CAR-NK Cells Production and Expansion From Patients With Lympho or Myeloproliferative Disorders and From Healthy Donors

The primary aim of the study is to manufacture and expand NK cells expressing CD19, CD79 and CD123 CARs from Peripheral Blood Mononucleated Cells (PBMCs) (cohort a) and from CD34+ cells (cohort b and c) of subjects affected by myelo or lymphoproliferative disorders or healthy donors. The secondary aim is to evaluate their cytotoxic activity in preclinical models. The main steps of CAR-NK cell manufacturing and preparation are the followings 1. Collection of peripheral venous blood (40 mL collected in EDTA tubes for cohort a, or 10 mL from patients with \>20 CD34+ cells/mcL for cohort b), or blood from bone marrow aspiration (5 mL of bone marrow collected in one EDTA tube for cohort b), or a part of LP (2 mL of PL in one EDTA tube for cohort c) from subjects with myelo/lymphoproliferative diseases and from Healthy Donors; 2. Selection of mononucleated cells through a density gradient centrifugation technique (Ficoll-Histopaque) (cohort a and b); 3. Selection of CD34 + Hematopoietic stem cells (HSCs) through magnetic activated cell sorting (cohort b) 4. Expansion NK cells from PBMCs (cohort a), using supplements such as recombinant cytokines, stimulatory antibodies, medicinal products and irradiated cells to support the process; 5. Expansion of CD34+ cells (cohort b and c), using supplements such as recombinant cytokines, stimulatory antibodies, medicinal products and irradiated cells to support the process, in order to promote differentiation to the NK cell lineage; 6. Engineering of NK cells through retroviral or lentiviral vectors (VL) that enable transduction with chimeric antigen receptors (CARs) specific for neoplastic cells. 7. Increasing the activation of NK cells through the down regulation of their inhibitory receptors (CRISPR Cas-9 technology and pharmacological modulation) or pharmacologic modulation of epigenetic, metabolic (HIF1-alpha) and immune-related pathways; 8. Testing, in in vitro and in vivo preclinical models, the persistence and the activity of the CAR-NK cells produced. 100 patients and 40 healthy donors will be prospectively enrolled over 3 years in order to perform 70 experiments of NK-cell expansion from PBMCs and 70 experiments from CD34+ cells (from PB or bone marrow or LP).