Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aβ) peptide were constructed and shown to display higher brain penetration than the parent anti-Aβ antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4–5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.

We report on two chondrosarcoma cell lines, FS and AQ, that may be used as models of multidrug resistance in chondrosarcoma. Multidrug resistance-1 expression was assayed with reverse transcription-polymerase chain reaction. Immunostaining for the multidrug resistance-1 product, P-glycoprotein, was performed with the monoclonal antibody C494. Intracellular levels of doxorubicin were measured by fluorescent emission at 590 nm after 1 hour of incubation with the agent and again after 1, 2, and 4-hour washout periods. Chemosensitivity was assayed by staining micropellet cultures of AQ and FS cells with fluorescein acetate before and after the cells were exposed to varying doses of doxorubicin for 48 hours. Cytotoxicity was assessed by comparison of computer-processed images before and after treatment. The FS cell line was positive for multidrug resistance-1 expression, stained heavily for P-glycoprotein, and had significantly lower intracellular levels of doxorubicin than the AQ cell line, which was negative for multidrug resistance-1 and P-glycoprotein. Chemosensitivity testing showed that the FS cell line was significantly more resistant to doxorubicin than was the AQ cell line at all doses tested. Our results show that multidrug resistance-1 expression in a human chondrosarcoma cell line results in resistance to doxorubicin in vitro.

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Summary:We report on two chondrosarcoma cell lines, FS and AQ, that may be used as models of multidrug resistance in chondrosarcoma. Multidrug resistance-1 expression was assayed with reverse transcriptionpolymerase chain reaction. Immunostaining for the multidrug resistance-1 product, P-glycoprotein, was performed with the monoclonal antibody C494. Intracellular levels of doxorubicin were measured by fluorescent emission at 590 nm after 1 hour of incubation with the agent and again after 1,2, and 4-hour washout periods. Chcmosensitivity was assayed by staining micropellet cultures of AQ and FS cells with fluorescein acetate before and after the cells were exposed to varying doses of doxorubicin for 48 hours. Cylotoxicity was assessed by comparison of computer-processed images before and after treatment. The FS cell line was positive for multidrug resistance-1 expression. stained heavily for P-glycoprotein, and had significantly lower intracellular levels of doxorubicin than the AQ cell line, which was negative for multidrug resistance-1 and P-glycoprotein. Chemosensitivity testing showed that the FS cell line was significantly more resistant to doxorubicin than was the AQ cell line at all doses tested. Our results show that multidrug resistance-1 expression in a human chondrosarcoma cell line results in resistance to doxorubicin in vitro.