Building on prior work tuning CAR activation [1], this design adds a FUS-like insert to encourage clustering while preserving ITAM1. An 11-residue insert adds tyrosine “stickers” for aromatic interactions [2] and an arginine for charge–aromatic interactions. Glycine and serine spacers are intended to maintain flexibility and limit hydrophobic collapse, leaving the surrounding sequence unchanged.

This design replaces the inter-ITAM spacers with two shorter TRAF2-binding segments to add costimulation while retaining all three ITAMs. One segment uses a consensus motif and the other a CD40-derived motif [1]; the aim is to recruit multiple TRAF complexes that act cooperatively. Basic flanking residues are intended to preserve charge character, while glycine provides flexibility near the ITAMs. Trimming the tail further reduces length.

This design retains a single ITAM1 for the activation input through ZAP-70 [1] [2], leaving room to replace the downstream region with a different costimulatory module. A CD40-derived fragment offers a route to TRAF6 rather than the TRAF2-associated route of 4-1BB, with the aim of engaging NF-κB and JNK/MAPK signaling. A short basic linker and acidic tail complete the compact design. The design aims to combine these two inputs to support survival and cytokine production and alter exhaustion.

This design places a short 4-1BB-derived TRAF-binding segment between the two tyrosine-containing halves of ITAM1. The aim is to use the same stretch for activation and costimulation, with an eight-residue spacer matching the spacing found in ITAMs [1]. A separate, intact ITAM3 provides a conventional activation motif, while most of the remaining 4-1BB and CD3ζ sequence is removed.

Structural studies of OX40–TRAF2 binding [1] and mutational studies of RANK–TRAF6 signaling [2] motivated this combination. The design pairs ITAM1 with short OX40- and RANK-derived fragments intended to recruit TRAF2 and TRAF6, respectively. Short connecting residues separate the motifs. The aim is to combine two costimulatory inputs in a compact tail, replacing ITAM2, ITAM3 and their intervening linkers.