Bioconjugate Chem. 2006, 17, 597−602
Effects of Cell-Permeating Peptide Binding on the Distribution of Fab Fragment in Rats
597
125I-Labeled
Shouju Kameyama,*,† Mayo Horie,† Takeo Kikuchi,† Takao Omura,† Toshihide Takeuchi,‡ Ikuhiko Nakase,‡ Yukio Sugiura,‡ and Shiroh Futaki‡,§ Research Planning, Bipha Corporation, Chitose, Hokkaido 066-0051, Japan, Institute for Chemical Research, Kyoto University, and PRESTO, JST, Uji, Kyoto 611-0011, Japan. Received August 24, 2005; Revised Manuscript Received January 5, 2006
The peptides comprising the sequence of HIV-1 Tat protein (positions 48-60), Antennapedia (positions 43-58), and HIV-1 Rev protein (positions 34-50) are known to be cell-permeating. In this study, we examined how the distribution of Fab fragments in rats is affected by conjugation with these peptides. Fab fragment was iodinated by a chloramine-T method and then chemically conjugated with cell-permeating peptide. The complex of 125IFab and cell-permeating peptide was administered to male rats intravenously at a dose of 1 mg/kg, and wholebody autoradiography was performed at 4 and 24 h after administration. The patterns of distribution of 125I-Fab exhibited remarkable variation depending on the cell-permeating peptide used. In particular, at 4 h, high concentrations of radioactivity were observed in the spleen, adrenal gland, renal medulla, and liver with Rev peptide-Fab complex, in the liver and spleen with Tat peptide-Fab complex, and in the spleen, adrenal gland, and liver with Antennapedia peptide-Fab complex. Even at 24 h, high concentrations of radioactivity were still observed in the spleen and renal medulla of rat with Rev peptide-Fab complex, and in the spleen and renal cortex of rat with Antennapedia peptide-Fab complex. These findings demonstrate that the patterns of distribution of peptide-125I-Fab complexes can be modulated by selection of cell-penetrating peptides. Moreover, the patterns of retention of peptide-125I-Fab complexes in internal organs also differed at 24 h after administration. These findings provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.
INTRODUCTION Antibodies are used in a variety of diagnostic methods and treatments for their high specificity and affinity to antigens. Their specificity of binding to viruses and bacteria, in particular, is associated with the prevention of viral and bacterial proliferation and infection, and antibody preparations are already available as therapeutic pharmaceuticals. However, although they are effective in blood, lymph, and similar tissues, antibodies are incapable of penetrating into cells and tissues and are thus nearly ineffective against latent viruses and bacteria. Recently, a method of delivering high-molecular-weight proteins into cells using a cell-permeating peptide was developed, and the intracellular delivery of various proteins is now being studied (1-9). One of the most well-known cellpermeating peptides is that derived from positions 48-60 of Tat protein, a transcription regulator protein of HIV-1 (TAT). Antennapedia-derived peptide corresponding to positions 4358 (ANP) has also been widely used (10-12). We have shown that the RNA binding site (positions 34-50: REV) of the HIV-1 Rev protein, which is involved in the cytosolic transport of viral mRNA, can act as a cell-permeating peptide (13-15). We are seeking to develop an innovative pharmaceutical by conjugating the REV peptide to an antibody. Although many individual studies of these cell-permeating peptides have been performed, there have been few comparative studies of them. In particular, there have been almost no such studies of the effects of * Corresponding author. Mailing address: 1007-124, Izumisawa, Chitose, Hokkaido, 066-0051, Japan. Phone: +81-72-856-9389. Fax: +81-72-864-2341. E-mail:
[email protected]. † Bipha Corporation. ‡ Kyoto University. § PRESTO, JST.
attachment of these cell-permeating peptides to binding partner proteins on patterns of distribution in vivo. In this study, we used REV, TAT, and ANP peptides as cellpermeating peptides. As a binding partner protein, we employed a Fab fragment (Fab), a functional low-molecular-weight antibody component. Each cell-permeating peptide was conjugated to iodinated Fab, radioisotope-labeled with 125I in advance, and each complex was intravenously administered to rats. The patterns of distribution of complexes were examined by wholebody autoradiography (ARG), a method commonly used to examine the kinetics of labeled drugs in animals. The Fab was prepared from a polyclonal antibody (16), and each conjugate of cell-permeating peptide and Fab was prepared by a convenient method based on chemical modification (13).
