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Development and in Vitro Characterization of a Cationized Monoclonal Antibody against @A4Protein: A Potential Probe for Alzheimer's Disease' Ulrich Bickel,',? Virginia M. Y. Lee,' John Q. Trojanowski,' and William M. Pardridget Department of Medicine and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024, and Medical Pathology Section, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104. Received August 3, 1993"
The blood-brain barrier (BBB) is impermeable to IgG. Therefore, a delivery strategy has to be applied in order to use monoclonal antibodies (mAb) as diagnostic or therapeutic agents in the brain. It has been demonstrated that cationization of IgG allows for the BBB penetration following peripheral administration. A cationized mAb against PA4-amyloid could be a sensitive and specific diagnostic tool for Alzheimer's disease (AD). The site-protected cationizationand radiolabeling with lllIn of the specific anti @-amyloidmAb, AMY33, is described. The binding affinity of the antibody was retained after these procedures (& = 3.1 f 0.5 nM), as determined by solid-phase immunoradiometric assay and immunocytochemistryon AD brain sections. The in vitro binding by isolated brain capillaries indicated that the cationized antibody may be delivered to the brain in vivo. The ability of the modified antibody to detect cerebral &amyloid deposits in vivo can now be evaluated using single photon emissioncomputed tomography (SPECT) and a suitable animal model for cerebral amyloidosis,such as non-human primates or aged canines.
INTRODUCTION Vascular amyloid deposits and senile plaques are among the neuropathologic hallmarks of Alzheimer's disease (AD).2 The main constituent of these lesions is the 39-43 amino acid 4.2 kD @ amyloid protein (pA4), which was originally characterized from meningeal blood vessels (1) and subsequently found in senile plaques (2)and cerebral cortical microvessels ( 3 ) . It is derived from the amyloid precursor protein (APP) (4), and PA4 derivatives are released in small quantities into biological fluids (plasma, cerebrospinal fluid) in vivo ( 5 ) . The APP consists of 695770 amino acids depending on whether the APP isoform contains a Kunitz protease inhibitor-like insert. Soluble forms of APP are released from the membrane-boundAPP pool, but these soluble forms do not contain the intact PA4 peptide moiety, which is found in the full-length form of membrane-bound APP. The measurement in CSF of the nonamyloidogenic, secreted form of APP, which appears to be the main product of physiologic APPprocessing, has recently been suggested as a potential diagnostic test for AD (6,7). However, despite the central role played by PA4 amyloid deposition in the etiology of Alzheimer's disease (491,there is currently no noninvasive, in vivo diagnostic test for the presence of amyloid deposits within the central nervous system.
* Address correspondenceto this author at UCLA c/o William M. Pardridge. + UCLA School of Medicine. Hospital of the University of Pennsylvania.
¶ . Abstract published in Advance ACS Abstracts, February 1, 1994. 1 This work has been presented in part at the 22nd Meeting of the Society of Neuroscience, Anaheim, CA, Oct 25-30, 1992. 2 Abbreviations used: AD, Alzheimer's disease;PA4, @amyloid protein; APP, amyloid precursor protein; BBB, blood-brain barrier; IEF, isoelectricfocusing;HMD, hexamethylenediamine; DTPA, diethylenetriaminepentaacetic acid; EDC, N-ethyl-N'[3-(dimethylamino)propyllcarbodiimide; SPECT,single photon emission-computed tomography; IRMA, immunoradiometric assay; mAb, monoclonal antibody.
The availability of highly specific anti-PA4 monoclonal antibodies (10-13) suggeststheir use in a diagnostic method such as radioimmunoimaging, analogous to the immunoscintigraphic methods in tumor diagnosis (14). Such a diagnostic method can be expected to be more specific and sensitive for AD than clinical criteria (15)or, e.g., the currently evaluated measurement of regional cerebral blood flow using 99mTc-HMPA0single photon emissioncomputed tomography (SPECT) (15,16). However, not only parenchymal PA4 but also the cerebrovascular amyloid are localized beyond the endothelial cells of cerebral vessels ( I 7), which comprise the blood-brain barrier (BBB) in vivo. Despite the demonstration of morphologic alterations of the BBB in AD (18,19), there is no convincing functional evidence for a significant leakiness of the BBB in this disease (19,20). Therefore, it cannot be anticipated that native antibodies or even Fab fragments will be useful in AD radioimaging (13), owing to the negligible transport of proteins through the brain capillary endothelial wall. On the other hand, it has been demonstrated that cationization of IgG allows for brain delivery of antibodies via absorptive-mediated transcytosis through the BBB following peripheral administration (21, 22). In the present paper we describe the cationization and in vitro characterization of the specific anti-PA4 monoclonal antibody, AMY33 (11). The cationized antibody was then radiolabeled with an isotope suitable for SPECT, namely ll1In. The BBB permeability of the labeled cationized antibody was evaluated in vitro by measuring the uptake by isolated bovine brain capillaries, which represent an in vitro model of the BBB (23, 24). EXPERIMENTAL PROCEDURES
Materials. /3A41-28corresponding to the first 28 amino acids of the P-amyloid sequence as reported by Masters et al. (2) was synthesized using solid-phase methodology by the UCLA Peptide Synthesis Facility. Chromatographically purified mouse IgG (mIgG) and the mouse IgG1, mAb MOPC21 was purchased from Cappel (Durham, NC). Chloramine T was from MCB Reagents
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(Cincinnati, OH). Cla-Sep Pak cartridges and Ultra-free 30 000 PLTK filtration units were obtained from Millipore (Bedford, MA). Superose 12HR fast protein liquid chromatography (FPLC) columns, protein G-Sepharose 4 fast flow, and ampholine PAG plate (pH = 3.5-9.5) isoelectric focusing (IEF) gels were purchased from Pharmacia (Piscataway, NJ). Maxisorp break-apart modules were obtained from Nunc (Naperville, IL). Biotinylated horse anti-mouse IgG and Vectastain ABC-elite reagents were obtained from Vector Labs (Brulingame, CA). Na125I was purchased from Amersham (Arlington Heights, IL) and "lInC13 from New England Nuclear (Boston, MA). Hexamethylenediamine (HMD) and diethylenetriaminepentaacetic acid (DTPA) cyclic dianhydride were from Aldrich Chemical Co. (Milwaukee, WI). Centricon concentrators were supplied by Amicon (Danvers, MA). N-Ethyl-" [3-(dimethylamino)propyl]carbodiimide (EDC), Pristane, and all other reagents were obtained through Sigma Chemical Co. (St. Louis, MO). Female BALB/c mice were obtained from the Department of Laboratory Animal Medicine (UCLA). Production of AMY33 Antibody. Pristane primed female BALB/c mice received lo7AMY33 hybridoma cells per animal ip. The IgG fraction was purified from the harvested ascites by affinity chromatography on a protein G Sepharose 4 fast flow affinity column. The antibody was eluted from the affinity column with 0.1 M glycine a t pH 2.5 and immediately neutralized to pH 7 with 1M Tris base. The elution from the column was monitored at 280 nm, and the IgG-containing fractions were pooled and dialyzed against 20 mM phosphate bufferi0.15 M NaCl (PBS, pH = 7.4). Protein was measured by the method of Lowry (25),and SDS-PAGE of the purified mAb was performed under reducing conditions followed by Coomassie blue staining. The nonspecific control IgGl antibody, MOPC21, was similarly affinity purified from ascites on protein G sepharose. Cationization of IgG. The cationization,as developed for albumin (24)and bovine IgG (211,was modified for the present experiments as follows: the IgG (AMY33, MOPC21, or nonimmune, chromatographically purified mouse IgG) was used at a concentration of 1mg/mL in 20 mM PBS (pH = 7.4). Solutions of EDC (100 mg/mL, 0.52 M) and 2 M HMD were prepared in H20. The HMD solution was adjusted to the desired pH for the cationization with concentrated HC1. The known amino acid composition of MOPC21 (26) was used to estimate the number of acidic amino acids (Asp, Glu) in the IgG molecules. There are 120 Asp and Glu residues per MOPC21 molecule. HMD and EDC were added to the IgG to give final molar ratios in the reaction mixture of HMD/EDC/COOH groups of 200:7:1 or 1000:35:1, respectively. The pH was adjusted to values of 7.8,6.8, and 6.0 with 1 M HC1. Cationization was performed with or without site protection. Cationization under site protection of the antibody was performed after an overnight preincubation of AMY33 in PBS, pH 7.4, with a 10-fold molar excess of the synthetic peptide PA41-28at 4 "C. For purification, the crude pA41-28peptide had been dissolved in 90% formic acid and evaporated to dryness. It was then redissolved in 5 M guanidine/l M acetic acid and injected onto a reversed-phase HPLC column (Vydac Cq, 10 X 250 mm). Elution was performed with a linear gradient of acetonitrile in 0.1 7c trifluoroacetic acid at a flow rate of 3 mL/min and was monitored at 214 nm. The peptide peak was pooled and lyophilized. The purity of the HPLC-purified peptide has previously been confirmed
Bickel et al.
