Preparation And Properties Study Of Targeted Biotechnology Drugs Based On EGFR Antibody

Anti-body drug conjugates(ADCs)as a novel biotechnological drug for cancer treatment have been approved by regulatory agencies such as FDA and NMPA.The specific targeting and drug delivery effects of ADCs for tumor treatment,as well as their relative safety characteristics,have been positively evaluated in clinical applications.Currently,in addition to the technical challenges of stability during distribution and effective drug release after entering target cells,the process of ADCs(D>10 nm and V>1800 nm~3)diffusing from blood into tissues and further penetrating tumor cells is determined by the potential energy factors driven by drug concentration gradients,resulting in an exponential decrease in drug concentration.It is known that most anti-tumor drugs can kill or eradicate one tumor cell per four molecules.Studies have shown that the typical ADCs with drug antibody ratio(DAR)≤3.Therefore,to achieve effective clinical outcomes,high-dose administration and personalized patient management are prominent considerations.The technical approach to solving this bottleneck problem involves significantly improving the DAR without compromising the high target specificity of ADC drugs,using epidermal growth factor receptor(EGFR)ADC-like drugs as an example in the paper.Based on genetic engineered antibodies and endogenous proteins of the body as raw materials,a biocompatible and highly targeted nanocarrier for drug delivery with dual-responsive release in the tumor microenvironment was designed and prepared.While maintaining the targeting characteristics of specific tumors,the drug loading capacity was effectively increased,and both in vitro and in vivo evaluations were performed.(1)Preparation and functional evaluation of humanized EGFR full-length antibody and its single-chain antibody(sc Fv)Plasmids expressing the full-length anti-EGFR antibody heavy and light chain genes(p FUSEss-CHIg-h G1 heavy and p FUSE2ss-CLIg-hk light)were simultaneously transfected into CHO-K1 cells.Monoclonal cell lines expressing full-length EGFR antibody(AE01)were screened and obtained.The expressed product AE01 had a molecular weight of approximately150 k Da.Using a similar method,CHO-K1 cells expressing EGFR single-chain antibody(hus A)were constructed,with hus A having a molecular weight of approximately 28 k Da.NIH/3T3mouse embryonic fibroblast cells,which are EGFR-negative,were used as a control.Flow cytometry analysis showed that both AE01 and hus A specifically bound to EGFR-positive human epidermoid carcinoma A431 cells and inhibited A431 cell proliferation in a concentration-dependent manner.Co-incubation with EGF showed that AE01 had an inhibitory effect on EGF-activated EGFR-mediated proliferation of A431 cells equivalent to the original therapeutic antibody panitumumab.Laser confocal quantitative analysis revealed that approximately 80%of AE01 and 60%of hus A were internalized by A431 cells after co-culture for 2 hours,indicating the potential of AE01 and hus A as targeted components of a nanodelivery drug system for intervening in EGFR overexpressing tumors.(2)Design and Preparation of EGFR Full-Length Antibody and Double-Layer Human Serum Albumin(HSA)Modified Mesoporous Silica Nanoparticles(FMSN-DOX-H2-AE01)Given that mesoporous silica nanoparticles have the characteristics of a large specific surface area,low toxicity,adjustable pore volume,pore size,and particle size,and the ability to easily encapsulate various tumor therapeutic drugs,this paper takes doxorubicin(DOX),a frontline anthracycline-type tumor therapeutic drug,as an example for implementation.Mesoporous silica nanoparticles(FMSN)with a diameter of 80 nm,labeled with fluorescein isothiocyanate(FITC),were prepared using a classic method.DOX was dissolved in methanol and loaded into FMSN particles at room temperature,achieving an encapsulation efficiency of2.4%.To avoid premature drug release during tissue distribution,FMSN-DOX was double-modified with human serum albumin(HSA)to obtain FMSN-DOX-H2.A dual-functional linker was used to connect AE01 to the surface of FMSN-DOX-H2,resulting in a nano drug delivery system,FMSN-DOX-H2-AE01,with a diameter of approximately 120 nm,decorated with 145 AE01 molecules and loaded with 2.95×10~4 DOX molecules.In vitro cell experiments revealed that FMSN-DOX-H2-AE01 nanoparticles did not show significant inhibitory effects on normal control cells at the given intervention dosage.The particles effectively enhanced the internalization capability of A431 cells with high expression of EGFR in a dose-dependent manner;no clear macrophage clearance of the particles was observed.In vivo experiments showed that the particles mainly accumulated in the transplanted tumor tissue,resulting in a significant slowdown of tumor growth and a higher proportion of tumor tissue death during the observation period,suggesting that these particles have the potential to interfere with tumor growth similar to ADCs.(3)Design and Preparation of EGFR Single-Chain Antibody hus A and Monolayer HSA-Modified Mesoporous Silica Nanoparticles(FMSN-DOX-H-hus A)The therapeutic efficacy of targeted drugs is closely associated with the drug concentration in the target tissue,and the drug concentration in the tissue is influenced by various factors.Reducing the particle size of drugs and maintaining a high DAR is one of the methods to improve particle quality.Previous experiments have observed that single-layer human serum albumin(HSA)-modified particles can achieve similar effects as double-layer modification.In this study,a 28 k Da humanized epidermal growth factor receptor(EGFR)single-chain antibody(hus A)was used as the targeting molecule to replace AE01,and HSA single-layer modified particles were used to reduce the effective diameter of the particles and improve the penetration rate of drug particles in tumor tissue.Using a similar method,FMSN-DOX-H-hus A nanoparticles with a diameter of 100 nm were prepared,obtaining nanoparticles carrying 151hus A molecules and 4.44×10~4 DOX molecules,which had 50%higher drug loading capacity than FMSN-DOX-H2-AE01.In vitro cell experiments showed that,at equivalent dosages,the cytotoxic effects of FMSN-DOX-H-hus A nanoparticles on A431,human cervical cancer epithelial cells(Hela),and human breast cancer ductal epithelial cells(MCF-7)were significantly better than those of FMSN-DOX-H2-AE01 nanoparticles,with a decrease in cell viability ranging from 5%to 40%.In vivo experiments revealed that,under equivalent dosage conditions,the distribution concentration of FMSN-DOX-H-hus A nanoparticles in tumor tissues was approximately 30%higher than that of FMSN-DOX-H2-AE01 nanoparticles.Both types of nanoparticles significantly slowed down tumor growth during the observation period,but histological observations showed a higher proportion of tumor tissue death with FMSN-DOX-H-hus A nanoparticles intervention.Taking the current limitations of improving the ADC of targeted drugs as a problem-oriented approach,the paper proposes a nanoparticle design that maintains specific targeting and improves DAR to achieve the desired functional expression,providing a feasible design idea for the utility and clinical treatment of targeted drugs.