Antifibrin Monoclonal Antibody Research Articles

Abstract 4849: Implications of cancer induced blood coagulation in cancer diagnosis and therapy

Abstract A large body of clinical evidence indicates that abnormal coagulation occurs in a variety of cancers, especially invasive cancers. If cancer clusters erode adjacent normal or tumor vessels, hemorrhage may occur, and fibrin clots immediately form in situ to stop the bleeding. The fibrin clots are subsequently replaced by collagen, similar to the case in the normal wound healing process. Although there are many points in common between cancer-induced stroma and normal wound healing, the critical difference between the two is that the pathophysiological condition in cancer lasts for as long as cancer cells survive in the body (Adv Drug Deliv Rev 2012). First, we successfully developed a mAb that reacts only with human insoluble fibrin, not human fibrinogen (Cancer Sci 2011). In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognizes an unexplored hole that is uncovered only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ-chain that interacted closely with a counterpart region on the γ-chain in the soluble state. New anti-Bβ and anti-γ mAbs specific for peptides lining the discovered hole appeared to bind exclusively to fibrin clots. Analysis of the kinetics of fibrin clot deposition using 102-10 in several disease models indicated that fibrin clot formation occurs only in the acute phase of the disease process, thrombosis, inflammation or trauma, and that these clots virtually disappear within a few weeks to be substituted by collagen. Then, we developed a new PET probe using 102-10. The radiolabelled mAb was injected into mice bearing chemically induced spontaneous tumors, and the probe showed clear and specific accumulation in the tumors. We named this detection strategy cancer stromal targeting (CAST) diagnois. Immunohistochemistry using this mAb revealed higher fibrin deposition in WHO grade 4 gliomas than in lower-grade gliomas. Because erosive tumors are highly likely to show micro-hemorrhages, even early asymptomatic tumors detected with radiolabeled 102-10 mAb may be aggressively malignant (Sci. Rep. 2013). In conclusion, CAST diagnosis and therapy based on anti-insoluble fibrin mAb may be a useful new strategy in the field of oncology. Citation Format: Yohei Hisada, Masahiro Yasunaga, Shingo Hanaoka, Shinji Saijou, Takashi Sugino, Atsushi Tsuji, Tsuneo Saga, Kouhei Tsumoto, Shino Manabe, Jun-ichiro Kuroda, Jun-ichi Kuratsu, Yasuhiro Matsumura. Implications of cancer induced blood coagulation in cancer diagnosis and therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2014-4849

Discovery of an uncovered region in fibrin clots and its clinical significance

Despite the pathological importance of fibrin clot formation, little is known about the structure of these clots because X-ray and nuclear magnetic resonance (NMR) analyses are not applicable to insoluble proteins. In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102–10) recognises an uncovered region that is exposed only when a fibrin clot forms. The epitope of the 102–10 mAb was mapped to a hydrophobic region on the Bβ chain that interacted closely with a counterpart region on the γ chain in a soluble state. New anti-Bβ and anti-γ mAbs specific to peptides lining the discovered region appeared to bind exclusively to fibrin clots. Furthermore, the radiolabelled 102–10 mAb selectively accumulated in mouse spontaneous tumours, and immunohistochemistry using this mAb revealed greater fibrin deposition in World Health Organization (WHO) grade 4 glioma than in lower-grade gliomas. Because erosive tumours are apt to cause micro-haemorrhages, even early asymptomatic tumours detected with a radiolabelled 102-10 mAb may be aggressively malignant.

A Fibrin-Specific Thrombolytic Nanomedicine Approach to Acute Ischemic Stroke

To develop a fibrin-specific urokinase nanomedicine thrombolytic agent. In vitro fibrin-clot dissolution studies were utilized to develop and characterize simultaneous coupling and loading of anti-fibrin monoclonal antibody and urokinase onto perfluorocarbon nanoparticle (NP) surface. In vivo pharmacokinetics and fibrin-specific targeting of the nanolytic agent was studied in dogs. Simultaneous coupling of up to 40 anti-fibrin antibodies and 400 urokinase enzymes per perfluorocarbon NP produced an effective targeted nanolytic agent with no significant surface protein-protein interference. Fibrin clot dissolution was not improved by increasing homing capacity from 10 to 40 antibodies/NP, but increasing enzymatic payload from 100 to 400/NP resulted in maximized lytic effect. Fluorescent microscopy showed that rhodamine-labeled urokinase nanoparticles densely decorated the intraluminal thrombus in canine clots in vivo analogous to the fibrin pattern, while an irrelevant-targeted agent had negligible binding. This agent offers a vascularly constrained, simple to administer, low-dose nanomedicine approach that may present an attractive alternative for treating acute stroke victims.