EXPERIMENTAL PROCEDURES General Procedures. The native Fab was prepared from a commercially available polyclonal antibody preparation (Venogloblin-IH, Mitsubishi Pharma) using the ImmunoPure Fab Preparation Kit (Pierce). Cell-permeating peptides were synthesized using solid-phase methods at the Peptide Institute (Osaka, Japan). We employed REV, TAT, and ANP peptides as cell-permeating peptides, as shown in Table 1. The purities of these peptides were 95% or more as determined by HPLC. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was measured using an Applied Biosystems Voyager-DE STR. Preparation of Cell-Permeating Peptide-125I-Fab. The Fab was first labeled with 125I by the chloramine-T method (17, 18). Iodinated Fab (125I-Fab) was adjusted to 1 mg/mL with nonlabeled Fab solution. One hundred twenty micrograms of N-(6-maleimidocaproyloxy) succinimide ester (EMCS) (1 molar equiv to Fab) was added to 1 mL of 125I-Fab solution, and this
10.1021/bc050258k CCC: $33.50 © 2006 American Chemical Society Published on Web 04/25/2006
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Table 1. Cell-Permeating Peptides Used in This Study purity (%)a
peptide
amino acid sequence
REV (HIV-1 Rev (34-50)) TAT (HIV-1 Tat (48-60)) ANP (Antennapedia (43-58))
NH2-TRQARRNRRRRWRERQR-GCCONH2 NH2-GRKKRRQRRRPPQ-C-CONH2
98.2
NH2-RQIKIWFQNRRMKWKK-GCCONH2
95.6
98.0
a HPLC conditions: column, Zorbax 300SB-C18 (4.6 mm i.d. × 150 mm); eluant, 1-60% CH3CN/ 0.1% TFA (25 min); temperature, 50 °C; flow rate, 1.0 mL/min; detection, 220 nm.
mixture was gently stirred at room temperature for 2 h. The free EMCS was removed with a PD-10 column (Amersham) previously equilibrated with phosphate-buffered saline (PBS). Subsequently, the column effluent was concentrated by centrifugation using Microprep (Millipore), and this concentrate was adjusted to a final volume of 1 mL with PBS. One molar equivalent of REV, TAT, or ANP peptide (230 µL) was added to this reaction mixture, and the mixture was gently stirred at room temperature for 2 h. After excess peptide was removed using a PD-10 column, the protein fraction was concentrated by centrifugation using Microprep to obtain the complexes of REV, TAT, or ANP peptide with 125I-Fab (REV-125I-Fab, TAT-125I-Fab, ANP-125I-Fab). Each peptide-125I-Fab complex was adjusted to a final protein concentration of 1 mg/mL and stored below -30 °C until use. Radiochemical Purity of Cell-Permeating Peptide-Conjugated 125I-Fab. The radiochemical purity of test substances was determined by radio-HPLC before administration. The analytical column used was a POROS R2/10 (PerSeptive Biosystems), with a nonlabeled complex used to confirm retention time. Subsequently, an aliquot of the dosing solution was separated and assayed for radioactivity before administration. After this mixed solution was centrifuged (3000 rpm, 15 min, 4 °C), the supernatant was removed, and the sediment was assayed for radioactivity. The ratio of the concentration of radioactivity of the TCA sediment fraction to that of the entire dosing solution was calculated. Radioactivities were determined using a γ counter (Cobra 5005, PerkinElmer). Incorporation of Cell-Permeating Peptide-Conjugated 125IFab into HeLa Cells. HeLa cells (in 24-well plates) were grown to subconfluence. After replacement of culture medium with 500 µL per well of fresh MEM medium, the cells were incubated at 37 °C for 3 h. Subsequently, 5 µL of peptide-125I-Fab was added and the cells were incubated at 37 °C for 30 min. After washing three times with PBS, 200 µL of trypsin-EDTA solution was added to each well and incubated at 37 °C for at least 15 min to completely detach the cells. The cells and cell lysate were recovered from each well into a polystyrene tube, and radioactivity was determined using a γ counter (COBRA Quantum, PerkinElmer). Preparation of Fluorescein-REV-Fab and Microscopic Observation. The