Table 1.
Antigenic and physical characteristics of engineered RSV F glycoprotein variants.
|
Mechanism of
stabilization |
RSV F variant |
Oligomeric
state# |
Yield
(mg/L)* |
Antibody KD value (nM)@ |
Physical characterization
(fractional D25 reactivity) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|
||||||||||||||||
| Site Ø | Site I | Site II$ | Site IV | Temp (°) | pH |
Osmolality
(mM) |
Freeze-
thaw |
||||||||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||||
| D25 | AM22 | 5C4 | 131-2a | Paliv | Mota | 101F | 50 | 70 | 3.5 | 10.0 | 10 | 3000 | 10& | ||||
| Cavity filling | K87F-V90L | Aggregate | 0.3 | >1000 | >1000 | >1000 | 7.6 | 1.7 | 0.17 | 1.6 | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| F137W-F140W | N.D. | <0.1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| F137W-F140W-F488W | N.D. | <0.1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| S190F-V207L (Cav1) | Trimer | 2.2 | 0.23 | <0.10 | 9.3 | >1000 | 43 | <0.01 | 2.9 | 0.8 | 0.1 | 0.7 | 0.8 | 1.0 | 0.7 | 0.6 | |
| S190F-V296F | Aggregate | 0.4 | >1000 | >1000 | >1000 | 4.2 | 1.7 | <0.01 | 1.6 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| V207L-V220L | Aggregate | 0.4 | >1000 | >1000 | >1000 | 2.8 | 0.99 | 0.014 | 0.69 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
|
D486H-E487Q-F488W-
D489H (TriC) |
Trimer | 0.8 | 0.012 | 1.0 | 34 | >1000 | 31 | 0.48 | 4.0 | 0.8 | 0.1 | 0.1 | 0.8 | 1.3 | 0.6 | 0.1 | |
| F488W | Trimer | 1.7 | 0.087 | 0.25 | 27 | >1000 | 32 | 0.1 | 4.7 | 0.9 | 0.1 | 0.1 | 0.7 | 1.1 | 0.5 | 0 | |
|
|
|||||||||||||||||
| Disulfide formation | S155C-S290C (DS) | Trimer | 1.4 | 0.29 | <0.01 | 35 | 3.4 | 2.8 | 0.043 | 2.2 | 0.3 | 0 | 0.1 | 0.8 | 1.3 | 0.8 | 0.3 |
| T357C-N371C | N.D. | <0.1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| S403C-T420C | Aggregate | 0.3 | >1000 | >1000 | >1000 | 3.05 | 3.3 | 0.05 | 1.85 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
|
|
|||||||||||||||||
| N-Glycan addition | V178N | Aggregate | <0.1 | >1000 | >1000 | >1000 | 7.2 | 3.1 | 0.11 | 1.64 | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
|
|
|||||||||||||||||
| Postfusion destabilization |
V185E | Aggregate | <0.1 | >1000 | >1000 | >1000 | 3.5 | 1.7 | 0.11 | 1.64 | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| I506K | Aggregate | <0.1 | >1000 | >1000 | >1000 | 4.6 | 1.7 | 0.054 | 1.39 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
|
|
|||||||||||||||||
| Acid patch neutralization |
D486H-E487Q-D489H | Aggregate | 0.1 | >1000 | >1000 | >1000 | >1000 | 9.5 | 0.57 | 12.7 | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
|
|
|||||||||||||||||
| Double combinations | DS-Cav1 | Trimer | 1.9 | 0.15 | <0.01 | 13 | >1000 | 23 | 0.041 | 3.2 | 0.9 | 0 | 0.8 | 0.9 | 1.0 | 0.8 | 0.7 |
| DS-TriC | Trimer | 0.6 | 0.17 | <0.01 | 33 | 2.0 | 4.8 | 0.055 | 3.1 | 0.9 | 0 | 0.3 | 0.9 | 0.5 | 0.9 | 0.5 | |
| Cav1-TriC | Trimer | 0.2 | 0.99 | 0.086 | 5.1 | >1000 | 33 | 0.17 | 3.1 | 0.9 | 0.1 | 0.3 | 0.8 | 0.6 | 0.5 | 0 | |
|
|
|||||||||||||||||
| Triple combinations | DS-Cav1-TriC | Trimer | 1.3 | 0.17 | 0.02 | 18 | >1000 | 20 | 0.10 | 3.2 | 0.9 | 0.1 | 0.6 | 0.9 | 0.6 | 0.6 | 0 |
Variants were often observed to exist in a mixture of oligomeric states on size chromatography. If a measureable trimeric fraction was observed, then this is indicated by the label “Trimer”, and the various properties of the trimer fraction are listed. If no trimeric fraction was observed, then the oligomeric state of the dominant oligomeric species is provided. If the total yield prior to size chromatography was <0.1 mg/l, then oligomeric state is listed as not determined (N.D.).
Yield shown is for the specified oligomeric state.
When no binding was observed for 1 μM Fab, the KD is shown as >1000 nM; when total yield was <0.1 mg/l, then the value for KD is not applicable (N/A).
Palivizumab (Paliv); motavizumab (Mota).
Ten cycles of freeze-thaw.