Journal: bioRxiv

Article Title: Engineering multivalent Fc display for FcγR blockade

doi: 10.1101/2024.01.20.576357

Figure Lengend Snippet: a, Domain organization of the scFc. Different domains and regulatory elements are colored and highlighted. Two hIgG1 Fc regions, each composed of the hinge region, the CH2, and the CH3 domains, were genetically fused via a flexible linker. SNAPtag and SpyTag were added to the N- and C-terminus, respectively. Protein expression in mammalian cells was driven by a cytomegalovirus promoter (CMV), and protein secretion was induced by an optimized signal peptide (SP). b, Cartoon representation of the predicted scFc model. The flexible linker connecting the SNAPtag (in cyan) and the Fc region (in red) as well as the Fc interdomain flexible linker are depicted in black. The Cys residues located at the reconstructed hinge region are shown in yellow. c, Schematics of the SPR experimental designs and corresponding sensorgrams. ECD-FcγRIIa, ECD-FcγRIIb, or ECD-FcγRIIIa were covalently attached to an activated dextran matrix, and scFc binding was assessed using single-cycle kinetic analysis, where the analyte was injected in three increasing concentrations (0.15, 0.44, and 1.3 µM). Black lines represent the measured curves and red lines represent the bivalent analyte binding model curve fits. One representative experiment of n=2 independent repeats is shown. The dissociation constant (K D ) and kinetic rate constants are shown as mean ± std. d, Schematic diagram of the scFc-VLP platform, consisting of SpyCatcher-VLP and SpyTag-scFc. Lysine and arginine residues present in SpyCatcher and SpyTag, respectively, spontaneously react to form covalent isopeptide bonds. e, Silver stained SDS–PAGE of VLP, scFc-VLP variants (s1, s2, s3, s4, and s5), and scFc. f, Conjugation efficiencies of scFc-VLP variants (s2, s3, s4, and s5) at 1 h, 24 h, 48 h, and 72 h reaction times, estimated using SDS-PAGE densitometry (n=1). g, DLS characterization of VLP and scFc-VLPs (n=3, mean ± std). D H , hydrodynamic diameter. h, Autocorrelation curves derived from FCS measurements on ATTO 488-labeled scFc-VLPs. One component fits are shown as dashed black lines. i, Estimation of the number of ScFc molecules per VLP based on the FCS molecular brightness analysis, for scFc-VLPs generated using scFc concentrations of 0.05 µM (s1), 0.1 µM (s2), 0.2 µM (s3), 0.5 µM (s4), and 1 µM (s5) (n=1).

Article Snippet: After surface adhesion, the cells were fixed with pre-warmed 4% PFA/PBS for 12 min at 25°C, washed 3x with PBS, blocked with blocking solution [3 % Bovine Serum Albumin (BSA)/0.1 % Triton X-100 in PBS] for 90 min at 25°C, and incubated with 5 μg/ml anti-human FcγRIIa (Origene, clone OTI9G5; 5 μg/ml) in blocking solution at 4°C overnight.

Techniques: Expressing, Binding Assay, Injection, Staining, SDS Page, Conjugation Assay, Derivative Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Engineering multivalent Fc display for FcγR blockade

doi: 10.1101/2024.01.20.576357

Figure Lengend Snippet: a, Schematic of an ensemble of energetically plausible SNAPtag conformers linked to the Fc domain (depicted in a ghostly white) of the scFc. The center of mass (COM) of each conformer is denoted by a dot. Red dots highlight SNAPtag conformers clashing with the FcγRIIa (depicted in red), while green dots indicate non-interfering conformers. b , Distribution of scFc length. The scFc length is estimated based on the distance between the Fc and SNAPtag domains of the scFc, determined by the sum of their radii of gyration and the distance between their COMs. The mean scFc length is estimated to be 9.7 ± 0.9 nm.

Article Snippet: After surface adhesion, the cells were fixed with pre-warmed 4% PFA/PBS for 12 min at 25°C, washed 3x with PBS, blocked with blocking solution [3 % Bovine Serum Albumin (BSA)/0.1 % Triton X-100 in PBS] for 90 min at 25°C, and incubated with 5 μg/ml anti-human FcγRIIa (Origene, clone OTI9G5; 5 μg/ml) in blocking solution at 4°C overnight.

