Identification Of KRAS Neoantigen Specific TCR And Structural Basis For TCR Recognition

Cancer is one of the leading causes of death in the world,and such malignancies are a huge drain on society's resources.Surgery,chemotherapy and radiation therapy(RT)have developed as the three pillars of cancer treatment.But the legacy of large wounds,drug resistance and tumor recurrence has exposed the drawbacks of traditional cancer treatments.On the other hand,immunotherapy to treat tumor formation by activating the anti-tumor function of the body's immune system has been making breakthroughs.TCR-T(T Cell Receptor-engineered T cell)therapies take advantages of recognizing most tumor antigens because they are able to recognize both extracellular and intracellular antigens.By August 2020,77 clinical trials related to TCR-T cell therapy have been conducted.KRAS(V-Ki-RAS2 Kirsten Rat Sarcoma Viral Oncogene Homolog)is a protooncogene that plays a key role in the development of many tumors.It is a major activator of a variety of cellular activation pathways essential for cell division and metabolism.Proteins in the Ras family all have vital GTPase(guanosine triphosphatase)activity,and they interact with GTPase activating protein(GAP)to facilitate this hydrolysis process into a GDP-bound inactive state.Mutations at the KRAS locus usually impair GTP-enzyme activity,resulting in continuous activation of downstream activation signals and abnormal cell proliferation.The most common mutation site for KRAS is the substitution of wild-type glycine by aspartic acid or valine in amino acid12.These two mutations alone occur in about 60-70% of pancreatic cancers and about20-30% of colorectal cancers.Due to the tumor-specific nature of this gene mutation,we attempted to target the mutation sites of KRAS with TCR by developing a TCR specific to KRAS mutations.The KRAS mutation specific TCR was obtained by immunizing mice in the early stage of the study.The expression vector was constructed by prokaryotic method,and the inclusion body was expressed according to the test expression result.Therefore,the misfolded protein was reopened by denaturation and renaturation,And then successfully purified 1-2C-TCR protein through 15 Q Source ion exchange column and B200 gel chromatography column,using the same method to renature A11-nobiotin/h?2m with KRAS-G12 V polypeptide,using AKTA Pure further purified the peptide-MHC complex protein through molecular sieves,and further obtained the complex protein of the two through the co-incubation of the two proteins.The growth condition was 1.0 M Ammonium citrate tribasic pH=7.0,0.1 MBIS-TRIS propane pH=7.0,15 mg/ml,18?,the crystal was selected,and the structure of the complex was successfully analyzed by using CCP4,Phenix,PyMOL and COOT software.Provide new treatment strategies for tumor immunotherapy,and provide theoretical basis for tumor immunotherapy based on KRAS-neoantigen.