Influence of circulating antigen on blood pool activity of a radioiodinated monoclonal antibody

Athymic mice with and without circulating CA 125 antigen were injected with 0.1–100μg of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment. Both the blood clearance of ¹³¹I activity and the change in serum CA 125 were monitored over 24 h. Influence of CA 125 on blood pool activity could be avoided only at the 100 βg dose. In patient studies, circulating CA 125 levels decreased for the first 2 h after injection of OC 125 F(ab′)₂ but generally returned to preinjection levels shortly thereafter. In vitro binding studies using the sera from patients injected with ¹³¹I-labeled OC 125 F(ab′)₂ suggest that circulating CA 125 could interfere with the tumor uptake of the labeled antibody.     

Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate

Two methods were investigated for the no-carrier-added synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). The first, an attempted nucleophilic aromatic substitution by [¹⁸F]fluoride on N-succinimidyl 4-nitrobenzoate was unsuccessful. The second method involved three steps; [¹⁸F]fluoride for trimethylammonium substitution on 4-formyl-N,N,N-trimethylanilinium triflate, oxidation to 4-[¹⁸F]fluorobenzoic acid, followed by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide to form S[¹⁸F]FB. Total synthesis and purification time was 100 min and the overall radiochemical yield was 25% (decay corrected). A monoclonal antibody F(ab′)₂ fragment could be labeled in 40–60% yield by reaction with S[¹⁸F]FB for 15–20 min. The tissue distribution in normal mice and in vitro tumor binding of the antibody F(ab′)₂ labeled by reaction with S[¹⁸F]FB were comparable to those observed for the fragment after radioiodination using N-succinimidyl 4-[¹²⁵I]iodobenzoate.     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

Infarcted heart uptake and biodistribution of radiolabelled anti-myosin monoclonal antibody in rat and dog myocardial infarct models

A new mouse monoclonal antibody that recognizes α- and β-heavy chains of human atrial and ventricular myosin and β-heavy chain of human slow skeletal muscle myosin was obtained. The ¹²⁵I- and ¹¹¹In-labelled antibody, and its F(ab′)₂ and Fab fragments localize in isoproterenol induced infarcted rat heart, with the F(ab′)₂ fragment showing the highest uptake. Comparison with ^99mTc-pyrophosphate uptake in infarcted dog heart, induced by selective obstruction of a coronary artery, suggest that the ¹¹¹In-labelled F(ab′)₂ localizes specifically in infarcted myocardium only.     

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.     

Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.     

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQ_m-1/pUQ_p-1 and pUQ_m-2/pUQ_p-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [¹²⁵I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [¹²⁵I]pUQs used, indicating that [¹²⁵I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.     

Interaction and release of soulble immune complexes from mouse B luymphocytes

Soluble immune complexes (¹²⁵I BSA-anti-BSA-C) bind to B lymphocytes and accumulate at one pole of the cells (“caps”). The complexes remain on the membrane after incubation of the cells at 37 °C in tissue culture medium for several hours. The ¹²⁵I BSA can be quantitatively removed from the cell surface by incubation with excess BSA but not with excess antibody to BSA or preformed BSA-anti-BSA-C complexes. The release of ¹²⁵I BSA is probably due to the removal of the complexes from the cell membrane and not to an exchange between unlabeled BSA in the medium and the labeled BSA present in the membrane-bound complexes. Release of ¹²⁵I BSA by excess BSA is temperature dependent. The membrane-bound complexes can also be removed by incubating the cells with papain fragments of rabbit antibody to mouse Ig (anti-γ₁, γ₂, and k Ig chains). However, after exposure to divalent [F(ab′)² or 7S Ig] rabbit antibodies to mouse Ig, the complexes remain associated with the cells. In addition, after such treatment the complexes cannot be removed by excess BSA or by Fab anti-Ig.     

