Molecular targeted therapy of non-small cell lung cancer / 国际肿瘤学杂志

At present,the treatment of non-small cell lung cancer (NSCLC) has entered the era of targeted therapy,and small molecule tyrosine kinase inhibitors are typical representatives,which have clear molecular targets and remarkable effects.But many patients without mutations are not able to benefit from them.Vascular endothelial growth factor and immune targeted therapy have become the new focus of NSCLC treatment.Inhibitors such as programmed cell death-1 and programmed cell death-ligand 1 have shown great applied potential in a series of clinical trials of NSCLC.

Effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis in mice / 重庆医学

Objective To investigate the effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis(PF) in mice.Methods The rats were divided into the normal group,control group and experimental group,6 cases in each group.The mice were performed the PF induction.The experimental group was treated with immunotoxin,the control group was given the contrast protein and the normal group was not treated.The mouse left lung was used for histological analysis,and the right lung was used for hydroxyproline analysi s.The effect of macrophage FRβ expression on bleomycin induced pulmonary fibrosis in mice and the pulmonary macrophage FRβ expression in the patients with idiopathic pulmonary fibrosis(UIP) were detected.Results The macrophage FRβ expression mainly existed in the patients with UIP and pulmonary fibrosis area of PF mice induced by bleomycin;the survival rate was significantly increased by giving the mice immunotoxin with nose(P=0.003),and the level of total hydroxyproline and fibrosis of PF mice induced by bleomycin was decreased(P=0.009,0.014);immunohistochemistry results showed that immunotoxin could reduce the cells number of lung tumor necrosis factor(TNF)-α,chemotactic CCL2 and CCL12 cells inPF mice induced by bleomycin(P=0.000).Conclusion The FRβ expression of macrophages plays a pathogenic role in IPF,and the targeted therapy of FRβ expression in macrophages may be an effective method for the treatment of IPF.

A breakthrough in antibody-drug conjugates in anti-immune rejection / 中国组织工程研究

BACKGROUND:The immunological rejection between host and graft is the leading cause of organ transplantation failure. The traditional immunosuppressive agents have been unable to meet the needs of clinical treatment. Antibody-drug conjugate, as a type of new drugs, may be hope for the treatment of immune rejection. OBJECTIVE:To comprehensively analyze the composition of antibody-drug conjugates, mechanism of action, clinical research progress as wel as the development trend. METHODS:A computer-based online retrieval was performed to search papers in CNKI and PubMed database using the key words of ADCs, immunosuppressive agents, immunotoxins, organ transplantation, graft rejection in Chinese and English. Recently published or published in the prestigious journals were selected in the same field. After excluding objective-independent papers and repeated studies, 42 papers were included for further analysis. RESULTS AND CONCLUSION:Antibody-drug conjugates, as highly effective and lowly toxic immunosuppressant, have achieved a breakthrough in treatment of targeting tumor, while the role of it in anti-immune rejection is stil at the exploratory stage. For islet transplantation, novel antibody-drug conjugates are required to block CD8+T effector by CD103/E-Cadherin pathway, and wil probably serve as a potential drug intervention for al ograft rejection.

Mecanismo de acción de la toxina alfa hemolisina de Escherichia coli / Action mechanism of Escherichia coli alpha-hemolysin toxin / Mecanismo de ação da toxina alfa-hemolisina de Escherichia coli

Escherichia coli es una de las bacterias anaerobias facultativas más predominantes en el intestino, siendo, en la mayoría de los casos, inocua para el huésped. Existen cepas que traslocan al torrente sanguíneo causando enfermedades extraintestinales como infecciones urinarias, septicemia y meningitis. Dentro de éstas se encuentran las cepas uropatogénicas (Uropathogenic Escherichia coli: UPEC), que secretan varios factores de virulencia. Estos últimos incluyen: toxinas, sistemas de adquisición de hierro, adhesinas y antígenos capsulares. Las principales toxinas secretadas son: alfa-hemolisina (HlyA) y el factor necrotizante citotóxico 1 (CNF-1). En esta revisión se presenta una descripción exhaustiva de HlyA, incluyendo su síntesis, maduración y exportación desde la bacteria. La acilación de la proteína en dos residuos internos de lisina la convierte en una toxina muy virulenta al exponer regiones intrínsecamente desordenadas que son esenciales en diferentes pasos del mecanismo de acción de la misma. Específicamente, la exposición de estas regiones está involucrada en interacciones proteína-proteína dentro del proceso de oligomerización. La formación del oligómero es responsable de la permeabilidad inducida en las células blanco. Finalmente, basado en los conocimientos acerca de las características estructurales y funcionales de HlyA, se presentan potenciales usos de HlyA en terapias basadas en toxinas.

