Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Drug targeting through the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, is possible with peptidomimetic monoclonal antibodies that undergo receptor-mediated transcytosis through the BBB (Pardridge, 1999). In the Old World primate, murine monoclonal antibodies (mAbs) to the human insulin receptor have been used to deliver drugs through the primate BBB in vivo (Wu et al., 1997). Mouse monoclonal antibodies to the rat transferrin receptor (TfR) have been used to deliver drugs through the rat BBB in vivo (Wu and Pardridge, 1999). These peptidomimetic mAbs act as brain drug delivery vectors because the mAb binds an exofacial epitope on the BBB peptide receptor at the luminal membrane of the capillary endothelium, and this binding triggers receptor-mediated transcytosis through the BBB on the peptide receptor system without interfering with the BBB transport of the endogenous peptide, e.g., insulin or transferrin (Bickel et al., 1994; Skarlatos and Pardridge, 1995; Descamps et al., 1996). Conjugation of drugs to brain drug delivery vectors is facilitated with the use of avidin-biotin technology, wherein the drug is monobiotinylated in parallel with the production of the mAb/avidin or mAb/streptavidin fusion protein (Shin et al., 1997; Li et al., 1999).

BBB transport vectors are generally species-specific. The OX26 mAb is a murine antibody to the rat transferrin receptor (Jefferies et al., 1985), and it is thought that the OX26 mAb is not active in other species such as mice, although this has not been documented. The anti-human insulin receptor mAb is specific for only Old World primates or humans and cannot be used in mice (Pardridge et al., 1995). The emergence of transgenic mouse models (Jaenisch, 1988) to examine the pathogenesis of disease creates the need for a delivery system that can be used for BBB drug transport in the mouse. Therefore, the purpose of the present investigations was to examine three different anti-TfR mAbs, including the murine OX26 mAb to the rat TfR. Two other antibodies analyzed in these studies are rat mAbs to the mouse TfR, the RI7-217 mAb (Lesley et al., 1984) and the 8D3 mAb (Kissel et al., 1998). The RI7-217 antibody is a rat IgG_2a to the mouse TfR and binds to TfR on mouse cells (Lesley et al., 1989), and cytokine/RI7-217 fusion proteins are biologically active (Dreier et al., 1998). The 8D3 antibody is also a rat IgG_2a to the mouse TfR, and immunocytochemistry shows specific binding of the 8D3 mAb to capillary endothelium in mouse brain (Kissel et al., 1998).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. ¹²⁵I-Na was obtained from Amersham (Arlington Heights, IL) and chloramine-T was supplied by MCA Reagents (Cincinnati, OH). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The ammonium sulfate precipitated 8D3 rat mAb to the mouse TfR was provided by Dr. Britta Engelhardt (W.G. Kerckhoff Institut, Bad Nauheim, Germany), and was purified by protein G affinity chromatography as described previously (Kang and Pardridge, 1994). The RI7-217 (designated RI7) rat hybridoma to the mouse TfR was obtained under Material Transfer Agreement from Dr. Jayne Lesley (Salk Institute, LaJolla, CA). Male BALB/c mice (~20-30 g) were obtained from Harlan Sprague-Dawley (San Diego, CA).

Production of RI7 mAb. The RI7 hybridoma was propagated in tissue culture from serum free hybridoma-conditioned media and was affinity purified with protein G affinity chromatography as described previously (Kang and Pardridge, 1994). The antibody was purified to homogeneity based on SDS-polyacrylamide gel electrophoresis, which showed a single heavy chain and double light chains, similar to the murine OX26 mAb (Yoshikawa and Pardridge, 1992).

Iodination of Antibody. The 8D3 mAb, RI7 mAb, or OX 26 mAb (0.33 nmol) was iodinated with 1 mCi of ¹²⁵I-Na and either Iodogen (Pierce Chemical Co., Rockville, IL) or chroramine-T and the reaction was stopped by adding sodium metabisulfite. The sample was applied to a Sephadex G-25 column to separate free iodine from ¹²⁵I-mAb in the presence of PBS and 0.1% BSA. The trichloroacetic acid (TCA) precipitability of ¹²⁵I-8D3 mAb, ¹²⁵I-RI7 mAb and ¹²⁵I-OX 26 mAb were 98, 99, and 99%, and the specific activity was 1.2, 8.0, and 4.9 µCi/µg, respectively.

