Antitumor efficacy and safety of unedited autologous CD5.CAR T cells in relapsed/refractory mature T-cell lymphomas.

ABSTRACT: Despite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.

Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.

Lip trill effects on vocal function, vocal pitch, and harmonics-to-noise ratio: A multiple baseline study of three vocally healthy females

Background@#The lip trill is a semi-occluded vocal tract exercise found to have positive outcomes on vocal parameters of healthy individuals after a single session. However, the effect of several lip trill sessions has not been studied.@*Objective@#This study investigated the effect of a 3- to 4-week lip trill exercise program on Maximum Phonation Time (MPT), Maximum Phonation Frequency Range (MPFR), Harmonics-to-Noise Ratio (HNR), and mean Speaking Fundamental Frequency (mean SFF).@*Methods@#Three vocally untrained healthy females with perceptually normal voices participated in a multiple-baseline, single-case experimental research. Five to seven direct training sessions were randomly assigned. Self-implementation was encouraged thereafter. Participants were assessed on all outcome measures for 15 sessions. Data were visualized and inspected. Cohen’s d was also derived using Standardized Mean Difference for single-subject research.@*Results@#Visual and statistical analyses revealed a decreased MPT when training was introduced, increased MPFR variability during the training phase, and increased stability in mean SFF post-training. Perceived effects include vocal freedom, decreased laryngeal stress, and vocal conditioning. Vocal outcomes in one participant yielded a significant effect on MPFR and mean SFF and a small effect on HNR.@*Conclusion@#There is evidence of lip trill effect on pitch control and vocal conditioning. Findings also revealed that motivation, exercise frequency, and adherence could contribute to positive gains in vocally healthy speakers. Further investigation in consideration of the study findings and limitations is warranted.

Engineered off-the-shelf therapeutic T cells resist host immune rejection.

Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.

Integrin αvß8-expressing tumor cells evade host immunity by regulating TGF-ß activation in immune cells.

TGF-ß is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-ß (L-TGF-ß) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-ß. Binding of L-TGF-ß to integrin αvß8 results in activation of TGF-ß. We engineered and used αvß8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect ß8 in human tumors. Inhibition of αvß8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. ß8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-ß, suggesting that tumor cell αvß8 serves as a platform for activating cell-surface L-TGF-ß presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to ß8 inhibition with major increases in chemokine and tumor-eliminating genes. High ß8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvß8 is a PD-1/PD-L1-independent immunotherapeutic target.

The Role of AIRE in the Immunity Against Candida Albicans in a Model of Human Macrophages.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1ß, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.

Role of IL-17A in murine models of COPD airway disease.

Small airway fibrosis is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Chronic inflammatory cells accumulate around small airways in COPD and are thought to play a major role in small airway fibrosis. Mice deficient in α/ß T cells have recently been shown to be protected from both experimental airway inflammation and fibrosis. In these models, CD4+Th17 cells and secretion of IL-17A are increased. However, a pathogenic role for IL-17 in specifically mediating fibrosis around airways has not been demonstrated. Here a role for IL-17A in airway fibrosis was demonstrated using mice deficient in the IL-17 receptor A (il17ra) Il17ra-deficient mice were protected from both airway inflammation and fibrosis in two different models of airway fibrosis that employ COPD-relevant stimuli. In these models, CD4+ Th17 are a major source of IL-17A with other expressing cell types including γδ T cells, type 3 innate lymphoid cells, polymorphonuclear cells, and CD8+ T cells. Antibody neutralization of IL-17RA or IL-17A confirmed that IL-17A was the relevant pathogenic IL-17 isoform and IL-17RA was the relevant receptor in airway inflammation and fibrosis. These results demonstrate that the IL-17A/IL-17 RA axis is crucial to murine airway fibrosis. These findings suggest that IL-17 might be targeted to prevent the progression of airway fibrosis in COPD.

TGF-ß-Dependent Dendritic Cell Chemokinesis in Murine Models of Airway Disease.

Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of Ag surveillance and Ag presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1ß) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 µm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvß8, a critical activator of TGF-ß. αvß8-Mediated TGF-ß activation is known to enhance IL-1ß-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvß8, ccl20, and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli.

A critical role for dendritic cells in the evolution of IL-1ß-mediated murine airway disease.

Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease and are refractory to current treatments. How and whether chronic inflammation contributes to airway fibrosis remain controversial. In this study, we use a model of chronic obstructive pulmonary disease airway disease utilizing adenoviral delivery of IL-1ß to determine that adaptive T cell immunity is required for airway remodeling because mice deficient in α/ß T cells (tcra(-/-)) are protected. Dendritic cells (DCs) accumulate around chronic obstructive pulmonary disease airways and are critical to prime adaptive immunity, but they have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor ccr6 both protect from adenoviral IL-1ß-induced airway adaptive T cell immune responses and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/ß T cells and driven by DCs, is critical to airway fibrosis.

Selective targeting of TGF-ß activation to treat fibroinflammatory airway disease.

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-ß (TGF-ß) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-ß is expressed in a latent form that requires activation. The integrin αvß8 (encoded by the itgb8 gene) is a receptor for latent TGF-ß and is essential for its activation. Expression of integrin αvß8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvß8 (B5) inhibited TGF-ß activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-ß activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvß8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvß8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-ß pathway to treat fibroinflammatory airway diseases.