OBJECTIVELeukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.

METHODS2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.

RESULTSAfter 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.

CONCLUSION2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD19 ; Apoptosis ; drug effects ; Cell Line, Tumor ; Flow Cytometry ; Genistein ; immunology ; pharmacology ; Humans ; Immunotoxins ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology

OBJECTIVE: To investigate the in vitro and in vivo anticancer efficacy of the immunotoxin DTAT and DTATEGF against globlastoma multiforme. METHODS: The in vitro cytotoxicity of DTAT and DTATEGF was measured using MTT assay. In vivo studies were performed in which 18 nude mice were randomly divided into 3 groups and the glioma xenograft intracranial mouse model was constructed with U87-luc cell line of human glioma. Then 1 μg of DTAT, or DTATEGF, or a control protein Bickel3 was delivered intracranially by convection-enhanced delivery (CED) via an osmotic minipump. The brain tumor fluorescence signal intensity was investigated by bioluminescent imaging (BLI). Microvessel density (MVD) was measured by immunchistochemistry SABC method in each group. RESULTS: In vitro DTAT and DTATEGF were found highly potent against U87-luc cell line, with IC(50) <0.01 nmol/L and IC(50)<1 nmol/L, respectively. In vivo BLI monitoring of the control group showed progressively increasing luminescence, while in the two treatment groups, luminescence was reduced on day 8, and increased slowly (P<0.05). The MVD of DTAT (31.6±5.2)/mm(2) and DTATEGF (25.1±6.5)/mm(2) groups had significant difference with that of the control group (51.3±7.4) /mm(2) (P<0.01). CONCLUSION Both DTAT and DTATEGF have potential in clinical application against globlastoma multiforme because of their ability to target the tumor cells and neovasculature simultaneously.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Brain Neoplasms ; drug therapy ; Cell Line, Tumor ; Glioblastoma ; drug therapy ; Glioma ; Humans ; Immunotoxins ; pharmacology ; Mice ; Mice, Nude ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the inhibitory effects of (188)Re-labeled herceptin on the proliferation in vitro of breast carcinoma cell line (SKBR-3) overexpressing HER-2/neu proto-oncogene.

METHODSHerceptin was radiolabeled with (188)Re through a direct labeling method. SKBR-3 cells were cultured with (188)Re-Herceptin at different radioactivity doses (3.7x10(4), 18.5x10(4), 37x10(4), 55.5x10(4) and 74x10(4) Bq/ml) or with (188)Re-nmIgG and (188)ReO(4)(-) for comparison. The cell proliferation inhibition was determined with MTT colorimetric assay.

RESULTS(188)Re-Herceptin could markedly inhibit the growth of SKBR-3 cells in a radioactivity dose-dependent fashion, while the effect of (188)Re-nmIgG and (188)ReO(4)(-) showed rather poor inhibitory effect in vitro. The 50% inhibition doses (IC(50)) of (188)Re-Herceptin, (188)Re-nmIgG and (188)ReO(4)(-) were 76.1x10(4) Bq/L, 139.2x10(4) Bq/L and 175x10(4) Bq/L, respectively.

CONCLUSION(188)Re-Herceptin can effectively inhibit the growth of in vitro cultured breast cancer cells overexpressing HER-2/neu, and shows much potential for clinical use in beast cancer radioimmunotherapy.

Antibodies, Monoclonal ; chemistry ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Immunotoxins ; pharmacology ; Radioisotopes ; chemistry ; pharmacology ; Receptor, ErbB-2 ; biosynthesis ; Rhenium ; chemistry ; pharmacology ; Trastuzumab

OBJECTIVEUsing monoclonal antibody (mAb) Fab' fragment to develop mAb immunoconjugates for cancer.

METHODSFab' fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DT), while Fab' fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by beta-mercaptoethanol. The conjugation between Fab' fragment and pingyangmycin (PYM), an antitumor antibiotic, was mediated by dextran T-40. Immunoreactivity of Fab'-PYM conjugates with cancer cells was determined by ELISA, and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay. Antitumor effects of the Fab'-PYM conjugates were evaluated by subcutaneously transplanted tumors in mice.

RESULTSThe molecular weight of Fab' fragment was approximately 53 kD, while the average molecular weight of Fab'-PYM conjugate was 170 kD. The Fab'-PYM conjugates showed immunoreactivity with antigen-relevant cancer cells and selective cytotoxicity against target cells. Administered intravenously, Fab'-PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb.

CONCLUSIONFab'-PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth, suggesting its therapeutic potential in cancer treatment.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antibodies, Monoclonal ; pharmacology ; Bleomycin ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Colonic Neoplasms ; pathology ; Female ; HT29 Cells ; Humans ; Immunoglobulin Fab Fragments ; pharmacology ; Immunotoxins ; pharmacology ; KB Cells ; Liver Neoplasms ; pathology ; Mice ; Tumor Cells, Cultured ; drug effects

