Development of integrated strategies for lentiviral particles purification

• Grant number: 24/01305-4
• Support Opportunities: Scholarships in Brazil - Master
• Start date: March 01, 2024
• Status: Discontinued
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Virginia Picanço e Castro
• Grantee: Camilly Melo Garcia Ferreira
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Associated research grant: 20/07055-9 - Nucleus of Cellular Therapy - NuTeC, AP.NPOP
• Associated scholarship(s): 24/22467-2 - Generation of CD19-Targeted CAR-NK Cells Derived from Primary Sources Using CRISPR/AAV, BE.EP.MS

Abstract

Our research group has dedicated efforts to the implementation of CAR T-cell therapy at the Blood Center-RP. With the support of FAPESP (NPOP project 2020/07055-9), we have made significant progress in the production of lentiviral vectors for the CAR-T cell project, for which later this year we will start the I/II clinical study approved by ANVISA. Since the inception of the NPOP project we have highlighted remarkable progress in the production of lentiviral particles. Currently our LV platform has the capacity to produce batches of up to 4 liters, which go through a downstream process for purification and concentration of LV particles. There is no doubt that the use of autologous T cells with chimeric antigen receptors (CAR) has revolutionized the treatment of hematologic malignancies. However, this therapy still faces challenges, such as high costs and complex manufacturing. NK cell immunotherapy offers the prospect of an allogeneic "off-the-shelf" approach that is more affordable and available to a larger number of patients. NK cells have intrinsic advantages, such as the ability to eliminate tumor cells without triggering graft-versus-host disease, a common concern in allogeneic T-cell therapies. Recently, our group developed the CAR. CD19-IL15/IL15R± (patent BR1020220083339) and the entire proof-of-concept was performed on lineage NK cells (NK-92) demonstrating that the cytokine IL-15 plays a key role in expansion and cytotoxicity (Silvestre et al., 2023). The next step is to test the effectiveness of this vector in primary NK. Primary NK stands out as promising candidates for immunotherapy, although ex-vivo expansion of these cells remains a challenge. Adoptive NK cell transfer, despite its advantages, faces obstacles, including difficulty in obtaining a sufficient number of cells for treatment, insufficient persistence, poor in-vivo expansion, and limited antitumor activity of infused cells. Therefore, the main challenge in this field lies in obtaining NK cells in adequate quantity and quality for adoptive immunotherapy. In this context, we present an innovative proposal for the large-scale production of primary NK cells while preserving their antitumor activity. Development of allogeneic therapies is foreseen in the NPOP project in the goal Medium-long term strategic innovations. This master's project aims to utilize our LV platform to produce our second CAR vector, the CAR. CD19-IL15/IL15Ra, specific for NK cells, following the attached activity plan. However, we intend to move forward with the expansion of primary NK cells and evaluate their transduction efficiency. In this project, in addition to the production of lentiviral particles, an appropriate culture method will be established for primary NK cells (NK-PB or NK-CB), with testing of different media, culture systems, supplementation and feeder cells. After expansion, the cells will be evaluated for their immunophenotypic profile and cytotoxic potential.After expansion, the cells will be evaluated for their immunophenotypic profile and cytotoxic potential. With the culture established, the NK cells will be modified with our lentiviral vector, containing the anti-CD19 CAR sequence co-expressing IL-15/IL-15R±, to potentiate the anti-tumor response. NK-CAR cells will be tested for their cytotoxic potential against CD19+ and CD19- tumor cells in-vitro. The effective establishment of this ex-vivo NK cell expansion system will represent a significant contribution to the clinical application of these cells.