Abstract
Development of therapeutic antibodies in oncology has attracted much interest in the past decades. More than 30 of them have been approved and are being used to treat patients suffering from cancer. Despite encouraging results, and albeit most clinical trials aiming at evaluating monoclonal antibodies directed against TRAIL agonist receptors have been discontinued, DR4 or DR5 remain interesting targets, since these receptors are overexpressed by tumour cells and are able to trigger their death. In an effort to develop novel and specific anti-DR4 and anti-DR5 antibodies with improved properties, we used genetic immunization to express native proteins in vivo. Injection of DR4 and DR5 cDNA into the tail veins of mice elicited significant humoral anti-DR4 and anti-DR5 responses and fusions of the corresponding spleens resulted in numerous hybridomas secreting antibodies that could specifically recognize DR4 or DR5 in their native forms. All antibodies bound specifically to their targets with a very high affinity, from picomolar to nanomolar range. Among the 21 anti-DR4 and anti-DR5 monoclonal antibodies that we have produced and purified, two displayed proapoptotic properties alone, five induced apoptosis after cross-linking, four were found to potentiate TRAIL-induced apoptosis and three displayed antiapoptotic potential. The most potent anti-DR4 antibody, C#16, was assessed in vivo and was found, alone, to inhibit tumour growth in animal models. This is the first demonstration that DNA-based immunization method can be used to generate novel monoclonal antibodies targeting receptors of the TNF superfamily that may constitute new therapeutic agents.
Highlights
TRAIL agonist receptors, DR4 and DR5, have for more than two decades been considered as potential targets for cancer therapy owing to their ability to trigger selective apoptosis in tumour cells while sparing normal cells[1,2]
The neomycin cassette of the pCR3-neo-hDR4-Fc and pCR3-neohDR5-Fc expression vectors was replaced by puromycin by assembling PCR fragments obtained using OR435/OR436 and OR439/OR446 from each vector to the cDNA encoding resistance to puromycin generated by PCR amplification from OM181, a retroviral vector derived from pMSCV-Puro, resulting in the pCR3-Puro-hDR4-Fc (OM1450) and pCR3-Puro-hDR5-Fc (OM1449) constructs
Mice were immunized with DR4 or DR5 plasmids and immune reactivity was evaluated by enzyme-linked immunosorbent assay (ELISA) on mice serum
Summary
TRAIL (tumour necrosis factor-related apoptosis inducing ligand) agonist receptors, DR4 and DR5, have for more than two decades been considered as potential targets for cancer therapy owing to their ability to trigger selective apoptosis in tumour cells while sparing normal cells[1,2]. Clinical evaluations of TRAIL, the natural ligand of DR4 and DR5, or anti-DR4/DR5 antibodies, alone or combined with chemotherapy, have, been. Dubuisson et al Cell Death and Disease (2019)10:101 within a supramolecular scaffold coined DISC for Death Inducing Signalling Complex[10]. Within this scaffold, the zymogen caspase gets activated by proximity[11] and released to the cytosol, where it can cleave other proteases such as caspase-3, another cysteine protease responsible for the execution phase of the apoptotic machinery. Antibodies assessed in the clinic have mostly focussed on DR5, with only one anti-DR4 evaluated in phase I/II4. Keeping in mind that DR4 prevails over DR5 in transducing TRAIL-induced cell death[12] or that anti-DR5 and anti-DR4 antibodies can potentiate TRAIL-induced cell death[13,14], development of antibodies targeting these receptors still holds interest in oncology.
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