Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy

Abstract

Assessment of anti-adeno-associated virus (AAV) antibodies in patients prior to systemic gene therapy administration is an important consideration regarding efficacy and safety of the therapy. Approximately 30%-60% of individuals have pre-existing anti-AAV antibodies. Seroprevalence is impacted by multiple factors, including geography, age, capsid serotype, and assay type. Anti-AAV antibody assays typically measure (1) transduction inhibition by detecting the neutralizing capacity of antibodies and non-antibody neutralizing factors, or (2) total anti-capsid binding antibodies, regardless of neutralizing activity. Presently, there is a paucity of head-to-head data and standardized approaches associating assay results with clinical outcomes. In addition, establishing clinically relevant screening titer cutoffs is complex. Thus, meaningful comparisons across assays are nearly impossible. Although complex, establishing screening assays in routine clinical practice to identify patients with antibody levels that may impact favorable treatment outcomes is achievable for both transduction inhibition and total antibody assays. Formal regulatory approval of such assays as companion diagnostic tests will confirm their suitability for specific recombinant AAV gene therapies. This review covers current approaches to measure anti-AAV antibodies in patient plasma or serum, their potential impact on therapeutic safety and efficacy, and investigative strategies to mitigate the effects of pre-existing anti-AAV antibodies in patients.

Keywords: efficacy; gene therapy; humoral immunity; neutralizing antibodies; recombinant adeno-associated virus; safety; seroprevalence; total antibody assay; transduction inhibition assay.

Graphical abstract

Figure 1

Mechanisms for the inhibition of vector transduction and transgene expression by neutralizing anti-AAV antibodies rAAV vector transduction and key steps in which transduction may be inhibited by neutralizing anti-AAV antibodies. Non-neutralizing anti-AAV antibodies bind to the rAAV capsid but do not prevent vector binding to cell-surface receptors or inhibit transduction. Of note, non-antibody neutralizing factors (not depicted) might also impact transduction. AAV, adeno-associated virus; rAAV, recombinant AAV.

Figure 2

Anti-AAV antibody seroprevalence ranges for different AAV serotypes and across geographical regions using the hemophilia A and B population as an example The published anti-AAV antibody seroprevalence rates vary widely according to a variety of factors, including (A) different AAV serotypes,^,,,,,,,,, and (B) across geographical regions (using AAV6 data from three separate reports as an example).^,, Other factors that may influence seroprevalence rates include assay type, donor health and age, and the use of some medications, such as immunosuppressants. ∗As these studies were performed in different regions/countries using different assay types, the seroprevalence rates cannot be directly compared. AAV, adeno-associated virus; TAb, total antibody; TI, transduction inhibition.

Figure 3

Principles of transduction inhibition and total antibody assays (A) (1) Serum samples are heat-inactivated and any potential precipitates removed by centrifugation. Patient and control serum dilutions are prepared. The TI assay is usually carried out in a 48- or 96-well plate format, permitting a high-throughput sample analysis. Typically, a reporter rAAV vector is combined with the serially diluted test sample before (2) being incubated with the cell line (in some cases, target cells are pre-infected with wild-type adenovirus to increase rAAV transduction). (3) The target cells are lysed, and reporter gene expression (luciferase or GFP activity) is measured as luminescence after the addition of the enzyme substrate (in the case of luciferase) or fluorescence (in the case of GFP). The presence of AAV NAbs and non-antibody neutralizing factors (not depicted in the figure for reasons of clarity) interferes with the transduction process and decreases the reporter gene expression when compared with the negative control. (4) Confirmatory steps to determine neutralization due to NAbs can be performed, although they are not always essential. This step may involve use of an irrelevant monoclonal non-AAV antibody to determine specificity, Ig fraction depletion, or competitive inhibition with empty vectors (or irrelevant transgenes). (B) TAb assays can be divided into antigen-capture and bridging formats, depending on the secondary reagents used. TAb assays can be developed using either co-incubation (homogeneous) or sequential incubation (heterogeneous) protocols based on the desired attributes, such as improved assay selectivity, antigen tolerance, or specificity (reviewed in Gorovits et al.⁴⁸). (1) rAAV capsids are immobilized on an ELISA or electrochemiluminescence (ECL) microtiter plate; (2) serum samples are added to allow binding of antibodies to the rAAV capsid; (3) after washing to remove unbound material, in the antigen-capture format, a secondary detection reagent such as a horseradish peroxidase-conjugated or ruthenylated anti-species antibody is added; in the bridging format, a labeled (e.g., biotinylated or ruthenylated) rAAV capsid is added⁴⁸). (4) In the case of a ruthenylated detection system, the assay signal is measured in luminescence units after the addition of the read buffer. For enzyme-conjugated detection systems, the enzyme substrate is added for colorimetric detection. Commercially available serotype-specific anti-AAV capsid monoclonal/polyclonal antibodies or proprietary antibodies may be used as a positive control, while pooled samples from TAb-negative donors can be used as a negative control. (5) Anti-AAV TAb screening assays typically involve an additional confirmatory assay to ensure the specificity of the signal obtained. AAV, adeno-associated virus; ECLA, electrochemiluminescence assay; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; Ig, immunoglobulin; NAb, neutralizing antibody; rAAV, recombinant AAV; TAb, total antibody; TI, transduction inhibition.

Figure 4

Readout of a semi-quantitative transduction inhibition assay These principles also apply to total antibody assay. (A) Reporter gene expression in serial one in three dilutions of serum samples of four patients and positive and negative controls. The negative control does not contain neutralizing anti-AAV antibodies (or non-antibody neutralizing factors); therefore, the rAAV vector transduces the target cells where the reporter gene is expressed (yellow well). The positive control contains neutralizing anti-AAV antibodies, often marginally above the screening titer cutoff. Patient serum samples one to four contain varying levels of neutralizing anti-AAV antibodies (or non-antibody neutralizing factors) that affect the level of reporter gene expression seen at different dilutions. (B) The degree of inhibition of reporter gene expression is plotted against the dilutions of the serum sample.^,, The 50% inhibition cutoff from the resulting curves is based on the highest sample dilution that achieves 50% inhibition of vector transduction in comparison with the negative-control sample. In this example, patient 3 has an anti-AAV NAb titer of 1:81, which will be compared with the screening titer cutoff to determine patient eligibility. If the patient 3 titer of 1:81 ≥ screening titer cutoff (e.g., 1:3), the patient would be ineligible for the rAAV GTx. AAV, adeno-associated virus; rAAV, recombinant AAV; TAb, total antibody; TI, transduction inhibition.