A closed, autologous bioprocess optimized for TCR-T cell therapies.

Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating engineered TCR-T cells. This bioprocess yielded 5-12 × 10e9 TCR-expressing T cells, transduced at low multiplicity of infections, within 7-10 days, and cells exhibited an enriched memory T-cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leukapheresed cells in a bioreactor without any T-cell or peripheral blood mononuclear cell enrichment steps had a high level of T-cell purity (~97%). Several critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale-up, lowering glycolysis through the addition of 2-deoxy- d-glucose, and modulating interleukin-2 levels, were investigated on their roles in regulating transduction efficiency, cell growth, and T-cell fitness such as T-cell memory phenotype and resistance to activation-induced cell death. The bioprocess described herein supports scale-out feasibility by enabling the processing of multiple patients' batches in parallel within a Grade C cleanroom.

Development of a QPatch-Automated Electrophysiology Assay for Identifying TMEM16A Small-Molecule Inhibitors.

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC50s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents, but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC50s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.

Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide Are Potent TMEM16A Antagonists That Fully Bronchodilate Airways.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor ß-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ∼580,000 compounds. The anthelmintics niclosamide, nitazoxanide, and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting ß-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the ß-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

Age-related homeostatic midchannel proteolysis of neuronal L-type voltage-gated Ca²âº channels.

Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment channels that separate but remain on the plasma membrane. This "midchannel" proteolysis is regulated by channel activity, involves the Ca(2+)-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment channels mimicking the products of midchannel proteolysis do not form active channels on their own but, when properly paired, produce currents with distinct biophysical properties. Midchannel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Midchannel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states.