convertibleCARs: A chimeric antigen receptor system for flexible control of activity and antigen targeting.

We have developed a chimeric antigen receptor (CAR) platform that functions as a modular system to address limitations of traditional CAR therapies. An inert form of the human NKG2D extracellular domain (iNKG2D) was engineered as the ectodomain of the CAR to generate convertibleCARTM-T cells. These cells were specifically directed to kill antigen-expressing target cells only in the presence of an activating bispecific adapter comprised of an iNKG2D-exclusive ULBP2-based ligand fused to an antigen-targeting antibody (MicAbodyTM). Efficacy against Raji tumors in NSG mice was dependent upon doses of both a rituximab-based MicAbody and convertibleCAR-T cells. We have also demonstrated that the exclusive ligand-receptor partnering enabled the targeted delivery of a mutant form of IL-2 to selectively promote the expansion of convertibleCAR-T cells in vitro and in vivo. By altering the Fv domains of the MicAbody or the payload fused to the orthogonal ligand, convertibleCAR-T cells can be readily targeted or regulated.

Evaluating the genomic and sequence integrity of human ES cell lines; comparison to normal genomes.

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count, and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation, using both microarray and sequence-based analyses, provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines, the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly, genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes, such as tumor suppressors and genetic disease genes, do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens, such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents.

5-benzyloxytryptamine as an antagonist of TRPM8.

5-Benzyloxytryptamine 19 was found to act as an antagonist of the TRPM8 ion-channel. For example, 19 had an IC(50) of 0.34 µM when menthol was used as the stimulating agonist. Related commercially-available tryptamine derivatives showed diminished, or no antagonist activity at TRPM8. The structural similarity of 5-benzyloxytryptamine to other literature TRPM8 antagonists was noted.

Oxime derivatives related to AP18: Agonists and antagonists of the TRPA1 receptor.

AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapoutian. However, no detailed structure-activity relationships around 1 have been disclosed. Thus, a small number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1 activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.

Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine.

The functional activity of a number of ion channels is highly sensitive to large changes in temperature. Foremost among these are the thermosensing TRP channels which include cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members. TRPV1, also known as the vanilloid receptor (VR1), is activated by ligands such as capsaicin, acidic pH, and heat (an increase in temperature to approximately 42 degrees C will lead to channel opening). Screening against the thermal gating of TRPV1 is generally performed using perfusion systems or water baths for temperature control, in conjunction with electrophysiology or Ca2 + influx readouts for direct functional assessment. These approaches are very useful, but have limited throughput or minimal thermo-temporal control. A standard real-time PCR machine with standard microplates allowed us to combine fluorescent Ca2 + detection with precise temperature manipulation to develop a homogeneous (Z' = 0.53), cell-based assay that uses temperature as the agonist. A temperature response curve of TRPV1 was obtained, which provided a T50 of 46.1 degrees C, and IC50 values against heat agonism were determined for known TRPV1 antagonists. Furthermore, we expanded this approach to a cold-activated ion channel, TRPM8. We developed and validated an analytical technique with broad applications for the study and screening of temperature-gated ion channels.

Efficient synthesis of a novel, twisted and stable, electroluminescent "twistacene".

[reaction: see text] A twistacene, 6,8,15,17-tetraphenyl-1.18,4.5,9.10,13.14-tetrabenzoheptacene (3), was synthesized using a mild and novel bisbenzyne precursor. It was characterized by X-ray crystallography, NMR, UV-vis, and IR spectroscopies, as well as cyclic voltammetry and DFT calculations. The heptacene derivative possesses a nonpropeller twist topology and is unusually stable for a highly conjugated oligoacene. In addition, it is fluorescent, with a quantum efficiency of 15%. Distortion from planarity, mostly due to the phenyl substituents, causes only marginal changes in electronic properties and is beneficial for redox reversibility, which is required for efficient OLED devices.