CHARACTERIZATION OF A CHEMICAL CONJUGATE BETWEEN A LOW-MOLECULAR WEIGHT FORM OF RECOMBINANT SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR (COMPRISING LEU144 THROUGH LEU411) AND F(AB')2-FRAGMENTS OF A FIBRIN D-DIMER-SPECIFIC MONOCLONAL-ANTIBODY

A recombinant low Mr derivative of single chain urokinase-type plasminogen activator consisting of amino acids Leu144 through Leu411 (rscu-PA-32k) was conjugated to F(ab′)2-fragments of a murine monoclonal antibody (MA-15C5), directed against fragment D-dimer of crosslinked human fibrin, by means of the crosslinking reagent N-succinimidyl 3-(2-pyridyldithio) propionate (rscu-PA/MA-15C5-F(ab′)2). The conjugate was purified by immunoadsorption on a monoclonal antibody directed against urokinase followd by affinity chromatography on insolubilised fibrin fragment D-dimer. Recoveries were 26±3.1% for rscu-PA-32k and 27±2.7% for total protein (mean±SD, n=3). The purified conjugate contained 1.0±0.03 mol of F(ab′)2-fragment per mol of rscu-PA-32k. The specific amidolytic activity on a chromogenic substrate for urokinase was <500 iu/mg u-PA antigen for both rscu-PA-32k/MA-15C5-F(ab′)2 and rscu-PA-32k, whereas the specific fibrinolytic activity on fibrin plates was 29000 iu/mg u-PA antigen in the conjugate as compared to 53000 iu/mg u-PA antigen in rscu-PA-32k. Activation of plasminogen by rscu-PA-32k/MA-15C5-F(ab′)2 obeyed Michaelis-Menten kinetics with a Km value of 0.71±0.26 μM and a k2 value of 0.00023±0.00010s-1 (n=3), as compared to Km=1.6 μM and k2=0.00028s-1 for rscu-PA-32k. Both rscu-PA-32k/MA-15C5-F(ab′)2 and rscu-PA-32k induced dose- and time-dependent lysis of a 125I-fibrin labelled human plasma clot immersed in citrated human plasma. Fifty per cent lysis in 2h was obtained with a concentration of 6.4 μg/ml (n=4) for rscu-PA-32k, whereas only 1.2μg u-PA/ml (n=4) was required of rscu-PA-32k/MA-15C5-F(ab′)2. We conclude that low Mr scu-PA can be targetted to a fibrin clot with the F(ab′)2-fragment of a fibrin-specific antibody, resulting in a 5-fold increased thrombolytic potency, which is comparable to that of intact scu-PA coupled to intact antibody. These fragments may serve as preferred moieties for the construction of recombinant chimaeric scu-PA/antibody proteins for fibrin-targetted thrombolytic therapy. © 1990 Longman Group UK Ltd.