Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90095, USA and 2 Institute for Molecular Genetics, University of Heidelberg, Heidelberg, Germany
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Abstract |
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Keywords: bloodbrain barrier/drug delivery/single chain antibody/streptavidin
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Introduction |
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The conjugation of drugs to BBB transport vectors is facilitated with the use of avidinbiotin technology (Pardridge, 1991). In this approach, the non-transportable drug is monobiotinylated in parallel with the production of a conjugate of the MAb with either avidin or streptavidin (SA). The MAbSA conjugate is generally prepared by chemical methods which involve the introduction of a thioether bond between the transport vector MAb and the SA (Pardridge, 1997
). The production of MAbSA or MAbavidin fusion genes and fusion proteins is also possible using genetic engineering methodology and previous studies have described the production of an antibodyavidin fusion gene and fusion protein (Shin et al., 1997
), where the antibody was directed against the hapten, 5-dimethylaminonaphthalene 1-sulfonylchloride. However, if the MAb moiety of an MAbavidin or MAbSA fusion protein is directed against a BBB receptor system so that MAb undergoes receptor-mediated transport through the BBB in vivo, then it should be possible to genetically engineer a MAbSA fusion protein that has the dual function of recognizing BBB receptor transport systems and binding biotinylated therapeutics, that normally do not undergo transport across the BBB in vivo. The production of SA fusion proteins and MAbSA fusion proteins in bacterial systems has been described (Sano et al., 1992
; Dubel et al., 1995
; Kipriyanov et al., 1996
, 1997
).
The OX26 murine MAb to the rat transferrin receptor binds an exofacial epitope on this receptor (Brandon et al., 1985), and undergoes receptor-mediated transcytosis across the BBB in vivo (Bickel et al., 1994
; Skarlatos et al., 1995
; Huwyler and Pardridge, 1998
). Therefore, the present studies describe the cloning of the genes encoding the variable region of the heavy chain (VH) and the light chain (VL) of the OX26 MAb from hybridoma-generated poly(A)+ mRNA using the polymerase chain reaction (PCR). The OX26 VH and VL genes were subcloned into a bacterial expression vector encoding the gene for the streptavidin core protein, and the expressed protein was purified from Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). Confocal microscopy is used to demonstrate selective binding to isolated rat brain capillaries, and the in vivo pharmacokinetics and brain uptake in rats are reported for the anti-rat transferrin receptor single chain Fv (scFv) antibodySA fusion protein.
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Materials and methods |
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Both male Sprague-Dawley (200300 g) and 1819 day pregnant female rats were obtained from Harlan/Sprague-Dawley (Indianapolis, IN). Rat transferrin was obtained from Cappel Laboratories. The 9E10 hybridoma (ATCC CRL 1729) secreting the mouse MAb to the c-myc epitope was obtained from the American Type Culture Collection (Rockville, MD). The OX26 mouse hybridoma to the rat transferrin receptor was originally produced in the laboratory of Dr Alan F.Williams (University of Oxford) and was obtained from Dr Arthur Like (University of Massachusetts Medical School, Worcester, MA). [3H]-Biotin (50 Ci/mmol) was obtained from Dupont-NEN (Boston, MA). Soluene-350 was obtained from Packard Instrument Company (Downers Grove, IL). FluoresceinPEG 2000biotin was custom synthesized by Shearwater Polymers, Inc. (Huntsville, AL), where PEG 2000 is polyethyleneglycol with a molecular weight