Exploration Of The Feasibility Of Peptide/Major Histocompatibility Complex (pMHC) As Antibody-drug Conjugates (ADCs) Target

Recently,antibody drug conjugate(ADCs)developing rapidly with the advantages of good targeting and high toxicity.Due to these advantages,ADCs rapidly arrived the tumor site and kill the tumor cells effectively.Until now,4 drugs have been on the market,and more than 80 kinds of drugs are undergoing clinical research.However,these ADCs all target tumor-associated antigens(i.e.HER2,EGFR)while tumor-specific antigens,which brings drug resistance and adverse effects.A new type of entigen may should be digged and developed for future ADCs.However,as the cost of high-throughput sequencing has decreased,more and more tumor samples have been sequenced.The results show that the vast majority of tumors have somatic mutations,the mutant proteins caused by these somatic mutations are tumor-specific antigens that distinguish tumors from normal tissues.However,most of the tumor-specific mutant proteins were present in cells,and antibody drugs could not be combined under normal conditions.But,studies also have shown that mutant peptides partially hydrolyzed by proteasomes in tumor cells could bind to major histocompatibility complex(MHC)class I molecules to generate peptide/major histocompatibility complex(pMHC)and be presented to the cell surface as a kind of tumor-specific target.Whether such tumor-specific pMHC can be a potential target for ADCs is still unknown.Hence,we firstly took 4 peptide/MHC I(pMHC)complexes of 3 melanoma-associated antigens as our targets for ADCs research.Based on the results,we found that pMHC complexes can be targeted by ADC,but the affinity of conjugates still low.Hence,we explored the sortase A mediated site-directed conjugating technique to generate a homogeneous ADC(DAR=4)with undamaged affinity and endocytosis.In vitro cell viability experiments,EA1 HL-vcMMAE effectively kill tumor cells with IC50=1.07 ?g/mL,which is far less than the corresponding IC50 value of EA1 antibody,fully reflects the high cytotoxicity advantages of ADCs.In addition,for the disadvantage of low density of HLA/MART-126-35 complex on cell surface,we used the MEK kinase inhibitor,trametinib,to effectively increase the expression of MHC class I molecules.Therefore,in themouse xenograft model,we evaluated the antitumor activity of EA1 monoclonal antibody,EA1 HL-vcMMAE,and trametinib in combination with EA1 HL-vcMMAE.The results showed that high-dose EA1 HL-vcMMAE effectively eliminate tumor cells in mice;and combination of trametinib and EA1 HL-vcMMAE effectively inhibit tumor growth at low doses,extended the survival time of tumor-bearing mice,showed better tumor inhibition than the same dose of EA1 HL-vcMMAE.Since the amount of pMHC complexes present on the cell surface varies from one cell to another.We took another approach,still target the four pMHC complexes selected in Chapter 3 by expressing as fusion proteins.After transfected into K562-A2 cells,the fusion protein was hydrolyzed by ubiquitin hydrolase to release peptides.Through flow sorting,four fusion proteins successfully transfected into K562-A2 cells and expressed,of which three pMHC complexes were detected on the respective cell surface.Through the selection of monoclonal cells,the three cells transfected with the fusion protein were successfully single-clone monoclonal cells that could present their antigenic peptides.In the cell line transfected with the MART-126-35 antigen peptide,two monoclonal cell with different presentation amounts of the two antigen peptides were selected with 3635 molecules/cell and 1456 molecules/cell,respectively.In vitro cell viability experiments,EA1 HL-vcMMAE was found to be effective in killing higher-matrix monoclonal cell lines with an IC50 =2.67 ?g/mL,which is much lower than that of monoclonal cells with a lower amount of presentation and negative cell line.It has been demonstrated that a cell model of the specific antigen peptide pMHC complex can be successfully constructed by the above-described transfection of the fusion protein followed by monoclonal-selection,and can be specifically killed by corresponding ADCs.It solves the problem of uneven drug delivery of different antigen peptide pMHC complex cell lines and difficult drug evaluation.Considering that MHC class I molecules can deliver tumor-specific antigens to the surface of cells,we hope to find this class of tumor-specific targets for ADCs.Based on the above purpose,725 cancer-ralated gene targeted depth sequencing was performed on a tumor sample and paired peripheral blood DNA from a patient with malignant mesothelioma of the peritoneum.Four somatic base-substitutions andl insert frameshift mutation were validated by the Sanger method at the transcriptional level.Meanwhile,10 mutant peptides were predicted to specifically bind to MHC I molecules with high affinity,and a neo 8-mer LPHLQGAF peptide of mutated BAP1 has the highest affinity with HLA-B35:42.This means that these tumor-specific mutated peptides originally present in tumor cells can be localized on the cell surface by MHC class I molecule presentation and become potential targets for tumor cell-specific ADCs.