NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity.

Abstract

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

• Authors: Angelo Corti, Anna Maria Gasparri, Angelina Sacchi, Barbara Colombo, Matteo Monieri, Eltjona Rrapaj, Andrés J M Ferreri, Flavio Curnis
• Journal: Molecular pharmaceutics (Mol Pharm) Vol. 17 Issue 10 Pg. 3813-3824 (10 05 2020) ISSN: 1543-8392 [Electronic] United States
• PMID: 32805112 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Peptides
• Recombinant Fusion Proteins
• TNF protein, human
• Tnf protein, mouse
• Tumor Necrosis Factor-alpha
• Serine
• CD13 Antigens

Topics

• Amino Acid Motifs (genetics)
• Animals
• Antineoplastic Agents (chemistry, pharmacology, therapeutic use)
• CD13 Antigens (metabolism)
• Cell Line, Tumor (transplantation)
• Disease Models, Animal
• Drug Stability
• Humans
• Mice
• Neoplasms (drug therapy, pathology)
• Peptides (chemistry, genetics, pharmacology, therapeutic use)
• Protein Engineering
• Protein Processing, Post-Translational (genetics)
• Protein Stability
• Proteolysis
• Recombinant Fusion Proteins (chemistry, genetics, pharmacology, therapeutic use)
• Serine (genetics, metabolism)
• Tumor Necrosis Factor-alpha (chemistry, genetics, pharmacology, therapeutic use)