Techniques:

Journal: bioRxiv

Article Title: Engineering multivalent Fc display for FcγR blockade

doi: 10.1101/2024.01.20.576357

Figure Lengend Snippet: a-c, Schematics of the SPR assay designs and sensorgrams. a, ECD-FcγRIIa, b, ECD-FcγRIIb, and c, ECD-FcγRIIIa were covalently attached to an activated dextran matrix and binding of VLP and scFc-VLPs (s1, s2, s3, s4, and s5) was assessed using single-cycle kinetic analysis. The analyte was injected in three increasing concentrations (0.15, 0.44, and 1.3 µM). One representative example of two independent experiments is shown. d, Comparison of K D values of scFc-VLP binding to the different FcγRs tested, estimated by bivalent analyte model fitted to data shown in a-c (n=2). e, Schematic of SPR assay and SPR traces of binding of VLP, scFc, and scFc-VLPs (s4 and s6) to human C1q complex (n=1). For the construction of s6 we used VLP at 4 µM and scFc at 2 µM.

Article Snippet: After surface adhesion, the cells were fixed with pre-warmed 4% PFA/PBS for 12 min at 25°C, washed 3x with PBS, blocked with blocking solution [3 % Bovine Serum Albumin (BSA)/0.1 % Triton X-100 in PBS] for 90 min at 25°C, and incubated with 5 μg/ml anti-human FcγRIIa (Origene, clone OTI9G5; 5 μg/ml) in blocking solution at 4°C overnight.

Techniques: SPR Assay, Binding Assay, Injection, Comparison

Journal: bioRxiv

Article Title: Engineering multivalent Fc display for FcγR blockade

doi: 10.1101/2024.01.20.576357

Figure Lengend Snippet: a and b, Binding traces of scFc-VLPs and scFc to CM5 chip immobilized with low levels (∼350 RU) (a) and high levels (∼1000 RU) (b) of ECD-FcγRIIa. Analyte concentration was 1.3 µ M. Each row represents an independent experiment. The scFc binding trace displayed an atypical behavior marked by the presence of an inflection point (annotated with a black arrow on the scFc sensorgrams), suggesting distinct interaction modes when the scFc binding was assessed on CM5 chips immobilized with high levels of FcγRIIa ECD. This behavior, distinct from that observed for scFc-VLPs, could be explained by the different accessibility of scFc to FcγRIIa immobilized on the dextran matrix compared with scFc-VLPs. c and d, Summary graphs of the scFc-VLPs response levels at 180 s (annotated as a dashed black line in the sensorgrams on a and b ) for low ( c) and high ( d) immobilization levels of FcγRIIa. (n=3, mean ± std). e, Avidity effects exhibited by scFc-VLPs on dextran-free C1 chip. Binding of scFc, VLP, and scFc-VLPs (s1, s2, s3, s4, s5) to ECD-FcγRIIa immobilized on a dextran free C1 sensor chip, suitable to probe the binding of multivalent and high molecular weight complexes to immobilized proteins. The scFc binding was negligible and the binding trace had a similar shape compared with the other samples. Two independent experiments are shown.

Article Snippet: After surface adhesion, the cells were fixed with pre-warmed 4% PFA/PBS for 12 min at 25°C, washed 3x with PBS, blocked with blocking solution [3 % Bovine Serum Albumin (BSA)/0.1 % Triton X-100 in PBS] for 90 min at 25°C, and incubated with 5 μg/ml anti-human FcγRIIa (Origene, clone OTI9G5; 5 μg/ml) in blocking solution at 4°C overnight.

