Combination of ImmTOR Tolerogenic Nanoparticles and IL-2 Mutein Induces Massive Expansion of Antigen-specific Regulatory T cells

We have previously demonstrated that ImmTOR nanoparticles encapsulating rapamycin induce selective immune tolerance to co-administered antigens.  Unlike Treg-selective IL-2 muteins, ImmTOR does not expand total Tregs; however, we hypothesized that ImmTOR could act synergistically with IL-2 muteins.  Here we demonstrate that a single dose of ImmTOR co-administered with IL-2 mutein resulted in increased and more durable total Tregs.  We next evaluated the expansion of antigen-specific Treg in mice with adoptively-transferred ovalbumin-specific OTII T cells.  As expected, ImmTOR + ovalbumin did not expand total Treg, but increased the percentage of Foxp3+ OTII cells from ~3% to ~15%.  IL-2 mutein + ovalbumin resulted in a modest increase that was similar to that observed with ovalbumin alone.  However, the combination of ImmTOR + IL-2 mutein + ovalbumin showed a profound synergistic effect, with ~45% of OTII cells expressing Foxp3.  We tested whether ImmTOR and IL-2 mutein would enable more durable inhibition of antibody responses to co-administered AAV gene therapy vectors.  Mice treated with AAV8 vector on Days 0 and 56 developed a robust anti-AAV IgG antibody response.  Treatment with IL-2 mutein showed only a modest reduction in anti-AAV IgG antibodies.  ImmTOR showed dose-dependent inhibition of anti-AAV antibodies; however some mice showed delayed development of antibodies.  In contrast, the combination of ImmTOR + IL-2 mutein completely inhibited antibody formation through Day 117, even at sub-optimal doses of ImmTOR.  These results show that the combination of ImmTOR and IL-2 mutein has the potential to provide more durable antigen-specific immune tolerance to mitigate unwanted immune responses.