Blood-brain barrier transport using a high-affinity, brain-selective VNAR (Variable Domain of New Antigen Receptor) antibody targeting transferrin receptor 1

Abstract

A one-sentence summary:Development of highly efficient, TfR1 specific, cross-species reactive blood-brain barrier (BBB) shuttle based on shark single domain VNAR antibody.

Brain delivery of biologics using a cross‐species reactive transferrin receptor 1 VNAR shuttle

“…TXB2 was expressed as a fusion protein with the human IgG1 Fc domain as reported previously. 16 A similar construct based on a VNAR clone, that binds to TfR1 with nanomolar affinity but does not penetrate brain, served as control (control VNAR-hFc). 16 VNAR-hFc formats were produced with VNARs at the N-terminal end of a human IgG1 Fc ( Figure 1A), where a series of substitutions (E233P, L234V, L235A, ΔG236, A327G, A330S, P331S) were introduced to attenuate effector functions.…”

“…16 A similar construct based on a VNAR clone, that binds to TfR1 with nanomolar affinity but does not penetrate brain, served as control (control VNAR-hFc). 16 VNAR-hFc formats were produced with VNARs at the N-terminal end of a human IgG1 Fc ( Figure 1A), where a series of substitutions (E233P, L234V, L235A, ΔG236, A327G, A330S, P331S) were introduced to attenuate effector functions. 18 For in vivo studies, in AβPP transgenic mice, TXB2 was fused to the Aβ binding humanized IgG1 antibody Bapi, attached to the N-terminal end of the antibody heavy chain via a 3xG4S linker (Figure 2A).…”

“…[13][14][15] Recently a single domain VNAR (TXB2) shark antibody, which binds to TfR1 in a variety of species was described. 16 In the present study, we wished to investigate the ability of this VNAR to shuttle a biologic cargo into the brain and chose to study the well described therapeutic antibody bapineuzumab (Bapi) which targets Aβ. 17 Positron emission tomography (PET) and radioactivity measurements were used to image and quantify the uptake and intrabrain distribution in wildtype mice and transgenic mice over-expressing human Aβ.…”

A single domain shark antibody targeting the transferrin receptor 1 delivers a TrkB agonist antibody across the blood brain barrier to provide full neuroprotection in a mouse model of Parkinson’s Disease

“…The TfR1 binding TXB2 VNAR can carry cargos, including a human IgG Fc domain (TXB2-hFc) as well as biologically active neurotensin, across the BBB. Importantly the TXB2-hFc does not compete for transferrin binding, clear the TfR1 from capillaries, or lyse reticulocytes 33 . TXB4 is a version of TXB2 with some modifications in the CDR3 that improved brain penetration (JLR and PS, manuscript in preparation).…”

“…There is considerable interest in the possibility of utilizing receptor mediated transcytosis pathways that exist on brain endothelial cells that form the BBB to carry biotherapeutics from the blood to the brain parenchyma with the transferrin receptor 1 (TfR1) being the most widely studied 32 . TXB4 is a single domain shark variable new antigen receptor (VNAR) antibody specific to TfR1 and was developed from previously reported parental TXB2 VNAR 33 by restricted randomisation of CDR3 domain that led to improved brain penetration (JLR & PS, manuscript in preparation). TXB2 when fused to human IgG1 Fc (TXB2-hFc) readily crossed the intact BBB, reaching low nanomolar concentrations with a half-life of several days in the brain where it can be found to localised to TfR1 expressing neurons.…”