Chemical design of radiolabeled antibody fragments for low renal radioactivity levels (original) (raw)
Related papers
Bioconjugate Chemistry, 2013
We previously demonstrated that Fab fragments labeled with 3′-[ 131 I]iodohippuryl N ε -maleoyl-L-lysine ([ 131 I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[ 131 I]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-L-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: L-tyosine O-methyl, Lasparagine, L-glutamine, and N ε -Boc-L-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3′-
2005
Renal localization of radiolabeled antibody fragments presents a problem in targeted imaging and radiotherapy. We recently reported that Fab fragments labeled with 3′-[ 131 I]iodohippuryl N -maleoyl-L-lysine (HML) demonstrated markedly low renal radioactivity levels from early postinjection in mice. Previous studies suggested that low renal radioactivity levels were attributable to cleavage of the glycyl-lysine sequence in HML by the action of renal brush border enzymes, followed by urinary excretion of the resulting m-iodohippuric acid. In this study, an in vitro system using brush border membrane vesicles (BBMVs) isolated from the rat kidney cortex was developed to estimate renal brush border enzyme s)-mediated cleavage of the peptide linkage. Low molecular weight HML derivatives, 3′-[ 125 I]iodohippuryl L-lysine (HL), 3′-[ 125 I]iodohippuryl N -tert-butoxycarbonyl-L-lysine (HBL), and their D-amino acid counterparts, were synthesized and incubated in BBMVs. Both [ 125 I]HL and [ 125 I]HBL generated m-[ 125 I]iodohippuric acid after incubation in BBMVs at 37°C while the latter liberated significantly higher amounts of the metabolite. [ 125 I]D-HL and [ 125 I]D-HBL failed to release the metabolite under similar conditions. The liberation of m-[ 125 I]iodohippric acid from [ 125 I]HL was significantly facilitated or completely inhibited by the addition of an activator or an inhibitor for carboxypeptidase M. The release of m-[ 125 I]iodohippuric acid from [ 125 I]HBL increased by the addition of the activator, whereas the inhibitor partially inhibited the release of the metabolite from [ 125 I]-HBL. The BBMV-mediated release of m-[ 125 I]iodohippuric acid from [ 125 I]HBL was not impaired by the addition of inhibitors for neutral endopeptidase or renal dipeptidase. These findings showed that the glycyl-L-lysine sequence in HML would be recognized and cleaved by metalloenzymes and nonmetalloenzymes on the renal brush border even when iodine was incorporated into a benzene ring and the N -amine residue of lysine was chemically modified, which supported the hypothesis that low renal radioactivity levels of HML-conjugated Fab fragments would be attributed to the release of m-iodohippuric acid by renal brush border enzymes. This study suggested that this in vitro system using BBMVs would be useful to estimate radiolabeling reagents of antibody fragments or peptides designed to reduce renal radioactivity with a variety of radionuclides. Figure 1. Chemical structures of [ 125/131 I]HML and [ 125/131 I]-HML-conjugated Fab fragments.
Cancer research, 1995
The renal uptake of radiolabeled antibody fragments and peptides is a problem in radioimmunodetection and radioimmunotherapy, especially with intracellularly retained radiometals. The aim of this study was to develop suitable methods to reduce this kidney uptake. BALB/c mice or nude mice bearing the human GW-39 colon carcinoma xenograft were given i.p. injections of basic amino acids or a range of different basic amino acid derivatives, amino sugars, as well as cationic peptides. The effect of these agents on the biodistribution of Fab' and F(ab')2 fragments of different mAbs radiolabeled with *9nlTc, 188Re, '"In, 88Y, or I25I was studied. Tumor and organ uptake was determined and compared to untreated mice. The kidney uptake of Fab' fragments was reduced 5-6fold in a dose-dependent manner as compared to untreated controls. The uptake in all other organs, as well as the tumor, was unaffected. A similar reduction in renal retention was seen for all other intracellularly retained isotopes, as well as for F(ab')2 fragments. D-and L-isomers of lysine were equally effective whether given i.p. or p.o. D-glucosamine was effective, but its /V-acetyl derivative was not. Basic polypeptides (e.g., poly-L-lysine) were also effective; their potency increased with increasing molecular weight. HPLC of the urine taken from treated animals showed the excre tion of intact Fab', in contrast to mostly low-molecular-weight metabolites in the control group. These studies indicate that a variety of basic com pounds is capable of inhibiting the tubular reabsorption of peptides and proteins, thus lowering the kidney uptake of antibody fragments signifi cantly. On a molecular basis, the effect seems to essentially rely on the presence of a positively charged amino group. By reducing renal retention of antibody fragments, their role as imaging and therapeutic agents may be expanded.