Pharmacokinetics and Organ Distribution. The mice were anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg) i.p.; 50 µl of PBS, pH 7.4/0.1% BSA containing 5 µCi of ¹²⁵I-8D3 mAb, 4 µCi of ¹²⁵I-RI7 mAb, or 6 µCi of ¹²⁵I-OX 26 mAb were injected into the jugular vein and arterial blood was withdrawn from the aorta at 0.25, 2, 5, 15, and 60 min after injection of the isotope solution. The blood samples were centrifuged and plasma was counted for both total radioactivity and TCA-precipitable radioactivity. Mouse organs (brain, liver, kidney, lung, spleen, and heart) were taken at 60 min after the isotope administration for gamma counting.

Capillary Depletion Analysis. The anesthetized mice were administered 50 µl of PBS containing 0.1% BSA and 5 µCi of ¹²⁵I-RI7 mAb into the jugular vein. Arterial blood and brain were collected at 0.5, 1, 6, and 24 h after injection of the isotope solution. After centrifugation of the blood samples, plasma was counted for total radioactivity and was assayed for TCA precipitation. The brain tissue was homogenized in ice-cold physiological buffer for capillary depletion analysis, as described previously (Triguero et al., 1990). The total brain homogenate was fractionated into the capillary pellet and the postcapillary supernatant by the capillary depletion technique. Vd values for the homogenate and the capillary pellet were calculated as follows:
where C_p(t) is the counts per minute per microliter of plasma at brain sampling time (t). For the postcapillary supernatant, the Vd was calculated by subtracting the Vd of capillary pellet from the Vd of homogenate. Statistical analysis was performed by Student's t test.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

The plasma profiles for the three monoclonal antibodies are shown in Fig. 1. The plasma concentration for the three antibodies are shown in Fig. 1 (left), and the plasma TCA precipitability of the ¹²⁵I-mAbs is shown in Fig. 1 (right). All three antibodies are relatively stable based on the high TCA precipitability during the experimental time period.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Plasma concentration (%ID/ml) versus time profile of 125I-8D3 mAb, 125I-RI7 mAb, and 125I-OX 26 mAb (left) in anesthetized BALB/c mice. Time course of TCA-precipitable plasma radioactivity (%) of 125I-8D3 mAb, 125I-RI7 mAb, and 125I-OX 26 mAb (right) after i.v. injection. Data are means ± S.E. (n = 3).

The plasma profiles in Fig. 1 were subjected to a pharmacokinetic analysis and these parameters are shown in Table 1. The plasma clearance of the RI7 mAb > the 8D3 mAb the OX26 mAb. The systemic volume of distribution (V_ss) of the OX26 mAb, 50 ± 2 ml/kg, approximates the plasma volume in mice (Table 1), whereas the V_ss of the 8D3 or RI7 mAb is ~3-fold higher (Table 1).

The organ uptake of the three mAbs in mice at 60 min after i.v. injection was measured and these data are shown in Table 2 for brain, liver, kidney, and heart. The organ Vd is shown for the three mAbs for the four organs in comparison with the plasma V₀ for each of the four organs. The V₀ was determined from the organ uptake of ¹²⁵I-labeled rat IgG_2B isotype control antibody, which is restricted to the plasma volume in mice (Skarlatos and Pardridge, 1995). There is virtually no brain uptake of the OX26 mAb by mouse brain because the brain Vd of this antibody is nearly identical with the brain plasma volume (Table 2). The brain uptake of the OX26 mAb is 0.06 ± 0.01% ID/g (Table 2). Conversely, the brain uptake of either the 8D3 or the RI7 mAb is high, 1.6 to 3.1% ID/g (Table 2). The 8D3 mAb is significantly transported into liver and kidney, whereas the RI7 mAb is not measurably taken up by these organs, and is selective for brain (Table 2).

To demonstrate that the RI7 mAb was effectively transcytosed through the BBB in vivo, a capillary depletion analysis was performed. The uptake of the ¹²⁵I-RI7 mAb was measured in mouse brain at 0.5, 1, 6, and 24 h after i.v. injection. The Vd was measured in the total brain homogenate, the capillary pellet, and the postvascular supernatant (Fig. 2). These data show that the RI7 mAb was effectively transcytosed through the BBB in vivo because the Vd of the postvascular supernatant increased with time and approximated 1000 µl/g at 24 h after i.v. injection (Fig. 2). The RI7 mAb was relatively stable in plasma as the plasma TCA precipitability at 0.5, 1, 6, and 24 h after i.v. injection was 96 ± 1, 93 ± 1, 86 ± 3, and 80 ± 2%, respectively (mean ± S.E., n = three mice per point).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Vd values of 125I-RI7 mAb in brain homogenate, capillary pellet, and postcapillary supernatant. Capillary depletion analysis of the brain tissue was performed at 0.5, 1, 6, and 24 h after i.v. injection of the isotope solution. Data are means ± S.E. (n = 3).

