Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins.

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.

Quantifying persistence in the T-cell signaling network using an optically controllable antigen receptor.

T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response, which is hypothesized to depend on T cells combining stimuli from multiple antigen-presenting cell interactions into a more potent response. To quantify the capacity for T cells to accomplish this, we have developed an antigen receptor that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling. We observe limited persistence within the T-cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 min, and directly show sustained proximal receptor signaling is required to maintain gene transcription. T cells thus primarily accumulate the outputs of gene expression rather than integrate discrete intracellular signals. Engineering optical control in a clinically relevant chimeric antigen receptor (CAR), we show that this limited signal persistence can be exploited to increase CAR-T cell activation threefold using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.

Dissecting cellular mechanics: Implications for aging, cancer, and immunity.

Cells are dynamic structures that must respond to complex physical and chemical signals from their surrounding environment. The cytoskeleton is a key mediator of a cell's response to the signals of both the extracellular matrix and other cells present in the local microenvironment and allows it to tune its own mechanical properties in response to these cues. A growing body of evidence suggests that altered cellular viscoelasticity is a strong indicator of disease state; including cancer, laminopathy (genetic disorders of the nuclear lamina), infection, and aging. Here, we review recent work on the characterization of cell mechanics in disease and discuss the implications of altered viscoelasticity in regulation of immune responses. Finally, we provide an overview of techniques for measuring the mechanical properties of cells deeply embedded within tissues.

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing.

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array.

The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly (13)C- and (15)N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR.

New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling.

Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae. We show that just like transmembrane arrays, cytoplasmic clusters contain trimers-of-receptor-dimers organized in 12-nm hexagonal arrays. In contrast to transmembrane arrays, however, cytoplasmic clusters comprise two CheA/CheW baseplates sandwiching two opposed receptor arrays. We further show that cytoplasmic fragments of normally transmembrane E. coli chemoreceptors form similar sandwiched structures in the presence of molecular crowding agents. Together these results suggest that the 12-nm hexagonal architecture is fundamentally important and that sandwiching and crowding can replace the stabilizing effect of the membrane. DOI: http://dx.doi.org/10.7554/eLife.02151.001.

Heat management strategies for solid-state NMR of functional proteins.

Modern solid-state NMR methods can acquire high-resolution protein spectra for structure determination. However, these methods use rapid sample spinning and intense decoupling fields that can heat and denature the protein being studied. Here we present a strategy to avoid destroying valuable samples. We advocate first creating a sacrificial sample, which contains unlabeled protein (or no protein) in buffer conditions similar to the intended sample. This sample is then doped with the chemical shift thermometer Sm2Sn2O7. We introduce a pulse scheme called TCUP (for Temperature Calibration Under Pulseload) that can characterize the heating of this sacrificial sample rapidly, under a variety of experimental conditions, and with high temporal resolution. Sample heating is discussed with respect to different instrumental variables such as spinning speed, decoupling strength and duration, and cooling gas flow rate. The effects of different sample preparation variables are also discussed, including ionic strength, the inclusion of cryoprotectants, and the physical state of the sample (i.e. liquid, solid, or slurry). Lastly, we discuss probe detuning as a measure of sample thawing that does not require retuning the probe or using chemical shift thermometer compounds. Use of detuning tests and chemical shift thermometers with representative sample conditions makes it possible to maximize the efficiency of the NMR experiment while retaining a functional sample.

Failure of the Durom Metasul acetabular component.

UNLABELLED: Large-diameter metal-on-metal articulations reportedly provide better stability and range of motion than smaller diameter bearings. We therefore asked whether a large-diameter (44- to 50-mm) metal-on-metal articulation (Durom) would eliminate dislocation and provide similar functional scores and clinical and radiographic failure rates as those with 28-mm articulation. We prospectively followed 181 patients (207 hips) who had a large-diameter articulation implanted between May 2006 and November 2007. We compared these patients with a historical control of 54 patients who had a small-diameter (28-mm Metasul) articulation. All patients had a Harris hip score and a self-assessment of outcome and radiographic followup. The minimum followup was 1 year (mean, 1.6 years; range, 1-2 years). During the followup period, we performed revisions on 29 patients (30 hips [15%]) with 21 of 29 (72%) having radiographic criteria of loosening. Thirteen retrieved cups and acetabular tissue were examined histologically. Twenty-eight of 151 unrevised patients had radiographic impending failure; 12 without revision had clinical failure. Eight patients (nine hips) had both clinical failure and impending radiographic failure. Cup inclination was 41.3 degrees +/- 5.4 degrees and anteversion was 20.2 degrees +/- 7 degrees. The revision rate and quality of clinical results were unacceptable as compared with our historical controls. We do not recommend use of the Durom implant. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Exposure to fall hazards and safety climate in the aircraft maintenance industry.

