<p>Administration of human therapeutic proteins such as monoclonal antibodies (mAb) to animals during preclinical drug development often leads to the development of anti-drug antibodies (ADA). ADA may reduce the systemic exposure of the mAb by enhancing its immune-complex mediated clearance. Thus, ADA may hinder the preclinical pharmacokinetic and toxicology assessments of mAbs. To mitigate this effect, we explored the ability of short-term administration of immunosuppressants to induce prolonged immune tolerance towards a human mAb, erenumab, in rats. In two studies, we investigated dosing regimens using the immunosuppressants methotrexate and tacrolimus/sirolimus combination, and compared them to non-immunosuppressed control groups. Each study comprised three phases: induction (weeks 1–4), washout (weeks 5–8), and rechallenge (weeks 9–12). Animals received mAb during the induction and rechallenge phase, while immunosuppression was limited to the induction and washout phase. Blood samples were collected at predefined time-points for erenumab and ADA quantification. The tacrolimus/sirolimus regimen, but not the tested methotrexate regimens, completely prevented ADA formation in all treated animals relative to the control groups. The tacrolimus/sirolimus treated animals not only remained ADA-negative with initial immunosuppression during the induction phase but remained ADA-negative even after erenumab rechallenge suggesting the induction of immune-tolerance beyond the immunosuppressive treatment period. Correspondingly, erenumab systemic exposures were maintained throughout the study period in all animals in the tacrolimus/sirolimus group and were similar to the erenumab exposures in ADA-negative animals of the control group. In contrast, ADA-positive animals in the control group exhibited a 60–80% reduction in erenumab exposures.</p> Graphical Abstract <p></p>