Poster CC11 - Achieving Recruitment and Activation of T cells Near Amyloid Beta Plaques Using Immunocytokines With Enhanced Affinity for the Effector T cell Interleukin-2 Receptor Complex

Introduction: As state-of-the-art medical practices become more effective at extending average life expectancy, the prevalence of age-related pathologies becomes increasingly more pronounced. Many illnesses seen at a higher frequency in geriatric populations result from the dysregulation of cellular processes carried out by post-mitotic cells; one such cell type is the neuron. Over time, amyloid precursor proteins that are securely anchored to the cell membranes of healthy neurons, may be detached by intracellular secretase proteins. Once solubilized, these peptides can form large oligomeric structures, termed amyloid-ß (Aß) plaques. Through mechanisms that have yet to be completely understood, accumulation of these large extracellular peptide tangles contribute to the symptoms observed in Alzheimer’s Disease (AD).
Recently, capitalizing on immune cell populations naturally present in the brain to treat AD has been of increasing interest, due to the inherent proteolytic and phagocytotic properties of these cells. Our team previously engineered a de novo computationally designed mimetic of the interleukin-2 (IL-2) cytokine, denoted Neo-2. Neo-2 shows improved stability and T cell affinity compared to the native human protein, making the peptide an attractive target for T cell recruitment in the brain. Additionally, a split, conditionally active version of Neo-2 was designed, such that colocalization of two fragments is required to successfully signal. Ultimately, we aim to achieve specific recruitment to and activation of T cells near Aß plaques in the brain by formulating immunocytokines consisting of an antibody single-chain variable fragment (scFv) specific to oligomerized Aß fused to each of the two fragments of split Neo2.

Materials and

Methods: Four immunocytokine plasmid constructs were designed, comprising each of the split Neo-2 fragments linked to Aß-targeted scFvs. Two different scFvs validated to recognize different binding epitopes on Aß oligomers, Bapineuzumab and Gantenerumab, were used in the fusion proteins and connected to the N-terminus of each Neo-2 fragment (denoted Neo2A and Neo2B). The developed immunocytokines were denoted Bapineuzumab-Neo2A (Bap-Neo2A), Bapineuzumab-Neo2B (Bap-Neo2B), Gantenerumab-Neo2A (Gant-Neo2A), and Gantenerumab-Neo2B (Gant-Neo2B). Genes encoding these constructs were cloned into a mammalian expression vector, which was then transfected into Expi293F human embryonic kidney cells. Secreted proteins were purified by affinity chromatography followed by size-exclusion chromatography on a fast protein liquid chromatography (FPLC) instrument.
Proteins are currently being characterized to ensure integrity of their binding and signaling functions in the immunocytokine format. The specificity and binding affinity of fusion proteins for human interleukin-2 receptor subunits ß and γ (IL-2Rß and IL-2Rγ), independently and as a heterodimer, is being measured by biolayer interferometry. In addition, the specificity and binding affinity of our fusion proteins for Aß oligomers is being measured via ELISA. We are also interrogating signaling activity by Neo-2 splits on IL-2-responsive YT-1 human natural killer cells, using phosphorylation of signal transducer and activator of transcription factor 5 (STAT5) as a readout for IL-2 stimulation. In forthcoming work, our fusion proteins will be conjugated to lipid delivery vehicles, and localization of our fusion proteins to Aß plaques will be measured in mouse brains. We will also interrogate T cell infiltration to determine the efficacy of our therapeutic.

Results, Conclusions, and Discussions: We have successfully designed plasmids encoding all 4 immunocytokines and demonstrated their stable expression of in a mammalian cell expression system. Size-exclusion chromatography successfully removed all aggregates, as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed high purity for all fusion proteins. Binding and signaling studies are underway to characterize the functional properties of the four Aß-targeted split IL-2 fusion proteins.
After completing these preliminary validation experiments, we have designed in vivo delivery studies to ask the following questions: (1) Can we successfully deliver our immunocytokines to mouse brains? (2) Can both Neo-2 fragments colocalize to Aß plaques to reconstitute IL-2 signaling at the site of disease? and (3) Does localization to Aß plaques and activation of IL-2 signaling lead to recruitment and stimulation of T cells in the brain? Ultimately, by demonstrating that these engineered proteins possess these functions, new artificial immunocytokines may be developed from native human peptides to target other immune cell populations in the brain, including microglia and perivascular macrophages. The key proof of concept for targeted and conditionally active immune modulation in the brain presents an exciting opportunity for treatment of AD and a host of other neurological conditions.

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