fluorescently labeled conjugates of Fab with cell-penetrating peptides were prepared similarly to cellpermeating peptide-125I-Fab conjugates. One hundred twenty micrograms of EMCS, together with 305 µg of fluorescein5(6)-carboxamidocaproic acid N-hydroxy succinimide ester (FLUOS), was added to 1 mL of nFab solution (1 mg/mL), and this mixture was gently stirred at room temperature for 2 h. Free EMCS and FLUOS were removed with a PD-10 column (eluate, PBS). The first main peak was concentrated by centrifugation using Microprep, and this concentrate was adjusted to a final volume of 1 mL with PBS. One molar equivalent of REV peptide (230 µL) to EMCS was added to this reaction mixture, and the mixture was gently stirred at room temperature for 2 h. After excess peptide was removed using a
PD-10 column, the protein fraction was concentrated by centrifugation using Microprep to obtain the conjugates of fluorescein-REV peptide with nFab (fluorescein-REV-Fab). Fluorescein-REV-Fab was stored below -30 °C until use. For microscopic assay, 2 × 105 cells were plated into 35mm glass-bottomed dishes (Iwaki) and cultured for 48 h. After complete adhesion, the culture medium was exchanged. The cells were then incubated at 37 °C with fresh medium (200 µL) containing the fluorescein-REV-Fab. The cells were washed five times with cold PBS. Distribution of fluorescein-REVFab in live cells was analyzed using a confocal scanning laser microscope FV300 (Olympus) equipped with a 40 × objective without fixation. Determination of Concentrations of Radioactivity in Blood. At 0.25, 0.5, 1, 3, 6, and 24 h after administration of test substance, blood (approximately 0.4 mL) was drawn from the caudal vein using a glass capillary with an inner wall treated with 500 units/mL heparin sodium in physiological saline. A 0.1 mL portion of each blood sample was separated for determination of blood concentrations and transferred to a polystyrene tube, and the radioactivity in the sample was determined using a γ counter. Subsequently, 1 mL of physiological saline and 1 mL of 20% trichloroacetic acid (TCA) were added to the assayed sample, and this solution was subjected to centrifugation (3000 rpm, 15 min, 4 °C). After the supernatant was removed, 1 mL of physiological saline was added to the sediment, followed by centrifugation in the same manner. The supernatant was removed, and the radioactivity in the TCA sediment fraction was determined. Whole-Body ARG. Animals receiving test substance were killed by ether anesthesia at 4 h after administration. After each animal body was clipped, an approximately 3% carboxymethylcellulose sodium salt (CMC-Na) solution was filled in the oral cavity, nasal cavities, earholes, and anus, and the animal was quickly frozen in a hexane-dry ice bath. The tail and extremities were then amputated, and the body was preserved in wrap at -20.4 to -17.0 °C for at least 24 h. An approximately 3% CMC-Na solution was applied to the body surface of the frozen fixed rat. Subsequently, the body was embedded in an approximately 5% CMC-Na solution and frozen in a hexane-dry ice bath to obtain a frozen block. Frozen sections 30 µm in thickness were prepared using a cryomicrotome. The sections were obtained in a median plane, in 2 planes containing kidney and other major tissues. Each section was freeze-dried at approximately -20 °C. The dry sections were applied to base paper and covered with Mylar film. The sections for ARG were brought into close contact with an imaging plate (IP), and exposed in a shield box for 12 h. After completion of exposure, the images were scanned using a bioimaging analyzer, and whole-body ARGMs were generated (gradation, 65536; resolution, 50 µm; sensitivity, 4000; latitude, 5, IP; BAS-MS2040). Images obtained by whole-body ARG were analyzed on a relative basis using the Image Gauge software program (version 3.46, Fuji Photo Film).
