In the present study a novel Ab-avidin fusion protein has been constructed to deliver biotinylated compounds across the blood brain barrier. This fusion molecule consists of an Ab specific for the transferrin receptor genetically fused to avidin. The Ab-avidin fusion protein (anti-TfR IgG3-CH3-Av) expressed in murine myeloma cells was correctly assembled and secreted and showed both Ab- and avidin-related activities. In animal models, it showed much longer serum half-life than the chemical conjugate between OX-26 and avidin. Most importantly, this fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate. We also delivered a biotinylated 18-mer antisense peptide-nucleic acid specific for the rev gene of HIV-1 to the brain. Brain uptake of the HIV antisense drug was increased at least 15-fold when it was bound to the anti-TfR IgG3-CH3-Av, suggesting its potential use in neurologic AIDS. This novel Ab fusion protein should have general utility as a universal vehicle to effectively deliver biotinylated compounds across the blood-brain barrier for diagnosis and/or therapy of a broad range of CNS disorders such as infectious diseases, brain tumors as well as Parkinson’s and Huntington’s diseases.

Efficient and specific targeting of an active agent to the desired site is a critical factor for the successful diagnosis and/or therapy of many diseases. One region of the body particularly difficult to target is the brain due to the presence of the high resistance blood-brain barrier (BBB)3 formed by tightly joined capillary endothelial cell membranes (1, 2, 3, 4, 5). The BBB effectively restricts transport from the blood of certain molecules, especially those that are water soluble and larger than several hundred daltons (6). In fact, the clinical utility of many proteins of therapeutic interest for the brain is limited by their inability to cross the BBB. In some cases neurotrophic factors have been administered to the brain by invasive neurosurgical procedures or grafting neurotrophin-producing cells into brain sites (7, 8, 9).

The BBB has been shown to have specific receptors that allow the transport from the blood to the brain of several macromolecules including insulin (10), transferrin (Tf) with iron attached (11), and insulin-like growth factors (IGFs) (12). Therefore, one noninvasive approach for the delivery of drugs to the brain is to attach the agent of interest to a molecule with receptors on the BBB, which would then serve as a vehicle for transport of the agent across the BBB (3, 13, 14). An alternative approach is the delivery of agents attached to an Ab specific for one of the BBB receptors. Indeed, both NGF and CD4 will cross the BBB when chemically conjugated to an Ab directed against the TfR (15, 16, 17).

Despite the fact that Abs normally are excluded from the brain (18), they can be an effective vehicle for the delivery of molecules into the brain parenchyma if they have specificity for receptors on the BBB. In fact, the i.v. injection of an anti-rat TfR Ab-nerve growth factor (NGF) chemical conjugate prevented the loss of striatal choline acetyltransferase-immunoreactive neurons in a rat model of Huntington’s disease and reversed the age-related cognitive dysfunction (19, 20). Recently a fusion protein with NGF attached to the N terminus of an Ab directed against human TfR using genetic engineering techniques (21) showed both Ag binding and NGF activity, suggesting its therapeutic utility. Although promising, this approach requires that unique chimeric molecules be constructed for each specific application and is cumbersome and sometimes can lead to the decrease or loss of activity of one or both of the covalently conjugated partners. To overcome these limitations, it is therefore desirable to develop a universal delivery system that eliminates the need to make a specific construct for each individual application.

The ideal brain delivery vehicle should be able to deliver many different compounds that are bound to the vehicle by high affinity noncovalent interactions such as those seen between avidin (Av) and biotin. Indeed Ab-Av chemical conjugates have been used to deliver a mono-biotinylated drug (22). However, an important drawback of the chemical coupling procedure is the difficulty in producing a reproducible and homogeneous product. Genetic engineering provides an alternative approach for large scale production of homogeneous Ab-Av fusion proteins. The present work describes the brain delivery characteristics of a TfR-specific Ab containing chicken Av and its initial application in delivery to the brain of anti-HIV peptide nucleic acid, an 18-mer antisense to the rev gene of HIV-1 with lysine and tyrosine at the 5′ end and biotin at the 3′ (biotin-PNA) (23). The fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate. In addition, the brain uptake of anti-HIV PNA was increased at least 15-fold when it was bound to the anti-TfR IgG3-CH3-Av. Since the brain is a shelter for HIV, the successful brain delivery of anti-HIV peptide nucleic acid (PNA) with the anti-TfR IgG3-CH3-Av may provide an effective treatment for cerebral AIDS.

