Stiudies On The Design Of Novel-carrier And Novel-target Based Antibody-drug Conjugates

Antibody-drug conjugates(ADCs)combine antigen-targeting mAb with potent cytotoxic drugs via stable linkers.Thus they can bring drugs specifically into tumor,which greatly improves the drug concentration in the site of tumor,and reduces the drug concentrations in other tissues so as to expand the therapeutic window of drugs.At present,ADCs are mainly based on full-length antibody carrier and tumor associated antigens(TAAs)targeting.While antibody is too large to permeate into inside of tumor,resulting in low utilization in vivo.In order to broaden the scope of the concept of ADC,some small antibody fragments such as ScFv and Fab,or even the smaller peptide,have been used as carrier in ADCs,to improve permeability and reduce immunogenicity.But still failed due to their short half-life and low affinity.Although TAAs are highly expressed in tumor cells,there are also low level of expression in some normal cells.Resulting the"on-target-off-tumor" side effect during the long period of circulation in vivo,which limits the clinical application of ADCs.In this paper,we sought out to study the design of novel-carrier and novel-target ADCs,hoping to expand the application of ADCs and improve ADCs' safety.As for the novel-carrier ADC,we chose the TNF related apoptosis-inducing ligand(TRAIL).TRAIL was conjugated with MMAE,termed ligand-drug conjugate(LDC),to increase the activity of TRAIL and overcome drug resistance.PEGylation was applied to prolong half-life of TRAIL-MMAE conjugate,and enhanced the accumulation of drug in tumor site.After comparing the binding affinity of different PEG/MMAE ratios of PEG-TRAIL-MMAE conjugates,we found that either over-loading of PEG or MMAE would attenuate TRAIL binding ability.And PEG had more influence than MMAE,indicating that reduce PEG payload appropriately might be better for TRAIL.In order to reach a balance between the pharmacokinetics(PK)and pharmacodynamics(PD),we evaluated the PK,antitumor activity and potentially toxicity in mice.As a result,PEG-TRAIL-MMAE conjugates with a PEG/MMAE ratio of 1:2 showed prolonged half-life in rat(from 0.7 h to 6.8 h),and the best antitumor activities in vitro(ICso 0.31 nM)and in vivo while no sign of toxicity in xenograft models,suggesting it's a promising novel antitumor LDC.To study the novel-target ADC based on tumor-specific mutant antigens(TSMA)presented by HLA,we chose the KRAS Gly-12 mutant/HLA-A0201 complex as a target.Since they are personal antigens only presented as particular mutation and particular HLA genotype,we called the antibody targeting this kind of antigens as personalized antibody,and personalized antibody-drug conjugate(PADC)for its conjugates.By evaluated the antigen recognition of antibodies generated from G12D peptide or G12V/HLA-A0201 complex binding screen,we concluded that antibody only binds to mutant peptide cannot recognize the neoantigens expressed on cell surface.While G12V/HLA-A0201 complex-binding antibody 2G1 and its variants recognized neoantigens specifically without cross-reacted to G12D mutant or KRAS wildtype.And this proofed the concept that single amino-acid mutation can be distinguished by antibody.Barely cytotoxicity was observed on SW480(HLA-A0201+9G12V+)cells when treated with PADCs directly,due to very low antigen expression on this cellline under general culture condition in vitro.Whilst the activity of PADCs improved when cells were pulsed with G12V peptide,which increased antigens presenting to a detectable level.We also constructed a K562-A2 HUP cellline,which stably express the ubiquitin-peptide recombination protein to improve antigen presented on the cell surface.It turned out that both PADCs can internalize into K562-A2 HUP upon binding to the G12V neoantigen,and then exert their cytotoxicity.And the potency of two PADCs with DARs of 1.85 or 3.65 was different,the IC50 of 2E8-MMAE(DAR 1.85)and 2A5-MMAE(DAR 3.65)were 214.6 nM and 112.8 nM respectively,suggesting that antitumor activity of PADCs can be enhanced through increasing their payload.Although it was still much weaker than traditional ADCs,that the IC50 was 100-fold higher compared under the same DAR.PADCs can be well tolerated by neoantigen-negative cells even at a very high concentration of 400 nM,demonstrating the highly specificity of our PADCs,and a better antitumor effect can be achieved via increasing doses without any cost of safety.In summary,we concluded that some important points should be thoroughly considered when designing a PADC,including high mutant expression,strong binding between peptide and HLA,a high specific and high affinity personalize antibody and more cytotoxins or a more powerful payload,and this could be helpful for the development of other PADCs.