Preliminary Identiifcation Of Molecular Markers Of Ovarian Cancer And Preparation Of New Targeting Antibody

Background: Ovarian cancer is a common gynecological malignancies, and it’salso the highest fatality cancer of all gynecological tumors. Because of the incidence ofoccult and lack of effective methods for early diagnosis,70%of patients are diagnosedwith advanced stage with the5-year survival rate of only20%-30%. At present, themain treatment is surgery and/or chemotherapy; however,60%to70%of patientsoccur resistance or recurrence in the course of treatment. Therefore, to find effectivemethods for early diagnosis or clinical treatment is essential to improve the survival rateof patients with ovarian cancer.CA125is currently the most widely used marker to detect ovarian cancers,however, CA125level also elevates in physiological period of ovulation, endometriosis,pelvic inflammatory disease, uterine adenomyoma and other benign gynecologicaldiseases in order to appear false-positive results, indicating the poor sensitivity andspecificity in the diagnosis of ovarian cancer. With the development of tumor molecularbiology, new molecular markers associated with ovarian cancer have been discoveredfor cancer diagnosis and/or clinical therapy. According to the2012NCCN guidelines,only anti-VEGF monoclonal antibody bevacizumab could be currently used as ananti-angiogenic compound for treating ovarian cancer. In addition, antibodies againstthe epidermal growth factor receptor family members, e.g. HER1, HER2, HER3, oragainst the insulin-like growth factors (IGF1R) have been reported as available potentialtargeted therapy drugs, in which HER-2is a classic target for breast cancer treatment,with two specific antibody drugs approved by FDA (Trastuzumab and Pertuzumab).Studies have shown that the expression of HER2in ovarian cancer is high, and HER2was related to the occurrence, development and poor prognosis of ovarian cancer. Objective: To preliminary identify the possible molecular markers closelyassociated with ovarian cancer in order to choose reasonable marker(s) for preparingpotential therapeutic antibody drug(s). By constructing large phage antibody library, anew optimized anti-HER2antibody was screened out and identified in vitro and in vivo;furthermore, the inhibitory mechanism of the new antibody was explored in ovariancancer cell line SKOV3.Materials and methods:1. Collect serum samples from68women with benigngynecological diseases, detecte the serum level of HE4and CA125, and evaluate thespecificity of HE4in the diagnosis of ovarian cancer;2. Using bio-chip technology, theexpression level of HER2, HER3or IGF1R was detected in ovarian cancer tissues foranalyzing the their relationship with ovarian tumors;3. using the Cre/LoxPrecombination system, we built a large-content single chain antibody (scFv) phagelibrary for screening new antibodies. The new antibodies were identified also in vitroand in vivo;4. using eukaryotic expression vector pCMV, the full-length antibody wasexpressed and purified. The antigen binding, affinity, epitope and antitumor effect of thenew antibody were identified.5. Using the ovarian cancer cell line SKOV3, themechanisms of the new antibody were identified, e.g. migration, apoptosis, ADCC andsignal transduction, etc.Results:1. Women with benign gynecological disease serum HE4levels increasedless than CA125, showing HE4had higher specificity in the diagnosis of ovarian cancerthan CA125;2. HER2, HER3, and IGF1R all expressed high in ovarian cancer tissuesand they were closely associated with tumor stage, indicating that they can be used aspotential therapeutic targets in ovarian cancer;3. Using the Cre-LoxP recombinantsystem, a large-capacity, epitope-specific scFv library was established based on thecomplex structure and binding mode of HER2and pertuzumab, from which a optimizedantibody was screened out with higher affinity and overlapped epitope. The antigenbinding capacity and the inhibitory ability of ovarian cancer in vivo were also testifiedsimilar to parent antibody;4. The full-length antibody, named as M5, was obtained byeukaryotic expression and affinity purification. M5showed similar epitope and higher affnity than pertuzumab, and its anti-tumor effect in vivo seemed better. The possibleanti-tumor mechanisms may include: inhibition of cell migration, induction of apoptosis,ADCC, and blocking the activation of HER2downstream signal pathway, etc.