of 2000 Da. XL1-Blue supercompetent cells were obtained from Stratagene (San Diego, CA). TrisHCl ready gels (12%), ultra-pure SDS, Coomassie brilliant blue-R250, prestained SDSPAGE low range and biotinylated SDSPAGE molecular weight standards, silver stain plus, mini-protein electrophoresis apparatus and mini-transblot apparatus were obtained from Bio-Rad Labs (Hercules, CA). BCA protein assay kit was purchased from Pierce Chemical Co. (Rockford, IL). PVDF Immobilon-P transfer membrane (0.45 µ) was purchased from Millipore Corp. (Burlington, MA). Biotinylated goat anti-rabbit IgG (H+L), biotinylated horse anti-mouse IgG, peroxidase labeled anti-mouse IgG (H+L) and Vectastain ABC peroxidase kit were obtained from Vector Labs, Inc. (Burlingame, CA). Bovine serum albumin, rabbit anti-streptavidin polyclonal antiserum, polyoxyethylenesorbitan monolaurate (Tween 20), 3,3'-diamiobenzidine (DAB), L-arginine, imidazole, isopropyl-ß-D-thiogalactopyranoside (IPTG), desferrioxamine and all other molecular biology grade reagents were purchased from Sigma Chemical Co. (St Louis, MO). Cyanogen bromide-activated Sepharose 4B, ABTS, Superose 6HR 10/30 FPLC columns, chelating Sepharose fast flow, oligo d(T)18 and 100 mM dNTPs were obtained from Pharmacia Biotech., Inc. (Piscataway, NJ). Guanidine hydrochloride was purchased from Fisher Scientific (Tustin, CA). Ultra-pure urea and tissue culture reagents were obtained from Gibco/BRI (Frederick, MD). Pefabloc SC and AMV-RT were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Centriprep-10 microconcentrators were obtained from Amicon, Inc. (Beverly, MA). Escherichia coli JM109 competent cells, and all restriction endonucleases were obtained from Promega (Madison, WI). AmpliTaq DNA polymerase was purchased from PE Applied Biosystems (Foster City, CA). Custom oligodeoxynucleotides were obtained from Biosource International (Camarillo, CA).
Purification of hybridoma-generated monoclonal antibodies
The OX26 hybridoma was grown to confluency in RPMI-1640 medium with 10% calf serum. The cells were propagated in serum-free medium (SFM, Gibco) and 6 l hybridoma-conditioned SFM was concentrated to 200 ml with an Amicon concentrator (membrane S1Y10) followed by purification of 50 ml aliquots with protein G affinity chromatography. The 9E10 hybridoma was treated with identical protocols. Both monoclonal antibodies were purified to homogeneity and only heavy chain and light chains were visible on SDSPAGE following Coomassie blue staining. The characteristic double light chains of the OX26 hybridoma-generated MAb were observed as reported previously (Yoshikawa and Pardridge, 1992). A typical yield of purified IgG from 6 l hybridoma-conditioned SFM was 75100 mg.
Isolation of RNA
Total RNA was isolated from 2.5x108 OX26 hybridoma cells using guanidinium thiocyanate/acid phenol and methods previously described (Boado and Pardridge, 1994). RNA yield was 3.6 mg and the ratio OD260:OD280 was 2.3. Poly(A)+ mRNA was selected with oligo d(T) cellulose, as described previously (Boado and Pardridge, 1991
), from an aliquot of 280 µg OX26 RNA, yielding 5.2 µg poly(A)+ material with an OD260:OD280 ratio of 2.3.
RTPCR
Reverse transcription of OX26 poly(A)+ RNA was performed with oligo d(T)18 and AMV-RT (Boado and Pardridge, 1994). PCR amplification of light and heavy chain variable regions (VL and VH, respectively) was carried out with 0.5 µg RNA-derived cDNA in a total volume of 100 µl with 2 µM VL and VH specific primers (Table I
), in the presence of 2.5 mM MgCl2, dNTPs and Taq polymerase (Boado and Pardridge, 1994
; Boado et al., 1998
). Amplification comprised 35 cycles of denaturation for 1 min at 94°C, followed by annealing for 2 min at 52°C, and extension for 2 min at 72°C. PCR products were analyzed by gel electrophoresis on 1% agarose, and identified by UV after ethidium bromide staining (Figure 1
).
