Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: A In vitro growth inhibitory activity (GIA) of monoclonal antibodies against the 3D7 clone of P. falciparum at 0.5 mg/ml, individual points indicate the value of each independent replicate, the bar indicates the mean, and errors bars indicate standard deviation across independent replicates ( n = 2, Cy.002, Cy.005 and Cy.010, n = 3 Cy.003, Cy.004, Cy.007, Cy.009). B GIA dilution curve starting at 2 mg/ml of each inhibitory antibody, including chimeric human c12 and 8A7. Points show mean of triplicates and error bars indicate standard deviation. Curve fit used a four-parameter dose-response curve with the upper bound constrained to 100% GIA. All GIA curves were repeated at least twice with a single representative dilution curve shown here. C Competing interactions between mAbs based on the data in panel ( D ). Red box contains inhibitory mAbs and black lines indicate mAbs that compete with one another. D Competition matrix of all nine anti-CyRPA mAbs. The value contained in each box is the mean OD 405 of a given mAb pair across triplicate; a value <0.25 was taken to be negative binding of the detection mAb. Competing combinations are highlighted in pink. Non-competing pairs are shown in blue. E Kinetic parameters for the binding of each antibody to CyRPA, as determined by surface plasmon resonance analysis.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: In Vitro, Activity Assay, Bioprocessing, Standard Deviation, Binding Assay, SPR Assay

Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: Crystal structures of Fab fragments of Cy.002 (green), Cy.003 (yellow), Cy.004 (dark pink), and Cy.007 (light blue), each bound to CyRPA (blue). Within the inset box (bottom right) we show three different views of CyRPA, each rotated by 90°. The upper row “cartoon” images show the six blades of the CyRPA β-propeller. The lower row shows the CyRPA surface (blue) with binding epitopes indicated using the same colours as the antibody structures.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: A Cy.004 Fab fragment (pink) bound to CyRPA (blue) with the two calcium ions shown as orange spheres. The inset shows a close up of the calcium-binding site with the coordinating residues labelled. B The absolute abundance of different metal ions within the Cy.004:CyRPA complex as determined by microPIXE measurements. Bars indicate the mean and standard deviation across n = 3 replicate measurements. C SPR sensorgrams of Cy.004 (immobilized) binding to CyRPA in TBS with or without 1 mM MgCl 2 or 1 mM CaCl 2 . Each curve shows the outcome of a two-fold dilution series of CyRPA from 100 to 3.1 nM. The black line indicates measured response while the red line indicates 1:1 curve fit.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: A Relative binding of 100 nM of each mAb to the reconstituted recombinant RCR complex divided by their binding to CyRPA at the same concentration, as determined by SPR analysis. Dotted line indicates relative binding of 1.0. B Crystal structure of RH5 (yellow) bound to Fab fragments of growth inhibitory mAb R5.016 (red) and CyRPA-blocking non-growth inhibitory mAb R5.015 (light blue). C Comparison of a model of the CyRPA:RH5 complex, derived from cryo-electron microscopy (PDB: 6MPV), with the structures of CyRPA bound to Cy.002 and RH5 bound to R5.015 shows that both Cy.002 and R5.015 will prevent the formation of the CyRPA:RH5 complex through a steric mechanism.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: Binding Assay, Recombinant, Concentration Assay, Blocking Assay, Comparison, Derivative Assay, Cryo-Electron Microscopy

Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: A Predicted growth inhibitory activity (GIA) based on Bliss additivity (red) compared to measured GIA in blue for a mAb combination where one was held at 30 μg/ml (title) and the other held at 20% GIA ( X -axis). Complete dilution curves can be seen in Supplementary Fig. . Combinations were measured twice with a single representative experiment shown here. Bar indicates the mean across triplicate measurements. B Heat map summary of the fold improvement over Bliss additivity from panel ( A ). C SPR sensorgrams of Cy.003 and Cy.007 binding to CyRPA and Fab:CyRPA complexes of Cy.002, Cy.004, or Cy.009 in TBS with 1 mM CaCl 2 . The black lines indicate the measured response while red lines indicate the curve fit. Each graph shows a five-step two-fold dilution curve starting from 500 nM. D GIA of Cy.009 compared to that of a Cy.003/Cy.009 1:1 mixture (i.e., 1 mg/ml = 1 mg/ml Cy.009 or 0.5 mg/ml Cy.009 + 0.5 mg/ml Cy.003). Each individual point is the mean of a triplicate measurement with error bars indicating the standard deviation. n = 5 and n = 6 independent experiments of complete dilution curves for Cy.009 and Cy.003/Cy.009 respectively. Curve fit used a four-parameter dose-response curve with the upper bound constrained to 100% GIA. EC 50 shift determined through extra-sum-of-squares F test ( F = 77.68, DFn = 1, DFd = 80), p < 0.0001. E Summary of changes in dissociation rate constant ( k d ) for SPR data shown in ( C ). Horizontal line indicates the mean and error bars show the standard deviation across n = 2 independent experiments.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: Activity Assay, Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

doi: 10.1038/s41467-022-28601-4

Figure Lengend Snippet: Crystal structure of CyRPA (blue) bound to Fab fragments of Cy.003 (yellow), Cy.004 (pink), and Cy.007 (light blue). The lower panels show close-up views of each of the interfaces between Cy.004 and Cy.007 (left), Cy.003 and Cy.007 (centre), and Cy.004 and Cy.003 (right). The residues forming heterotypic interactions are labelled and bonds are indicated with yellow dashed lines.

Article Snippet: After 60 min, the well contents were discarded and then washed 3× with PBS-T. Next, 50 μL of the “competing” anti-CyRPA biotinylated mAb (biotinylated using antibody biotinylation kit, according to the manufacturer’s instructions (Thermo Fisher Scientific)) at 10 μg/ml was added to each well and incubated for 60 min, followed by 3× wash steps with PBS-T. 50 μL streptavidin-alkaline phosphatase (Thermo Fisher Scientific) diluted 1/1000 in PBS-casein, was then added, incubated for 15 min, and washed 6× with PBS-T.

Techniques: