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Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene's family usage in naïve rats have been used to validate the frequency's distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399-233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

Blood vessel micro-angioarchitecture plays a pivotal role in tumor progression, metastatic dissemination and response to therapy. Thus, methods able to quantify microvascular trees and their anomalies may allow a better comprehension of the neovascularization process and evaluation of vascular-targeted therapies in cancer. To this aim, the development of a restricted set of indexes able to describe the arrangement of a microvascular tree is eagerly required. We addressed this goal through 3D analysis of the functional microvascular network in sulfo-biotin-stained human multiple myeloma KMS-11 xenografts in NOD/SCID mice. Using image analysis, we show that amounts, spatial dispersion and spatial relationships of adjacent classes of caliber-filtered microvessels provide a near-linear graphical "fingerprint" of tumor micro-angioarchitecture. Position, slope and axial projections of this graphical outcome reflect biological features and summarize the properties of tumor micro-angioarchitecture. Notably, treatment of KMS-11 xenografts with anti-angiogenic drugs affected position and slope of the specific curves without degrading their near-linear properties. The possibility offered by this procedure to describe and quantify the 3D features of the tumor micro-angioarchitecture paves the way to the analysis of the microvascular tree in human tumor specimens at different stages of tumor progression and after pharmacologic interventions, with possible diagnostic and prognostic implications.

In the last decade, the use of flexible biosensors for neuroprosthetic and translational applications has widely increased. Among them, the polyimide (PI)-based thin-film electrodes got a large popularity. However, the usability of these devices is still hampered by a non-optimal tissue-device interface that usually compromises the long-term quality of neural signals. Advanced strategies able to improve the surface properties of these devices have been developed in the recent past. Unfortunately, most of them are not easy to be developed and combined with micro-fabrication processes, and require long-term efforts to be testable with human subjects. Here we show the results of the design and in vitro testing of an easy-to-implement and potentially interesting coating approach for thin-film electrodes. In particular, two biocompatible coatings were obtained via covalent conjugation of a laminin-derived peptide, CAS-IKVAV-S (IKV), with polyimide sheets that we previously functionalized with vinyl- and amino- groups (PI_v and PI_a respectively). Both the engineered coatings (PI_v+IKV and PI_a+IKV) showed morphological and chemical properties able to support neuronal adhesion, neurite sprouting, and peripheral glial cell viability while reducing the fibroblasts contamination of the substrate. In particular, PI_v+IKV showed promising results that encourage further in vivo investigation and pave the way for a new generation of peptide-coated thin-film electrodes.