The anti-TfR IgG3-CH3-Av H chain vector was constructed by the substitution of the variable region of anti-dansyl (5-dimethylamino naphthalene 1-sulfonyl chloride) IgG3-CH3-Av fusion H chain (24) with the variable region of the H chain of anti-rat TfR mAb OX-26 (25) (Fig. 1). The anti-TfR κ L chain expression vector containing the Escherichia coli gpt gene for eukaryotic selection and the anti-TfR IgG3-hinge-transferrin fusion H chain expression vector containing the hisD gene for eukaryotic selective marker were constructed (S.-U. Shin, manuscript in preparation). The IgG3-CH3-Av H chain specific for dansyl was available in the laboratory (24). The IgG3-hinge-transferrin DNA fragment (between Age I and BamHI) of the anti-TfR IgG3-hinge-transferrin fusion H chain expression vector containing anti-TfR variable region was replaced with the Age I-BamHI DNA fragment (IgG3-CH3-Av H chain gene) of the anti-dansyl IgG3-CH3-Av H chain expression vector.

FIGURE 1.

Schematic diagram of the construction and expression of the anti-TfR IgG3-CH3-Av fusion protein. Using convenient restriction sites, the anti-TfR IgG3-CH3-Av H chain expression vector was constructed by substituting the variable region of the anti-dansyl IgG3-CH3-Av H chain with that of an Ab specific for the rat TfR (OX-26). TAUD3.1, a transfectant of P3 × 63Ag8.653 expressing a L chain with the OX-26 variable region, was used as a recipient for transfection of the anti-TfR IgG3-CH3-Av H chain expression vector.

FIGURE 1.

Schematic diagram of the construction and expression of the anti-TfR IgG3-CH3-Av fusion protein. Using convenient restriction sites, the anti-TfR IgG3-CH3-Av H chain expression vector was constructed by substituting the variable region of the anti-dansyl IgG3-CH3-Av H chain with that of an Ab specific for the rat TfR (OX-26). TAUD3.1, a transfectant of P3 × 63Ag8.653 expressing a L chain with the OX-26 variable region, was used as a recipient for transfection of the anti-TfR IgG3-CH3-Av H chain expression vector.

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All cells were cultured in DMEM (Life Technologies, Grand Island, NY) with 5% calf serum (HyClone, Logan, UT). A cell line that produces high levels of the anti-TfR κ L chain, TAUD3.1, was obtained by transfecting P3 × 63Ag8.653 by electroporation with a chimeric mouse/human k L chain gene with the variable region of OX-26 (Fig. 1), selecting with 0.33× HXM (30× HXM contains 3.3 mM hypoxanthine, 49.3 mM xanthine, 0.52 mM mycophenolic acid, and 0.1 N NaOH) and detecting stable transfectants secreting L chain by ELISA (26). One L chain-expressing transfectant, TAUD3.1, was electroporated (26) with the gene encoding anti-rat TfR IgG3-CH3-Av H chain; stable transfectants were selected with 5 mM histidinol (Sigma, St. Louis, MO) and screened by an ELISA for the secretion of H chain (26). The fusion protein biosynthetically labeled with [35S]methionine (ICN, Irvine, CA) was immunoprecipitated using rabbit anti-human IgG and a 10% suspension of staphylococcal protein A (IgGSorb; The Enzyme Center, Malden, MA) and then analyzed by SDS-PAGE with/without 2-ME. The fusion protein was purified from culture supernatants using protein G immobilized on Sepharose 4B fast flow (Sigma). Purity was assessed by Coomassie blue staining of SDS-PAGE gels. Protein concentrations were determined by bicinchoninic acid-based protein assay (BCA Protein Assay; Pierce, Rockford, IL) and ELISA.

The binding of anti-TfR IgG3-CH3-Av to the TfR was studied by flow cytometry using the rat myeloma cell line Y3-Ag1.2.3. Cells (1 × 106) were incubated with 1 μg of anti-TfR IgG3-CH3-Av, anti-DNS IgG3-CH3-Av (negative control), or anti-rat TfR IgG3 (positive control) (M. J. Coloma et al., manuscript in preparation), in a volume of 100 μl for 2 h at 4°C, washed, incubated 2h at 4°C with FITC-labeled goat anti-human IgG (PharMingen, San Diego, CA) and analyzed by flow cytometry (Becton Dickinson, Mountain View, CA).

