Preliminary Study On The Feasibility Of Tumor Specific Presented Antigens As Targets For Antibody Drugs

It is very important to select appropriate targets for tumor diagnosis and treatment.At present,great progress has been made in antibody-based anti-tumor drugs.However,the targets of these drugs are mainly tumor associated antigens(TAAs),including HER2,CD20,EGFR,CD19 and other membrane proteins.TAAs are not unique to tumor cells,so therapies targeting this kind of antigen would result in non-specific killing,that is,while killing tumor cells,it would also have certain killing effect on normal cells,resulting in inevitable toxic and side effects.There is an urgent need to find targets with high tumor specificity to improve the safety of tumor therapy.There are a large number of somatic mutations identified in tumor cells,which can be used to distinguish tumor cells from normal cells.But most of the mutated proteins are expressed intracellularly and cannot be drugged by conventional antibodies.Through the antigen-presentation mechanism,these mutated proteins are hydrolyzed into peptides and bind to the major histocompatibility complex(MHC)class I molecules to generate peptides/major histocompatibility complex(pMHC),which are delivered to the cell surface and recognized by T cell receptor(TCR).At present,it has been proved that pMHC can also be used as an antibody recognition target.However,the feasibility of tumor-specific pMHC as a tumor therapeutic target has not been systematically studied.By means of the selection of peptide delivery sequence,specific antibody screening and rational design of antibody drugs,this study conducted a more comprehensive discussion on antibody drugs targeting tumor-specific pMHC for the first time,aiming to provide a rational design scheme for development of such antibody drugs.First of all,we took the tumor-specific fusion protein BCR-ABL as an example to explore the identification method of peptide presentation.The candidate pMHCs(peptide A9K or peptide K9R presented by HLA-A*03:01)were obtained by the presentation prediction software,and were confirmed by protein refolding and liquid chromatography-mass spectrometry assay.Then we screened the specific antibodies with a mouse phage immune library,and we obtained two specific antibodies against A9K-pMHC.We developed antibody-drug conjugates(ADC)and bispecific antibodies based on the two screened clones,and studied the related factors affecting their antitumor activity.We found that the anti-tumor activity of ADCs on antigen-positive A431-A9K cells was unsatisfactory with the EC₅₀ higher than 3.5?g/m L.The bispecific antibodies killed antigen negative cells in a non-specific manner,which might be related to the non-specific binding of antibodies to pMHC.To verify this infer,we chose the highly specific pMHC targeting antibody 2G1(targeting HLA-A*02:01/KRAS G12V)with the reported sepuence for further study on the antitumor activity of the bispecific antibody(2G1/CD3-Ig G1).The 2G1/CD3-Ig G1 antibody can mediate T cells to effectively kill antigen-positive tumor cells,while avoiding killing antigen-negative cells.In addition,2G1/CD3-Ig G1 can effectively inhibit the growth of transplanted tumor in mice,demonstrating its great anti-tumor activity and specificity in vitro and in vivo.In conclusion,tumor-specific pMHC can be used as a target for antibody drugs.Compared with the target of traditional antibody,the low expression of tumor-specific pMHC makes the target identification very difficult.Through the combination of software presentation prediction and experimental verification,we can effectively identify the positive presentation peptide.In addition,the expression of antigens is too low,which puts forward higher requirements for the molecular form of antibody drug.We evaluated ADC and bispecific antibodies respectively,and proposed that bispecific antibodies could play a good anti-tumor activity against such targets by activating T cells.Bispecific antibodies have high requirements for the specificity of targeting tumor cells.However,the antibody specific recognition epitope of tumor-specific pMHC is only in the peptide part,which increases the difficulty of antibody acquisition.We established a rapid antibody screening and evaluation method through the construction and screening of mouse immune-phage library,the obtained antibodies can be used for preliminary studies.In this study,we systematically studied the antibody drugs targeting tumor-specific pMHC from the aspects of target identification,antibody screening,drug design and activity evaluation,and revealed the related factors affecting their activity.We further demonstrated the advantage of bispecific antibodies targeting tumor-specific pMHC.We proposed the feasible strategy and proved great potential of targeting tumor-specific pMHC in the treatment of tumors.