BackgroundIn 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.Methods7NP2 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.ResultsBiodistribution 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.ConclusionsThe results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

Mesenchymal stromal cells (MSCs) have been successfully employed in clinical applications. In most studies, autologous MSCs from the bone marrow (bmMSCs) were used, and others employed autologous adipose tissue-derived stromal cells (ADSCs). Recently, clinical feasibility studies provided evidence that MSCs from human term placenta (pMSCs) can be used for homologous therapy facilitating access to regenerative cells in emergency situations, when autologous cells are not available or not suitable. We therefore investigated the expression of MSC stemness marker CD146 and the expression of neuro- and myoregenerative cytokines by human pMSCs after expansion in three different media compliant with good manufacturing protocols (GMP) in comparison to pMSCs expanded in a commercial MSC expansion media. To replace xenobiotic serum in the GMP-compliant media employed in this study, either human serum, human serum plus platelet lysate (PLL), or human plasma plus PLL was used. We report that enrichment of media with PLL accelerates pMSC proliferation but reduces the expression of the stemness marker CD146 significantly, while PLL deprivation enhanced the CD146 expression. In contrast, the reduced expression of CD146 by PLL deprivation was not observed on bmMSCs. The expression of the cytokines investigated was not modulated significantly by PLL. We conclude that accelerated expansion of pMSCs in GMP-compliant media enriched by PLL reduces the expression of stemness marker CD146, but does not influence the expression of neuro- and myoregenerative cytokines.

Programmed cell death protein 1 (PD‐1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti‐PD‐1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti‐PD‐1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD‐1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single‐chain fragment variable (scFv) format, based on the IGHV3‐23*03 (VH) and IGKV1‐39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross‐reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD‐1/PD‐L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD‐1 were elucidated by x‐ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N‐terminus of PD‐1 following D12 binding, as well as partial overlap with the binding site for the cognate PD‐L1 and PD‐L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD‐1 activity in vitro and in vivo.