Table 1.
Statistics of X-ray data collection and structural model refinement.
Figure 1.
Cross reactivity of ATN-658 with uPAR from various species.
A & B. The specificity of ATN-658 was measured by direct binding assays using uPAR expressing mouse melanoma (B16), human prostate carcinoma (PC-3) and African green monkey (COS-1) cells and biotin-labeled ATN-658. B. FACS analysis was performed using HeLa cells (expressing human uPAR), D-17 lung carcinoma cells (expressing canine uPAR and COS-1 cells. Each cell line was incubated with normal rabbit serum (NRS), a rabbit polyclonal antibody raised against a fragment of human uPAR (rDIIDIII), mouse IgG (mIgG) or ATN-658 and the appropriate FITC labeled secondary antibody.
Figure 2.
Immunoprecipitation of AGM suPAR clones using ATN-615 and ATN-658.
A. Single point mutants were introduced into AGM suPAR such that a single amino acid was changed from the AGM sequence to the human uPAR sequence and the proteins expressed and purified as described in the Materials and Methods. Each clone is identified by number (1–9) with the point mutation identified below the number using the single letter amino acid code. Except for the single point mutation, the rest of the suPAR sequence is that of AGM. suPAR mutants were expressed in S2 cells and supernatants incubated with either ATN-615 or ATN-658 followed by immunoprecipitation as described in Materials and Methods. suPAR was then detected by western blot using a rabbit polyclonal antiserum raised against human suPAR. B. Capture ELISA assays were used to measure the binding affinity of ATN-658 for the various suPAR clones described in (A). Capture on the ELISA plate was through the V5 tag and detection used biotin-ATN-658. C. Biotin-ATN-617 was used in a similar ELISA format as described in (B) to confirm that introduction of the single point mutation did not have a global effect on suPAR conformation.ATN-617 cross-reacts with monkey suPAR and all clones appeared to retain this reactivity after introduction of the mutation.
Table 2.
Amino acid sequence differences in DIIDIII of uPAR from human, African green monkey (AGM) and cynomolgus monkey (crab eating macaque).
Table 3.
Sequence alignment of the ATN-658 epitope in primates and lower order mammals.
Figure 3.
EXSAR difference map between ATN-658 bound and free suPARDIIDIII.
EXSAR analysis was carried out as described in Material and Methods.
Figure 4.
X-ray structure of ATN-658-uPAR-ATF-SMB tertiary structure.
(A) The 1.6 Å structure of the ATN-658 Fab at two orthogonal views. Light chain is shown in light blue and heavy chain in dark blue. (b) Stereo view of the ATN-658 Fab in complex with suPAR (magenta) in the presence of ATF (cyan) and SMB (green) of vitronectin. All figures were made by PyMOL. (c) Interaction of uPAR–ATN-658 Fab in stereoview. Selected contacting residues in stick representation; hydrogen bonds are shown in dashed lines. (d) Open-book view of the interface between suPAR (left) and atn658 Fab (right). The Fab heavy and light chains are pink and yellow, respectively, whereas the suPAR, ATF and SMB are rose, green and cyan, respectively. The binding interface between suPAR and Fab are colored as red for atn658, blue for atn615 and orange for the overlapping epitope of these two antibodies.
Figure 5.
Sequence and 3-dimensional structural similarity between αM binding loops and ATN-658 CDR loops.
(A) Sequence alignment of integrin αM loops to ATN-658 CDR loops. (B) Similar spatial arrangement between integrin αM loops and the ATN-658 CDR loops. Structure of αM was a homology model built from the known integrin structures (http://prosite.expasy.org/cgi-bin/pdb/get-pdb.pl?1a8x).
Figure 6.
ATN-658 inhibits α5β1 mediated binding to uPAR and adhesion to fibronectin.
(A) ATN-658, but not ATN-615, inhibits coIP of uPAR with α5. HT1080 cells were lysed in 1% Triton X-100 lysis buffer and the lysates were immunoprecipitated with indicated antibodies, followed by separation on SDS-PAGE and blotting for uPAR (R2) and integrin α5. Data shown are representative of three independent experiments. White lines indicate that intervening lanes have been spliced out. (B) ATN-658, but not ATN-615, interferes with H1299 uPAR-expressing cells adhesion on Fn in the presence of RGD peptide. H1299 cells pre-treated with RGD or RAD peptides (500 µM) were seeded onto Fn-coated 96-well plates together with antibodies ATN-615 or ATN-658. After incubation for 1 hr at 37°C, plates with triplicate determinations were washed, and attached cells were fixed and stained with Giemsa. All the above experiments were performed at least three times with similar results.
Figure 7.
Schematic of ATN-658 disruption of uPAR signalosome.