Monoclonal Antibodies to uPAR

mAbs are important tools for functional, structural, and localization studies as well as for quantification of the antigen in question. mAbs were initially raised against uPAR(I-III) purified from U937 cell lysates by DFP-uPA affinity chromatography [1]. One of these mAbs, R3 was used to show that uPA binding to uPAR is required for cell-induced potentiation of plasminogen activation [1]. These mAbs react in Western blotting with uPAR under nonreducing conditions, but not after chemical reduction, indicating that the mAbs recognize conformational but not linear epitopes [16]. The domain reactivity of these mAbs was initially determined using immunoprecipitation of I125-labeled individual domains of uPAR(I-III) [1]. Among the mAbs obtained from this fusion, 3 are specific for domain I whereas the 14 others bind mainly to domain III. For two of the domain I-specific mAbs, R3 and R5, the functional epitopes have been determined using alanine-scanning mutagenesis and analysis of the biomolecular interaction by surface plasmon resonance [8, 65]. The functional epitope of mAb R5 comprises the uPAR residues R2, E16, A18, L19, and G20 and R5 functions as a noncompetitive inhibitor of uPA binding, displacing uPA from uPAR(I-III). The mAb R3 is a competitive inhibitor of uPA binding to uPAR(I-III) with the functional epitope localized to uPAR residues E33, L61, and K62 [8, 26]. Neither R3 nor R5 have epitopes overlapping with the uPA-binding site consisting of amino acids R53, L55, Y57, and L66, even though the amino acids important for R3 binding are in proximity to the uPA-binding site [65]. Another inhibitory mAb, R23, requiring both domains II and III for binding, was obtained using recombinant suPAR1-277 as the antigen [108]. It has not been clarified whether the epitope recognized by R23 is located in the linker region between domains II and III or if the epitope is composed of amino acids in both domains II and III. It will be of great interest to map the R23 epitope and locate it on the three-dimensional structure in order to elucidate the inhibitory mechanism of this mAb.

mAbs reacting with uPAR have also been obtained by immunizing mice with human peripheral blood mononuclear cells. These mAbs were directed against differentiation markers called Mo antigens, for which the expression was induced during the myeloid-monocyte-macrophage sequence of differentiation [109-111]. Comparing the cDNAs for Mo3 and uPAR(I-III) revealed the identity of these two proteins, and two of the Mo3-specific mAbs were subsequently found to prevent uPA binding to uPAR(I-III) [112]. mAbs were also raised against cells of the human monocytic cell line THP-1 [113, 114]. One of these mAbs, VIM-5 binds to domain I and prevents uPA binding to uPAR(I-III) [115].

For studies on uPAR cleavage and glycosylation, it has been important to visualize the reduced and alkylated forms of uPAR. mAbs recognizing these forms of the receptor were generated by immunization of mice with reduced and alkylated suPAR1-277 [17]. These mAbs do not react with native uPAR forms but have been useful for detection of the enzymatically degly-cosylated forms of uPAR. Another collection of 12 mAbs specific for the reduced forms of uPAR was obtained by immunizing with recombinant, nonglycosylated uPAR(I-III) consisting of amino acids 1-284 and produced in Escherichia coli [28]. All of these mAbs reacted with uPAR under reducing conditions, whereas seven also reacted with uPAR under nonreducing conditions. Three of the mAbs were directed against domain I, and their epitope locations were mapped using overlapping peptides representing linear sequences in uPAR. mAb IIIF10 is a competitive inhibitor of uPA binding and the epitope was localized to amino acids 52-60 of uPAR(I-III). This sequence includes three of the four amino acids present in the functional epitope for uPA as determined by alanine scanning. Although no domain II-specific but several domain III-specific mAbs were obtained from the fusion, where uPAR(I-III) from U937 cells was used as immunogen [1, 116], four mAbs reacting with amino acids 125-132 of domain II were generated when the immunogen was recombinant uPAR, expressed by E. coli [28]. The mAbs described above are those that have been most frequently used in uPAR studies, but the list is not exhaustive.

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