RESULTS Preparation of Cell-Permeable Peptide-Fab Conjugates. When the in vivo distribution of cell-permeable peptide-Fab conjugates is examined, contamination of free Fab in the conjugates may make the analysis of results difficult, since the methods of biodistribution of conjugates may differ from that of free Fab. We therefore first established conditions in which no free Fab remained in the conjugates, with use of various amounts of the cross-linking agent EMCS and the peptides (Figure 1). SDS-PAGE of REV-Fab and nFab showed that the ranges of molecular weights of the conjugates shifted depending on the amounts of cross-linking agent (EMCS) and
Distribution of
125I-Labeled
Bioconjugate Chem., Vol. 17, No. 3, 2006 599
Fab Fragment in Rats
Figure 1. SDS-PAGE of REV peptide and Fab conjugate. (A) Lanes 1-9 correspond to conjugates prepared by the treatment of nFab with 0, ×1.5, ×3, ×6, ×12 (1/2 sample volume), ×12, ×24, ×36, and ×48 equiv of EMCS and REV peptide, respectively, at room temperature for 2 h. When ×12 equiv of these are employed, substantially no free Fab is observed in the sample (B). Lane 1, nFab, and lane 2, REVFab, correspond to samples prepared by the same conditions as samples administered to rats. A 4-12% gradient gel (NOVAX) was employed for the analysis. Table 2. Stoichiometric Ratios of Fab and Cell-Permeable Peptide Conjugates molecular weight complexb samples nFab REV-Fab TAT-Fab ANP-Fab
peptidea (A) 2 597 1 821 2 406
main 48 000 61 000 57 000 60 000
diffc (B)
stoichiometric ratio (B/A)
Figure 2. The cellular uptake of the fluorescently labeled REV-Fab conjugate by HeLa cells. The cells were observed without fixation. (A) Fluorescently labeled REV-Fab. (B) Fluorescently labeled nFab. Table 3. Radiochemical Purities of the Cell-Permeable Peptide-Conjugated 125I-Fabs sample
13 000 9 000 12 000
5.0 4.9 5.0
a Molecular weight determined by electrospray ion mass spectroscopy (ESIMS). b Molecular weight determined by TOFMS. c The difference of molecular weight between peptide-Fab and nFab.
the peptides employed (Figure 1A). We selected use of ×12 equiv of EMCS and peptides each to Fab for preparation of the conjugates, with which no notable bands corresponding to unmodified nFab were observed on SDS-PAGE (Figure 1B). Since the cationic cell-penetrating peptides may neutralize the negative charges of SDS and may affect the mobility of samples in the gel, we further analyzed the molecular weights of the conjugates using MALDI-TOFMS. Table 2 shows the stoichiometric ratios of cell-penetrating peptides and Fab in the conjugates prepared using the same conditions as determined above, which were also employed for chemical conjugation of 125I-Fab in subsequent study. The average peptide:Fab ratios were thus estimated to be about 5:1 for each conjugate. In addition, no notable signals corresponding to the free Fab were observed (data not shown). Cellular Uptake of Fluorescently Labeled REV-Fab Conjugate in HeLa Cells. Figure 2 shows results of confocal
125I-Fab
REV-125I-Fab TAT-125I-Fab ANP-125I-Fab
radioactivity (MBq mg-1 mL-1) 1.27 1.45 1.49 1.27
radiochemical purity HPLC (%) TCA insoluble (%)a 97.3 98.5 98.4 98.5
95.1 96.5 96.2 94.3
a HPLC conditions: column, POROS R2/10 (4.6 mm diameter × 50 mm length); eluant, 1-100% CH3CN/0.1% TFA (5.5 min); temperature, 23 °C; flow rate, 3.0 mL/min; detection, 280 nm.
