Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies

Abstract

Adeno-associated viruses (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of eight new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors in development for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism.

High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors

“…Novel anti-AAV5 capsid antibody HL2476 targets the 3-fold capsid region. A novel MAb directed against the AAV5 3-fold capsid region was generated by immunization of mice with AAV5 VLPs and their splenocytes used to generate hybridoma cell clones as previously described for AAV8 and AAV9 (24). To select for a hybridoma cell line expressing a MAb targeting the 3-fold region of the AAV5 capsid, a native dot immunoblot analysis was performed with AAV5 capsids pretreated with either phosphate-buffered saline (PBS) or the AVB nanobody prior to addition of the hybridoma supernatant ( Fig.…”

“…Animals and cell culture. Six-week-old female CD-1 mice were used for the production of anti-AAV5 monoclonal antibodies (MAb) at the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) Hybridoma Core Laboratory, as previously reported (24). Use of animals in the UF Hybridoma Core Laboratory occurred under the guidelines of the Institutional Animal Care and Use Committee.…”

Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction

“… 13 , 14 When anti-capsid antibodies target viral epitopes critical for cellular entry, they can block virus infectivity and are deemed neutralizing antibodies (NAbs). 15 , 16 , 17 NAbs can have a profound effect on the efficiency of tissue transduction with AAV vectors, as even low titers of NAbs can result in lack of efficacy following gene transfer. 9 , 18 Depending on the AAV serotype and the age of the donor, the proportion of subjects positive for anti-AAV antibodies can be up to ∼60%, with only a small window of time after birth during which the vast majority of individuals are naive to the virus.…”

Thermal Stability as a Determinant of AAV Serotype Identity

“…Furthermore, although mouse monoclonal antibodies are available for some of the AAV serotypes, which can identify their capsids, this is not the case for all available AAVs. 14 , 15 , 16 , 17 The engineering of AAVs for various reasons, including escape from pre-existing neutralizing host antibodies or improved tissue targeting, which modifies antigenic regions, compounds this problem. An alternative for capsid identification of purified rAAV preparation is mass spectrometry.…”