Alexandra M. Joseph

Immune tolerance induction to enzyme-replacement therapy by co-administration of short-term, low-dose methotrexate in a murine Pompe disease model.

Clinical investigations of recombinant human acid α‐glucosidase for the treatment of Pompe disease often reveal the appearance of therapy‐specific antibodies. These antibodies could potentially interfere with recombinant human acid α‐glucosidase efficacy and induce immunological consequences. Several immunosuppressive agents, including methotrexate, mycophenolate mofetil and cyclosporin A with azathioprine, were evaluated for their potential to induce immune tolerance to recombinant human acid α‐glucosidase. Methotrexate was the only agent that reduced recombinant human acid α‐glucosidase‐specific antibody responses in acid α‐glucosidase knock‐out mice. A 3‐week, low‐dose methotrexate regimen controlled recombinant human acid α‐glucosidase‐specific antibody levels throughout 8 months of weekly recombinant human acid α‐glucosidase treatment. The success of this methotrexate regimen appears to require methotrexate administration within the first 24 h of recombinant human acid α‐glucosidase treatment. In an attempt to understand the benefit of methotrexate within the first day of recombinant human acid α‐glucosidase administration, the immune response 24 h following intravenous recombinant human acid α‐glucosidase treatment was investigated. A consistent expansion of peritoneal B1 B cells was observed. Control over this B1 B cell response may be part of the complex mechanism of action of methotrexate‐induced immune tolerance.

Transient Low-Dose Methotrexate Generates B Regulatory Cells That Mediate Antigen-Specific Tolerance to Alglucosidase Alfa

Biologic drugs, including enzyme-replacement therapies, can elicit anti-drug Abs (ADA) that may interfere with drug efficacy and impact patient safety. In an effort to control ADA, we focused on identifying regimens of immune tolerance induction that may be readily available for clinical use. Data generated in both wild-type mice and a Pompe disease mouse model demonstrate that single-cycle, low-dose methotrexate can be as effective as three cycles of methotrexate in providing a long-lived reduction in alglucosidase alfa-specific ADA. In addition, we show that methotrexate induces Ag-specific tolerance as mice generate similar Ab responses to an irrelevant Ag regardless of prior methotrexate treatment. Methotrexate-induced immune tolerance does not seem to involve cell depletion, but rather a specific expansion of IL-10– and TGF-β–secreting B cells that express Foxp3, suggesting an induction of regulatory B cells. The mechanism of immune tolerance induction appears to be IL-10 dependent, as methotrexate does not induce immune tolerance in IL-10 knockout mice. Splenic B cells from animals that have been tolerized to alglucosidase alfa with methotrexate can transfer tolerance to naive hosts. We hypothesize that methotrexate induction treatment concomitant with initial exposure to the biotherapeutic can induce Ag-specific immune tolerance in mice through a mechanism that appears to involve the induction of regulatory B cells.

Transient Low-Dose Methotrexate Induces Tolerance to Murine Anti-Thymocyte Globulin and Together They Promote Long-Term Allograft Survival

Rabbit anti-thymocyte globulin (Thymoglobulin) effectively treats transplant rejection but induces anti-rabbit Ab responses, which limits routine readministration. Aiming to tolerize anti-rabbit responses, we coadministered a brief methotrexate regimen with a murine version of Thymoglobulin (mATG) for effects on anti-mATG Abs and cardiac allotransplantation in mice. Although both single and three courses of methotrexate could significantly inhibit anti-drug Ab titers to repeated mATG treatment, surprisingly, the single course given at the first mATG administration was most effective (>99% reduction). The transient methotrexate treatment also significantly improved pharmacokinetics and pharmacodynamics of repeated mATG administration. In the cardiac allograft model, the combination of transient mATG and methotrexate given only at the time of transplant dramatically improved allograft survival (>100 d) over either agent alone (<30 d). Anti-drug Ab titers were reduced and mATG exposure was increased which resulted in prolonged rather than enhanced mATG-mediated effects when combined with methotrexate. Moreover, methotrexate administration significantly reduced alloantibodies, suggesting that methotrexate not only decreases anti-drug Ab responses but also reduces Ab responses to multiple tissue-derived alloantigens simultaneously. These data suggest that mATG and methotrexate together can provide long-term allograft survival potentially through the induction of immune tolerance.

Approaches to Mitigate the Unwanted Immunogenicity of Therapeutic Proteins during Drug Development

ABSTRACTAll biotherapeutics have the potential to induce an immune response. This immunological response is complex and, in addition to antibody formation, involves T cell activation and innate immune responses that could contribute to adverse effects. Integrated immunogenicity data analysis is crucial to understanding the possible clinical consequences of anti-drug antibody (ADA) responses. Because patient- and product-related factors can influence the immunogenicity of a therapeutic protein, a risk-based approach is recommended and followed by most drug developers to provide insight over the potential harm of unwanted ADA responses. This paper examines mitigation strategies currently implemented and novel under investigation approaches used by drug developers. The review describes immunomodulatory regimens used in the clinic to mitigate deleterious ADA responses to replacement therapies for deficiency syndromes, such as hemophilia A and B, and high risk classical infantile Pompe patients (e.g., cyclophosphamide, methotrexate, rituximab); novel in silico and in vitro prediction tools used to select candidates based on their immunogenicity potential (e.g., anti-CD52 antibody primary sequence and IFN beta-1a formulation); in vitro generation of tolerogenic antigen-presenting cells (APCs) to reduce ADA responses to factor VIII and IX in murine models of hemophilia; and selection of novel delivery systems to reduce in vivo ADA responses to highly immunogenic biotherapeutics (e.g., asparaginase). We conclude that mitigation strategies should be considered early in development for biotherapeutics based on our knowledge of existing clinical data for biotherapeutics and the immune response involved in the generation of these ADAs.

An immune tolerance approach using transient low-dose methotrexate in the ERT-naïve setting of patients treated with a therapeutic protein: experience in infantile-onset Pompe disease.

PurposeTo investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients.MethodsNewly diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and <51,200 within 12 months), or low (LT; ≤6400 within 12 months), were compared with those of 37 CRIM-positive IOPD historic comparators receiving rhGAA alone.ResultsFourteen IOPD TLD-MTX recipients at the median age of 3.8 months (range, 0.7–13.5 months) had a median last titer of 150 (range, 0–51,200) at median rhGAA duration ~83 weeks (range, 36–122 weeks). One IOPD patient (7.1%) developed titers in the SIT range and one patient (7.1%) developed titers in the HSAT range. Twelve of the 14 patients (85.7%) that received TLD-MTX remained LT, versus 5/37 HSAT (peak 51,200–409,600), 7/37 SIT (12,800–51,000), and 23/37 LT (200–12,800) among comparators.ConclusionResults of TLD-MTX coinitiated with rhGAA are encouraging and merit a larger longitudinal study.