title: A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75(NTR.).
abstract: The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.
authors.affiliation: Department of Human Physiology; Centre for Neuroscience; Flinders University School of Medicine; Adelaide, Australia; Current affiliation: Department of Neurology and Neurological Sciences; Stanford University School of Medicine; Stanford, CA USA.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tani
authors.affiliation: MedImmune Ltd. (Formerly Cambridge Antibody Technology); Cambridge, UK.
authors.forename: Jane K
authors.initials: JK
authors.lastname: Osbourn
authors.affiliation: MedImmune Ltd. (Formerly Cambridge Antibody Technology); Cambridge, UK.
authors.forename: Edward H
authors.initials: EH
authors.lastname: Walker
authors.affiliation: Department of Human Physiology; Centre for Neuroscience; Flinders University School of Medicine; Adelaide, Australia.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Rush
authors.affiliation: Department of Human Physiology; Centre for Neuroscience; Flinders University School of Medicine; Adelaide, Australia.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Ferguson
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001812:Blood-Brain Barrier; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D058990:Molecular Targeted Therapy; D009046:Motor Neurons / Q000378:metabolism* / Q000821:virology; D019151:Peptide Library; D051381:Rats; D017207:Rats, Sprague-Dawley; D020800:Receptor, Nerve Growth Factor / Q000276:immunology; D012584:Sciatic Nerve / Q000378:metabolism* / Q000821:virology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism* / Q000821:virology; D053586:Virus Internalization
chemical_list: D019151:Peptide Library; D020800:Receptor, Nerve Growth Factor; D057127:Single-Chain Antibodies
keywords: blood-brain barrier; internalizing antibodies; p75NTR; phage display; retrograde transport; scFv
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