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OX26 VL and VH PCR generated fragments (Figure 1A) were subcloned into the cohesive ends of the pOPE51 expression vector (Dubel et al., 1993
) to produce OX26 scFv (Figure 1
). PCR products (i.e. ~360 bp VL and ~380 bp VH; Figure 1
) were extracted with phenolchloroform and precipitated with ethanol (Boado et al., 1996
). An aliquot equivalent to 75% of the original VHPCR product and 1 µg pOPE51-phox (Dubel et al., 1993
) were double digested with NcoI and HindIII. OX26VH and pOPE51VL phox (without VH phox) were purified by agarose gel electrophoresis/Spin X centrifugation (Boado et al., 1996
). VH was ligated into pOPE51-NcoI/HindIII with T4 ligase (Boado et al., 1996
). Following transformation of E.coli JM109 competent cells, clones containing OX26VH in the open reading frame (ORF) with the c-myc tag at the C-terminus (Dubel et al., 1993
) were identified by plate-colony screening with the 9E10 MAb following induction with IPTG (Dubel et al., 1993
). Positive clones were further confirmed by BglII restriction endonuclease mapping. A pool of seven positive pOPE51VH OX26VL phox and OX26 VL (75% of the original VHPCR product) were double digested with MluI and NotI, and purified as described above for OX26VH. Following ligation and transformation of E.coli, clones in ORF with the c-myc tag were identified as described above for VH.
In order to functionally characterize clones expressing the OX26 scFv, an ELISA with rat TfR was developed. Rat TfR was purified as described below, and aliquots of 1 µg TfR per well were bound to 96-well Nunc-Immuno MaxiSorb plates in 100 µl 0.1 M NaH2CO3 (pH 8.3), overnight at 4°C. Wells were washed 3x200 µl PBS and blocked with 200 µl 1% BSA/PBS for 60 min at 22°C. Aliquots of OX26 MAb (0.1 µg/well), OX26 scFv (comprising periplasmic and osmotic shock fractions, see below), or IgG2a (negative control, 1 µg/well) were added to the wells as primary antibody in 200 µl blocking buffer and incubated for 60 min at 22°C. Wells were washed as described above, and incubated with anti-c-myc 9E10 MAb 60 min at 22°C as linker antibody. Following washing, wells were incubated with 1.5 µg/ml biotinylated horse anti-mouse IgG for 60 min at 22°C as secondary antibody, washed as described above, and developed with 0.2 mg/ml ABTS in 50 mM citric acid (pH 4.0), containing 0.05% H2O2, for 15 min at 22°C. Results were quantified by OD readings at 450 nm. The OX26 scFv clone, named clone 21, produced a greater than sixfold increase in the OD450 values compared with the IgG2a negative control. Therefore, this clone was used for subsequent studies and DNA sequencing.
Construction of OX26 scFv-SA expression vector
The OX26 scFv cDNA was released from pOPE51 clone 21 with NcoI and NotI double digestion and ligated into the pSTE expression vector (Dubel et al., 1995) at the same cohesive ends, to form pSTE-OX26 (Figure 1
). Isolation of DNA, ligation and colony screening of the c-myc ORF with the 9E10 MAb were performed as described above.
DNA and amino acid sequencing
Sequencing of the OX26-SA scFv cDNA was performed in both directions with forward and reverse primers (Table I) at the UCLA-DNA Sequencing Core Facility, and confirmed by manual DNA sequencing as described previously (Boado and Pardridge, 1990
). In addition, DNA sequencing corresponding to the VH region of the OX26-SA scFv was also performed at the Keck Biotech Resource Lab., Yale University (New Haven, CN), using an Applied Biosystems 377 DNA Sequencer and VH forward and reverse primers (Table I
). Amino-terminal amino acid sequence analysis of purified OX26 scFv-SA was performed by the micro-sequencing technique on PVDF-immobilized material at the UCLA-Peptide Sequencing Core Facility. Amino-terminal amino acid sequence of the OX26 hybridoma generated heavy chain (HC) and light chain (LC) was performed on PVDF-blotted OX26 MAb by the City of Hope Beckman Research Institute (Duarte, CA).
