Retroviral transfer of multidrug transporter to murine hematopoietic stem cells.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
I Pastan
Publications and source records attributed to I Pastan.
Retroviral transfer of human MDR1 gene into human T lymphocytes.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Recombinant Fv immunotoxins and Fv fragments as novel agents for cancer therapy and diagnosis.
Recombinant immunotoxins are new agents being developed for cancer therapy. They are composed of Fv fragments of antibodies that bind to cancer cells fused to a truncated form of a very potent bacterial toxin. The antibody moiety directs the toxin to cancer cells, which are killed, while normal cells are not recognized and thus survive. The excellent preclinical results in vitro and in vivo have led to the initiation of several clinical trials.
Animals↗
Similarities in the biodistribution of iodine-labeled anti-Tac single-chain disulfide-stabilized Fv fragment and anti-Tac disulfide-stabilized Fv fragment.
We evaluated the biodistribution and pharmacokinetics of two different iodine-labeled Fv fragments of anti-Tac monoclonal antibody (MAb) in normal and tumor-bearing nude mice. One was a disulfide-stabilized Fv fragment (dsFv), and the other was a single-chain disulfide-stabilized Fv fragment (scdsFv). The scdsFv is a newly developed type of Fv fragment superior to the dsFv in which the VH and VL are linked by covalent bonds through a spacer arm and by an internal disulfide bond. These modifications increase the yield of scdsFv. Both reagents recognize the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The biodistribution of the Fv fragments was evaluated in normal mice co-injected with 50 mg of L-lysine and in a no-lysine control group. Biodistribution was also evaluated in nude mice bearing subcutaneous tumor xenografts derived from IL-2Ralpha-positive ATAC4 cells and receptor-negative A431 cells. These mice were co-injected with 125I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 131I-labeled anti-Tac dsFv (2 microCi/0.7 microg) or with 131I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 125I-labeled anti-Tac dsFv (4 microCi/0.7 microg). The biodistribution of 125I-labeled anti-Tac scdsFv and 131I-labeled anti-Tac dsFv was very similar in all organs and the tumors. The renal uptake of both reagents was blocked effectively (<93%) and similarly by lysine. The scdsFv cleared slightly faster from the circulation than did the dsFv because there were more aggregates of dsFv than of scdsFv (3% vs. 1%, respectively). The scdsFv-to-dsFv ratio ranged from 0.79 to 1.20 in all organs at all time points we examined. In conclusion, the first biodistribution study of an scdsFv molecule shows that the scdsFv had a biodistribution very similar to that of the dsFv and seems to be a good alternative to the dsFv because of its higher production yield.
Animals↗
Characterization of an MDR1 retroviral bicistronic vector for correction of X-linked severe combined immunodeficiency.
X-linked severe combined immunodeficiency (XSCID) is a hereditary disorder characterized by severe T cell lymphopenia and abnormal B cell function. The disease is caused by mutations in IL2RG, the gene encoding the interleukin-2 receptor common gamma chain (gamma c) shared by several interleukin receptors. A Harvey retroviral bicistronic vector containing an IL2RG cDNA and cDNA encoding the multidrug transporter (MDR1) was constructed to investigate the correction of XSCID. Translation of the MDR1 cDNA is achieved from an internal ribosome entry site (IRES). Mouse fibroblasts transfected or transduced with the vector expressed both membrane proteins as detected with specific monoclonal antibodies by fluorescence activated cell sorting. Two human XSCID B cell lines were transduced using a filter concentration method in combination with phosphate depletion. Significant expression of both proteins was detected by Western blot analysis. This construct might be particularly useful if high expression of gamma c is required, as might be achievable through in vivo selection for drug resistance of recipient lymphocytes.
3T3 Cells↗
Epitope blocking: positive and negative effects on the biodistribution of 125I-labeled anti-Tac disulfide-stabilized Fv fragment of two antibodies against different epitopes of the circulating antigen.
