Poster N4 - Immunomodulatory Potential of Glatiramoid Peptide Nanofibers

Introduction: Glatiramoids are non-biologic complex drugs composed of random co-polymer mixtures of amino acids alanine (A), lysine (K), glutamic acid (E), and tyrosine (Y). One such glatiramoid is glatiramer acetate (GA) an active ingredient in Copaxone® approved for the treatment of relapsing Multiple Sclerosis (MS). GA treatment in MS patients is associated with increased levels of neuroprotective factors and the induction of anti-inflammatory Th2 cytokines and Tregs. GA also effectively reverses molecular neuropathology and behavioral phenotypes in cell culture and mouse models of Parkinson Disease (PD) as well as Huntington’s Disease and Alzheimer’s Disease (AD). Prior studies have reported that conjugation of randomized K-E-Y-A repeats to a self-assembling peptide domain result in a fibrillar form of GA, which acts as an adjuvant, inducing antibody and T cell responses with an anti-inflammatory Th2 bias. Glatiramoid quality is intrinsically linked to its manufacturing process, as evidenced by comparative studies between Copaxone® and generic GA products. Here we report the development of glatiramoid nanofibers (GNFs) with a GNF core composed of the self-assembling peptide KFE8 (FKFEFKFE) and functionalized on the N-terminus with (KEYA)20. Our data indicated that KEYA20-KFE8 significantly reduced LPS-induced inflammation in THP-1 cells compared to (KEYA)20 or Copaxone®. Our findings suggest that fibrillar forms of KEYA more effectively suppress immune cell activation, with the potential to enhance immunogenicity of selected peptide epitopes with a Th2-bias.

Materials and

Methods: Peptides were synthesized using standard Fmoc chemistry, with the molar ratios of each amino acid in the mixture were 6.1(A):4.7(K):1.9(E):1.0(Y) similar in composition to Copaxone®. This mixture was used in 20 sequential couplings to generate a random KEYA20 epitope on the N-terminus of KFE8 resulting in (KEYA)20-KFE8. The crude peptide was washed twice with HCl to remove residual trifluoroacetic acid (TFA) from cleavage. Fibril morphology was imaged using transmission electron microscopy (TEM). THP-1 cells were incubated with LPS (activated) or media (control) as well as Copaxone, KEYA, KFE8-KEYA for 24 hours, prior to subsequent incubation with IFNy (activated) or media (control) for 48 hours. Cell soups were collected for flow cytometry to determine HLA-DR expression by THP1 cells, quantified by the mean fluorescence intensity (MFI). Cytokines secreted in the supernatant of dendritic cells (DCs) treated with Copaxone were quantified by Isoplexis innate immune chip.

Results, Conclusions, and Discussions: TEM showed successful formation of fibrillar structures in both synthesized peptides thus KFE8-(KEYA)20 and (KEYA)20, although the GNF exhibited more consistent networks of fibrils compared to unconjugated (KEYA)20, confirming that nanofiber self-assembly remains intact for the GNF. DCs treated with Copaxone secreted Th2 cytokines, including elevated IL-13, and IL-5 levels, consistent with previous literature. Treatment of activated THP-1 cells with KFE8-(KEYA)20 suppressed HLA-DR upregulation compared to both no treatment and Copaxone, indicating an enhanced anti-inflammatory effect in the conjugated peptide. The anti-inflammatory effect could be beneficial in combating many diseases resulting from an imbalance in the immune system. Future studies are required to explore the DC cytokine response to KFE8-(KEYA)20 treatment to confirm Th2 biasing. Another prospective experiment could involve systematically polymerizing the GNF peptide to elucidate how ordered sequences influence immunomodulation.

Acknowledgements (Optional): This work was supported with funds from ROI AI168918-01A1 (Jai Rudra) and PJ000027470 (Cory Berkland).
Many thanks to the members of the Rudra and Berkland labs at Washington University in St. Louis for their technical assistance.