geneart strings dna fragments  (Thermo Fisher)

Journal: Nature Communications

Article Title: Balancing the efficacy and safety of chimeric antigen receptor T-cell therapy by affinity combination

doi: 10.1038/s41467-026-71354-7

Figure Lengend Snippet: a Schematic of the scFv design for periplasmic expression in E. coli and CAR construct structures for retroviral gene delivery. b Surface plasmon resonance (SPR) analysis of scFv binding kinetics, including dissociation constant ( K D ), association rate ( k on ) and dissociation rate ( k off ). Each dot represents an individual replicate ( n = 3; JCAR021 n = 4, dash indicates the mean measured with the capture of 50 nM scFv for 60 s with a 10 µL/min flow rate). c Schematic representation of epitope mapping to determine similarities in the recognition sites of JCAR017 and JCAR021 scFvs. d SPR sensorgram showing soluble CD19 binding to JCAR021 scFv in the presence or absence of JCAR017 scFv. e Representative flow cytometry plots of transduction efficiency (EGFRt + ) and surface expression level (STII + ) of JCAR017, JCAR021 and reference CAT CAR in primary human T cells. f Quantification of Nur77-tdTomato expression in CAR-transduced Jurkat cells stimulated with CD19 + GFP + Raji tumor cells at an effector-to-target (E:T) ratio of 1:1 or cultured in medium, background-subtracted. Each dot represents the mean of technical triplicates. Bars indicate the mean + SD across biological replicates ( n = 7; CAT n = 5). Additionally, representative histograms displaying Nur77-tdTomato signal are shown. g Intracellular cytokine production quantified after 5 h co-culture with CD19 + GFP + Raji cells at the indicated E:T ratios. Each dot represents the mean of technical triplicates of one independent biological replicate ( n = 3). PMA/ionomycin was used as a positive control. h Representative xCelligence impedance-based killing curves with CD19 + (left) or wildtype (WT) (right) HEK cells, including quantification by the area under the curve (AUC), are shown. Each dot represents a technical replicate of one out of three independent biological experiments. Data is presented as mean + SD ( n = 3). Statistical analyses were performed for ( b , f ) using one-way ANOVA for multiple comparisons between the individual scFvs ( b ) or with JCAR017 as reference ( f ). * p < 0.05, ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: scFv DNA strings were cloned into a pASG-IBAwt2 vector (IBA Lifesciences) and expressed in electrocompetent E. coli JM83 (IBA Lifesciences).

Techniques: Expressing, Construct, Retroviral, SPR Assay, Binding Assay, Flow Cytometry, Transduction, Cell Culture, Co-Culture Assay, Positive Control

Journal: Nature Communications

Article Title: Balancing the efficacy and safety of chimeric antigen receptor T-cell therapy by affinity combination

doi: 10.1038/s41467-026-71354-7

Figure Lengend Snippet: a Schematic of the scFv sequence used to generate fluorescently labeled scFv FLEXamers along with the CAR sequence for retroviral transduction to construct the affinity library derived from the JCAR021 framework. Mutagenesis regions are indicated by stars. b Principle of the flow cytometry-based assay to assess monomeric scFv:epitope dissociations. c Quantification of scFv dissociation kinetics ( k off -rate) by one-phase exponential decay curve fitting, expressed as dissociation half-life ( t 1/2 ) ( n = 3, 30 min 20 °C). d Comparative analysis of TCR and scFv koff-rates ( n ≥ 3, 30 min 4 °C). e Transduction efficiency (EGFRt + ) (upper left), CAR-expression (STII + ) (upper right) and representative flow cytometry plots (below) in primary human T cells ( n = 3). f Fold change in Nur77-tdTomato expression of CAR-transduced Jurkat cells upon stimulation with CD19 + GFP + Raji cells (E:T 1:1) or incubation in medium for 3 h ( n ≥ 5). Representative flow cytometry plots are shown on the right. g Intracellular cytokine production in CAR-engineered primary human T cells following 5 h co-culture with CD19 + GFP + Raji cells at an E:T of 4:1 ( n ≥ 3) with representative flow cytometry histograms. h Impedance-based xCelligence killing curves (below) and quantification of CAR-T-cell killing (upper) as area under the curve (AUC) ( n ≥ 2). i Heatmap summarizing the functional properties of JCAR021, CAR mfunct and CAR mlowfunct in vitro. f – i Data are normalized to the average value of JCAR017. Data are expressed as mean ± SD. Each dot represents the mean of technical replicates for JCAR017 and JCAR021 and the mean of an independent experiment for the JCAR021 mutants. Statistical analyses were performed in ( e – h ) using a one-way ANOVA test for multiple comparisons with JCAR017 as reference. * p < 0.05, ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: scFv DNA strings were cloned into a pASG-IBAwt2 vector (IBA Lifesciences) and expressed in electrocompetent E. coli JM83 (IBA Lifesciences).

Techniques: Sequencing, Labeling, Retroviral, Transduction, Construct, Derivative Assay, Mutagenesis, Flow Cytometry, Expressing, Incubation, Co-Culture Assay, Functional Assay, In Vitro