Prior in vivo studies using the 125I-labeled anti-Tac disulfide-stabilized variable region fragment (125I-anti-Tac dsFv) of monoclonal antibody in the presence of the circulating soluble alpha subunit of the interleukin-2 receptor (sIL-2R alpha) have shown formation of complexes which interfere with biodistribution. In this study we evaluated the effects of preinjecting HuTac and 7G7/B6, two immunoglobulin Gs (IgGs) that recognize different epitopes of sIL-2R alpha, on the biodistribution of 125I-anti-Tac dsFv in mice bearing SP2/Tac tumor xenografts, which produce sIL-2R alpha, or on nude mice injected with 500 ng of sIL-2R alpha. We also evaluated the biodistribution in mice of 125I-labeled sIL-2R alpha injected alone or with HuTac and 7G7/B6. Injection of either HuTac or 7G7/B6 resulted in complexes with the sIL-2R alpha in serum. Injection of HuTac before 125I-anti-Tac dsFv, in SP2/Tac tumor-bearing mice, resulted in faster clearance of the dsFv from the blood (7.6% ID/g at 30 min), compared to 23.2% ID/g for the no-antibody control; preinjection of 7G7/B6 prolonged the retention of 125I-anti-Tac dsFv to 35.3% ID/g, with more complexes in serum. In mice pre-injected with 7G7/B6 the concentration of 125I-anti-Tac dsFv in tumor was lower (5.2 +/- 0.3% ID/g) than in mice preinjected with HuTac (7.9 +/- 1.2% ID/g) or in the control group (5.6 +/- 0.7% ID/g). In conclusion, while both IgGs formed complexes with sIL-2R alpha and prolonged its retention, preinjection of 7G7/B6 was detrimental, because the increased circulating sIL-2R alpha still had the epitope recognized by the dsFv available for binding and neutralized the anti-Tac dsFv upon injection, whereas preinjection of HuTac blocked the epitope.
Animals↗
Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein.
BACKGROUND: 3B3 is a high-affinity anti-gp120 antibody that neutralizes a wide range of primary and laboratory isolates of HIV-1. The parental antibody was isolated from a combinatorial phage display library constructed from bone marrow RNA of an HIV-infected individual. We have generated a highly active immunotoxin using the 3B3 single-chain Fv (scFv) which can specifically kill lymphocytes infected by HIV-1. MATERIALS AND METHODS: We used recombinant DNA technology to clone the Fv fragment of 3B3 and produce a single-chain Fv (scFv). 3B3 scFv was then fused to a truncated version of Pseudomonas exotoxin A (PE38), giving rise to a recombinant immunotoxin 3B3(Fv)-PE38 that was expressed in E. coli and purified to near homogeneity. RESULTS: 3B3(Fv)-PE38 binds with the same affinity as the parental Fab antibody to the MN strain of gp120. The immunotoxin specifically kills a gp120-expressing transfected cell line and a chronically HIV-infected lymphocytic cell line. The immunotoxin is very stable at 37 degrees C, retaining 80% of its original activity after 24 hr. CONCLUSIONS: Potent immunotoxins such as 3B3(Fv)-PE38 could be utilized in combination with multidrug cocktails that limit viral replication to help reduce viral reservoirs in patients with AIDS.
Anti-HIV Agents↗
Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo.
Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.
Animals↗
Evaluation of the in vivo biodistribution of yttrium-labeled isomers of CHX-DTPA-conjugated monoclonal antibodies.
UNLABELLED: We evaluated the in vivo stability and biodistribution of four isomers (CHX-A', CHA-A", CHX-B' and CHX-B") of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriaminepentaaceti c acid (CHX-DTPA), a recently developed backbone-substituted derivative of DTPA. METHODS: The ligands were conjugated to monoclonal antibody B3, a murine IgG1 kappa, and labeled with 88Y at 55.5-66.6 MBq/mg (1.5-1.8 mCi/mg). Nontumor-bearing nude mice were injected intravenously with 55.5-66.6 kBq (1.5-1.8 microCi) of 88Y-labeled B3 conjugates and with 125I-labeled B3 as an internal control. The mice were then killed at 6, 24, 48, 96 and 168 hr postinjection. RESULTS: At 168 hr, the concentration of 88Y in processed bone of either CHX-A' [4.6% injected dose (ID)/g] or CHX-A" (4.0%ID/g) was less than that of either the CHX-B' (21.9%ID/g) or B" (12.1%ID/g) ligands. The two ligands CHX-B" and CHX-B' were not acceptable for yttrium labeling of antibody because of their high and progressive bone accumulation. The accumulation of 88Y in bone of CHX-B' was five times greater than that of CHX-A' at 168 hr. The CHX-A" cleared from the circulation slightly faster than CHX-A' without releasing the yttrium and showed the lowest uptake by bone of any of the four isomers. The accumulation in the other normal organs was similar for all four isomers of 88Y-CHX-B3 conjugates. CONCLUSION: Although the CHX-B" and CHX-B' were not acceptable for labeling with yttrium, the CHX-A' and CHX-A" were suitable, indicating that differences in stereochemistry can greatly influence stability of radionuclide in the chelate.