In Vivo Penetration of Experimentally Produced Clots by Monoclonal Antibodies

Antifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of two antifibrin mabs (NIB 1H10 and NIB 12B3) to penetrate occlusive clots in vivo was investigated. Both mabs react with human fibrin but not with human fibrinogen nor with the fibrin or fibrinogen from the species used in this study. Two heterologous animal (sheep and rabbit) thrombus models were used. Clots in both cases were made within isolated vein segments using a mixture of human and native fibrinogen. The clots in sheep veins were observed radiographically and found to be occlusive for a mean of 4.2 +/- 2.2 days and thereafter appeared only partially occlusive. When targeted in their occlusive phase (131)I labelled mab accumulated in the clot reaching a maximum ratio of 1.82 +/- 0.42 when compared to counts in homologous sheep clots in the contralateral limb. It was confirmed in the rabbit jugular vein model that total occlusivity did not prevent antibody accumulation in the heterologous clot by injecting the fibrin specific mab 1H10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments (125)I labelled mab 1H10 was used and detected using autoradiography. Both sets of experiments indicated that penetration of occlusive clots by the antibody occurred and that considerable accumulation was present at 6 and 24 h. The results indicate that a circulating antibody can readily gain access to experimentally produced clots in occluded veins.

New thrombolytic drugs.

Thrombolytic drugs reduce mortality from myocardial infarction and research is focused on newer drugs with improved clinical efficacy. The ideal thrombolytic drug should be effective in dissolving fresh and older thrombi, be rapid in its action with complete dissolution of the thrombus, be able to be given as a bolus and be safe without hypotensive or allergic or immunogenic reactions. The types of agents being developed fall into five broad categories: * mutants or variants of single chain urokinase type plasminogen activator; * mutants or variants of tissue type plasminogen activator; * recombinant chimaeric plasminogen activators; * conjugates of plasminogen activators and anti-fibrin monoclonal antibodies; * compounds derived from haemophagous animals. Within the mutant and variant groups there may be single point mutations which increase the half life or deletion of various amino acid sequences to increase resistance to plasma protease inhibitors or cause more selective binding to fibrin. The recombinant chimaeric plasminogen activators are designer drugs where the kringle regions, the growth hormone domain region, the finger domain region and the serine protease part of the molecules are all cleaved and recombined in various ways to improve potency. The conjugates of plasminogen activators and anti-fibrin and monoclonal antibodies improve the targeting of the agent to fibrin clot. Monoclonal antibodies are commonly used against the seven aminoterminal residues of the beta chain of fibrin. These are cross linked and bound to plasminogen activators. A variety of substances from haemophageous animals are currently being studied including salivary plasminogen activator from vampire bats, venom from southern copperhead snakes and staphylokinase from bacteria. Currently available thrombolytic agents have many limitations, but the novel thrombolytic agents have yet to be tested in clinical trials. With few exceptions, they all act via the plasminogen system and it may be in the future that combinations of anti-platelet and thrombolytic agents may prove to be more efficacious than thrombolytics and aspirin alone.

A Bispecific Antifibrin-antiplatelet Urokinase Conjugate (BAAUC) Induces Enhanced Clot Lysis and Inhibits Platelet Aggregation

