Endocytic Mechanisms Of The Antibody-Drug Conjugates Targeting HER2-Positive Breast Cancer Cell

Breast cancer is emerging as an important threat in women’s lives and health, and the number of women suffering from breast cancer is increasing, of which 20-30% are HER2 overexpression patients. Although monoclonal antibody has seen great success in application to malignancies with high target-specifity, low self-heterologous, minimal non-specific cell killing and low side effects. However, a part of HER2-posisitive patients are not sensitive to monoclonal antibody therapy, accompanied by the development of drug-resistance, providing a challenge for clinical theraputic. A strategy that combine shigh cell-killing potenty of cytotoxic agents with high target-specificity of antibodies would represent a potentially new paradigm in cancer treatment.ADCs provide such an choice, where the antibody component provides specific target tumor antigen and the drug moiety confers the cytotoxicity. The mechanism of ADCs is that the antibody binding to tumor targets, ADC lysed in tumor cells to release the effector molecule which caused apoptosis. The effeciency of ADCs depend on their ability to specifically bind the target cell, and properly release the cytotoxic agents. So it is important to study endocytosis, intracellular transport and clearance of ADCs. However, HER2-mediated endocytosis and intracellular transport remain poorly understand.Most recently, Kadcyla(ado- trastuzumab emtansine, T-DM1), which combines the humanized antibody trastuzumab and a potent antimicrotubule cytotoxic agent DM1 with a highly stable linker, was approved for the treatment of patients with Her2-positive breast cancer. P-DM1 was connected Pertuzumab with DM1 through a nonreducible thioether linkage SMCC. P-MMAE was designed using a valine-citrulline dipeptide linker to bridge the Pertuzumab antibody and an antimicrotubule cytotoxic agent MMAE. WBP265 conjugated effectively and specifically 265 SD which is introducing unnatural amino acid to the trastuzumab with MMAF, resulting site-specific the ADC. By monitoring key factors such as the monoclonal antibody, cytotoxic drugs, linker on ADCS-receptor interaction, and their in vitro binding properties to verify that these factors on their endocytosis rate and removal rate impact, further elaborated their mechanism of action.Our work is divided into two parts: study the interaction between ADCs and HER2 receptor, and study the endocytosis mechanism of ADCs.At First, we studied the interaction between ADCs and HER2 receptor. We used capture method,specific experimental methods is that amine coupling kit amino Anti-human Ig G(Fc) antibody conjugated to dextran CM5 sensor chip surface of the substrate. Firstly ADCs diluted by freshly prepared HBS-EP + buffer was injected to the detection channel; secondly HER2 receptor solution was injected on chip surface to the detection channel and reference channel; thirdly chip was regenerated by injection of 3M Mg Cl2 to prepare for the next cycle. The bonding reaction at 25 ℃. Biacore T200 Control Software SPR signal acquisition and save the resulting curve using 1: 1 Lamgmuir model to describe the dynamics model fitting. Biaevaluation analysis software for data processing, computing affinity and kinetics for the test product. Smaller KD values indicate stronger antibody-antigen affinity.In this experiment, Biacore T200 detected that KD value of antibody-drug conjugates or positive drugs(monoclonal antibody)with the HER2 receptor is about 10-10,which has high affinity and high activity. According to Biacore criteria, generally considered that KD values is diciation by less than five times, and capacity can be determined as no difference. Affinity of T-DM1(KD value of was 133.9 p M) was corresponded with Trastuzumab(KD value of was 94.2 p M); P-DM1, P-MMAE(KD value of P-DM1 was 722.1 p M, P-MMAE was 326.7 p M) had greater affinity than Pertuzumab(KD value was 1191 p M), wherein P-MMAE is stronger than the P-DM1; while affinity with ligand, WBP265 is equivalent with 265 SD monoclonal antibody(KD value of 265 SD was 606.6 p M, KD value of WBP265 was 577.3 p M).Second, we used immunofluorescence method to observe directly the binding properties between ADCs/mab with HER2 in vitro, flow cytometry experiments observed a saturated binding between the receptor and the ligand. Ligand and receptor binding is a dose-effect relationship, the concentration range from 0.3125-20 the μg/ml, with increasing concentrations, MFI increased gradually, till reach the concentration of 20 μg/ml. By ADCs, HER2 monoclonal antibody binding curves were found: T-DM1 binding HER 2 capacity was slightly weaker than Trastuzumab; P-DM1, P-MMAE binding HER 2 ability was weaker than Pertuzumab, but P-MMAE slightly stronger than P-DM1; WBP265 binding HER 2 ability was weaker than 265 SD. Comparison among different monoclonal with same other structures : from the binding curves of T-DM1 and P-DM1, P-DM1 slightly stronger than T-DM1. Comparison binding capacity of three m Ab, 265 SD was strongest, followed by Pertuzumab and Trastuzumab.The second part was to study in vitro endocytosis of antibody drug conjugates. Within the antibody-drug conjugates after swallowing enters the intracellular degradation in lysosomes. First, the use of laser scanning confocal microscopy in real-time detection of ADCs drug transporters and endocytosis rate of SKBR3 cells in negative cells MCF, the endocytosis phenomenon was not observed. In the positive cells SKBR3, ADCs or monoclonal antibody firstly binded with cell surface, endocytosis into the cell a period of tim, after arriving lysosomal degradation, indicating the test by receptor-mediated endocytosis to enter to lysosomes. After the test-positive cells after treatment, showing T-DM1 by endocytosis after 4 hours into lysosomes, and monoclonal antibody trastuzumab for 2 hours to reach lysosomes, indicating that T-DM1 endocytosis rate of less than Tratuzumab; the P-DM1 and 6 hours after coculture swallow lysosomes, P-MMAE treated within eight hours to reach the lysosome endocytosis, and after 4 hours pertuzumab treatment endocytosis reach lysosomes, Description P-DM1, P-MMAE endocytosis rate of less than Pertuzumab; and antibody conjugates WBP265 and 265 SD monoclonal antibody and cell co-cultured for 8 hours to reach the lysosomes are described both the same endocytic rate. Comprehensive found that endocytosis rate of anti-HER2 antibody-drug conjugates is less than the rate of endocytosis of its monoclonal antibody.Then immunofluorescence Methods was applied to assay in vitro clearance rate of ADCs and MAb. Flow cytometry resultes showed that the four different antibody drug conjugates in SKBR3 breast cancer cell depletion was in a time- dose dependent manner. With prolonged incubation fluorescence intensity weakened, compared to under 4℃ treatment conditions have remained unchanged in the MFI, show that within antibody conjugate into the cell after swallowing degradation. At the same time, it can be drawn from the T-DM1 clearance less than trastuzumab clearance; clearance rate of P-DM1, P-MMAE were less than pertuzumab; clearance rate of WBP265 was less than clearance rate of 265 SD. That antibody drug conjugates clearance less than universal monoclonal antibody clearance. The results of this study was consistent with binding properties and in vitro internalization experiments.In summary, we used SPR technology to study the interactions of antibody-drug conjugates and their corresponding monoclonal antibodies with the receptor HER2, to investigate their binding affinity and kinetic features; and we also used immunofluorescence in vitro experiment to verifiy their binding properties, therefore providing an important theoretical basis for pharmacokinetic and pharmacodynamic evaluations of the ADC drugs. The understanding of the mechanisms of ADCs in targeted breast cancer therapy at molecular and cellular level provided a reasonable guide for treatment and drug development in the future. This work, by comparing its clearance rate and endocytosis, put new sight in the understanding of cell internalization and the mechanism of the ADC drugs, providing a useful guider for antibody-drug conjugates optimization for future clinical applications.