Polypeptide composition of an adenovirus type 5 used in cancer gene therapy

Area of Science:

• Oncology
• Virology
• Biochemistry

Background:

• Recombinant adenovirus serotype 5 (RPR/INGN201) is engineered for cancer gene therapy by incorporating the human tumor suppressor gene p53.
• Understanding the precise protein composition of viral vectors is crucial for optimizing gene therapy efficacy and safety.

Purpose of the Study:

• To comprehensively characterize the proteome of the RPR/INGN201 viral vector.
• To identify and analyze post-translational modifications of viral proteins.
• To establish a robust analytical method for monitoring viral vector integrity and manufacturing consistency.

Main Methods:

• Proteins from RPR/INGN201 virions were separated using reversed-phase High-Performance Liquid Chromatography (HPLC).
• Individual proteins and their tryptic fragments were identified using electrospray time-of-flight mass spectrometry and N-terminal sequencing.
• Quantitative analysis was performed to determine the contribution of identified peptides to the overall viral protein content.

Main Results:

• Twenty-five peptide components, including fiber, were identified and characterized, representing over 0.25-0.5% of the viral protein content.
• Fiber protein was confirmed to be partially glycosylated, with both non-glycosylated and monoglycosylated forms detected.
• Protein V and protein IIIa were identified as phosphorylation derivatives, existing in non-phosphorylated, monophosphorylated, and diphosphorylated states.

Conclusions:

• The applied analytical methodology provides a detailed understanding of the polypeptide repertoire in infectious virions.
• This approach enables rapid monitoring of structural modifications arising from manufacturing process changes.
• The method is effective for characterizing diverse adenoviral constructs used in gene therapy.