No abstract

Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aβ antibody, which uses a monovalent binding mode to the TfR, increases β-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.

The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.

No abstract

The pleiotropic cyto/lymphokine tumor necrosis factor (TNF) exerts its functions by binding to specific cell-surface receptors. We have prepared two sets of monoclonal antibodies (mAbs) against TNF-binding proteins from the HL-60 (htr-mAb series) and U-937 (utr-mAb series) cell lines.The htr antibodies inhibit the binding of '25I-labeled TNF-a to HL-60 cells only partially, whereas they block the TNF-a binding to several adenocarcinoma cell lines (HEp-2, HeLa, and MCF7) almost completely. In contrast, the utr antibodies have no effect on TNF-a binding to the adenocarcinoma cell lines but partially inhibit TNF-a binding to HL-60 and U-937 cells. However, htr-9 and utr-1 antibodies in combination fully inhibit the TNF-a binding to HL-60 and U-937 cells. The binding of TNF-13 to HEp-2 and U-937 cells is also inhibited by htr and utr antibodies. Neither htr nor utr mAb has an effect on the TNF-sensitive murine cell lines L929 and WEHI 164. Flow cytometry studies show that mAbs htr-9 and utr-1 detect two distinct TNF-binding sites on human cell lines. Immunologic blot and immunoprecipitation analyses indicate that mAbs htr-9 and utr-1 recognize proteins of =55 kDa and 75 kDa, respectively. These data provide evidence for the existence of two distinct TNF receptor molecules that contribute to varying extent to the TNF binding by different human cells.