All steps were conducted in PBS, and plates were washed six times between each step with the same buffer. Ninety-six-well plates were coated with 50 ml/well biotinylated-BSA (Sigma) (biotin:BSA ratio = 11:1, 5 μg/ml) overnight at 4°C, then blocked with 100 ml/well 3% BSA (overnight at 4°C) (24). All fusion proteins (by duplicate) were diluted and applied in a volume of 50 μl/well, and, after overnight incubation at 4°C, goat anti-human k alkaline phosphatase conjugate (Sigma) was added, followed by 50 μl of the substrate p-nitrophenyl phosphate at 0.5 mg/ml in diethanolamine buffer (pH 9.6) (Sigma). The OD was read at 410 nm. To determine whether anti-TfR IgG3-CH3-Av could be removed with biotin acrylic beads, varying concentrations of the fusion protein (0.5–250 nM) were preincubated with biotin acrylic beads (Sigma) (5 μl) at room temperature for 30 min. After brief centrifugation, the presence of the fusion protein in the supernatants was quantified by ELISA as described above. For a competition ELISA, anti-rat TfR IgG3-CH3-Av (2.5 nM) was preincubated with various concentrations of biotin-BSA (35.4 pM-36.3 nM) at 37°C for 2 h, and then ELISA was performed as described.

Male Sprague Dawley rats (three rats per group) weighing 220 to 230 g purchased from Samyook Experimental Animals (Buann, Korea) were anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg) by i.m. injection. The left femoral vein was cannulated with PE50 tubing and injected with 0.2 ml HEPES (pH 7.4) containing 0.1% native rat serum albumin and 5 μCi (0.1 nmol) of [3H]biotin (DuPont NEN Research Products, Bukyungsa, Korea) mixed with 20 μg of Ab-fusion proteins (0.1 nmol) or chemical conjugate (OX-26/Av). Five microcuries of [125I]biotin-PNA mixed with 20 μg of anti-TfR IgG3-CH3-Av or 20 μg of [125I]anti-TfR IgG3-CH3-Av PNA, an 18-mer antisense to the rev gene of HIV type 1, was custom synthesized by Millipore (Millipore Corporation, Bedford, MA) such that the 5′ end was biotinylated, and tyrosine and lysine were placed at the amidated 3′ end (biotin-CTCCGCTTCTTCCTGCCA-Tyr-Lys-CONH2) (23). OX-26 was labeled with [3H]succinimidyl propionate (Amersham, Arlington Heights, IL) as described previously (27), and PNA was directly labeled with [125I] as described previously (23). Blood samples (0.3 ml) were collected via a heparinized PE50 cannula implanted in the left femoral vein at 0.25, 1, 2, 5, 15, 30, and 60 min after the i.v. injection. After each blood sampling, the blood volume was replaced with the same volume of normal saline, and plasma was separated by centrifugation. The animals were decapitated after 60 min, and the brain was removed and weighed. The plasma and brain samples were solubilized with Soluene-350 (Packard Instrument, Saehan, Korea) and neutralized with glacial acetic acid before liquid scintillation counting. The other peripheral tissues, such as liver, kidney, lung, and heart, were also removed and weighed, and their radioactivities were counted. The pharmacokinetic parameters were calculated by fitting plasma radioactivity data to a mono- or bi-exponential equation, as described previously (22). The BBB permeability-surface (PS) area product of [3H]biotin or [125I]biotin-PNA bound to anti-TfR IgG3-CH3-Av was calculated as described (22) from the plasma concentrations, the apparent brain volume of distribution (VD), and the plasma volume in brain (10 μl/g). The percentage injected dose (ID) delivered per gram brain was computed from the PS product and the 60-min area under the plasma area under the curve (AUC), as described previously (28).

The serum stability of the [3H]biotin anti-TfR IgG3-CH3-Av complex was examined by fast protein liquid chromatography (FPLC) using a Superose 6HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden). A 50-μl aliquot of either 60-min serum samples, or of an in vitro preparation containing 7.5 μCi of [3H]biotin and 30 μg of anti-TfR IgG3-CH3-Av as a control (injectate) was injected into the column. The samples were passed through the column in the presence of 0.01 M PBS (pH 7.4) containing 0.05% Tween 20 at a flow rate of 0.25 ml/min. Fractions (0.5 ml) were collected, and the radioactivity of each fraction was counted on a Packard Liquid Scintillation Analyzer (Model A2100 TR).

