Design,Synthesis And Biological Evaluation Of Antibody-Drug Conjugates Based On Novel Linkers

Cancer is the leading cause of death in humans and the number of new cases and deaths per year is still on the rise.At this stage,antitumor drugs mainly include monoclonal antibodies(mAb)and small molecule chemotherapy drugs.Although the antibody drugs have good targeting characteristic,but there are still problems of insufficient efficacy and drug resistance.Chemotherapy drugs have advantages of high cell killing activity and variety mechanisms of action,but the adverse effects caused by poor targeting still restricted its clinical application.Antibody-drug conjugate(ADC)includes three components:antibody,cytotoxin and linker.It combined the advantages of the mAb with the cytotoxic drug,which use mAb to target tumor and release cytotoxins inside the cells to induce tumor apoptosis.ADC have the characteristics of high activity,small toxic side effects and long acting time,which had greatly enhanced the therapeutic index of chemotherapy cytotoxins.There are currently four ADCs in the market(Mylotarg?,Adcetris?,Kadcyla?,and Besponsa?),and nearly a hundred of ADCs are now in the stage of clinical research.ADC has become another hot topic in the development of antitumor drugs besides immunotherapy now.The construction of linker is a key factor restricting the development of ADC,which were used as hubs for the part of ADC.The basic requirement for linker including two requirements:one is ensure ADC’s stability during the circulatory system,and another is that cytotoxin can be efficiently released after the ADC enter into the tumor cells.Existing linkers can be divided into enzymatic cleavable linkers,chemical cleavable linkers,and non-cleavable linkers three categories by their drug release mode,and there are still several challenges that need to be addressed:(1)The succinimide,which is widely used in the listed or in research ADC linkers,may cause off-target inactivation of about 50%of the cytotoxins since its inherent stability problems.Many pharmaceutical giants are looking for solutions with yet no result and challenges remained.(2)For the most mainstream enzymatic linkers,their drug release mechanism currently is extremely simple and only include cathepsin B andβ-glucuronidase,which are normal lysosomal enzymes,leaving tumor-specific enzymatic drug released study of ADC is still blank.(3)For chemical cleavable linkers,there are obvious limitations in the cleavage structure fragment,which only involved the imine,disulfide and carbonate bond etc,for which chemical space is still need to be further expanded.(4)There are still plenty of space for the improvement in terms of the drug-resistance and preparation of ADC that can be benefit from linkers.In view of the above problems,this study give full play to the subject advantages of medicinal chemistry,starting from the construction of novel linkers,we designed and synthesized five types of ADCs and conducted targeted research and evaluation:1.In view of the ubiquitous cytotoxin off-targetting problem from the maleimide in the traditional linker of ADC at this stage,one monomethyl maleate based linker was first developed.Such linkers are simple and readily available with which metabolites are clear,and can remarkably improve the stability of the ADC while maintaining the good reaction kinetics of original linkers.3 ADCs(YM03～YM05)based on this type of linkers are obtained,and YM04’s stability,efficacy and drug safety studies show the overall performance superior to the ADC with traditional maleimide linker.This new class of linkers expected to provide a simple and effective solution to the long-awaited ADC cytotoxin off-target challenge.2.In view of the problems of single enzymolysis type and non-specific tumor drug released mechanism,this thesis first explored new mechanism of drug release based on specific aryl nitro reductase in solid tumor anoxic environment,and three ADCs(YM06～YM08)were designed and synthesized.The drug resistance of YM08 was systematically evaluated from several aspects,such as in vitro stability,selectivity of enzymolysis,drug release at cellular level,pharmacodynamics in vitro and in vivo and drug safety.The evaluation results basically accord with the design expectation,and this strategy not only enriches the mechanism of enzymolysis and drug release,but also expected to provides a better choice for the development of solid tumor ADC.3.Aiming at the single structure problem of chemical cleavable ADC,this study for the first time designed a silylether acid sensitive cleavable ADC(YM09)that with the mainstream cytotoxin MMAE as the payload.The evaluation results of YM09,which including in vitro stability,acid-dependent drug release performance,efficacy and safety and other systemic tests,validates the potential of this drug release model to some extent.This study is expected to provide a suitable guide for broadening the model of ADC release.4.Furthermore,a new linker containing Val-Ala(VA)dipeptide was designed to replace Val-Cit(VC)for ADC that with MMAE as the payload,which has the advantages of readily available raw materials,simple preparation process and so on.Two VA-based ADCs(YM10 and YM11)were prepared,and one VC-based ADC(YM12)was prepared used as control.Compared with YM12,system comparison studies show that YM11 can give full play to the original medicinal properties and improve the aggregation of the product while exerting the advantages of VA dipeptide raw materials and processes.For this type of alternative linkers,we are conducting further research to expand their potential applications.5.In addition,we also designed and synthesized an ADC(YM13)using the dominant cytotoxic MMAE with non-cleavable conjugation pattern,which efficacy in vivo and in vitro and safety were further evaluated.Preliminary studies have shown that YM13 has good efficacy and safety in vivo.This type of non-cleavable MMAE based ADC has the value of further research,and expected to provide new possibilities for improving the stability and safety of the MMAE-ADC.In summary,from the perspective of linker construction,we designed and synthesized 5 new types of ADCs in total of 11,and 13 linker-MMAE conjugates,17 linkers,1 MMAE substitute and 5 linker-MMAE substitute conjutates also have been synthesized.The above ADCs meet the requirements of a number of indicators such as concentration,degree of polymerization,DAR,drug distribution,endotoxin content before carrying out biological evaluations,and the corresponding linkers and related conjugates were confirmed by 1H-NMR and MS.The above works not only provide solutions to solve the problems existing in ADC at this stage,but also provide some references for enriching and improving the current conjugating technology of ADC at the same time.