Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL

Abstract

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent “induced DCs” (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14 + monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34 + cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

“…Transient transfection for packaging of RVs/LVs became an important technology as it bypasses the need of selecting, expanding and characterizing different master packaging cell lines carrying different constructs. RVs/LVs obtained after transient transfection were validated for different types of clinical applications such as gene modification of HSPCs for correction of defective genes (1), for harnessing dendritic cells for active immunotherapy against cancer (34,35) and, more prominently, for gene modification of T cells for different types of adoptive immunotherapies (21).…”

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18

“…Genomic DNA was isolated from 1 x 10 6 transduced or untransduced cells (-20°C frozen cell pellets) with the QIAamp ® DNA blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The determination of the VCN by qPCR was performed as recently described ( 35 ).…”

In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution

“…High titers of the concentrated vectors were obtained, in the ranges of 15 µg/mL HIV-p24 equivalents for LV-G2α/gB and 22 µg/mL HIV-p24 equivalents for LV-DR4/fLuc (Figure 1C). In our laboratory, 1 µg of HIV-p24 equivalents correspond to about 1 × 10 7 infective LV particles measured by quantitative real-time PCR analyses [19].…”

“…LV particles were produced as a third-generation packaging system in transiently transfected 293T cells. Transfection of the transfer plasmid and three packaging plasmids (pMD.G expressing VSV-G; pRSV/Rev expressing Rev; pMDLg/pRRE expressing Gag, Pol, and RRE) was performed as previously reported [19,27]. Briefly, 293T cells were seeded into culture flasks (Sarstedt, Nümbrecht, Germany), and transfection was performed on the next day using polyethylenimine (PEI, Polysciences, Warrington, PA, USA).…”

“…Remarkably, administration of iDCs shortly after HCT profoundly improved the development of lymph node-like structures in HIS-mice, including the presence of follicular T helper (FTh) cells, maturation of antigenspecific terminal effector (TE) T cells, and IgG + B cells [14][15][16][17]. iDCs are currently in clinical development as an adoptive cell therapy to accelerate the reconstitution of the adaptive immunity of immune-compromised patients after transplantation, in order to protect them against human cytomegalovirus (HCMV) reactivation and leukemia relapse [17][18][19]. For research utilization, however, the production of iDCs is technically and logistically demanding.…”