(530b) Design of an IL-2 Based Immunocytokine for the Targeted Expansion of Muscle Regulatory T Cells

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by loss-of-function mutations in the dystrophin gene that promote muscle degeneration and weakness¹. Recombinant adeno-associated viral (rAAV) microdystrophin gene therapy has demonstrated significant promise in treating DMD.² However, this therapy induces rAAV- and dystrophin-specific humoral and cell-mediated immunity that prevent repeat dosing and thus limit its therapeutic potential.^2–5 This creates a considerable need to improve the safety and tolerability of rAAV gene therapy for targeted treatment in patients with DMD. The naturally occurring cytokine interleukin-2 (IL-2) has shown tremendous potential in treating autoimmune conditions through promotion of immunosuppressive activities. IL-2 is a pleiotropic cytokine that orchestrates the differentiation, proliferation, survival, and activity of immune cells by signaling through a high-affinity heterotrimeric receptor (IL-2 receptor-α [IL-2Rα], IL-2Rβ, and γ_c chains) or an intermediate-affinity heterodimeric receptor (IL-2Rβ and γ_c chains) (Fig. 1). Since IL-2Ra is highly expressed on regulatory T cells (Tregs), but virtually absent from naïve immune effector cells, low-dose IL-2 administration preferentially stimulates polyclonal expansion of Tregs over immune effector cells to elicit an immunosuppressive response. This effect has been leveraged as a therapeutic strategy to treat autoimmune disorders.^6,7 However, despite the cytokine’s proclivity towards Tregs, IL-2 can still activate immune effector cells. Co-administration of IL-2 with the anti-cytokine antibody F5111 selectively biases IL-2 activity towards Tregs, presenting an enticing opportunity for targeted cytokine therapy.⁸ Our lab engineered a single-agent human IL-2/F5111 fusion protein (termed F5111 immunocytokine (IC)), which selectively expands Tregs and protects against autoimmune diseases in mice.⁹ Previous work by Villalta and colleagues showed that Tregs are capable of suppressing antigen-specific effector T cells in a mouse model of DMD.¹⁰ Thus, we hypothesize that F5111 IC-induced expansion of Tregs will suppress the undesired immunostimulatory activities of rAAV- and dystrophin-specific immunity, thereby enhancing the performance of gene therapy strategies (Fig. 2). We engineered an innovative bispecific biologic, consisting of a human alpha-dystroglycan (α-hDAG1) monoclonal antibody fused to a single chain variable fragment format of F5111 IC (denoted mini F5111 IC), to achieve muscle specific delivery of IL-2 and Treg expansion.¹¹These novel fusion proteins will prevent undesired immune activity and enhance tolerance to gene therapy by targeting the immunosuppressive effects of F5111 IC to muscle tissue, presenting an enticing opportunity to drastically improve patient outcomes in DMD.

The α-hDAG1 mAb-miniF5111 IC fusion proteins were designed to fuse mini F5111 IC at either the C-terminus of the heavy chain of the α-hDGC mAb (denoted DAG-mini-HC) or at the C-terminus of the light chain (denoted DAG-mini-LC). DNA encoding the IC fusion protein was cloned by PCR and then transfected into Expi293 human embryonic kidney cells and purified via protein G chromatography followed by size-exclusion chromatography. (Fig. 3). Bio-layer interferometry studies were performed to measure the binding affinity of the α-hDAG1-mini-LC fusion protein against immobilized human dystroglycan. Preliminary studies showed that DAG-mini-LC retained binding to human dystroglycan with comparable affinity compared to the α-hDAG1 parent antibody (Fig. 4). IL-2 receptor binding studies, cell signaling assays, and in vivo immune characterization and therapeutic experiments are ongoing. Overall, we have engineered fusion proteins comprised of the α-hDAG1 antibody and mini F5111 IC that will induce targeted Treg stimulation in muscle cells to revolutionize the treatment of DMD.

References:

1 Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy. Nat Rev Dis Primers 2021;7:13. https://doi.org/10.1038/s41572-021-00248-3.

2 Mendell JR, Sahenk Z, Bowles D, Galloway G, Clark KR, McPhee SW, et al. Dystrophin Immunity in Duchenne’s Muscular Dystrophy. The New England Journal of Medicine 2010.

3 Mueller C, Chulay JD, Trapnell BC, Humphries M, Carey B, Sandhaus RA, et al. Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression. J Clin Invest 2013;123:5310–8. https://doi.org/10.1172/JCI70314.

4 Nance ME, Duan D. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy. Human Gene Therapy 2015;26:786–800. https://doi.org/10.1089/hum.2015.107.

5 Leborgne C, Latournerie V, Boutin S, Desgue D, Quéré A, Pignot E, et al. Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients. Cellular Immunology 2019;342:103780. https://doi.org/10.1016/j.cellimm.2018.03.004.

6 Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 2012;12:180–90. https://doi.org/10.1038/nri3156.

7 Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 2013;38:13–25. https://doi.org/10.1016/j.immuni.2013.01.004.

8 Spangler JB, Tomala J, Luca VC, Jude KM, Dong S, Ring AM, et al. Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity 2015;42:815–25. https://doi.org/10.1016/j.immuni.2015.04.015.

9 VanDyke D, Iglesias M, Tomala J, Young A, Smith J, Perry JA, et al. Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection. Cell Reports 2022;41:111478. https://doi.org/10.1016/j.celrep.2022.111478.

10 Villalta SA, Rosenthal W, Martinez L, Kaur A, Sparwasser T, Tidball JG, et al. Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy. Sci Transl Med 2014;6:258ra142. https://doi.org/10.1126/scitranslmed.3009925.

11 Humphrey EL, Lacey E, Le LT, Feng L, Sciandra F, Morris CR, et al. A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients. Neuromuscular Disorders 2015;25:32–42. https://doi.org/10.1016/j.nmd.2014.09.005.