Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: Atomic model and SAXS parameters of the TrYbe ®. A ) Ribbon representation of a TrYbe® homology model based on TrYbe® A showing the IL-17A binding Fab variable (purple) and constant (gray) regions, with the anti-TNF ds-scFv (blue) and anti-albumin ds-scFv (green) attached to the respective Fab light (pale) and heavy (dark) chain. Structural parameters Rg, porod volume (Vp) and D max obtained from SAXS analysis are displayed (see Table S2). B ) A surface representation of the TrYbe® format highlighting orientation of the CDRs (solid purple) and modeled S(G 4 S) 2 linkers (gray spheres). Figure 2 shows two models of the TrYbe® as deduced by SAXS analysis. Part A is a three-dimensional model showing the beta sheet orientations of the TrYbe®’s Fab, constant and ds-scFv domains within a dotted circle. The SAXS parameters ( Rg, Vp , and D max ) are indicated on this figure. These are mathematical measurements where Rg indicates the degree of compactness of a TrYbe®, Vp is the volume occupied by a TrYbe® and D max is the largest interparticle distance reached by a TrYbe®. Rg is represented by a dotted arrow starting in the midpoint of the TrYbe® and extends to the left toward the dotted circle. The value of Rg is indicated as 41.2 Å; Vp is represented by a dotted line connected to the dotted circle at 180° angle and extends inwards toward the TrYbe®. The value of Vp is given as 157,140 Å; D max is represented by a double arrowed dotted line that cuts across the diameter of the dotted circle from top to bottom at a 45° angle. The value of D max is given as 130 Å. Part B of the figure shows a model of the 3-D surface of the TrYbe® model where CDRs of each domain are highlighted.

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: Binding Assay

Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: TrYbe® A binding kinetics and simultaneous binding responses to TNF, IL-17A and HSA.

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: Binding Assay

Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: Human rheumatoid arthritis synoviocytes seeded in the lower chamber of a 24-well trans well plate was stimulated with Th17 supernatants for 24 h and in the presence of isotype IgG as a negative control ( ), monospecific neutralizing antibodies alone, a proprietary anti-IL-17A binding IgG ( ) or with the anti-TNF-α-specific Etanercept ( ) or a combination of the anti-IL-17A antibody and anti-TNF-α antibody (Etanercept) ( ), whilst TrYbe® A ( ) was added alone. An unstimulated control ( ) representative of unstimulated human RA synoviocytes was also set up. To evaluate the inhibition of neutrophil migration, human leucocytes were seeded onto the upper transwell insert and after 6 h of incubation, neutrophils in the lower chamber were enumerated by flow cytometry. Statistical analysis was performed using a One-way Anova with either a Dunnet post-test using the negative control as the comparator; p < 0.0001 or Sidak post-test for direct group comparisons; p < 0.001. GraphPad prism v6 was used for statistical analysis.

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: Negative Control, Binding Assay, Control, Inhibition, Migration, Incubation, Flow Cytometry

Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: Bispecific antibody formats targeting TNF-α and IL-17A The antibody formats are (a) TrYbe® A, (b) DVD-IgG, ABT-122 and (c) a FynomAb, COVA322. Binding specificities are annotated as: anti-IL-17A (blue), anti-TNF (magenta), anti-HSA (green).

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: Binding Assay

Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: Antibody:antigen complex formation with antigens added independently or in combination DLS was used to measure complex volume (nm 3 ) formed when molar ratios of the TrYbe®, DVD-IgG and FynomAb were combined with (a, i) TNF, (b, i) IL-17A or (c, i) both TNF and IL-17A. Plots designed as (ii) represent zoomed-in versions of the respective plots a-c to focus on the smaller complex volumes of TrYbe® and FynomAb, where applicable. The antibody formats are indicated as follows: TrYbe® A ( ), ABT-122, DVD-IgG ( ) and COVA322, FynomAb ( ). The complex volume (nm 3 ) was calculated from the Z mean. Each data point represents the average of n = 5 DLS measurements per sample on the instrument. Figure 6b shows the range of complexes formed with increasing ratios of TNF or IL17-A alone or a combination of both antigens to the antibody in vitro as measured by DLS. In all scenarios, the TrYbe® being a monovalent format, produces the smallest complexes compared to the bivalent DVD-IgG and FynomAb formats.

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: In Vitro

Journal: mAbs

Article Title: TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life

doi: 10.1080/19420862.2022.2160229

Figure Lengend Snippet: UV-vis spectroscopic analysis of antibody:antigen complexes A molar 1:1 ratio of the TrYbe® (TrYbe® A), DVD-IgG (ABT-122) or the FynomAb (COVA322) complexed with TNF ( ), with IL-17A ( ) or at 1:1:1 ratio with both TNF and IL-17A ( ), were analyzed by UV-vis spectroscopy at 340 nm. A sample containing the cytokine(s) with no antibody ( ) was included for each panel as a control. Data represents the mean ±SD of 3 replicates.

Article Snippet: Antibodies against the human TNF and IL-17A were raised in Female Sprague Dawley rats using immunization and screening processes similar to those described previously.

Techniques: UV-Vis Spectroscopy, Control