Project Funding Details

Clinical risk and choice of therapy in colorectal cancer (CRC) are based on histology, tumor staging, Kras/Nras/Braf mutations; however, this classification is limited in estimating outcome of patients and response to post-surgical therapy. The evaluation of immune cell infiltrate in the tumor, named immunoscore, has revealed a more significant prognostic value. Innate immunity effectors, including natural killer (NK) and γδT cells, can exert anti-tumor activity in CRC. γδT lymphocytes respond to pyrophosphate products of the mevalonate pathway; accumulation of pyrophosphates in cancer cells can be enhanced by amino-bis-phosphonates, including zoledronic acid (ZA). We have demonstrated that mesenchymal stromal cells/tumor-associated fibroblasts (MSC/TAF) and tumor cells in CRC express the butyrophilin 3A1 (BTN3A1) molecule involved in the exposure of pyrophosphates. BTN3A1+cells localize close to areas infiltrated by Vδ2(γδ)T lymphocytes: ex-vivo BTN3A1+MSC/TAF and CRC cells primed with ZA stimulate proliferation and anti-tumor cytotoxicity of γδT cells. An immunoscore based on the number and type (MSC/TAF or CRC or monocyte/macrophages, Mo/Mφ) of BTN3A1+ cells, together with the quantitative evaluation of γδT cell infiltrate, can define CRC where ZA would trigger such anti-cancer immune response. This response can be amplified with therapeutic mAbs, e.g. the anti-EGFR cetuximab (Cet), able to target tumor cells and trigger the antibody-dependent cellular-cytotoxicity (ADCC) exerted by FCγRI/II/III+Mo/Mφ, NK or γδT lymphocytes. A stronger effect would be achieved by targeting CRC with: 1. Antibody drug conjugate (ADC) of ZA linked to Cet; 2. Spherical Polymeric Nanoconstructs (SPNs) loaded with ZA and conjugated with Cet. 1. Definition of BTN3A1 expression and γδT cell infiltrate as immunoscore parameters. 2. Selection of patients eligible for a ZA-based immunostimulating therapy. 3. Synthesis of ZA/Cet ADC and ZA/Cet SPNs for a precision medicine therapeutic approach. 1. The number of γδT cells and expression of BTN3A1 on CRC, MSC/TAF or Mo/Mφ will be analyzed, by digital pathology, in CRC sections and related to Kras/Nras/Braf mutational status and staging. 2. ZA/Cet ADC will be synthesized by linking a ZA-thioether-succinimide to interchain disulfide bonds of Cet; ZA/Cet SPNs will be obtained loading ZA onto SPNs with aqueous core, enclosed in polymer matrix and lipid monolayer, conjugated with Cet. 3. Spheroids and organoids, both in static and dynamic conditions, will be used to test the expansion and anti-CRC activity of γδT/NK lymphocytes and Mo/Mφ in response to ZA/Cet ADC and ZA/Cet SPNs. 1. Contribution of infiltrating γδT cells and BTNA3A1+CRC, MSC/TAF or Mo/Mφ to immunoscore; identification of CRC patients potentially responsive to ZA as immunostimulating drug. 2. ZA/Cet ADC and ZA/Cet SPNs chemical conjugation. 3. Amplification of γδT, NK cell and Mo/Mφ response to CRC with ZA/Cet ADC and/or ZA/Cet SPNs, demonstrated in static and dynamic 3D-culture pre-clinical models. CRC accounts for >12% of all tumors, with 50% 5-year survival rate, despite improvement of early diagnosis and surgery. This raises the need of novel/complementary post-surgery therapies. We propose a precision medicine approach based on a new strategy: amplify anti-cancer immune-response, refine drug delivery, enhance single drug effects. This can be achieved with two novel feasible conjugates of ZA and Cet.