European Journal of Nuclear Medicine and Molecular Imaging, 1998
Elevated renal uptake and prolonged retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. Over recent years, one of the focuses of research has therefore been to develop suitable methods to reduce this renal uptake, and to evaluate whether the resulting methodology will benefit therapy with antibody fragments and peptides. In these studies it has been shown that the kidney uptake of antibody fragments in animals can be reduced in a dose-dependent manner by almost one order of magnitude by the systemic administration of cationic amino acids and their derivatives, whereas the uptake in all other organs, as well as the tumor, remains unaffected. A similar reduction in renal retention is achieved for all intracellularly retained radionuclides (e.g., radiometals) or radioiodinated immunoconjugates, as well as for smaller peptides. Lysine is usually the preferred agent, and its d- and l-isomers are equally effective whether given intraperitoneally or orally. Amino sugars are effective, but their N-acetyl derivatives, lacking the positive charge, are not. Basic polypeptides are also effective, and their potency increases with increasing molecular weight (i.e., the amount of positive charges per molecule). Urine analysis of treated individuals shows the excretion of unmetabolized, intact fragments or peptides, in contrast to mostly low-molecular-weight metabolites in untreated controls. In therapy studies using radiometal-conjugated Fab fragments, the kidney is the first dose-limiting organ. Administration of cationic amino acids results in a substantial increase in the maximum tolerated dose of such Fab fragments. No biochemical or histological evidence of renal damage has been observed under these conditions. As was the case in animal studies, in pilot clinical trials the renal uptake in patients injected with Fab′ fragments and given amino acids could be decreased significantly, whereas the uptake by all other organs remained unaffected. These recent studies indicate that a variety of basic compounds are capable of inhibiting the tubular reabsorption of peptides and proteins, thus significantly lowering the renal uptake of antibody fragments or peptides in both animals and patients. On a molecular basis, the effect seems to rely essentially on the presence of a positively charged amino group. Thus, radiation nephrotoxicity of antibody fragments and peptides can be overcome successfully; this may provide new prospects for cancer therapy with radiolabeled antibody fragments and peptides.
Bioconjugate Chemistry, 2010
We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R 8 ) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R 8 with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R 8 -conjugated NuB2 was then reacted with succinimidyl meta-[ 125 I]iodobenzoate to prepare [ 125 I]SIB-NuB2 I (0.92 R 8 /NuB2) and [ 125 I]SIB-NuB2 III (3.38 R 8 /NuB2). Both SIB-NuB2 I and SIB-NuB2 III exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2 I also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2 III registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [ 125 I]SIB-NuB2 I and [ 125 I]SIB-NuB2 III exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [ 125 I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [ 125 I]SIB-NuB2 I and [ 125 I]SIB-NuB2 III exhibited significantly higher cell-associated radioactivity than [ 125 I]SIB-NuB2. In biodistribution studies in normal mice, while both [ 125 I]SIB-NuB2 I and [ 125 I]SIB-NuB2 exhibited similar biodistribution profiles, [ 125 I]SIB-NuB2 III showed faster clearance from the blood and higher hepatic radioactivity levels than [ 125 I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [ 131 I]SIB-NuB2 I exhibited significantly higher radioactivity in xenografts than those of [ 125 I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R 8 modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R 8 , which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2018
This study was undertaken to evaluate the renal radioactivity levels of a newly designed Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of Ga-NOTA-MVK-Fab into mice was compared with Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates Ga-NOTA-Met (Ga-NOTA-MI-Fab) or a conventional thiourea linkage (Ga-NOTA-Fab). The MVK linkage remained stable in plasma and was recognized by enzyme...
Monoclonal antibodies (mAbs) that internalize following binding to cell-surface receptors require radiolabeling approaches that minimize loss of radioactivity from the cell after intracellular processing. One class of internalizing mAbs ofgreat interest for imaging and radioimmunotherapy are those specific for EGERvIII, a truncated form ofthe epidermal growth factor receptor found on gliomas, non-small cell lung carcinomas, breast carcinomas, and ovarian carcinomas. Because lysosomes are known to retain positively charged compounds, N-succinimidyl 5-iodo-3-pyridine carboxylate (SLPC) might be ideal for radioiodination of these mAbs because of the positive charge on its pyridine ring. To investigate this hypothesis, the anti-EGFRvIII mAb LSA4 was labeled using SLPC, and internalization assays were performed usiag the EGFRvIII-positive cell lines HC2 20 di and NR6M. Compared with LSA4 labeled using lodogen or N-succinimidyl 3-iodobenzoate, SIPC increased infracellular retention of activity by up to 65%. Reverse-phase high-performance liquid chro matography analyses indicated that a significantly higher fraction of the low molecular weight catabolites from mAbs labeled via SIPC were retained within cells (SH'C, 28.1%; lodogen, 7.6% at 1 h). With SWC, the primary labeled species in cell lysates was the 5-iodonicotinic acid (INA) lysine conjugate, whereas in the supernatant, both INA.lysine and INA were seen. A 3-4-fold higher percentage of these catabolites were charged at lysosomal pH in comparison with those from mAb labeled using Nsuccinimidyl 3-iodobeazoate, in concert with the differences In cellular retention observed between these two labeling methods. In mice bearing HC2 20 d2 xenografts, a significant improvement in tumor retention of radioiodine and tumor:normal tissue ratios was seen when L8A4 was labeled using SIPC instead of the lodogen method. These results suggest that SIPC is a promising reagent for the radioiodination of anti-EGFRvIII L8A4and, possibly,otherinternalizingmAbs.
Antibody labeling with radioiodine and radiometals
Methods in molecular biology (Clifton, N.J.), 2014
Antibodies have been conjugated to radionuclides for various in vitro and in vivo applications. Radiolabeled antibodies have been used in clinics and research for diagnostic applications both in vitro as reagents in bioassays and in vivo as imaging agents. Further, radiolabeled antibodies are used as direct therapeutic agents for cancer radioimmunotherapy or as tracers for studying the pharmacokinetics and biodistribution of therapeutic antibodies. Antibodies are labeled with radiohalogens or radiometals, and the choice of candidate radionuclides for a given application is dictated by their emission range and half-life. The conjugation chemistry for the coupling of MAbs with the radiometals requires a chelator, whereas radiohalogens can be incorporated directly in the antibody backbone. In this chapter, we describe the commonly used methods for radiolabeling and characterizing the antibodies most commonly used radiohalogens (125I/131I) and radiometals (177Lu/99mTc).