This panel of antibodies allows better characterization of antigenic epitopes on AAV8 and AAV9 capsids with the goal of mitigating patient immune response to the existing AAV based gene therapy. Adeno-associated viral (AAV) vectors are currently the tool-of-choice for gene therapy, due to their unique lifecycle and versatility in targeting different tissues. Some recent successful applications of AAV-based gene therapy include treatments for ophthalmologic and neurodegenerative diseases, among others. Although the use of AAV vectors is very promising, several challenges remain. One of the biggest challenges facing gene therapy is, overcoming host immune response to AAV-based gene delivery. Estimates claim that approximately 40-70 percent of the general population has been pre-exposed to AAVs and carries antibodies against AAV. ??Researchers at the University of Florida have developed a panel of monoclonal antibodies against AAV8 and AAV9 to facilitate characterization of their capsid-antibody interactions. AAV8 and AAV9 have become the choice vectors in targeting liver and central nervous system, but some immunogenicity has been observed in human and animal studies. The panel of antibodies against AAV8 and AAV9 developed at the University is an excellent tool for identification of potential mutation sites to allow next generation AAV vectors to evade the immune response and improve the efficacy of gene therapy. Application • Can be used to determine antigenic regions on the capsids of AAV8 and AAV9 • Potential to use in an ELISA tittering kit • Potential to use in an immuno-dot blot assay • Can be used in a neutralization assay • These antibodies can be coupled to chromatography resins for improved binding and purification of AAV8 and AAV9 over current chromatography resins Advantages • To design a vector that can escape this host immune response has been an ongoing challenge in the field of gene therapy. This panel of monoclonal antibodies can be used as a tool to study the antigenicity of the AAV8 and AAV9 capsids • These antibodies can also aid in characterizing variants being developed for improved therapeutic efficacy • AAV capsid epitope of each antibody has been mapped Technology Using existing hybridoma technology, UF researchers generated two anti-AAV8 (designated HL2381 and HL2383; HL stands for Hybridoma Lab) and four anti-AAV9 (HL2368, HL2370, HL2372, and HL2374) IgG antibodies. The HL2372 antibody cross-reacts with AAV8. The clones were selected from fused hybridoma hybrids after capsid ELISA and native dot immunoblot screenings. All the six antibodies generated only bound to intact capsids, not the linear viral proteins, when tested. The identified isotypes of the two new anti-AAV8 antibodies, HL2381 and HL2383, are both IgG3. The identified isotypes of the four anti-AAV9 antibodies, HL2368, HL2370, HL2372, and HL2374, are IgG3, IgG2a, IgG2a, and IgG3, respectively. The two anti-AAV8 antibodies, HL2381 and HL2383, were able to recognize AAV8 as predicted, but also cross-reacted with AAV3B. During the clonal selection, one anti-AAV9 clone, HL2372, exhibited binding to both AAV8 and AAV9. This cross-reactivity was confirmed by dot immunoblot assay. All other anti-AAV9 antibodies, HL2368, HL2370, and HL2374, bound to AAV9 only.
