Isothiocyanate-trigalactose: application for antibody-targeted delivery of diagnostic and therapeutic agents.

Radiolabeled monoclonal antibodies (MAb) and MAb-streptavidin conjugates exhibit slow blood clearance which impedes radioimmunoimaging and radioimmunotherapy. To control blood clearance and lower background levels, lesion-specific targeting proteins can be modified with galactose derivatives for liver uptake via the hepatocyte galactose receptor. In this study, an isothiocyanate-trigalactose derivative (ITC-Tgal) designed for direct coupling to protein amino groups, was synthesized and characterized.

Two-step immunological approaches for imaging and therapy.

Radiolabeled monoclonal antibodies and their F(ab')2 and Fab fragments have been successful for imaging and therapy. However, their prolonged circulation and nonspecific accumulation in metabolic organs have resulted in high background radioactive levels and delayed imaging times. Current approaches are two- and three-step procedures consisting of first, the injection of a targeting moiety, which has specific binding affinity for both a lesion and a small molecular weight radiolabeled agent, and, second, a subsequently injected radioactive diagnostic or therapeutic agent.

Biochemical modification of streptavidin and avidin: in vitro and in vivo analysis.

Unlabelled: The high affinity streptavidin (or avidin)/biotin system is being investigated for imaging and radiotherapy procedures. Streptavidin (SA) and avidin exhibit markedly different pharmacokinetics, with avidin clearing from the blood much faster than SA. To optimize blood clearance kinetics, SA and avidin were biochemically modified and analyzed in vitro and in vivo.

Galactose-modified streptavidin-GC4 antifibrin monoclonal antibody conjugates: application for two-step thrombus/embolus imaging.

Diagnostic and therapeutic procedures utilizing the high affinity streptavidin (SA)/biotin system are being investigated for in vivo use. We are developing a rapid two-step imaging technique for the diagnosis of deep venous thrombosis and pulmonary embolism. The optimal SA-bound targeting moiety would circulate adequately for sufficient lesion accumulation, but nonbound reagent would clear in a reasonably short time before the injection of radiolabeled biotin.

Synthesis, pharmacokinetics, and biodistribution of 67GA deferoxamineacetyl-cysteinylbiotin.

The exceptionally high affinity of streptavidin for biotin may be exploited for two-step in vivo approaches for delivering radiolabelled biotin derivatives to lesion-bound streptavidin-conjugated monoclonal antibodies. A radiolabeled biotin derivative was prepared, and its characterization, stability, pharmacokinetics, and biodistribution studies are presented. This derivative contains deferoxamine, a chelating moiety with high affinity for trivalent metals suitable for imaging and therapy.

Quantification and lowering of serum biotin.

An enzyme-linked assay was developed to quantify serum biotin concentrations in experimental animals and humans. With this assay the effect on serum biotin concentration after intravenous injection of streptavidin or the addition of avidin to food was studied in rabbits and dogs. Intravenous injection of streptavidin reduced serum biotin values quickly but temporarily, in a dose-dependent manner in both species.

Pharmacokinetics and biodistribution of radiolabeled avidin, streptavidin and biotin.

The extraordinary high affinity of avidin and streptavidin for biotin may be exploited in a two-step approach for delivering radiolabeled biotin derivatives suitable for imaging and therapy to target-bound streptavidin or avidin conjugated monoclonal antibodies (MAbs). The in vivo pharmacokinetics and biodistribution of radiolabeled avidin, streptavidin (SA) and DTPA-biocytinamide (DTPA-biotin) were studied in the rabbit and dog. SA circulated in the blood similar to other 60 kDa proteins, avidin cleared immediately and DTPA-biotin exhibited plasma clearance by glomerular filtration.

Plasma stability and pharmacokinetics of radiolabeled deferoxamine-biotin derivatives.

The extraordinary high affinity of biotin for streptavidin may be exploited in a two-step in vivo approach for delivering radiolabeled biotin derivatives suitable for imaging and therapy to lesion-bound streptavidin-conjugated monoclonal antibodies. Compared to the use of directly radiolabeled monoclonal antibodies, the two-step approach is desirable because of the fast renal clearance of radiobiotin, which reduces in vivo background levels and radiation dose. Deferoxamine binds with high-affinity trivalent metals useful for imaging and radiotherapy.

Thrombus imaging: a comparison of radiolabeled GC4 and T2G1s fibrin-specific monoclonal antibodies.

Radioimmunoimaging of experimentally-induced canine thrombi has previously been achieved with iodine-131- and indium-111-labeled (131I and 111In) anti-fibrin T2G1s monoclonal antibody (MAb). We now compare T2G1s to another anti-fibrin MAb, designated GC4, for imaging fresh and aged canine thrombi. GC4 is specific for a neoepitope exposed on fibrin later in the thrombolytic process after plasmin digestion.

Immunoreactivity of 111In and 131I fibrin-specific monoclonal antibody used for thrombus imaging.

Immunoreactivity of radiolabeled F(ab')2 fragment of anti-fibrin T2G1s monoclonal antibody was determined by affinity chromatography using fibrin-coated Sepharose. This preparation is useful for thrombus detection in vivo by gamma camera imaging, provided a high percentage of immunoreactivity is retained after labeling. For 111In labeling, DTPA/F(ab')2 molar ratios were varied from 1000 to 6600/1, with little effect on immunoreactivity.

Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments.

We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131I-labeled F(ab')2 and Fab and 111In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG.

Imaging fresh venous thrombi in the dog with I-131 and In-111 labeled fibrin-specific monoclonal antibody and F(ab')2 fragments.

The authors report success in imaging thrombi using labeled monoclonal antibody or antibody fragments. An In-111 labeled antibody fragment appears to be the best imaging agent studied to date.

Aged venous thrombi: radioimmunoimaging with fibrin-specific monoclonal antibody.

Radioimmunoimaging of fresh canine venous thrombi with a murine monoclonal antibody specific for human and dog fibrin has been reported. Successful imaging of canine deep venous thrombi 1, 3, and 5 days old at the time of antibody injection is reported. Images were positive in all dogs, and the uptake of fibrin-specific antibody was equivalent to that of fresh thrombi.

Purification of fibrin-specific monoclonal antibody from ascites fluid by preparative isoelectric focusing.

With the increased use of site-directed monoclonal antibodies (MAbs) for the detection and localization of antigens (Ags) in vivo, an efficient, noninjurious MAb purification procedure is essential [1-9]. In this study MAb was purified by both immunoaffinity chromatography (IAFC) and preparative isoelectric focusing (PIEF). For anti-fibrin MAb purified by each method, the purification efficiency, blood clearance, and in vivo localization in thrombi were compared.

Radioimmunoimaging of venous thrombi using iodine-131 monoclonal antibody.

Murine monoclonal antibody (Mab) specific for the NH2-terminal region of human fibrin, but not cross-reactive with fibrinogen, was used in radioimmuno-imaging of fresh, induced venous thrombi in three dogs. Iodine-131-labeled Mab was injected intravenously, with iodine 125-labeled polyclonal murine gamma-G globulin (IgG) simultaneously injected as a control. Images were strongly positive at 24 and 48 hours in all three animals, with thrombus-to-blood and thrombus-to-muscle ratios of 8.