The strategy for the expression of anti-TfR IgG3-CH3-Av is illustrated in Fig. 1. Clones expressing anti-TfR IgG3-CH3-Av fusion proteins were identified by an ELISA, and transfectants faithfully express up to 1 μg/106 cells/24 h. Purified anti-TfR IgG3-CH3-Av fusion proteins were stable at 4°C in PBS for 1 yr. Anti-TfR IgG3-CH3-Av fusion proteins were biosynthetically labeled by growth in the presence of [35S]methionine. SDS-PAGE analysis of the secreted [35S]methionine-labeled proteins under nonreducing conditions (Fig. 2,A) showed the anti-TfR IgG3-CH3-Av to have a molecular mass of ∼200 kDa, the size expected for a complete Ab with 2 molecules of Av attached. Following reduction, H and L chains of the expected m.w. were observed (Fig. 2 B). Anti-TfR IgG3-CH3-Av purified from culture supernatants using affinity chromatography was also shown to be ∼200 kDa (data not shown). Anti-TfR Ab-Av fusion proteins of the expected m.w. are faithfully produced and secreted as the H2L2 form.

FIGURE 2.

SDS-PAGE analysis of the anti-TfR IgG3-CH3-Av fusion protein. Secreted anti-TfR IgG3-CH3-Av biosynthetically labeled with [35S]methionine was immunoprecipitated using anti-human IgG and staphylococcal protein A and analyzed by SDS-PAGE under nonreducing (A) and reducing (B) conditions. Included for comparison are anti-TfR IgG3 without attached Av, OX-26 (the murine IgG2a anti-TfR that donated the variable regions), and a previously characterized anti-dansyl IgG3-CH3-Av. The positions of the m.w. standards are indicated at the side.

FIGURE 2.

SDS-PAGE analysis of the anti-TfR IgG3-CH3-Av fusion protein. Secreted anti-TfR IgG3-CH3-Av biosynthetically labeled with [35S]methionine was immunoprecipitated using anti-human IgG and staphylococcal protein A and analyzed by SDS-PAGE under nonreducing (A) and reducing (B) conditions. Included for comparison are anti-TfR IgG3 without attached Av, OX-26 (the murine IgG2a anti-TfR that donated the variable regions), and a previously characterized anti-dansyl IgG3-CH3-Av. The positions of the m.w. standards are indicated at the side.

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Flow cytometry using the rat myeloma cell line Y3-Ag1.2.3 showed that anti-TfR IgG3-CH3-Av bound to the TfR expressed on the cell surface to the same extent as the anti-TfR Ab with the same variable region but lacking Av (Fig. 3). An irrelevant Ab (anti-hapten) fused to Av failed to bind. Anti-TfR IgG3-CH3-Av also bound to biotinylated BSA coated on the surface of a microtiter plate in a dose-dependent manner (Fig. 4,A). This binding activity could be removed by preincubation with biotin acrylic beads. In addition, soluble biotin-BSA inhibited the binding of anti-TfR IgG3-CH3-Av to coated plates with 50% inhibition seen at an inhibitor concentration of 0.4 nM (Fig. 4 B). Thus, the anti-TfR Ab-Av fusion protein retains its specificity for rat transferrin receptors and ability to bind to biotin.

FIGURE 3.

Flow cytometry demonstrating the specificity of the anti-rat TfR IgG3-CH3-Av for the TfR expressed on the surface of rat Y3-Ag1.2.3 cells. The cells were incubated with either negative control anti-DNS IgG3-CH3-Av (A), positive control anti-rat TfR IgG3 (B), or the anti-rat TfR IgG3-CH3-Av fusion protein (C), followed by FITC-labeled goat anti-human IgG. These results are representative of four studies that have been done.

FIGURE 3.

Flow cytometry demonstrating the specificity of the anti-rat TfR IgG3-CH3-Av for the TfR expressed on the surface of rat Y3-Ag1.2.3 cells. The cells were incubated with either negative control anti-DNS IgG3-CH3-Av (A), positive control anti-rat TfR IgG3 (B), or the anti-rat TfR IgG3-CH3-Av fusion protein (C), followed by FITC-labeled goat anti-human IgG. These results are representative of four studies that have been done.

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FIGURE 4.

The binding of anti-TfR IgG3-CH3-Av to biotinylated BSA-coated microtiter plates. A, Anti-TfR IgG3-CH3-Av was added at varying concentrations with (○)/without (•) previous incubation with biotin acrylic beads and the bound protein detected using anti-κ conjugated with alkaline phosphatase. B, Anti-TfR IgG3-CH3-Av (2.5 nM, •) preincubated with varying concentrations of biotinylated BSA was added to the biotinylated BSA-coated microtiter plates, and bound Ab was detected using anti-κ conjugated with alkaline phosphatase. The values shown are mean (n = 3). These results are representative of three studies that have been done.

