Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment

Abstract

Lysosomal storage diseases are characterized by deficiencies in lysosomal enzymes, allowing accumulation of target substrate in cells and eventually causing cell death. Enzyme replacement therapy is the principal treatment for most of these diseases. However, these therapies are often complicated by immune responses to the enzymes, blocking efficacy and causing severe adverse outcomes by neutralizing product activity. It is thus crucial to understand the relationships between genetic mutations, endogenous residual enzyme proteins (cross-reactive immunologic material), development of neutralizing antibodies and their impact on clinical outcomes of lysosomal storage diseases. For patients in whom neutralizing antibodies may cause severe adverse clinical outcomes, it is paramount to develop tolerance inducing protocols to preclude, where predictable, or treat such life-threatening responses.

Figure 1

Impact of neutralizing antibody on enzyme entry and activity in ERT. (a) There are several crucial steps by which therapeutic enzymes for LSDs mediate effectiveness. First, lysosomal enzyme (E) is taken up by target cells in an M6PR-dependent fashion, endocytosed by clathrin-coated vesicles (CCV) and fused with endosomes and then with lysosomes, where enzymatic activity is exerted on accumulated substrate. During this migration, the full-length enzyme may be processed into an intermediate form in the late endosomes or lysosomes, and then into the fully activated species in a proteinase-dependent manner. There are several possible outcomes of the binding of lysosomal enzyme–specific antibodies (yellow ‘Y’): blockade of enzyme uptake through M6PR by binding to the receptor binding or uptake domain(s) (UD; domains containing exposed M6P); blockade of enzyme uptake by M6PR and suppression of enzymatic activity by binding to epitopes near the receptor binding domain and the enzymatic activity domain (AD; theoretical); blockade of both uptake and activity domains by separate antibodies specific for each site; degradation of the enzyme by catalytic antibody (red ‘Y’); reduction of enzymatic activity by targeting the enzymatic domain; prevention of enzyme maturation by targeting the enzyme protease processing sites (PS); and targeting of other sites (OS) of the enzyme, resulting in conformational or trafficking changes. (b) Binding of antibodies to enzyme may redirect the enzyme to FcR-expressing cells, such as macrophages and B cells. Enzyme–antibody complexes internalized through FcRs may prevent proper translocation of functional enzymes to the lysosome. Binding of antibody to other domains of the enzyme may change pharmacokinetics or redirect the enzyme to FcR-expressing cells. IC, immune complex.

Figure 2

Neutralization assays. (a) Uptake neutralization assay. The assay format presented here is based on flow cytometry and uses enzymes labeled with fluorescent probes and live cells. The specificity of the receptor-mediated internalization of the labeled enzyme should be shown by competition with excess unlabeled enzyme. Approaches using other types of labels could also be developed. Sera (taken before treatment and at various time points after treatment) from all patients who either have detectable binding antibody or clinical decline should be tested with a validated assay. (b) Enzyme activity neutralization assay. This can be done without the use of cells. The neutralizing capacity of the patient’s serum is measured directly on enzyme activity.