Post-infusion CAR TReg cells identify patients resistant to CD19-CAR therapy.

Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4+Helios+ CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (TReg) cells. Validation cohort analysis upheld the link between higher CAR TReg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR TReg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans.

CD22-directed CAR T-cell therapy induces complete remissions in CD19-directed CAR-refractory large B-cell lymphoma.

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins.

Membrane proteins are responsible for conducting essential biological functions that are necessary for the survival of living organisms. In spite of their physiological importance, limited structural information is currently available as a result of challenges in applying biophysical techniques for studying these protein systems. Electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study the structural and dynamic properties of membrane proteins. However, the application of EPR spectroscopy to membrane proteins in a native membrane-bound state is extremely challenging due to the complexity observed in inhomogeneity sample preparation and the dynamic motion of the spin label. Detergent micelles are very popular membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is important to test whether the protein structure in a micelle environment is the same as that of its membrane-bound state. Lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have been introduced as a potentially good membrane-mimetic system for structural studies of membrane proteins. Recently, we reported on the EPR characterization of the KCNE1 membrane protein having a single transmembrane incorporated into lipodisq nanoparticles. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the more complicated membrane protein system human KCNQ1 voltage sensing domain (Q1-VSD) having four transmembrane helices using site-directed spin-labeling EPR spectroscopy. Characterization of spin-labeled Q1-VSD incorporated into lipodisq nanoparticles was carried out using CW-EPR spectral line shape analysis and pulsed EPR double-electron electron resonance (DEER) measurements. The CW-EPR spectra indicate an increase in spectral line broadening with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated a superior quality of distance measurement with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles when compared to proteoliposomes. These results are consistent with the solution NMR structural studies on the Q1-VSD. This study will be beneficial for researchers working on investigating the structural and dynamic properties of more complicated membrane protein systems using lipodisq nanoparticles.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.