| NK/T cell lymphoma（NKTCL）is a non-Hodgkin’s lymphoma（NHL）that originated from NK/T cells.Its clinical and pathological manifestations are unique.Tumor cells exhibit multiple morphologies.Tumor infiltration often centers on blood vessels combined with destruction and focal necrosis of blood vessels.In2001,NKTCL was officially listed in the new classification of malignant lymphoma by the WHO as an independent clinical pathological classification.This type of lymphoma can invade all extranodal organs,and is commonly found in the nose and nasal cavity,skin,gastrointestinal tract,and upper respiratory tract.Similar to most nasopharyngeal carcinomas,NKTCL is highly associated with EBV infection,and it has distinct geographical distribution difference and ethnic specificity.It is highly prevalent in Asia,especially in Southeast Asia and southern China.NKTCL is vitally invasive and resistant to chemical drugs.Although chemotherapy including CHOP and SMILE are employed,there are no acknowledged therapeutic protocols for clinical application and NKTCL often leads to poor prognosis.At present,the treatment of this type of lymphoma is still at an exploratory stage and it is of great importance to find new therapeutic strategies for NKTLC treatment.The molecular targeted therapy of tumors utilizes specific structural molecules possessed by tumor tissues or cells as targets,and uses drugs that specifically bind to these target molecules to intentionally kill tumor cells.Antibody drugs have become the research focus in the field of targeted tumor therapy because of their strong targeting ability,low side effects and significant efficacy.The key of targeted antibody therapy is to identify the suitable target antigen.The target of an ideal monoclonal antibody should be an antigen that is specifically expressed or highly expressed on the surface of tumor cells but not expressed or lowly expressed in normal tissues.The EBV-encoded latent membrane protein 1（LMP1）has attracted much attention for its role in promoting epithelial cell transformation and carcinogenesis,and is currently recognized as a substantial oncogene.Previous studies have also shown that LMP1 plays an important role in the occurrence and development of NKTCL.As far as we know,LMP1 is highly expressed in NKTCL tissues,and the high expression of LMP1 is positively correlated with the malignant biological behavior and poor prognosis of NKTCL,which makes it a potentially appropriate target for NKTCL therapy.In the previous experiments,our research group has screened several strains of high-binding-activity anti-LMP1-Fab from the completely human Fab antibody library and clone LMP1-Fab 36 showed higher antigenic affinity and neutralized activity.In this study,we prepared and expressed a full length human anti-LMP1-Ig G with neutralizing activity through genetic engineering techniques,performing functional identification of the expressed and purified antibodies.Subsequently,we tested the inhibitory-effectiveness of NKTCL by LMP1-Ig G and preliminarily explore its mechanism of action,laying the foundation for further exploration of its role in the treatment of NKTCL.Materials and methods:1.Construction of recombinant anti-LMP1-Ig G antibody eukaryotic expression vector:Optimize the LMP1-Fab variable region sequence and design heavy and light chain primers of anti-LMP1-Ig G antibody according to Infusion PCR principles.The PCR product was cloned into the eukaryotic expression plasmid of the antibody and transformed into E.coli DH5α.The positive clones were screened and identified by enzyme digestion.The clones that had correct enzyme digestion identification were tested to confirm that the sequence was correctly connected.2.Expression,purification,and identification of anti-LMP1-Ig G antibodies:The correctly sequenced p FUSE-CHIg-h G1-LMP1-VH（p TH-LMP1-VH）,p FUSE-CLIg-hk-LMP1-VK（p TH-LMP1-VK）recombinant plasmids were transfected into 293Freestyle（293F）cells,after the cell culture and the supernatants were collected.After filtration,the protein purification system and Hitrap Protein A prepacked column were used for purification.The characteristics of binding and affinity of anti-LMP1-Ig G were tested by enzyme-linked immunosorbent assay,immunoblot assay,affinity assay,and immunohistochemistry assay.3.MTT and CCK-8 assays were performed to detect the inhibitory effect of LMP1-Ig G on NKTCL cell lines:MTT assay:Collect logarithmic phase NKTCL cells into cell culture plate,then add different concentration gradient LMP1-Ig G,MTT solution