Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody.

BACKGROUND: In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS: 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS: Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS: The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas.

Certain cytokines synergize in activating anti-cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of cytokine pairs to the tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-cytokine biopharmaceutical featuring the sequential fusion of interleukin-2 (IL2) with the XE114 antibody in scFv format and a tumor necrosis factor mutant (TNFmut). The fusion protein recognized the cognate antigen (carbonic anhydrase IX, a marker of hypoxia and of renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed cytokine activity in in vitro tests and preferentially localized to solid tumors in vivo. The product exhibited a partial growth inhibition of murine CT26 tumors transfected for carbonic anhydrase IX. When administered to Cynomolgus monkey as intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1,500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 h. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic clear-cell renal cell carcinoma, since these tumors are known to be sensitive to IL2 and to TNF.

Multivalent supramolecular dendrimer-based drugs.

Supramolecular complexes consisting of a hydrophobic dendrimer host [DAB-dendr-(NHCONH-Ad)(64)] as well as solubilizing and bioactive guest molecules have been synthesized using a noncovalent approach. The guest-host supramolecular assembly is first preassembled in chloroform and transferred via the neat phase to aqueous solution. The bioactive guest molecules can bind to a natural (serotonin 5-HT(3)) receptor with nanomolar affinity as well as to the synthetic dendrimer receptor in aqueous solution, going toward a dynamic multivalent supramolecular construct capable of adapting itself to a multimeric receptor motif.

Further studies on imidazo[4,5-b]pyridine AT1 angiotensin II receptor antagonists. Effects of the transformation of the 4-phenylquinoline backbone into 4-phenylisoquinolinone or 1-phenylindene scaffolds.

The 4-phenylquinoline fragment of novel AT(1) receptor antagonists 4 based on imidazo[4,5-b]pyridine moiety was replaced by 4-phenylisoquinolinone (compounds 5) or 1-phenylindene (compounds 6) scaffolds to investigate the structure-activity relationships. Binding studies showed that most of the synthesized compounds display high affinity for the AT(1) receptor. Because of the in vitro high potency of carboxylic acids 5b,f, they were evaluated in permeability (in Caco-2 cells) and in pharmacokinetic studies in comparison with quinoline derivatives 4b,i,j,k. The studies showed that these compounds are characterized by rapid excretion, low membrane permeability, and low oral bioavailability. The structure optimization of the indene derivatives led to compounds 6e,f possessing interesting AT(1) receptor affinities. Optimization produced polymerizing AT(1) receptor ligand 6c, which forms a thermoreversible polymer (poly-6c) and is released from the latter by a temperature-dependent kinetics. The results suggest the possibility of developing novel polymeric prodrugs based on a new release mechanism. Finally, a set of 34 AT(1) receptor antagonists was used as a new test for the evaluation of the predictive capability of the previously published qualitative and quantitative pharmacophore models.