microscopic observation of HeLa cells treated with the fluorescently labeled REV-Fab conjugate (1 M) at 37 °C for 1 h. Significant internalization of the conjugate was observed, whereas internalization of the free, unconjugated Fab was poor. Radiochemical Purity of Peptide-125I-Fab Complexes. Table 3 shows the concentrations of radioactivity in and radiochemical purities of the samples given to rats. Rev peptide conjugated 125I-Fab (REV-125I-Fab), Tat peptide conjugated 125I-Fab (TAT-125I-Fab), and Antennapedia peptide conjugated 125I-Fab (ANP-125I-Fab) each had radiochemical purities above 97% as determined by HPLC. The radiochemical purities of the TCA sediment fractions of these samples were also 94% or higher. Incorporation of Cell-Permeating Peptide Conjugated with 125I-Fab into HeLa Cells. REV-125I-Fab, TAT-125I-Fab, ANP-125I-Fab, or 125I-Fab was added to the culture medium
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Figure 3. Effects of peptide binding on cellular uptake of 125I-Fabs in HeLa cells. Values are the mean ( SD of the results for 4 wells.
Figure 5. Whole-body autoradiograms at 4 h after intravenous administration of peptide-125I-Fab complexes to rats: 1, kidney; 2, spleen; 3, liver; 4, skin; 5, gastric contents; 6, bone marrow; 7, adrenal gland; 8, lung; 9, eyeball; 10, testis; 11, intestinal contents; 12, heart; 13, blood; 14, spinal cord; 15, brain; 16, thyroid gland; 17, urinary bladder.
Figure 4. Concentrations of radioactivity in blood after intravenous administration of 125I-Fab or REV-125I-Fab to male rats. Values are the mean ( SD of results for 3 animals.
for HeLa cells, and uptake into cells was determined. Figure 3 shows findings for these peptides at 5 and 30 min after addition of samples. The level of incorporation of 125I-Fab was very low at both times. On the other hand, the cell-permeating, peptideconjugated Fabs each exhibited high intracellular incorporation. The order of cellular uptake was REV-125I-Fab > TAT-125IFab g ANP-125I-Fab at both time points. Concentrations of Radioactivity in Blood Following Intravenous Administration. Figure 4 shows the time course of radioactive concentration in rat blood following intravenous administration of 125I-Fab or REV-125I-Fab. We measured the concentrations of radioactivity in whole blood and the TCAinsoluble fractions. 125I-Fab without cell-permeating peptide exhibited a slow biphasic decline in concentration of radioactivity. On the other hand, a rapid decrease in concentration of radioactivity was observed for REV-125I-Fab as early as within 1 h after administration, though the rate of decline subsequently slowed, with a plateau at 3 to 6 h after administration.
