In vitro prediction of the immunogenicity of therapeutic proteins: state of the art and current challenges and perspectives.

INTRODUCTION

Immunogenicity of therapeutic proteins is a major concern because it may compromise their clinical use and efficacy. Preclinical prediction of anti-drug antibody development using in vitro assays is therefore a challenge for candidate biologics.

AREAS COVERED: This review, based on a thorough literature search of the PubMed database up to December 2025, provides an overview of in vitro assays predicting biologic immunogenicity, including antigen internalization assay, dendritic cell activation assay, peptide-MHC II affinity measurement, MHC‑associated peptide proteomics (MAPPs) assay, and T cell activation/proliferation assays. The main features and applications of these assays are summarized, with special emphasis on their advantages and limitations.

EXPERT OPINION: Although in vitro immunogenicity assays are proposed to discriminate between low- and high-immunogenic biologics, they face numerous challenges. These include selecting the most appropriate test(s), lack of standardization in methods and technical parameters, defining positivity thresholds, high costs, the need for specialized equipment and trained personnel, the complexity and labor-intensive nature of assays, and the difficulty of translating in vitro results into clinical immunogenicity outcomes. Additional assay validation studies are undoubtedly required to better define the implementation and robustness of in vitro immunogenicity prediction for biologics, which could be advantageously combined with in silico approaches.