A high‐affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo‐vasculature in vivo

Abstract

The alternatively spliced extra-domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra-domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody-based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (K D 5 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody-based targeted anti-cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over-expression is detectable not only in solid tumors, but also in hematological malignancies.

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

“…The recombinant catalytic domain of CA IX was used for the isolation of human monoclonal antibodies from the ETH-2-Gold phage antibody library (Silacci et al, 2005). One of the clones isolated from the library ('A11') ( Table 1), which was shown to be non-inhibitory in the enzymatic assay described above (data not shown), was affinity-matured by combinatorial mutagenesis of residues in the CDR1 loops of VH and VL domains according to a procedure recently developed by our group (Villa et al, 2008), yielding clone A3. Additional mutagenesis of CDR2 loops led to the isolation of the daughter antibody clone CC7 (Table 1).…”

“…In this study, we have used antibodies in SIP format (Borsi et al, 2002;Villa et al, 2008), as this format and similar mini-antibody formats have extensively been shown to offer distinctive advantages both for imaging applications (Leyton et al, 2009);von Lukowicz et al, 2007;Wei et al, 2008) and for radioimmunotherapy of cancer (Berndorff et al, 2005;Tijink et al, 2006;Kenanova et al, 2007;Sauer et al, 2009). Recent publications suggest that smaller high-affinity ligands (MW o 2000 Da) may enjoy a much more rapid tissue distribution compared with antibodies and antibody fragments (Low et al, 2008).…”

“…Antibody residues are numbered according to (Tomlinson et al, 1992) and (Williams et al, 1996). Mutations at positions 31, 32, 33 of VH and 31, 31a, 32 of VL were introduced by PCR using the same partially degenerate primers (Eurofins MWG Operon, Ebersberg, Germany) as described previously (Villa et al, 2008). A single round of selection on biotinylated antigen (final concentration 10 À7 M) was carried out eluting bound phage with 100 mM triethylamine, as described previously (Silacci et al, 2005).…”

High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer

“…Application of an optimized phage display selection protocol and subsequent screening methods resulted in the selection of scFvD2 showing a high affinity for FGFR1 with a low dissociation constant of 18 nmol/L. It is also worth noting that we subjected scFvD2 to affinity maturation by randomization of several residues located in the CDR1 of the heavy and light chains as described by others (39), but none of the resulted clones showed an improvement in the kinetic constants. It is likely that the amino acid motifs present in the CDR1 regions of the parental clone support the observed strong binding of scFvD2 to FGFR1.…”

“…The diversification of the CDR1 of both heavy and light chains was introduced as described by Villa and colleagues (39). An "off-rate" phage-selection strategy was applied to eliminate low affinity binders.…”