by amino acid sequencing (27). In addition, HPLC analysis following radioiodination yields a single, sharp peak (27). The cationization was allowed to proceed for 3 h a t room temperature under constant mixing, after which the reaction was quenched by the addition of a 20-fold (relative to EDC) excess of glycine (1 M in HzO, pH = 6.0 with HCl), followed by incubation for 1h a t room temperature. The final reaction volume was then adjusted to pH 2.5 with 1M HC1, filtered, and injected onto a Superose 12HR FPLC column. The elution was performed with 0.1 M glycine at pH 2.5 a t a flow rate of 0.5 mL/min. Elution under acidic conditions is required to separate the cationized AMY33 and the pA41-28peptide. The elution was monitored at 280 nm, and the fractions corresponding to the molecular weight of IgG were immediately neutralized by the addition of an appropriate volume of 1M Tris base. Isoelectric focusing (IEF) on polyacrylamide slab gels was used as described (21)to determine the isoelectric point (PI) of the native and cationized IgGs. Solid-Phase Immunoradiometric Assay (IRMA). The binding affinity of the native and cationized antibodies to pA41-28was tested in a solid-phase IRMA. The binding of lz51-labeledAMY33 to the peptide antigen, which was absorbed to the surface of polystyrene microtiter wells, was competed with increasing concentrations of unlabeled IgG. Native AMY33,cationized AMY33 with and without site protection, native MOPC21, and cationized MOPC2l in concentrations between 0 and 67 nM were used as competitors. HPLC-purified /3A41-28was coated to NUNC Maxisorp microtiter wells overnight at 4 "C with 50 pL per well of a 10 pg/mL peptide solution in 0.1 M NaHC03 at pH 9. The peptide solution was removed by aspiration. Blocking of nonspecific binding was performed by incubating the wells for 2 h at room temperature with an aqueous 0.25% polylysine (126 kDa) solution. The wells were then washed three times with 200 p L of 10 mM PBS (pH = 7.4). Iodination of native AMY33 was achieved with a chloramine T technique: 20 l g (0.13 nmol) of IgG in 50 mM PBS (pH 7.4) was labeled with 0.5 mCi Na 1251 by addition of 2 X 0.85 nmol chloramine T in 1-minintervals. The total reaction volume was 40 pL. The reaction was stopped by addition of 2.5 nmol of NazSz05 in 10 pL of HzO. The iodinated antibody was purified by gel filtration on a 0.7- X 28-cm Sephadex G25 column and eluted with 50 mM PBS containing0.01'36 bovine serum albumin. The specific activity of the tracer was calculated as 500 Ci/ mmol. For the binding experiments, the tracer was diluted to 50 000 dpm per 50 pL (0.9 nM) in assay buffer (PBS with 0.5% gelatin, 0.1 % Tween 20 and 10 U/mL heparin) and incubated in the microtiter wells in the presence or absence of unlabeled competitors overnight a t 4 "C. After aspiration of the tracer solution, the wells were washed five times with 200 pL of PBS, and the bound radioactivity was counted in a y-counter. The binding data were expressed as fraction of total tracer bound to the wells and were evaluated by nonlinear regression analysis. The computer program used for the regression analysis employs the BMDP 3R program (28). It was developed for the evaluation of ligand binding assays, and one specific binding site and nonspecific binding was assumed (29). Nonspecific binding and binding capacities were assumed to be independent of the ligand species but were rescaled within each experiment to account for minor differences in antigen plating density of the microtiter wells between assays. Immunocytochemistry on Alzheimer's Disease
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Cationized Monoclonal Antiamyloid Antibody