Escherichia coli is one of the predominant species of facultative anaerobes in the human gut, and in the majority of the cases it is harmless to the host. Some strains of this species can translocate to blood and cause infection such as urinary infection, septicemia and meningitis. These are the uropathogenic E. coli strains (UPEC) that secrete a number of virulence factors. The latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens. Secreted toxins include HlyA, the cytotoxic necrotizing factor-1 (CNF-1). In this review an exhaustive description of the toxin has been delineated, including its synthesis, maturation, and export from the bacteria. The acylation of the protein at two internal lysine residues gives the toxin its virulence, by exposing intrinsic disordered regions that are essential in different steps of the toxin's mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsG-ible for the permeability induced in all the target cells. Based on the already known structural and functional characteristics of HlyA, the potential use in toxin-based therapy is presented.

Escherichia coli é uma das bactérias anaérobias facultativas mais predominantes no intestino, sendo na maioria dos casos inócua para o hóspede. Há cepas que passam ao torrente sanguíneo causando doenças extraintestinais como infecção urinária, septicemia e meningite. Dentro destas se encontram as cepas uropatogênicas (Uropathogenic Escherichia coli: UPEC) que secretam varios fatores de virulência. Estos últimos incluem: toxinas, sistemas de aquisição de ferro, adesinas e antígenos capsulares. As principais toxinas secretadas são: alfa hemolisina (HlyA) e o fator necrotizante citotóxico 1 (CNF-1). Nesta revisão apresenta-se uma descrição exaustiva de HlyA incluindo sua sintese, seu amadurecimento e exportação a partir da bactéria. A acilação da proteína em dois residuos internos de lisina a transforma numa toxina muito virulenta ao expor regiões intrinsecamente desordenadas que são essenciais em diferentes passos do mecanismo de ação da mesma. Especificamente, a exposição destas regiões esta envolvida em interações proteína-proteína dentro do processo de oligomerização. A formação do oligômero é responsável pela permeabilidade induzida nas células alvo. Finalmente, com base nos conhecimentos acerca das características estruturais e funcionais de HlyA, apresentam-se potenciais usos de HlyA em terapias baseadas em toxinas.

The construction and expression of Luffin-β-targeted fusion immunotoxin as well as its inhibitory effect on non-small cell lung cancer cells / 肿瘤

Objective: To investigate the inhibitory effect of Luffin-β-targeted fusion immunotoxin, which contains endoplasmic reticulum-resident signal fragment KDEL (Lys-Asp-Glu-Leu-COOH) and the uPAcs (cleavage site of urokinase-type plasminogen activator), on NSCLC (non-small cell lung cancer) cells. Methods: Luffin-β gene was cloned by RT-PCR (reverse transcriptase-polymerase chain reaction). Fused gene Luffin-β-KDEL-uPAcs was constructed by primer extension method, and inserted in pET-32a(+) vector to form the recombinant vector pET-32a(+)/Luffin-β-KDEL-uPAcs. After pET-32a(+)/Luffin-β-KDEL-uPAcs imported into Escherichia coli, the exression of fusion protein TELKP (Trx-EK-Luffin-β-KDEL-uPAcs) was induced, and then the TELKP was purified. EK (enterokinase) was used to digest TELKP to produce the targeting fused immunotoxin LKP (Luffin-β-KDEL-uPAcs). CCK-8 (cell counting kit-8), RT-PCR and Western blotting were employed to test the cytotoxic effect of LKP cleavaged by uPA (urokinase-type plasminogen activator) for setting free immunotoxin Luffin-β on NSCLC cells in vitro. Results: The recombinant vector pET-32a(+)/Luffin-β-KDEL-uPAcs could be induced to express the fusion protein TELKP, which contained Trx tag of vector pET-32a(+) and its relative molecular mass was about 4.88×104. The immunotoxin protein LKP, which contained 290 amine acids and its relative molecular mass was about 3.1 8×1104, could be produced after the fusion protein TELKP was digested by EK. The immunotoxin Luffin-β, which possessed cytotoxic effect on tumor cells, could be released from LKP protein cleavaged by uPA in vitro, and its cytotoxic effect was dose-dependent. Conclusion: Fused gene Luffin-β-KDEL-uPAcs and its prokaryotic express vector are successfully constructed. Recombinant fusion immunotoxin LKP, whose relative molecular mass is about 3.1 8×104, is successfully prepared. The immunotoxin Luffin-β, which possesses cytotoxic effect on tumor cells, can be released from LKP protein cleavaged by uPA in vitro. Copyright © 2013 by TUMOR.