Techniques: Binding Assay, Concentration Assay, High Molecular Weight

Journal: bioRxiv

Article Title: Engineering multivalent Fc display for FcγR blockade

doi: 10.1101/2024.01.20.576357

Figure Lengend Snippet: a, Confocal microscopy images of FcγRII-stained THP-1 cells treated with VLP, scFc-VLPs (s2, s4, s5), and scFc for 30 min at 37°C. HAIgG, IC, or IgG treatment served as controls. Arrows indicate large receptor aggregates. Each image is representative of at least 10 images from n=3 independent experiments. Cyan: DAPI stained cell nuclei; Yellow: FcγRII stained with PE-labeled anti-FcγRII antibody. Scale bar: 5 µm. b, Schematic of DNA-PAINT method used to image FcγRIIa in THP-1 cells. The DNA-PAINT signal is generated from transient interactions between short, fluorescently labeled DNA probes and complementary strands conjugated to secondary nanobodies bound to a FcγRII specific antibody (one or two nanobodies can bind to one FcγRII antibody). c, Number of detected clusters per 10 µm detected in THP-1cells left untreated (control) or treated with VLP, scFc-VLPs (s2, s4, s5), scFc, HAIgG, IC, or IgG for 30 min at 37°C. Horizontal lines indicate the median for each condition. P-values determined by a two-tailed Mann– Whitney test. d, Number of localizations per cluster detected in THP-1cells left untreated (control) or treated with VLP, scFc-VLPs (s2, s4, s5), scFc, HAIgG, IC, or IgG for 30 min at 37°C. Horizontal lines indicate the median for each condition. e, Immunoblotting analysis of FcγRII in THP-1 cells treated with VLP, scFc-VLPs (s2, s4, s5), scFc, HAIgG, and IgG for 30 min at 37°C. Untreated cells serve as controls. Cleaved FcγRII product denoted by *. The gel is representative of n=3 independent experiments. f, Phospho-flow cytometry of THP-1 cells for pSyk after a 3 min treatment with VLP, scFc-VLPs (s2, s4, s5), and scFc. Untreated cells served as a control (n=4, median ± std). g, NFAT activation in FcγRII Jurkat NFAT-luc reporter cells stimulated with VLP, scFc-VLPs (s2, s4, s5) and scFc. Luciferase activity measured after 8 h incubation. Untreated cells used as a control (n=3, median ± std). h, Percentage of FITC-labeled IgG-coated latex beads phagocytosed by THP-1 cells after 30-min treatment with VLP, scFc-VLPs (s1, s2, s3, s4, s5), and scFc (n=3, median ± std). Statistical analysis for c and d was performed by two-tailed Mann-Whitney test and statistical analysis for f-h was performed using one-way ANOVA with Tukey’s multiple comparisons post-hoc test. P-values calculated based on the comparison between the control and the indicated group.

Article Snippet: After surface adhesion, the cells were fixed with pre-warmed 4% PFA/PBS for 12 min at 25°C, washed 3x with PBS, blocked with blocking solution [3 % Bovine Serum Albumin (BSA)/0.1 % Triton X-100 in PBS] for 90 min at 25°C, and incubated with 5 μg/ml anti-human FcγRIIa (Origene, clone OTI9G5; 5 μg/ml) in blocking solution at 4°C overnight.

Techniques: Confocal Microscopy, Staining, Labeling, Generated, Control, Two Tailed Test, MANN-WHITNEY, Western Blot, Flow Cytometry, Activation Assay, Luciferase, Activity Assay, Incubation, Comparison

Journal: Experimental Hematology & Oncology

Article Title: Preclinical investigations and a first-in-human phase 1a trial of JS007, a novel anti-CTLA-4 antibody, in patients with advanced solid tumors

doi: 10.1186/s40164-024-00567-7

Figure Lengend Snippet: JS007 binding affinity to CTLA-4 protein and Fc receptor by Biacore

Article Snippet: The affinity of JS007 for Fc receptors was determined using Biacore (GE Healthcare Life Sciences), in which anti-His tag antibody was coupled to a CM5 chip surface, followed by capturing the His-tagged recombinant human FcγRIIIa (CD16a) V176 (Junmeng Biomedical, 20200421), FcγRIIIa (CD16a) F176 (Junmeng Biomedical, 20200421), FcγRIIa (CD32a) R167 (Sino Biological, 10734-H08C), FcγRI (CD64) (Sino Biological, 10256-H08S), and FcRn (Sino Biological, CT009-H08H).

Techniques: Binding Assay, Recombinant