PROBLEM: Falls represent a significant occupational hazard, particularly in industries with dynamic work environments. This paper describes rates of noncompliance with fall hazard prevention requirements, perceived safety climate and worker knowledge and beliefs, and the association between fall exposure and safety climate measures in commercial aircraft maintenance activities. METHODS: Walkthrough observations were conducted on aircraft mechanics at two participating facilities (Sites A and B) to ascertain the degree of noncompliance. Mechanics at each site completed questionnaires concerning fall hazard knowledge, personal safety beliefs, and safety climate. Questionnaire results were summarized into safety climate and belief scores by workgroup and site. Noncompliance rates observed during walkthroughs were compared to the climate-belief scores, and were expected to be inversely associated. RESULTS: Important differences were seen in fall safety performance between the sites. The study provided a characterization of aircraft maintenance fall hazards, and also demonstrated the effectiveness of an objective hazard assessment methodology. Noncompliance varied by height, equipment used, location of work on the aircraft, shift, and by safety system. DISCUSSION: Although the expected relationship between safety climate and noncompliance was seen for site-average climate scores, workgroups with higher safety climate scores had greater observed noncompliance within Site A. Overall, use of engineered safety systems had a significant impact on working safely, while safety beliefs and climate also contributed, though inconsistently. IMPACT ON INDUSTRY: The results of this study indicate that safety systems are very important in reducing noncompliance with fall protection requirements in aircraft maintenance facilities. Site-level fall safety compliance was found to be related to safety climate, although an unexpected relationship between compliance and safety climate was seen at the workgroup level within site. Finally, observed fall safety compliance was found to differ from self-reported compliance.

Energetics of subdomain movements and fluorescence probe solvation environment change in ATP-bound myosin.

Energetics of conformational changes experienced by an ATP-bound myosin head detached from actin was studied by all-atom explicit water umbrella sampling simulations. The statistics of coupling between large scale domain movements and smaller scale structural features were examined, including the closing of the ATP binding pocket, and a number of key hydrogen bond formations shown to play roles in structural and biochemical studies. The statistics for the ATP binding pocket open/close transition show an evolution of the relative stability from the open state in the early stages of the recovery stroke to the stable closed state after the stroke. The change in solvation environment of the fluorescence probe Trp507 (scallop numbering; 501 in Dictyostelium discoideum) indicates that the probe faithfully reflects the closing of the binding pocket as previously shown in experimental studies, while being directly coupled to roughly the early half of the overall large scale conformational change of the converter domain rotation. The free energy change of this solvation environment change, in particular, is -1.3 kcal/mol, in close agreement with experimental estimates. In addition, our results provide direct molecular level data allowing for interpretations of the fluorescence experiments of myosin conformational change in terms of the de-solvation of Trp side chain.

Impact of nerve sparing technique on patient self-assessed outcomes after radical perineal prostatectomy.

PURPOSE: We investigated the impact of nerve sparing technique on erectile function, urinary continence and health related quality of life after radical perineal prostatectomy using a validated self-assessment questionnaire. MATERIALS AND METHODS: The Expanded Prostate Cancer Index Composite questionnaire was administered preoperatively and at defined intervals after surgery to 265 patients who underwent radical perineal prostatectomy at 2 institutions between January 2001 and December 2004. Of these patients 153 (57.7%) and 112 (42.3%) underwent nonnerve sparing and nerve sparing approaches, respectively. Kaplan-Meier analysis was used to determine time to recovery of erectile function (erections firm enough for intercourse) and urinary continence (0 pads per day). RESULTS: Median patient age was 60.6 years. Median followup was 15 months. In multivariate analysis preoperative erectile function (p = 0.005) and preservation of the neurovascular bundle (p = 0.018) were independent predictors of earlier recovery of erectile function, with hazard ratios of 2.3 (95% CI 1.2-4.6) and 4.0 (95% CI 1.5-10.3), respectively. Median time to recovery of urinary continence was 4.8 months in the nerve sparing group and 6.1 months in the nonnerve sparing group (p = 0.001). In multivariate analysis nerve sparing technique (p = 0.001, HR 1.4, 95% CI 1.1-1.9) and age (p = 0.012, HR 1.7, 95% CI 1.3-2.2) were independent predictors of recovery of continence. CONCLUSIONS: This analysis suggests that nerve sparing radical perineal prostatectomy is associated with improved recovery of urinary continence and favorable health related quality of life scores and, therefore, should be considered a viable alternative to other nerve sparing approaches.

The anatomic radical perineal prostatectomy: an outcomes-based evolution.

