SEUNG-UON SHIN*, PHILLIP FRIDENt, MARJORIE MORANt, TRAcY OLSONt, YOUNG-SOOK KANGt,

In the present study, the receptor binding potential oftransferrin (Tf) was linked to an antibody binding specificity. Human Tf was fused to mouse-human chimeric IgG3 at three positions: at the end of heavy chain constant region 1 (CHi), after the hinge, and after CH3. The resulting Tf-antibody fusion proteins were able to bind antigen and the Tf receptor. The CH3-Tf fusion protein showed no complement-mediated cytolysis but possessed IgG receptor I (Fc'yRI) binding activity. Most importantly, all of the fusion proteins demonstrated significant uptake into brain parenchyma, with 0.3% ofthe injected dose ofthe hinge-ITfusion protein rapidly targeted to the brain. Recovery of iodinated CH3-Tf fusion protein from the brain parenchyma demonstrated that the fusion proteins can cross the blood-brain barrier intact. The binding specificity of these fusion proteins can be used for brain delivery of noncovalently bound ligands, such as drugs and peptides, or for targeting antigens present within the brain. Morbidity caused by brain dysfunction affects more than 50 million persons in the United States. Although various neuropharmaceutical drugs have the potential for treating specific brain diseases, their effective delivery remains a problem. In vivo drug delivery to the brain is limited by poor transport of water-soluble drugs through the brain capillary endothelial wall that makes up the blood-brain barrier (BBB). Brain capillary endothelial tight junctions form a very highresistance barrier to the uptake of circulating solutes (1, 2). There are many possible strategies for drug delivery to the brain. Disruption of the BBB by hyperosmolar shock (3) or by the administration of vasoactive substances such as leukotrienes (4) provides transient access to the brain. However, this approach allows uncontrolled access of blood-borne compounds to the brain, and when the BBB is repetitively opened, neuropathologic changes take place in the brain (5). Direct intraventricular administration of drug by using surgical techniques provides another approach (6). However, intraventricularly administered drugs are distributed primarily to the surface of the brain, and penetration into the parenchyma is limited by diffusion (7). In addition, the drug will be rapidly removed from the intraventricular space by cerebrospinal fluid bulk flow. Peptide lipidization, which involves the attachment of lipid-soluble moieties, has been useful for small peptides (8) but does not appear useful for peptides of >5 amino acids (7). Liposomes, a standard approach to drug delivery, are too large to traverse the BBB and have not proved to be effective as neuropharmaceutical drug delivery vehicles for the brain (7). Specific receptors on the BBB are used to transport required nutrients and growth factors such as insulin (9), transferrin (Tf) (10), and insulin-like growth factors (11) from the blood to the brain. These receptors provide potential avenues for transport into the brain. For example, immunoglobulin molecules are normally excluded from the brain (12); however, an antibody to the Tf receptor (TfR) has been shown to be transported into the brain parenchyma and to be effective as a drug delivery vehicle (13). In the present study, we have used the ligands to the brain endothelial receptors, instead of an anti-receptor antibody, to direct molecules to the brain and have constructed proteins consisting of Tf fused to antibody. Tf was chosen as the receptor-targeting ligand because TfRs are abundant on the vascular endothelium of the brain capillaries (14). Antibodies were chosen because their binding specificity can be used either for delivery of agents to the brain or for secondary targeting once the fusion protein is within the brain. Fusion proteins consisting of human Tf sequences fused to the C terminus of full-length and truncated antibody sequences were constructed and found to retain both antigen and TfR binding activity. Additionally, depending on where the Tf is joined to the antibody, molecules were produced that retain some of the antibody effector functions. Our results show that Tf can indeed be used to target fusion proteins to the brain. MATERIALS AND METHODS Cell Lines. The human histiocytic lymphoma U937, which expresses Fc and C3 receptors (15, 16), was grown in RPMI 1640 medium with 10% (vol/vol) fetal calf serum. Sp2/0 and P3X63Ag8.653 cells were cultured in Iscove's modified Dulbecco's medium with 5% (vol/vol) calf serum. Construction and Characterization of the IgG3-Tf Fusion Proteins. Site-directed mutagenesis was used to generate unique restriction enzyme cleavage sites in the human IgG3 heavy chain gene as described (17, 18). The human Tf cDNA was cloned at the 3' end of the heavy chain constant region 1 (CH1) exon (CH1-Tf), immediately after the hinge (H) (H-Tf) (17), or at the 3' end of the CH3 exon (CH3-Tf) in the mouse-human IgG3 heavy chain gene specific for the hapten dansyl (DNS, 5-dimethylaminonaphthalene-1-sulfonyl) (18). The IgG3-Tf fusion heavy chain gene and the chimeric mousehuman K light chain specific for DNS (pSV184AHneo) (19,20) were transfected into P3X63Ag8.653 or Sp2/0 by electroporation, transfectomas were selected, and those producing the fusion proteins were identified as described (21). Cells proAbbreviations: BBB, blood-brain barrier; Tf, transferrin; TfR, transferrin receptor; DNS, dansyl; CH, heavy chain constant region; Fc'yRI, IgG receptor I; BSA, bovine serum albumin; SRBC, sheep red blood cell; H, hinge. §To whom reprint requests should be addressed. 2820 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natt Acad Sci USA 92 (1995) 2821 ducing the IgG3-Tf fusion proteins were biosynthetically labeled with [35S]methionine, and the proteins were immunoprecipitated and analyzed by SDS/PAGE with or without 2-mercaptoethanol. The IgG3-Tf fusion proteins were purified from culture supernatants by affinity column chromatography (22). Purity was determined by Coomassie blue staining of SDS/PAGE. Receptor Binding Assays. IgG receptor I (FcyRI) binding was assayed essentially as described (23) with U937 cells. TfR was purified from freshly obtained full-term human placentas (24) by affinity chromatography on a human Tf-Sepharose column. The resulting receptor was 98% pure and of the expected molecular weight. The relative affinity of the various fusion proteins for the TfR was estimated by a solid-phase competition assay on microtiter plates coated with TfR incubated with a mixture of 4 nM 125I-labeled Tf and various concentrations of unlabeled competitor (100 ,ul per well). Bound radioactivity was determined by using a y counter. Nonlinear curve fitting to determine 50% inhibition points was performed by using SIGMA PLOT. Capillary Depletion. Capillary depletion experiments were performed essentially as described (13). Fusion proteins were labeled with [3H]succinimidyl propionate and the control IgG was labeled with [14C]acetic anhydride. For all experiments, the radiolabeled compounds were injected as a 400-,ul bolus into the tail vein of female Sprague-Dawley rats (100-125 g) under halothane anesthesia, and the animals were sacrificed at the appropriate time by using a lethal dose of anesthetic. Radiolabeled IgG was coinjected with the labeled fusion protein to serve as a control for nonspecific radioactivity recovered in the brain due to residual blood. All data were corrected for residual blood in the brain by using the coinjected 14C-labeled chimeric IgG3 as a blood marker. Data are expressed as the percent of the injected dose in the parenchyma or capillary fractions. Internal Carotid Artery Perfusion. CH3-Tf (40 ,ug) iodinated by the chloramine T method was used within 24 hr for internal carotid artery perfusion with male Sprague-Dawley rats (300-325 g) as described (25). The perfusion was initiated by closure of the ipsilateral common artery and infusion of