e.g. be based on a) targeted therapy, b) immune checkpoint inhibitors, and c) cell-based therapies which are divided into 4 distinct groups, namely T infiltrating lymphocyte (TIL) therapy, T cell receptor (TCR)-engineered lymphocyte therapy, chimeric antigen receptor (CAR) T cell therapy, and CAR natural killer (NK) cell therapy. All of these adoptive cell therapies are based on a similar approach. In the current era, CAR T cell therapy represents a new pillar of cancer immunotherapy (Figure 1). CAR T cells are generated to specifically recognize a target antigen, that is highly, homogenously, and stably expressed by tumor cells but not by normal tissues. In this revolutionary approach, T cells are isolated from a patient by leukapheresis and genetically modified to express a specific artificial receptor, comprised of an extracellular binding domain that is fused to intracellular co-stimulatory and activation domains. Modified and activated CAR T cells are then reinfused back into the patient and will eliminate the target antigen expressing tumor cells.
In spite of the tremendous promising outcomes of applying CAR T cell therapy in haematological malignancies, similar success in solid tumors has not been achieved yet. Main obstacles in solid tumors are a) high heterogeneity and/or lack of appropriate antigens, b) inadequate T cell infiltration into the tumor mass, and c) the presence of an immunosuppressive microenvironment.
On the other hand, CAR T cells can be more functional and effective than other immune cells in cancer treatment. Firstly, the majority of tumor associated antigens are self-antigens which means that the immune system in the normal situation is unable to recognize and mount an immune response against these antigens. CAR T cells, however, can be designed to recognize and eliminate cells expressing tumor associated (self-)antigens. Secondly, CAR T cells can not only target surface proteins, but also glycoproteins, glycolipids and lipids. Thirdly, tumor cells have a tendency to downregulate their major histocompatibility complex class I (MHC-I) expression, however, CAR T cells are MHC independent and do not require antigen presentation by MHC. In conclusion, CAR T cell therapy can be one of the best cancer therapies, if we are able to overcome the aforementioned obstacles in solid tumors.
Several CAR T cells have been generated against antigens overexpressed by breast tumor cells, such as mesothelin (MSLN), human epidermal growth factor receptor 2 (HER2), epithelial cell adhesion molecule (EPCAM), carcinoembryonic antigen (CEA) and receptor tyrosine kinase-like orphan receptor 1 (ROR1). Although these CAR T cells indicated to have a profound impact on cancer treatment in clinical trials, however, unfortunately, none of them have been approved by the FDA yet which can be related to heterogeneous tumor antigen expression, as well as inhibitory molecules in tumor microenvironment (TME).
The aim of this study is to develop CAR T cells that target the vasculature of solid tumors, rather than the tumor cells themselves. Targeting tumor vasculature offers multiple advantages. Not only the main source of tumor nutrition can be destroyed which leads to tumor eradication and metastasis inhibition, but also tumor endothelial cells (TEC)s are genetically more stable than tumor cells , ensuring stable target antigen expression. Furthermore, effective targeting TECs instead of tumor cells with CAR T cells may be accomplished using a lower number of CAR T cells, which will decline the risk for cytokine release syndrome (CRS) and also other severe side effects. In previous studies, we showed that TECs overexpress and secrete vimentin, a type III intermediate filament protein. The extracellular form of vimentin stimulates tumor angiogenesis and confers immune suppression, and targeting extracellular vimentin counteracted this. Extracellular vimentin (eVim) can be considered as an ideal antigen due to its overexpression in the tumor vasculature of many epithelial cancers, such as breast cancer, but absence in normal healthy blood vessels and healthy tissues. Moreover, targeting accessible antigens in the tumor vasculature overcomes a barrier in T cell homing as the CAR T cells do not need to infiltrate into the immunosuppressive tumor microenvironment to find their target antigen and be activated.
In the past decade immunotherapy has revolutionized the field of cancer treatment. Cancer immunotherapy can e.g. be based on a) targeted therapy, b) immune checkpoint inhibitors, and c) cell-based therapies which are divided into 4 distinct groups, namely T infiltrating lymphocyte (TIL) therapy, T cell receptor (TCR)-engineered lymphocyte therapy, chimeric antigen receptor (CAR) T cell therapy, and CAR natural killer (NK) cell therapy. All of these adoptive cell therapies are based on a similar approach. In the current era, CAR T cell therapy represents a new pillar of cancer immunotherapy (Figure 1). CAR T cells are generated to specifically recognize a target antigen, that is highly, homogenously, and stably expressed by tumor cells but not by normal tissues. In this revolutionary approach, T cells are isolated from a patient by leukapheresis and genetically modified to express a specific artificial receptor, comprised of an extracellular binding domain that is fused to intracellular co-stimulatory and activation domains. Modified and activated CAR T cells are then reinfused back into the patient and will eliminate the target antigen expressing tumor cells.
