Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration

Abstract

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.

FIGURE 1.

Sequence homology for uPAR-binding Fabs identified by phage display. A, the heavy and light chain protein sequences of the 22 unique clones were aligned to generate a percent identity tree diagram. The number of identical clones is indicated in parentheses for redundant Fab sequences. The Fab subgroups, defined by their light chain identity (κ or λ), are labeled. The vertical line indicates the 82% sequence identity threshold. Sequences that branch to the right of the 82% cut-off are considered equivalent. B, the sequences of the CDR loops of each unique Fab were aligned and shaded to indicate sequence identity. The name of each CDR loop is indicated above the alignment. Fab heavy and light chain protein sequences with greater than 82% sequence identity were grouped together (box). A representative clone was selected from each group based on expression levels in E. coli Rosetta-gami^TM B cells and is indicated to the left of the box. Asterisks indicate Fab clones that did not express in Rosetta cells.

FIGURE 2.

Binding of uPA to uPAR in the presence of Fab. uPA was added to a uPAR-coated plate in the absence and presence of each Fab. The presence of uPA was determined by the amount of bound proteolytic activity and is reported as the initial velocities from the progress curves. Maximal uPA binding was determined by incubating uPA without Fab and is labeled no Fab. Data is plotted left to right from Fabs that do not compete with uPA for uPAR binding to Fabs that show maximal competition. Inset, for 1A8 and 2B1, the amount of Fab bound to uPAR in the presence and absence of uPA was determined by ELISA. The ratio of bound Fab in the presence of uPA to bound Fab in the absence of uPA is reported as a percentage.

FIGURE 3.

IgG expression by transient transfection. A, Fab sequences were grafted onto an IgG1 scaffold by independently subcloning the heavy (HC) and light chain sequences into pTT5-SP-H1. The plasmid map of this transient expression vector is shown. For a given antibody, both the pTT5-SP-H1 heavy chain vector and pTT5-SP-H1 light chain vector were co-transfected into HEK-293-EBNA1 cells for expression. CMV, cytomegalovirus. IgG HC Const, IgG Heavy Chain Constant Fc Region. B, SDS-PAGE analysis of purified antibodies is shown. The λ light chain of 2E9 runs at a higher apparent molecular weight than the κ light chain of the other antibodies.

FIGURE 4.

Equilibrium affinity determination of uPAR antibody interaction. Percent of maximal surface plasmon resonance response during analyte (uPAR) injection versus analyte concentration is shown. Curve fitting for 2E9 (open circles), 1A8 (open squares), 2G10 (closed diamonds), and 2B1 (×) yielded K_D values that are summarized in the table.

FIGURE 5.

Detection of cell surface uPAR with human anti-uPAR antibodies. A—D, white profiles represent staining with control whole human IgG; shaded profiles represent staining with human anti-uPAR antibody. The identity of the human anti-uPAR antibody is indicated within the shaded profile (A = 1A8; B = 2B1; C = 2E9; D = 2G10). To quantify the relative staining intensities of the human anti-uPAR antibodies, the same gate (horizontal line) was applied to each sample. The % of cells staining positive for uPAR expression is indicated above the gate.

FIGURE 6.

Inhibition of uPA/uPAR mediated invasion and signaling in H1299 cells. A, H1299 cells were pretreated with antibodies (10 μg/ml), 2E9, 2G10, 2B1, and 1A8, before they were allowed to invade Matrigel for 24 h. The cells that migrated through and attached to the bottom of the filter were fixed, stained with Giemsa, and extracted with 10% acetic acid. Cell invasiveness is evaluated by measuring A₅₉₅ nm. The results are expressed as percent inhibition of that observed with no treatment control. B, H1299 cells expressing endogenous uPAR were serum-starved, acid-washed, pre-treated with antibodies (10 μg/ml), and then incubated with pro-uPA (10 nm). The lysates were immunoblotted with anti-pERK (top panel) and anti-total ERK (bottom panel).

FIGURE 7.

Determination of 3C6 as a putative uPAR/β1 integrin antagonist. A, H1299 cells were serum-starved, acid-washed, pre-treated with Fabs (10 μg/ml), 2B1, 2B7, 2B11, 2D5, 2E9, 2G10, 2G12, 3C6, and 4C1, and cultured on a FN-coated surface (10 μg/ml) for 30 min before lysis. The lysates were immunoblotted with anti-pERK (top) and anti-total ERK (T-ERK, bottom). B, H1299 cells were seeded on FN-coated (10 μg/ml) or VN-coated (5 μg/ml) 96-well plates with or without anti-uPAR antibody and RGD or RAD peptide. Shown here is a direct comparison between 2G10 (uPAR/uPA antagonist) and 3C6, now identifiable as an uPAR/β1 integrin antagonist. C, shown is a normalized graph comparing the adhesion for each antibody treatment on the two different ECM coatings, obtained by dividing the average reading from RGD-treated wells by that from RAD-treated wells. Note that 3C6 treatment disrupts uPAR-mediated integrin adhesion at least 4-fold more than 2G10 treatment.

FIGURE 8.

3C6 binds to cell surface uPAR and abrogates uPAR association with α5β1 integrin. A, HEK-293 cells overexpressing uPAR were stained with 3C6 and 2G10 to confirm the 3C6 ability to bind cell surface uPAR. The dashed white profile represents staining with 2G10 Fab; the shaded profile represents staining with 3C6 Fab; the solid white profile represents no Fab staining but inclusion of the AlexaFluor 488-conjugated secondary. B and C, H1299 lysates were incubated with anti-uPAR Fab (2G10 or 3C6), Penta-His antibody, and Protein A/G-agarose. The presence of β1 integrin (B) or α5 integrin (C) and uPAR was probed with the respective antibodies in Western analysis. Ctl, control; IP, immunoprecipitate.

FIGURE 9.

Combined 2G10 and 3C6 treatment of H1299 cells significantly decreases invasive potential through Matrigel/Collagen I and Collagen I. H1299 cells were pretreated with antibodies (2G10, 3C6, and 2G10/3C6 at 5–10 μg/ml) before seeding on the Collagen I-coated (A) or Matrigel/Collagen I-coated (B) top membrane of a 24-well Transwell plate (105 cells/well in triplicate). Cells were incubated for 24 h. The cells that migrated through and attached to the bottom of the filter were fixed, stained with Giemsa, and extracted with 10% acetic acid. Cell invasiveness is evaluated by measuring A₅₉₅ nm. The results are expressed as a percentage of inhibition observed in the no-treatment control. Note, the inhibition potential of combined 2G10 (5 μg/ml) and 3C6 (5 μg/ml) is significantly stronger than either antibody alone (10 μg/ml), suggesting an additive effect.