Rutkowski, J. L. | Search bioRxiv

bioRxiv2021

Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro

Single domain shark VNAR antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the SARS-CoV-2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant ACE2 receptor in vitro. In addition, 3 antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies due to their low complexity, small size, unique epitope recognition and formatting flexibility should be a useful adjunct to existing antibody approaches to treat COVID-19.

bioengineering↗

bioRxiv2019

High efficiency blood-brain barrier transport using a VNAR targeting the Transferrin Receptor 1 (TfR1)

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high-affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. MethodsA synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. Phage formatted VNARs cross-reactive to recombinant human and mouse TfR1-ECD were fused to Fc domain of human IgG1 (hFc) and tested for TfR1-ECD binding by ELISA and surface plasmon resonance. The pharmacokinetics and biodistribution of VNAR-hFcs were studied in mice by ELISA and immunolabeling following intravenous (IV) injection and cardiac perfusion. Functional activity was measured by body temperature reduction following the IV injection of neurotensin fused to a TXB2-hFc (TXB2-hFc-NT). ResultsTXB2 was identified as a high-affinity, species cross-reactive VNAR antibody against TfR1-ECD, that does not to compete with transferrin or ferritin for receptor binding. IV dosing of TXB2-hFc at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc-NT at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. ConclusionsA species cross-reactive and brain-selective VNAR antibody to TfR1 was identified by a combination of in vitro and in vivo phage selection. As a high-affinity, bivalent Fc fusion protein, TXB2 rapidly crossed the BBB and exhibited a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

neuroscience↗