RESUMO
Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed. We hypothesize that gene modification of haematopoietic stem cells (HSC) with an anti-CD19 chimeric antigen receptor (CAR) will produce a multi-lineage, persistent immunotherapy against B-lineage malignancies that can be controlled by the HSVsr39TK suicide gene. High-titer third-generation self-inactivating lentiviral constructs were developed to deliver a second-generation CD19-specific CAR and the herpes simplex virus thymidine kinase HSVsr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC were transduced with such lentiviral vectors and evaluated for function of both CAR and HSVsr39TK. Satisfactory transduction efficiency was achieved; the addition of the suicide gene did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. NSG mice transplanted with gene-modified human HSC showed CAR expression not significantly different between transduced cells with or without HSVsr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage. Treatment with ganciclovir led to significant ablation of gene-modified cells in mouse tissues. Haematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies; following HSC collection, a portion of the cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Linfoma de Células B/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Antígenos CD28/imunologia , Ganciclovir/administração & dosagem , Humanos , Imunoterapia , Células Jurkat , Lentivirus/genética , Linfoma de Células B/imunologia , Camundongos , Recidiva Local de Neoplasia/terapia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Transdução GenéticaRESUMO
Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity.