Chemical conjugates of fibrin-specificantibodies and plasminogen activators. Urokinase or tPA were linked covalently toamonoclonal antibody specific for the amino terminus of the beta chain of human fibrin (59D8) by means of the unidirectionalcross-linking reagent SPDP. The fibrinolytic potency of the conjugates at equal amidolytic activities was compared to the native plasminogen activators in an assay measuring lysis of 1251-fibrin monomer covalently linked to Sepharose CL-4B. Urokinase was least potent, tPA exhibited a 10fold increase in fibrinolysis whereas both the urokinase and tPA antibody conjugates and a urokinase-Fab conjugate were 250fold more potent than urokinase and 25 fold more potent than tPA. Enhanced fibrinolysis was fully inhibited by b peptide indicating its dependence on antigen binding. In a plasma assay conjugates of tPA orUK to antibody produced a 3.2- to 4.5-fold enhancement in clot lysis in human plasma over that of the respective unconjugated plasminogen activator. However, the UK-59D8 conjugate was only as potent as tPAalone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasminand plasminogen than did the unconjugatedactivators, at equipotent thrombolytic concentrations. In a quantitative rabbit thrombolysis model, the activity of the purified conjugate was compared with that oftPA alone and that of a conjugate betweentPA and a digoxin-specific monoclonal antibody. After correction for spontaneous lysis, tPA-59D8 was shown to be 2.8 to,9.6times more potent than tPA alone. Unconjugated tPA and tPA-digoxin were equipotent.At equivalent thrombolytic concentrations, tPA-59D8 degraded less fibrinogen and consumed less alpha 2-antiplasmin than did tPA alone. These results suggest that tPA can be efficiently directed to the site of a thrombus by conjugation to an antifibrin monoclonal antibody, resulting in both more potent and more selective thrombolysis.A recombinant fusion protein comprising a fibrin-specific antibody site and theB chain of tPA. The rearranged 59D8 heavychain gene was cloned and combined in theexpression vector pSV2gpt withsequence coding for a portion of the Gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinitychromatography and analyzed with Western blots. These revealed a 65-kD heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kD disulfide linked dimer. A chromogenic substrate assay showed the fusion protein to have 70 percent of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus by recombinant techniques we have produced a novel hybrid protein capable of high affinity fibrin binding andplasminogen activation.Chemical conjugates between a fibrin-specific and a tPA-specific antibody. A heteroantibody duplex (duplex) with specificities for both tPA and fibrin was synthesized by conjugating iminothiolane-modified anti-tPA monoclonal antibody (TCL8) toantifibrin antibody 59D8. Addition of both duplex and tPA to a plasma clot assay gave more lysis (200 units produced 23.1 lysis; 400 units, 29.5 lysis) than did tPAalone (200 units, 1.8% lysis; 400 units,19% lysis). Despite increased potency associated with duplex addition, fibrinogen and alpha-2-antiplasmin levels at equal tPA concentrations did not differ. Thus, itis possible to concentrate tPA (added separately) to the site of a thrombus in plasma using a heteroantibody duplex with specificities for both tPA and fibrin.Biosynthetically produced heteroduplexantibodies that are both fibin and tPA-specific. The bispecific antibodies were prepared in two ways. First, polyethylene glycol-mediated fusions were performed with two different hybridoma cell lines: anti-fribrin b chain producer, 59D8 and anti-tPA producer, TCL8. TCL8 cells were selected for HPRT-minus variants and then fused with TK-deficient 59D8 cells. One cell line, F36.23, possessed both anti-human fibrin and anti-human t-PA immunoreactivities. A second method yielded another bispecific antibody, F32.1. This cell line was selected after fusing TCL8 (HPRT-minus) cells with spleen cells from a mouseimmunized with a fibrin-like peptide corresponding to the amino terminus of fibrinalpha-chains. Affinity-purified F32.1 andF36.23 retained anti-fibrin and anti-t-PAactivity and enhanced fibrinolytic potency of tPA by a factor of 10.
Identification of a neutralizing epitope in the βE–βF loop of VP1 of equine rhinitis A virus, defined by a neutralization-resistant variant