CD103 molecule-mediated pancreatic islet allograft injury induced by CD8 T cells / 中华器官移植杂志

Objective To test whether the CD103 molecule mediates CD8+ T lymphocytes on allogeneic islet graft immune injury. Methods By using flow cytometry, the expression of CD103 in peripheral CD8+ T lymphocytes in wild-type C57BL/6 mice was detected. Allogenic islet transplantation models were made using Balb/c donor mice and C57BL/6 recipient mice. Recipients were divided into 3 groups: M290-SAP-treated mice were injected with CD103 immunotoxin M290-SAP; M290-treated mice were injected with CD103 monoclonal antibody M290; untreated mice were only transplanted islet without any drug treatment. CD3, CD8, CD44 and CD103 positive cells were counted in islet allograft infiltrative lymphocytes. CD3, CD8, and CD103 positive cells were measured in the mesenteric lymph node. The islet allografts were removed and subjected to HE staining and immunohistochemical staining at the time of graft loss or the end of the observation period. Results 44. 06% peripheral CD8+ T cells expressed CD103 in wild-type C57BL/6 mice. 29 % CD8+ T cells expressed CD103 in the infiltrative lyrnphocytes of islet allografts in the untreated mice. In M290-SAP-treated mice, the lymphocytes had no CD103 expression and the absolute number of CD8+ lymphocytes was decreased as well The blood glucose was maintained stable for more than 100 days (13 days in untreated group, P＜0.05) in the M290-SAP-treated mice. Moreover, the transplanted islets retained intact. Conclusion CD103 expression is required for destruction of pancreatic islet allograft by CD8+ T cells. CD103 might provide a novel target for therapeutic intervention in islet allograft rejection.

Inhibitory effects of cytotoxin IL-2-PE40 on corneal allograft rejection / 中华器官移植杂志

Objective To examine the inhibitory effect of IL-2-PE40 on the mouse corneal allograft rejection. Methods A mouse corneal graft model was set up by using C57BL/6 mice as donors and Balb/c mice as recipients. In the treatment group, IL-2-PE40 (0.6 μg/g body weight) was intraperitoneally injected from the day of surgery every 12 h until rejection happened. In the control group, the equal volume of PS was injected intraperitoneally at the corresponding time points. The transplanted cornea was observed under slit-lamp twice a week and the transplanted corneal opacity and neovascularization were rated according to Horis grading standards. It led to the determination of rejection response. The survival of transplanted cornea was regarded to be stopped when the rejection occurred. The operated eyes were observed historically on the 10th, 15th, 25th and 35th day after the surgery, and the peripheral blood was collected for measurement of T cell subgroups and T lymphocyte colonies. Results The survival time of cornea in the treatment and control groups was (30.2±2.9) days and (15.1±2.1) days respectively. In the control group, rejection occurred on the 15th day after the surgery, CD4~+ cells started rise right after the surgery and increased most obviously on the 15th day [(63.9±4.0)%] and decreased afterwards. CD4~+ cells in the treatment group were increased slightly [(42.6±4.0)%] on the 15th day. The number of CD4~+ cells in the treatment group was obviously less than in the control group (P<0.01). No significant changes in CD8~+ cells were observed in both groups. The number of T lymphocyte colonies in the control group was increased at the beginning and dropped then. No obvious change was found in the treatment group. The number of T lymphocyte colonies in the treatment group was significantly less than in the control group (P＜0.01). Conclusion IL-2-PE40 is a highly specific immunosuppressive agent. It can delay the development of corneal graft rejection and reduce the percentage of T-helper cells significantly. It also works to weaken the forming capacity of the peripheral T lymphocyte colonies.