FIGURE 4.

The binding of anti-TfR IgG3-CH3-Av to biotinylated BSA-coated microtiter plates. A, Anti-TfR IgG3-CH3-Av was added at varying concentrations with (○)/without (•) previous incubation with biotin acrylic beads and the bound protein detected using anti-κ conjugated with alkaline phosphatase. B, Anti-TfR IgG3-CH3-Av (2.5 nM, •) preincubated with varying concentrations of biotinylated BSA was added to the biotinylated BSA-coated microtiter plates, and bound Ab was detected using anti-κ conjugated with alkaline phosphatase. The values shown are mean (n = 3). These results are representative of three studies that have been done.

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Rats were injected i.v. with OX-26 (IgG2a anti-TfR) labeled with tritium, or with OX-26 chemically conjugated to Av or anti-TfR IgG3-CH3-Av labeled by incubation with [3H]biotin, and the radioactivity was followed for 60 min. (Fig. 5). [3H]biotin bound to the OX-26/Av chemical conjugate was removed rapidly from the plasma compartment, while the rate of removal of [3H]biotin bound to anti-TfR IgG3-CH3-Av is similar to that of [3H]-labeled OX-26 (Fig. 5). The corresponding pharmacokinetic parameters obtained by fitting the data to a mono- or bi-exponential equation are given in Table I. These data show that [3H]biotin bound to anti-TfR IgG3-CH3-Av is cleared from the peripheral compartment 5.8-fold more slowly than [3H]biotin bound to the OX-26/Av chemical conjugate. The plasma “area under the plasma concentration curve” (AUC) of [3H]biotin bound to the anti-TfR IgG3-CH3-Av for the period of 0 to 60 min was increased by a factor of 2.8 compared with that of [3H]biotin bound to the OX-26/Av conjugate, as a consequence of both a longer half-life of elimination (80.6 ± 4.8 min vs 20.5 ± 2.2 min) and an increased “mean residence time” (MRT) (114 ± 7 min vs 16.0 ± 1.3 min). Brain uptake of [3H]biotin bound to anti-TfR IgG3-CH3-Av was increased by a factor of 6.1 compared with that of the OX-26/Av conjugate (Table I) reflecting both a 2.6-fold increase in the BBB PS product (2.25 ± 0.65 μl · min−1·g−1 vs 0.85 ± 0.02 μl · min−1·g−1) and the higher AUC. These results showed that the fusion protein has much longer serum half-life than the chemical conjugate between OX-26 and avidin, and most importantly this fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate.

FIGURE 5.

. Plasma clearance of proteins. The plasma profiles of [3H]OX-26 and of [3H]biotin bound to either the OX-26/Av conjugate, or anti-TfR IgG3-CH3-Av fusion protein were analyzed. The filled circles (•) represent anti-TfR IgG3-CH3-Av, the open circles (○) anti-TfR OX-26/Av conjugate, and the open triangles (▵) [3H]OX-26. % ID/ml, Represents percentage of injected dose per milliliter of plasma. The values shown are mean ± SE (n = 3 rats per point). These results are representative of three studies that have been done.

FIGURE 5.

. Plasma clearance of proteins. The plasma profiles of [3H]OX-26 and of [3H]biotin bound to either the OX-26/Av conjugate, or anti-TfR IgG3-CH3-Av fusion protein were analyzed. The filled circles (•) represent anti-TfR IgG3-CH3-Av, the open circles (○) anti-TfR OX-26/Av conjugate, and the open triangles (▵) [3H]OX-26. % ID/ml, Represents percentage of injected dose per milliliter of plasma. The values shown are mean ± SE (n = 3 rats per point). These results are representative of three studies that have been done.

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Table I.

Pharmacokinetic parametersa for [3H]OX-26 and [3H]biotin bound to the OX-26/Av conjugate, or anti-TfR IgG3-CH3-Av 60 min after i.v. injection in the rat