In spite of the tremendous promising outcomes of applying CAR T cell therapy in haematological malignancies, similar success in solid tumors has not been achieved yet. Main obstacles in solid tumors are a) high heterogeneity and/or lack of appropriate antigens, b) inadequate T cell infiltration into the tumor mass, and c) the presence of an immunosuppressive microenvironment.
On the other hand, CAR T cells can be more functional and effective than other immune cells in cancer treatment. Firstly, the majority of tumor associated antigens are self-antigens which means that the immune system in the normal situation is unable to recognize and mount an immune response against these antigens. CAR T cells, however, can be designed to recognize and eliminate cells expressing tumor associated (self-)antigens. Secondly, CAR T cells can not only target surface proteins, but also glycoproteins, glycolipids and lipids. Thirdly, tumor cells have a tendency to downregulate their major histocompatibility complex class I (MHC-I) expression, however, CAR T cells are MHC independent and do not require antigen presentation by MHC. In conclusion, CAR T cell therapy can be one of the best cancer therapies, if we are able to overcome the aforementioned obstacles in solid tumors.
Several CAR T cells have been generated against antigens overexpressed by breast tumor cells, such as mesothelin (MSLN), human epidermal growth factor receptor 2 (HER2), epithelial cell adhesion molecule (EPCAM), carcinoembryonic antigen (CEA) and receptor tyrosine kinase-like orphan receptor 1 (ROR1). Although these CAR T cells indicated to have a profound impact on cancer treatment in clinical trials, however, unfortunately, none of them have been approved by the FDA yet which can be related to heterogeneous tumor antigen expression, as well as inhibitory molecules in tumor microenvironment (TME).
The aim of this study is to develop CAR T cells that target the vasculature of solid tumors, rather than the tumor cells themselves. Targeting tumor vasculature offers multiple advantages. Not only the main source of tumor nutrition can be destroyed which leads to tumor eradication and metastasis inhibition, but also tumor endothelial cells (TEC)s are genetically more stable than tumor cells , ensuring stable target antigen expression. Furthermore, effective targeting TECs instead of tumor cells with CAR T cells may be accomplished using a lower number of CAR T cells, which will decline the risk for cytokine release syndrome (CRS) and also other severe side effects. In previous studies, we showed that TECs overexpress and secrete vimentin, a type III intermediate filament protein. The extracellular form of vimentin stimulates tumor angiogenesis and confers immune suppression, and targeting extracellular vimentin counteracted this. Extracellular vimentin (eVim) can be considered as an ideal antigen due to its overexpression in the tumor vasculature of many epithelial cancers, such as breast cancer, but absence in normal healthy blood vessels and healthy tissues. Moreover, targeting accessible antigens in the tumor vasculature overcomes a barrier in T cell homing as the CAR T cells do not need to infiltrate into the immunosuppressive tumor microenvironment to find their target antigen and be activated.
In this study, second generation CARs with two different costimulatory receptors, CD28 and 4-1BB separately, were designed and investigated. The single chain variable fragment (scFv), named TE6, derived from an in-house developed anti-vimentin monoclonal antibody was cloned into the CAR constructs with CD28-CD3z and 4-1BB-CD3z separately. Having produced retroviruses, encoding the CARs, we transduced activated T cells, isolated from PBMCs. The CAR was detected on the surface of T cells by flow cytometry. Subsequently, vimentin secreting target cells (HMEC-1 and MDA-MB-231) and vimentin negative cells (MCF-7) were co-cultured with CAR T cells and functional assays were performed, including proliferation, cytotoxicity assays. Our results show that anti-eVim-CAR T cells were able to specifically identify and bind to eVim, secreted by HMEC-1 and MDA-MB-231 cell lines, and were immediately activated to eradicate the target cells. In addition, tumors were grafted on the chicken chorioallantoic membrane, as an animal model, followed by injection of CAR T cells to investigate if this in vivo model could be used to assess in vivo targeting of the tumor vasculature.
In AngioCAR we show for the first time proof of concept that extracellular vimentin can be targeted by CAR T cells. This achieved outcome is a major contribution to the state of the art in the field of CAR T cell therapy and will lead to the development of improved CAR T cell therapy for the treatment of solid tumors.