Antitumor Effect And Mechanism Of Multiple Immunotoxins In Nude Mouse Transplanted Tumor Model

BackgroundImmunotoxins are a class of targeted cytotoxin fusion protein produced by attaching the antibody to the toxin using a chemical linker or using genetic recombination techniques expression from a prokaryotic system^[1,2].Immunotoxins have good antitumor effects in clinical experiments but suffer from a series of problems such as short half-life,immunogenicity,and off-target toxicity.To address these problems,we constructed a series of novel immunotoxins,aiming to investigate the antitumor effects and mechanisms of novel immunotoxins.Our study is divided into two parts,the first part is the study of the therapeutic effect and mechanism of action of dual-targeted immunotoxin against Human epidermal growth factor receptor 2（HER2）positive tumors;The second part is the study of the therapeutic effect and mechanism of novel immunotoxins with low immunogenicity and off-target toxicity on trastuzumab-resistant tumor cells.Part 1 Therapeutic effect of long half-life bispecific immunotoxin IHPP targeting HER2 and PDGFRβon HER2-positive tumorsObjectives1.Construction of a bispecific immunotoxins that target both HER2 and Platelet-derived growth factor receptorsβ（PDGFRβ）;2.Detecting the dual-targeting effect of bispecific immunotoxins;3.Exploring the tumor suppressive effects and potential mechanisms of action of bispecific immunotoxins in vitro and in vivo.Methods1.Construction and expression of a bispecific immunotoxins capable of targeting both HER2 and PDGFRβ;2.Enzyme-Linked Immuno Sorbent Assay（ELISA）assay for the analysis of the binding capacity of bispecific immunotoxins to HER2 and PDGFRβ;3.The targetability of the bispecific immunotoxins was analyzed by immuno-fluorescence assay;4.Establishing NCI-N87 nude mice xenograft tumor model and evaluating the tumor suppressive effect of bispecific immunotoxin by immunohistochemistry and tumor volume;5.Transcriptome sequencing after antitumor therapy to analyze the potential mechanism of bispecific immunotoxins.Results1.A bispecific immunotoxin,IHPP,which recognizes both HER2 and PDGFRβ,and a control group of single targeted immunotoxins,IHP and IPP,were successfully constructed and purified.The cytotoxicity test of Ig G combined with immunotoxin showed that the antitumor activity of immunotoxin was not affected by Ig G,and it still had a good antitumor effect;2.IHP and IHPP were found to have similar antigen affinities for HER2 by ELISA assay.Immunofluorescence assay found that the bispecific immunotoxin could target both HER2and PDGFRβin tumor tissue;3.The antitumor effect of the bispecific immunotoxin IHPP was found to be significantly better than the control single targeting immunotoxins IHP and IPP by calculating the Ki67 positive proportion and measuring the tumor volume size;4.In this study,high-throughput transcriptome sequencing was used to sequence NCI-N87 transplanted tumor nude mice tumor tissues after immunotoxin treatment to explore the reasons for the different therapeutic effects of IHPP and IHP.The differentially expressed genes after IHPP and IHP treatment were compared based on the mouse genome.The transcriptome results suggested that IHPP may exert a strong antitumor effect through the interferon signaling pathway;5.Differential gene expression in transcriptome sequencing results was examined by quantitative reverse transcription polymerase chain reaction（q RT-PCR）,while Interferon-α（IFN-α）and Interferon-γ（IFN-γ）in the blood of the mice were detected by ELISA,and.The results showed that the levels of IFNαand IFNγin the blood of the mice were significantly increased after being treated with the bispecific immunotoxin IHPP.Conclusion1.Successfully constructed IHPP,a bispecific immunotoxin that can target and recognize both HER2 and PDGFRβ;2.Bispecific immunotoxins showed good antitumor effects in vitro and in vivo;3.IHPP might play a significant antitumor role through the interferon signaling pathway.Part 2 Therapeutic effect of immunotoxin IHP25-BT with low immunogenicity and off-target toxicity on trastuzumab-resistant tumor cellsObjectives1.Obtaining trastuzumab-resistant tumor cells NCI-N87-TR and BT474-TR;2.Evaluating the tumor suppressive effects of novel immunotoxins on trastuzumabresistant tumor cells in vitro;3.Evaluating the tumor suppressive effect of the novel immunotoxin in vivo on a nude mouse model of NCI-N87-TR xenograft tumor.Methods1.Long-term culture and screening of trastuzumab-resistant tumor cells NCI-N87-TR and BT474-TR;2.Novel immunotoxins IHP25-B,IHP25-T,and IHP25-BT were constructed by protein structure optimization and amino acid point mutation modification.The tumor suppressive effect of these novel immunotoxins against trastuzumab-resistant tumor cells NCI-N87-TR and BT474-TR in vitro was determined by 5-ethynyl-2 ’-deoxyuridine（Ed U）and flow cytometry.The immunogenicity of these immunotoxins in mice was detected by ELISA,and the off-target toxicity of immunotoxin was assessed by measuring Alanine aminotransferase（ALT）and Aspartate aminotransferase（AST）in serum of mice;3.A NCI-N87-TR nude mice xenograft tumor model was established and the in vivo antitumor effect of IHP25-BT was evaluated by Ki67 immunohistochemistry and tumor volume.The potential mechanism of inhibiting tumor metastasis by immunotoxin IHP25-BT was analyzed by Hematoxylin-eosin（H＆E）staining and transcriptome sequencing after antitumor treatment.Results1.In this study,trastuzumab-resistant tumor cells NCI-N87-TR and BT474-TR were obtained by long-term culture and screening,in vitro assays revealed that the proliferation rate of trastuzumab-resistant cells was increased compared to wild-type cells,meanwhile,Western blot（WB）results showed that the expression of HER2 on the surface of resistant cells BT474-TR was significantly increased;2.The proliferation inhibition effect of immunotoxin IHP25-BT on wild-type NCI-N87 and BT474 and trastuzumab-resistant cells was examined by Ed U in vitro,and the results found that the novel immunotoxin IHP25-BT was able to effectively inhibit the proliferation of trastuzumab-resistant cells.Flow cytometry results indicated that the immunotoxin IHP25-BT promoted apoptosis of trastuzumab-resistant tumor cells,and trastuzumabresistant tumor cells were more sensitive to the killing effect of the immunotoxin compared to wild-type cells;3.Compared with wild-type NCI-N87,IHP25-BT can better inhibit the growth of trastuzumab-resistant tumor cell NCI-N87-TR in mice.The results of H＆E staining of mouse liver showed that IHP25-BT could reduce the distal metastasis of trastuzumab resistant tumor cells compared with trastuzumab.Using transcriptome sequencing technology to sequence the tumor tissues of NCI-N87-TR transplanted tumor nude mice after immunotoxin treatment,we tentatively hypothesized that the immunotoxin IHP25-BT may have a potential mechanism of action by inhibiting the Extracellular matrix（ECM）process of the cells,thereby reducing distal metastasis of tumor cells.Conclusion1.Trastuzumab-resistant tumor cells NCI-N87-TR and BT474-TR were successfully obatained;2.The novel immunotoxins showed good antitumor effects against trastuzumabresistant tumor cells in vitro and in vivo;3.It is tentatively hypothesized that the immunotoxin IHP25-BT may have a potential mechanism of action by inhibiting the ECM process of the cells and thereby reducing tumor cell distal metastasis.