Preparation Of Monoclonal Antibody Against 15KD-human Augmenter Of Liver Regeneration And Its Effect On Multiple Myeloma

Introduction : Multiple myeloma(MM)is the second most common haematological malignancy.Although modern combination therapies induce rapid,deep and sustainable responses,and prolong survival,MM remains an incurable disease and the majority of patients relapse and require treatment.Thus,novel targets for treating MM are urgently needed.Monoclonal antibodies are produced by B cells and binding to antigen specificity.Monoclonal antibodies are now widely used in the diagnosis,prevention,treatment and study of diseases.Augmenter of Liver Regeneration(ALR),also known as GFER,is a vital protein for various life processes.ALR occurs in two isoforms;a long form comprising 205 amino acid residues with a molecular weight of 23 k D that is present in the intermembrane space of mitochondria,and a short of 125 amino acids with a molecular weight of 15 k Da that is secreted by hepatocytes and is present in serum.Different isoforms of ALR have been associated with different subcellular locations,and therefore specific functions,but despite considerable research on the overexpression and inhibition of 23KD-ALR and 15KD-ALR,their specific functions remain unclear.Overexpression and inhibition of ALR will affect normal tissues.The use of monoclonal antibodies to bind extracellular 15 KDALR does not affect the 23 KDALR,which is conducive to studying the role of 15 KDALR alone and also reduces the impact on normal tissues.Therefore,we block extracellular15 KDALR by using monoclonal antibodies to research the role of15 KDALR on myeloma and hope to find a new target for the treatment of multiple myeloma.Methods: The 15KD-recombinant human ALR(rh ALR)protein was prepared by our laboratory.After immunisation,anti-ALR monoclonal antibody was prepared using hybridoma technology.Supernatants of growth-positive wells were screened for the presence of antibodies by enzyme-linked immunosorbent assay(ELISA).ELISA and western blot were used to verify the monoclonal antibodies.Ascites were prepared for larger-scale m Ab production.Negative hybridoma cells were also prepared as negative control ascites.Ascites containing Anti-ALR Mc Ab were added to the medium as the treatment group.Ascites produced by negative hybridoma cells constituted the negative control group,and PBS was used for the blank group.Cell count and CCK8 were used to observe the Cell viability.The apoptosis rates of human multiple myeloma cells were determined using flow cytometry.The express of apoptosis-related proteins were detected by western blot,and Cell proliferation was measured by Ed U assay.In vivo,xenograft model was established and treated with ascites containing Anti-ALR Mc Ab,ascites produced by negative hybridoma cells and PBS respectively.Tumor volume and quality were recorded after treatment.RNA-seq technology was used to analyze the gene expression difference between 15 KDALR monoclonal antibody treatment group and negative ascites treatment group.Gene Ontology(GO)analysis was performed to predict the biological functions of differentially expressed genes.Pathway analysis was used to identify significant pathways related to the differential genes according to the KEGG database.Protein-protein interaction network analysis of differential genes was performed by the STRING online database,The top 10 hub genes were identified by the cyto Hubba plugin app of Cytoscape software.Path-Act-Network was employed to select genes in enriched biological pathways,and Cytoscape was used for graphical representation of pathways.The results of RNA-seq were verified by RT-PCR,western blot,and cell cycle detection,which further confirmed the mechanism of monoclonal antibodies affecting myeloma.RESULTS:After immunizing with 15 KDrh ALR,the antibody titer in the serum reached 104.After cell fusion,two hybridoma clones stably secreting anti-ALR m Ab were successfully selected.Ascites were prepared for larger-scale m Ab production.The titer of m Ab reached 106.The anti-ALR m Ab was verified by ELISA and Western blot.The subtype of the monoclonal antibody is Ig M.Expression of 15KD-ALR in different MM cell lines was investigated,and U266 cell lines displayed the highest expression levels among the three MM cell lines,hence this cell line was chosen for further study.To construct a cell model,U266 cells were treated with different m Ab concentrations(1:100,1:50,and 1:10 ratio of ascites to medium)for different durations(24,48 and 72 h).The CCK-8 assay results showed that at a 1:10 concentration,cell viability was decreased in a dose-and time-dependent manner,and cell viability was significantly lower than at the other two concentrations at 72 h.Apoptosis in U266 cells were investigated by flow cytometry.When treated with m Abs for 72 h,the proportion(%)of apoptotic cells detected by flow cytometry did not differ between the three groups.We also measured expression levels of the proapoptotic protein Bax and the anti-apoptotic proteins Bcl-2,and there was no difference in the ratio of the two proteins between the three groups.Similarly,there were no differences in caspase-3 and cleaved caspase-3protein levels.However,following cell injury induced by epirubicin treatment,the proportion of apoptotic cells increased after blocking extracellular ALR with m Ab.In vivo,the tumor growth rate was significantly slower in anti-ALR15 KDALR m Ab group than that of the negative ascites and PBS groups.After the treatment,the tumor volume and mass in treatment group were also significantly smaller than those in the control group.RNA-Seq was performed to identify differentially expressed RNAs between anti-ALR Mc Ab and negative ascites groups.The results showed revealed 289 and 138 significantly up-and downregulated genes,respectively.GO enrichment revealed that these differential genes were mainly associated with the cell cycle,cell division and mitosis,all related to cell proliferation.The protein-protein interaction network based on the identified differentially expressed genes is shown the top 10 genes were CDK1,SMC3,KIF11,SMC4,NDC80,NCAPG,TTK,CENPE,NUF2 and CENPU.Pathway-act network analysis suggested the MAPK,cell cycle and Jak-STAT were the key pathways.The top 10 hub genes were validated by RT-PCR,the changes in hub genes were consistent with the microarray results.However,changes in TTK and CENPU were not statistically significant.Signal pathways were verified by western blotting.Expression of ERK1/2 and p-ERK1/2 was downregulated in the treatment group,while p38 and p-p38 showed no significant difference in expression,and JNK was downregulated in the treatment group,while p-JNK was upregulated.Expression of STAT3 was downregulated slightly in the treatment group,while p-STAT3 was significantly downregulated,as were cdc2 and cyclin D1.Cell cycle analysis revealed cell cycle arrest at the S phase in the anti-ALR Mc Ab group,but not in the PBS or negative control groups.Conclusion:Our results provide evidence that blocking extracellular15 KD ALR inhibited the proliferation of U266 MM cells through cell cycle arrest without increasing apoptosis in the absence of injury factors.ERK1/2and JNK branches of the MAPK pathway,STAT3,and the cell cycle were involved in the possible mechanism.Thus,15 KD ALR may be a new target for myeloma treatment.