Rational
It is now well known that cancer is essentially a disease of the genome. The mutations that will eventually lead to the transformation of normal cells into malignant cells progressively accumulate within the genome. Moreover, the characteristics that make two distinct forms of cancer so different can also be found within the genome. More specifically, these characteristics are identified in the combination of the different genetic alterations that DNA undergoes during the process of transformation and tumor progression. In the cancer genome, therefore, important information can be found to better understand the biology of tumors, i.e. the characteristic traits that allow us to identify with precision the different forms, to predict the clinical course, and to recognize the critical elements for the identification of specific targets to design and develop highly selective drugs. Ultimately, genetic tumor profiling is an essential element for modern precision cancer medicine. In this context, the IOV has been actively involved in both the development of cellular neoplasm models able to reproduce those same molecular traits found in the tumors of interest, and in the genetic and molecular profiling of the patients, with the general objective to determine those genetic differences that allow the identification of the right sequence or combination of therapies to be used for a multiple attack on multiple targets, able to overcome the limitations of a single therapeutic strategy. These aspects are currently being heavily implemented thanks, in particular, to the acquisition of advanced technologies for genomics studies, such as the latest-generation DNA sequencing method (NGS), which can sequence the entire genome in a very short time.
On the other hand, not only do the aspects related to the mutational evolution of the genome impact tumor transformation and progression, they also have important implications in the field of immunology and immunotherapy. In fact, the armamentarium of cancer therapies has recently been enriched with the addition of monoclonal antibodies (mAbs), which essentially act by “activating” the endogenous immune responses to the tumor. These so-called immunological checkpoint inhibitors (ICIs) are mAbs able to prevent feedback inhibition of activated T cells, with the result of favoring an immune response to the tumor. Moreover, recent data indicate that, in responding patients, ICIs are able to stimulate protective and therapeutic T cell responses directed against tumor neoantigens deriving from mutations of normal neoplastic cell proteins, which are thus behaving as highly tumor-specific antigens. These observations may well lead to the possible identification of specific individual antigenic expression profiles on the basis of which to propose customized immunotherapeutic interventions, as well as the conception of combination therapies with ICI and vaccine approaches and / or adoptive therapies with T cells able to favor the expansion and functionality of highly effective effector populations.
Finally, closely related to cancer immunogenetic profiling, cellular profiling of the tumor-stroma context, to obtain a qualitative-quantitative evaluation (immunoscore) of the infiltrating immune populations, represents another cornerstone of modern immuno-oncology research – the identification of predictive response biomarkers.
Genomic and immunogenetic sequencing, cellular immunoprofiling, and clinical data to correlate results, all constitute what is known in technical jargon as big data, huge amounts of data that are produced and collected systematically with the help of computers. This database represents a precious source of information, which requires a careful interrogation to get the desired answers. In this context, bioinformatics is opening its doors to extremely interesting scenarios, placing itself at the crossroads of DNA sequencing techniques and the development of software capable of analyzing big data, fundamental aspects for a large step forward towards precision cancer medicine. In fact, in oncology, the large-scale analysis of big data is considered the basis for a real revolution in the treatment of the disease, thanks to the possibility of analyzing molecular and clinical details with a precision that has never before been reached.
Although tumor genomics is being rapidly implemented in our country and in our Institute, centers specifically dedicated to high throughput analysis are still relatively rare and poorly interlaced with structural bioinformatics. On the purely clinical and clinical-pathological front, the introduction of ICIs in current therapeutic practice is drastically modifying treatment expectations in some oncological fields, but at the same time, the identification of predictive response biomarkers able to guide the selection of patients are urgently needed, to rationalize and personalize therapies for better toxicity profiles and cost effectiveness for the National Health System. Furthermore, on the immunological and immunotherapeutic level, these considerations are further aggravated by the relative absence of a scientific culture focused on the development of new techniques of adoptive cellular immunotherapy and of adequate structures for the production of cellular drugs for therapeutic use.
The IOV has promoted translational research studies aimed at:
- implementing highly personalized genomic approaches to tumors;
- identifying biomarkers of response to ICI therapy;
- integrating the data produced in the field of bioinformatics;
- developing potential adoptive cell immunotherapy approaches that can be translated into clinical practice.
Specifically, research is being supported in the main oncology areas that have invested in the use of ICI, such as melanoma, lung, breast, colorectal and prostate cancer. Thanks to the “Capital Grant” programs 2013 and 2015, the IOV has obtained a sophisticated high-throughput genomic analysis platform; the main areas of intervention are genetic and immune profiling in peripheral blood and neoplastic tissue, to identify gene expression profiles and molecular immunoscores predictive of response and / or toxicity.
