Neoantigen mRNA vaccine designTCRshows

String several neoantigen peptides into a single open reading frame, pick a linker, add a signal peptide and MITD — and then do the step this kind of design most often skips: enumerate every 8–11mer created across the junctions, and reorder the peptides so that as few of those as possible carry an HLA anchor motif.

1Neoantigen peptides

One per line, optionally with a >name FASTA header. Personalised vaccines usually use 25–27 aa long peptides with the mutation centred; 8–11 aa short epitopes work too.

Example:

2Linker and terminal elements

The linker is the first gate on junctional epitopes: in a flexible GS linker, G and S are poor anchor residues for most HLA supertypes, which by itself makes junction peptides unlikely to bind well.

Anchor-motif screening is a heuristic, not a binding predictor. It only looks at which residues the common HLA supertypes prefer at the two main anchor positions, P2 and PΩ, and uses that to say whether a junction peptide is worth a closer look. For quantitative binding affinity use a predictor such as netMHCpan; for the self-proteome comparison use this site’s off-target epitope scan — the buttons above send a junction peptide straight there.
Why junctions deserve their own step. In a string-of-beads construct, every pair of adjacent peptides creates sequence that is present in none of the inputs. Two kinds of risk follow. One is that a junction peptide becomes an immunodominant decoy, pulling the response away from the real neoantigens. The other is that it happens to match a self protein, which is an autoimmunity risk. The first is suppressed by the choice of linker and the peptide order; the second can only be settled by a proteome comparison. All results are for research use only.

Sources: the MITD and the MHC-I signal peptide are taken from the feature annotations of UniProtKB P04439 (HLA-A α chain) — signal peptide 1–24, transmembrane 309–332, cytoplasmic 333–365. The anchor-position motifs are the accepted preferences of the common HLA supertypes and are meant for qualitative screening only.