Whole-Body ARG. Single intravenous administration of REV-125I-Fab, TAT-125I-Fab, ANP-125I-Fab, or 125I-Fab to male rats was performed, and whole-body ARGMs were generated 4 and 24 h later. At 4 h after administration of 125IFab, particularly high radioactivity was observed in the thyroid, renal cortex, and gastric contents, and relatively high radioactivity in the skin. However, little radioactivity was detected in the brain or spinal cord (Figure 5A). At 24 h after administration, particularly high radioactivity was observed in the thyroid and relatively high radioactive concentrations were observed in the renal cortex, gastric contents, and skin, but almost no radioactivity was detected in any other tissue (Figure 6A). In the case of REV-125I-Fab, particularly high radioactivity was observed in the spleen, adrenal gland, and liver at 4 h after administration, and relatively high radioactivity was observed in the renal medulla (Figure 5B). At 24 h after administration, relatively high radioactivity was observed in the spleen and renal medulla (Figure 6B). For TAT-125I-Fab, particularly high radioactivity was observed in intestinal contents at 4 h after administration, and relatively high radioactivities were observed in the liver, spleen, and small intestinal contents (Figure 5C). At 24 h after administration, relatively high radioactivity was observed in intestinal contents, but almost no radioactivity was observed in any other tissue (Figure 6C). At 4 h after administration of ANP-125I-Fab, relatively high radioactivities were observed in the spleen, adrenal gland, and liver (Figure 5D). At 24 h after administration, relatively high radioactivity was observed in the spleen and renal cortex (Figure 6D).
DISCUSSION Whole-body ARG has been used for accurate analysis of the kinetics of labeled drugs and bioactive proteins in animals, and it yields precise findings useful for characterizing patterns of
Distribution of
125I-Labeled
Fab Fragment in Rats
Figure 6. Whole-body autoradiograms at 24 h after intravenous administration of peptide-125I-Fab complexes to rats: 1, kidney; 2, spleen; 3, liver; 4, skin; 5, gastric contents; 7, adrenal gland; 8, lung; 9, eyeball; 10, testis; 11, intestinal contents; 12, heart; 13, blood; 14, spinal cord; 15, brain; 16, thyroid gland; 17, urinary bladder.
distribution as well. Development of new antibody pharmaceuticals with the potential for penetration into cells is a challenge in antibody therapy. Determination of in vivo patterns of distribution is a very important part of this development. We therefore examined the patterns of distribution of peptideantibody complexes in rat using whole-body ARG. In the present study, we used REV, TAT, and ANP peptides as cell-permeating peptides, which were chemically conjugated to Fab as an antibody component. The patterns of distribution of the complexes were compared with each other. Contamination by free, unmodified Fab would make assessment of biodistribution of the peptide conjugates difficult due to the presence of two radioiodinated species (125I-Fab and peptide-125I-Fab); in this case, the biodistribution profiles of each peptide-125I-Fab conjugate would be the sum of those of the two radiolabeled species. We therefore established conditions in which free Fab should not contaminate cell-permeating peptide-Fab conjugates in significant amounts (Figure 1). TOFMS analysis revealed that each peptide-Fab conjugate had a peptide:Fab stoichiometric ratio of about 5:1. Therefore, the differences in biodistribution of conjugates can be attributed to the nature of each peptide. It would be ideal to use conjugates for which carrier peptides are modified to specific sites with a predefined stoichiometry. Generally speaking, it is not easy to obtain these by chemical modification. They may be obtained using genetically engineered proteins. However, considering the lack of information on antibody delivery using peptide carriers, we decided to employ chemical modification as a first step in our study. Confocal microscopic observation of HeLa cells showed that cellular uptake of Fab was promoted by conjugation with the cell-permeating peptides. Others have reported similar observations (23, 24). Using 125I-labeled Fab, we then examined the cellular uptake of REV-125I-Fab, TAT-125I-Fab, ANP-125IFab, and unconjugated 125I-Fab by HeLa cells. Each complex of 125I-Fab conjugated with a cell-permeating peptide was
