Table 2.
Summary of previously reported cases to deliver macromolecule therapeutics through the BBB
| Therapeutic target/disease | Ab/protein design/engineering/key characterization | Biological effects (uptake/therapeutic effects) | Status (pre-clinical, clinical stages, and approval) | Ref |
|---|---|---|---|---|
| Transcytosis receptor: InsR | ||||
| IDUA/Hurler’s syndrome | HIR Mab-IDUA fusion (valanafusp alpha), bivalent antibody-enzyme fusion, high-affinity HIR binding, unaltered antibody binding, and enzyme activity upon fusion | In Rhesus monkeys, brain uptake of HIR Mab-IDUA fusion showed >12-fold increase versus IDUA as measured by radioactivity. In a 52-week, open label phase 1–2 trial on the safety and efficacy in pediatric MPSI patients with cognitive impairment, the pediatric subjects had CNS involvement with a mean enrollment Development Quotient (DQ) of 36.1 ± 7.1. The DQ, and the cortical gray matter volume of the brain, were stabilized by valanafusp alpha treatment. Somatic manifestations were stabilized, or improved, based on urinary glycosaminoglycan levels, hepatic and spleen volumes, and shoulder range of motion. | Phase 2 clinical trial | [101–104] |
| IDS/Hunter’s syndrome | HIR Mab-IDS fusion, bivalent antibody-enzyme fusion, high-affinity HIR binding, unaltered antibody binding and enzyme activity (363 ± 37 U/μg) upon fusion, uptake by Hunter fibroblasts | In Rhesus monkeys, brain uptake of HIR Mab-IDS fusion showed >10-fold increase versus GDNF (BBB impermeable marker) as measured by radioactivity. | Preclinical in Rhesus monkey | [105,106] |
| ASA/MLD | HIR Mab-ASA fusion, bivalent antibody-enzyme fusion, high affinity HIR binding EC50 = 0.34 ± 0.11 nM, retains high ASA enzyme activity, 20 ± 1 units/mg. Endocytosis in MLD fibroblasts. | In Rhesus monkeys, brain uptake of HIR Mab-ASA fusion showed >25-fold increase versus control IgG as measured by radioactivity. Enhanced brain distribution was confirmed also by radioautography. | Preclinical in Rhesus monkey | [107] |
| SGSH/Sanfilippo type A syndrome | HIR Mab-SGSH fusion, bivalent antibody-enzyme fusion, high affinity HIR binding EC50 = 0.33 ± 0.05 nM, retains high ASA enzyme activity, 4712 ± 388 units/mg. Endocytosis in MPSIIIA fibroblasts. | In Rhesus monkeys, brain uptake of HIR Mab-SGSH fusion showed >35-fold increase versus control IgG as measured by radioactivity. | Preclinical in Rhesus monkey | [108] |
| NAGLU/MPSIIIB | HIR Mab-NAGLU fusion, bivalent antibody-enzyme fusion, high-affinity HIR binding, unaltered antibody binding and enzyme activity upon fusion, uptake in MPSIIIB fibroblasts normalizes intracellular NAGLU enzyme activity | In Rhesus monkeys, brain uptake of HIR Mab-NAGLU fusion showed >15-fold increase versus control IgG as measured by radioactivity. | Preclinical in Rhesus monkey | [109] |
| PON1/atherosclerosis | HIR Mab-PON1 fusion, bivalent antibody-enzyme fusion, and fusion protein were rapidly removed from the blood, primarily by the liver | In Rhesus monkeys, brain uptake HIR Mab-PON1 fusion showed >25-fold increase versus control IgG as measured by radioactivity. | Preclinical in Rhesus monkey | [110] |
| EPO | HIR Mab-EPO fusion, bivalent antibody-protein fusion, high affinity HIR binding EC50 = 0.21 ± 0.05 nM, retains high EPO activity, ED50 = 0.30 ± 0.01 nM. | In Rhesus monkeys, brain uptake HIR Mab-EPO fusion showed >16-fold increase versus EPO as measured by radioactivity. | Preclinical in Rhesus monkey | [111] |
| GDNF/PD | HIR Mab-GDNF fusion, bivalent antibody-protein fusion, high affinity HIR binding EC50 = 0.87 ± 0.13 nM, retains high GDNF activity in binding GFPα1, ED50 = 1.68 ± 0.17 nM. | In a rat middle, cerebral artery occlusion model stroke volume was reduced by 77%. In the Rhesus monkey, no improvement of Parkinsonian motor symptoms and induced a dose-dependent hypersensitivity reaction | Preclinical in Rhesus monkey (failure) | [112,113] |
