Table 1.
Structure-based designs to abrogate pre-existing ADA binding to VHH C-terminus.
Fig 1.
Structural analysis of the VHH C-terminus to aid the design of variants with abrogated pre-existing ADA binding.
(A) Cartoon representation of a representative VHH structure. N- and C-termini are indicated and the three CDRs are color coded in blue (CDRH1), green (CDRH2), and red (CDRH3). (B) Close-up view of the residues constituting the VHH C-terminus and their structural interactions with surrounding residues. Hydrogen-binding interactions are shown as yellow dashes. Main chain hydrogen bonds are formed from Thr110 to Leu11 and Ser112 to Val12. In addition, the side chain of Gln13 forms a bidentate hydrogen bond to the C-terminal carboxy group of Ser113 and an additional hydrogen bond to the main chain carbonyl of Ser112. Furthermore, there are hydrophobic packing interactions between the side chains of Val111 and Val12 as well as Thr110 and Leu11.
Fig 2.
Structure models of various VHH variants with pre-existing ADA designs.
(A) Computational model of the non-modified VHH MC6.1 in a ribbon representation. (B) Structure superimposition of computational models of the non-modified VHH MC6.1 (grey) and the VHH variants with various C-terminal pre-existing ADA designs (color). The C-terminal amino acid substitutions, truncations and additions result in a wide variety of conformational arrangements, all of which are limited to the C-terminal portion of the molecule and do not appear to impact the overall VHH structural integrity.
Table 2.
Ellipro-based prediction of conformational B cell epitopes.
Table 3.
BepiPred-based prediction of linear B cell epitopes.
Fig 3.
Expression and thermal stability of VHH variants.
(A) Expression of VHH variants in transient CHO cell culture supernatants visualized by reducing SDS-PAGE. 10μl of supernatant were loaded into each lane. Molecular weight markers are in the further leftmost lanes on both gels. MC6.1 is the unmodified VHH, MC6.40 to MC6.58 are the various VHHs with pre-existing ADA designs. All VHH variants expressed comparably to the unmodified VHH MC6.1. (B) Mean melting temperature (Tm) for each VHH variant in a thermal shift assay. The unmodified VHH MC6.1 and most variants exhibit Tms of approx. 75–76°C (mean Tm of 75.3°C indicated by a black dashed line) except for MC6.41 and M6.46 which feature 1.5- and 3.5°C-reduced thermal stability.
Table 4.
SPR kinetic parameters of MC6.1 and VHH variants binding to human serum albumin.
Fig 4.
Screening ELISAs to identify the designs that most effectively abrogate pre-existing ADA binding.
(A) Schematic representation of the ELISA assay used to evaluate pre-existing ADA binding to the VHH variants. N-terminal His-tagged VHH variants (blue) are captured on a Ni-NTA plate. The plate is then incubated with either a pool of or with individual human donor serums. Pre-existing ADA that recognize and bind to the VHH variants (red) are detected with an HRP-conjugated anti-human Fc Ab (black). (B) Binding of pre-existing ADA from a pool of sera of 10 naïve human individuals to VHH variants at 3 capture concentrations (0.5, 2, 8ug/ml). Different VHH variants have various levels of pre-existing ADA binding ranging from high for the unmodified MC6.1 and MC6.47 to low levels of pre-existing ADA binding (MC6.43, 44, 51, 52) comparable to the buffer control (no VHH captured).
Fig 5.
Pre-existing ADA binding from individual sera of 70 human donors to the unmodified VHH MC6.1 and six VHH variants.
Heat map representation of the signals in pre-existing ADA binding ELISAs for six VHH variants screened against 70 individual sera from naïve donors. The selected six variants represent different design rationales that had low levels of pre-existing ADA binding in a primary screen against pooled sera. A wide variety of pre-existing ADA binding responses are observed between the different sera. Sera with significant adherence to the non-coated plate are indicated by red asterisks, while sera that exhibit no apparent pre-existing ADA reactivity are marked with green asterisks. Approximately 55% of all donors have pre-existing ADA reactivity against the unmodified MC6.1 VHH.
Fig 6.
Comparison of pre-existing ADA reactivity of the unmodified VHH MC6.1 and six VHH variants.
(A) ELISA absorbance values (X-axis) for each of the 70 individual sera is plotted for each respective VHH variant. ELISA absorbance signals were used to quantitate and compare pre-existing ADA binding. Mean and standard deviation are shown for each dataset, and relative mean values are indicated on top of each dataset. The unmodified VHH MC6.1 exhibits approximately a 4-fold higher mean signal as compared to buffer, while the control VHH MC6.40 as well as MC6.51, MC6.52, and MC6.56 exhibit approximately 1.5-fold higher mean signal. MC6.43 has quantitative pre-existing ADA binding comparable to the buffer control (~1.2), indicating that most pre-existing ADA reactivity has been successfully eliminated by the addition of two extra proline residues at the VHH C-terminus. (B) Structure model of the C-terminal portion of the unmodified VHH MC6.1 in cartoon and surface presentation compared to (C) MC6.43 with the pre-existing ADA design addition of amino acids Pro114 and Pro115 (cyan).