Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Abstract

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CART cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

Assessment of anti-CD20 antibody pre-treatment for augmentation of CAR-T cell therapy in SIV-infected rhesus macaques

“…The genes were subcloned into the pMSGV1 gammaretroviral vector and gammaretroviruses were produced by lipofectamine-mediated transfection of 293T cells. CD4-MBL CAR/CXCR5-T cells were manufactured using the CD4-MBL CAR/CXCR5 gammaretrovirus as outlined previously ( 46 , 47 ). Briefly, the PBMCs were thawed and stimulated with plate-bound anti-CD3 (clone FN18) and soluble anti-CD28 (clone 28.2) for two days prior to retronectin-mediated transduction with gammaretroviral vector at an MOI of 0.5.…”

CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection

“…Gammaretroviruses were produced by lipofectamine-mediated transfection of 293T cells 61 . CD4-MBL CAR/CXCR5-T cells were manufactured using the CD4-MBL CAR/CXCR5 gammaretrovirus as outlined previously 70,98 . Prior to infusion, cells were stained with CTV, an intracellular fluorescent dye, resuspended at a density of 2 × 10 7 cells/mL in RPMI for T0 and in PBS containing 10% autologous serum for all other animals, packed on ice and transported to the WNPRC.…”

The Landscape of CAR-T Cell Clinical Trials against Solid Tumors—A Comprehensive Overview

“…Sixteen clinical trials target several antigens at the same time [ 35 , 36 ], and two clinical trials do not disclose the targeted antigen ( Table 1 ). The top six targeted antigens that are expressed on solid tumors in many different organs are (1) EGFR [ 37 , 38 , 39 , 40 , 41 ] (14 different organs), (2) NKG2D-ligands [ 42 , 43 ] (11 different organs), (3) HER2 [ 12 , 44 , 45 , 46 , 47 , 48 , 49 , 50 ] (11 different organs), (4) B7-H3 (10 different organs), (5) MUC1 (9 different organs), and (6) CEA [ 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ] (9 different organs) ( Table 1 ).…”

“…CAR-T cells can also be engineered to express homing molecules to target these cells to specific tissue locations. A clinical trial performed by the Sun Yat-sen University, Guangzhou, China in collaboration with Bio-gene Technology Co., Ltd., Guangzhou, China uses CAR-T cells specific for EGFR which were additionally transduced with the lymphoid follicle homing molecule CXCR5 [40]. Alternatively, to circumvent problems with homing of CAR-T cells to the tumor site, these cells can be directly infused into the tumor [55,154,196,197].…”