An Exploration Of Novel Antibody-drug Conjugate Targeting VEGF And EGFR In The Treatment Of Glioma

Background: Glioma is the most common malignant tumor of the central nervous system,known for its high invasiveness and generally poor prognosis.Glioblastoma(GBM)is the most common and most malignant type of glioma,with a median survival period of only 14.6 months.Existing treatments,including surgery and radiochemotherapy,have not been able to achieve complete cure of glioma.Antibody-drug conjugate(ADC)use monoclonal antibodies as the ‘guidance system’ to specifically bind to tumor cell surface antigens,and potent cytotoxic drugs as the "warhead" to target and kill tumor cells.Thus,ADC are often referred to as ‘biological missiles’.ADC possess target specificity and efficacy,making them promising in cancer treatment.In the design of ADC,the selection of targets is crucial.In the development of novel anti-glioma ADC,we have chosen two therapeutic targets,VEGF and EGFR.The malignancy of glioma is closely associated with abnormal tumor angiogenesis.Vascular endothelial growth factor(VEGF)plays a vital role in promoting endothelial cell proliferation,forming new blood vessels,and is a key therapeutic target in anti-glioma treatment.Bevacizumab is an FDA-approved VEGF-specific antibody used in clinical treatment of glioma;however,it has not significantly prolonged the overall survival(OS).In this study,by utilizing the specificity of bevacizumab to VEGF and the potent cytotoxicity of monomethyl auristatin E(MMAE),a novel anti-glioma ADC(AGBM-22)was developed using the dipeptide linker Valine Alanine(VA).Moreover,epidermal growth factor receptor(EGFR)is a transmembrane protein kinase that plays a crucial role in the proliferation and growth of tumor cells.Overactivation of EGFR is closely associated with the development of tumors.Cetuximab is a specific antibody to EGFR with high affinity to tumor cell surface EGFR.It was approved by the FDA in 2004 for clinical treatment of metastatic colorectal cancer and advanced squamous cell carcinoma.Research has shown that EGFR is overexpressed in approximately 40%-70% of GBM patients.Although cetuximab specifically targets and binds to EGFR in GBM cells,it does not show inhibitory effects on the growth of GBM cells in vitro.Therefore,we utilized the specificity of cetuximab to EGFR and the cytotoxicity of MMAE,linked through VA,to create a novel EGFR-based anti-glioma ADC(AGCM-22).Objective: 1.Using chemical conjugation techniques,novel glioma ADC(AGBM-22 and AGCM-22)with anti-glioma effects were synthesized,and their structures were characterized and quality controlled.2.Evaluate the anti-tumor effects and metabolic properties of AGBM-22 and AGCM-22.3.Investigate the specific mechanisms by which AGBM-22 and AGCM-22 exert antiglioma effects.Methods: 1.Using chemical conjugation techniques,two anti-glioma ADC(AGBM-22 and AGCM-22)were synthesized.2.The structures of AGBM-22 and AGCM-22 were characterized by proton nuclear magnetic resonance spectroscopy(1H-NMR)and mass spectrometry(MS).3.The DAR of AGBM-22 and AGCM-22 were determined by reverse phase high performance liquid chromatography(RP-HPLC)and MS.4.The targeting abilities of AGBM-22 and AGCM-22 were evaluated through the establishment of in situ glioma xenograft mouse models.5.The in vitro metabolic properties of AGBM-22 and AGCM-22 were assessed through co-incubation experiments.6.The inhibitory effects of AGBM-22 and AGCM-22 on glioma cell proliferation in vitro were measured through crystal violet and CCK-8 assays.7.The anti-glioma effects of AGBM-22 and AGCM-22 in vivo were evaluated in human and murine glioma xenograft models,along with assessing in vivo safety.8.The mechanisms underlying the antitumor effects of AGBM-22 and AGCM-22 were explored through flow cytometry,Western blot,and immunofluorescence experiments.Results: Through chemical conjugation techniques,we successfully synthesized two ADC targeting glioma,named AGBM-22 and AGCM-22.The structures of AGBM-22 and AGCM-22 were characterized by 1H-NMR and MS;and the DAR of AGBM-22 and AGCM-22 were determined to be 4.7 and 3.9,respectively,providing a rational basis for the anti-glioma efficacy of AGBM-22 and AGCM-22.AGBM-22 and AGCM-22 exhibited excellent tumor targeting capabilities in in vitro and in vivo experiments,along with high stability in non-tumor tissues,thus minimizing off-target effects.In vitro experiments showed significant proliferation inhibition of glioma cells by AGBM-22 and AGCM-22,and markedly demonstrated anti-tumor effects in an orthotopic glioma mouse model.AGBM-22 and AGCM-22 induced G2/M phase arrest in glioma cells by reducing the expression levels of tubulin proteins,thereby inhibiting cell proliferation;and promoted cell apoptosis while inducing incomplete autophagy,leading to glioma cell death.Conclusion: This study synthesized two novel antibody-drug conjugates,AGBM-22 and AGCM-22,using ADC strategy.In vivo and in vitro experimental results indicated that AGBM-22 and AGCM-22 exhibited high specificity towards glioma cells with high expression of VEGF and EGFR.They demonstrated a strong anti-tumor effect in orthotopic glioma mouse models and showed good safety profiles.AGBM-22 and AGCM-22 exerted anti-tumor effects by inducing cell cycle arrest to inhibit glioma cell proliferation,promoting cell apoptosis,and inducing cell autophagy to facilitate glioma cell death.In conclusion,AGBM-22 and AGCM-22 are promising ADC that provide research references for future safe and effective drug treatments for glioma.Innovation Point: 1.Two novel ADC(AGBM-22 and AGCM-22)have been developed.2.AGBM-22 and AGCM-22 exhibit targeting ability and good stability.3.Both AGBM-22 and AGCM-22 demonstrate efficient anti-glioma effects in vitro and in vivo activity evaluations,with good safety profiles.4.AGBM-22 and AGCM-22 exert anti-tumor effects by inducing cell cycle arrest,promoting apoptosis,and inducing autophagy to facilitate glioma cell death.