# Protocol to generate human stem cell-derived CD70-directed allogeneic CAR-NKT cells for treating renal cell carcinoma

## TL;DR

This paper presents a protocol to generate CAR-NKT cells from stem cells for treating kidney cancer by targeting CD70.

## Contribution

A novel protocol for generating allogeneic CAR-NKT cells from cord blood stem cells targeting CD70 in renal cell carcinoma.

## Key findings

- AlloCAR70-NKT cells were successfully generated from CD34+ hematopoietic stem cells.
- The cells showed cytotoxic activity against RCC cell lines in vitro.
- A clinically guided culture method was used for ex vivo differentiation.

## Abstract

CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) (AlloCAR70-NKT) cells derived from cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the therapeutic efficacy of AlloCAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro.

For complete details on the use and execution of this protocol, please refer to Li et al.1

•Generation of AlloCAR70-NKT cells from hematopoietic stem and progenitor cells•Ex vivo differentiation of AlloCAR70-NKT cells via a clinically guided culture method•Evaluation of antitumor capacity of AlloCAR70-NKT cells against renal cell carcinoma

Generation of AlloCAR70-NKT cells from hematopoietic stem and progenitor cells

Ex vivo differentiation of AlloCAR70-NKT cells via a clinically guided culture method

Evaluation of antitumor capacity of AlloCAR70-NKT cells against renal cell carcinoma

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) (AlloCAR70-NKT) cells derived from cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the evaluation of the therapeutic efficacy of AlloCAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro.