“Active” Cancer Immunotherapy by Anti-Met Antibody Gene Transfer

Abstract

Gene therapy provides a still poorly explored opportunity to treat cancer by “active” immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing (“cell autonomous activity,” in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface (“bystander effect,” in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer–based immunotherapy strategy. [Cancer Res 2008;68(22):9176–83]

Monovalency Unleashes the Full Therapeutic Potential of the DN-30 Anti-Met Antibody

“…An additional possibility is to compensate higher plasmatic clearance by gene transfermediated, continuous production of the therapeutic Fab. This genetic approach has already proved to be successful for DN-30 mAb, using a bidirectional lentiviral vector (18). A similar approach is now under study for the DN-30 Fab fragment.…”

“…Molecular Biology and Lentiviral Vectors-The cDNA corresponding to the N-terminal portion of the DN-30 heavy chain (the VH and the CH1 domains) was obtained by PCR amplification using the bidirectional lentiviral vector encoding for DN-30 mAb as template (18). The following oligonucleotides were used as primers: 5Ј-TATACCCGGGCCACCATGG-GATGGAGCTATATCATCC-3Ј (forward) and 5Ј-GGCTT-GATGCTAGCCCCTCTGG-3Ј (reverse).…”

“…Met Activation Analysis-Subconfluent A549 human lung carcinoma cells (European Cell Culture Collection) were incubated in serum-free medium for 48 h and then stimulated for 10 min with the indicated concentrations of recombinant HGF (R&D Systems) or purified DN-30 mAb/Fab as described (18). Following stimulation, cells were immediately lysed and processed as described (21).…”

“…Using the DN-30 heavy chain cDNA as template (18), we PCR-amplified the region corresponding to the variable (VH) and the first constant (CH1) domains. The amplified cDNA was subcloned into a bidirectional LV expressing both the light chain and the truncated heavy chain (18). A nucleotide sequence encoding for a FLAG epitope and a polyhistidine tail was inserted at the C terminus to facilitate protein detection and purification (Fig.…”

MET signalling: principles and functions in development, organ regeneration and cancer

“…Unlike CBL-mediated endosomal degradation, regulated proteolysis of MET is ligand-and ubiquitin-independent and does not require the kinase activity of the receptor: the mechanism occurs basally and affords MET signalling with chronic, low-grade attenuation under steady-state conditions. The shedding of MET that is catalysed by ADAM metalloproteases can be acutely enhanced by various agents such as phorbol esters, suramin, lysophosphatidic acid and monoclonal antibodies (mAbs) against the MET ectodomain 94,95,96,97,98,99,100,101,102 . Importantly, the extracellular shedding of MET not only decreases the number of receptor molecules on the cell surface but also generates a decoy moiety that interacts with both HGF (by sequestering the ligand) and full-length MET (by impairing dimerization and transactivation of the native receptor) to further inhibit MET signalling 103,104 .…”