Donald J. Hnatowich

Preparation and use of NHS-MAG3 for technetium-99m labeling of DNA

The chelator mercaptoacetylglycylglycylglycine (MAG3) is on of several amidothiols that have been used successfully to radiolabeled proteins and other molecules with 99mTc. Prior to radiolabeling, the sulfur in these amidothiols is usually protected by a benzoyl group (i.e. S-benzoyl MAG3) which requires extreme alkaline pH or boiling water temperatures for rapid deprotection. As a result, the benzoyl-protected chelator is radiolabeled prior to conjugation (i.e. preconjugation labeling) in the case of carriers such as proteins or polypeptides which cannot withstand harsh conditions. We have employed a simple, two-step, synthesis of the N-hydroxysuccinimide ester of MAG3 in which the sulfur is protected with an acetyl group (i.e. S-acetyl NHS-MAG3). A single-stranded amine-derivitized DNA was coupled with NHS-S-acetyl MAG3. Radiolabeling was accomplished at room temperature and neutral pH by transchelation from 99mTc-tartrate. In comparison to labeled SHNH-DNA, the labeled MAG3-DNA was unstable to cysteine transchelation, however, in contrast to SHNH-DNA, no evidence for serum protein binding of the labeled MAG3-DNA was observed. We conclude that the S-acetyl NHS MAG3 bifunctional chelator may prove to be an attractive alternative method of radiolabeling DNA and other biologically important molecules with 99mTc.

Pretargeting using peptide nucleic acid

Pretargeting studies in animals and humans have usually involved (strept)avidin and biotin. Depending on the particular strategy, endogenous biotin can adversely influence localization when these molecules are used.

Can a cysteine challenge assay predict the in vivo behavior of 99mTc-labeled antibodies?

Recent investigations have shown that transchelation to cysteine in a principal mode of in vivo instability of 99mTc-labeled antibodies. In this investigation, a cysteine challenge assay was used to measure the in vitro instability of 99mTc directly labeled to two IgG antibodies (B72.3 and C110) via two established direct labeling methods employing mercaptoethanol and stannous ion for antibody reduction and by a novel method using glutathione for this purpose. For both antibodies, the greatest instability to cysteine occurred with stannous ion reduction. The stability of glutathione-reduced B72.3 was indistinguishable from mercaptoethanol-reduced B72.3 whereas glutathione-reduced C110 showed stability roughly intermediate between that of the other reducing agents for this antibody. Results obtained in normal mice were in the direction predicted by the assay: for both antibodies, urinary clearance of 99mTc was fastest in mice receiving antibodies labeled via stannous ion reduction, presumably because of the increased transchelation of label to cysteine in vivo. Urinary clearance was slower and identical in mice receiving B72.3 labeled via glutathione or mercaptoethanol whereas clearance in the case of glutathione-reduced C110 was intermediate between that of the other two reducing agents. At both time points, higher radioactivity levels were observed in kidneys and lower levels in blood and most other tissues for both antibodies in the case of stannous ion reduction as expected for the label of greatest instability. In the B72.3 case, with only one exception, tissue and blood levels following administration of glutathione-reduced antibody were indistinguishable from that following administration of mercaptoethanol-reduced antibody. In the C110 case, significant differences in activity levels were observed in several tissues between glutathione- and mercaptoethanol-reduced antibodies. In conclusion, the relative in vivo behaviour of 99mTc when administered to mice while labeled to two IgG antibodies were successfully predicted based on the results of an in vitro cysteine challenge assay.

Effect of endogenous biotin on the applications of streptavidin and biotin in mice

The use of streptavidin-conjugated antibody to pretarget tumors in animals and patients, prior to administration of radiolabeled biotin, has provided encouraging results, in part because of the high affinity of biotin for streptavidin and the rapid whole-body clearance of biotin. However, binding of endogenous biotin to streptavidin may interfere with the clinical potential of this approach. This report evaluates the effect of endogenous biotin on an antibody-streptavidin conjugate in a mouse tumor model. Tumored nude mice were depleted of endogenous biotin by sequential intraperitoneal injections of streptavidin. The assay of serum biotin levels indicated less than 0.5 ng of biotin per mL of serum in treated mice versus 4 ng per mL in untreated animals. Flow cytometric analysis was used on single-cell suspensions of tumor from animals receiving streptavidin-conjugated IgG to detect the presence of the antibody on the cell membrane (with fluoroisothiocyanate-conjugated goat anti-mouse antibody), and to detect biotin binding sites on streptavidin (with biotin-phycoerythrin). Both treated and untreated mice demonstrated the presence of antibody on tumor cells through 48 h postadministration, but only in treated animals were biotin binding sites observed. These results in the mouse model suggest that the small concentration of streptavidin delivered to a tumor via a specific antibody may be saturated with endogenous biotin and therefore not able to be targeted subsequently with radiolabeled biotin.

