David A. Goodwin

Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions.

Preparation of the chelating agent (S)-4-[2,3-bis[bis(carboxymethyl)am ino]propyl]phenyl isothiocyanate is reported. Procedures for conjugation of this and (S)-N-4-[2,3-bis[bis-(carboxymethyl)amino] propyl]phenyl bromoacetamide to monoclonal antibodies and other proteins are described. The conjugates may be purified quickly by centrifugation through Sephadex G-50. The number of protein-bound chelating groups may be measured by titration with standard 57Co2+, using thin-layer chromatography to monitor binding. The labeled products retain their immunoreactivity, as illustrated by experiments in vivo with chelate-conjugated antibody to mouse I-AK antigen.

Chelating agents for the binding of metal ions to macromolecules

POLYAMINOCARBOXYLATE chelating agents such as ethylene-diaminetetraacetic acid (EDTA) form stable chelate complexes with the ions of many heavy metals. These metal ions exhibit a wide range of useful spectral and radioactive properties, such as electronic absorption, scattering of electrons and X rays, electron paramagnetic resonance spectra, the production of line-broadening and chemical shifts in nuclear magnetic resonance spectra, the emission of correlated gamma-ray cascades and various radioactive lifetimes and nuclear radiations. The preparation of chelating agents whose complexes can interact, in some selected manner, with biological macromolecules could make possible several new applications of metal ions as probes of biological systems.

Advances in pretargeting biotechnology.

A major focus of current drug research is to improve drug targeting to internal target sites such as to solid tumors or specific organs. The objective of drug targeting, especially for cancer chemotherapy and radioimmunotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects in nontarget sites. Although tumor targeting has been obtained with large long-circulating radiolabeled antibody molecules, normal organ activity, especially in the blood kidneys, liver, and bone marrow is a significant problem. Over the last 20 years, studies to improve the therapeutic use of antibodies have included the use of antibody fragments, chase molecules, metabolizable linkers, antibody-directed enzyme prodrugs (ADEPT), local delivery, and pretargeting. Here, we will review the most interesting recent advances in pretargeting biotechnology.

Use of specific antibody for rapid clearance of circulating blood background from radiolabeled tumor imaging proteins

AbstractA major problem that arises when radiolabeled serum proteins are used for tumor imaging is the presence of a large amount of circulating background activity that persists for several days. This delays imaging for at least 2 days following injection and necessitates computer subtraction of simulated background (second radiopharmaceutical injection) which introduces artifacts that are difficult to control. We propose here the injection of specific antibody immediately before imaging as an alternate way of reducing blood background through clearance of the immune complex by the liver. 111In-alkyl human transferrin and IgG were injected IV in BALB/c tumor mice, and followed in 18 h by anti-human transferrin and anti-human IgG antibody IV. Two hours later, the tumor and organ distribution of activity was compared with control mice not receiving antibody. 111In-transferrin blood activity was reduced to 1/48 of control with no decrease in tumor concentration: as a result, the tumor to blood ratio increased from 1.4:1 to 78:1. 111In-IgG blood activity was reduced to 1/17 of control, again with no decrease in tumor. The tumor to blood ratios increased from 0.7:1 to 17:1. The liver picked up most of the blood activity with none of the complex going to spleen, bone marrow, or kidney. Dog experiments showed clearance of blood was 90% complete in less than 15 min following antibody injection. Simultaneous scintillation images showed complete clearance of activity from the heart and great vessels in the chest and neck, and over the abdomen, with a concomitant increase in liver activity but no increase in spleen, kidney, or bone marrow activity. These studies show the feasibility of using specific antibody to lower the blood background just minutes prior to tumor imaging procedures using radiolabeled proteins.

Reagent and method for radioimaging leukocytes

Leukocytes are radioimmunoimaged by injecting patients with an immunoreactive nonleukocidal conjugate of an anti-leukocyte antibody and a gamma-emitting radioactive metal chelate, waiting for the conjugate to localize on the leukocytes, injecting the patient with an antibody to the conjugate to clear the blood of background nonlocalized conjugate, and visualizing the leukocytes by scintillation scanning.

Decomposition rates of radiopharmaceutical indium chelates in serum

The rates at which six small aminopolycarboxylate chelates of trivalent111In and three protein-bound chelates of111In deliver indium to the serum protein transferrin have been studied in sterile human serum at pH 7.3, 37°C. Sterically hindered chelates containing a substituent on an ethylene carbon of EDTA decompose with rates in the range 0.03 to 0.11% per day—one to two orders of magnitude slower than other chelates. Only small differences are observed between rates of decomposition for low-molecular-weight chelates and for protein-bound chelates having analogous structures.

Metabolizable 111in chelate conjugated anti-idiotype monoclonal antibody for radioimmunodetection of lymphoma in mice

The relative biological properties of 111In-labeled monoclonal antibodies (MoAb) coupled with a conventional bifunctional chelate (BC) and a new, enzyme metabolizable, bifunctional chelate (BCM) were investigated. A rat IgG2a MoAb against idiotype from a mouse B-cell lymphoma was utilized. Mice bearing B-cell lymphomas in the subcutaneous tissues of the flank were given IV-injections of labeled MoAb and imaged or killed for organ counting at 24 h or 48 h. Rat anti-dinitrophenyl IgG2a MoAb and non-specific polyclonal mouse IgG were used as controls. Compared to BC, the use of BCM resulted in a substantial decrease in blood background activity, a shorter biological half-life and an increase in tumor to blood ratio at the expense of a moderate decrease in absolute tumor uptake. The versatile chemistry of these C-1 substituted bifunctional chelates provides a variety of possible enzyme cleavable moieties for further investigation.

The attachment of metal-chelating groups to proteins: Tagging of albumin by diazonium coupling and use of the products as radiopharmaceuticals

The ability to attach firmly chelated metal ions or powerful chelating agents to sites on biological molecules can enhance the utility of a number of physical techniques now used in the study of biological systems. A “bifunctional” chelating agent, containing both an EDTA group and a diazonium group, has been prepared and coupled to human serum albumin. The extent of labeling under various conditions and the amino-acid sidechains labeled have been investigated. The reaction of protein-bound chelating groups with added metal ions has been studied, with the finding that only about 40–50% of these groups are available to bind metal ions. Proteolysis of the products leads to recovery of full metal-binding capacity. Properties of the products in vivo are discussed.

Monoclonal antibody hapten radiopharmaceutical delivery.

One hundred μg of monoclonal antibody (MoAb) CHA255 with a binding constant Kb of 4X109 was complexed with indium-111 labelled BLEDTA II, BLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-h tumour and organ distribution of BALB/c mice bearing KHJJ tumours was studied for each compound alone, the antibody complex, and 3 h following a chelate chase of the antibody complex. Whole body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-h whole body counts dropped 20 to 60% and blood concentration fell over 89% within 3 h of administering the chelate chase. Theoretical equivalent human organ doses were calculated from the 24-h organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads mCi-1. The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labelled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies enhances tumour imaging and improves the radiation dosimetry.

The use of indium-111-labeled leukocytes for abscess detection.

A case is reported in which a labeled white cell scan was helpful in the diagnosis of a periappendiceal abscess. The method of labeling is described and the usefulness of the technique discussed.