Maureen McTigue

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.

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.

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.

Rapid synthesis and quality control of 68Ga-labeled chelates for clinical use

Abstract Rapid synthesis of radiopharmaceuticals labeled with 68 Ga obtained from a 68Ge68Ga generator is described. Approximately 70% of the available 68 Ga activity was obtained in 4 mL 1 N HCl and evaporated to dryness in 5 min in a Rotovap system. Activity was reconstituted in 0.01 N HCl and 0.5 M NaAc, MPO added and pH adjusted to 6.7–7.0 with NaOH. Previously prepared human platelets were labeled with 68 Ga and reinjected within 1 h of eluting the generator.

Metal decomposition rates of 111In-DPTA and EDTA conjugates of monoclonal antibodies in vivo

We have studied the metal chelate decomposition rates in vivo in both 111In-labelled benzyl EDTA and DTPA (bicyclic anhydride) conjugates of monoclonal anti-IAk IgG2a with identical Ka = 1 x 1011M-1 in both Ag+ve and Ag-ve mice. Twenty μCi was given i.v. and whole body counting done immediately and daily for 10 days, with six to eight mice in each group. Half the mice in each group received i.p. injections of 5.0 mg CaNa2 EDTA chase (Versenate) to facilitate urinary excretion of free 111In. 50% of control 111In-citrate remained at nine days but only 8% with chase. No significant loss of 111In with chase occurred with C1 substituted EDTA conjugates. A 19% increase in excretion was demonstrated with the chase in mice given DTPA conjugates (1.9% per day). While this will not interfere with radioimmunoimaging up to 24 h after injection, waiting periods of a week or longer will produce significant background of free 111In in the reticuloendothelial system, RES. 111In-EDTA stability was important in accurate metabolic rate measurements of anti-IAk; T1/2 = 7.0 days in Ag-ve mice, T1/2 = 9.3 days in Ag-ve mice. It will be important to measure the in vivo rates for each new metal complex, especially those intended for therapy such as Y-90.

Viability and biodistribution of 68Ga MPO-labelled human platelets.

The viability and biodistribution of 68Ga-mercaptopyridine-N-oxide (MPO)-labelled autologous platelets was studied in 10 patients. The average platelet labelling yield was 36 ± 12% and injected activity was 2.0 ± 0.9 mCi 68Ga. The % activity in platelets per ml whole blood was 64 ± 20% at 15 min-1.0 h postinjection and 76 ± 14% at 2–4 h. The average recovery of platelets (% injected platelets circulating in peripheral blood) was 31 ± 21% at 15 min-1 h and 39 ± 20% at 2–4 h. The positron emission tomographic (PET) images showed high circulating vascular background. Two patients had technically inadequate scans, and six were false negative due to high blood background. One patient with a massive pulmonary embolus occurring 24 h prior to scanning had marked uptake of 68Ga platelets in a large clot in the superior branch of the right main pulmonary artery. A second patient, with 68Ga platelets circulating during angioplasty of a left posterior tibial artery stenosis, had intense uptake in the lesion shown on the PET scan obtained 4 h following the procedure. These results indicate good viability of 68Ga-MPO-labelled autologous human platelets, but poor visualization of clots by PET imaging, due to the high blood background at early times.

Pretargeting: Improved Pharmacokinetics and Therapeutic Ratio

Pre-clinical studies of three step pretargeting of an anti-hapten mAb were done in BALB/c mice with KHJJ mouse adenocarcinoma. An 80% reduction in whole body radiation burden and a 10 fold increase in Therapeutic Ratio was achieved, compared to directly labeled mAb in this model.

68Ga MPO Platelets in Human Pet Thrombus Imaging

The excellent immediate results of various interventional techniques for recanalization of peripheral and coronary arterial lesions has highlighted the persistant problem of restenosis, occurring in as many as 30% of patients in 1 year and 66% in 2 years1. Platelet deposition and its sequellae are among the most important possible causes of restenosis, and intensive research on the prevention of restenosis has involved many new anti-platelet drugs2. A method of measuring platelet deposition in vivo, would better define the effectiveness of the various anti-platelet protocols, help select patients and permit monitoring of anti-platelet drug activity3.