Parameterb[3H]OX-26[3H]Biotin Carrier
OX-26/Av conjugateAnti-TfR IgG3-CH3-Av
A1 (%ID/ml) 2.99 ± 0.38 6.75 ± 0.43 2.91 ± 0.32 
A2 (%ID/ml) – 0.62 ± 0.20 2.75 ± 0.58 
K1 (min−10.011 ± 0.001 0.25 ± 0.02 0.58 ± 0.07 
K2 (min−1– 0.035 ± 0.003 0.0087 ± 0.0005 
t1/21 (min): distribution 65 ± 5 2.82 ± 0.22 1.24 ± 0.15 
t1/22 (min): elimination 81 ± 5 20.5 ± 2.2 80.6 ± 4.8 
AUC0–60min (% IDmin/ml) 132 ± 19 48.5 ± 4.0 134 ± 29 
AUC0–∞ (% IDmin/ml) 282 ± 52 50.4 ± 5.0 332 ± 89 
Vss (ml/kg) 133 ± 15 143 ± 17 172 ± 25 
CLss (ml/min/kg) 1.45 ± 0.23 8.94 ± 0.61 1.54 ± 0.29 
MRT (min) 93 ± 6 16.0 ± 1.3 114 ± 7 
Brain VD (mg/min/g) 169 ± 3 401 ± 48 91 ± 8 
PS (ml/g) 1.92 ± 0.06 0.85 ± 0.02 2.25 ± 0.65 
Brain uptake (% ID/g) 0.27 ± 0.04 0.041 ± 0.004 0.25 ± 0.09 
Parameterb[3H]OX-26[3H]Biotin Carrier
OX-26/Av conjugateAnti-TfR IgG3-CH3-Av
A1 (%ID/ml) 2.99 ± 0.38 6.75 ± 0.43 2.91 ± 0.32 
A2 (%ID/ml) – 0.62 ± 0.20 2.75 ± 0.58 
K1 (min−10.011 ± 0.001 0.25 ± 0.02 0.58 ± 0.07 
K2 (min−1– 0.035 ± 0.003 0.0087 ± 0.0005 
t1/21 (min): distribution 65 ± 5 2.82 ± 0.22 1.24 ± 0.15 
t1/22 (min): elimination 81 ± 5 20.5 ± 2.2 80.6 ± 4.8 
AUC0–60min (% IDmin/ml) 132 ± 19 48.5 ± 4.0 134 ± 29 
AUC0–∞ (% IDmin/ml) 282 ± 52 50.4 ± 5.0 332 ± 89 
Vss (ml/kg) 133 ± 15 143 ± 17 172 ± 25 
CLss (ml/min/kg) 1.45 ± 0.23 8.94 ± 0.61 1.54 ± 0.29 
MRT (min) 93 ± 6 16.0 ± 1.3 114 ± 7 
Brain VD (mg/min/g) 169 ± 3 401 ± 48 91 ± 8 
PS (ml/g) 1.92 ± 0.06 0.85 ± 0.02 2.25 ± 0.65 
Brain uptake (% ID/g) 0.27 ± 0.04 0.041 ± 0.004 0.25 ± 0.09 
a

For the pharmacokinetic parameters, the subscript 1 represents the distribution phase and the subscript 2 the elimination phase. A indicates the intercept value on the y-axis in Fig. 5, K the transfer rate, and CL the plasma clearance rate. AUC0–60 and AUC0–∞ are the first 60 min and steady-state area under the plasma concentration curve, respectively. Vss is the systemic volume of distribution, MRT the mean residence time, and VD the brain volume of distribution.

b

Calculated from the data in Fig. 5 for a 60 min period; therefore, the t1/22 is considered as an estimate.

Systemic clearance of [3H]biotin bound to anti-TfR IgG3-CH3-Av is mainly by the liver, which cleared 5.6 ± 0.7% ID/g within 60 min following an i.v. injection, while its renal clearance is minor with 0.37 ± 0.18% ID/g (Table II). This means that the binding of [3H]biotin to anti-TfR IgG3-CH3-Av is very stable in serum. The serum stability of the [3H]biotin/anti-TfR IgG3-CH3-Av fusion protein complex was examined by FPLC (data not shown). Examination of the FPLC profile indicated that more than 90% of the plasma radioactivity ([3H]biotin) eluted as the anti-TfR IgG3-CH3-Av complex 60 min after injection, with little free [3H]biotin detected in the serum. These results suggest that it should be possible to use the Ab-Av fusion protein as a vehicle to deliver biotinylated compounds to the brain.

Table II.

Organ clearance and delivery of [3H]biotin bound to the anti-IgG3CH3-Av fusion proteina

OrganOrgan Clearance (μl/min/g)Uptake (% D/g)
Brain 2.25 ± 0.65 0.25 ± 0.09 
Lung 2.54 ± 0.78 0.30 ± 0.06 
Heart 1.18 ± 0.49 0.14 ± 0.05 
Kidney 2.44 ± 0.69 0.37 ± 0.18 
Liver 46.4 ± 12.8 5.60 ± 0.69 
OrganOrgan Clearance (μl/min/g)Uptake (% D/g)
Brain 2.25 ± 0.65 0.25 ± 0.09 
Lung 2.54 ± 0.78 0.30 ± 0.06 
Heart 1.18 ± 0.49 0.14 ± 0.05 
Kidney 2.44 ± 0.69 0.37 ± 0.18 
Liver 46.4 ± 12.8 5.60 ± 0.69 
a

Measurements were made 60 min after i.v. injection. Data are mean ± SE (n = 3, rats).

