Research About Anti-CD71 MAb Conjugate's Targeted Anti-tumor Effects And The Genetic Analysis Of Vκ Chains Of Its Hybridoma.

The project designed to take curcumin as anti-cancer drug and photosensitizer in the light condition. We conjugated modified-curcimin with anti-TfR antibody collaborative to cure cancer. In order to extend drug half-life, to further explore the optical properties of curcumin, we compared different conjugate methods of antibody in combination with the photosensitizer and assessed the coupling efficiency and in vitro testing, discussed the mechanism. Based on the former research, we constructed nano- curcumin and anti-TfR monoclonal antibody conjugate. This work gave experimental evidence for multiple tumor imaging and targeted therapy. Based on analysis of three light chains from the same hybridoma which serecting against TfR mAb, the efforts lay solid foundation for the realization of genetically engineered and genetic engineering targeted anti-tumor effect.[Methods]1. Fabricate and assessment the conjugate of antibody and curcumin.(1) Evaluated tumor cells growth condition in light/non-light by cell count, and determined tumor cell killing effect of photosensitizatied curcumin. Fluorescence microscope to observe HepG2 cells endocytose curcumin.(2) Depended on carbodiimide as the condensing agent, preparated Cur-Ab immunoconjugate chemical, calculated conjugates'antibody titers by indirect ELISA assessed their IC50 by the MTT method.(3) Preparation photosensitizated curcumin-anti-TfR monoclonal antibody actively nanoparticles and preliminary assessment its functions. 1) With a thin film and the injection method to prepare liposomes, and assessed the peak rate of packets.2) Modified curcumin liposomes with TfR mAb.3) Particle morphology studied using scanning electron microscopy.4) FCM detection Cur-NP-PEG-Ab binding rate with cancer cells.5) FCM detection Cur-NP-PEG-Ab in the light and non-light situations on proliferation/apoptosis, cell cycles, etc.6) Used DCFH-DA and ROS kits to assess the changes of mitochondrial potential and ROS.2. Analysis the variable chains of TfR antibody.(1) PCR amplification of VL and VH genes from hybridoma which secertin anti-TfR antibody.(2) Cloning and sequencing of the VH and VL gene by IMGT and NCBI database.(3) Molecular modeling by Insight II to simulate Fabs of against TfR mAb.[Results]1. Fabricated and assessed anti-tumor effect of the conjugate of antibody and curcumin.(1) Normal white light had no effect on cell culture, curcumin can emission khaki-colored fluorescence by electron microscopy, and curcumin killing tumor cells power will be greatly enhanced in the light irradiation, and Curcumin had been endocytosed by HepG2 cells.(2) Coupled of anti-TfR monoclonal antibody and curcumin conjugate in chemical method, the conjugate's antibody titer decreased. But the killing activity of conjugate super than curcumin, anti-TfR monoclonal antibody on hepatocellular carcinoma cells the killing effect was similar to curcumin+antibody mixture.(3) Active targeted nanoparticles of photosensitizated curcumin conjugate anti-TfR monoclonal antibody characteristics are in line with nano-level:1) The liposome encapsulation has higher stability by film dispersion method. 2) Scanning electron microscopy showed that Cur-NP-PEG-Ab form stable, belonging to nano-level.3) FCM showed that Cur-NP-PEG-Ab has similar binding capacity with the parental murine monoclonal antibody.4) FCM detected under illumination condition, Cur-NP-PEG-Ab can affect hepatoma cells cycle, and promoted their apoptosis.5) Cur-NP-PEG-Ab impacted on mitochondrial potential and ROS, made mitochondrial potential decreased and higher ROS.2. Analysis the variable chains of TfR antibody.Amplified VH and VL genes of anti-TfR monoclonal antibody by PCR, the fragment size in line with the theoretical value, DNA sequencing also showed their are correct sequences. We got the functional light chain different from the former result, belonged to IGKV6-17*01 family has 324bp, and 60bp signal. In order to ensure the accuracy of tests, we tried several amplifications and sequence analyses, confirmed that the light chain was amplified by the primer VL-f3, and the previous sequence in line with VL-f2 primer. So we use a single primer amplification, respectively. We got three different light chain genes from one hybridoma: non-functional endogenous light chain of MOPC21 amplified byVL-fl primers; the primer VL-f2 amplified the variable VL genes was classified to IGKV4-59*01 family has 318bp, and 66bp signal peptide; the primer VL-f3 hybridized the sequence is not the same as in previous one, belonging to different family. Molecular simulation showed the Fabs of the two functional light chains and heavy chain both have binding grooves。[Conclusions]This study was prepared biodegradable mPEG-PLGA-CUR-NPs nanoparticles and preliminary studied the modified nanoparticles biological effects in vitro, obtained meaningful results, studies demonstrated that:(1)Preparation of the Cur-NP-PEG-Ab nanoparticles with high drug loading and encapsulation efficiency complied the nano-level; (2)Cur-NP-PEG-Ab had similar binding rate with target cells (HepG2) to the parental murine antibody; (3)Cur-NP-PEG-Ab enhanced tumor cell killing effect significantly in the light, suggesting that the nanoparticles in the completion of target killing of tumor, can play as a photosensitizer at the same time;(4)the paper first time reported that curcumin in the Cur-NP-PEG-Ab as a herb photosensitizer, liposome-modified-curcumin nanoparticles can play longer in the body metabolism, and after circulated through the body, curcumin coated off liposomes, can then play their anti-cancer effects of Chinese medicine. Based on this analysis of here light chain genes from the same hybridoma, and laid the foundation for the transformation of antibodies.