The saturation of the organ uptake of the labeled 8D3 antibody is shown in Table 3. The brain uptake, percentage of ID/g, is reduced 6-fold and the BBB PS product is reduced 95% by a 4-mg/kg dose of unlabeled 8D3 antibody (Table 3). This dose increases the plasma AUC ~3-fold. In addition to brain, the uptake of the labeled 8D3 antibody by heart, lung, and spleen is inhibited by the 4 mg/kg-dose of unlabeled antibody, whereas the uptake systems in liver and kidney are less inhibited (Table 3).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The results of these investigations are consistent with the following conclusions. First, the OX26 mouse mAb to the rat TfR is not significantly taken up by mouse brain in vivo. Second, both the 8D3 and the RI7 rat mAbs to the mouse TfR are effectively transported into brain in vivo. Third, the organ selectivity for the RI7 and 8D3 mAbs are different because the 8D3 mAb is taken up by liver, and to a lesser extent, kidney, whereas the RI7 mAb is not taken up by these organs and is selective for brain (Table 2). Fourth, the brain uptake of the 8D3 mAb is saturable (Table 3), consistent with a receptor-mediated process.

These data show clearly that the murine OX26 mAb to the rat TfR cannot be used for brain drug delivery studies in mouse models because the uptake of this murine mAb by mouse brain is not different from the brain uptake of a brain plasma marker (Table 2). Conversely, the brain Vd of either the 8D3 or RI7 rat mAb to the mouse TfR is >10-fold higher than the brain Vd of the OX26 mAb (Table 2). The brain uptake, expressed as %ID/g brain, for either the 8D3 or RI7 mAb is nearly 50-fold greater than the brain uptake of the OX26 mAb (Table 2). The data in Table 2 allow for comparison of the uptake by mouse brain of either the 8D3 or RI7 mAb, and the brain uptake of the OX26 mAb in rats. The BBB PS product for the OX26 mAb in the rat at 60 min after i.v. injection, 1.6 ± 0.1 µl/min/g (Bickel et al., 1993), is not significantly different from the BBB PS products for either the 8D3 or RI7 mAb in mouse brain at this time period (Fig. 3). In contrast, the brain uptake (%ID/g), for either the 8D3 or the RI7 mAbs in mouse brain, 1.6 to 3.1% ID/g (Table 2), is up to 12-fold greater than the brain uptake of the OX26 MAb by rat brain in vivo, 0.26% ID/g (Bickel et al., 1993). This higher uptake in mouse brain, normalized per gram organ weight, is directly proportional to the pharmacokinetic parameter, i.e., the plasma AUC. For example, the plasma AUC for the 8D3 mAb (Table 1) is 12-fold higher in mice, 2059 ± 117% ID · min/ml (Table 1) compared with the 60-min AUC value for the OX26 mAb in rats, 168 ± 76% ID · min/ml (Fig. 3). The plasma AUC for the RI7 mAb is 7-fold higher in mice relative to the plasma AUC of the OX26 mAb in rats. The plasma AUC is inversely proportional to the approximately 10-fold lower body weight of mice relative to rats because the blood volume (ml) in mice is 10-fold lower than in rats. Therefore, the higher brain uptake of the mAb in mice, expressed as %ID/g brain, is proportional to the higher plasma AUC in this species, and this is expected based on the much lower body weight of mice relative to rats. The pharmacokinetic estimates are semiquantitative because the experimental time was only 60 min (Fig. 1), but the plasma t_1/2 of all three mAbs is longer (Table 1). In addition, the measurements at the early time points of plasma clearance are restricted to 0.25 min (see Experimental Procedures). However, the initial plasma concentration (A, percentage of ID/ml, Table 1) for the 8D3 or RI7 mAb is only ~40% of the corresponding value for the OX26 mAb (Table 1). This suggests a very early and rapid phase of clearance of the 8D3 and RI7 mAbs occurs immediately after i.v. administration.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Comparison of the BBB PS product, AUC, and brain uptake, percentage ID/g brain, at 60 min after i.v. injection for the RI7-217, 8D3, and OX26 mAbs in the mouse, and for the OX26 mAb in the rat. The rat OX26 mAb data are from Bickel et al. (1993).

