3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αß T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αß-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Tc-99m Radiolabeled Peptide p5 + 14 is an Effective Probe for SPECT Imaging of Systemic Amyloidosis.

PURPOSE: Systemic peripheral amyloidosis is a rare disease in which misfolded proteins deposit in various organs. We have previously developed I-124 labeled peptide p5 + 14 as a tracer for positron emission tomography imaging of amyloid in patients. In this report, we now document the labeling efficiency, bioactivity, and stability of Tc-99m labeled p5 + 14 for single-photon emission computed tomography (SPECT) imaging of amyloidosis, validated in a mouse model of systemic amyloidosis. PROCEDURES: Radiochemical yield, purity, and biological activity of [(99m)Tc]p5 + 14 were documented by instant thin-layer chromatography (ITLC), SDS-PAGE and a quantitative amyloid fibril pulldown assay. The efficacy and stability were documented in serum amyloid protein A (AA) amyloid-bearing or wild-type (WT) control mice imaged with SPECT/X-ray computed tomography (CT) at two time points. The uptake and retention of [(99m)Tc]p5 + 14 in hepatosplenic amyloid was evaluated using region of interest (ROI) and tissue counting measurements. RESULTS: Tc-99m p5 + 14 was produced with a radiochemical yield of 75 % with greater than 90 % purity and biological activity comparable to that of radioiodinated peptide. AA amyloid was visualized by SPECT/CT imaging with specific uptake seen in amyloid-laden organs at levels ∼5 folds higher than in healthy mice. ROI analyses of decay-corrected SPECT/CT images showed <20 % loss of radiolabel from the 1 to 4 h imaging time points. Biodistribution data confirmed the specificity of the probe accumulation by amyloid-laden organs as compared to non-diseased tissues. CONCLUSION: [(99m)Tc]p5 + 14 is a specific and stable radiotracer for systemic amyloid in mice and may provide a convenient and inexpensive alternative to imaging of peripheral amyloidosis in patients.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aß and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aß- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aß and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 µM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

Investigating the structural impact of the glutamine repeat in huntingtin assembly.

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington's disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ42P10) and compared the results with those obtained for the wild-type (NtQ22P10). Our results show that the aggregation pathway of NtQ22P10 is very different from that of NtQ42P10, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ42P10 are monomer and dimer. The divergent pathways ultimately result in NtQ22P10 fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ42P10 fibrils present a better fit to the Perutz ß-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.

Human anti-Aß IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Soluble non-fibrillar assemblies of amyloid-beta (Aß) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aß immunotherapy is a promising and advanced therapeutic strategy, but the precise Aß species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aß conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aß dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aß extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aß's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aß monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aß NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aß NAbs are warranted.

Anti-amyloidogenic activity of IgGs contained in normal plasma.

INTRODUCTION: We have previously shown that a subpopulation of naturally occurring human IgGs has therapeutic potential for the amyloid-associated disorders. These molecules cross-react with conformational epitopes on amyloidogenic assemblies, including amyloid beta (Abeta) protein fibrils that are a pathological hallmark of Alzheimer's disease. MATERIALS AND METHODS: Using our europium-linked immunosorbant assay, we established that approximately 95% of 260 screened donor plasma samples had amyloid fibril-reactive IgGs and Abeta conformer-reactive IgGs with minimal binding to Abeta monomers. Anti-amyloidogenic reactivity was diverse and attributed to Abeta targeting multiple fibril-related binding sites and/or variations in multidentate binding. RESULTS AND DISCUSSION: There was no correlation between anti-fibril and anti-oligomer reactivity and donor age (19 to 60 years old) or gender. These findings demonstrate the inherent but diverse anti-amyloidogenic activity of natural IgGs contained in normal plasma. CONCLUSION: Our studies provide support for investigating the clinical significance and physiological function of this novel class of antibodies.