EMBO/soda-nel huggingface dataset:
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 1

Figure 1. Vitreous concentrations of angiopoietins in patients newly diagnosed with retinal diseases and cell model of barrier breakdown testing the interaction of VEGF-A and angiopoietins.

A) Box blot of vitreal ANG-1 levels from newly diagnosed patients diagnosed with wAMD, DR, proliferative DR, and RVO compared to controls (macular whole).

B) Box blot of vitreal ANG-2 levels from patients as in A). The interquartile range of the data is indicated by the box. A non-parametric Kruskal-Wallis analysis (**, P = 0.029) followed by Dunn's method for multiple comparisons was used to show significant differences of the groups to control which are indicated by an asterisks. ANG-1 levels did not differ significantly, but ANG-2 levels were significantly different:control vs AMD (*, P = 0.0451), vs DR (*, P < 0.0001), pDR (*,P < 0.0001) and RVO (P < 0.0001).

C) Schematic of cellular experiment to measure transendothelial resistance in human endothelial cells using transwell filters and CellZscope technology.

D) 24-h concentration of ANG-2 in supernatants of the basal site of the culture stimulated with 5 ng/ml of VEGF-A, 200 ng/ml of ANG-1 or the combination. Error bars show SEM with one sided ANOVA (****, P < 0.0001) and Tukey’s multiple T-test for five independent experiments indicating significance for control vs VEGF-A (**, P = 0.0028); VEGF-A vs ANG-1 (****, P <0.0001); ANG-1 vs VEGF-A & ANG1 (**, P = 0.0061); VEGF-A vs VEGF-A & ANG-1 (ns, P = 0.0625).

E) Human endothelial cells plated on filters were assessed for endothelial barrier function over time after addition of 5 ng/ml VEGF-A alone or in combination with 200 ng/ml ANG-1 or 10 μg/ml anti-ANG-2 or all three test items. The final time point was used for statistical analysis. Error bars show SEM with one sided ANOVA (****, P < 0.0001) and Tukey’s multiple T-test for three independent experiments indicating significance of VEGF-A vs untreated (****, P < 0.0001); vs VEGF-A & ANG-1 & anti-ANG-2 (***, P = 0.0004); vs VEGF-A & anti-ANG-2 (P = 0.038); vs VEGF-A & ANG-1, (P = 0.040). Furthermore, VEGF-A & anti-ANG-2 & ANG-1 is significantly different vs VEGF-A & ANG-1 (*, P = 0.0451) and VEGF-A & anti-ANG-2 (*, P = 0.0474) and untreated is significantly different from vs VEGF-A & ANG-1 (*, P = 0.0009) and VEGF-A & anti-ANG-2 (*, P = 0.0008).

ANOVA, analysis of variance; SEM, standard error mean; ANG-2, angiopoietin-2; DR, diabetic retinopathy; RVO, retinal vein occlusion; wAMD, wet age-related macular degeneration, pDR proliferative diabetic retinopathy; vs, versus
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 3

Figure 3. Reduction of edema by dual VEGF-A and ANG-2 inhibition in late interference model in the retina of JR5558 mice at D60 as revealed by OCT.

A) Bar graph showing the relative change in OCT-measured volume between the outer nuclear layer and the RPE layer following intervention with test articles. Anti-VEGF-A and anti-ANG-2 were given at 5 mg/kg and anti-VEGF-A/ANG-2 at 1, 5, and 10 mg/kg (low, mid, and high dose).

B) Representative OCT images converted to heat maps with the scaling grade given on the right of the figure delineating fluid filled areas of the retina. Baseline (D46) and 14 days treatments of IgG control, anti-VEGF-A, anti-ANG-2, and bispecific anti-VEGF-A/ANG-2 at the end of the experiment (D60) are shown. For the region of interest, the corresponding FA image at D60 is also shown as comparator.

SEM is shown as error bars with n = 6 animals per group, significance is shown by *using one sided ANOVA (**,P = 0.0048) followed by Tukey’s multiple t-test. Significant changes are shown with anti-VEGF-A/ANG-2 high vs anti-ANG-2 (**, P = 0.0069) and IgG control (*, P = 0.018). In addition the comparison of anti-VEGF-A vs anti-VEGF-A/ANG-2 (high) approaching significance (NS, P = 0.072))

FA, fluorescein angiography; NS, not significant; OCT, optical coherence tomography; RPE, retinal pigment epithelium; VS, versus
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 4

Figure 4. Dual inhibition of VEGF-A and ANG-2 reduced the number of IBA-1+ macrophages in the model of sCNV in JR5558 mice.