Brain Sections. Immunocytochemistryon 5-pm paraffin sections of human AD brains were performed as described (30). Following deparaffinizationin xylene and decreasing ethanol steps, the slides were incubated in 90% formic acid for 15 min followed by a tap water rinse (10 min) and treatment with 0.3 % H202 and again tap water for 5 min. After 5 min in TBS (50 mM Tris, pH 7.4,0.9% NaCl), the sections were covered with 3% normal horse serum for 30 min. The excess serum was blotted from the sections, and the primary antibody was applied for 90 min at room temperature in concentrations of 5,20, and 100 pg/mL in TBS. Biotinylated horse anti-mouse IgG was used as secondary antiserum at a concentration of 50 pg/mL for 30 min. The avidin-biotin-peroxidase complex (Vectastain ABC-Elite) was used according to the manufacturer's instructions. 3-Amino-9-ethylcarbazote(AEC) was used as a chromagen. DTPA Conjugation of Antibodies and Labeling with "'In. A modification of the method described by Sakahara et al. (31) was used. Native and cationized antibodies were dialyzed against 0.1 M NaHC03 and adjusted to a concentration of 1 mg/mL. DPTA cyclic dianhydride was dissolved in DMSO (5 pmol/mL) and added to the antibodies in a 30:l molar excess. After 60 min at room temperature, the free, unconjugated DTPA was removed by repeated ultrafiltration on Centricon 30 filtration units (molecular weight cutoff 30 kDa). The initial reaction volume was concentrated to 10% ,diluted 10-fold with 0.2 M Na citrate, and again concentrated to 10%. The DTPA-conjugated IgG was finally taken up into 0.2 M Na citrate a t a concentration of 1 mg/mL. 5 mCi of ll1InC13 was diluted in 500 pL of Na-citrate and added to the DTPA-conjugated antibody (200-300 pg of IgG). After 60 min at room temperature, free lllIn was removed by ultrafiltration on Centricon 30 concentrators as describedabove. 0.9 % NaCl with 0.1 % bovine albumin was used as a washing fluid. Binding Studies with Isolated Bovine Brain Capillaries. Bovine brain capillaries were isolated by a mechanical homogenization technique as described (32). The cortex of fresh bovine brains was scraped off after removal of the pial membrane. Following addition of a &fold volume excess of Ringer-HEPES buffer (10 mM HEPES, 151 mM NaC1, 4 mM KC1, 2.8 mM CaC12) containing 1%BSA, the tissue was homogenized with a hand-held Teflon homogenizer. The homogenate was then suspended in an equal volume 26% dextran (molecular weight = 60-90 kDa), followed by centrifugation at 5800g for 15 min at 4 "C. The vascular pellet was resuspended in buffer, and the microvessels were purified from nuclei and red cells by filtration over a 210-pm nylon mesh and passage over a glass bead filtration column. The microvesselswere finally resuspended in 0.25 M sucrose with 0.02 M Tris (pH = 7.4) containing 2 mM dithiothreitol and stored in liquid nitrogen. The binding assays were performed as described (21). Isolated capillaries corresponding to approximately 200 pg of protein were incubated in a final volume of 450 pL of Ringer-HEPES buffer and 1% BSA with 40 000 cpm radiolabeled antibodies at 37 "C for incubation times from 5 s to 30 min. At the end of the incubation, the mixture was microfuged at lOOOOg for 45 s. The supernatant was discarded,and the capillaries were solubilized in 0.5 mL of 1M NaOH. Bound radioactivity was measured in a y counter, and protein measurements were performed by the method of Lowry (25). RESULTS
A total of 50 mg of AMY33 antibody was obtained by affinity chromatography on protein G Sepharose from 31
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IEF OF ANTI-PA4 MAb 1
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+ + 8.15 + 8.65 8.45
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Figure 1. Coomassie blue staining of a polyacrylamide isoelectric focusing gel showing different degrees of cationization of AMY33 (lanes 3 and 4) as obtained by the reaction conditions indicated in the inset. The molar ratios are based on the amino acid composition of the mIgGl mAb, MOPC21, and refer to the number of Asp and Glu residues in the antibody. The reaction time was 3 h a t room temperature. PI standards (lane l),native AMY33 (lane 2).