Anti-cancer effects of immunotoxins and its improvement strategy / 国际肿瘤学杂志

Immunotoxin is composed of chemical crosslinked mixture of specific monoclonal antibody or growth factor and toxin. Immunotoxin exerts its anti-cancer effects through inhibiting protein synthesis in cancer cells and promoting cancer cell apoptosis. Although immunotoxin is characterized by high toxicity and specificity which may provide remarkable potential in tumor therapy compared with traditional anti-cancer drugs, there are a lot of limitations for its clinical application.

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.

Progression of anti-tumor targeted drugs / 肿瘤研究与临床

Targeted drug is becoming a hot point for anti-tumor research.The paper reviews the cate- gory,speciality,principle of action and clinical application.The review also focus on the advance of anti-tu- mor targeted drug and forsee the prospect of it.

Genetic Construction and Characterization of Murine Interleukin-18 Immunotoxin with a Truncated Pseudomonas Exotoxin / 航天医学与医学工程

Objective To construct a new recombinant immunotoxin expression vector fused with a murine interleukin18(IL18) gene and a truncated pseudomonas exotoxin (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli (E. coli). Method Murine IL-18 (mIL-18) cDNA was cloned from murine liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was ligased with a PE38 gene carried by PRKL expression vector through T4 DNA ligase and constructed into fusion protein expression plasmid PRKL-IL18-PE38. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and the molecular weight and specificity were determined by SDS-PAGE and Western-blotting. Result The new recombinant immunotoxin expression vector was constructed successfully. DNA sequencing revealed that the mIL-18 and PE38 gene were consistent with NCBI Gene Bank. The IL-18-PE38 fusion protein was expressed in E.coli BL21, and Western-blotting analysis indicated that the molecular weight of the expression product is about 56 kDa, and could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cells and may have some potential value in the treatment of Th1 cell-mediated autoimmune diseases.

Effects of Recombinant Chimeric Toxin Dsg3EC_(1-2)PE40 on T and B Lymphocytes from Pemphigus Vulgaris / 中华皮肤科杂志

Objective To study the effects of recombinant chimeric toxin Dsg3EC_(1-2)PE40 on T and B cells from PV patients. Methods The recombinant protein Dsg3EC_(1-2)PE40 was expressed on BL21TrxB (DE3) cells, then identified and purified. ELISPOT assay was used to detect and quantitate autoantibody-producing B cells in different concentrations of recombinant chimeric toxin, and MTT assay and ~3H-TdR assay to observe the metabolism and proliferation of T cells from PV patients in vitro. Results The purity of expressed protein Dsg3EC_(1-2)PE40 was up to 80%. The number of anti-Dsg3 antibody-producing B cells in PBMC from PV patients decreased by 40% with treatment of Dsg3EC_(1-2)PE40, which was significantly lower (P

Conjuncting on targeting toxin of vascular endothelial growth factor a antibody-diphtheria toxin mutation / 中华检验医学杂志

0.05). It is decrease greatly, than those in anti-VEGF control (P0.05).The toxicity of CRM9 control are markedly higher than those in blank control groups (P

Primary determination for activity and expression of Stx 2a’-LHRH chimeric toxin / 中国病理生理杂志

AIM: To express chimeric toxin Stx2a’-LHRH a nd to investigate the cytotoxic activity of recombinant toxin Stx2a’-LHRH to huma n carcinoma cells.METHODS: Stx2a’-LHRH sequences that added the res tri ction endonucleases NcoⅠ and EcoRⅠ at the 5' and 3 ends were amplified by PCR a nd digested with appropriate restriction enzymes. The digested fragment was subc loned into the vector obtatined by digestion of plasmid pET-28a(+) with NcoⅠ an d EcoRⅠ. E. coli BL21 (DE3) cells were transformed with plasmids of interst and cultured in LB medium containing ampicillin. Expression of the recombinant protein was induced by the addition of isopropylthio-?-D-galactoside (IPTG). T h e cytotoxity of Stx2a’-LHRH to Hep-2 cells was observed under the microscop y. RESULTS: Recombitant plasmid pET-SL was constructed successfu lly and the clones expressing pET-SL stablely were obtained. A special electroph oretic band in SDS-PAGE (a glycoprotein of 28kD) was noted. Stx2a’-LHRH killed He lp-2 cells clearly. CONCLUSION: In this study, construction of c himeric toxin Stx2a’-LHRH and its expression were described. Moreover, it has o bvious cytotoxity to Hep-2 cell. These finding could open up new vistas in the s tudy of targeted durgs.

