RESUMO
The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.
Assuntos
Linfócitos T CD4-Positivos , Imunoterapia Adotiva , Leucemia , Receptores de Antígenos Quiméricos , Antígenos CD19/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Humanos , Leucemia/imunologia , Leucemia/terapia , Receptores de Antígenos Quiméricos/imunologia , Fatores de TempoRESUMO
Chimeric antigen receptor (CAR)-engineered T cells have demonstrated remarkable success in the treatment of B cell malignancies. FDA approval of these therapies represents a watershed moment in the development of therapies for cancer. Despite the successes of the last decade, many patients will unfortunately not experience durable responses to CAR therapy. Emerging research has shed light on the biology responsible for these failures, and further highlighted the hurdles to broader success. Here, we review the recent research identifying how interactions between cancer cells and engineered immune cells result in resistance to CAR therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/imunologia , Imunoterapia Adotiva/efeitos adversos , Neoplasias/tratamento farmacológico , Receptores de Antígenos Quiméricos/imunologia , Humanos , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/imunologiaRESUMO
CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling. Significance: CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.
Assuntos
Leucemia Linfocítica Crônica de Células B , Receptores de Antígenos Quiméricos , Humanos , Linfócitos T , Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Antígenos Quiméricos/genética , Linfócitos B , Ligante de CD40/genéticaRESUMO
Chimeric antigen receptor (CAR) T-cell therapies have evolved from a research tool to a paradigm-shifting therapy with impressive responses in B cell malignancies. This review summarizes the current state of the CAR T-cell field, focusing on CD19- and B cell maturation antigen-directed CAR T-cells, the most developed of the CAR T-cell therapies. We discuss the many challenges to CAR-T therapeutic success and innovations in CAR design and T-cell engineering aimed at extending this therapeutic platform beyond hematologic malignancies.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Antígenos CD19 , Humanos , Imunoterapia Adotiva , Neoplasias/terapia , Linfócitos TRESUMO
BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq. CONCLUSION: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.