OBJECTIVE: Radical prostatectomy is the most effective treatment for localized prostate cancer. With increasing use of minimally invasive treatment methods, clinical outcomes are becoming important assessment tools to compare one option to another. Perineal prostatectomy is modified to incorporate contemporary surgical ideas, including preservation of cavernosal nerve bundles, sphincteric urethra at the prostatic apex, and the bladder neck. METHODS: Objective parameters and physician-reported clinical outcomes are collected prospectively on 704 consecutive patients undergoing radical perineal prostatectomy (RPP) by one surgeon. The technique described herein is the current state of evolution of RPP. The enclosed digital video is edited from two recent nerve-sparing RPPs. RESULTS: Freedom from prostate-specific antigen (PSA) detectability by stage is 94.5%, 80.0%, and 81.5% for organ-confined, specimen-confined, and margin-positive disease with actual 5-yr follow-up. Margins are positive in 18% of cases. By 1, 3, 6 mo and 1 yr, 52%, 71%, 85%, and 94% of the men are free from using pads. Although >97% of nerve-spared patients have spontaneous erections, >80% can penetrate to complete intercourse. CONCLUSIONS: This method of prostatectomy is able to achieve complete cancer resection while preserving urinary and sexual function in the majority of men presenting with clinically localized prostate cancer. The simplicity and minimally invasive nature of this procedure contribute to a short recovery and low overall cost of therapy. The anatomic RPP is a cost-efficient, outcome-effective minimally invasive method of treating men with localized prostate cancer.

Early patient self-assessed outcomes of nerve-sparing radical perineal prostatectomy.

OBJECTIVES: To compare the early recovery of erectile function and urinary continence in patients undergoing nerve-sparing and non-nerve-sparing radical perineal prostatectomy (RPP) using a validated patient self-assessment instrument. METHODS: Eighty-eight patients who underwent RPP by a single surgeon between January 2002 and July 2003 were given a validated patient self-assessment questionnaire, the Expanded Prostate Cancer Index Composite, preoperatively and subsequently at 3- to 6-month intervals. Of the 88 patients, 40 underwent non-nerve-sparing and 48 nerve-sparing RPP. The time to recover erectile function and urinary continence, as well as sexual and urinary health-related quality of life was analyzed using the Kaplan-Meier method and a multivariate Cox proportional hazards model. RESULTS: Nerve-sparing RPP was a predictor of the time to recover erections sufficient for intercourse (P = 0.013). In a multivariate regression model adjusting for baseline erectile function, age, and prostate size, preservation of the neurovascular bundle (P = 0.007) was an independent predictor of the time to recover erectile function sufficient for intercourse (hazard ratio 5.3; 95% confidence interval 1.2 to 23.1). The median time to recover urinary continence, defined as no pad use, was 4.7 months in the nerve-sparing group compared with 5.6 months in the non-nerve-sparing group (P = 0.021). In multivariate analysis, nerve-sparing status was an independent predictor (P = 0.003) of an earlier recovery of continence (hazard ratio 2.2; 95% confidence interval 1.3 to 3.6). CONCLUSIONS: Patient self-assessed outcomes support a benefit of nerve-sparing RPP for the postoperative recovery of erectile function and urinary continence. Future studies with larger patient numbers and longer follow-up are necessary to validate these findings.

Radical perineal prostatectomy: cost efficient, outcome effective, minimally invasive prostate cancer management.

OBJECTIVE: Localized prostate cancer is a common disease for which minimally invasive treatment methods are being explored. Perineal prostatectomy, as a historical open procedure, is modified to incorporate contemporary surgical ideas. There is relatively little in the literature regarding modern adaptations of perineal prostatectomy. This method of anatomic radical perineal prostatectomy has been developed to accomplish a minimally invasive method of achieving goals of disease control and preservation of genito-urinary functions. METHODS: Prospective outcome data is accumulated on 508 consecutive radical perineal prostatectomies by a single surgeon. Pathologic stage and PSA detectability are measures of cancer control. Pad use and ability to complete intercourse measure urinary and sexual function. General complications and other outcome measures are evaluated. RESULTS: Freedom from PSA detectability by pathologic stage is 96.3%, 79.4%, and 69.4% for organ confined, specimen confined and margin positive in the absence of seminal vesical invasion with an average 4 years follow up (3-114 months). Margins are positive in 18% of cases. The average cancer size is 9.4g and 36% of cases have extracapsular invasion. By the first, third, sixth months and one year, 38%, 65%, 88% and 96% are free of pad use and report being dry. While over 80% of nerve-spared patients enjoy the return of spontaneous erectile function, the men with bilateral nerve preservation note earlier and more complete return of function. There are no cardiopulmonary complications or deaths. Transfusions occurred in 1%, none in the past 400 cases. Average total hospital charges are USD$4889.00 in 1999 and 2000. Anterior urethral strictures, anastomotic strictures and fecal urgency/stress flatus occur 2%, 2% and 2-4%, respectively. CONCLUSIONS: This method of prostatectomy is able to achieve complete cancer resection while preserving urinary and sexual function as well as laparoscopic or retropubic prostatectomy. The simplicity and minimally invasive nature of this procedure contribute to a short recovery and low overall cost of therapy. The anatomic radical perineal prostatectomy is a cost-efficient, outcome effective minimally invasive method of treating men with localized prostate cancer.