RESUMEN
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Inmunoterapia/métodos , Linfoma de Células B/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Células Cultivadas , Epítopos/inmunología , Humanos , Células Jurkat , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
PURPOSE: The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. EXPERIMENTAL DESIGN: A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. RESULTS: Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. CONCLUSIONS: DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent in vivo antitumor efficacy.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/trasplante , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales de Origen Murino/genética , Línea Celular Tumoral , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Dominios Proteicos/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To determine whether the expression of either interleukin-10 (IL-10) or tumor necrosis factor (TNF) inhibitor genes in transduced rabbit lacrimal gland epithelial cells suppresses lymphocyte proliferation in an autologous mixed cell reaction, an apparent in vitro model of autoimmune dacryoadenitis. METHODS: Purified lacrimal gland epithelial cells, transduced with an adenovirus vector carrying either viral IL-10 or TNF-inhibitor genes, were used to study their effects on the proliferation of autologous lymphocytes as monitored by 3H-thymidine incorporation in a mixed cell reaction. After transduction, both epithelial cells and lymphocytes were cultured separately for 2 days and then epithelial cells were irradiated. Equal numbers of both cell types were then cocultured together for 5 days. Cocultures were pulsed with 3H-thymidine and isotope incorporation was determined. Gene expression was detected by enzyme-linked immunosorbent assay and Western blots. RESULTS: Lymphocyte proliferation was stimulated by epithelial cells and 3H-thymidine incorporation was significantly greater in these cocultures than in controls. The proliferation was significantly diminished in the presence of transduced cells producing either IL-10 or TNF inhibitor. CONCLUSIONS: Transduction of lacrimal gland epithelial cells with adenovirus vectors encoding for either IL-10 or TNF-inhibitor proteins leads to expression of functional proteins capable of suppressing lymphocyte proliferation. Thus, lacrimal gland epithelial cells are a plausible target for gene therapy methods meant to produce immunoregulatory proteins.