Tengonermin

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.

Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is the standard treatment of diffuse large B-cell lymphoma (DLBCL). Primary DLBCL of the central nervous system (CNS) (primary central nervous system lymphoma [PCNSL]) is an exception because of the low CNS bioavailability of related drugs. NGR–human tumor necrosis factor (NGR-hTNF) targets CD13+ vessels, enhances vascular permeability and CNS access of anticancer drugs, and provides the rationale for the treatment of PCNSL with R-CHOP. Herein, we report activity and safety of R-CHOP preceded by NGR-hTNF in patients with PCNSL relapsed/refractory to high-dose methotrexate-based chemotherapy enrolled in a phase 2 trial. Overall response rate (ORR) was the primary endpoint. A sample size of 28 patients was considered necessary to demonstrate improvement from 30% to 50% ORR. NGR-hTNF/R-CHOP would be declared active if ≥12 responses were recorded. Treatment was well tolerated; there were no cases of unexpected toxicities, dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with confirmed tumor response in 21 patients (75%; 95% confidence interval, 59%-91%), which was complete in 11. Seventeen of the 21 patients with response to treatment received consolidation (ASCT, WBRT, and/or lenalidomide maintenance). At a median follow-up of 21 (range, 14-31) months, 5 patients remained relapse-free and 6 were alive. The activity of NGR-hTNF/R-CHOP is in line with the expression of CD13 in both pericytes and endothelial cells of tumor vessels. High plasma levels of chromogranin A, an NGR-hTNF inhibitor, were associated with proton pump inhibitor use and a lower remission rate, suggesting that these drugs should be avoided during TNF-based therapy. Further research on this innovative approach to CNS lymphomas is warranted. The trial was registered as EudraCT: 2014-001532-11.