Experiments were then performed to determine whether the anti-TfR IgG3-CH3-Av fusion protein can be used to deliver a biotinylated 18-mer antisense specific for the rev gene of HIV-1 (biotin-PNA), a molecule with therapeutic potential against HIV, to the brain. [125I]Biotin-PNA was injected i.v. into rats with or without anti-TfR IgG3-CH3-Av, and the brain uptake was analyzed as described above (Table III). The brain uptake of unconjugated [125I]biotin-PNA was negligible, with a PS product of 0.12 ± 0.01 μl · min−1·g−1 and a brain uptake of 0.0083 ± 0.0009% ID/g. In contrast, the brain uptake of [125I]biotin-PNA bound to anti-TfR IgG3-CH3-Av was 0.12 ± 0.01% ID/g at 60 min after an i.v. injection, and its BBB PS product was 0.67 ± 0.09 μl · min−1·g−1. The PS product for the [125I]biotin-PNA was increased 5.6-fold, and brain uptake was increased 14.5-fold when the [125I]biotin-PNA was bound to anti-TfR IgG3-CH3-Av. Thus, this novel Ab-Av fusion protein can deliver the biotinylated antisense drug anti-HIV PNA across the BBB, suggesting that brain delivery of anti-HIV PNA with the anti-TfR IgG3-CH3-Av may provide an effective treatment for cerebral AIDS.

Table III.

Brain uptake of biotin-PNA with or without anti-TfR IgG3-CH3-Ava

InjectatePS Product (μl/ml/g brain)Brain Uptake (% ID/g brain)
[125I]-Biotin-PNA 0.12 ± 0.01 0.0083 ± 0.0009 
Anti-TfR IgG3-CH3-Av/[125I]- biotin-PNA 0.67 ± 0.09 0.12 ± 0.03 
InjectatePS Product (μl/ml/g brain)Brain Uptake (% ID/g brain)
[125I]-Biotin-PNA 0.12 ± 0.01 0.0083 ± 0.0009 
Anti-TfR IgG3-CH3-Av/[125I]- biotin-PNA 0.67 ± 0.09 0.12 ± 0.03 
a

Measurements were made 60 min after i.v. injection. Data are mean ± SE (n = 3, rats).

Following i.v. injection, biotin bound to Av is rapidly removed from plasma with a half-life of 1.3 min (24). This rapid rate of plasma clearance has been attributed to the attached carbohydrate and the cationic charge of Av, which has 9 lysine and 8 arginine residues leading to an isoelectric point of 10. It is not surprising that chemical conjugation of Av to OX-26 leads to a reduced plasma AUC and a marked reduction of brain targeting compared with OX-26 (Table I) (29). It was therefore unexpected that genetic fusion of Av to human IgG3 would result in a protein with a half-life similar to that of unconjugated OX-26. In related studies, we have shown that the half life of anti-TfR IgG3-CH3-Av is similar to that of anti-TfR IgG3 (M. J. Coloma et al., manuscript in preparation).

It is difficult to explain why the Ab chemically conjugated to Av has such different pharmacokinetic properties compared with the Ab genetically fused to Av. Perhaps the chemical treatment per se partially denatures the conjugate, leading to its more rapid clearance. Alternatively, the site of Av addition may make important contributions to the pharmacokinetic properties. The fusion proteins are homogeneous with one Av attached at the end of the H chain. The conjugated proteins would be expected to be heterogeneous, varying both in the site and number of attached Av. The IgG-Av fusion protein behaves similar to the IgG-CD4 immunoadhesin, which is an IgG-CD4 fusion protein (30). Free CD4, a cationic protein like Av, is rapidly removed from the bloodstream (30). However, the plasma clearance of CD4 is greatly reduced when the protein is administered in the form of an IgG-CD4 fusion protein (30).

The amount of a drug delivered to the brain is typically expressed as the % ID/g, which is a function of the BBB permeability-surface area (PS) product and the plasma AUC (28). The more efficient brain uptake of [3H]biotin bound to anti-TfR IgG3-CH3-Av (compared with the chemical conjugate) with an accumulation of 0.25% ID/g at 60 min after the i.v. bolus reflects both its improved PS and AUC. This brain concentration is 3-fold higher than the brain uptake after 60 min of the classical neuroactive alkaloid morphine (0.081% ID/g) (28) and is comparable to that of OX-26.

