RESUMO
Using gene modification of hematopoietic stem cells (HSC) to create persistent generation of multilineage immune effectors to target cancer cells directly is proposed. Gene-modified human HSC have been used to introduce genes to correct, prevent, or treat diseases. Concerns regarding malignant transformation, abnormal hematopoiesis, and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. Truncated epidermal growth factor receptor (EGFRt) was tested as a suicide gene system co-delivered with anti-CD19 chimeric antigen receptor (CAR) to human HSC. Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. An antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used, incubating target cells with leukocytes and monoclonal antibody cetuximab to determine the percentage of surviving cells. In vivo, gene-modified HSC were engrafted into NSG mice with subsequent treatment with intraperitoneal cetuximab. Persistence of gene-modified cells was assessed by flow cytometry, droplet digital polymerase chain reaction (ddPCR), and positron emission tomography (PET) imaging using 89Zr-Cetuximab. Cytotoxicity was significantly increased (p = 0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1 µg/mL compared to EGFRt+ cells without cetuximab and non-transduced cells with or without cetuximab, at all effector:target ratios. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p = 0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging confirmed ablation with a decrease of an average of 82.5% after cetuximab treatment. These results give proof of principle for CAR-modified HSC regulated by a suicide gene. Further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers, should also be evaluated.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Cetuximab/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos/genética , Antígenos CD19/genética , Antígenos CD19/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunoterapia , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/terapia , Tomografia por Emissão de Pósitrons , Receptores de Antígenos Quiméricos/genética , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells.