The RI7 and 8D3 mAbs have comparable BBB PS products (Tables 2 and 3); the higher PS product for the 8D3 antibody in the experiments reported in Table 3 is about twice the PS product recorded in the experiments reported in Table 2. The specific activity of the ¹²⁵I-8D3 mAb used in the experiments reported in Table 3 is 10-fold higher than the specific activity of the ¹²⁵I-8D3 mAb used in the experiments reported in Table 2 (see Experimental Procedures). Therefore, the dose of 8D3 mAb (in micrograms) was 10-fold higher in the experiments reported in Table 2. The PS product was reduced in these experiments due to the saturation of the BBB transport of the anti-TfR mAbs (Table 3). Because the BBB PS products for the 8D3 and RI7 antibodies are comparable, the higher brain uptake of the 8D3 mAb (Fig. 3) is due solely to the higher plasma AUC of the 8D3 mAb compared with the RI7 antibody (Table 1). The higher plasma AUC for the 8D3 mAb is surprising because this mAb is targeted to the liver, whereas the RI7 mAb is not taken up by liver (Table 2). The plasma AUC should be inversely related to the hepatic clearance of the mAb. The absence of significant hepatic uptake of the RI7 mAb in mice is also unexpected given the high level of transferrin receptor in liver (Rudolph et al., 1988). The selective uptake of the RI7 mAb by brain, but not liver, is not due to organ-specific gene expression because there is a single mouse TfR gene (Stearne et al., 1985). The RI7 mAb may recognize an epitope that is fully expressed at the BBB TfR, and perhaps other organs such as bone marrow or spleen, but is masked in part at the TfR in mouse liver. Alternatively, TCA-precipitable metabolites of the RI7 mAb may be exported from the liver to blood. The BBB PS product for the RI7 mAb may actually be about twice the value shown in Fig. 3 because the RI7 hybridoma secretes mAb with endogenous light chain (see Experimental Procedures), similar to the OX26 hybridoma (Yoshikawa and Pardridge, 1992). The endogenous light chain would combine with the RI7 heavy chain and this mixed mAb is presumed to have a much reduced affinity for the TfR. Genetically engineered forms of the OX26 mAb have been recently expressed from cloned OX26 light and heavy chain genes, which eliminates the problem of endogenous light chain secretion (Li et al., 1999), and similar genetically engineered forms of either the RI7 or 8D3 mAb could be produced.

The OX26 mAb undergoes receptor-mediated transcytosis through the BBB in rat brain and this has been demonstrated by capillary depletion analysis, thaw mount autoradiography, and immunogold electron microscopy (Bickel et al., 1994; Skarlatos and Pardridge, 1995; Broadwell et al., 1996). The anti-TfR mAbs undergo receptor-mediated transcytosis through the BBB similar to transferrin (Fishman et al., 1987; Skarlatos and Pardridge, 1995). The RI7 mAb also undergoes receptor-mediated transcytosis through the mouse BBB in vivo based on the capillary depletion studies shown in Fig. 2. The Vd of the postvascular supernatant for the RI7 mAb approximates 800 µl/g brain at 24 h after i.v. injection and this is ~3-fold greater than the vascular pellet volume of distribution for the RI7 mAb (Fig. 2). The receptor-mediated transport of the anti-TfR mAbs across the BBB explains the saturation of this process by increasing doses of coinjected unlabeled antibody (Table 3). Similar to results on the brain uptake of the OX26 mAb by the rat (Kang et al., 1994), the mouse brain uptake of the 8D3 antibody is fully inhibited by a dose of unlabeled antibody of 4 mg/kg (Table 3).

In summary, these studies compare the mouse brain uptake of three different anti-TfR mAbs and show that the OX26 mAb used for rat brain transport studies cannot be used in mice. Either the 8D3 or the RI7 mAb may be used in mouse brain uptake studies, and each mAb has particular advantages. The 8D3 mAb has a higher plasma AUC and higher brain uptake and percentage ID/g compared with the RI7 mAb (Fig. 3), but the RI7 mAb is selective for brain with no measureable uptake by liver or kidney (Table 2). The availability of these rat mAbs to the mouse TfR will make possible future studies of brain drug delivery in the mouse, including transgenic mouse models.