Study of Anti-RRM-2 Antibody Drug Generation and Their Inhibitory Mechanisms

Ribonucleotide reductase (RR) possesses the enzymatic activity to convert the ribonucleoside diphosphate (NDP) to 2-deoxyribonucleoside diphosphate (dNDP) for DNA replication and cell proliferation. RR is composed of one large subunit of RRM1 and 2 small subunits including RRM2 and RRM2B molecules. Previous studies showed that RRM1 up-regulation is associated with better prognosis in patients with non-small cell lung cancer. Similarly, Up-regulation of RRM2B inhibits the invasive ability of colorectal cancer cells (CRC) whereas the suppression of its expression increases cell migration. In addition, RRM2B level in the metastatic tissues of CRC patients is significantly lower in the primary ones. These results suggested that RRM2B possesses the inhibitory effect on the growth and metastasis of tumor cells. Interestingly, although RRM2 shows an 80% similarity in amino acid sequences with those of RRM2B, its expression in malignant tissues is relatively higher compared to the surrounding normal tissues in CRC patients, suggesting that this protein might play a crucial role in the development and metastasis of cancerous cells. RR inhibitors have been extensively applied for the treatment of many malignant diseases. However, their therapeutic applications in clinics are significantly limited due to the occurrence of drug resistance and side effects. Considering the suppressive effect of both RRM1 and RRM2B on tumorigenesis, RRM2 presents a most suitable target for developing immunotherapeutic agents. Thus, mono-specific anti-RRM2 antibodies with inhibitory effects on tumor proliferation and metastasis will be important for the development of antibody drugs and for understanding the mechanisms of tumorigenesis. Given the wide application of nanotechnology in many scientific areas, we will prepare gold nano-particle probe with surface-modified anti-RRM2 antibodies to detect the presence of RRM2 in the patient sea. In our preliminary study, we have obtained purified recombinant RRM2 protein and generated polyclonal anti-RRM2 IgY antibodies in chickens. Several phages displaying anti-RRM2 scFv (single chain variable fragment) on their surface have been cloned by panning 2 established antibody libraries. Similarly, polyclonal IgG and monoclonal scFv antibodies will be made in mice. Combined together, all the antibodies will be applied for the development of therapeutic antibody drugs and diagnostic reagents in the future. Thus, the objectives of this research proposal are as the following: 1. Cloning, expression and purification of recombinant human RRM2 and RRM2B proteins. RRM2B protein is mainly used to eliminate the cross-reactive anti-RRM2 antibodies in absorption experiments. 2. Production and characterization of polyclonal IgG and IgY antibodies in mice and chickens, respectively. 3. Production and characterization of monoclonal anti-RRM2 scFv antibodies using phage display antibody technology. 4. Investigation of the potential inhibitory effect of these antibodies on tumorigenesis. 5. Study of the mechanisms involved in the inhibitory function of these antibodies. 6. Development of a highly sensitive method for detecting the presence of RRM2 protein or anti-RRM2 antibodies in the patient sera. 7. Modification of anti-RRM2 scFv antibodies into chimeric or humanized antibodies with high affinity using antibody engineering technology