An engineered CRM197 variant with enhanced stability and efficacy.

Cross-Reacting Material 197 (CRM197), a non-toxic mutant of diphtheria toxin, is structurally similar and immunologically cross-reactive with the native toxin. It is extensively utilized as a carrier protein in conjugate vaccines to enhance T-cell-dependent immunity and also displays antitumor properties via interaction with heparin-binding epidermal growth factor (HB-EGF). Nevertheless, the structural instability of CRM197 restricts its broader use, causing accelerated in vivo degradation, diminished immunogenicity, and challenges in recombinant expression and purification. Here, we engineered a CRM197 mutant with enhanced stability and superior immunogenicity. As a diphtheria immunogen or conjugate carrier, it elicits rapid, potent, and sustained immunity surpassing the wild-type, and maintains effective HB-EGF-binding activity for tumor growth inhibition. Structural insights reveal a rigidified activation site loop (residues 38-52), a stabilized receptor domain through additional hydrogen bonds at site 511, and strengthened adjuvant adsorption from an increased negative charge as the underlying mechanisms.