Expression and purification of OX26SA scFv
An IPTG dose response study was performed by transforming an 0.4 ml aliquot of XL-1 Blue cells with pSTE-OX26 overnight; this was used to inoculate 20 ml LB medium containing 50 µg/ml ampicillin and 100 mM glucose (LBG). The culture was incubated at 37°C until an OD600 of 0.8 was reached, and bacteria were obtained by centrifugation (1500 g, 10 min, 20°C). Cells were suspended in LB medium containing graded concentrations of IPTG (51000 µM), and incubated for 17 h at 24.5°C. Cells were harvested by centrifugation at 5000 g for 10 min at 4°C, lysed in SDSPAGE loading buffer, and the abundance of OX26 scFv-SA was determined by Western blot with the 9E10 MAb or an anti-SA antiserum as described below.
Large-scale expression was performed as follows: 25 ml overnight culture of XL-1 Blue cells were transformed with pSTE-OX26 scFv-SA and used to inoculate 1000 ml LBG. Cells were grown with vigorous shaking at 37°C until an OD600 of 0.8. Bacteria were collected by centrifugation at 1500 g for 10 min at 20°C, and resuspended in 1000 ml LB containing 70 µM IPTG. Following 17 h incubation at 24.5°C, cells were harvested by centrifugation at 5000 g for 10 min at 4°C, and scFv was isolated from periplasmic inclusion bodies. The cellular pellet was resuspended in 50 ml ice cold extraction buffer (50 mM TrisHCl, 20% sucrose, 1 mM EDTA pH 8.0) and incubated on ice for 1 h. The soluble fraction was removed by centrifugation at 30 000 g for 30 min at 4°C. The pellet was resuspended in 33 ml extraction buffer and was lysed with sonication (8x35 s on ice). The soluble cytoplasmic fraction was removed by centrifugation at 30 000 g for 30 min at 4°C. The pellet was resuspended in 33 ml of 0.05 M TrisHCl, 3 M urea (pH 7.0) and the soluble fraction was removed by centrifugation as described above. The pellet was resuspended into 25 ml 0.1 M TrisHCl, 6 M guanidine HCl (pH 7.0) and was mixed continuously overnight at 4°C. The soluble inclusion bodies were clarified by centrifugation at 30 000 g for 1 h at 4°C.
Immobilized metal affinity chromatography (IMAC)
Because pOPE and pSTE expression vectors introduce a (His)5 tail at the C-terminus of the recombinant OX26 scFvSA (Dubel et al., 1993; Kipriyanov et al., 1994
; Dubel et al., 1995
), immobilized metal affinity chromatography (IMAC) was used to purify OX26 scFvSA to homogeneity as previously described (Kipriyanov et al., 1994
), with minor modifications. IMAC was performed with 5 ml Chelating Sepharose (Pharmacia) charged with Ni2+ and equilibrated with 6 M guanidine, 0.1 M TrisHCl (pH 7.0). After the solubilized inclusion bodies were passed through the column, the column was washed with 10 column volumes of 6 M guanidine, 0.1 M TrisHCl (pH 7.0) followed by 10 bed volumes of 6 M urea, 50 mM TrisHCl, 10 mM imidazole and 20 bed volumes of 6 M urea, 50 mM TrisHCl, 50 mM imidazole. OX26SA scFv was eluted with 20 ml (4 bed volumes) of 6 M urea, 50 mM TrisHCl, 250 mM imidazole, and was then dialyzed in TEA (0.4 M L-arginine, 0.1 M TrisHCl, 2 mM EDTA, pH 7.0) overnight. Protein concentrations were determined by the BCA protein assay. The purified proteins were stored at 20°C. One liter cultures generated 175 mg periplasmic inclusion body protein and 4.8 mg protein of purified OX26 scFvSA fusion protein after IMAC.
