Effects of Conformational Changes in Peptide–CRM₁₉₇ Conjugate Vaccines

Abstract

Conjugate vaccines prepared with the cross-reactive material 197 (CRM197) carrier protein have been successful in the clinic and are of great interest in the field of immunotherapy. One route to preparing peptide–CRM197 conjugate vaccines involves an activation–conjugation strategy, effectively coupling lysine residues on the protein to cysteine thiolate groups on the peptide of interest using a heterobifunctional linker as an activation agent. This method has been found to result in two distinct populations of conjugates, believed to be the result of a conformational change of CRM197 during preparation. This report explores the factors that lead to this conformational change, pointing to a model in which the unintentional alkylation of histidine-21 by the activating agent promotes the “opening” of the monomeric protein. This exposes a new set of lysine residues that are modified by additional activation agents. Subsequent peptide ligation to these sites results in the two conformers. This is the first time that a specific chemical modification is demonstrated to induce a defined conformational change for this carrier protein. Importantly, alternative conditions and reagents have been found to minimize this effect, improving the conformational homogeneity of peptide–CRM197 conjugates.

Survival Impact of Anti-GD2 Antibody Response in a Phase II Ganglioside Vaccine Trial Among Patients With High-Risk Neuroblastoma With Prior Disease Progression

“…6 However, adjuvants for cancer vaccines currently in use or in development are all parenteral 7,8 and do not fulfill all the benchmarks for efficacy: high seroconversion rates, robust and rapid immunoglobulin G (IgG) response, impact on disease outcome, and sustained immunological memory. 9 To boost helper T cells, vaccines can be conjugated to highly immunogenic protein scaffolds like keyhole limpet hemocyanin (KLH) 10 or the nontoxic diphtheria toxin CRM197 11 and then combined with subcutaneous (sc) immune adjuvants like Quillaja saponaria (QS)-21. 12,13 Despite conjugating GD2 to KLH, no anti-GD2 response was induced using adjuvant monophosphoryl lipid A.…”

Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents

“…The typical strategies to afford fluorescent VLPs for cell and animal imaging have involved bioconjugation of synthetic dyes, which can be expensive, will occupy valuable reactive residues on protein surfaces, and can alter their structure and antigenicity. 29 Non-covalent methods of dye conjugation are sparse, though several elegant method using genetically encoded protein-peptide interactions has produced a modular method to encapsulate green fluorescent protein (GFP) within the hollow capsid of viral nanoparticles. [30][31] Here, we utilize a supramolecular strategy to capture a proteinaceous far-red fluorescent probe for the production of near-infrared fluorescent VLPs (Scheme 1).…”

Production of recombinant SARS-COV-2 proteins and diphtheria toxoid CRM197-based fusion

“…In these vaccines, low-immunogenic bacterial oligosaccharides are chemically conjugated to the surface of the carrier molecule. CRM197 can also be used to enhance the immune response to peptide antigens attached to its molecule by chemical methods [11].…”