Such libraries may yield clones which are homogenous in terms of their biophysical properties and amino acid sequence, with some additional desirable properties, such as protein A binding for affinity capture applications. Over the last 15 years, we have described and extensively validated human antibody synthetic libraries, which featured antibodies in scFv format capable of binding to protein A affinity supports. These antibody libraries have been used as sources of useful binding specificities, including the monoclonal antibodies F8, L19 and F16, specific to the alternatively spliced EDA and EDB domain of O4I1 fibronectin and the A1 domain of tenascin-C, respectively. The three antibodies, which have been shown to selectively recognize stromal and neovascular structures in cancer and inflammation, are able to preferentially localize at sites of pathological angiogenesis in vivo and are currently being investigated in Phase I and Phase II clinical trials. Here, we describe the design and construction of a very large antibody phage display library, containing over 40 billion human antibodies. This is the largest antibody library ever produced in our lab and one of the largest synthetic antibody libraries described in the literature. The new library was highly functional, as revealed by the observation that.90% randomly picked antibody clones can be expressed at acceptable levels. A side-by-side comparison of antibody selections, performed with the PHILODiamond library and with other libraries against more than 15 antigens, revealed that various binding specificities could be isolated against structurally diverse targets. The PHILODiamond library differs from other synthetic libraries in terms of size and modular design, facilitating affinity maturation procedures. Furthermore, all antibody clones bind to protein A, thus facilitating purification and detection procedure. We introduced a S52N mutation in the VH domain, since position 52 is the most frequently mutated solvent exposed residue in the CDR2 loop and since asparagine may favor both donor and NVS-CECR2-1 acceptor hydrogen bonding interactions. Position 52 is often mutated into an asparagine residue in naturally occurring antibodies. In order to demonstrate library performance for biomedical research applications, we raised human monoclonal antibodies against the alternatively spliced BCD segment of tenascin-C, a highly conserved alternatively spliced protein fragment comprising the three fibronectin type-III homology domains B, C and D, which displays 86% sequence identity between mouse and man, respectively. The binding properties of clone G5, originating directly from library selections without affinity-maturation procedures, were characterized in vitro by Biacore analysis and ex vivo, by immunofluorescence analysis of human and mouse tumor sections.