mL of ascites fluid produced in nine mice. SDS-PAGE confirmed the purity of the IgG by the demonstration of two sharp bands representing the heavy and light chain. The isoelectricpoints (PI)of native and cationizedAMY33 are shown in the IEF in Figure 1. Native AMY33 yielded two bands corresponding to a PI of approximately 7. Cationization at pH 7.8 with a 2007:l molar ratio of HMD/ EDC/COOH groups did not result in detectable cationization (data not shown). At pH 6.8 and with the same molar ratios, a partial cationization was observed with PI values ranging from 7 to 8. In contrast, molar ratios of 100035:l (HMD/EDC/COOH groups) at pH 6.0 led to a homogeneous cationization resulting in a PI of 9 (lane 4 in Figure 1). These reaction conditions were applied in the subsequent experiments for AMY33, MOPC21, and the chromatographically purified mIgG. Cationization with or without site protection resulted in identical PI values. Native MOPC21 had a PI value of 6.5 and was cationized to a PI of > 9.5. Purified mIgG from mouse serum displayed a broad PI range between 5.5 and 7.5, which was converted by cationization to a homogeneous band corresponding to a PI of > 9.5. Purification of cationized AMY33, MOPC21, and mIgG on Superose 12HR did not reveal the presence of a significant peak in the high molecular weight range, indicating that there was no formation of high molecular weight aggregates due to the cationization procedure. 95% of the IgG eluted in a peak between 12 and 15mL, correspondingto the retention volume of native IgG, as determined in standard chromatograms. In the case of the site-protected cationization of AMY33,the gel filtration chromatography also provided
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(Figure 3B),and the immunocytochemicalresults obtained with the derivatized AMY33 were nearly identical to those described earlier with native AMY33 ( 1 1 , 33). When compared to native AMY33, the staining intensity was slightly decreased. A concentration of 20 pg/mL of cationized AMY33 resulted in the same staining intensity as a concentration of 5 pg/mL of native AMY33. There was a moderate increase in diffuse nonspecificbackground staining with the cationized AMY33 (Figure 3B). A comparable level of nonspecific background staining was present in control sections stained with the DTPAconjugated cationized mIgG. However, the nonimmune mIgG did not label any amyloid deposits. Incubation with lllh-labeled cationized AMY33 and cationized nonimmune mIgG with isolated bovine brain capillaries resulted in a time-dependent increase in tracer binding to the capillaries as compared to the native antibody (Figure 4). After 30 min, 71 % of the cationized mIgG and 26% of the cationized AMY33 were bound per mg protein compared to 6 % of the native AMY33.
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Figure 2. (A) Principle of the solid-phase IRMA used for the measurement of the binding affinity of native and cationized AMY33. (B) Competition curves in the solid-phase IRMA for 12SI-AMY33binding to synthetic pA41-28peptide. SP and NSP refer to cationization of AMY33 with or without site protection, respectively. IgGl refers to the mAb MOPC21. Data points are means of duplicates, and the curves were fitted by nonlinear regression analysis.