A) Bar graph of number of IBA-1+ cells in the retina of IgG control, anti-VEGF-A, anti-ANG-2 (all 5 mg/kg IP), and bispecific anti-VEGF-A/ANG-2 (at 10, 5, and 3 mg/kg)-treated mice in the late interference model at D60.B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E–G) anti-VEGF-A/ANG-2 low, mid and high: representative micrographs of whole-mount eyecup preparations stained with isolectin-B4-positive (green) to stain vessels and anti-IBA1 (red) to stain for macrophages show a decrease both in absolute numbers and clustering of subretinal IBA-1-expressing cells associated with neovascular complexes in anti-VEGF-A/ANG-2 treatment groups. Error bars show SEM with n = 6 animals per group (Scale bar, 200µm). All significant differences are shown by * after one sided ANOVA (***, P < 0.0008) and Tukey’s multiple t-test. IgG control is significant against anti-VEGF-A/ANG-2 low (*, P < 0.0134), -mid (**, P < 0.0078) and –high (**, P < 0.0042); furthermore anti-ANG-2 against anti-VEGF-A/ANG-2 mid (*, P < 0.0353).
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 5

Figure 5. Dual inhibition of VEGF-A and ANG-2 reduced the number of TUNEL-positive cells and improved visual functionality, as revealed by ERG, in a model of sCNV in JR5558 mice.

A) Bar graph of TUNEL-positive cells in the retina of mice treated with 5 mg/kg anti-VEGF-A, anti-ANG-2, and 3, 5, and 10 mg/kg anti-VEGF-A/ANG-2. Error bars show SEM with n = 6 animals per group and * denote all significant changes after one sided ANOVA (**, P = 0.0041) and Tukey’s multiple t-test. IgG control is significantly different from anti-VEGF-A/ANG-2 mid (**, P = 0.0077) and –high (**, P = 0.0028). B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E–G) anti-VEGF-A/ANG-2 low, mid, high: representative micrographs of whole-mount retinae preparations display the reduction of TUNEL-positive photoreceptor cells (green) clustered around focal neovascular lesions in anti-VEGF-A/ANG-2 (E–G) and, to a lesser extent, anti-VEGF-A- (C) and anti-ANG-2 (D)-treated groups compared with IgG-treated controls (B), scale bar 200 μM.

H) ERG was used to assess retinal function in response to large-field flash stimuli under both scotopic and photopic conditions. Representative photopic (light-adapted) and scotopic (dark-adapted) flash response series show increased amplitude and reduced latency after treatment with bispecific VEGF-A/ANG-2 compared with IgG control (both 3 mg/kg). ERG shows reduced depression of B-wave responses in JR5558 mice treated with anti-VEGF-A/ANG-2 compared to IgG control.

I) Maximum photopic ERG amplitude was increased using the rodent crossreactive bispecific VEGF-A/ANG-2 compared to single anti-VEGF-A or anti-ANG-2 treatments (all at 3 mg/kg). There was a significant increase in amplitude at stimuli >2 log (cd*s/m2) between the bispecific VEGF-A/ANG-2 and IgG control group under scoptopic and phototopic conditions. *Denotes significance after ANOVA and Dunnett’s multiple t-test compared to IgG control compared for each stimuli separately, error bars show SEM with n = 8 animals per group. Under phototopic conditions anti-VEGF-A/ANG-2 reached significance at 2.58 and 2.30 cd*s/m2 with P = 0.0498 and *P = 0.0479, respectively. Under scotopic conditions all stimuli reached significance for anti-VEGF-A/ANG-2 with P-values of -2.3 cd*s/m2 (P = 0.0498); -1.93 cd*s/m2 (P = 0.0035); -1.33 & -1.03 cd*s/m2 (P < 0.0001); -0.73 cd*s/m2 (P = 0.0002); -0.43 cd*s/m2, (P = 0.0004); -0.13 cd*s/m2, (P = 0.0024) and 0.18 cd*s/m2 (P = 0.0031). In addition anti-ANG-2 treatment reached significance at -0.13 cd*s/m2 (P = 0.011) and 0.18 cd*s/m2 (P = 0.005), respectively.

ERG, electroretinogramme; sCNV spontaneous choroidal neovascularisation.
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 6

Figure 6. Structural characteristics and functional binding properties of CrossMAb RG7716.

A) Structural presentation of CrossMAb RG7716 with substituted amino acids highlighted and colored. Amino acids corresponding to individual point mutations ensuring correct and efficient heavy chain heterodimerization (‘knobs-into-holes’ [green] and additional disulfide bridge [yellow]), abolishing Fc-receptor functionality (Fc-gamma receptors I, II, and III, colored in pink) and Fc-Rn (colored in blue) are highlighted.