Concurrently, the 2015 “Capital Grant” program is allowing the IOV to obtain sophisticated equipment for “digital and quantitative pathology”, which allows the visualization and quantification of multiple immunofluorescent signals of the same sample (different lymphocyte subset, immunosuppressive pathway markers of functionality / activation, components of innate immunity, ICI ligands); therefore, the IOV will also be able to conduct retrospective multicenter clinical studies on archived material. In fact, while on the one hand, evidence supports the existence of a positive association between the presence of tumor-infiltrating lymphocytes (TIL) and a better prognosis in different types of solid tumors, on the other hand, the determination of the expression, at the level of both the neoplastic component and the infiltrating lymphocyte, of the critical molecules (CTLA-4, PD-1, PD-L1) that represent the ICI target is assuming an increasingly important role.
The need to give a biological meaning to the enormous mass of information being produced, and the integration and correlation of this information with clinical data is another critical objective of the project that also aims to support structural bioinformatics, which is currently being implemented at our Institute thanks to private contributions.
Moreover, these private contributions have supported studies aimed at implementing adoptive cell immunotherapy approaches, based on tumor-specific T cells obtained by genetic engineering with TCR or chimeric antigen receptors (CARs), or on cytotoxic genetically unmodified cells characterized by high antineoplastic activity (NK cells, CIK cells). The large-scale production and expansion of these cells is simplified at a technical level and at a low cost. Given the national relevance of the CAR-T program, further details are reported below.
CAR-T program
Currently, T-lymphocytes armed with CAR represent one of the most advanced frontiers of onco-hematology research. The development of CAR-T therapies in solid tumors, on the other hand, is still a challenge; so far, it has proved much more difficult to achieve the same positive therapeutic effects observed in hematologic malignancies. At the IOV, efforts in this area are focused on prostate cancer (PC) and prostate-specific membrane antigen (hPSMA). hPSMA is one of the most promising biomarkers in the diagnosis and treatment of PC, and its clinical relevance is currently evaluated in several immunotherapy studies. hPSMA is a 100 kDa type II integral membrane protein with a cytoplasmic tail of 19 amino acids, predominantly located on epithelial cells of the prostate gland. In normal prostate epithelial cells, there is a low expression of hPSMA, which instead increases markedly in high-grade, metastatic, and androgen-insensitive prostate tumors. The importance of hPSMA as the optimal target of therapeutic interventions also lies in the evidence that it is absent in normal endothelial cells and is instead expressed in the neo-vascularization endothelium of numerous tumors of different histotypes.
On the basis of accessibility to one of the best anti-hPSMA (D2B) mAb present at the international level, projects have been developed aimed at using the variable regions of this mAb for the construction of the single chain variable fragment (scFv), to be used both as a recombinant protein for diagnostic / therapeutic purposes and for the development of a CAR for adoptive immunotherapy. With reference to this last aspect, our first project dealt with the creation and development of a second-generation CAR.
A second research project was devoted to the development of a third-generation receptor. The approach involved the insertion of a second co-stimulatory molecule, 4-1BB, between CD28 and the zeta chain. This CAR is fully functional and studies are still in progress aimed at comparatively evaluating the in vitro and in vivo efficacy with respect to the second-generation version. In onco-immunology, the phenomenon of immunoediting is well known; the selective pressure of the immune system is able to “sculpt” the neoplasm favoring the onset of clonal variants, which lose the expression of antigens recognized as an escape and survival mechanism. Since this could also occur in treatments with CAR-T, a third approach that we are pursuing involves the construction of a lentiviral vector that allows the coordinated and simultaneous expression of two CARs by the transduced T lymphocytes. We then generated a second chimeric receptor against another antigen: hPSCA (Prostate Stem Cell Antigen). The hPSCA is a 123 aa protein anchored to the membrane as a GPI molecule and belonging to the Thy-1 / Ly-6 family, with a presumed role in signal transduction and / or cell adhesion; it is present at low levels on normal basal / secretory cells of the prostate, but its expression increases in PC tumor cells and in bone metastases from prostate cancer. The experimental evidence collected so far is very encouraging and shows that the transduced T cells express both CARs (anti-hPSMA and anti-hPSCA) in a coordinated manner, and exert a high and specific lytic activity against PSCA + and / or PSMA + tumor lines.