Possibility for tumor detection with fatty acid analogs

Newly synthesized 17-[¹⁸F]fluoro-3-methylheptadecanoic acid ([¹⁸F]BMHDA), 16-[¹⁸F]fluoropalmitic acid ([¹⁸F]PA) and 15-(p-[¹²⁵I]iodophenyl)-3-R,S-methylpentadecanoic acid ([¹²⁵I]BMIPP), fatty acid tracers, were examined for the possibility of tumor imaging using 10 tumor models in rats and mice. The highest tumor/muscle ratios with [¹⁸F]BMHDA were 2.3 using rat tumor AH109A and 1.9 using mouse tumor B16F1 (tumor accumulation 0.41 ± 0.05, 4.29 ± 0.77% ID/g, respectively). Tumor/muscle ratios with [¹²⁵I]BMIPP and [¹⁸F]PA were lower than those with [¹⁸F]BMHDA. Labeled fatty acids seem to have a lower potential for tumor detection.     

N-(m-[125I]iodophenyl)maleimide: an agent for high yield radiolabeling of antibodies

In an effort to radiolabel antibodies, N-(m-[¹²⁵I]iodophenyl)maleimide (m-[¹²⁵I]IPM) was prepared by the demetallation of an N-[m-tri-(n-butyl)stannylphenyl]maleimide intermediate. The unlabeled intermediate was synthesized in ⩾ 75% yield using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline, followed by reaction with maleic anhydride and ring annulation. All products were confirmed by NMR and elemental analysis. Labeling with ¹²⁵I was carried out in a biphasic mixture containing chloramine-T (radiochemical yield ⩾ 70%). Rabbit IgG modified with the heterobifunctional crosslinking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and bovine serum albumin were conjugated with m-[¹²⁵I]IPM (yield: 40 and 80%, respectively). In addition, m-[¹²⁵I]IPM was conjugated to rabbit IgG subunits (HL) in 70% yield. The in vitro stability of the radiolabeled proteins in serum showed < 1% deiodination over 24 h.     

Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with ¹⁸F (t_½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for ¹⁸F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [¹⁸F]AlF-NOTA moiety with [¹⁸F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[¹⁸F]fluoronicotinyl-PEG₄-methyltetrazine to provide [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB) also were synthesized. Radiochemical purity, affinity (K_D) and immunoreactive fraction of [¹⁸F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [¹⁸F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[¹²⁵I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [¹⁸F]FN-GK-5F7 were 3–4 times higher than those for co-injected iso-[¹²⁵I]SGMIB-5F7 as well as those observed for the ¹⁸F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [¹⁸F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [¹⁸F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.     

m-[125I]iodoaniline: a useful reagent for radiolabeling biotin

Biotinyl-m-[¹²⁵I]iodoanilide (BIA) was synthesized by coupling biotin to m-[¹²⁵I]iodoaniline via a mixed anhydride reaction. m-[¹²⁵I]Iodoaniline was produced from the tin precursor, which was prepared using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline. The radioiodinated BIA derivative is characterized by a stable amide and/or intact ureido group on the biotin molecule; it may thus be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors.     

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.     

Imaging of systemic Candida albicans infections with a radioiodinated monoclonal antibody: Experimental study in the guinea pig

Guinea pigs intravenously infected with Candida albicans were scanned to evaluate the use of radioiodinated monoclonal antibodies (MAb) to fungal antigens for detecting tissue infection sites. A total of 18 infected and 8 uninfected animals were used. MAb and F(ab′)₂ fragments directed against cell wall glycoproteins of C. albicans were labeled with ¹³¹I. Another MAb directed against a Schistosoma mansoni glycoprotein was labeled with ¹²⁵I and used as a nonspecific control. Radiolabeled MAbs were injected at a dose of 12.5 μg (500 kBq) per animal. Images were acquired 24 h later. Animals were then killed and the dissected organs were separately gamma-counted. The number of C. albicans colony forming units (cfu) per gram was determined in each organ. A clear relationship was found between the anatomic distributions of C. albicans and ¹³¹I. The biodistribution of ¹³¹I radioactivity associated with anti-Candida MAb was greater in infected animals than in healthy animals and increased with the number of cfu per g in each organ. The distribution was highly specific in animals with Candida endophthalmitis, a pathognomic feature of organ involvement during hematogenous dissemination. In contrast, the distribution of ¹²⁵I radioactivity associated with the nonspecific MAb was similar in healthy and infected animals. In infected animals, it was totally independent of the intensity of fungal infection.     

