Lentiviral vector purification using genetically encoded biotin mimic in packaging cell

Lentiviral vectors (LVs) are powerful tools in gene therapy that have recently witnessed an increasing demand in both research and clinical applications. Current LVs purification represents the main bottle neck in their application as several methods are employed which are time consuming, cumbersome and yield low recoveries. The aim of this project was to develop a one-step method to specifically and efficiently purify LVs, with high vector yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging 293T cells were genetically engineered with biotin mimicking synthetic peptides and different cell membrane anchoring strategies for optimal streptavidin binding were tested. We have identified a flanked disulphide-constrained peptide, termed Ctag (ECHPQGPPCIEGRK), displayed on a CD8α stalk to be the most promising. LVs were modified with Ctag by its random incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. The expression of Ctag on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As Ctag binds streptavidin in the nanomolar range, we hypothesised that gentle elution from streptavidin matrix should occur by biotin’s competitive binding. Accordingly, addition of micromolar concentrations of biotin to captured LVs resulted in an overall yield of ≥60%. Analysis of eluted LVs revealed high purity levels, with a ≤3-log and 2-log reduction of DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using streptavidin monolith affinity chromatography and preliminary results were encouraging with 20% overall yield. In conclusion, we developed a single-step affinity chromatography which allows specific purification and concentration of infectious vectors modified with a biotin mimic. Based on intended usage, efficient LV purification can be achieved using both magnetic beads and column chromatography. This method will be of valuable use for both research and clinical applications of LVs

Large-scale manufacturing of base-edited chimeric antigen receptor T cells

Base editing is a revolutionary gene-editing technique enabling the introduction of point mutations into the genome without generating detrimental DNA double-stranded breaks. Base-editing enzymes are commonly delivered in the form of modified linear messenger RNA (mRNA) that is costly to produce. Here, we address this problem by developing a simple protocol for manufacturing base-edited cells using circular RNA (circRNA), which is less expensive to synthesize. Compared with linear mRNA, higher editing efficiencies were achieved with circRNA, enabling an 8-fold reduction in the amount of RNA required. We used this protocol to manufacture a clinical dose (1 × 108 cells) of base-edited chimeric antigen receptor (CAR) T cells lacking expression of the inhibitory receptor, PD-1. Editing efficiencies of up to 86% were obtained using 0.25 μg circRNA/1 × 106 cells. Increased editing efficiencies with circRNA were attributed to more efficient translation. These results suggest that circRNA, which is less expensive to produce than linear mRNA, is a viable option for reducing the cost of manufacturing base-edited cells at scale

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials

Functional antibody and T-cell immunity following SARS-CoV-2 infection, including by variants of concern, in patients with cancer: the CAPTURE study

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study (NCT03226886) integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2-positive, 94 were symptomatic and 2 patients died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies, 82% had neutralizing antibodies against WT, whereas neutralizing antibody titers (NAbT) against the Alpha, Beta, and Delta variants were substantially reduced. Whereas S1-reactive antibody levels decreased in 13% of patients, NAbT remained stable up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment-specific, but presented compensatory cellular responses, further supported by clinical. Overall, these findings advance the understanding of the nature and duration of immune response to SARS-CoV-2 in patients with cancer

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Immunogenicity of COVID-19 Vaccination in Patients With Axial Spondyloarthritis: A Monocentric Prospective Belgian Cohort Study.

info:eu-repo/semantics/publishe

Waning humoral immunity of SARS-CoV-2 vaccination in a rheumatoid arthritis cohort and the benefits of a vaccine booster dose.

We aimed to assess SARS-CoV-2 spike-specific antibody kinetics postvaccination and the benefit of a mRNA vaccine booster dose in rheumatoid arthritis (RA) patients treated with immunosuppressive drugs.info:eu-repo/semantics/publishe

Association de différents profils fonctionnels de cellules T spécifiques d’antigènes mycobactériens à la tuberculose active et latente.

info:eu-repo/semantics/nonPublishe

MyéLome multiple révélé par deux infections invasives à Streptococcus pneumoniae :À propos d'un cas

We here report the case of a 45-year-old patient who presented 2 invasive infections with Streptococcus Pneumoniae in 9 months period (two bacteriemias, associated with pneumonia then meningitis), in which the workup of a possible immunodeficiency led to the diagnosis of multiple myeloma, an usual pathology at this age. Although S. Pneumoniae infections are a frequent complication of multiple myeloma, they do not usually lead to its diagnosis. The mechanisms of this greater susceptibility to encapsulated germs as S. pneumoniae in multiple myeloma are discussed. In clinical practice, this case reminds us that the occurrence of at least two severe bacterial infections is a criterion to perform a search for immunodeficiency.SCOPUS: ar.jinfo:eu-repo/semantics/publishe