Technetium-99m antibodies labeled with MAG3 and SHNH: an in vitro and animal in vivo comparison.

The in vitro stability and animal pharmacokinetics of 99mTc bound to Sandoz and C110 IgG antibodies via a modified MAG3 has been compared with the hydrazino nicotinamide (SHNH) moiety as standard. For both antibodies, the stabilities of the label to challenge at up to 50:1 cysteine: IgG molar ratio were comparable, but at higher molar ratios, MAG3 showed greater instabilities. For the Sandoz antibody, size-exclusion HPLC analysis of 37 degrees C serum incubates and plasma samples from injected mice showed no clearly distinguishable differences. In the C110 case, some increased high molecular weight radioactivity was apparent with MAG3. Biodistributions in normal mice showed significant differences only in liver (Sandoz) and liver, spleen, intestines, stomach, and blood (C110), with SHNH usually providing higher levels. Thus, for two different antibodies and under the conditions of this study, the MAG3 chelator provided a 99mTc label with properties similar to that of SHNH moiety.

Use of indium-111-labeled OC-125 monoclonal antibody in the detection of ovarian cancer

Abstract This is a preliminary study to evaluate the utility of using the monoclonal antibody (CO-125) labeled with indium-111 to image recurrences of ovarian cancer. This technique has been investigated in 23 patients with ovarian cancer and the results have been compared with blood OC-125 levels, CT scans, and findings at second-look surgery. Following infusion of 1 mg of F(ab′) 2 fragments (1–2 mCi 111 In), quantitative SPECT and planar imaging was obtained daily for 72 hr along with analysis of serum. The nuclear medicine scans of the tumor site recurrences were technically excellent. When compared to second-look laparotomy, there were 2 true negatives, 2 false positives, 14 true positives, and 2 false negatives by nuclear imaging. CT scans correlated less well with surgery, but serum OC-125 levels correlate more closely with nuclear scans and second-look surgery. Those with multiple small metastatic implants showed a pattern of diffuse uptake which increased with time, whereas those with nodal or larger recurrences showed a more focal uptake. The combination of favorable biodistribution and positive images, especially in patients with normal antigen levels and negative CT scans, suggests a role for OC-125 monoclonal antibody imaging in their clinical management. However, further investigation is needed to determine whether nuclear scans can replace second-look surgery. If it can show that enough 111 In-labeled antibody accumulates in the tumor site to justify radioimmunotherapy, then 90 Y (a pure beta emitter) could be exchanged for 111 In. This is potentially a method of radioimmunotherapy for recurrent ovarian carcinoma.

Improved tumor localization with (strept)avidin and labeled biotin as a substitute for antibody

Because of its short physical half life, the use of anti-tumor antibodies radiolabeled with 99mTc has necessitated early (i.e. 2-6 h post-administration) imaging. It is possible that at these early times localization of antibodies in certain tumors may be largely due to non-specific processes. If so, other proteins or agents may be preferred for early imaging of solid tumors. We have investigated tumor localization with labeled biotin administered subsequent to unlabeled and unconjugated streptavidin. Nude mice bearing anti-CEA tumors (LS174T) received 10 micrograms of 111In-labeled anti-CEA antibody (C110) or 111In-labeled streptavidin with sacrifice 5 h later. In an examination of pretargeting, other animals received 50 micrograms of unlabeled streptavidin followed 3 h later with 1 micrograms of 111In-labeled biotin (EB1) and sacrifice 2 h later. The biodistribution of labeled streptavidin was similar to that of labeled specific antibody except for lower blood and higher kidney levels. Tumor levels were also lower with labeled streptavidin but, because of still lower levels in liver and blood, the tumor/normal tissue ratios were improved. When unlabeled streptavidin was administered and followed by labeled biotin (pretargeting), tumor levels were further reduced modestly; however, normal tissue levels were greatly reduced such that the tumor/blood and tumor/liver ratios were 10.6 and 2.2 vs 1.5 and 0.5 for the specific antibody. Improvements were seen in all tissues sampled with the exception of kidney and muscle. A further control of labeled biotin alone (without the preinjection of streptavidin) showed minimal accumulations in all tissues with the exception of kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