Western blot analyses
Escherichia coli lysates, periplasmic fractions and inclusion bodies, which had been dialyzed in TEA before electrophoresis, or purified OX26 scFvSA, were resolved in 12% SDSPAGE (Bio-Rad) under reducing conditions for Western blotting (Pardridge et al., 1990). Gels were stained with Coomassie brilliant blue, or electro-blotted onto a PVDF membrane. Immunoblot analysis was carried out with the 9E10 mouse MAb to the c-myc epitope or rabbit anti-streptavidin polyclonal antiserum (Sigma) as primary antibodies. Peroxidase labeled horse anti-mouse IgG (Vector Labs) was used as secondary antibody for 9E10 and results were visualized with DAB. In experiments with the anti-streptavidin antiserum, results were visualized with biotinylated goat anti-rabbit IgG (Vector labs) and the ABC Elite detection kit (avidin, biotinylated peroxidase) with DAB.
Western blot with rat TfR was used to determine the binding activity of purified scFv. Rat TfR was electrophoresed in SDSPAGE and blotted as described above. The membrane was sequentially incubated with the OX26 scFvSA (primary antibody), the 9E10 MAb (linker antibody), biotinylated horse anti-mouse IgG (secondary antibody) and avidin and biotinylated peroxidase (Kipriyanov et al., 1994).
Purification of rat placental transferrin receptor
Pregnant Sprague-Dawley rats (18 days) were anesthetized and 151 placentas were removed from 11 rats for purification of the rat transferrin receptor as described by Bowen and Morgan (1987). The placental extract was applied to a rat transferrin affinity column, which was prepared by conjugating 10 mg rat transferrin to 0.6 g cyanogen bromide-activated Sepharose 4B, as described by van Driel et al. (1984). After the placental extract was applied, the column was washed at 4°C with 50 ml buffer D (0.15 M Na acetate/pH 5.0/0.5% Triton X100) or until the A280 of the column eluate was low, and then with 15 ml buffer E (0.1 M Na citrate, 0.5% Triton X-100, 50 µg/ml desferrioxamine). The transferrin receptor was then eluted with 10 ml buffer F (0.1 M NH4HCO3, pH 7.8, 0.5% Triton X100, 50 µg/ml desferrioxamine). The transferrin receptor in the column eluate was detected with SDSPAGE.
ELISA
Purified rat transferrin receptor (5 µg) was diluted in 5 ml 0.05 M NaHCO3 (pH 8.3), and aliquoted (100 µl) to wells of a NUNC MaxiSorb 96 well ELISA plate and incubated for 60 min at room temperature. The excess transferrin receptor was removed by aspiration and wells were washed with 180 µl/well HBST (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% Tween 20). The wells were blocked with a 100 µl/well 2% horse serum (30 min at room temperature) followed by aspiration, followed by addition of 100 µl per well of primary antibody. The primary antibody consisted of either hybridoma-generated purified OX26 MAb, mouse IgG2a isotype control or OX26 scFvSA fusion protein in 0.033, 0.10, 0.33, 1.0 or 3.0 µg/ml concentrations. The secondary antibody was biotinylated horse anti-mouse antibody.
