Successful induction of immune tolerance to enzyme replacement therapy in canine mucopolysaccharidosis I

Abstract

Immune responses can interfere with the effective use of therapeutic proteins to treat genetic deficiencies and have been challenging to manage. To address this problem, we adapted and studied methods of immune tolerance used in canine organ transplantation research to soluble protein therapeutics. A tolerization regimen was developed that prevents a strong antibody response to the enzyme alpha-l-iduronidase during enzyme replacement therapy of a canine model of the lysosomal storage disorder mucopolysaccharidosis I. The tolerizing regimen consists of a limited 60-day course of cyclosporin A and azathioprine combined with weekly i.v. infusions of low-dose recombinant human alpha-l-iduronidase. The canines tolerized with this regimen maintain a reduced immune response for up to 6 months despite weekly therapeutic doses of enzyme in the absence of immunosuppressive drugs. Successful tolerization depended on high plasma levels of cyclosporin A combined with azathioprine. In addition, the induction of tolerance may require mannose 6-phosphate receptor-mediated uptake because alpha-l-iduronidase and alpha-glucosidase induced tolerance with the drug regimen whereas ovalbumin and dephosphorylated alpha-l-iduronidase did not. This tolerization method should be applicable to the treatment of other lysosomal storage disorders and provides a strategy to consider for other nontoleragenic therapeutic proteins and autoimmune diseases.

Fig. 1.

Immune tolerance induction regimen. The tolerance regimen is diagrammed including CsA plus Aza dosing and rhIDU infusions. On day 0, full-dose CsA plus Aza are initiated (CsA, 25 mg/kg per day divided two times per day; Aza, 5 mg/kg every other day) and are later tapered to one-half the initial dose on day 32 and one-fourth the initial dose on day 46 and terminated on day 60. Low-dose rhIDU (0.056 mg/kg) infusions begin as indicated on day 18 and continue weekly thereafter.

Fig. 2.

Immune response to rhIDU in canines. The ELISA titer (OD units/μlof serum) of anti-rhIDU antibodies are plotted vs. weeks of rhIDU infusions for canines treated with the tolerance regimen (open symbols) and for those treated without the tolerance regimen (filled symbols) as indicated in Table 1. The data show that 8 canines treated with the optimum regimen have low anti-rhIDU titers for up to 6 months whereas 11 canines infused with rhIDU, with inadequate or no tolerance regimen, mounted substantial immune responses. Preimmune serum was drawn at week 0, 18 days after starting the CsA plus Aza drug regimen and before the first infusion was administered. Canine JH was treated with the every-other-day CsA regimen and has gray symbols to distinguish it from the other tolerant canines. The nontolerant canines BI, BC, and BO are included for completeness although they received 5 weeks or less of rhIDU and are not included in Table 1 for this reason.

Fig. 3.

Immune tolerance to full-dose rhIDU in treated MPS I canines. Seven MPS I canines were treated with rhIDU infusions, two with no tolerance regimen (filled symbols), and five with the tolerance regimen (open symbols) for the first 60 days ending at week 7. Through week 12, the five treated MPS I canines had low anti-rhIDU titers compared with the two untreated canines. After this initial tolerization period at week 13, the dose of rhIDU was doubled each week and finally reached 0.58 mg/kg each week (full therapeutic dose) at week 15. The tolerant MPS I canines (open symbols) did not respond to the increased rhIDU dose whereas the control canines had increasing anti-rhIDU titers.

Fig. 4.

Immune response to rhIDU after a hiatus from antigen exposure. Canines PE, RO, NI, and JH (open symbols) were previously tolerized with rhIDU infusions and the tolerance regimen and were then rechallenged with rhIDU at 5 weeks, 6 months, 6 months, and 2.5 yr, respectively, after the last exposure to the enzyme. All but NI showed an absent or muted response to enzyme after a hiatus from exposure. The control canine RU showed a typical anamnestic response after a hiatus from enzyme exposure, rising from a titer of <25 to >300 after a single dose. The data show that the tolerant state is retained for weeks to months without continuous antigen exposure.

Fig. 5.

Immune responses to other antigens. (a) Immune response to ovalbumin. Canines SM (open triangles) and TR (open diamonds) were treated with the effective tolerance regimen by using ovalbumin as the toleragen; canine VS (filled squares) was the control. A strong immune response is induced in all canines regardless of the drug regimen. Canine NI (filled circles) received ovalbumin as a second antigen without the tolerance regimen and showed a similar degree of immune response. The data show that ovalbumin is not a tolerizing antigen with the regimen, unlike rhIDU. (b) Immune responses to α-glucosidase. Canine SC (open circles) was treated with the CsA plus Aza regimen by using α-glucosidase as the toleragen and showed no significant immune response (<1 OD/μl). Canine ST (filled circles) was given the same enzyme without the regimen and showed a >100 OD/μl response to the enzyme. The data suggest that α-glucosidase is a tolerizing antigen with the regimen although the data are limited to one canine.

Fig. 6.

Dephosphorylated rhIDU is ineffective as toleragen. Dephosphorylated rhIDU was administered to canine WI and XO by weekly infusions at a dose of 0.056 mg/kg per wk with the CsA plus Aza tolerance regimen. A low but significant titer was observed in both canines. After week 12, the canines were then challenged weekly with either a full dose of 0.58 mg/kg rhIDU immediately (WO) or a ramped dose of rhIDU (XO), and both showed a strong immune response, reaching a titer of >100 OD/μl. The data suggest that dephosphorylated rhIDU is not tolerizing to the same degree as native rhIDU, consistent with a role for the mannose 6-phosphate receptor in the tolerizing uptake of the antigen.