Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.     

A bispecific antibody enhances the fibrinolytic potency of single-chain urokinase.

A monoclonal antibody specific for an epitope at the amino terminus of the beta chain of fibrin and a monoclonal antibody that binds both one- and two-chain high molecular weight urokinase were chemically cross-linked [using N-succinimidyl 3-(2-pyridyldithio)propionate and 2-iminothiolane]. The chemically modified material was heterogeneous, ranging in molecular size from tetramers to monomers and containing the two antibodies in various ratios. Nevertheless, fractions of a molecular size larger than a monomer were capable of binding fibrin and urokinase simultaneously in a radioimmunoassay. These fractions also enhanced fibrinolysis by high molecular weight single-chain urokinase (scuPA) by 50-fold and plasma clot lysis by 5-fold. Whereas scuPA significantly decreased the concentration of fibrinogen in plasma clot assay supernatants, scuPA in association with the bispecific antibody did not.     

A monoclonal antibody to human brain-type creatine kinase. Increased avidity with mercaptans.

1. A monoclonal antibody (subclass immunoglobulin G1) has been raised against human brain-type creatine kinase (CK-BB). This antibody did not cross-react with either muscle-type creatine kinase (CK-MM) or heart-type creatine kinase (CK-MB). 2. The binding constant measured with native antibody was 6 X 10(8) M-1. In the presence of 2mM-dithiothreitol this constant was some 40-50-fold greater. 3. Partial reduction and alkylation showed that the increased binding was due to a direct effect on the antibody and was associated with concomitant cleavage of the heavy-heavy interchain disulphide bonds. The binding constant measured with Fab' fragments produced from reduced and alkylated antibody was similar to that shown by the native, unreduced antibody. 4. The molecular weight of the complex found in the absence of mercaptans was consistent with one antibody and one CK-BB molecule, whereas the molecular weight estimated with reduced and alkylated antibody was consistent with a complex of two antibodies and two CK-BB molecules. 5. It is proposed that mercaptans increase the flexibility of the hinge region of the antibody molecule, allowing the formation of a higher-order complex with increased avidity for the CK-BB dimer.     

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.     

Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen

The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a (99m)Tc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab' fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab')(2) fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG x Fab' < F(ab')(2) x Fab' < Fab' x Fab' conjugate). The IgG x Fab' bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab')(2) x Fab' conjugate allowed for superior tumor accretion of the (99m)Tc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab' x Fab' conjugate, albeit tumor uptake was lower than with the F(ab')(2) x Fab' conjugate. Because the IgG x Fab' bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG x Fab' bsMAb remaining in the blood to take advantage of its 3-4-fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalent-DTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG x Fab' conjugate (time of maximum tumor accretion for this bsMAb), (99m)Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the (99m)Tc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG x Fab'/blocking agent combination and the F(ab')(2) x Fab' (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab')(2) x Fab'. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab')(2) x Fab' by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.     

Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Affinity enhancement of bispecific antibody against two different epitopes in the same antigen.

For the enhancement of antibody binding affinity, a bispecific antibody against two different epitopes in human chorionic gonadotropin hormone, one is in alpha-subunit and the other is in beta-subunit, was prepared by chemical recombination using 5,5'-dithiobis(2-nitrobenzoic acid). The epitopes recognized by antibodies were investigated by competitive radioimmunoassay, two-site sandwich radioimmunoassay and additivity assay and a proper epitope pair was chosen for preparation of the bispecific antibody. This bispecific antibody has dual specificity and as much as 17.2-fold higher affinity than that of monoclonal antibody with higher affinity by dual antigen binding radioimmunoassay and Scatchard plot analysis.     

Prolonged in vivo residence times of antibody fragments associated with albumin

Antibody fragments can be expressed at a high level in microbial systems, but they may have limited therapeutic value because they are rapidly eliminated from the body. We demonstrate here that site-specific conjugation or binding of bacterially derived Fab' to the long-lived protein serum albumin allows full retention of the antibody's binding characteristics while imparting the albumin's longevity in vivo. In rats the area under the curve for Fab' conjugated to rat serum albumin was 17-fold greater than for the control of Fab' conjugated to cysteine. Again, a bispecific F(ab')(2) with specificity for rat serum albumin showed an area under the curve about 8-fold greater than did a F(ab')(2) without specificity to albumin. Genetic fusions of scFv to albumin were similarly long-lived and could be expressed in yeast to provide the basis of a cost-effective production system.     

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.     

Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro

Tissue plasminogen activator (tPA) was covalently linked by disulfide bonds to a monoclonal antibody specific for the amino terminus of the beta chain of fibrin (antibody 59D8). The activity of the tPA-59D8 conjugate was compared with that of tPA, urokinase (UK), and a UK-59D8 conjugate. For lysis of fibrin monomer, tPA was 10 times as potent as UK, whereas both UK-59D8 and tPA-59D8 conjugates were 100 times as potent as UK and 10 times as potent as tPA. Conjugation of tPA or UK to antibody 59D8 produced a 3.2-4.5-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 tPA alone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasmin, and plasminogen than did the unconjugated activators, at equipotent fibrinolytic concentrations. Antibody targeting thus appears to increase the concentration of tPA in the vicinity of a fibrin deposit, which thereby leads to enhanced fibrinolysis.     

Luminescent detection method for immunodot, Western, and Southern blots

An anti-peroxidase-anti-biotin hybrid hybridoma rat cell line, capable of producing a bispecific monoclonal antibody, has been derived to explore its use in conjunction with a luminol immunodetection system. Luminescence was detected using x-ray film. The method was sufficiently sensitive and effective, but was less sensitive than autoradiographic methods using high-specific-activity 32P-labeled probes. Exposure times, on the other hand, were of the order of seconds rather than days. The direct binding of both peroxidase and biotin by the bispecific monoclonal antibody is simpler but less sensitive than the more conventional indirect method using a commercial peroxidase coupled with anti-rat antibody as a developing antibody.     

Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells.

To investigate whether the retargeting of resting CTL can benefit from cooperative signaling between the TCR/CD3 complex and various accessory molecules, such as CD2, CD4, CD5, and CD8, we have constructed a series of trispecific F(ab')3 derivatives. Each derivative was designed to engage effector T lymphocytes with two Fab' arms, and tumor cells with a single Fab' arm. They were constructed by selective coupling of three mAb Fab' fragments, primarily via their hinge-region sulfhydryl groups, using the cross-linker o-phenylenedimaleimide. En route to the production of trispecific F(ab')3 antibodies a range of bispecific F(ab')2 derivatives was first prepared which could bind simultaneously to two different receptor molecules on T cells. Bispecific derivatives containing specificities for (CD2 (T11(1)) x CD3), (CD3 x CD4), (CD3 x CD8) or two epitopes on CD2, ((T11(1) x (T11(3)), all yielded two to three times the uptake of [3H]thymidine with fresh PBMC to that seen with intact IgG from anti-CD3 (OKT3). The exception to these findings was a bispecific F(ab')2 derivative with specificities for (CD3 x CD5) which caused slightly less proliferation than the control reagent, OKT3 IgG. When these bispecific antibodies were converted into trispecific antibodies (TsAb) by the addition of a Fab' from anti-CD37 they were then all able to retarget resting, unprimed, T cells from fresh PBMC for lysis of CD37+ tumor cells. However, the cytotoxic activity of these reagents fell into two distinct groups: group one, containing (anti-CD3 x anti-CD4 x anti-CD37), (anti-CD3 x anti-CD5 x anti-CD37), and (anti-CD3 x anti-CD8 x anti-CD37), gave minimal lysis and behaved in a similar way to the BsAb, (anti-CD3 x anti-CD37), i.e., no evidence of cooperative signaling for lysis; and group two, containing (anti-T11(1) x anti-CD3 x anti-CD37) and (anti-T11(1) x anti-T11(3) x anti-CD37), which were highly cytotoxic and gave up to 80% specific 51Cr-release. The failure of group one TsAb, in particular (anti-CD3 x anti-CD8 x anti-CD37) which should recruit CD8+ CTL, to give efficient lysis despite having anti-T cell arms that were mitogenic as a bispecific antibody, indicates that the cooperative signaling for proliferation is probably distinct from the signal(s) provided by group two TsAb that activate for both proliferation and lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

Molybdate-stabilized glucocorticoid receptor: evidence for a receptor heteromer

The composition of the molybdate-stabilized glucocorticoid receptor (GR) complex has been investigated with a monoclonal antibody against the steroid-binding Mr 94 000 (94K) GR protein. It was concluded that one antibody molecule binds one 94K GR molecule. This finding constituted the basis for calculating the number of antibodies bound to the molybdate-stabilized nonactivated GR complex, which has an Mr of 302 000 (302K). Gel filtration on Sephacryl S-400 and density gradient centrifugation showed that only one antibody molecule bound to the molybdate-stabilized GR complex (calculated relative molecular mass for the antibody--molybdate-stabilized GR complex, 456 000; relative molecular mass for one antibody molecule, 157 000). Furthermore, experiments performed with a second antibody immunoprecipitation assay in the presence of an excess of both antibody and GR confirmed the above results. The possibility of steric hindrance not allowing more than one antibody molecule to bind to the molybdate-stabilized GR complex could be excluded. These results suggest that the molybdate-stabilized GR complex with an Mr of 302K only contains one steroid-binding 94K GR molecule and therefore represents a heteromeric complex.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.     

Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin.

We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.     

Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.