Thrombolysis is well established in the treatment of acute myocardial infarction. However, clinical application of thrombolytic agents has limitations with respect to efficacy and specificity. To achieve highly effective and at the same time clot-selective plasminogen activation urokinase was coupled to a bispecific antibody consisting of the monovalent Fab' from the antifibrin monoclonal antibody 59D8 and the monovalent Fab' from the anti-glycoprotein GPIIb/IIIa monoclonal antibody 7E3. The bispecific antifibrin-antiplatelet urokinase conjugate (BAAUC) was synthesized and characterized. Assays with either immobilized platelets, GPIIb/IIIa or fibrin showed an increase in plasminogen activation compared to uncoupled urokinase by 10-fold, 58-fold and 13-fold, respectivley (p < 0.0001 each). In vitro clot lysis was performed on platelet-rich and fibrin-rich clots and revealed an up to 5-fold higher potency of BAAUC compared to uncoupled urokinase (p < 0.0001). In vitro platelet aggregation was effectively inhibited by the hybrid molecule, whereas urokinase had no effect. BAAUC and two monospecific urokinase-conjugates, UK-59D8-IgG and UK-7E3-(Fab')2 were compared with each other with regard to similar tests. In vitro clot assays with platelet-rich and platelet-poor clots were performed. BAAUC achieved a significantly higher plasminogen activation compared to each of the monospecific conjugates (p < 0.05, respectively). We conclude that BAAUC, a bispecific plasminogen activator with antifibrin and antiplatelet properties has the potency to lyse both fibrin-rich and platelet-rich thrombi with high efficacy and to effectively inhibit platelet aggregation.

Abstracts of Literature

Abstracts of Literature

A novel site-targeted ultrasonic contrast agent with broad biomedical application.

In this work, we report a novel targetable ultrasonic contrast agent with the potential to noninvasively define and localize myriad pathological tissues for diagnosis or therapy. The agent is a biotinylated, lipid-coated, perfluorocarbon emulsion that has low inherent echogenicity unless bound to a surface or itself. In study 1, emulsions with and without biotin were suspended in buffered saline and imaged with a 7.5-MHz linear-array transducer. Neither emulsion manifested significant ultrasonic backscatter until avidin was added. Avidin-induced aggregation produced a marked enhancement in backscatter from the biotinylated but not from the control emulsion. In study 2, porcine fibrin clots in vitro were pretargeted with biotinylated antifibrin monoclonal antibodies and then exposed to avidin and then to biotinylated or control perfluorocarbon emulsions. The basal acoustic reflectivity of clots imaged with a 7.5-MHz linear-array transducer was uniformly low and was increased substantially by exposure to the targeted biotinylated emulsion. In study 3, partially occlusive arterial thrombi were created in dogs and then exposed to antifibrin antibodies and avidin in situ. Biotinylated or control emulsion was administered either in situ or systemically. At baseline, all thrombi were undetectable with a 7.5-MHz linear-array transducer. Thrombi exposed to antifibrin-targeted contrast exhibited increased echogenicity (P < .05); control thrombi remained acoustically undetectable. These data provide the first in vivo demonstration of a site-specific ultrasonic contrast agent and have potential for improved sensitivity and specificity for noninvasive diagnosis of thrombi and other pathological diseases.

Preparation of an antifibrin thrombus-specific murine/human chimeric monoclonal antibody fab fragment in escherichia coli

We have successfully developed a murine antifibrin monoclonal antibody designated SZ-63 with property of binding specifically with thrombus both in vitro and in vivo. In order to reduce its immunogenicity and molecular weight, a murine/human chimeric Fab fragment of the antibody was preparaed. mRNA was selected on oligo (dT) cellulose from total RNA isolated from SZ-63 hybridoma cells. cDNAs coding for heavy and light variable regions were amplified by reverse transcriptase polymerase chain reaction. The amplified fragments were cloned and sequenced. The nucleotides of SZ-63 VH and VL are 354 and 321 respectively. The variable genes were then linked with human IgG γ1 CH and κ CL genes. Expression vector pHEN1-63 Fab/Hu was thereby constructed and chimeric Fab fragment was expressed in E. coli HB2151 cells in soluble form. Western blot and ELISA results showed that it remained the same capability of binding with cross-linked fibrin as the murine SZ-63 antibody, the content in culture is about 125 ug/L.

