GMP-Compliant Manufacturing of NKG2D CAR Memory T Cells Using CliniMACS Prodigy

Abstract

Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA− memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA− cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.

SARS-CoV-2-Specific Memory T Lymphocytes From COVID-19 Convalescent Donors: Identification, Biobanking, and Large-Scale Production for Adoptive Cell Therapy

“…In previous studies, this population of CD45RA – memory T cells showed no alloreactivity when compared with the CD45RA + counterpart ( Fernández et al, 2017 ). These cells were mainly CD4+ ( Fernández et al, 2019 ) and showed effectiveness against viral infections ( Triplett et al, 2015 ). Phenotypically, we found that CD45RA – memory T cells were fully capable of producing IFN-γ in the presence of SARS-CoV-2-specific peptides.…”

Transposon-Based CAR T Cells in Acute Leukemias: Where Are We Going?

“…However, most of them still need to be coupled to different devices for the sequential steps of the production process [72][73][74][75][76]. A fully automated closed system, the CliniMACS prodigy (Miltenyi Biotec, Germany), has been recently proposed and validated for lentiviral transduced CAR T-cells in several clinical experimental settings [77][78][79].…”

Chimeric antigen receptor-engineered natural killer cells for cancer immunotherapy

“…Use of CAR T cells created using spacers with suboptimal length results in off-target effects. The downstream CAR component(s) are co-stimulatory domain(s) that can be shared between CAR NK and CAR T cells [ 42 , 43 ], followed by CD3ζ. With the rapid development of DNA synthesis technology, many CAR sequences are now synthesized by a vendor if the DNA sequences encoding the scFv are known.…”