Targeted killing effect of mucin-1 single chain antibody targeting, lentivirus-mediated suicide gene therapy and ganciclovir in mucin-1~+ ovarian epithelial carcinoma cells / 中华妇产科杂志

Objective To investigate the targeted killing effect of mucin 1 single chain antibody targeting, lentivirus mediated suicide gene therapy and ganciclovir(GCV) in mucin 1 + ovarian epithelial carcinoma cells Methods Mucin 1 single chain antibody targeting lentivirus produced by packaging cell line 293T transduced herpes simplex virus thymidine kinase (HSV tk) gene into the ovarian epithelial cancer cell line SKOV3 (MUC1 +) The infection effect was observed through fluorescence microscopy Polymerase chain reaction (PCR) and reverse transcription PCR (RT PCR) were resorted to demonstrate the successful transduction and transcription of the HSV tk gene After administration of GCV, changes of those cells were observed through optical microscopy The cytotoxicity efficacy of HSV tk/GCV system was evaluated by methyl thiazolyl tetrazolium method Results It was observed through fluorescence microscopy that anti MUC1 directed lentivirus can specificly infect MUC1 + ovarian cancer cells A fragment of 600 bp was generated through PCR and RT PCR which indicated successful transduction and transcription of the HSV tk gene in SKOV3 cells Changes of cells followed by administration of GCV were observed with optical microscopy Significant cytotoxicity efficacy of GCV to SKOV3 was observed Conclusions The HSV tk gene can be targetedly transducted into MUC1 + ovarian cancer cell line under the mediation of anti MUC1 directed lentivirus, and such HSV tk/GCV system has targetingly killing effect on MUC1 + cancer cells

Effects of recombinant immunotoxin hIL-2-LuffinP1 on lymphocyte proliferation in vitro / 解放军医学杂志

Objective To observe the biological activity of the recombinant immunotoxin hIL-2-LuffinP1 derived by gene engineering in vitro.Methods To observe the inhibitory effect on lymphocytes in vitro,the splenocytes isolated from C57 and BALB/c mice were used for mixed mouse lymphocyte response(MLR),and the lymphocyte proliferation was observed in vitro with BALB/c mice splenocytes stimulated by concanavalin A(ConA).Different concentrations of hIL-2-LuffinP1 was added into the reaction system,with LuffinP1 adopted as control.3H-TdR incorporation assay was used to detect the effects of hIL-2-LuffinP1 on lymphocyte proliferation,and its effects on CTLL-2 cells(IL-2 dependent cells) were also observed.The experiments consisted of six groups(PBS,IL-2,LuffinP1,LuffinP1+IL-2,hIL-2-LuffinP1 and hIL-2-LuffinP1+IL-2).CTLL-2 cells were cultured for 12h after being treated with different agents,and then the cells were stained by trypan blue to estimate the dead cells in every 100 cells.Results It was showed that CPM declined correspondingly to the gradually increased concentration of hIL-2-LuffinP1,and cell proliferation was inhibited obviously.The inhibition rate was up to 97% at the concentration of 10-6mmol/L of hIL-2-LuffinP1.The activity of inhibiting lymphocyte proliferation was proportional to the dosage.On the contrary,no obvious inhibition to lymphocyte proliferation was found in the control group treated with LuffinP1,the inhibition ratio only reached to 14% with the same concentration of hIL-2-LuffinP1.It was also found that,when hIL-2-LuffinP1 and LuffinP1 in different concentrations added to spleen cells stimulated by ConA,hIL-2-LuffinP1 still exhibited strong ability of inhibition on the splenocyte proliferation,while LuffinP1 has no obvious inhibiting effect on the lymphocyte proliferation in the two reaction systems mentioned above.MLR showed the same results.Furthermore,in the experiment of cytotoxic effects of IL-2-LuffinP1 on CTLL-2 cells,the inhibition rates of hIL-2-LuffinP1+IL-2 group and hIL-2-LuffinP1 group were 29.6% and 47.4% respectively,the difference was significant compared with IL-2 group(P