Generation of CAR-NK Memory-Like NKG2A KO cells using CRISPR-Cas9

• Grant number: 24/03044-3
• Support Opportunities: Scholarships in Brazil - Master
• Start date: February 01, 2025
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Rodrigo Nalio Ramos
• Grantee: Maria Paula Oliveira Lima
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated scholarship(s): 25/05497-8 - Generation of Cytokine-Induced Memory-Like NK Cells derived from hiPSCs for Immunotherapy Applications, BE.EP.MS

Abstract

Multiple Myeloma (MM) is characterized by the uncontrolled proliferation of malignant monoclonal plasma cells in the bone marrow, affecting the production of blood cells. New treatment strategies use Natural Killer (NK) cells, mainly due to their ease of manipulation in vitro, generating NK cells with an enhanced profile for tumor control. In the presence of the cytokine combination of IL-12, IL-15, and IL-18, NK cells exhibit properties similar to immune memory (memory-like NK), such as increased production of IFN-³ and greater persistence. Additionally, preclinical and clinical studies with genetically modified NK cells with a chimeric antigen receptor (CAR-NK) are promising for use in allogeneic immunotherapy, due to their increased proliferation capacity, safety, and persistence against tumor cells, especially when directed against the B-cell maturation antigen (BCMA), predominantly expressed in MM plasma cells. However, different immune checkpoint receptors involved in NK cell dysfunction are being investigated to prevent tumor cell evasion from immunosurveillance, such as NKG2A, which suppresses NK cell activity by binding to HLA-E, a molecule expressed by MM cells at relatively high levels. A favorable approach for the genetic engineering of immune cells is the CRISPR/Cas9 technology, which allows for the insertion or deletion of genes at specific locations in the cellular genome. In this context, this project proposes the genetic editing of memory-like NK cells with CRISPR/Cas9 aiming at the concomitant deletion of the NKG2A immune checkpoint and the insertion of an anti-BCMA CAR sequence as a cell therapy product for MM. Once generated, CAR-NK NKG2A KO cells will be characterized for their phenotypic and functional profile in vitro against BCMA+HLA-E+ MM cells. Thus, we propose to generate genetically modified NK cells optimizing their functional profile, aiming, at the same time, to direct them to a specific target and overcome immunosuppression by HLA-E in malignant MM cells.