| TNFR/inflammation | HIR Mab-TNFR fusion, bivalent antibody-protein fusion, high-affinity HIR binding, unaltered antibody binding, and TNFR activity upon fusion | In Rhesus monkeys, brain uptake HIR Mab-TNFR fusion showed >30-fold increase versus TNFR-Fc as measured by radioactivity. | Preclinical in Rhesus monkey | [114] |
| Aβ peptide/AD | HIR Mab-scFv fusion, bivalent antibody-scFv fusion, high affinity HIR binding EC50 = 1.9 ± 0.1 nM, unaltered antibody binding with Aβ1–40 upon fusion with ED50 = 2.0 ± 0.8 nM | Rapidly cleared from plasma and was transported across the primate BBB. In vivo reached 1% ID/100 g tissue, HIR Mab-scFv fusion showed >10-fold increase versus anti-Aβ Mab as measured by radioactivity. | Preclinical in Rhesus monkey | [115,116] |
| Transcytosis receptor: TfR | ||||
| BACE1/AD | Anti-mouse TfR/anti-BACE1, KiH, monovalent binding. Bispecific antibody maintains the activity of BACE1 antibody in inhibiting Aβ production. High-affinity anti-TfR was found to induce TfR lysosomal degradation, causing reduced TfR surface level and curbing brain accumulation. Antibody effector function and complement binding were found to be responsible for acute toxicity and reticulocyte reduction upon injection. | For anti-TfR/BACE1 bispecific using therapeutic dosing, 3–5-fold increased brain uptake (vs. control IgG) 24 hr after injection as measured by sandwich ELISA. Single IV injection at 25 or 50 mg/kg reduced brain Aβ concentration by more than 50% within 24–48 h. | Preclinical in mouse | [117–119] |
| Aβ/AD | IgG-sFab, KiH, monovalent binding to TfR, and bivalent binding to Aβ. Monovalent sFab design showed lower TfR degradation than bivalent dFab design. | sFab design showed 55-fold higher plaque staining versus dFab design after intravenous injection into PS2APP mice. Weekly sFab design injection for 3 months showed a significant decrease of plaque number in both cortex and hippocampus in APPPS2 mice (vs. parent Aβ antibody mAb31). | Preclinical in mouse | [120] |
| BACE1/AD | Anti-human/cyno TfR/anti-BACE1, KiH, monovalent binding. Non-competing with Tf, binding TfR at apical domain. Optimal affinity is required since either high affinity or low affinity showed lower brain uptake. Compared to high-affinity anti-TfR, optimal affinity against TfR showed no degradation of TfR upon injection. Effector-less antibodies with N297G mutation showed no toxicity in monkeys. | TfR/anti-BACE1 with the optimal affinity of the anti-TfR arm showed a 3–5-fold increase in brain uptake in both human TfR KI mice and cynomolgus monkeys. In addition, therapeutic effects were observed as reduced Aβ concentration in both plasma and CNS (brain of mice and CSF of monkeys). | Preclinical in cynomolgus monkey | [121] |
| BACE1/AD | Directed-evolution engineered Fc fragment binding apical domain of human TfR, which did not impede Tf-TfR or Fc-FcRn interactions. ATV:BACE1 has monovalent binding to TfR and bivalent binding to BACE1. The ATV molecules have a native IgG structure and stability. Dose-dependent cellular internalization. | ATV:BACE1 showed >20-fold increase in brain IgG uptake than BACE1 antibody in human TfR KI mice. The increased brain uptake also showed >2-fold decrease in brain Aβ load. ATV:BACE1 showed a 50% reduction of CSF Aβ40 in cynomolgus monkeys and maintained the effects for 14 days after a single IV injection. In comparison, the BACE1 antibody showed no CSF Aβ40 reduction. | Preclinical in cynomolgus monkey | [122] |
