Functional Analysis of primary CAR-NK cells armored with cytokines targeting CD19 and ROR1

• Grant number: 25/01430-6
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 01, 2025
• End date: May 31, 2026
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Virginia Picanço e Castro
• Grantee: Alison Felipe Bordini Biggi
• Supervisor: Fernando de Souza Fonseca Guimaraes
• 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
• Institution abroad: University of Queensland, Brisbane (UQ), Australia
• Associated to the scholarship: 23/09711-9 - Use of anti-CD19 CAR-NK cells expressing IL-15/IL-15R± and IL-27 cytokines as an allogeneic treatment for B-cell cancers, BP.DR

Abstract

CAR-NK cells represent a potentially safer and "off-the-shelf" alternative to CAR-T cells, offering broad tumor recognition without HLA restriction, intrinsic antitumor activity, and a lower risk of cytokine release syndrome (CRS), without causing graft-versus-host disease (GVHD). During my PhD, we tested CAR constructs coexpressing IL-15, IL-15R¿, and IL-27 in NK cells. These constructs significantly enhanced CAR-NK cell proliferation and cytotoxicity. In parallel, our group designed CARs incorporating the 2B4 signaling domain as a costimulatory module, which demonstrated promising functionality in NK-92 cells. Thus far, all evaluations have been conducted in NK-92 cells, and the next step is to validate these constructs in primary NK cells. In this project, our first objective is to generate primary NK cells expressing anti-CD19 CARs with IL-15 and IL-27, using a royalty-free feeder cell line provided by Professor Guimarães. This feeder system enables a sustainable and scalable method for NK cell expansion. Our second goal is to combine the 2B4 costimulatory domain with cytokine coexpression to develop a novel CAR targeting ROR1. ROR1 is overexpressed in multiple malignancies, including hematological cancers, and targeting it could expand the therapeutic reach of CAR-NK therapy beyond CD19, potentially addressing both hematologic and solid tumors. As specific aims, we will perform multi-omics analyses of anti-CD19 CAR-NK cells expressing IL15 and IL27, and the novel anti-ROR1 construct. We will also evaluate the impact of NKG2A knockout in primary NK cells, both alone and in combination with CAR expression, to assess potential enhancements in cytotoxicity. This collaborative project will be instrumental in enabling multi-omics profiling of primary CAR-NK cells and in the development and functional validation of our new CAR construct. Ultimately, it will provide a strong foundation for advancing CAR-NK cell therapies as a safer and more adaptable platform in cancer immunotherapy.