B) Summary table of experimental binding affinities of the parental components targeting VEGF-A (ranibizumab) and ANG-2 (LC10) as well as of RG7716 measured by isothermal titration calorimetry. RG7716 binds VEGF-A121 and -165 with an affinity comparable to ranibizumab (3.3 vs. 3.1 nM). ANG-2 binding of RG7716 is slightly decreased to 22 nM compared to α-ANG-2 IgG1 (LC10) with 4.1 nM. No binding to ANG-1 was detectable for RG7716 or α-ANG-2 IgG1 (LC10).

C) Inhibition of ANG-1 induced endothelial cell survival was tested in the presence of increasing concentrations of LC10 (anti-ANG-2 IgG, ANG-2 binding part in RG7716) and LC08 (ANG-1 and ANG-2 binding antibody). An average of three independent experiments is shown with SEM.

D) The ability of RG7716 to bind to ANG-2 and VEGF-A simultaneously and independent is demonstrated by a surface plasmon resonance experiment. RG7716 is captured via an anti-Fc antibody on the chip surface. The green curve depicts the experiment with ANG-2 in the first and VEGF-A in the second binding event. The red curve shows the binding events in the other sequence.
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 7

Figure 7. Binding of CrossMAbs with modification of the Fc part to FcγR- I, II, and III, as well as to FcRn in SPR interaction assays.

Time-dependent binding of RG7716 is shown in pink, anti-VEGF-A/ANG-2 without modification of the Fc- part in red (wild-type), anti-VEGF-A/ANG-2 with modification of the FcRn binding site only in yellow (FcRn-), and anti-VEGF-A/ANG-2 with modification of the FcγR binding in grey (FcγR-).

A) Shows SPR binding profile over time to immobilized FcγR-I, B) to immobilized FcγR-II, C) to immobilized FcγR-III, D) to immobilized FcRn. E) Summary data table of binding data for RG7716 in comparison to anti-VEGF-A/ANG-2 CrossMAbs without modifications in the Fc-part.

SPR, surface plasmon resonance
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 8

Figure 8. No platelet aggregation by RG7716 when antibody is complexed with VEGF-A and heparin in vitro and pharmacokinetic properties of RG7716 in comparison to wild-type anti-VEGF-A/ANG-2.

A) Bar graph demonstrating platelet aggregation induced by complexes of heparin, VEGF-A165, and anti-VEGF-A (IgG1) in vitro using washed human platelets. Introduction of modification of the Fc-region in RG7716 does not allow platelet aggregation to happen as compared to anti-VEGF-A as wild type IgG1. All three components are needed as heparin/anti-VEGF-A alone does not activate platelet aggregation. Error bars show SEM of three independent experiments and **** denotes significance using ANOVA (P < 0.0001) and Dunnett’s multiple t-test comparing to IgG control with P < 0.0001 of Heparin/VEGF-A/anti-VEGF-A against IgG control.

B) Kinetic presentation of mean serum, vitreous, and aqueous humor concentrations after single intravitreal administration of 0.5 mg of RG7716 in cynomolgus monkeys.

C) Summary table of key experimental PK parameters of RG7716 and anti-VEGF-A/ANG-2-FcγR- (with intact FcRn binding).

AUC, area under the curve; Cmax, maximum concentration; t1/2, half-life; tmax, time to maximum serum concentration; tlast, time of collection of the last of a series of blood samples; F, bioavailability; NA, not analyzed; PK, pharmacokinetic;
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 9

Figure 9. Efficacy of RG7716 in a laser-induced CNV model using Cynomolgus monkeys, comparing RG7716 to anti-VEGF-A (ranibizumab) and anti-ANG-2 (LC10), as well as an IgG control.

A) Schematic representation of the experimental setup including treatment, sampling, and efficacy readouts.

B) Inhibition of neovascularization measured in severity grades, change of severity from baseline is shown for each treatment; all treatment significantly reduced the severity grade compared to IgG control. In addition, efficacy of RG7716 (150 kDa molecule at 90 µg) was significantly better at equal molar concentration of binding sites than anti-VEGF-A (ranibizumab, 50 kDa molecule at 30 µg), anti-ANG-2 and the low dose of RG7716 (30 µg).

C-G) Representative figures of fluorescence fundus angiograms at day 28 and H-L) of cross sections of a hematoxylin staining of a spot at the end of the experiment from eyes treated with IgG control, anti-VEGF-A, anti-ANG-2 and RG7716 at 30 and 90 μg, respectively.