an efficient separation from the low molecular weight antigen /3A41-28,which eluted in a retention volume of 19 mL. Figure 2A shows the principle of the solid-phaseIRMA, and Figure 2B shows the competition curves of native AMY33, cationized AMY33, and control antibody, MOPC21. The & of native AMY33 was 1.36 f 0.26 nM. Cationization of AMY33 with and without site protection resulted in Kd values of 3.06 f 0.49 nM and 4.20 f 0.67 nM, respectively. Neither native nor cationized MOPC21 inhibited the specific binding of the l25I-AMY33 tracer within the covered concentration range. 111In labeling of the DTPA conjugated native and cationized antibodies resulted in specific activities of 1.5 mCi/nmol IgG. Challenge of the complex binding of lllIn with an 18 000-fold molar excess of EDTA, followed by ultrafiltration on Ultrafree 30 000 PLTK filters, resulted in the recovery of less than 2.5% of the radioactivity in the ultrafiltrate. Immunocytochemistry on AD brain sections with the native AMY33 antibody resulted in a strong specific staining of vascular amyloid deposits at all three concentrations of antibody used in these experiments. There was virtually no nonspecific background staining (Figure 3A). DTPA-conjugated native AMY33 exhibited the same specific staining pattern as unconjugated AMY33 at the same concentration, indicating that the DTPA conjugation had no effect on the binding affinity of the antibody. With cationized AMY33 and DTPA-conjugated cationized AMY33, the same vascular amyloid deposits could be detected on serial sections as with the native antibody
In the present study the mouse mAb, AMY33, was used, which has been raised against a synthetic peptide corresponding to the first 28 amino acids of the PA4 sequence (2). The specificity of this antibody for @-amyloidprotein in neuritic and diffuse plaques and cerebrovascular deposits has previously been demonstrated (11, 33). Sufficient quantities of the monoclonal antibody for the evaluation of the chemical modifications and subsequent in vivo studies were produced by ascites generation in pristane-primed mice, followed by affinity purification on protein G Sepharose. SDS-PAGE confirmed the purity of the obtained antibody. Cellular uptake of IgG in general and transcytosis through the BBB in particular can be considerably enhanced by cationization, i.e., an increase of the PI of the molecule into the basic range (21,221. Like other cationic proteins, such as cationized albumin (241, histone, and CD4 (34),cationized IgG penetrates the BBB by absorptive-mediated transcytosis. Cationized albumin also undergoes enhanced transport through the blood-cerebrospinal fluid barrier (35). It is apparently the process of cationization which enhances capillary uptake. In our experience, the absolute PI value alone is not crucial, but rather some structural modification introduced into the protein by cationization. As an example, there was no cellular uptake of a native mouse mAb generated against the ras oncogene, despite a PI of 8.8 (unpublished data). Therefore, even naturally basic antibodies cannot be expected to show BBB permeability, which may confound brain imaging attempts based on unmodified antibodies or Fab fragments (13). The degree of the cationization involvingthe conversion of EDC-activated carboxyl groups on the protein into extended primary amino groups by HMD depends on the pH of the reaction (36),the availability of surface carboxyl groups (34),and the molar ratio of the reagents. In order to obtain significant cationization of AMY33, which has a neutral PI of 7, relatively higher concentrations of EDC and HMD and a lower pH value were required compared to the cationization of polyclonal bovine IgG (21). The number of amino groups added to the antibody molecule was not measured in the present study. However, estimates can be made based on the results obtained with another protein. The cationization of rat serum albumin, which resulted in a PI shift from 5 to 8.5 (comparable to the shift from 6.5 t o >9.5 for MOPC21), introduced 30
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Figure 3. Immunocytochemistry on serial sections from AD brain with native AMY33 (A) and DTPA-conjugated cationized AMY33 (B). The arteriole in the center shows circular amyloid disposition. Antibody concentration was 20 pg/mL in both samples. B was lightly counterstained with hematoxylin and A is without counterstain. Scale bar = 50 pm.
