Chimeric antigen receptor (CAR) T-cell therapy was envisioned as a mechanism to re-direct effector T-cells to eliminate tumor cells. CARs are composed of the variable region of an antibody that binds ...a native cancer antigen coupled to the signaling domain of a TCR and co-stimulatory molecules. Its success and approval by the U.S. Food and Drug Administration for the treatment of B-cell malignancies revolutionized the immunotherapy field, leading to extensive research on its possible application for other cancer types. In this review, we will focus on the evolution of CAR-T cell therapy outlining current technologies as well as major obstacles for its wide application. We will highlight achievements, the efforts to increase efficacy and to evolve into an off-the-shelf treatment, and as a possible future treatment for non-cancer related diseases.
Expression of the transcription factor Zbtb1 is required for normal lymphoid development. We report in the present study that Zbtb1 maintains genome integrity in immune progenitors, without which ...cells undergo increased DNA damage and p53-mediated apoptosis during replication and differentiation. Increased DNA damage in Zbtb1-mutant (ScanT) progenitors was due to increased sensitivity to replication stress, which was a consequence of inefficient activation of the S-phase checkpoint response. Increased p53-mediated apoptosis affected not only lymphoid but also myeloid development in competitive bone marrow chimeras, and prevention of apoptosis by transgenic Bcl2 expression and p53 deficiency rescued lymphoid as well as myeloid development from Zbtb1-mutant progenitors. Interestingly, however, protection from apoptosis rescued only the early stages of T cell development, and thymocytes remained arrested at the double-negative 3 developmental stage, indicating a strict requirement of Zbtb1 at later T cell developmental stages. Collectively, these results indicate that Zbtb1 prevents DNA damage in replicating immune progenitors, allowing the generation of B cells, T cells, and myeloid cells.
NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK ...effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model. Lack of in vivo efficacy correlated with downregulation of CD3 levels in target T cells after coculture with CD3-CAR effector cells. The CAR-NK framework greatly improved the efficacy of CARs leading to increased degranulation, cytokine secretion and elimination of the tumor xenograft by CD5-CAR-NK effector cells. Finally, all CAR constructs were similarly effective to eliminate malignant T cells in vitro. Our results show that the NK-CAR framework improves the activity of CARs in NK cells and that CD5 would be a better target than CD3 for T-cell malignancies, as dynamic downregulation of target expression may affect in vivo efficacy.
Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive ...pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist.
A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP.
Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient's surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature.
Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted.
NCT, number NCT02859454 , registered August 9, 2016.
A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during ...differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRalpha locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4(lo)CD8(lo). These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4(lo)CD8(lo) stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4(lo)CD8(lo) stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo.
Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4+ gamma delta NKT cells. In this study, we show that PLZF is ...also required for the development and function of IL-17+ gamma delta T cells. We observed that PLZF is expressed in fetal-derived invariant V gamma 5+ and V gamma 6+ gamma delta T cells, which secrete IFN- gamma and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of V gamma 6+ cells, leading to reduced numbers of mature CD27-CD44+ phenotype capable of secreting IL-17. Although PLZF was not required for V gamma 5+ gamma delta T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to V gamma 6+ gamma delta T cells. The impaired effector differentiation of PLZF-deficient V gamma 6+ gamma delta T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27+CD44- V gamma 6+ gamma delta T cells, which was required for their differentiation into mature CD27-CD44+ IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4+ T cells, but it also controls the development of IL-17+ gamma delta T cells.
The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a ...role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5'-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes.
Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for ...deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.
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