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rapidly incorporated into HeLa cells, though 125I-Fab was not. These results are consistent with those of previous microscopic studies using fluorescently labeled conjugates (12, 13), suggesting that radioisotope labeling did not decrease the cellular permeability of these peptides. We subsequently performed whole-body ARG for cellpermeating peptides conjugated to 125I-Fab following intravenous administration to rats. The times for performance of wholebody ARG were determined based on the time courses of blood concentration of radioactivity following intravenous administration. 125I-Fab exhibited a slow biphasic decline in the blood, whereas REV-125I-Fab initially exhibited a rapid decline in blood following intravenous administration and then slowly entered a continuous phase about 3 to 6 h after administration. This initial rapid reduction in radioactivity in blood may be due to the quick penetration of REV-125I-Fab into tissue. One of the most successful studies of in vivo delivery using TAT and other cell-penetrating peptides is that by Schwarze et al. (4), who reported that intraperitoneal injection of conjugate of TAT and β-galactosidase to mouse resulted in distribution of the conjugates to various tissues including brain. They included tissue distributions at 4 h after administration. There have been several other in vivo studies, the majority of which present distributions in the body from 1 to 24 h (20, 21, 2527). In order to compare our results with the preceding findings, we performed whole-body ARG at 4 and 24 h. The latter time point was also selected to assess the duration of the conjugates in tissues; this information is very important for designing effective delivery systems with low toxicity. Conjugation of the cell-permeating peptides yielded remarkable changes in distributions of 125I-Fab. At 4 h after administration, nonconjugated 125I-Fab was present in the thyroid, gastrointestinal contents, renal cortex, and skin. On the other hand, peptide-conjugated 125I-Fabs were distributed not only to these organs but to others as well, including the liver, spleen, and adrenal gland. Interestingly, distinct patterns of penetration were observed for each of the cell-permeating peptides. For REV-125I-Fab, the highest concentrations of radioactivity were observed in the liver, spleen, and adrenal gland. TAT-125IFab was nearly unobservable in the adrenal gland, although it was observed in the liver and spleen. The pattern of tissue distribution of ANP-125I-Fab in tissues was similar to that of REV-125I-Fab, though REV-125I-Fab was characterized by greater penetration into the liver, spleen, and adrenal gland. On whole-body ARG at 24 h after administration, the earliest elimination was observed for TAT-125I-Fab, the levels of which had decreased nearly to those of 125I-Fab in almost all organs examined. The finding of high levels of radioactivity in intestinal and gastric contents suggests that TAT-125I-Fab might be excreted in the form of low-molecular-weight components. For REV-125I-Fab, high retention compared to that of 125I-Fab was observed in the spleen and renal medulla. Retention of ANP125I-Fab was observed in the spleen and renal cortex. These differences among peptides in localization and retention of Fab conjugates in vivo provide valuable information for the development of organ-specific antibody delivery systems. The distribution of TAT-125I-Fab at 4 h after administration in this study was similar to those of [99mTc]Tat peptide and 125I-scFV-Cys-TAT in previous studies. Previous studies on the distribution of TAT peptide alone or complexes of it with proteins have revealed accumulation in the liver and spleen (4, 19-22), consistent with the present findings. It should be noted, however, that although relatively high pulmonary penetration was observed in previous studies, almost no pulmonary accumulation of TAT-125I-Fab was observed in this study. This difference in findings might be due in part to differences among studies in binding partner proteins used or in methods used for
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conjugation. In addition, although the existence of β-galactosidase activity in brain was reported by Schwarze et al. (4), none of the Fab conjugates tested in this study exhibited high levels of accumulation in brain or spinal cord. Schwarze et al. administered test samples intraperitoneally, whereas we employed the intravenous route for administration. This difference in route of administration might have affected passage through the blood-brain barrier. In conclusion, this study has clearly demonstrated that appropriate selection of cell-penetrating peptides yields differences in in vivo distribution of Fab, with large changes in distribution observed in organs such as the liver, spleen, adrenal gland, and kidney. Moreover, conjugation of Fabs with cellpenetrating peptides was found to have profound effects on retention of such Fabs in internal organs. Further study using cell-penetrating peptides, especially on the effects of peptide: Fab stoichiometry and the precise kinetics/time course of biodistribution, will provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.
ACKNOWLEDGMENT We are grateful to Dr. Takatoshi Nakamura (Shin Nippon Biomedical Lab., Japan) for helpful discussions.
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