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

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 the Blood-Brain Tumor Barrier with Tumor Necrosis Factor-α.

The blood-brain tumor barrier represents a major obstacle for anticancer drug delivery to brain tumors. Thus, novel strategies aimed at targeting and breaching this structure are of great experimental and clinical interest. This review is primarily focused on the development and use of a derivative of tumor necrosis factor-α (TNF) that can target and alter the blood-brain-tumor-barrier. This drug, called NGR-TNF, consists of a TNF molecule fused to the Cys-Asn-Gly-Arg-Cys-Gly (CNGRCG) peptide (called NGR), a ligand of aminopeptidase N (CD13)-positive tumor blood vessels. Results of preclinical studies suggest that this peptide-cytokine fusion product represents a valuable strategy for delivering TNF to tumor vessels in an amount sufficient to break the biological barriers that restrict drug penetration in cancer lesions. Moreover, clinical studies performed in patients with primary central nervous system lymphoma, have shown that an extremely low dose of NGR-TNF (0.8 µg/m2) is sufficient to promote selective blood-brain-tumor-barrier alteration, increase the efficacy of R-CHOP (a chemo-immunotherapy regimen) and improve patient survival. Besides reviewing these findings, we discuss the potential problems related to the instability and molecular heterogeneity of NGR-TNF and review the various approaches so far developed to obtain more robust and homogeneous TNF derivatives, as well as the pharmacological properties of other peptide/antibody-TNF fusion products, muteins and nanoparticles that are potentially useful for targeting the blood-brain tumor barrier. Compared to other TNF-related drugs, the administration of extremely low-doses of NGR-TNF or its derivatives appear as promising non-immunogenic approaches to overcome TNF counter-regulatory mechanism and systemic toxicity, thereby enabling safe breaking of the BBTB.

Ways to enhance lymphocyte trafficking into tumors and fitness of tumor infiltrating lymphocytes.

The tumor is a hostile microenvironment for T lymphocytes. Indeed, irregular blood flow, and endothelial cell (EC) anergy that characterize most solid tumors hamper leukocyte adhesion, extravasation, and infiltration. In addition, hypoxia and reprograming of energy metabolism within cancer cells transform the tumor mass in a harsh environment that limits survival and effector functions of T cells, regardless of being induced in vivo by vaccination or adoptively transferred. In this review, we will summarize on recent advances in our understanding of the characteristics of tumor-associated neo-angiogenic vessels as well as of the tumor metabolism that may impact on T cell trafficking and fitness of tumor infiltrating lymphocytes. In particular, we will focus on how advances in knowledge of the characteristics of tumor ECs have enabled identifying strategies to normalize the tumor-vasculature and/or overcome EC anergy, thus increasing leukocyte-vessel wall interactions and lymphocyte infiltration in tumors. We will also focus on drugs acting on cells and their released molecules to transiently render the tumor microenvironment more suitable for tumor infiltrating T lymphocytes, thus increasing the therapeutic effectiveness of both active and adoptive immunotherapies.