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showed that AMY33 does not recognize the peptide when the peptide is 1251-labeled at either the TyrlO with chloramine T or Lys16 with 1251-Bolton-Hunter reagent. Together with the previous finding that AMY33 has no cross-reactivitywith the peptide fragments P A W 2 and PA411-28 (11) and with the fact that post-translational modifications of Asp1 or Asp7 of PA4 present in AD brain (37) apparently do not affect the recognition of PA4 by this mAb, this further proves the high specificity of AMY33 against the midregion of PA41-28. The ELISA system resulted in high nonspecific background levels for the cationized IgG. This is due to nonspecific absorption of cationized proteins to solid-phase surfaces and has been described in other ELISA studies (38,39). These high background levels prevented the quantitative evaluation of the ELISA for cationized AMY33. In contrast, nonspecific binding of the cationized antibody should not significantly interfere with the evaluation of the solidphase IRMA as applied here. The unlabeled native or cationized antibody competes for binding to the solidphase absorbed synthetic peptide antigen with the iodinelabeled native AMY33 tracer. Nonspecific binding of the cationized unlabeled competitor would not affect the signal, as long as the total free concentration of the unlabeled competitor is not significantlylowered. In this case, the determination of the Kd value would underestimate the true value. Therefore, the true & value of cationized AMY33 could be even closer to the & value of native AMY33. Site protection during the cationization reaction led to a decreased reduction of binding affinity compared to cationizationwithout site protection (& 3.06 f 0.49 nM versus 4.20 f 0.67 nM, respectively). In any case, the dissociation constant in the low nM range of the cationized AMY33 demonstrates that it is possible to cationize monoclonal antibodies without appreciable loss of affinity. Previously, this had only been demonstrated for polyclonal antibodies (40,41). The beneficial effect of site-protection has to be evaluated for each individual monoclonal antibody. The relatively small & difference
! o io 20
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[min] Figure 4. Binding of lllIn-labeled antibodies by isolated bovine brain capillaries (approximately 200 pg of capillary protein per tube). Time course of the binding of native AMY33, cationized AMY33, and cationized nonspecific mIgG in serum-free assay buffer. The binding is normalized to percent bound per mg capillary protein. Data are means of duplicates, and the SE was below 5 5%.
new primary amino groups as determined with the amine detection reagent, 2,4,6-trinitrobenzenesulfonic acid (unpublished results). Because the cationization is a random process which could potentially lower the affinity of the antibody in the presence of crucial Asp or Glu residues in the antigen binding site, the affinity of the cationized antibody has to be determined. It was necessary to develop the IRMA system with the unlabeled solid-phase absorbed antigen, pA41-28, because two alternative systems (solution phase RIA and ELISA, data not shown) proved to be unsuitable for this purpose. The solution-phase RIA experiments
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between site-protected and non-site-protected cationization of AMY33 may be due to the absence of crucial carboxyl side chains in the antigen binding site. Similarly, cationization did not decrease the binding affinity of the monoclonal antibody MAblll against the rev-protein of HIV (42),while this procedure lead to a loss of antigen recognition of the mAb B72.3, which is directed against a colon carcinoma antigen (unpublished observations). The choice of "'In as radioisotope was based on its photon energy and half-life, which are suitable for SPECT imaging (43). Although the liver accumulation of lllIn and "'In-labeled antibodies may present problems in the imaging of abdominal organs (43),this would not interfere with the intended use for brain SPECT. Reaction conditions for the DTPA conjugation, such as those applied in the present study, have been shown to result in the incorporation of approximately one DTPA molecule per antibody molecule without affecting the antigen binding activity of monoclonal antibodies (31). Immunocytochemistry on AD brain sections provided evidence for retained binding affinity of DTPA-conjugated cationized AMY33 for tissue amyloid deposits. Therefore, the modified antiamyloid antibody not only reacts with the synthetic peptide antigen pA41-28but is also able to recognizethe full-length PA4 protein, which will be the target in the in vivo application. Uptake of the radiolabeled antibodies by isolated bovine brain capillaries was used as an in vitro model system for the BBB uptake of cationized proteins. This system only indicates binding to and endocytosis by endothelial cells, but not transcytosis. Nevertheless, in all cases of cationized proteins investigated so far, isolated capillary studies were predictive of BBB transcytosis in vivo (21,24,34,44).The species from which the capillaries are isolated appears to be unimportant for binding experiments with cationized proteins, as can be concluded from studies with cationized rat serum albumin and isolated capillaries from rat or bovine brain (44). The binding values for native AMY33 were in the range previously found for native IgG (21), and they can be explained by physical trapping of the tracer inside the patent vascular lumen (23). The results for the cationized AMY33 and the cationized nonspecific mouse IgG are consistent with the previous data with lZ5Ilabeled cationized bovine IgG (21). The cationized antibodies showed rapid initial binding to the capillaries which approximates a plateau after 30 min. The differences in the uptake between cationized AMY33 and cationized mouse IgG can be attributed to a higher degree of cationization of the latter. Similarly, cationization of bovine serum albumin to PI 2 10resulted in a 5-fold higher capillary uptake compared to mild cationization (PI = 8.59) (24). The lower in vitro uptake of cationized AMY33 may, however, not translate into a smaller brain uptake in an in vivo experiment. Tissue uptake in vivo is a function of both intrinsic organ clearance, represented by the permeability surface area (PS)product of the respective organ, and of the area under the plasma concentrationtime curve (34). Previous studies have shown that highly cationic proteins with pI values above 10 are rapidly cleared from the plasma compartment. This is mainly due to uptake by peripheral organs such as liver, kidney, and lung and results in low AUC values (34). In conclusion, the lllIn-labeled cationized AMY33 has been developed as a tool for radioimmunoimaging of cerebral amyloid deposits using SPECT technology. The feasibility of the approach may now be evaluated in aged canines (45) or nonhuman primates (461, which develop brain lesions morphologically equivalent to human AD.