| IDS/Hunter’s syndrome | Directed-evolution engineered Fc fragment binding apical domain of human TfR. IDS fusion to the N′ of engineered Fc fragment (ETV:IDS) was chosen due to unimpeded TfR binding. ETV:IDS maintains the biological activity of IDS in MPS II patient fibroblasts. | ETV:IDS showed 3–5-fold higher brain uptake than Fc:IDS in human TfR KI mice. Single or multiple doses of ETV:IDS reduced brain and CSF sGAG levels by 3–5-fold in comparison to IDS. ETV:IDS significantly reduced GAGs in neurons, astrocytes, and microglia and reduced lysosomal lipid accumulation in the brain. | Preclinical in mouse | [123,124] |
| PGRN/frontotemporal dementia | Directed-evolution engineered Fc fragment binding apical domain of human TfR. PGRN fusion to the C′ of engineered Fc fragment (PTV:PGRN) was chosen due to the need for a free PGRN C-terminus to interact with sortilin. Affinity to huTfR is 130 nM, PGRN activity is retained: EC50s sortilin 2 nM, prosaposin 5 nM. | PTV:PGRN rescued various Grn−/− phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn−/− CNS, including microgliosis, lipofuscinosis, and neuronal damage. | Preclinical in mouse | [125] |
| TNFR/inflammation | Anti-mouse TfR-TNFR fusion (cTfRMAb-TNFR), bivalent antibody-protein fusion, high-affinity TNFα binding Kd = 96 ± 34 pM | In mice, brain uptake of cTfRMAb-TNFR showed >45-fold increase versus control IgG after IV injection as measured by radioactivity. Neuroprotection in mouse MCAO model with 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively, upon 1 mg/kg intravenous injection. | Preclinical in mouse | [126,127] |
| GDNF/PD | Anti-mouse TfR-GDNF fusion (cTfRMAb-GDNF), bivalent antibody-protein fusion. No chronic toxicity in mice after 2 mg/kg twice-weekly IV injection for 12 consecutive weeks. Chronic injection induced no anti-drug antibodies. | Chronic cTfRMAb-GDNF treatment showed no impact on brain uptake of cTfRMAb-GDNF. In comparison to the OX26 antibody, a 30-fold increase in brain uptake was observed. In a mouse PD model, chronic cTfRMAb-GDNF injection significantly reduced symptoms of PD. | Preclinical in mouse | [128,129] |
| Aβ/AD | cTfRMAb-scFv fusion, bivalent antibody-scFv fusion targeting both mouse TfR and Aβ. | Daily sc injections of 5 mg/kg of the cTfRMAb-ScFv fusion protein for 12 consecutive weeks reduced amyloid plaque in the cortex and hippocampus 1-year-old PS1/APP AD double transgenic PSAPP mice. | Preclinical in mouse | [130] |
| EPO/MCAO and AD | cTfRMAb-EPO fusion, bivalent antibody-EPO fusion targeting both mouse TfR and EPOR. Retains high EPO activity, ED50 = 0.33 ± 0.04 nM. | The brain uptake of the cTfRMAb-EPO fusion protein was 2.0 ± 0.1% ID/g of the brain following iv administration. cTfRMAb-EPO showed an 81% reduction in hemispheric stroke volume and a 78% reduction of the neural deficit in a permanent mouse MCAO model after a 1 mg/kg IV injection. APP/PS1 mice treated with cTfRMAb-EPO fusion protein showed significantly lower cortical and hippocampal Aβ peptide number and immune-positive area, decreased hippocampal synaptic loss, and cortical microglial activation and improved spatial memory compared with APP/PS1 saline controls. | Preclinical in mouse | [131–133] |
| IDUA/Hurler’s syndrome | cTfRMAb-IDUA fusion, bivalent anti-mouse TfR antibody-IDUA. Unaltered IDUA enzyme activity upon fusion 776 ± 79 units/μg. | Treatment of the MPSI mice with the cTfRMAb-IDUA reduced intracellular lysosomal inclusion bodies by 73% in the brain (versus saline-treated mice) with IV injections twice a week for 8 weeks at a 1 mg/kg dose. | Preclinical in mouse | [134] |
