Figures & data
Figure 1. The TrYbe® and potential applications. An illustrative representation of the TrYbe® format with three target binding regions (Fab region 1, ds-scFv region 2 and ds-scFv region 3). The TrYbe® is composed of a Fab fragment scaffold at its core and two ds-scFvs connected to the C-termini of the respective Fab light and heavy chain via S(G4S)2 linkers. DSBs are represented as – . Potential mechanisms of action of the TrYbe® are outlined. Abbreviations: neonatal Fc receptor, FcRn; Transferrin receptor, TfR; blood brain barrier, BBB. Figure 1 shows an inner circle in the center of the figure, containing a cartoon of the TrYbe® with the constant regions and antigen-binding regions indicated. Seven, equidistant arrows extend outwards from the circle and point to cartoons indicating a different mechanism of action of the TrYbe®: 1) immune modulation is represented by TrYbes® shown to co-engage with two different receptors on a cell and initiate a signal into the cell to indicate receptor activation or receptor inhibition. In another scenario a TrYbe® is shown to co-engage with receptors which indices a novel function via signaling or bind to two paratopes on the same receptor to elicit a biparatopic response. Jagged arrows are used to denote a signal inside the cell from each engaged TrYbe®-receptor complex; 2) co-receptor clustering is represented by a TrYbe® binding to two distally separated receptors on a cell. An arrow points to another TrYbe® which is now engaging two clustered receptors. A jagged arrow denotes a signal elicited inside the cell from the engaged TrYbe®-receptor complex; 3) Ligand- receptor co-localization is represented by a TrYbe® binding to a soluble ligand and a surface receptor on a cell. A jagged arrow denotes a signal inside the cell, elicited from the engaged TrYbe®-receptor complex; 4) Recruitment of immune cells shows a TrYbe® engaging a surface receptor on a target cell by one ds-scFv and another receptor on an effector cell by the other ds-scFv. A jagged arrow denotes a signal inside the cell, elicited from the engaged effector cell; 5) Piggybacking (FcRn recycling) is represented by a cell containing an endosome which is signified by a circle with receptors on the inner side. A TrYbe® is shown to engage to a FcRn recyclable ligand and subsequently is endocytosed into the cell. An arrow then shows the same TrYbe® in the endosome where the FcRn recyclable ligand also engages a endosomal receptor on the inner side whilst still bound to the TrYbe®. An arrow then shows the TrYbe® being exocytosed from the cell on the apical side whilst still being bound to the ligand; 6) Similar to 5), a TrYbe® is shown to engage a TfR receptor on the cell surface on the apical side. An arrow points to the TrYbe® now in an endosome, followed by another arrow point showing the TrYbe® transcytosed from the cell on the basolateral side; and 7) dual blockade is represented as a TrYbe® blocking the binding of soluble ligands to a cell and in another scenario, a TrYbe® blocking ligands binding to two receptors.
Figure 2. 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 Dmax 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(G4S)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 Dmax) 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 Dmax 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; Dmax 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 Dmax 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.
Figure 3. Disulfide stabilization and improved TrYbe® stability upon storage. TrYbes® B (
) and C (
, 
) composed of disulfide-stabilized scFvs (filled symbols) or non-stabilized scFvs (open symbols) at 5 mg/mL in PBS, pH7.4 were incubated at 5°C for up to 28 days and monitored by SEC for HMWS on days 0, 8, 15, 22 and 29 days. Figure 3 is a time course plot with time from 0 to 30 days on the X-axis and percentage HMWS on the Y-axis. Two lines are shown broadly parallel to the X-axis and represent TrYbes® with disulfide-stabilized scFvs. Another two lines that appear to show a linear increase represent TrYbes® with non-disulfide stabilized scFvs. The line representing TrYbe® B is shown to contain 4% of HMWS on day 0 which progressively increases to > 5% by 28 days. Whist the line representing TrYbe® C shows a much steeper slope, registering ~5% of % HMWS on day 0 increasing to 13% HMWS on day 28.
Table 1. TrYbe® A binding kinetics and simultaneous binding responses to TNF, IL-17A and HSA.
Figure 4. 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 (
) 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.
Figure 5. Inhibition of the p38 B cell signaling pathway by CD79b/CD22 -specific formats. In IgM-stimulated B cells, phosphorylation of the p38 B cell signaling pathway was inhibited by TrYbe® D (
) Mixtures of CD22-Fab Y and CD79b Fab-Y (
) and an isotype IgG (
) were used as controls. Data shown is represented as mean (±SD) of three technical replicates. Figure 5 shows a typical sigmoidal response curve to inhibit p38 signaling with increasing dose of the two antibody formats. Lines corresponding to the two antibody formats correlate well with each other and show a broadly linear increase in % inhibition from 0 to 100% at a starting dose of ~1 x 10−2 nM and ~5 x 10−4 nM for the respective TrYbe® and Fab-KD-Fab formats.
Figure 6a. 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).
Figure 6b. Antibody:antigen complex formation with antigens added independently or in combination DLS was used to measure complex volume (nm3) 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 (
) and COVA322, FynomAb (
). The complex volume (nm3) 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.
Figure 6c. 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 (
) 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.
Table 2. Pharmacokinetic parameters of TrYbe® E and F in cynomolgus monkeys.
Figure 7. PK of TrYbes® in cynomolgus monkey. Two treatment groups (n = 3) were dosed with a TrYbe® at 10 mg/kg either intravenously (i.v.) or subcutaneously (s.c.) and are represented as follows: TrYbe® E, i.v. (
), TrYbe® F, i.v. (
), TrYbe® F, s.c. (
). Blood samples were collected pre-dose and at 15 min, 6 h, 24 h, 2 d, 4 d, 7 d, 11 d, 14 d, 22 d and 28 d following administration. The mean serum concentration of the two TrYbe® molecules in plasma samples was measured at the multiple time points and detected by LC-MS for peptides specific to at least two of the binding arms of the TrYbe®. PK parameters were determined based on a 2-compartment analysis of the individual serum concentration-time profiles (Phoenix 64 v8.1.0, Certara, NJ, USA). Figure 7 is a time course plot of the serum concentration in μg/mL measured in the blood (on a semi log scale on the Y-axis) up to 28 days (X-axis) of two TrYbes® dosed intravenously or subcutaneously in cynomolgus monkeys. Four lines representing the serum concentration of the TrYbes® with respect to the dosing regimen, show an inverse-proportional relationship with time. The serum concentration of the intravenous dosing starts at the midpoint between 100 and 1000 μg/mL whilst the subcutaneous dosing starts between 1 and 100 μg/mL concentration, otherwise all four lines have the same slope.
Figure 8. Downstream processing of TrYbes®. The product recovery (%) of TrYbes® E and F following each DSP step is shown. This follows a typical 3-column step process with: protein-A affinity capture, followed by proprietary intermediate and polishing steps. Percentage product recovered (
) are represented. Figure 8 shows a stacked column chart of the product recovery and product-related impurities following a three-step downstream process of two TrYbes®. Percentage product recovered is represented by a column which starts at 100% after the protein-A capture step, ~90% following the intermediate steps and ~76% after the polishing step. Overall, an average of 70% of the drug substance is recovered. Similarly, the product-related impurities represented as a stack on each column show its gradual removal following each step: starting with 20% impurities after protein A-capture and decreasing to < 1.5% in the final drug substance.
Table 3. Stability of highly concentrated TrYbes®.