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The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.

A procedure for coupling liposomes to a target specific macromolecular species, anticanine cardiac myosin antibodies, was developed. The anticanine cardiac myosin antibodies covalently coupled to liposomes were shown to retain the specific antibody activity in vitro for the homologous radioiodinated antigen. Radioisotopes can be transported inside such liposome-antibody complexes and were shown experimentally to localize in acute canine myocardial infarction. This new method of coupling of liposome to target-specific agents could provide a novel and highly specific means of transporting radioisotopes or pharmaceuticals protected within the liposomes to selected target organs.

Polyethyleneimine or polylysines of differing molecular sizes were substituted with either EDTA or DTPA and then with succinic acid groups. These polymers were then reacted with the amino groups on myosin-specific monoclonal antibody or its Fab using a water soluble carbodiimide. The polymer-antibody complexes were capable of binding up to 150 di- or trivalent ions per mole (Mn++, Gd , or 111In ) without attendant loss of antigen binding. The polylysine derivatives of the intact antibody were rapidly cleared and sequestered in the liver, whereas the polylysine 14-kilodalton (kd) derivative of Fab was cleared from the circulation with minimal hepatic and kidney sequestration. This differed from the biodistribution of intact antimyosin or its Fab labeled with 111In via direct attachment of DTPA to the epsilon amino group of the lysyl residues. Applications in magnetic resonance and nuclear imaging are envisioned.

Monoclonal antibody R11D10 to human cardiac myosin, which also cross-reacted with canine cardiac myosin, was used to demonstrate in vivo localization and visualization by gamma scintigraphy of experimental myocardial infarction. R11D10 Fab with a Ka of 5 X 10(8) M-1 was labeled with technetium-99m (99mTc) by the dithionite reduction method of technetium pertechnetate, via a bifunctional chelating agent, diethylene triamine pentaacetic acid (DTPA). Uptake of 99mTc R11D10 Fab in the infarct can be visualized as early as 2 h after intravenous administration. Comparison of R11D10 uptake to thallium-201, an analogue of potassium which is sequestered by normal myocardium, showed an inverse relation (r = -0.75, -0.87, -0.89), similar to that obtained with 125I labeled polyclonal antimyosin Fab. Ratios of R11D10 Fab in the infarct to normal myocardium were as high as 30:1 where access of antibody to antigen was not blood flow limited. However, with severe blood-flow restriction, the ratios were lower at about 10:1. Despite the theoretical limitation of a single epitope per myosin molecule available for binding by R11D10 Fab, the immense excess of myosin in the infarcted myocardium allowed adequate concentration of radiolabeled R11D10 for visualization of the infarct by external gamma scintiscanning.