This study was aimed to investigate the clinical outcome of ricin-immunotoxin mediated T cell partially depleted HLA/MLC mismatched allogeneic hematopoietic stem cell transplantation. 13 patients with hematological malignancies were treated by ricin-immunotoxin mediated T cell partially depleted allogeneic hematopoietic stem cell transplantations from HLA/MLC mismatched donors, including 6 cases of CML in CP(1), 1 case of ALL in CR(1), 1 case of ALL in CR(2), 1 case of ALL in relapse, 2 cases of AML in CR(1), 1 case of AML in CR(2), 1 case of MDS-RAEBT-AML (M(4)) in CR(1). The results showed that 8 cases were engrafted successfully, 2 cases of them developed grade II acute GVHD and 2 cases developed grade III-IV acute GVHD. Within following-up of 8 - 90 months, 2 patients who experienced grade III-IV acute GVHD died early after transplantation; 1 patient died of late onset of infection; the other 5 patients survived free from diseases. After failure at first infusion, 4 patients were given reinfusion of peripheral blood hematopoietic stem cells from the same donor. 3 out of 4 cases failed to engraft and only one patient got engraftment but died of related complications of transplantation. One patient was performed a second transplantation from a syngeneic donor and survive free of disease until now. In conclusion, T cell partially depleted HLA/MLC mismatched allogeneic hematopoietic stem cell transplantation by ricin-immunotoxin decreases the occurrence of severe acute GVHD but with high risk of rejection, which clinical outcome still needs further evaluation.
Adolescent ; Adult ; Child ; Female ; Graft vs Host Disease ; epidemiology ; Hematopoiesis ; Hematopoietic Stem Cell Transplantation ; mortality ; Humans ; Immunotoxins ; pharmacology ; Lymphocyte Depletion ; methods ; Male ; Ricin ; pharmacology ; T-Lymphocytes ; drug effects ; Transplantation, Homologous

OBJECTIVE: To investigate the in vitro and in vivo anticancer efficacy of the immunotoxin DTATEGF against human NSCLC brain metastatic tumor PC9-BrM3 cell line. METHODS: The effect of the immunotoxin DTATEGF was tested for its ability to inhibit the proliferation of PC9-BrM3 cells in vitro by MTT assay. The cell cycle and the apoptosis of cells with 1 pmol/L DTATEGF were examined by flow cytometry. In vivo, 2 μg of DTATEGF or control Bickel3 was given intratumor to nude mice with established PC9-BrM3 xenografts on their hips, and tumor volumes were measured and tumor samples were investigated by immunchistochemistry SABC method. The microvessel density (MVD) was measured in each group. RESULTS: In vitro, DTATEGF killed PC9-BrM3 cells and showed an IC50 of 1 pmol/L. The apoptotic rate in the 1 pmol/L DTATEGF group was (64.0±0.5)% , significantly higher than that in the control group (1.5±0.4)% (P<0.01). The cell cycle was obviously inhibited by DTATEGF in a dose-dependent manner. The percentage of cells treated with 1 pmol/L DTATEGF in SubG0/G1 phase was (32.0±1.5)%, significantly higher than that in the control group (2.0±0.4)% (P<0.01). In vivo, DTATEGF significantly inhibited the growth of PC9-BrM3 hip tumors (P<0.05). The MVD of the DTATEGF group was (15.6±4.6)/mm2, significantly lower than that of the control group (31.2±5.4)/mm2 (P<0.01). CONCLUSION DTATEGF inhibits the growth of the PC9-BrM3 cell line and induces its apoptosis. It is highly efficacious against human metastatic NSCLC brain tumor and against neovascularization.
Animals ; Antibodies, Bispecific ; pharmacology ; Apoptosis ; Brain Neoplasms ; drug therapy ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cell Cycle ; Cell Line, Tumor ; Humans ; Immunotoxins ; pharmacology ; Mice ; Mice, Nude ; Neovascularization, Pathologic ; prevention & control ; Xenograft Model Antitumor Assays

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology

ADP Ribose Transferases ; chemistry ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Bacterial Toxins ; chemistry ; pharmacology ; Exotoxins ; chemistry ; pharmacology ; Female ; HMGN2 Protein ; chemistry ; pharmacology ; HeLa Cells ; Humans ; Immunotoxins ; pharmacology ; Mice ; Mice, Inbred BALB C ; Protein Structure, Tertiary ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; Virulence Factors ; chemistry ; pharmacology

OBJECTIVETo prepare anti-human IgG-dextran-adriamycin conjugate for immunotargeting of S180 sarcoma and assess its effects on the tumor weight and survival time of the tumor-bearing mice.

METHODSAnti-human IgG-dextran- adriamycin was synthesized by conjugating dextran and adriamycin with anti-human IgG. The immunoactivity of anti-human IgG-dextran-adriamycin was measured by enzyme-linked immunosorbent assay (ELISA), and the cytotoxicity of anti-human IgG, adriamycin, and the IgG-dextran-adriamycin conjugate against the tumor cells in vitro was evaluated using MTT assay. In mice bearing S180 sarcoma, the agents were tested for their effects against tumor cell growth and the survival time of mice.

RESULTSThe molar ratio of anti-mouse IgG, dextran, and adriamycin was 1:2.5:38 in the conjugate. The conjugates were shown to retain the immunoactivity of anti-human IgG, and possessed cytotoxicity to S180 cells in vitro. Administration of the conjugate and intratumor injection of human IgG resulted in a tumor suppression rate of 17.72%in mice bearing S180 sarcoma, but did not prolong the survival time of the mice.

CONCLUSIONThe anti-human IgG-dextran-adriamycin conjugate shows targeted antitumor effect against S180 sarcoma in mice.

Animals ; Antibodies, Anti-Idiotypic ; administration & dosage ; pharmacology ; Antibodies, Monoclonal ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cell Survival ; drug effects ; Dextrans ; administration & dosage ; pharmacology ; Doxorubicin ; administration & dosage ; pharmacology ; Female ; Immunoglobulin G ; administration & dosage ; pharmacology ; Immunotoxins ; administration & dosage ; pharmacology ; Mice ; Sarcoma 180 ; drug therapy ; pathology ; Survival Analysis ; Tumor Burden ; drug effects

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Cell Division/drug effects ; Cell Line ; Cells, Cultured ; Corneal Stroma/*cytology/drug effects ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2/*pharmacology ; Humans ; Immunotoxins/pharmacology ; *N-Glycosyl Hydrolases ; Plant Proteins/*pharmacology ; Rabbits ; Ribosome Inactivating Proteins, Type 1