New indium-111 labeled biotin derivatives for improved immunotargeting

Investigations into the use of streptavidin-conjugated antibodies and labeled biotin to improve radioimmunotargeting have shown background levels drastically reduced over the conventional approach. Nevertheless, accumulation of 111In-biotin in normal tissue as well as streptavidin-independent accumulation in tumor, was observed. In this work, the effect of altering the biotin molecule to reduce this nonspecific uptake without decreasing specific localization has been investigated. Three EDTA and DTPA derivatives of biotin have been synthesized and investigated along with a commercial biotin derivative (DTPA-B2). The labeled biotin chelates were administered i.p. to normal mice implanted with avidin beads in one thigh. A wide variation in biodistribution was seen among the biotin derivatives. The most favorable results were obtained with biotinyl-hydrazino-EDTA (EDTA-B1), which showed the lowest accumulation in normal tissues but equivalent uptake in the target with respect to the other compounds. Averaged over 8 tissues sampled, the target-to-nontarget ratio was 140 vs 9 for EDTA-B1 vs DTPA-B2 (N = 6) at 24 h post administration. Similar observations have been made in culture with two tumor cell lines: positive accumulation of both DTPA-B2 and EDTA-B1 was measured in tumor cells independent of streptavidin-antibody conjugate, however in the case of the latter derivative, this accumulation was 3-5 fold lower. These studies show that modification of the biotin species can alter accumulation in normal tissues as well as the antibody-streptavidin independent accumulation in tumor tissue.

Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with 111In and 90Y. Serum stability studies at 37 degrees C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with 90Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to stimulate the attachment to protein. The serum stability for both 111In and 90Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with 90Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 +/- 0.5, N = 8) was significantly (P less than 0.001) lower than that of the DTPA-conjugated protein (9.4 +/- 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.

NHS–MAS3: a bifunctional chelator alternative to NHS–MAG3

This laboratory uses an N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS-MAG3) to conjugate amines for subsequent labeling with 99mTc. However, the synthesis from triglycerine is general and not restricted to this tripeptide. We had earlier selected a small number of alternative tripeptides and synthesized the corresponding NHS derivatives. Each was then evaluated in a search for bifunctional chelators with properties superior to NHS-MAG3, such as lower serum protein binding or improved stability to cysteine challenge. Based on these preliminary results, NHS-S-acetylmercaptoacetyltriserine (NHS-MAS3) was selected for further investigation. We have now conjugated this bifunctional chelator to an biocytin and to an amine-derivatized peptide nucleic acid (PNA). Both carriers were also conjugated with NHS-MAG3 under identical conditions and all were labeled with 99mTc at neutral pH and at boiling temperature while the conjugated PNAs were radiolabelled at neutral pH and at room temperature. Regardless of the chelator, reverse phase HPLC radiochromatograms of the labeled biotins and PNAs after purification showed a single peak. However, by size exclusion HPLC, the radiochromatograms always showed several peaks even after purification, but the MAS3 radiochromatograms were less complicated. For biotin and PNA both, radiolabeling via MAS3 showed improved 99mTc stability in 37 degrees C serum and in cysteine solution. The four preparations were administered to mice implanted in one thigh with avidin beads (biotins) or complementary PNA beads (PNAs). At 5 h post-administration, no significant differences were observed in the targeting of PNA beads between the two chelators, however the target thigh/normal thigh ratio was significantly higher for MAS3-biotin compared to MAG3-biotin. We conclude that labeling biocytin and amine-derivatized PNA with NHS-MAS3 compared to NHS-MAG3 provides simpler radiochromatographic profiles, improved stability of the label in serum and cysteine solution and can improve in vivo targeting.