Rat brain capillary isolation and confocal microscopy
Capillaries were isolated from rat brain using a mechanical homogenization technique described previously (Pardridge et al., 1985). The final capillary pellet was resuspended in an 0.5 ml volume of 0.01 M Tris, 0.15 M NaCl, pH 7.4, 0.1% bovine serum albumin (BSA) and an 0.25 ml volume of the capillaries was added to 1.0 ml OX26 scFv/SA (64 µg protein/ml) in buffer that consisted of 0.01 M Tris, 0.2 mM EDTA, 40 mM L-arginine/0.1% Tween 20, 0.15 M NaCl pH 7.0, and 1.2 mg biotinPEG 2000fluorescein. A parallel 0.25 ml aliquot of rat brain capillaries was added to 1.0 ml of the same solution that contained recombinant streptavidin (30 µg/ml), but no scFvSA. Following incubation at room temperature for 60 min, the capillaries were centrifuged at a 1000 g for 3 min at 4°C and the supernatant was discarded. The capillary pellet was washed 3 times with 1 ml ice cold TBSH buffer (0.01 M Tris, pH 7.4, 0.15 M NaCl, 5% BSA) and the final pellet was resuspended in 400 µl TBSH buffer and cyto-centrifuged in quadruplicate to glass slides using a Shandon cyto-centrifuge (300 r.p.m. for 5 min at room temperature). The glass slide containing the unfixed cyto-centrifuged capillary pellet was mounted with 5% n-propylgallate in 100% glycerol for confocal microscopy as described previously (Huwyler and Pardridge, 1998
). A Fluovert FU inverted fluorescent microscope (Leica) with a Leitz NP1 40x or 63x objective (numerical aperture of 1.4) and a Leica confocal laser scanning adapter utilizing a krypton/argon laser with fluorescine band pass filters was used. Optical sections (0.5 µm, resolution 200 nm) were obtained sequentially in the z plane of each sample. Photomicrographs were processed with Photoshop 2.51 on a Power Macintosh microcomputer. In parallel, freshly isolated rat brain capillaries were stained with o-toluidine blue and photographed with an Olympus light microscope. The confocal studies employed biotinPEG 2000fluorescein, rather than biotinfluorescein, because the latter does not undergo fluorescence upon binding to SA (Gruber et al., 1997
). The introduction of the flexible, extended PEG 2000 linker restores fluorescence upon binding to SA (Gruber et al., 1997
).
Pharmacokinetics and brain uptake measurements
Male Sprague-Dawley rats were anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (2 mg/kg) for pharmacokinetic studies as described previously (Pardridge et al., 1994). The femoral vein was cannulated with a PE50 cannula and 0.3 ml buffer containing 2 µCi [3H]-biotin and 50 µg OX26 scFvSA was injected. Prior to injection, the complex of [3H]-biotin and OX26 scFvSA was purified on a 1.5x18 cm column of Sephadex G25 in TBST (0.01 M Tris, 0.15 M NaCl, pH 7.4, 0.25% Tween 20). Pharmacokinetic parameters were calculated by fitting the plasma radioactivity concentration data to either a mono-exponential or a bi-exponential equation (Gibaldi and Perrier, 1982
) using a derivative free-nonlinear regression analysis (PARBMDP, Biomedical Computer P series, developed at the UCLA School of Medicine). The data were weighed using the equation
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Gel filtration fast protein liquid chromatography (FPLC)
The metabolic stability of the complex of [3H]-biotin and the OX26 scFvSA fusion protein was determined by gel filtration FPLC using a Superose 6HR 10/30 FPLC column (Pharmacia). A 25 µl aliquot of 30 min terminal serum was pooled from each of three rats and injected onto the column followed by elution with PBST buffer (0.01 M Na2HPO4, 0.15 M NaCl pH 7.4, 0.05% Tween 20) at 0.25 ml/min. The fractions (1.0 ml) were then solubilized for [3H] liquid scintillation counting.
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Results |
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The induction of the OX26 scFvSA fusion protein by IPTG in E.coli is shown in Figure 2. Figure 2A
is a Western Blot of total cell lysate using the 9E10 MAb as a primary antibody, which reacts with the c-myc epitope in the C-terminus of the fusion protein (Figure 1B and C
). The 9E10 antibody reacts with a principal band that migrates at the expected size for the scFvSA fusion protein of 46 kDa, and a minor band that migrates at 40 kDa in total E.coli lysates. The anti-SA primary antibody reacts with a 46 kDa protein, as well as minor bands migrating at 40 and 60 kDa, in total cell lysates. The 40 kDa minor band reacted with both the 9E10 and the anti-SA primary antibodies, but the 60 kDa band only reacted with the anti-SA antibody. Coomassie blue staining of the E.coli lysates demonstrated many proteins (Figure 2C
, lane 2), but the 46 kDa scFvSA fusion protein was purified to homogeneity (Figure 2C
, lane 3) following IMAC. Only the 46 kDa band was detected with Western blotting using either the 9E10 MAb (Figure 2C
, lanes 5 and 6) or the anti-SA (Figure 2C
, lanes 8 and 9) as primary antibody and the 40 and 60 kDa minor products were not present in either the final purified product (Figure 2C
, lane 3) or the periplasmic inclusion bodies (Figure 2C
, lanes 5 and 8).
