Incorporation of a repeated polypeptide sequence in therapeutic antibodies as a universal masking procedure: A case study of T cell-engaging bispecific antibodies

N Biotechnol. 2023 Nov 25:77:80-89. doi: 10.1016/j.nbt.2023.07.004. Epub 2023 Jul 17.


Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies.

Keywords: Bispecific antibody; CD3; Epidermal growth factor receptor; Masking sequence; PAS; Prodrug.

MeSH terms

  • Antibodies, Bispecific*
  • Humans
  • Neoplasms* / drug therapy
  • Prodrugs* / therapeutic use
  • Single-Chain Antibodies*
  • T-Lymphocytes
  • Tumor Microenvironment


  • Antibodies, Bispecific
  • Prodrugs
  • Single-Chain Antibodies