Animals↗
Clinical applications of immunotoxins. Introduction.
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Animals↗
Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system.
Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr1a (also known as mdr3) and mdr1b (also known as mdr1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [125I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Targeted therapy of cancer with recombinant immunotoxins.
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ADP Ribose Transferases↗
Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein.
Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.
3T3 Cells↗
Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors.
Many B-cell malignancies express the CD22 antigen on their cell surface. To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin. These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas. To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4. The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct. The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE. The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity. RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37 degrees C and exhibited selective cytotoxic activity for CD22+-cultured cell lines. Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma. CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin. Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.
Amino Acid Sequence↗
Antisense inhibition of CAS, the human homologue of the yeast chromosome segregation gene CSE1, interferes with mitosis in HeLa cells.
We have analyzed the effects on HeLa cells of reduction of the CAS protein, the human homologue to yeast chromosome segregation protein CSE1. Expression of CAS antisense cDNA decreases the amount of CAS protein in HeLa cells and perturbs progression from G2 (retards transition from G2) to G1 in the cell cycle. Increased levels of cyclin B in CAS antisense transfected cells correlated with an arrest in G2 phase or mitosis. This arrest upon CAS attenuation is consistent with observations that yeast with CSE1 mutations are defective in mitosis and cyclin B degradation.
Cellular Apoptosis Susceptibility Protein↗
Amphiregulin antisense oligonucleotide inhibits the growth of T3M4 human pancreatic cancer cells and sensitizes the cells to EGF receptor-targeted therapy.
Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin. It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth. Therefore, we used an amphiregulin antisense oligonucleotide (AR-AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells. These cells express high levels of EGFR and amphiregulin. AR-AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose-dependent manner. Exogenous amphiregulin reversed AR-AS-mediated growth inhibition. A random oligonucleotide (AR-R) did not alter either cell growth or cellular amphiregulin immunoreactivity. AR-AS also increased cellular EGFR protein levels and enhanced the growth-inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor-alpha coupled to Pseudomonas exotoxin that internalizes into cells via EGFR. These findings indicate that there is an important EGFR/ amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR-targeted therapy.
Amino Acid Sequence↗
Recombinant toxins containing human granulocyte-macrophage colony-stimulating factor and either pseudomonas exotoxin or diphtheria toxin kill gastrointestinal cancer and leukemia cells.
The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is a potential target for toxin-directed therapy, because it is overexpressed on many leukemias and solid tumors and apparently not on stem cells. To investigate the potential therapeutic use of GM-CSF toxins, we fused human GM-CSF to truncated forms of either Pseudomonas exotoxin (PE) or diphtheria toxin (DT) and tested the cytotoxicity of the resulting GM-CSF-PE38KDEL and DT388-GM-CSF on human gastrointestinal (GI) carcinomas and leukemias. Toward gastric and colon cancer cell lines, GM-CSF-PE38KDEL was much more cytotoxic than DT388-GM-CSF, with IC50s (concentration resulting in 50% inhibition of protein synthesis) of 0.5 to 10 ng/mL compared with 4 to 400 ng/mL, respectively. In contrast, toward leukemia lines and fresh bone marrow cells DT388-GM-CSF was more cytotoxic than GM-CSF-PE38KDEL. The cytotoxicity of both GM-CSF-PE38KDEL and DT388-GM-CSF toward the human cells was specific, because it could be competed by an excess of GM-CSF. Binding studies indicated that human GM-CSF receptors were present on all of the human GI and leukemic cell lines tested, at levels of 540 to 3,700 sites per cell (kd = 0.2 to 2 nmol/L), and the number of sites per cell did not correlate with the cell type. A similar pattern of cytotoxicity was found with recombinant immunotoxins binding to the transferrin receptor, in that anti-TFR(Fv)-PE38KDEL was much more cytotoxic than DT388-anti-TFR(Fv) toward GI cells, but both were similar in their cytotoxic activity toward leukemia cells. The fact that PE is more effective than DT in killing GI but not leukemic tumor cells targeted by GM-CSF indicates a fundamental difference in the way PE or DT gains access to the cytosol in these cells. GM-CSF-PE38KDEL and DT388-GM-CSF deserve further evaluation as possible treatments for selected tumors.
Cell Death↗
Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers.
The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers. We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb). Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein. Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore). Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG. The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies.
Amino Acid Sequence↗