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Discussion |
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The OX26 hybridoma produces a single heavy chain and two discrete light chains that are visualized on SDSPAGE (Yoshikawa and Pardridge, 1992). Therefore, it is possible that PCR will amplify the aberrant light chain. Nevertheless, the correct heavy and light chain and functional scFv should be selected with a suitable colony expression screening method, and this was made possible with isolation of rat placental transferrin receptor (Materials and methods). Clone 21 (Materials and methods) gave a high signal in the ELISA of the colony screening and the cloning of the appropriate VH and VL genes for the OX26 MAb was verified by direct amino acid sequencing of the hybridoma generated heavy and light chains (Figure 4
). In addition, the amino acid sequence of the aberrant light chain was shown by searching the Genbank database to be the endogenous MOPC21 light chain (Materials and methods).
The OX26 scFvSA fusion protein was not secreted into the bacterial periplasm in significant amounts, which has been observed with several other systems involving bacterial expression of cloned scFv genes (Huston et al., 1991; Johnson and Bird, 1991
; Cho et al., 1995
). Similarly, the OX26 scFv (without the adjoining SA core protein) produced by the pOPE51 plasmid (Figure 1
) was not secreted significantly into the periplasm (unpublished observations). The OX26 scFvSA fusion protein secreted into the periplasmic inclusion bodies was not processed to remove the pelB leader peptide (Figure 4
), since amino acid sequencing of the N-terminus of the fusion protein through 21 cycles demonstrated 100% correlation with the pelB amino acid sequence (Results). However, it was possible to purify to homogeneity on SDSPAGE (Figure 2
) the OX26 scFvSA fusion protein from inclusion bodies using IMAC and guanidine denaturationrenaturation, whereby the protein was renatured while still bound to the nickel column (Kipriyanov et al., 1994
).
There is evidence that the affinity of the OX26 scFv for the rat transferrin receptor is not as high as the hybridoma generated OX26. The BBB PS product in the rat in vivo for the hybridoma generated OX26 MAb is 1.1±0.1 µl/min/g (Bickel et al., 1993), and the BBB PS product for [3H]-biotin bound to OX26/NLA is 0.84±0.04 µl/min/g (Kang and Pardridge, 1994
), which exceeds the BBB PS product for [3H]-biotin bound to the OX26 scFvSA fusion protein (Figure 6
). There are two possible reasons for the reduced affinity of the scFv compared with the hybridoma generated MAb. First, the presence of the intact pelB leader sequence may adversely affect affinity, although the N-terminus of the VH chain can form protein fusions without adversely affecting antibody affinity (McGrath et al., 1997
). Second, the denaturation of the inclusion bodies with 6 M guanidine followed by renaturation may affect the affinity of the scFv. The bacterial leader peptide and the need for 6 M guanidine denaturation can be circumvented in future studies using alternative expression systems, such as baculovirus (Laroche et al., 1991
) or eukaryotic expression systems (Jost et al., 1994
) for producing scFvSA fusion proteins.
