MMAE-loaded PLGA nanomedicine with improved biosafety to achieve efficient antitumor treatment

Monomethyl auristatin E(MMAE)is a derivative of the marine peptide Dolastatin 10,which has therapeutic effects against various cancers according to its antimitotic activity in multiple clinical trials.The antibody drug conjugate(ADC)of MMAE is currently used in clinical practice.However,the safety issues of MMAE-based ADC,such as high drug toxicity and poor bioavailability,still exist when using it for anticancer therapy.A sustained release of drug delivery approach should be used to reduce toxicity and achieve sufficient anticancer effects.Herein,PLGA-b-PEG 2000 with excellent biocompatibility and slow degradation ability was adopted to construct MMAE-loaded nanoparticles for safe and effective chemotherapy.The sustained release effect and the immunogenic cell death(ICD)effect of PLGA-MMAE nanoparticles were assessed by in vitro experiments.The PLGA-MMAE nanoparticles effectively accumulated in the tumor through the enhanced permeability and retention(EPR)effect,inducing cell apoptosis and causing a certain degree of immune response.The sustained drug release of PLGA-MMAE improved the bioavailability and effectively reduced the toxicity and development of the tumor compared to the effect of free MMAE or ADC.Overall,this study provides a safe and effective chemotherapeutic approach,as well as a simple and effective synthetic process for MMAE-based nanoparticles,improving their therapeutic efficacy and safety.

UPLC-MS/MS法同时测定大鼠血浆中一甲基澳瑞他汀E及其前药的浓度及药动学研究

目的 建立超高效液相色谱串联质谱法同时测定大鼠血浆中一甲基澳瑞他汀E(monomethyl auristatin E,MMAE)及其酶敏感性脂肪酸前药—一甲基澳瑞他汀E-缬氨酸-瓜氨酸-对氨基苄氧羰基-亚油酸(MMAE-valine-citrulline-PABC-linoleic acid, MMAE-V-C-LA)的质量浓度,并将该方法用于比较MMAE和MMAE-V-C-LA纳米粒在大鼠体内的药动学行为。方法 采用Phenomenex Kinetex XB-C_(18)(50 mm×2.1 mm, 2.6μm)色谱柱,甲醇-体积分数为0.1%甲酸溶液为流动相,采用梯度洗脱,流速为0.2 mL·min^(-1),采用电喷雾电离(ESI)源,正离子模式进行检测,采用沉淀蛋白法处理血浆样品,选择紫杉醇为内标。结果 大鼠血浆中MMAE及MMAE-V-C-LA的线性范围分别为5.000~2 000、10.00~10 000μg·L^(-1),最低定量下限分别为5.000μg·L^(-1)和10.00μg·L^(-1),日内、日间精密度的RSD在0.040%~14%内,准确度的RE在-1.3%~2.3%内,提取回收率在89.5%~95.7%内,内标归一化的基质因子在89.8%~97.7%内。结论 该方法准确可靠,符合生物样品测定要求,可用于MMAE和MMAE-V-C-LA纳米粒在大鼠体内的药动学研究。

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Highly efficient tumor-targeted nanomedicine assembled from affibody-drug conjugate for colorectal cancer therapy

Affibody is a new class of small non-immunoglobulin affinity proteins that possesses high affinity at the picomole level to several tumor overexpressed receptors.Owing to the simple framework,affibody is flexible for modification with payload,but the relatively low molecular weight of this construction simultaneously results in short half-life time which hinders its application in cancer therapy.In this work,we prepared a nanomedicine self-assembled from the conjugate of affibody(ZPDGFRβ:09591,PDGFRβ:platelet-derived growth factor receptorβ)with monomethyl auristatin E(MMAE)through cathepsin B cleavable dipeptide linker for targeted colorectal cancer therapy.The nanoscale characteristics of ZPDGFRβ:09591-MMAE affibody-drug conjugate nanomedicine(ZPDGFRβ:09591-M ADCN)resulted in enhanced pharmacokinetics,improved drug accumulation,and promoted biosecurity performance than those of free drugs.As a result,ZPDGFRβ:09591-M ADCN exhibited excellent antitumor efficacy with tumor inhibition rates(TIR)over 99.0%in PDGFRβ-positive tumor models with small solid tumors(~150 mm^(3))or large established tumors(~500 mm^(3)),indicating that ZPDGFRβ:09591-MMAE ADCN is promising for the clinic application in colorectal cancer therapy.

海洋细胞毒药物与抗体偶联物的制备及生物活性评价

目的以抗体N-糖链为偶联位点,制备曲妥珠单抗与海洋细胞毒药物单甲基澳瑞他汀E(MMAE)偶联物,并研究其对乳腺癌细胞BT474的杀伤作用。方法合成含叠氮和炔基修饰的唾液酸化寡糖,采用化学酶法制备含叠氮或炔基修饰的曲妥珠单抗,利用生物正交反应分别制备相应的抗体药物偶联物。利用SDS-PAGE对转糖基及偶联过程进行监测,表面等离子共振技术(SPR)对叠氮或炔基修饰前后抗体与FcγRIIIa亲和力进行检测。利用CCK-8法评价2种抗体药物偶联物对乳腺癌细胞株BT474的杀伤作用。结果成功制备了含叠氮或炔基修饰的曲妥珠单抗及N-糖链唾液酸位点抗体药物偶联物,修饰抗体与FcγRIIIa的亲和力较曲妥珠单抗增加,2种抗体MMAE偶联物对BT474有比曲妥珠单抗更明显的生长抑制作用。结论基于抗体糖链末端唾液酸位点偶联的方法可有效应用于海洋细胞毒抗体偶联药物的开发。