Similarly, cationized mAb targeted against other APP domains or cytoskeletal proteins found in senile plaques of AD (47)may be evaluated as diagnostic tools. Cationized proteins administered to humans may be pathogenic based on the immunogenicity of these modified proteins (48). However, the pathogenicity of cationized proteins has been observed in heterologous systems, wherein a protein with preexisting immunogenicity is cationized. Our previous studies have shown that cationized homologous proteins are not pathogenic and are weakly immunogenic (44). Therefore, the "humanization" of murine monoclonal antibodies prior to mAb cationization may facilitate the use of these proteins as neurodiagnostic or therapeutic agents in humans (49). ACKNOWLEDGMENT
Harry V. Vinters (Department of Pathology, UCLA School of Medicine, Los Angles) kindly provided human autopsy brain samples. Jody L. Buciak and Jing Yang provided expert technical assistance. Sherri J. Chien skillfully prepared the manuscript. This work was supported by California Department of Health Service Grant No. 90-11099. U.B. is recipient of a research stipend from the Deutsche Forschungsgemeinschaft. LITERATURE CITED (1) Glenner, G. G., and Wong, C. W. (1984) Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120, 885-890. (2) Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, L. A., and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sei. U.S.A. 82, 4245-4249. (3) Pardridge, W. M.; Vinters, H. V., Yang, J., Eisenberg, J., Choi, T. B., Tourtellotte, W. W., Huebner, V., and Shively, J. E. (1987) Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels. J.Neurochem. 49,13941401. (4) Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987) The precursor of Alzheimer's disease amyloid & protein resembles a cell-surface receptor. Nature 325, 733-736. (5) Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., and Schenk,D. (1992)Isolation and quantification of soluble Alzheimer's @-peptidefrom biological fluids. Nature 359,325327. ( 6 ) Farlow, M., Ghetti, B., Benson, M. D., Farrow, J. S., Van Nostrand, W. E., and Wagner, S. L. (1992) Low cerebrospinal fluid concentrations of soluble amyloid 0-protein precursor in hereditary Alzheimer's disease. Lancet 340, 453-454. (7) Van Nostrand, W. E., Wagner, S. L., Shankle, W. R., Farrow, J. S., Dick, M., Rozemuller, J. M., Kuiper, M. A., Wolters, E. C., Zimmerman, J., Cotman, C. W., and Cunningham, D. D. (1992) Decreased levels of soluble amyloid &protein precursor in cerebrospinal fluid of live Alzheimer disease patients. Proc. Natl. Acad. Sei. U.S.A. 89, 2551-2555. (8) Joachim, C. L., and Selkoe, D. J. (1992) The seminal role of @-amyloidin the pathogenesis of Alzheimer disease. Alz. Dis. Assoc. Disorders 6, 7-34. (9) Hardy, J., and Allsop, D. (1991) Amyloid deposition as the central event in the aetiology of Alzheimer's disease. Trends Pharmacol. Sei. 12, 383-388. (10) Wisniewski, H. M., Bancher, C., Barcikowska, M., Wen, G. Y., and Currie, J. (1989) Spectrum of morphologicalappearance of amyloid deposits in Alzheimer's disease. Acta Neuropathol. 78, 337-347. (11) Stern, R. A., Otvos, L., Jr., Trojanowski, J. Q., and Lee, V. M.-Y. (1989) Monoclonal antibodies to a synthetic peptide
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