"Don't Eat me" Signal in Hematological Malignancies: CD24 as New Target to Improve Anti-cancer Immunity.

MCL is an aggressive B-NHL with dismal prognosis with high probability of relapse after conventional immunochemotherapeutic regimen. CLL is instead an incurable chronic B-cell malignancy with higher probability of relapse after several lines of conventional therapies and a non-negligible risk of progression into an aggressive lymphoma. Further approaches are needed to provide valid alternatives for these relapsed and refractory diseases. In these two settings, TME is known to be involved in cancer cell survival and confers a negative impact on standard treatment efficacy and adoptive T cell immunity, for which new immunotherapeutic agents could be an alternative approach in case of poor prognostic settings. Within TME, TAM are involved in improving tumor growth survival in MCL and CLL, dampening immunotherapy as well. Anti-CD47 mAb turned out to be a consistent DEMs that, once blocked, is able to improve phagocytosis and clearance of tumor cells in blood cancer. CD24 has been recently validated as another DEMs in preclinical analysis in solid cancer. Since CD24 is expressed during early phase of B-cell differentiation, MCL and CLL might take advantage of the blockade of this signal, since TAM and NCLs are strictly interconnected with their corresponding tumoral cells, providing tumor cell proliferation and immune escape.

To fulfill the purposes of this project, collection of leftover patient-derived samples will be routinary performed at the time of diagnosis and at the time of relapse of the disease, in this latter case with the aim to check any CD24, CD47 and CD20 surface expression modification on B-cell blasts (e.g., clone selection with CD24/CD47 upregulation after conventional regimen), SIRP-α and Siglec-10 expression on patient-derived Monocyte/Macrophage system (expressing higher levels of DEMs ligands as potential mechanism of resistance after conventional therapy).

Furthermore, phagocytosis assays with samples obtained at the time of relapse will be compared with the equivalent experiments performed with the samples collected at the time of diagnosis to evaluate any potential differences.

As previously described, phagocytosis analysis will be based on a co-culture process that involves the use of the anti-CD24 antibody (alone or in combination with other antibodies described in the secondary endpoint) incubating with macrophages (from healthy donor or from patient) and patient tumor cells (MCL or CLL), this latter labeled with a fluorescent substance (i.e., CFSE). Macrophage staining (with anti-CD11b-PE antibody) and subsequent flow cytometric analysis will be performed: the ratio between double stained macrophages (CD11b-PE+/CFSE+) and single stained macrophages (CD11b-PE+) will provide the entity of phagocytosis, which will be compared with the negative control and the other experimental conditions (antibody combinations described later in the secondary endpoints). The double staining of macrophages provides indirect results of phagocytosis (if the macrophage phagocytes CFSE+ cells, it will also acquire this staining in addition to that due to the anti-CD11b-PE antibody).

Finally, donor-derived macrophages will be isolated from PBMC obtained from leftover peripheral blood which remained within the circuit used for plateletpheresis of healthy donors. As a matter of fact, residual blood (almost 15 ml) can be found in the reservoir of this circuit that is normally trashed after the procedure. Donors will sign informed consent for use of leftover material for research purposes and this project in particular.