Error bars show SEM of n= 6 cynomologus monkeys and nine spots per eye in the group; * denotes significance after one sided ANOVA and Tukey’s multiple t-test. IgG control is significantly different from anti-VEGF-A (****, P < 0.0001), anti-ANG-2 (***, P = 0.0003), RG7716, 30 μg (****, P < 0.0001) and RG7716, 90 μg (****, P < 0.0001). Furthermore RG7716, 90 μg is significantly different from RG7716, 30 μg (****, P < 0.0001), anti-ANG-2 (***, P = 0.0003) and anti-VEGF-A (***, P < 0.0004).

D,day; CNV, choroidal neovascularization
Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 10

Figure 10. Aqueous concentrations of VEGF-A, ANG-2, IL-6, and IL-8 samples 1 day after intravitreal antibody delivery (D16) and at the end of the experiment (D30) in non-human primate laser-induced CNV.

Error bars show SEM, +* denote significance using one sided ANOVA and Tukey’s multiple t-test with n = 6 cynomologus monkey. Data are presented in linear scale, for the statistical analysis data were transformed logarithmically to normalize distribution and allow parametric ANOVA testing. D16 (+) and D30 (*) were analyzed independently.

A) VEGF-A levels only slightly increased from D16 to D30 in the IgG control. At D16 levels of IgG control are significantly higher compared to anti-VEGF-A (++++, P < 0.0001); RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001). Anti-ANG-2 levels are comparable to IgG control and therefore significantly different from anti-VEGF-A (++++, P < 0.0001); RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001). At D30 the levels of VEGF-A are significantly reduced in the RG7716, 90 μg group are compared to IgG control (*, P = 0.0309). Anti-ANG-2 remains at levels comparable to IgG control and is therefore significantly higher than RG7716, 30 μg (*, P = 0.0107), RG7716, 90 μg (**, P = 0.002) and anti-VEGF-A (*, P = 0.0162).

B) ANG-2 levels increased from D16 to D30 in the IgG control. The same patterns, with a weaker total increase, were seen for the ANG-2 and both RG7716 treatment groups. ANG-2 levels were significantly higher at D16 in the anti-VEGF-A treated group compared to IgG control (++, P = 0.002), to anti-ANG-2 (+++, P = 0.0004), RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001).

C) The reductions of treatments compared to IgG control at day 30 did not reach significance. IL-6 levels only very slightly increased from D16 to D30 in the IgG control; while for all other treatment groups there was a trend to reduced IL-6 levels at D30 compared to baseline with the high dose of RG7716 reaching significance, IgG control vs RG7716, 90 μg (*, P = 0.023).

D) IL-8 levels increased from D16 to D30 in the IgG control group. Compared to the IgG control the levels of IL-8 were reduced in the anti-VEGF-A (*, P = 0.0262), RG7716, 30 μg (*, P = 0.0188) and RG7716, 90 μg (*, P = 0.0107) treatments at at D30.

IL-6, interleukin-6; IL-8, interleukin-8
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 1

Figure 1 - Deletion of concavin leads to rounding of sporozoites

A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.

B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM.

C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.

D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.

E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.

F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time.

G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.

H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.

I. Selected trajectories of manually tracked sporozoites.
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 2

Figure 2 - Concavin-GFP localizes to the periphery of ookinetes and sporozoites

A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.

B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.

C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µm

D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.

E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm. F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 3

Figure 3 - FRAP reveals dynamics of concavin-GFP and stasis of PhiLl1-GFP

A, B. Time series of concavin-GFP (A) PhiL1-GFP (B) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates. * indicates time of bleaching. Shown is the mean ± SD.

C. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594 (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.

D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm.

E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm.

F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm.

Data information: Asterisks in D-F indicate apical part of the sporozoite.
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 5

Figure 5 - Limited impact of potential palmitoylation for shape maintenance

A. Cysteine 7 is predicted to be palmitoylated and was changed into alanine in the concavinC7A-GFP mutant.

B. Expression and localization of concavinC7A-GFP in blood stage parasites. Scale bar 5 µm.

C. Oocysts number in infected mosquitoes. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-value calculated using the Mann Whitney test.

D. Quantification of concavinC7A-GFP cell shapes at the indicated days from midgut (MG) or salivary gland (SG) derived sporozoites. Numbers above bars indicate investigated sporozoites.

E. Average speed of salivary gland sporozoites. Data points represent individual sporozoites from 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.

F. Localization of concavinC7A-GFP in normal and deformed sporozoites (arrowheads point to deformations). Scale bar: 5 µm.