Citation: Clarke, E.; Stocki, P.; Sinclair, E.H.; Gauhar, A.; Fletcher, E.J.R.; Krawczun-Rygmaczewska, A.; Duty, S.; Walsh, F.S.; Doherty, P.; Rutkowski, J.L. A Single Domain Shark Antibody Targeting the Transferrin Receptor 1 Delivers a TrkB Agonist Antibody to the Brain and Provides Full Neuroprotection in a Mouse Model of Parkinson’s Disease. Pharmaceutics 2022, 14, 1335. https://doi.org/10.3390/ pharmaceutics14071335 Academic Editor: William M. Pardridge Received: 30 May 2022 Accepted: 22 June 2022 Published: 24 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article A Single Domain Shark Antibody Targeting the Transferrin Receptor 1 Delivers a TrkB Agonist Antibody to the Brain and Provides Full Neuroprotection in a Mouse Model of Parkinson’s Disease Emily Clarke 1 , Pawel Stocki 2 , Elizabeth H. Sinclair 2 , Aziz Gauhar 2 , Edward J. R. Fletcher 1 , Alicja Krawczun-Rygmaczewska 1 , Susan Duty 1 , Frank S. Walsh 2 , Patrick Doherty 1 and Julia Lynn Rutkowski 2, * 1 King’s College London, Institute of Psychiatry, Psychology and Neuroscience, Wolfson Centre for Age-Related Disease, Guy’s Campus, London SE1 1UL, UK; emily.clarke@kcl.ac.uk (E.C.); edward.fletcher@ucl.ac.uk (E.J.R.F.); alicja.krawczun-rygmaczewska@kcl.ac.uk (A.K.-R.); susan.duty@kcl.ac.uk (S.D.); patrick.doherty@kcl.ac.uk (P.D.) 2 Ossianix, Inc., Gunnels Wood Rd., Stevenage SG1 2FX, UK; pawel@ossianix.com (P.S.); liz@ossianix.com (E.H.S.); aziz@ossianix.com (A.G.); walsh@ossianix.com (F.S.W.) * Correspondence: rutkowski@ossianix.com; Tel.: +1-(610)-291-1724 Abstract: Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells have been used to shuttle antibodies and other cargos across the blood brain barrier (BBB) to the brain. For these studies the TXB4 brain shuttle was fused to a TrkB neurotrophin receptor agonist antibody. The TXB4-TrkB fusion retained potent agonist activity at its cognate receptor and after systemic administration showed a 12-fold increase in brain levels over the unmodified antibody. Only the TXB4-TrkB antibody fusion was detected within the brain and localized to TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc), where it was associated with activated ERK1/2 signaling. When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson’s disease (PD), TXB4-TrkB, but not the unmodified antibody, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 brain shuttle allows a TrkB agonist antibody to reach neuroprotective concentrations in the brain parenchyma following systemic administration. Keywords: TrkB; agonist antibody; variable new antigen receptor (VNAR); neuroprotection; transferrin receptor 1 (TfR1); blood-brain barrier (BBB); 6-OHDA; Parkinson’s disease 1. Introduction The interaction of neurotrophins (NGF, BDNF, NT3 and NT4) with their cognate Trk receptors (TrkA, TrkB and TrkC, respectively) protects neurons from naturally occurring cell death during development [1,2]. Their ability to nurture developing neurons spawned numerous studies to determine if they can promote the survival of adult neurons, particu- larly in the context of neurodegenerative disease or acute brain injury [3,4]. In this context, promising results have been found with BDNF which, by activating the TrkB receptor, can protect neurons from death in, for example, preclinical models of PD [5], Alzheimer’s disease (AD) [6,7], and ischemic lesions [8–11]. In addition, BDNF can promote func- tional recovery of injured neurons following spinal cord injury [12–14] and stimulate the production of new neurons in the adult brain [15,16]. The loss of BDNF has also been sug- gested as a contributory factor to the progression of PD [17–19], AD [20] and Huntington’s disease [21–23], as well as to conditions such as depression [24,25]. However, the therapeutic potential of BDNF in neurodegenerative diseases, acute brain injury and other neurological conditions has not been realized in the clinical setting due in part to a short plasma half-life in vivo [26], exclusion from the brain parenchyma Pharmaceutics 2022, 14, 1335. https://doi.org/10.3390/pharmaceutics14071335 https://www.mdpi.com/journal/pharmaceutics