and dimethyl sulfoxide.OD490nm was used to measure the absorbance of each well.CCK-8 assay:After the NKTCL cell suspension was prepared and added into different concentration gradients of LMP1-Ig G.After incubation,CCK-8 solution was added.The absorbance of each well was measured at OD450 nm.4.Annexin V/PI assay was used to detect the effect of LMP1-Ig G on apoptosis of NKTCL cells:NKTCL cells treated with LMP1-Ig G were digested with trypsin,and the suspended cells were collected.After washing,Annexin V-FITC and PI labeling were conducted.The detection was performed by flow cytometry.5.LDH release assay was employed to detect ADCC and CDC effects of LMP1-Ig G on NKTCL cells:ADCC:NKTCL cells were co-cultured with different concentrations of LMP1-Ig G.PBMCs from healthy volunteers were co-cultured with NKTCL cells with different effect-target ratios.LDH release value was measured at OD570nm.CDC:NKTCL cells were co-cultured with different concentrations of LMP1-Ig G and normal human serum and heat-inactivated human serum were added respectively.LDH release value was measured at OD570nm as described above.6.Research of targeted inhibition mechanism of LMP1-Ig G on NKTCL:RNA interference technology was used to construct LMP1-si RNA plasmid and transfect NKTCL cells to detect the effect of LMP1-Ig G on JAK/STAT signaling pathway in NKTCL cells.Results:1.Construction of anti-LMP1-Ig G eukaryotic expression vector and expression,purification and identification of anti-LMP1-Ig GA 360-bp VH gene and a 321-bp VK gene were amplified by PCR,respectively.p TH-LMP1-VH and p TH-LMP1-VK plasmids were obtained by Infusion PCR and transformed into E.coli,and then the eukaryotic expression of anti-LMP1-Ig G was performed with 293 Free style Expression System.The results of SDS-PAGE showed that the purified antibodies showed right bands on 55k D and 25k D,respectively,which were the heavy chain and light chain of anti-LMP1-Ig G,respectively.Cell ELISA and Western blot experiments showed that anti-LMP1-Ig G can recognize LMP1 antigen in NKTCL cells.For affinity detection,the affinity KD of anti-LMP1-Ig G was 3.175×10–9M.Immunohistochemistry experiments showed that the recognition of LMP1 expression in the NKTCL tissues was not statistically different between anti-LMP1-Ig G and a commercial LMP1 antibodies.2.Targeted inhibitory effect of anti-LMP1-Ig G on NKTCL cellsCCK8 and MTT assays respectively demonstrated that LMP1-Ig G had a significant killing effect on LMP1-positive NKTCL cells,and this killing function was concentration-dependent and time-dependent.The tumor inhibition rate was up to approximately 40%（CCK8）and 60%（MTT）.The IC50 values of LMP1-Ig G were7.421μg/ml（SNK-6 cells）and 17.68μg/ml（SNT-8 cells）,respectively.Annexin/V PI double staining experiments showed that LMP1-Ig G had a pro-apoptotic effect on LMP1-positive NKTCL cells in a concentration-dependent and time-dependent manner.LDH release assays showed that LMP1-Ig G could significantly induce the ADCC and CDC effects in LMP1-positive NKTCL cells,but there were no effects of ADCC and CDC on LMP1-negative NKTCL cells.3.Mechanism research of the inhibitory effect of anti-LMP1-Ig G on NKTCL cells LMP1-Ig G inhibited the phosphorylation of STAT3,but the inhibition of STAT5phosphorylation was not obvious.The phosphorylation of JAK3 was inhibited,but the inhibitory effects of phosphorylation of JAK1,JAK2 and TYK2 were not critical.The inhibitory ability of LMP1-Ig G on JAK3/STAT3 phosphorylation was also concentration-dependent and time-dependent.The RNAi interference technology was used to construct LMP1-si RNA plasmid and transfect NKTCL cells.si RNA had successfully inhibited the expression of LMP1 in NKTCL cells and the inhibitory effect of LMP1-Ig G on STAT3 phosphorylation could be rescued while LMP1expression was knockdown.Conclusions:1.The full length anti-LMP1-Ig G eukaryotic expression vector was successfully constructed,and the expressed and purified whole-molecular anti-LMP1-Ig G illustrated convincing biological activity.2.It was initially proved that the anti-LMP1-Ig G can inhibit the growth of NKTCL cells growth,induce cell apoptosis as well as exert ADCC/CDC effectiveness,which demonstrates the potential targeted therapeutic value for LMP1-positive tumors.3.The phosphorylation of JAK3 and STAT3 can be inhibited by anti-LMP1-Ig G and this inhibitory activity of JAK3/STAT3 signaling pathway may be one of the mechanisms of the anti-tumor effect of anti-LMP1-Ig G. |
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