Preparation Of Anti-Human CD105Murine Monoclonal Antibody And In Vitro Antitumor Effect Of Paclitaxel Polymer System

Background：Ovarian cancer is a common high-risk cancer, and the docetaxel (Docetaxel，TXT) is thefirst-line chemotherapy for ovarian cancer. Commonly TXT solvent can cause severe allergicreactions and has no targeting ability that affects drug efficacy, therefore require a newoptimal load system modified with TXT to prevent the allergic reactions and enhance drugeffectiveness. CD105is an important marker of ovarian cancer tumor angiogenesis, and as thetarget of the chemotherapy drugs which can enhance the specificity and efficacy. Polylacticacid (PLA) as the main chain joint combined with polyethylene glycol (PEG) and biotinnanoparticles (the TXT-BPLA NPs) is a good vector for TXT. Therefore, with the previousresearch (development of specific anti-CD105monoclonal antibody hybridoma cell lines), thespecific CD105monoclonal antibody has been identified and biotinylated. While cooperationwith Chongqing University research group, we bonded CD105to the TXT-BPLA NPScarrier, and investigate the characterization of TXT-BPLA NPS. Then we researched theanti-tumor effect new TXT-BPLA NPS in vitro with human ovarian cancer SKOV-3celllines. Our goal is to explore its inhibition mechanisms of tumor growth and pro-apoptotic, andto provide new ideas and experimental evidence to reduce side effects and improve the effect.Methods：We inoculated the method of injection in mouse ascites to generate anti-human CD105murine monoclonal antibody, and selected the highest affinity antibody-labeled biotin. PLA,as the main chain combined with PEG and biotin was synthesised to BPLA NPs. BPLA NPspackaged TXT through self-emulsification/solvent diffusion, and was prepared as a newbiotin-graft modified polylactic acid-docetaxel nanoparticles (TXT-BPLA NPs). We useddifferent doses of TXT-BPLA NPs (1,10,100,1000,10000nmol/L) to treat SKOV3cellsat different times (12,24,48,72,96,120h), and tested CCK-8growth inhibition rate, cell apoptosis by flow cytometry, caspase-3expression by Western blot.Results：1. The total amount of six purified antibody were in the range of70to121mg, theaverage yield of2.2to3.1mg/mL. The titer of1-H9,2-G4and G9were beyond1:1000000,and2-G9affinity was3.36×1010.2-G9antibodies strains binded biotin at the rate of1.9.2. Average particle size of BPLA NPs was161.6nm (range:100to300nm), and PDIdispersion coefficient was0.117.The average Zeta potential was-37.56mV. BPLA NPs TXTaverage parcel rate was54.91%, and the average drug loading was11.03%. TXT TXT PPLA-containing nanoparticles2h cumulative release rate was25%, and did not appear burstrelease. At8h the cumulative release rate was51%, which reaching half of the release. Themedium-term diffusion stage was12h after drug release, and the release rate was lower.96hrelease rate was64%. The antibody–BPLA could be combined with CD105in vitro.3. In the concentration range of10to10,000nmol/L, at the time, the SKOV3cell growthinhibition rates of TXT and TXT BPLA NPs were dose-dependent. In the same concentration,SKOV3cell growth inhibition rate, the apoptosis rate and Capase-3expression in TXTgroup at24h and48h were significantly higher than the TXT-BPLA NPs group (P <0.05).After72h, SKOV3cell growth inhibition rate, apoptosis rate and capase-3expression inTXT-BPLA NPs group were significantly higher than TXT group (P <0.05).Conclusion：1. Ascites inoculation could produce specific anti-CD105murine monoclonal antibodyeffectively.2. Because of its high affinity for specific markers of tumor angiogenesis, anti-humanCD105mouse monoclonal antibody can be used as targeting boot agent of anticancer drugs.3. BPLA nanoparticle has no cytotoxic effect in vitro, and the appropriate uniform sizeand charge characteristics make it a safe and effective drug carrier of TXT.4. BPLA NPs is able to effectively load TXT and releases TXT continuously in vitro.Drug loading capacity and encapsulation efficiency are also satisfying.5. BPLA NPs is able to bind to anti-CD105murine monoclonal antibody with avidin.The BPLA NPs and anti-CD105antibody complex still has specific binding ability toCD-105in vitro.6. TXT-BPLA NPs shows obvious inhibition of proliferation and induces apoptosis in human ovarian cancer SKOV-3cells lines, and its drug release effect on ovarian cancer cellproliferation inhibition and apoptosis is stable, durative, security.