| IL-1/neuropathic pain | Anti-mouse TfR-IL-1RA fusion. Anti-TfR used affinity engineered 8D3 rat anti-mouse TfR. Bivalent anti-mouse TfR 8D3-IL-1RA fusion as chimeric human IgG1 molecules with the S239D/A330L/I332E triple mutation significantly enhances cellular cytotoxicity. | Lower-affinity 8D3 variants showed 30–50-fold increased brain antibody uptake, and the 8D3 variant with 130 nM affinity to mouse TfR showed the maximum brain uptake. IL-1RA-anti-TfR fusions showed dose-dependent inhibition of IL-1β-induced IL6 secretion in NIH-3 T3 cells. 8D3130-IL-1RA fusions showed a significant reversal of partial nerve ligation–induced mechanical hyperalgesia. | Preclinical in mouse | [135] |
| TREM2/AD | A TREM2 antibody in TVD-Ig format with C′ scFv of anti-mouse TfR. Monovalent TfR engagement was confirmed to have broad brain parenchyma distribution. | Weekly treatment of 5XFAD mice (a model of AD) with Ab18 TVD-Ig/αTfR showed a considerable reduction of amyloid burden with increased microglia migration to and phagocytosis of amyloid plaques, improved synaptic and neuronal marker intensity, improved cognitive functions, reduced endogenous tau hyperphosphorylation, and decreased phosphorylated neurofilament H immunostaining. | Preclinical in mouse | [136,137] |
| Transcytosis receptor: CD98hc | ||||
| BACE1/AD | Anti-mouse CD98hc/BACE1 KiH is a bispecific antibody with monovalent binding. CD98hc was identified to be highly expressed in BECs via proteomic study. The ideal CD98hc/BACE1 bispecific antibody has IC50 = 164.4 nM against CD98hc in a competition assay for therapeutic dosing (50 mg/kg). Antibody showed no interference of CD98hc expression, stability, and function. | The lower affinity (CD98hc, IC50 = 164.4 nM) CD98hc/BACE1 bispecific antibody showed a 3-fold increase in brain concentration (measured by ELISA) at therapeutic dosing 50 mg/kg. Both high- and low-affinity CD98hc/BACE1 bispecific antibodies showed a 50% reduction in brain Aβ concentration | Preclinical in mouse | [138] |
| Transcytosis receptor: an unknown receptor that binds FC5 and FC5-derived antibodies | ||||
| Neuropeptides dalargin and neuropeptide Y/Inflammatory pain | Neuropeptides dalargin and neuropeptide Y are chemically conjugated with VHH FC5. FC5 was previously identified to cross both human and mouse BBB by phage-display using llama single-domain library. In the current design, bivalent FC5 fusion to human Fc was used for conjugation. FC5 was previously shown to bind luminal alpha (2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. | 30-fold enhanced apparent brain exposure of FC5-conjugates compared with control domain Antibody-Fc fusions after systemic dosing in rats. FC5 conjugates showed dose-dependent inhibition of thermal hyperalgesia in the Hargreaves model after a single systemic injection. | Preclinical in rat | [139–141] |
| mGluR1/thermal hyperalgesia | mGluR1-BBB was the fusion between FC5 and antibody targeting mGluR1 (mGluR1-IgG). Fusion protein showed unaltered mGluR1 antagonist activity in comparison to the parent mGluR1-IgG. mGluR1-BBB showed dose-dependent binding to rat (SV-ARBEC) and mouse (bEnd.3) BECs. | mGluR1-BBB showed up to a 10-fold increase in CSF concentration versus parent mGluR1-IgG. Significant inhibition of thermal hyperalgesia by BBB-mGluR1 treatment in rats with induced chronic inflammatory pain. | Preclinical in rat | [142] |
| IL-1RA/mechanical hyperalgesia | The BBB targeting antibody was screened by phage display in scFv format for competition with FC5 for binding mouse BEC line, bEND.3 (named as BBBt). | The BBBt-IL-1RA showed a 3–4-fold increase in brain concentration versus control IgG. Single IV injection of BBBt-IL-1RA showed long-lasting reversal of mechanical hyperalgesia in the Seltzer model of neuropathic pain versus control IgG-IL-1RA. | Preclinical in mouse | [143] |
EPO, erythropoietin.