Service Project #10: Nanoimmunotherapy for Chronic Immune-Mediated Diseases

Collaborating Investigator: David Horwitz
Affiliation: General Nanotherapeutics, LLC
Funding Status: NIH  R41AI170413   
Project Period: 07/14/22-06/30/23

Summary

The goal of SP #10 is to design nanoparticles that engage and expand both CD4+ and CD8+ regulatory T cells (Tregs) in vivo as a novel treatment for the autoimmune disease systemic lupus erythematosus (SLE). The Horwitz Lab has previously formulated nanoparticles loaded with interleukin-2 (IL-2) and transforming growth factor (TGF)-b that induce both CD4+ and CD8+ Treg proliferation to mitigate SLE. An engineered interleukin-2 (IL-2)-based Treg-biasing antibody/cytokine fusion protein (immunocytokine, IC) designed in TR&D 3 will be substituted for IL-2 in these nanoparticles to determine whether this biased approach leads to favorable therapeutic outcomes in humanized mouse models of SLE. For SP #10, the NCBIB will push our engineered Treg–potentiating immunocytokine denoted F5111 IC (TR&D 3) to General Nanotherapeutics, LLC so that they may incorporate our technology into their therapeutic nanoparticles and establish that these nanoparticles lead to superior disease control in animal models of SLE. 

Approach

Aim 1. Production and functional validation of F5111 IC-loaded nanoparticles. F5111 IC will be produced recombinantly by the NCBIB using a mammalian cell expression system and purified by protein G chromatography followed by size-exclusion chromatography using a fast liquid protein chromatography (FPLC) machine. The purified F5111 IC will be functionally validated through biolayer interferometry-based binding titrations against IL-2 and its cognate receptor subunits using an Octet® machine, as well as through IL-2 signaling studies on commercial human peripheral blood mononuclear cells. The validated protein will then be loaded into polylactic co-glycolytic acid (PLGA)-based nanoparticles coated with anti-CD3 antibodies by General Nanotherapeutics, LLC.

Specific Aim 2. Demonstrate that F5111 IC-loaded nanoparticles activate Tregs in vitro and stimulate Treg expansion in vivo. General Nanotherapeutics, LLC will evaluate in vitro proliferation of CD4+ and CD8+ Tregs within a mixed population (human peripheral blood mononuclear cells) in response to F5111 IC-loaded PLGA-based nanoparticles from Aim 1. These nanoparticles will also be administered in the murine lupus nephritis model to demonstrate the therapeutic potential for these engineered nanoparticles.

General Nanotherapeutics, LLC recently described a new approach to rebalance immune homeostasis in autoimmunity that engages both CD4+ and CD8+ Tregs using T cell-targeting antibody-coated  PLGA nanoparticles loaded with IL-2 and TGF-b. Building on these promising preliminary data, we will work with General Nanotherapeutics, LLC to upgrade their nanoparticle technology through integration with a Treg-biased IC from TR&D 3. General Nanotherapeutics, LLC found that TGF-b delivery is not required for therapeutic efficacy of their nanoparticles; thus, they will only encapsulate an IL-2-based molecule. To specifically direct the effects of their nanoparticles towards Tregs, the natural IL-2 cytokine, which activates both immune effector cells (i.e., CD4+ and CD8+ effector T cells and natural killer [NK] cells) will be replaced with the Treg-biased F5111 IC generated in TR&D 3 (produced and characterized by the NCBIB). The resulting nanoparticles will be evaluated in human peripheral blood mononuclear cells and subsequently in mouse models of SLE. We hypothesize that use of our engineered IL-2 IC will lead to more effective prevention and treatment of disease, establishing the utility of our novel immunocytokine technology in this prevalent autoimmune disorder.