Design,Synthesis And Biological Evaluation Of Novel Antibody-Drug Conjugates With Anti-Tumor Effects

Antibody-drug conjugate（ADC）has become a hot topic in the research and development of anti-tumor drugs,which creates a novel situation for tumor treatment.This type of drug is an organic whole composed of three components:antibody,linker and cytotoxin.It can deliver the small molecule cytotoxin to the target tissue and release it by the targeting ability of antibody,so as to effectively kill the tumor cells and realize the strong combination of antibody and small molecule chemotherapy drugs in terms of the advantage.Currently,6 ADCs have been approved for marketing in the world.Although the concept of ADC is simple,the ideal ADC design is very complex,and the change of each component may affect it as a whole.There are still many challenges in the further development of ADC:（1）At present,the antibodies used in ADC with Monomethyl Auristatin E（MMAE）as cytotoxin are all non-immune antibodies,which are relatively single.The design concept is mainly to use the targeting of antibodies to deliver cytotoxins to the tumor site.However,it has not been fully considered how to coordinate the other therapeutic effects of antibodies.（2）The traditional ADC linker design strategy only considers two points.One is to ensure the stability of the linker substructure in the blood.Second,linkers in tumor tissues depend on enzymes and other factors to degrade and release cytotoxins.However,these enzymes and other factors often exist in normal tissues,which can make ADC produce non-specific drug release.（3）ADC has high requirements for cytotoxins,which usually requires the effective concentration at the picomolar level.Only a few cytotoxins can be used in ADC.For example,microtubulin inhibitors MMAE and DM1,but these only act on tumor cells in the division phase.Therefore,it is necessary to further explore new ADC cytotoxins with different mechanisms and high activity.In this paper,a series of innovations and improvements were carried out to solve the problems of the above three components of ADC.Four kinds of new ADCs with anti-tumor effect were designed,and their synthesis and targeted biological evaluation were carried out.1.In order to solve the problem of single effect of MMAE-ADC antibody,in this paper,we combine the advantages of current immunotherapy antibodies with ADC technology to conjugate cytotoxin MMAE with programmed cell death ligand-1（PD-L1）antibody through enzyme-cleavable dipeptide linker,and design and synthesize four novel MMAE-ADC（class I）based on immune synergy mechanism.This strategy can take advantage of the high expression of PD-L1 antigen in a variety of tumor cells to achieve targeting ability,but also can activate immune function of tumor-infiltrating T cells by blocking the PD-1/PD-L1 signaling pathway,and synergize with ADC to kill tumor cells to enhance the therapeutic effect.The biological function and in vitro and in vivo activity of this kind of ADC were evaluated.The results showed that the prepared ADC 3 had similar antigen affinity and internalization ability to PD-L1 antibody.It has good inhibitory activity and selectivity to many kinds of tumor cell lines with high expression of PD-L1,and the minimum IC₅₀ value can reach 9.75 nM.Meanwhile,ADC 3 can significantly enhance the activity of T cells,retain the immune activation function of PD-L1 antibody,and play a synergistic role with ADC in killing tumor cells.In the mouse xenograft model,it shows better anti-tumor effect than PD-L1 antibody,and has great application potential.2.Aiming at the problem of non-specific drug release of linkers,this paper attempts to solve this problem through two innovative design strategies.（1）Strategy 1:design a new type of ADC based on the drug release mechanism controlled by ultraviolet（UV）light（classⅡ）.In this paper,we consider whether exogenous light conditions can be used to regulate the release of cytotoxin in tumor site without relying on the traditional non-specific drug release mode.Specifically,two target ADCs were designed and synthesized by skillfully replacing the o-nitrobenzyl（ONB）structure that belongs to a UV-activated group with the p-aminobenzyl spacer on the traditional ADC linker.The drug release,in vitro efficacy and in vivo targeting were further evaluated.The results showed that ADC 5 and 6 could release MMAE rapidly after transient UV irradiation,and effectively killing Hepceptin resistant tumor cells,and had the best inhibitory activity on BT474-Her DR cells.IC₅₀ values were all 0.04 nM,and the inhibitory activity was 51 times and 54 times higher than that in the absence of UV irradiation.At the same time,through in vivo fluorescence imaging experiments in mice,the ADC showed good tumor targeting ability and organ specificity.（2）Strategy 2:design a new type of ADC based on the drug release mechanism of nitro reductase（classⅢ）.The expression of nitroreductase（NTR）was up-regulated in the hypoxic environment of solid tumors,but down-regulated in normal tissues.In our previous work,p-nitrobenzyloxycarbonyl（PNBC）was first introduced into the ADC linker by using the specific reduction mechanism of NTR in solid tumors,and obtained two novel ADCs based on NTR release mechanism.In the enzyme and cell drug release experiments,ADC 7 and 8 could release the cytotoxin MMAE under hypoxia,showing significant NTR drug release specificity.At the same time,ADC 7 and 8 showed the same inhibition level to the tumor cells with human epidermal growth factor receptor 2overexpression as the cytotoxin MMAE.Further in vivo studies are ongoing.The above studies preliminarily prove that the novel ADC strategy of the above two drug release mechanisms is feasible.3.Aiming at the problem of ADC cytotoxins with few types and single mechanism of action,ADC with Talazoparib as a new mechanism cytotoxin was designed for the first time（classⅣ）,and conducted a preliminary exploration on whether Talazoparib can become an ADC cytotoxin.Talazoparib is a poly（ADP ribose）polymerase（PARP）inhibitor with an IC₅₀ of 0.57 nM.The drug can inhibit DNA repair and induce apoptosis of BRCA deficient tumor cells through synergistic lethal effect,which has a mechanism different from other cytotoxins of ADC.In this paper,using the above design strategy of linkers,one UV-activated prodrug 47 and five NTR hypoxia-activated prodrug 59-63 were synthesized by blocking the active site of Talazoparib with ONB group and PNBC group.This prodrug strategy can avoid the release of highly toxic talazoparib to normal tissues before reaching the tumor site,and can complete the specific release of the cytotoxin under special conditions such as UV light or hypoxia.The biological evaluation of prodrug 47 was carried out.It has good PBS stability.Compared with talazoparib,the inhibitory activity was reduced by 380 times and 658times respectively in PARP-1 enzyme test and PARylation test,which proved that the activity was blocked successfully.On the other hand,in cytotoxicity test,prodrug 47could quickly restore its inhibitory activity against BRCA gene deficient MX-1 and Capan-1 tumor cells after short UV irradiation,with IC₅₀ values of 0.577μM and 0.092μM,respectively.It has the potential to be used as an ADC cytotoxin.The normoxic in vitro pharmacodynamic experiment of prodrug 59-63unexpectedly found that the inhibitory activity of the prodrug 59-63 was similar to that of the original drug Talazoparib at the enzyme and cell level,indicating that it can be developed as an independent new ADC cytotoxin.At present,the work is under way to connect the above two kinds of prodrugs with different linkers and then conjugate with antibodies to prepare ADC.To sum up,in the paper,four types of novel ADCs have been designed.Simultaneously synthesized four new ADC molecules,four key linkers,four linkers-cytotoxin payloads and six prodrugs with potential to become ADC cytotoxins.In view of the problems existing in the design strategy of ADC,the innovation is conducted from antibody,linker and cytotoxin,respectively.In addition,these design strategies are combined with each other,and the preliminary exploration and verification are carried out.These work provide some new ideas for the further study of ADC.