Use of D-Amino acids and chemical modificationsto minimize imMUNOlogical anti-drug antibody response to therapeutic peptide candidates – DAMUNO

The DAMUNO project aims to transform four animal venom-derived peptides into therapeutic candidates by minimizing their intrinsic immunogenicity, a significant hurdle for their clinical development. We have selected four molecules with distinct canonical folds—Kunitz, three-finger, and knottin—which are drug candidates for treating pain, renal diseases, cardiovascular diseases, and ischemia. Given that the sequence of these animal peptides cannot be humanized, we propose three innovative chemical strategies to mitigate their immunogenicity.
Firstly, we plan to incorporate D-amino acids into the peptides. Our recent research demonstrated that introducing a single D-amino acid (D-AA) into a peptide markedly reduces its affinity for HLA-DR molecules and its ability to prime specific T cells. D-AAs disrupt the extended conformation of peptides bound to HLA-DR molecules, offering a promising solution to overcome HLA polymorphism. We have already shown that for one therapeutic peptide candidate, D-AA reduces its capacity to prime a T cell response in humans. However, D-AA introduction imposes structural constraints on peptides, which can disrupt their structure and biological functions. We will use molecular modeling to identify permissive positions for D-AA introduction and select the D-AA variants to synthesize.
The second strategy is based on the formation of selenocysteines and ether bridges to create stable disulfide bond surrogates. It has been shown that the reduction of disulfide bonds is necessary for the degradation of three-finger fold snake peptides by proteases and their presentation to T cells. We anticipate that preventing disulfide bond reduction will block the release of T cell epitopes from the structure and their presentation to T cells.
The third strategy aims to prevent the processing of the peptides in antigen-presenting cells by inhibiting their unfolding. This involves introducing molecular staples to tether side chains of amino acids from different regions of the peptide, thereby stabilizing its structure. This internal cross-linking will be performed using a click chemistry strategy.
All chemically modified variants will be synthesized and characterized. The preservation of their pharmacological properties will be assessed in vitro, as well as their ability to stimulate human T cells. This innovative project, based on the chemical modification of animal peptides to control undesired immunogenicity, should provide a generic approach to de-immunization by identifying permissive modifications in canonical folds and offering non-immunogenic therapeutic peptide variants. This approach could be extended to other synthetic peptide candidates.