High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Surface Functionalised Optical Fibre for Detection of Hydrogen Sulphide.

Dysregulated production of hydrogen sulphide in the human body has been associated with various diseases including cancer, underlining the importance of accurate detection of this molecule. Here, we report the detection of hydrogen sulphide using fluorescence-emission enhancement of two 1,8-naphthalimide fluorescent probes with an azide moiety in position 4. One probe, serving as a control, featured a methoxyethyl moiety through the imide to evaluate its effectiveness for hydrogen sulphide detection, while the other probe was modified with (3-aminopropyl)triethoxysilane (APTES) to enable direct covalent attachment to an optical fibre tip. We coated the optical fibre tip relatively homogeneously with the APTES-azide fluorophore, as confirmed via x-ray photoelectron spectroscopy (XPS). The absorption and fluorescence responses of the control fluorophore free in PBS were analysed using UV-Vis and fluorescence spectrophotometry, while the fluorescence emission of the APTES-azide fluorophore-coated optical fibres was examined using a simple, low-cost optical fibre-based setup. Both fluorescent probes exhibited a significant increase (more than double the initial value) in fluorescence emission upon the addition of HS- when excited with 405 nm. However, the fluorescence enhancement of the coated optical fibres demonstrated a much faster response time of 2 min (time for the fluorescence intensity to reach 90% of its maximum value) compared to the control fluorophore in solution (30 min). Additionally, the temporal evolution of fluorescence intensity of the fluorophore coated on the optical fibre was studied at two pH values (7.4 and 6.4), demonstrating a reasonable overlap and confirming the compound pH insensitivity within this range. The promising results from this study indicate the potential for developing an optical fibre-based sensing system for HS- detection using the synthesised fluorophore, which could have significant applications in health monitoring and disease detection.

In-situ detection scheme for EGFR gene with temperature and pH compensation using a triple-channel optical fiber biosensor.

An advanced multi-parameter optical fiber sensing technology for EGFR gene detection based on DNA hybridization technology is demonstrated in this paper. For traditional DNA hybridization detection methods, temperature and pH compensation can not be realized or need multiple sensor probes. However, the multi-parameter detection technology we proposed can simultaneously detect complementary DNA, temperature and pH based on a single optical fiber probe. In this scheme, three optical signals including dual surface plasmon resonance signal (SPR) and Mach-Zehnder interference signal (MZI) are excited by binding the probe DNA sequence and pH-sensitive material with the optical fiber sensor. The paper proposes the first research to achieve simultaneous excitation of dual SPR signal and Mach-Zehnder interference signal in a single fiber and used for three-parameter detection. Three optical signals have different sensitivities to the three variables. From a mathematical point of view, the unique solutions of exon-20 concentration, temperature and pH can be obtained by analyzing the three optical signals. The experimental results show that the exon-20 sensitivity of the sensor can reach 0.07 nm nM-1, and the limit of detection is 3.27 nM. The designed sensor gives a fast response, high sensitivity, and low detection limit, which is important for the field of DNA hybridization research and for solving the problems of biosensor susceptibility to temperature and pH.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Fluorescence-based aluminum ion sensing using a surface-functionalized microstructured optical fiber.

The first microstructured optical fiber-based sensor platform for aluminum ions using a surface-attached derivative of lumogallion (3), a known fluorescence-based indicator, has been fabricated. These fibers allow for strong evanescent field interactions with the surrounding media because of the small core size while also providing the potential for real-time and distributed measurements. The fluorescence response to aluminum ions was first demonstrated by applying the procedure to glass slides. This was achieved through the covalent attachment of the fluorophore to a polyelectrolyte-coated glass surface and then to the internal holes of a suspended-core microstructured optical fiber to give an effective aluminum sensor. Whereas the sensor platform reported is fabricated for aluminum, the approach is versatile, with applicability to the detection of other ions.

[3 + 2] Cycloaddition of acetylenes with azides to give 1,4-disubstituted 1,2,3-triazoles in a modular flow reactor.

The cycloaddition of acetylenes with azides to give the corresponding 1,4-disubstituted 1,2,3-triazoles is reported using immobilised reagents and scavengers in pre-packed glass tubes in a modular flow reactor.

Intensive induction chemotherapy and delayed irradiation in the management of parameningeal rhabdomyosarcoma.

PURPOSE: To evaluate local control, event-free survival, and overall survival for patients with parameningeal (PM) rhabdomyosarcoma (RMS) treated with intensive chemotherapy and delayed irradiation. PATIENTS AND METHODS: Thirteen consecutive patients with PM RMS were treated with an institutional protocol from 1992 to 1998 at the University of Washington/Children's Hospital and Regional Medical Center and Deaconess Medical Center. Patients received intensive chemotherapy consisting of vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide prior to radiotherapy. Irradiation was delayed, in contrast to current Intergroup Rhabdomyosarcoma Study Group (IRSG) recommendations. RESULTS: Median follow-up was 39 months. Eleven patients had high-risk features, including five with intracranial extension. All patients responded to the intensive chemotherapy, with 38% exhibiting a complete response and the remaining 62% a partial response. Radiation was administered a median of 21 weeks from initiation of chemotherapy. The Kaplan-Meier estimate of 5-year local control was 92%, with event-free survival and overall survival rates of 83%. CONCLUSIONS: With intensive induction chemotherapy, delayed irradiation for PM RMS does not compromise local control. Event-free survival and overall survival rates compare favorably with recently IRSG trials employing early irradiation. Delaying irradiation allows for intensification of chemotherapy and could permit response-based radiation volume and/or dose modifications, which could decrease treatment-related morbidity.