Research On Inhibition On Herceptin-resistant And Functional Optimization Of Anti-HER3 Antibody

With the further understanding of the pathogenesis of cancer on the molecular level, targeted therapy that target cell receptor, the key genes and regulatory elements has begun to play an important role in cancer treatment. With the advantages of specific antibody drugs targeting, safety and clinical effectiveness, as anti-cancer target, HER family is getting more and more attention.As member of the HER family, HER2 is over-expressed in many tumors, such as ovarian and breast cancer.There have been many antibody drugs and ADCs on the market, such as Herceptin, Perjeta, T-DM1, etc., which gradually become the first-lineclinical drugs in treatment of HER2-positive tumors. Although HER2 antibody has achieved a good anti-tumor effect in the treatment of HER2-positive tumors, the response rate in HER2-positive breast cancer is only 20 to 30%, that to say, late Herceptin drug-resistance phenomenon limits the antibody targeted therapeutic range and efficiency.Therefore, to clear the antibody resistance mechanism and to identify the predictor of the efficacy of antibody biomarkers havevery important significance in optimizing treatment and improving patient outcomes.HER3 is a member of epidermal growth factor receptor(HER) family, which is an activation key factor in PI3 K / AKT signaling pathway. A series of studies found the increased expression of the HER3 and increased rate of phosphorylation when EGFR and HER2 inhibitor are used for the treatment of a variety of tumors. As a result, the role of HER3 in anti-tumor is getting more and more attention.As HER3 inhibitors, the monoclonal antibody is the most effective and promising,which block the binding including the interaction of the ligand HRG and HER3, and the heterodimers between HER3 and the other HER family members. And then, the activation of downstream signaling pathways on HER3 was blocked in therapeutic tumor. Currently, anti-HER3 monoclonal antibody drugs, such as Duligotuzumab, Patritumab, have entered the phase II study, and the other drugs including Seribantuma, DL11, Seribantumab, LJM716, RG7116, etc., are in pre-clinical studies.Therapeutic antibodies have the advantages of high specificity, long half-life, little side effects and so on. However, with the antibodies’ large molecular weight, the mono-therapy treatment is limited. In recent years, an antibody drug conjugate(antibody-drug conjugate, ADC) technology is developing rapidly. ADC drug binds the advantages of both antibodies and cytotoxic drug medications, possesses specificity as antibody and strong cytotoxic as cytotoxic drugs, therefore, achieve better results in the treatment of tumors. Nowadays ADC is considered to be a new generation of targeted therapies drug. Successful listing of Mylotarg TM、Adcetris TM,Kadcyla TM draw more and more attention on the ADC drugs.According to the previous work in our lab, we obtained the trastuzumab resistance in ovarian cancer cell line SKOV3 / T.Then we compared thedifferences in proliferation ad signaling betweencells in trastuzumab resistance in ovarian cancer cell line SKOV3 / T and tumorigenesis, and made a deep discussion on the abnormal expression of HER3 in SKOV3 / T.Targeting HER3,we designed, optimized, filtered and expressed a fully human monoclonal antibody targeting HER3, and made an identification of its biological activity in vitro and in vivo.Based on this, the research work included the following three sections:I.The research on abnormal expression of HER3 in the trastuzumab resistant cell lines SKOV3 / TReasonable evaluation on cell proliferation and tumorigenicity in the trastuzumab resistant cell lines SKOV3/T.CCK8 cell proliferation assay experiments showed that the proliferation speed of SKOV3/T was faster significantly than that of the normal ovarian cancer cell SKOV3. Furthermore, in tumor model, SKOV3 / T cells grew faster than that the normal ovarian cancer SKOV3 cells.In addition, the expression level of HER1, HER2, HER3 in SKOV3 / T and SKOV3 was analyzed using flow cytometry and Western blot methods. The experimental results showed that in SKOV3 / T cell, the gene express of HER1 did not changed significantly, that of HER2 had a significantly down-regulated, while that of HER3 has increased compared with SKOV3 cell lines.II.The research on the molecular mechanisms of targeting HER3 antibody inhibition played in trastuzumab resistant tumor cell linesIn order to verify the role of HER3 in trastuzumab resistance, we expressed and synthesized Lm Ab3, a specific anti-HER3 antibody, and verifiedits biological activity. The results showed that Lm Ab3 could specifically recognize recombinantly expressed HER3 antigen and native HER3 molecules, and its affinity Kon value is 2.46E-10.- 7-Besides, Lm Ab3 could specifically inhibit phosphorylation of HER family members, and inhibit tumor cell proliferation in HER3-positive MCF7.Next, we used specific anti-HER3 antibody Lm Ab3 as a tool to investigate the role of HER3 in trastuzumab resistance.In three-dimensional culture experiment, we judged the role of HER3 played in SKOV3 / T cell proliferation. The experiment showed that the SKOV3 / T cell proliferation was inhibited when using the anti-HER3 antibody. Using anti-Her3 antibody to treat the mice with SKOV3 / T cells, the tumor growth was suppressed, while there was no significant inhibition in the normal saline control group and the Herceptin treatment group.To further investigate the mechanism of HER3 antibodies in drug-resistant cell lines, through Western blot method, the degree of phosphorylation of the key signaling molecules HER1, HER2, HER3 in the tumor cells stimulated by HRG were detected. The research resultsshowed that the phosphorylation of HER1, HER2, HER3, AKT in the tumor cells were inhibited treating with anti-HER3 antibody.III.The research on optimization and biological function evaluation of anti-HER3 antibodyDealing with computer-aided molecular simulation technology, the novel, reasonable anti-HER3 antibodies were designed and optimized.By using of reversed translation, codon suitability and secondary structure optimization, thegene sequences of the novel antibody were determined. And then, by expression and purified we obtained the humanized anti-HER3 antibody FD001. The experimental results showed that the FD001 has the ability to bind HER3 with high specificity and affinity.Next, dealing with the antibody- conjugating technology, coupling FD001 and poison small molecule DM1, we get the new ADC drug, FD001-DM1, which is targeted to HER3. The experimental study in vitro and in vivo showed that FD001-DM1 possessed good effect in inhibition of tumor cell proliferation.The innovation points of the research work was embodied in the followings: Confirmed the abnormal expression of HER3 in trastuzumab resistant cell lines SKOV3 / T; Revealed the effect of anti-HER3 antibody in inhibition of tumor cell proliferation in trastuzumab resistant cell lines inhibited; Designed and obtained a novel, high affinity anti-HER3 antibody, named as FD001; Constructed and evaluated the novel ADC, i.e. FD001-DM1. The ADC possessed good effect in vivo inhibition of tumor growth, which would be lay the foundation for clinical cancer therapy.