Evaluation of the Fibrin Binding Profile of two Anti-fibrin Monoclonal Antibodies

Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1H10 had a higher affinity for fragment-E (KD = 8.04 x 10(-9) M) than MAb 5F3 (KD = 1.13 x 10(-8) M). Individual association and dissociation rate constants of 7.97 x 10(5) M-1s-1 and 3.97 x 10(-3)s-1, respectively, for MAb 1HAb 1H10, and 5.16 x 10(5) M-1s-1 and 3.62 x 10(-3)s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-A alpha, E-B beta, E-gamma) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with 90Y-labeled monoclonal antifibrin antibody Fab fragment or with 90Y-labeled OC125-monoclonal antibody F(ab')2 fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.

99Tcm-labelled anti-fibrin Fab' fragment for evaluating the course of venous thrombosis in the rabbit.

Immunoscintigraphy was performed using the Fab' fragment of 99Tcm-labelled T2G1s anti-fibrin monoclonal antibody in a rabbit experimental model of venous thrombosis of known and increasing age (1, 3 and 7 days). Scans were positive in 3/7 cases for 1-day thrombosis (fibrin-poor network) and in 7/7 cases for 3-day thrombosis (fibrin-rich network). In rabbits with 7-day thrombosis, the scan was positive (2/4 cases) only when connective tissue was not present in the clot. Scintigraphic results were concordant with those of biodistribution studies showing the mean percentage of injected dose per gram 99Tcm-T2G1s present in the thrombosed vein wall (0.043, 0.082 and 0.07 for thrombi at 1, 3 and 7 days, respectively). Mean thrombosed vein wall-to-blood ratios were 1.027, 2.291 and 1.301, respectively. Immunoscintigraphy with T2G1s anti-fibrin monoclonal antibody thus enabled hematological status to be evaluated.

The labeling of proteins and LDL with 99mTc: a new direct method employing KBH 4 and stannous chloride

A new direct labeling technique for proteins with technetium-99m ( 99mTc) has been developed and makes use of borohydride and stannous chloride. The method is simple and reproducible and gives a high labeling efficiency and high retention of biological activity for proteins, including polyclonal immunoglobulin (Ig), antifibrin monoclonal antibody, tissue type plasminogen activator, fibrinogen and low density lipoprotein (LDL). This method can be used in kit-format and takes about 20 min preparation time at room temperature. Both in vitro and in vivo studies indicate good stability of the label. In vivo, mice and rabbit images show significant accumulation of 99mTc-Ig at an inflammation area, 99mTc-antifibrin at a thrombus site and 99mTc-LDL in atherosclerotic lesions. The method is attractive for routine research and clinical purposes.

Immunodetection of human atherosclerotic plaque with 125I-labeled monoclonal antifibrin antibodies

To test the affinity of a new F(ab′) 2 monoclonal antibody (TRF1) against human fragment D dimer of cross-linked fibrin for atherosclerotic plaques free of detectable thrombi, 6 atherosclerotic segments of carotid and femoral artery, and as a control 5 segments of atherosclerosis-free internal mammary artery, were drawn from 11 male patients undergoing bypass surgery. All segments were carefully washed in order to remove possible endoluminal thrombi, and cut to obtain pairs of intimal fragments of similar weight, containing either plaques ( n = 16), or fatty streaks ( n = 12), or normal endothelium ( n = 20). Each fragment underwent a direct binding test to TRFI, or to a non-specific antibody, both labeled with 125I. The activity in each fragment was measured after 3 h of incubation at 37°C, and after washing the fragments every hour for 3 h. TRF1 binding (as percentage of initial activity) was significantly higher ( P < 0.001) in atherosclerotic than in normal fragments (26% ± 11.5%, vs. 9.2% ± 3.9% in fatty streaks, and 1.9% ± 0.6% in normal endothelium), and indirect immunofluorescence confirmed TRF1 uptake within the plaque wall. By contrast, the non-specific antibody did not show any significant binding. These preliminary results demonstrate the high specific affinity of TRF1 for atherosclerotic plaques, probably due to the hemorheologic phenomena that activate platelets and provoke the formation of fragment D dimers of cross-linked fibrin on the plaque surface.

