The Design Of Antibody-drug Conjugates And Studies On Their Specific Antitumor Activities

Recent successes in the development of ADCs for targeted cancer therapy,such as Brentuximab vedotin(Adcetris(?),SGN-35)and Trastuzumab emtansine(Kadcyla(?),T-DM1),have proven that ADCs can be potent weapons used in the battle against cancer.All approved ADCs are produced by chemical conjugation that covalently attaches the toxic payload to the antibody through surface-exposed lysine or cysteine residues generated by reducing intrachain disulfide bonds of the antibody,which leads to heterogeneous mixtures in terms of position and stoichiometry of the payload coupled to the antibody(defined as "drug to antibody ratio",DAR).Consequently,site-specific conjugation technologies urgently need to be developed to produce homogeneous ADCs.1 Direct enzymatic approachSortase A(SrtA)-mediated ligation is a highly specific platform for conjugation that relies on the specificity of SrtA for short peptide sequences(LPXTG and GGG).SrtA retains its specificity while accepting a wide range of potential substrates,but its broad use is limited by the wild-type enzyme's poor kinetics,which requires large amounts of SrtA and extended reaction time for efficient conjugation.We previously have used the wild-type SrtA(AN59)to conjugate anti-CD20 ofatumumab(OFA)with GGG-vc-PAB-MMAE to yield the site-specific OFA-vcMMAE.Recently,a further improved SrtA variant,SrtA(7M),with increased efficiency was reported.We expressed the wild-type SrtA(?N59)and the mutant SrtA(7M),and explored their coupling efficiency between the LPETG-tagged OFA and oligoglycine-modified MMAE.The wild-type SrtA(?N59)was more efficient in our system,so we used SrtA(AN59)to yield approximately 70%of modified heavy chain for OFA-vcMMAE(DAR=1.4).However,the conjunction was very inefficient for the light chain,which may be not accessible to the light chain for the enzyme and the substrate due to steric hindrance.2 Chemo-enzymatic approachIn order to improve the coupling efficiency,we presented a chemo-enzymatic approach using sortase-mediated ligation and Cu(I)-free click reaction to generate OFA-GPN-vcMMAE.Firstly,we engineered the anti-CD20 antibody by introducing a flexible short peptide linker(GGGGS)between the C-terminus of the light/heavy chains and the LPXTG recognition sequence to reduce steric hindrance.Moreover,the larger oligoglycine-functionalized toxins(GGG-vc-PAB-MMAE)are less reactive than the small molecular weight isotype control substrates,so we introduced an easy-to-synthesize bifunctional oligoglycine-modified small molecule(GGG-PEG-N3,GPN)comprising a bio-orthogonal azide functional group at the C-terminus through enzymatic catalysis to accomplish site-specific covalent attachment of functional molecules to the C-terminus of OFA,and subsequently reacted with a suitable MMAE derivative(DBCO-PEG-vc-PAB-MMAE)via strain-promoted azide-alkyne cycloaddition(SPAAC)with a 1:2 molar ratio of azide to alkynyl groups to generate the desired OFA-GPN-vcMMAE(DAR=3.3).The chemo-enzymatic approach could effectively control the position and stoichiometry of the payload coupled to the antibody,and the homogeneity of OFA-GPN-vcMMAE was higher than general ADCs constructed by traditional lysine or cysteine ligation.In addition,the chemo-enzymatic approach not only increased the DAR in comparison with the direct enzymatic approach,but also obviously reduced the dosage of MMAE which is more suitable for industrial large-scale production,since toxins are usually expensive and may cause hazardous waste during the manufacturing procedure.3 Specific antitumor activities in vitro and in vivoCD20 is a non-internalization antigen.Previous studies have showed that anti-CD20 antibodies linked to doxorubicin,ricin chain A,or to immunoliposomes containing doxorubicin were ineffectual avenues for drug delivery to the cell interior.For the two prepared anti-CD20 ADCs,we investigated their specific antitumor effects at the cellular level and in the xenograft models of human B-lymphoma,respectively.OFA-vcMMAE prepared direct enzymatic approach and OFA-GPN-vcMMAE prepared by chemo-enzymatic approach basically retained the binding affinity to cell surface CD20,and could be internalized into the CD20-positive cells and trafficked to the lysosome to release the highly toxic MMAE to exert antitumor effects.OFA-GPN-vcMMAE had a better efficacy and safety in vivo compared with OFA-vcMMAE.