Development of a model system to evaluate methods for radiolabeling monoclonal antibodies.

We have developed a model system that can be used to evaluate methods for radiolabeling monoclonal antibodies. In this system, dinitrophenyl (DNP) is used as a model antigen, and HDP-1 (a monoclonal antibody specific for DNP) is used as a model monoclonal antibody. DNP-coupled agarose beads are injected into the femoral veins of rats, after which they localize in the animals' lungs. Radiolabeled antibody can then be injected and its biodistribution monitored. We describe here the development and initial testing of the model with radioiodinated antibody.

Magnetic resonance imaging using gadolinium labeled monoclonal antibody.

Gadolinium was attached to antibodies and tested in vitro and in vivo for its effect on proton relaxation enhancement. Using the cyclic anhydride method, diethylenetriaminepentaacetic acid (DTPA) was attached to albumin, IgG and anti-CEA monoclonal antibody. Gadolinium (Gd) was then chelated to the protein complexes forming protein-DTPA-Gd complex. With this technique approximately 9 atoms of Gd could be attached to each albumin molecule, 4 to each IgG molecule and 1.5 to each monoclonal antibody molecule. The minimal in vitro concentration of Gd in the form of IgG-DTPA-Gd necessary to produce proton relaxation enhancement at 0.35 tesla was 10(-1) mM. An in vivo experiment using anticarcinoembryonic antigen (CEA) monoclonal antibody-DTPA-Gd in hamsters implanted with human colon carcinoma resulted in a tumor concentration of Gd of less than 10(-4) mM. No enhancement of the tumors was detected at that concentration. For monoclonal antibodies to function as selective MR contrast agents, substantial advances in technology must occur.

Methods to label monoclonal antibodies for use in tumor imaging.

The use of radiolabeled monoclonal antibodies as a diagnostic tool in nuclear medicine has grown rapidly over the past several years. Early studies utilized antibodies labeled with radionuclides of iodine (i.e. 125I and 131I) although these radionuclides are not ideal for use in tumor imaging. Recent advances and the development of new chemical methods has made it possible to label antibodies with other radionuclides (i.e. 77Br, 111In and 99mTc). The advantages and disadvantages associated with all of the different radionuclides and labeling methods will be discussed.

Evidence for a saturable clearance mechanism for 111In-labeled monoclonal antibodies.

We have studied the dose effect of 111In-labeled HDP-1 on lung and liver uptake in rats. At low doses (much less than 25 micrograms) the lung/liver ratios are quite low. Higher doses result in dramatically increased ratios, suggesting that an uptake or metabolic process in the liver was becoming saturated. These results are compared with those previously obtained with 125I-labeled HDP-1.

Comparative studies using 125I- and 111In-labeled monoclonal antibodies.

HDP-1 monoclonal antibody was labeled with 111In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with 125I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab')2 and Fab' fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the 111In-labeled molecules were, however, slightly lower than those observed for the corresponding 125I-labeled molecules. High uptake was also observed in the liver or kidneys when the 111In-labeled molecules were used; no such results were obtained with the 125I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.

Antibody fragments labeled with fluorine-18 and gallium-68: in vivo comparison with indium-111 and iodine-125-labeled fragments.

Although monoclonal antibodies have been radiolabeled with many different radionuclides, the application of positron emission tomography (PET) to the imaging of radiolabeled antibodies has been limited to the investigation of a small number of long-lived radionuclides. In this study, we labeled F(ab')2 fragments of a mouse monoclonal antibody (BB5-G1) specific for a human parathyroid surface antigen with the positron emitting radionuclides, gallium-68 and fluorine-18. The biodistribution of the fragments was evaluated in a nude mice model and the results were compared to those obtained with fragments labeled with iodine-125 and indium-111 using conventional labeling techniques. All labeled fragments bound to human parathyroid tissue implanted in nude mice, with parathyroid-to-muscle ratios reaching as high as 10:1, 4 h after administration. A major difference was observed in the uptake and clearance of the various labeled fragments through the kidney. The halogen activity cleared, but the metal radioactivity was retained in the kidney. The results indicate that the fluorine-18 or gallium-68 labeled fragment may be useful for parathyroid imaging with positron emission tomography.

Human parathyroid antigen: characterization and localization with monoclonal antibodies.

A cell surface antigen on human parathyroid cells was identified by monoclonal antibodies. The antigen, called parathyroid antigen (PTA), is found on both of two major polypeptides (190 kDa and 160 kDa) apparently associated exclusively with parathyroid cells. To determine whether PTA was a suitable target for in vivo imaging, 125I-labeled anti-PTA was injected into nude mice bearing human parathyroid xenografts. IgG1 anti-PTA antibody showed excellent radiolocalization of the grafts, whereas IgM anti-PTA, containing the same variable domains as the IgG1 antibody, showed little specific binding. These results suggest that antibody isotype is an important parameter for the in vivo immunoscintigraphy of specific cellular antigens.

A potential new radiopharmaceutical for parathyroid imaging: radiolabeled parathyroid-specific monoclonal antibody--I. Evaluation of 125I-labeled antibody in a nude mouse model system.

Radioiodinated BB5-G1, a parathyroid-specific monoclonal antibody, and its F(ab')2 and Fab fragments were characterized using a nude mouse model system. Blood clearance studies indicated that the most slowly clearing species was the 125I-BB5-G1 intact antibody, while the most rapidly clearing one was the 125I-Fab fragment. 125I-F(ab')2 retained its capacity to localize in the human parathyroid tissue implants with the uptake at 24 h being similar to that observed with the intact antibody. Poor localization was observed with the Fab fragment. These results suggest that BB5-G1 or its F(ab')2 fragment may be useful for parathyroid imaging.

A potential new radiopharmaceutical for parathyroid imaging: radiolabeled parathyroid-specific monoclonal antibody--II. Comparison of 125I- and 111In-labeled antibodies.

The biodistributions of 111In-BB5-G1 and 111In-F(ab')2 were compared with the biodistributions of the corresponding 125I-labeled molecules. For BB5-G1 intact antibody, the relative uptake of the 111In- and 125I-labeled molecules in human parathyroid tissue implants was similar at 24 h, but by 96 h the uptake of the 111In-BB5-G1 %ID/g was four times greater than that observed with the 125I-labeled antibody. For the F(ab')2 fragments, the relative parathyroid uptake of the two preparations was similar at all times tested. The uptake by the clearance organs was significantly higher when the 111In-labeled molecules were used. Imaging results suggest that 111In-BB5-G1 or 111In-F(ab')2 may be a useful radiopharmaceutical for parathyroid radioimmunodetection.