Radioimmunotherapy with90Y-Labeled Monoclonal Antibodies in a Nude Mouse Ovarian Cancer Model

Tumor stroma contains much fibrin, and so monoclonal antifibrin antibody can accumulate in tumors. We treated nude mice bearing human ovarian carcinoma xenografts with 90Y-labeled monoclonal antifibrin antibody Fab fragments administered intratumorally. The survival time vs. a control group was significantly prolonged and tumor growth rate was decreased. Another group of animals was treated with 90Y-labeled OC 125-monoclonal antibody; these mice received the antibodies intratumorally, intraperitoneally or intravenously. The survival time was longest in the intratumorally treated group. There was no significant difference in survival between 90Y-labeled OC 125 and antifibrin in the intratumorally treated animal groups. The tissue activity distribution studies revealed that bone marrow is the critical organ. Intratumorally injected monoclonal 90Y-antifibrin antibodies were retained at least 36 h (up to 50% of injected activity per gram tumor tissue) in the xenograft after one treatment, causing cell death. Beta-camera imaging and immunohistochemistry were performed for studies of the correlation between 90Y activity and fibrin distribution in tumor specimens. These results were in concordance. In conclusion, intratumoral administration seems suitable for radioimmunotherapy, with an antibody that targets stromal structures. The accumulation can be successfully monitored by a beta-camera.

Thrombus Imaging with 99mTc-Hmpao-Labeled Platelets and 111In-Labeled Monoclonal Antifibrin Antibodies

Eighteen patients with suspicion of deep venous thrombosis (DVT) in the lower extremities were imaged both with autologous 99mTc-HMPAO-labeled platelets (Tc-PLT) and 111In-labeled monoclonal antifibrin antibodies (In-MoAbs) on the same day. Presence or absence of thrombosis was verified by venography. Tc-PLT was given i.v. followed after 30 min by In-MoAbs. Anterior and posterior projections of the lower extremities were obtained with a large field-of-view gamma camera at 5 to 25 min, 2 h, 4 to 6 h, and 20 h after administration of the marker. Both Tc-PLT and In-MoAbs detected DVT well but less frequently than venography. Thrombi were visualized at 2 to 4 h after injection. The quality of images was better with Tc-PLT than with In-MoAbs. In the patients treated during the study, heparin significantly (p < 0.01) inhibited the uptake of Tc-PLT but not of In-MoAbs. We conclude that both Tc-PLT and In-MoAbs are suitable agents for the detection of DVT especially in patients without anticoagulation.

Remaining perspectives of mutant and chimeric plasminogen activators.

Potential approaches to improve thrombolytic agents comprise the construction of mutants and variants of tissue-type plasminogen activator (tPA) or of single chain urokinase-type plasminogen activator (scuPA, pro-urokinase), of chimeric plasminogen activators and of conjugates of plasminogen activators with monoclonal antibodies. tPA mutants have been constructed with altered pharmacokinetic properties or altered functional properties, including binding to and stimulation by fibrin, resistance to plasmin and to protease inhibitors. Mutants of tPA described to date, obtained by deletion/substitution of functional domains or of single amino acids, have markedly reduced clearances, but usually also reduced specific thrombolytic potencies. Mutants of scuPA with improved thrombolytic potencies have thus far not been reported. Chimeric molecules containing functional domains of both tPA and scuPA have intact enzymatic properties of uPA and some fibrin affinity of tPA. Surprisingly, chimeras endowed with fibrin affinity usually have unaltered or reduced thrombolytic potencies. However, a chimera consisting of amino acids 87-274 of tPA and amino acids 138-411 of scuPA, with negligible fibrin affinity, has a 10-fold higher thrombolytic potency than scuPA in animal models of venous thrombosis, as a result of a delayed in vivo clearance and a relatively maintained specific thrombolytic activity. Plasminogen activators conjugated with antifibrin or antiplatelet monoclonal antibodies, either chemically or by recombinant DNA technology, are targeted to blood clots, resulting in a 5- to 10-fold increased thrombolytic potency. Thus, it is possible to develop plasminogen activators with improved thrombolytic potency. Whether such agents will be clinically useful remains to be established.

Evaluation of thrombus detection in a rabbit model using a technetium-99m-labelled anti-fibrin monoclonal antibody.

