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Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood–brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

Delivery of biotherapeutics across the blood–brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.SummaryBrain delivery, biodistribution and pharmacodynamics of a lysosomal enzyme fused to a moderate-affinity transferrin receptor-directed blood-brain barrier enzyme transport vehicle are superior to a traditional high-affinity anti-TfR monoclonal antibody fusion.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-β-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment resulting from deficiency in one or more lysosomal enzymes. Treatment of neuronopathic LSDs remains a considerable challenge, as the recombinant enzymes approved to treat these conditions are ineffective in modifying CNS disease because they do not effectively cross the blood–brain barrier (BBB). To address this unmet need, we developed the Enzyme Transport Vehicle (ETV), a lysosomal enzyme fused to an engineered Fc domain that binds to the transferrin receptor and enables receptor-mediated transcytosis across the BBB. ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme whose deficiency leads to Mucopolysaccharidosis II (MPS II), were generated that have high intrinsic activity and effectively degrade accumulated substrates in IDS-deficient cells and in vivo mouse models of MPS II disease. We establish that ETV:IDS, through its interactions with TfR, significantly improves delivery of IDS to the brain without affecting the native properties of the enzyme necessary for efficient lysosomal targeting and function. This translates to robust reductions of accumulated substrates in both the brain and peripheral tissues of IDS-deficient mice and supports the potential of ETV:IDS as an intravenous monotherapy to treat peripheral and CNS manifestations of MPS II. Further, these data, together with the inherent modularity of the platform, suggest that the ETV can be harnessed more broadly to enhance brain delivery of additional lysosomal enzymes, yielding a new class of biotherapeutics for the treatment of neuronopathic LSDs.

Gastric cancer is the second leading cause of death from cancer worldwide. Amplification of the FGFR2 gene has been reported in ∼ 5 to 7% of gastric cancers, and several studies have also reported overexpression of the FGFR2 protein using immunohistochemistry (IHC). FGFR2 gene amplification and protein overexpression have also been shown to correlate with an aggressive disease phenotype, making FGFR2 an attractive therapeutic target. We have reported elsewhere at this meeting that the b-isoform of FGFR2 is predominantly expressed in these tumors and have developed a therapeutic antibody, FPA144, which specifically targets this isoform. Here we report initial development of a separate monoclonal antibody, FPT mAb, for use as a companion IHC diagnostic (CDx) for selecting patients with high levels of FGFR2b protein. In specificity studies using cells transfected with various FGFRs we show that FPT mAb specifically detects the b-isoform of FGFR2. In contrast, 7 commercially available anti-FGFR2 antibodies showed cross-reactivity with other FGF receptors. In an assay using cells with inducible FGFR2 gene expression, FPT mAb also was more sensitive for detection of FGFR2b than any of the commercially available antibodies. Furthermore, FPT mAb was able to detect FGFR2b protein expression in gastric cancer cases without FGFR2 gene amplification. We examined FGFR2b protein expression by IHC using the FPT mAb and FGFR2 gene amplification using FISH in 186 gastric cancer samples. 6% of samples showed amplification of the FGFR2 gene as measured by FISH (FGFR2:CEN10 >3), compared to 16.7% of samples positive for FGFR2b expression as measured by IHC (2-3+ staining in >50% of the tumor cells). In conclusion, we show that the FPT mAb is sensitive and specific for detecting by IHC the b-isoform of FGFR2 expressed in gastric cancers. Further, we demonstrate that more gastric tumors overexpress FGFR2b protein than bear amplifications of the FGFR2 gene. These results suggest that a potentially larger percentage of patients with gastric cancer may be appropriately identified as candidates for FGFR2b-specific therapies. Citation Format: Amit M. Deshpande, Servando Palencia, David I. Bellovin, Abigael T. Gemo, Tina Giese, Bradley Stohr, Kristen L. Pierce, Gerrit Los. Expression of FGFR2b in gastric cancer as measured by immunohistochemistry with a highly specific monoclonal antibody. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2845. doi:10.1158/1538-7445.AM2014-2845

e15074 Background: Amplification of the Fibroblast Growth Factor Receptor 2 (FGFR2) gene is reported in ~5% of gastric cancer patients and leads to the specific overexpression of the FGFR2b isoform...