TABLE 2.
Epitope-mapping and kinetic rate constants for selected anti-uPAR mAbs
| mAb | Domain reactivitya | Hotspot residuesb | konc | koffc | KDc | IC50d |
|---|---|---|---|---|---|---|
| 105m−1s−1 | 10−4s−1 | 10−9m | 10−9m | |||
| R3 | DI, site 1 | Glu33, Leu61, Lys62 | 1.4 ± 0.6 | 0.52 ± 0.22 | 0.37 | 2.4 |
| R21 | DI, site 1 | Thr59, Gly60, Leu61, Lys62 | 4.1 ± 1.0 | 3.2 ± 0.3 | 0.78 | 1.2 |
| VIM-5 | DI, site 1 | Leu61, Lys62, Ile63 | 1.9 ± 0.5 | 16.3 ± 0.8 | 8.6 | 0.20 × 103 |
| R5 | DI, site 2 | Arg2, Glu16, Leu19, Gly20 | 3.6 ± 0.6 | 10.8 ± 0.04 | 3.1 | 0.22 × 103 |
| R9 | DI, site 2 | Arg2, Glu16, Asp74 | 1.4 ± 0.9 | 9.5 ± 0.03 | 7.7 | 0.20 × 103 |
| R20 | DI, site 2 | Glu16, Leu19, Asp22, Asp74 | 2.9 ± 1.5 | 10.7 ± 0.03 | 4.6 | >10 × 103 |
| mR1 | DI, site 2 | Leu19, Asp22 | 2.1 ± 0.2 | 1.1 ± 0.07 | 0.54 | 0.11 × 103 |
| R4 | DII(DIII) | Arg192, Asp214, Gly217, Ser269 | 4.5 ± 1.3 | 2.8 ± 0.4 | 0.62 | >10 × 103 |
| R8 | DII(DIII) | Arg192, Asp214, Gly217, Ser269 | 6.5 ± 2.1 | 2.4 ± 0.3 | 0.43 | >10 × 103 |
| R2 | DIII | Asp275, Leu276 | 3.2 ± 0.6 | 0.06 ± 0.03 | 0.02 | NAe |
| R24 | DIII | Asp275 | 6.7 ± 1.9 | 6.1 ± 0.08 | 1.0 | NAe |
a The overall domain reactivity for these mAbs was established by surface plasmon resonance using intact uPAR (1–283), uPAR DI (1–92), uPAR DII+DIII (88–283), and uPAR DIII (182–283); see supplemental Table SI. In Table 2, mAbs are clustered in four different bins displaying non-overlapping epitopes between groups but not within groups, as revealed by pairwise binding.
b Functional hotspot residues in epitopes for these mAbs are defined as the 1–4 residues, where alanine substitutions have the greatest impact on the dissociation rate constants (koff) as measured by surface plasmon resonance, as illustrated in Figs. 5 and 6 and supplemental Figs. 4–7.
c The kinetic constants for the interaction between intact soluble uPAR (residues 1–283) and the immobilized mAbs (∼7 fmol/mm2) were measured at 20 °C at a flow rate of 50 μl/min as specified under “Materials and Methods.” Data for R3 were, however, collected at 5 °C because of the lower inherent protein stability of this mAb. Supplemental Fig S1 demonstrates the data quality typically acquired for these analyses, as illustrated for the interaction between immobilized R21 and uPARwt and uPARK62A.
d The IC50 values of the uPA-uPAR interaction were determined by a time-resolved immunofluorescence assay, where the binding of 2.5 nm uPAR to immobilized pro-uPA was detected by Eu3+-labeled R2 as a function of preincubation with a 3-fold dilution series of the respective mAbs.
e NA, not applicable, as these mAbs interfere with the detecting Eu3+-labeled R2.