A novel anti-B7-H3 chimeric antigen receptor from a single-chain antibody library for immunotherapy of solid cancers

B7-H3 (CD276) has emerged as a target for cancer immunotherapy by virtue of consistent expression in many malignancies, relative absence from healthy tissues, and an emerging role as a driver of tumor immune inhibition. Recent studies have reported B7-H3 to be a suitable target for chimeric antigen receptor-modified T cell (CAR-T) therapy using CARs constructed from established anti-B7-H3 antibodies converted into single-chain Fv format (scFv). We constructed and screened binders in an scFv library to generate a new anti-B7-H3 CAR-T with favorable properties. This allowed access to numerous specificities ready formatted for CAR evaluation. Selected anti-human B7-H3 scFvs were readily cloned into CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine, and proliferation assays. Two binders with divergent complementarity determining regions were found to show optimal antigen-specific cytotoxicity and cytokine secretion. One binder in second-generation CD28-CD3ζ CAR format induced sustained in vitro proliferation on repeat antigen challenge. The lead candidate CAR-T also demonstrated in vivo activity in a resistant neuroblastoma model. An empirical approach to B7-H3 CAR-T discovery through screening of novel scFv sequences in CAR-T format has led to the identification of a new construct with sustained proliferative capacity warranting further evaluation

'We don't learn democracy, we live it!' : consulting the pupil voice in Scottish schools

As the education for citizenship agenda continues to impact on schools, there is a need to begin the discussion around examining the kind of initiatives that can push it forward. In Scotland the proposals should, it is argued, permeate the curriculum throughout the school. Yet there is the fear that the responsibility of all can become the responsibility of none. This paper examines, through case study research carried out by the authors, initiatives in schools designed to take forward the citizenship agenda in the light of children's rights. The first two relate to firstly the impact of pupil councils in primary schools and secondly the impact of discussing controversial issues in the primary classroom. The third outlines the impact on values and dispositions of developing more participatory, democratic practice in the classroom. The paper concludes by calling for both more initiatives of this type and more evaluation of their worth

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) combine T cell activation with antibody-mediated tumor antigen specificity, bypassing the need for T cell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using T cells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2(+) T cells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection

Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically

Anti-tumor activity without on-target off-tumor toxicity of GD2-Chimeric Antigen Receptor T cells in patients with neuroblastoma

The reprogramming of a patient’s immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity

Redirection to the bone marrow improves T cell persistence and antitumor functions

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15–dependent homeostatic expansion and promoted the differentiation of memory precursor–like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells

NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells

Epicutaneous application of Aldara cream containing the TLR7 agonist imiquimod (IMQ) to mice induces skin inflammation that exhibits many aspects of psoriasis, an inflammatory human skin disease. Here we show that mice depleted of B cells or bearing interleukin (IL)-10-deficient B cells show a fulminant inflammation upon IMQ exposure, whereas ablation of NFATc1 in B cells results in a suppression of Aldara-induced inflammation. In vitro, IMQ induces the proliferation and IL-10 expression by B cells that is blocked by BCR signals inducing NFATc1. By binding to HDAC1, a transcriptional repressor, and to an intronic site of the Il10 gene, NFATc1 suppresses IL-10 expression that dampens the production of tumour necrosis factor-α and IL-17 by T cells. These data indicate a close link between NFATc1 and IL-10 expression in B cells and suggest NFATc1 and, in particular, its inducible short isoform, NFATc1/αA, as a potential target to treat human psoriasis

Modified titania nanomaterials for sunscreen applications - reducing free radicals generation and DNA damage

Inorganic UV absorbers such as titanium oxide and zinc oxide are in common use in formulations of sunscreens and cosmetics. These materials strongly absorb UV, and show no UV induced degradation over time. However, the dissipation mechanism of the UV energy is not often considered. It is demonstrated that the primary de-excitation mechanism of inorganic sunscreen components is via the surface of the particle and leads to the creation of free radicals. Substantial free radical generation is observed under simulated solar energy with all sunscreen grades of titanium oxide and zinc oxide. Free radicals are implicated in a number of potential health issues such as skin aging. Doping trap centres into titanium oxide and zinc oxide results in the total elimination of free radical generation under simulated solar energy. The trap centres provide sites within the oxide via which it is energetically favourable for photogenerated charge carriers to de-excite. Thus the charge is confined on sites within the host lattice and cannot migrate to the particle surface and create free radicals. The absorbed UV energy is converted to long wavelength light and heat. This effect is demonstrated by electron spin resonance, a primary measure of free radical generation, and by a plasmid nicking assay. The plasmid nicking assay demonstrates the effect of free radicals on supercoiled DNA