The pharmacokinetic study in vivo in rats suggests the presence of the SA moiety in the fusion protein delays rapid removal of the scFv from the plasma compartment. Previous studies in rodents have shown that scFv antibodies are cleared more than 10 times faster than the native IgG (Milenic et al., 1991). Apparently, the results in Figure 6
are the first pharmacokinetic study of an scFvstreptavidin fusion protein, and the study indicates the OX26 scFvSA fusion protein is not cleared significantly faster from the blood stream than are neutral avidin conjugates of hybridoma derived OX26 MAb. The clearance of [3H]-biotin bound to the OX26 scFvSA fusion protein, 2.3±0.1 ml/min/kg (Table II
), is comparable to the clearance in rats of [3H]-biotin bound to a conjugate of OX26 and NLA, 1.1±0.1 ml/min/kg (Kang and Pardridge, 1994
), to the systemic clearance of a biotinylated [125I] peptide nucleic acid (PNA) bound to a conjugate of OX26 and recombinant streptavidin, 1.3±0.1 ml/min/kg (Pardridge et al., 1995
), or to the systemic clearance of [3H]-OX26 MAb, 0.50 ml/min/kg (Bickel et al., 1993
). Therefore, the OX26 scFvSA fusion protein is cleared from blood at rates comparable to that of conjugates of the hybridoma generated OX26 MAb and recombinant streptavidin, which are formed via a stable thioether linkage between the OX26 MAb and the SA (Pardridge et al., 1995
). The fact that the scFvSA fusion protein is not cleared rapidly from plasma has distinct advantages for brain drug delivery where the overall percent of injected dose delivered per gram of brain is inversely related to the plasma clearance rate of the scFvSA fusion protein from blood (Pardridge, 1997
). In contrast, in imaging studies using avidinbiotin technology, it is desirable to have a rapid rate of removal of the avidin fusion protein from blood (Rusckowski et al., 1996
; Samuel et al., 1996
). Rapid plasma clearance of the avidin fusion protein optimizes regional imaging, whereas delayed plasma clearance of the avidin fusion protein optimizes organ drug delivery (Pardridge, 1997
).
The bi-functionality of the OX26 scFvSA fusion protein is retained as both biotin and transferrin receptor binding is observed. With regard to biotin binding, the gel filtration FPLC of rat serum shows the retention of [3H]-biotin binding to the fusion protein in vivo (Figure 6). In vitro, the OX26 scFvSA fusion protein binds the biotinPEG 2000fluorescein to enable specific binding of this fluorescein conjugate to isolated rat brain capillaries in confocal microscopy studies (Figure 5
). The evidence for binding of the OX26 scFvSA fusion protein to the rat transferrin receptor is derived from (i) the Western blotting and the ELISA using purified rat placental transferrin receptor (Figure 3
), (ii) the confocal microscopy study showing specific binding of the OX26 scFvSA fusion protein bound to biotinPEG 2000fluorescein to isolated rat brain capillaries (Figure 5
), and (iii) the in vivo organ uptake study showing selective targeting of [3H]-biotin bound to the OX26 scFvSA fusion protein to those organs (liver, BBB) that are enriched in microvascular transferrin receptor (Table III
, Figure 6B
), but not to organs (heart, kidney) that lack abundant microvascular transferrin receptor (Table III
). Previous studies using electron microscopy of gold conjugates of the OX26 MAb (Bickel et al., 1994
) or autoradiography (Skarlatos et al., 1995
) have demonstrated the OX26 MAb undergoes transcytosis through the BBB in vivo and enters brain interstitial fluid.
In summary, these studies demonstrate that genetically engineered receptor-specific single chain antibodystreptavidin fusion proteins may be produced, expressed and purified, and used for brain drug delivery in vivo. Future studies may involve the optimization of expression using systems other than E.coli, so that purification of the fusion protein can be achieved without denaturation. Such receptor specific scFvSA fusion proteins may have clinical applications since both avidin and streptavidin have been given to humans without immunological sequelae (Rusckowski et al., 1996; Samuel et al., 1996
). The immunogenicity of the scFv portion of the fusion protein may be minimized with `humanization' whereby the mouse framework region amino acids are replaced by amino acids corresponding to human framework regions (Winter and Milstein, 1991
). The brain delivery of transferrin and insulin peptidomimetic MAb's are 0.30.4% of injected dose in the rat and 34% of the injected dose in the Rhesus monkey, respectively (Pardridge, 1997
). These levels of brain uptake exceed by many-fold the brain uptake of morphine, a neuroactive small molecule, which is 0.1% of injected dose in the rat (Pardridge, 1997
).
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Acknowledgments |
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Notes |
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3 To whom correspondence should be addressed
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References |
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Received February 18, 1999; revised April 27, 1999; accepted May 3, 1999.