Antisense oligodeoxynucleotides such as anti-HIV PNA may provide an effective therapy for HIV type 1 present in cerebral AIDS. Indeed, antisense oligonucleotides administered by intracerebroventricular injection or infusion have actually demonstrated selective inhibition of in vivo gene expression in the brain (31, 32). However, it would be desirable to have a noninvasive method of administering the oligonucleotides, but unfortunately they show negligible transcellular transport (33). In the present study, the brain uptake of free biotin-PNA (biotinylated anti-HIV PNA) injected i.v. was negligible (0.0083% ID/g). When biotinylated PNA was bound to the OX-26/streptavidin (SA) chemical conjugate, the brain uptake of systemically administered biotin-PNA was enhanced to about 0.075% ID/g (23). However, when anti-TfR IgG3-CH3-Av was used as the delivery vehicle, the brain uptake of biotinylated PNA increased to 0.12% ID/g, a 15-fold increase compared with free biotin-PNA. Thus, the brain uptake of biotin-PNA with the genetically engineered anti-TfR IgG3-CH3-Av is higher than that of biotin-PNA with the OX-26/SA chemical conjugate. Nevertheless, the brain uptake of biotin-PNA bound to anti-TfR IgG3-CH3-Av was half that of biotin bound to anti-TfR IgG3-CH3-Av. The PS product (0.67 μl/min/g brain) of anti-TfR IgG3-CH3-Av/biotin-PNA decreased to 30% of the PS product (2.25 μl/min/g brain) of anti-TfR IgG3-CH3-Av/biotin. The decreased brain uptake may reflect the poor intrinsic intracellular permeability of the PNA moiety in the complex.

A major concern is whether sufficient quantities of drugs can be delivered using anti-TfR IgG3-CH3-Av to have a therapeutic effect. Recent studies have demonstrated that the brain uptake of BDNF-polyethylene glycol (PEG)-biotin conjugated to OX26/SA was 0.144 ± 0.004% ID/g (34). Thus, the brain uptake of BDNF is ∼2-fold greater than that of morphine. When BDNF-PEG-biotin bound to OX26/SA was administered i.v. daily to rats for 1 wk after a 12-min period of transient forebrain ischemia, the hippocampal CA1 neuronal density was normalized; unconjugated BDNF or OX26 had no effect (35). Our studies suggest that anti-TfR IgG3-CH3-Av is even more effectively taken up into the brain than OX26/SA conjugates. Therefore, it would be expected to be an even more effective drug delivery vehicle capable of delivering therapeutic levels of drugs to the brain.

Our studies have indicated that anti-TfR IgG3-CH3-Av may be able to serve as a universal vehicle for targeting the brain with a vast array of different compounds, including chemicals, proteins, and DNA. In particular we have demonstrated that anti-TfR IgG3-CH3-Av can enhance the brain uptake of anti-HIV PNA and may provide a treatment for cerebral AIDS. Although we have focused our discussion on targeting to the cerebral hemisphere, the anti-TfR IgG3-CH3-Av can also be useful for targeting other structures of the CNS such as the cerebellum and spinal cord, which are also limited by the BBB. Therefore, the results presented here suggest that our novel universal vehicle will have a large number of potential applications in the diagnosis and/or therapy of various CNS disorders.

We thank Dr. Hwa Jeong Lee for technical assistance.

1

This work was supported in part by the Academic Research Fund (GE1997-019-D0019) of the Ministry of Education (Republic of Korea), Grant IM-754 from the American Cancer Society, Grant 3CB-0245 from the University of California Breast Cancer Research Program, Cancer Center Core Grant CA-16042, Grant AI-29470 from the National Institutes of Health (NIH), Grant 981-0717-132-2 from the Korea Science and Engineering Foundation (to Y.S.K.), and NIH Grant RO1-NS-34698 (to W.M.P.).

3

Abbreviations used in this paper: BBB, blood-brain barrier; AUC, area under the plasma concentration curve; Av, avidin; FPLC, fast protein liquid chromatography; ID, injected dose; NGF, nerve growth factor; PS, permeability-surface area; PNA, peptide nucleic acid; biotin-PNA, biotinylated anti-HIV PNA; SA, streptavidin; Tf, transferrin; BDNF, brain-derived neurotrophic factor.

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