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.

Targeting Tissue Factor to Tumor Vasculature to Induce Tumor Infarction

“…Although it might be of theoretical advantage to target tTF-NGR to two intratumoral binding sites, to have a clear and predictable mode of action, we have excluded this phenomenon by modifying the GMP process, storage and use of the molecule. Interestingly, A. Corti’s group has shown that some NGR-peptide degradation and post-translational modifications can be reduced by the introduction of an N-terminal serine [ 104 ].…”

Role of Immune Checkpoint Inhibitor Therapy in Advanced EGFR-Mutant Non-Small Cell Lung Cancer

“…While preclinical models support the ability of TNF-α to also enhance ICI therapy ( 103 ), there are no active clinical trials involving NGR-hTNF and ICI therapy in NSCLC due to the manufacturer of NGR-hTNF discontinuing the product after a Phase III mesothelioma trial did not meet its primary endpoint ( 104 ). Importantly, an NGR-TNF derivative with an additional serine at the N-terminus that demonstrates increased stability, S-NGR-TNF, has been recently developed ( 105 ). It will be intriguing to see if TNF-α strategies such as S-NGR-TNF can restore TIL trafficking, enhance ICI therapy, and augment chemotherapy delivery to EGFR m NSCLC tumors.…”

Targeting the Blood–Brain Tumor Barrier with Tumor Necrosis Factor-α

“…TNF is a homotrimeric protein. Structural studies of human and murine NGR-TNF, produced by genetic engineering technology in Escherichia coli ( E. coli ) cells, have shown that these products consist of trimers made by subunits with different molecular weights, including subunits with the expected molecular weight (17939.39 Da and 17844.25 Da, respectively) and subunits with −17, +1, +42 and +58 Da than expected [ 62 ]. The molecular heterogeneity of human and murine NGR-TNF is related to post-translational modifications of their N-terminal domains, including N-terminal acetylation and oxidation (+42 and +58 Da), asparagine deamidation (+1 Da), and formation of 6–7 membered rings between cysteine α-amino group and asparagine side-chain (−17 kDa).…”

“…The molecular heterogeneity of human and murine NGR-TNF is related to post-translational modifications of their N-terminal domains, including N-terminal acetylation and oxidation (+42 and +58 Da), asparagine deamidation (+1 Da), and formation of 6–7 membered rings between cysteine α-amino group and asparagine side-chain (−17 kDa). These post-translational modification and degradation reactions need a cysteine (C) in the first position (with a free α-amino group) and an asparagine (N) in second position, as in the CNGRCG domain of NGR-TNF [ 62 ].…”