Technetium-99 m (99mTc)-labelled conjugates of an anti-fibrin monoclonal antibody, DD-3B6/22, have been assessed for their detection of vascular thrombi in a rabbit model. DD-3B6/22 binds to a D-dimer epitope present on cross-linked fibrin but absent from the fibrin monomer or fibrinogen. Injection of a 99mTc-labelled Fab' fragment of DD-3B6/22 allowed delineation of model thrombi as early as 30 min postinjection (p.i.) with optimal localization at 4-5 h. Thrombus label uptake at 4 h p.i. was 0.304 +/- 0.106% injected dose/g (% ID/g) compared with 0.022 +/- 0.001% ID/g after the injection of a control Fab' fragment. These results suggest that the 99mTc-labelled Fab' fragment of DD-3B6/22 has excellent potential for scintigraphic detection of vascular thrombi in humans.

Evaluation of Indium-111 Antifibrin Monoclonal Antibody Imaging in Deep Venous Thrombosis Diagnosis

Objective: To evaluate Indium-111 antifibrin monoclonal antibody in the diagnosis of deep vein thrombosis. Design: Prospective study in twelve patients undergoing major surgical treatment, including hip surgery, who would be considered at high risk of developing subsequent deep vein thrombosis. Setting: Departments of Angiology and Vascular Surgery, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Patients: Twelve patients receiving surgical treatment. Interventions: Bilateral contrast phlebography was performed in all patients, combined with an ***immunoscintigraphic test of an Indium-111 labelled monoclonal ***antibody to fibrin. Patients with deep vein thrombosis were treated with full anticoagulation using heparin. Main outcome measures: The presence of thrombosis on phlebography and the presence of isotopic labelling of suspected deep vein thrombosis on immunoscintigraphic scanning. Results: Phlebography demonstrated venous thrombosis at 23 sites in veins, of which 22 were also detected by immunoscintigraphic scanning. Conclusions: Indium-111 labelled monoclonal antibodies to fibrin may provide an alternative method of investigation for suspected deep vein thrombosis as an alternative to other non-invasive diagnosis methodologies.

Noninvasive arterial thrombus imaging with 99mTc monoclonal antifibrin antibody.

The T2G1s monoclonal antifibrin antibody binds specifically to fibrin but not to fibrinogen. In a canine model of acute arterial thrombosis, we determined the feasibility of imaging thrombi using a 99mTc-labeled Fab' fragment. In 14 dogs, 10 carotid and 13 femoral artery thrombi were produced using 2-hour temporary occlusion, crush injury, and local thrombin injection methods. A sham-operated carotid artery served as control. Antifibrin antibody was injected intravenously at the end of temporary occlusion. Serial planar radionuclide images were obtained immediately and at 1 and 2 hours. Following killing the dogs at 2 hours, we measured antibody uptake ex vivo in 5-mm-long segments of thrombus, the adjacent injured artery, and a control artery. Antibody was cleared from the blood with a mean +/- SD t1/2 of 121 +/- 23 minutes. The thrombi weighed 218 +/- 140 mg. Antibody uptake in the thrombi was patchy, and the thrombi were closely adherent to the injured arterial wall. In the segment with maximal ex vivo antibody uptake, the ratio of control artery to blood counts/g/sec was 0.65 +/- 0.46, the injured artery-to-blood ratio was 2.35 +/- 1.01 (p less than 0.0001 versus control), and the thrombus-to-blood ratio was 4.24 +/- 2.58 (p less than 0.0001 versus control). In three dogs, an isotype-matched ovarian tumor antibody labeled with 111In was injected with T2G1s but was not taken up in the thrombus or the adjacent arterial wall. Visual analysis of the in vivo carotid radionuclide images showed uptake by 2 hours in all 10 carotid thrombi. Quantitative image analysis, measured as the thrombus-to-opposite carotid artery ratio, showed increasing uptake over time with ratios of 1.1 +/- 0.3, 1.6 +/- 2.0, and 2.2 +/- 1.3 on the immediate, 1-hour, and 2-hour images, respectively. All quantitative ratios of 1.3 or greater were visually identified. 99mTc-labeled Fab' fragments of the T2G1s antibody are taken up specifically by acute arterial thrombi after intravenous injection. Uptake is progressive over a 2-hour period, and all thrombi are detected by radionuclide imaging at 2 hours. These results show that it is feasible to noninvasively detect arterial thrombi within 2 hours of formation.