Synthesis and in vitro evaluation of radioiodinated indolequinones targeting NAD(P)H: Quinone oxidoreductase 1 for internal radiation therapy

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase and is highly expressed in many human solid cancers. Because NQO1 can be induced immediately after exposure to ionizing radiation, we aimed to develop an NQO1-targeted radiolabeled agent to establish a novel internal radiation therapy that amplifies the therapeutic effects when combined with external radiation therapy. We designed three NQO1-targeted radioiodinated compounds including two ether linkage compounds ([¹²⁵I]1 and [¹²⁵I]2) and a sulfide linkage compound ([¹²⁵I]3) based on the selective binding of indolequinone analogs to the active site of NQO1 by the stacking effect. These compounds were successfully prepared using an oxidative iododestannylation reaction with high radiochemical yields and purity. In NQO1-expressing tumor cells, [¹²⁵I]1 and [¹²⁵I]2 were readily metabolized to p-[¹²⁵I]iodophenol or m-[¹²⁵I]iodophenol and [¹²⁵I]I⁻, whereas over 85% of the initial radioactivity of [¹²⁵I]3 was observed as an intact form at 1 h after incubation. The cellular uptake of [¹²⁵I]3 was significantly higher than those of [¹²⁵I]1 and [¹²⁵I]2. The uptake of [¹²⁵I]3 was specific and was dependent on the expression of NQO1. These data suggest that the novel NQO1-targeted radioiodinated compound [¹²⁵I]3 could be used as a novel internal radiation agent for the treatment of cancer.     

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with K_i values ranged from 0.11 to 4.64 nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [¹²⁵I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [¹²⁵I]10 and [¹²⁵I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [¹²⁵I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [¹²⁵I]10 and [¹²⁵I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [¹²⁵I]13 exhibited high brain uptake (3.42% ID/g at 2 min) and rapid clearance from the brain (0.53% ID/g at 60 min), while [¹²⁵I]10 showed lower brain uptake (0.87% ID/g at 2 min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [¹²⁵I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.     

Regional distribution study of brain perfusion and metabolic agents in normal and tumour bearing rat brains using autoradiographic technique

Regional distribution of brain perfusion imaging agents, [¹³¹I]N,N,N′-trimethyl-N′-[2-hydroxy-3-methyl-5-iodobenzyl]1,3 propanediamine (HIPDM) and [¹³¹I]N-isopropyl-p-iodoamphetamine (IMP), was compared with the distribution of patterns of [¹⁴C]l-methionine and [¹⁴C]d-glucose in normal and tumour bearing rat brains using autoradiographic technique. There was higher concentration of the radiopharmaceutical in grey than white matter in normal rat brain. Autoradiographs of brain tumour sections showed very low uptake of [¹³¹I]HIPDM and [¹³¹I]IMP as compared to normal brain tissue. There was moderate concentration of [¹⁴C]d-glucose and avid uptake of [¹⁴C]l-methionine in tumours. Autoradiographic study is useful for evaluating distribution patterns of radiopharmaceuticals.     

Triaryl (Z)-olefins suitable for radiolabeling with iodine-124 or fluorine-18 radionuclides for positron emission tomography imaging of estrogen positive breast tumors

A group of (Z)-1,2-diphenyl-1-[4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]but-1-enes were synthesized using methodologies that will allow incorporation of a [¹²⁴I]iodine substituent at the para-position of either the C-1 phenyl ring or the C-2 phenyl ring, or a [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-2 phenyl ring. These [¹²⁴I] and [¹⁸F] radiotracers are designed as potential radiopharmaceuticals to image estrogen positive breast tumors using positron emission tomography (PET).     

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

In this report, we have employed macroautoradiography to compare the tumor targeting of ¹²⁵I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to ¹²⁵I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab′)₂ and Fab′ fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3–7 days post-treatment. Treatment with F(ab′)₂ and Fab′ fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background ¹²⁵I-radioactivity which was well advanced by 6–24 h and complete by 24–48 h after injection. Localization of ¹²⁵I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab′)₂ and Fab′ fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments.