Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 217 records · Page 12Linked to original sources

PubMed1995

Effects of phosphorylation of P-glycoprotein on multidrug resistance.

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to, their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Acquisition of multiple copies of a mutant topoisomerase IIalpha allele by chromosome 17 aneuploidy is associated with etoposide resistance in human melanoma cell lines.

Mutants of the human melanoma cell line, FEM-X, selected in multiple steps with VP- 16 (etoposide), are cross resistant to the epipodophyllotoxins and doxorubicin. Complementary DNA's for topoisomerase IIalpha were cloned from both FEM-X and FVP3, the most resistant mutant. Deletion of nucleotides 1320-1322 (or Ala429 from the resulting topoisomerase IIalpha protein) was unique to the cDNA from the drug resistant cell line. Expression of the mutant mRNA increases in parallel with VP-16 resistance in this series of cell lines. Restriction analysis and Southern analysis with allele-specific oligonucleotide probes were used to quantify the ratio of wild-type to mutant topoisomerase IIalpha alleles present in DNA amplified by PCR from both FEM-X and the drug resistant sublines. This analysis shows that in cell lines of increasing drug resistance, the number of mutant topoisomerase IIalpha alleles increases incrementally along with a concomitant decrease in the number of wild-type alleles. By quantitative Southern analysis of genomic DNA the total number of topoisomerase IIalpha alleles in FVP3 is approximately 2-fold that in the parental cells. Fluorescence in situ hybridization with a chromosome l7 paint reveals that amplification of the topoisomerase IIalpha locus in FVP3 correlates with an increase in the number of chromosome 17's, specifically the long arm. Cytogenetic analysis demonstrates that FEM-X contains three copies of chromosome 17, two of which are morphologically normal. During drug selection, FVP3 has gained 2-3 additional copies of the long arm of chromosome 17, the chromosomal location of the topoisomerase IIalpha locus. In this subline it is likely that three copies of the topoisomerase IIalpha gene are found on normal chromosome 17's and two on an isochromosome of the long arm of 17. By pulsed field gel electrophoresis, we were able to detect changes in the restriction pattern of the region of the long arm of chromosome 17 around the topoisomerase IIalpha locus that correlate with observed cytogenetic changes in FVP3. These results suggest that the acquisition of the mutant allele of topoisomerase IIalpha confers a selective advantage to cells in the presence of VP-16. As the drug concentration increased during the selection process, surviving sublines show preferential expression of the mutant topoisomerase IIalpha mRNA over that of the wild-type which is associated with a concomitant increase in the number of mutant topoisomerase IIalpha alleles.

Alleles↗

PubMed1995

Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog.

Recombinant DNA techniques now allow the production of "mini-antibodies" called Fv fragments. These have been produced either as the independent variable domains of the heavy and light chains non-covalently associated in one-to-one stoichiometry or as single-chain gene products with the two domains linked by an intervening peptide sequence. Although Fv fragments can have excellent binding properties, they are often difficult to produce in good yield and lack the characteristic stability of whole antibodies. To improve the stability of the Fv molecule, we have introduced a cysteine residue into conserved framework regions of both the heavy and light variable domains from the anti-Tac antibody at positions compatible with the formation of an interdomain disulfide linkage (i.e. VH-44 and VL-99). The mutant subunits form a disulfide-bonded Fv molecule, which binds to the alpha-subunit of the IL2 receptor (IL2R alpha) with an affinity identical to that of humanized anti-Tac IgG. This disulfide-stabilized Fv (dsFv) proved to be substantially more resistant to denaturation by heat or urea treatment than the single-chain Fv (scFv). Furthermore, the yield of dsFv is -four-fold higher than that of the single-chain analog.

Amino Acid Sequence↗

PubMed1995

Cytotoxicity of TGF alpha-PE40 and correlation to expression of epidermal growth factor receptor.

TGF alpha-PE40 is a chimeric protein composed of transforming growth factor alpha (TGF alpha) linked to a modified Pseudomonas exotoxin (PE40). We tested the in vitro cytotoxicity of TGF alpha-PE40 on 23 different solid human tumour xenografts established in nude mice and human bone marrow cells from healthy donors, utilising a modified clonogenic assay. In order to distinguish non-specific toxicity from the targeted effects of TGF alpha-PE40, epidermal growth factor receptor (EGFR) expression of the tumours studied was assessed by Northern blot, slot blot and immunohistochemistry. TGF alpha-PE40 demonstrated differential cytotoxicity on human tumour xenografts in the clonogenic assay. No toxicity on human bone marrow cells was observed. In vitro activity of TGF alpha-PE40 showed a significant correlation with the expression of EGF receptors as determined by immunohistochemistry and slot blot. Further studies will be performed in order to determine the in vivo activity of this compound in tumour-bearing nude mice.

Animals↗

PubMed1995

Construction of a functional disulfide-stabilized TCR Fv indicates that antibody and TCR Fv frameworks are very similar in structure.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions. We now design and produce a disulfide-stabilized Fv of a T cell receptor. It is composed of V alpha and V beta variable domains of the 2B4 TCR stabilized by a disulfide bond between framework residues of the TCR Fv at a site corresponding to that used for disulfide stabilization of antibody Fvs. For ease of production and detection, the TCRdsFv was fused to a truncated form of Pseudomonas exotoxin (PE38). The TCR(dsFv) retains its native conformation and is much more stable than a TCR scFv. Moreover, it is functional in biological assays. Because successful disulfide stabilization of the TCR Fv by the positions used for antibody Fv stabilization would not occur unless the mutated residues in TCR Fv are at positions closely similar to those in antibody Fvs, most likely within less than 1.5 A, these results provide very strong experimental evidence for the structural similarity between immunoglobulin and TCR antigen-binding variable domains.

Amino Acid Sequence↗

PubMed1995

Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.

Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.

Amino Acid Sequence↗

PubMed1995

Retroviral coexpression of a multidrug resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease.

Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.

3T3 Cells↗

PubMed1995

Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation.

Using molecular modeling technology we have recently identified positions in conserved framework regions of Fvs which can be used to stabilize antibody Fvs by an interchain disulfide bond engineered in between the structurally conserved framework positions of the variable domains of heavy (VH) and light (VL) immunoglobulin chains (disulfide-stabilized Fv; dsFv). The computer model indicated the existence of other potential sites in the framework regions that might be suitable for disulfide bond formation between VH and VL. The possibility of obtaining dsFvs using these positions is evaluated here experimentally by constructing dsFv immunotoxins in which the Fv moiety is fused to a truncated form of Pseudomonas exotoxin. We analyzed the extent of dsFv formation and the activity of the resulting dsFv immunotoxins, and compared various dsFv molecules with the scFv immunotoxin. Our results demonstrate that position H44-L105 is the only one which gives high production yields of active dsFv. All other positions gave either low yields and activity or completely failed to produce active dsFv. With one exception, the formation and activities of the dsFvs corresponded to the C alpha-C alpha distance between the VH and VL positions, with an optimal distance of 5.7 A producing the best dsFv. Distances of 6.0-6.9 A resulted in a low yield of protein that was still capable of binding antigen, whereas distances > 7.0 A resulted in molecules in which dsFv formation was not obtained.

Amino Acid Sequence↗

PubMed1995

Genetic analysis of the multidrug transporter.

The analysis of how human cancers evade chemotherapy has revealed a rich variety of cell-based genetic changes resulting in drug resistance. One of the best studied of these genetic alterations is increased expression of an ATP-dependent plasma membrane transport system, known as P-glycoprotein, or the multidrug transporter. This transporter actively effluxes a large number of natural product, hydrophobic, cytotoxic drugs, including many important anticancer agents. This review focuses on the genetic and molecular genetic analysis of the human multidrug transporter, including structure-function analysis, pre- and posttranslational regulation of expression, the role of gene amplification in increased expression, and the properties of transgenic and "knock-out" mice. One important feature of the MDR gene is its potential for the development of new selectable vectors for human gene therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Biochemical characterization of human GCF transcription factor in tumor cells.

GCF is a transcriptional regulator that was found to repress transcription of the epidermal growth factor (EGF) receptor and several other genes and is encoded by a 3-kb mRNA (R. Kageyama and I. Pastan, Cell, 59: 815-825, 1989; A. C. Johnson et al., J. Biol. Chem., 267: 1689-1694, 1992). To identify and characterize the GCF gene product at the cellular level, we have developed antibodies against a bacterially expressed GCF fusion protein. GCF antibodies recognize GCF present in extracts from human cells and causes a "supershift" of a protein DNA complex containing a GCF oligonucleotide binding site. The major form of GCF has a molecular weight of approximately M(r) 97,000, identical to that of GCF transiently expressed in CV1 cells by the vaccinia virus system. In addition, other less abundant species with slightly higher and lower apparent molecular weight are specifically recognized, suggesting extensive posttranslational modification. GCF is highly expressed in EGF receptor-negative human cell lines (HUT102, U266, and CA46) and in lower amounts in several EGF receptor-expressing cells (KB, A431, TMK, and HeLa). Cell fractionation studies indicate that GCF is predominantly localized in the nucleus. GCF is a stable protein with a relatively long half-life. In addition, GCF is a phosphoprotein, and the phosphorylated form is found to be associated with the nuclear compartment in both HUT102 and KB cells. Phosphorylation occurs on serine and threonine residues and is stimulated by okadaic acid, phorbol myristate acetate, and cyclic AMP, but not vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1995

Expression cloning of cDNAs that render cancer cells resistant to Pseudomonas and diphtheria toxin and immunotoxins.

BACKGROUND: Several immunotoxins in which antibodies are coupled to plant or bacterial toxins are now in clinical trials for the treatment of cancer. One of these is B3-LysPE38 in which MAb B3 which reacts with many human cancers, is coupled with a genetically modified form of Pseudomonas exotoxin (PE). MATERIALS AND METHODS: To investigate how cells can become resistant to PE-derived immunotoxins, we constructed an immunotoxin-sensitive MCF-7 breast cancer cell line that contains SV40 T antigen and allows episomal replication of SV40 origin containing plasmids. We transfected a pCDM8/HeLa cDNA expression library into these cells, thereby causing over-expression of the plasmid-encoded genes. The transfected cells were treated with immunotoxin to select for resistance-mediating plasmids, which were reisolated from these cells and amplified in Escherichia coli. The resulting plasmid pool was transfected into cells for two further rounds of selection and plasmid reisolation. RESULTS: Several plasmids that caused immunotoxin resistance were enriched by this selection procedure. Four plasmids were stably transfected into MCF-7 cells and found to increase their resistance to PE-derived immunotoxins by 5- to 20-fold. These plasmids also confer resistance to native PE and to diphtheria toxin but not to ricin or cycloheximide. Thus, they appear to specifically interfere with the action of ADP-ribosylating toxins. CONCLUSION: Cancer cells can become resistant to immunotoxins by deregulated expression of normal genes. The clinical significance of this type of resistance will be evaluated in clinical trials.

ADP Ribose Transferases↗

PubMed1995

Transfer of the MDR1 (multidrug resistance) gene: protection of hematopoietic cells from cytotoxic chemotherapy, and selection of transduced cells in vivo.

Expression of the drug efflux pump P-glycoprotein, encoded by the multidrug resistance (MDR1) gene, has been identified as an impediment to successful chemotherapy of neoplastic diseases. More recently, its potential use for gene therapy has been analyzed. Expression of a full-length MDR1 cDNA in hematopoietic cells renders them resistant to various anticancer drugs, as first shown in a transgenic mouse model. Similarly, mouse hematopoietic progenitor cells in bone marrow or peripheral blood are protected from the toxicity of anticancer chemotherapy by retroviral transduction of the MDR1 gene. Furthermore, cells engineered to express P-glycoprotein survived after the administration of cytotoxic drugs, indicating that the gene could function as a selectable marker in vivo. Recently, MDR1 transduction into isolated pluripotent hematopoietic stem cells has been demonstrated. Clinical studies on MDR1 gene transfer into hematopoietic cells of cancer patients are being planned. Transfer of the MDR1 gene into hematopoietic precursor cells may allow the introduction and selection of otherwise non-selectable genes in bone marrow. The ability to select transduced cells can circumvent the low transduction efficiency that has hampered efficient gene therapy. Recently, fusion genes in which the MDR1 cDNA is fused to genes that correct genetic disorders have been constructed to facilitate gene therapy of inherited metabolic disorders.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin.

Recently, we have demonstrated that a spectrum of human adenocarcinoma cell lines express binding sites for interleukin 13 (IL-13). These cells are killed by a chimeric protein composed of human (h) IL-13 and a derivative of Pseudomonas exotoxin, PE38QQR (Debinski et al., J. Biol. Chem., 270: 16775-16780, 1995). The cell killing was hIL-13- and hIL-4-specific, indicating that a common binding site for the two cytokines is present in several solid tumor cell lines. Herein, we report that an array of established glioma cell lines is killed by very low concentrations of hIL-13-PE38QQR, often reaching <1 ng/ml (<20 pM). Glioma cells express up to 30,000 molecules of IL-13 receptor/cell which has intermediate affinity toward hIL-13. hIL-13-PE38QQR is more active (up to 3 logs difference in cytotoxic activities) than are the corresponding chimeric toxins containing hIL-4 or hIL-6. The cytotoxic action of hIL-13-PE38QQR is blocked by an excess of hIL-13 on all cell lines studied, and it is not neutralized by hIL-4 on some of these cells. Our results show that human brain cancers richly express receptors for IL-13. Furthermore, the interaction detected previously between receptors for IL-13 and IL-4 on solid tumors cell lines is of a qualitatively different character in U-251 MG and U-373 MG glioma cells. The receptor for IL-13 may represent a new marker of brain cancers and an attractive target for anticancer therapies.

ADP Ribose Transferases↗

PubMed1995

Immunotoxins containing Pseudomonas exotoxin that target LeY damage human endothelial cells in an antibody-specific mode: relevance to vascular leak syndrome.

Vascular leak syndrome (VLS) was originally found to be a major dose-limiting toxicity in humans with cancer treated with several immunotoxins (ITs) containing ricin A chain or blocked ricin. Recently, VLS has also been observed in patients treated with an IT containing the murine monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant truncated form of Pseudomonas exotoxin (PE) A. Antibody B3 (IgG1k) recognizes LewisY and related carbohydrate epitopes present on many human solid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen. In the clinical trial, the development of VLS has prevented the administration of the amount of IT necessary to achieve blood levels required for good therapeutic responses. We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechanism of VLS induced by ITs containing PE. To assess the cytotoxic effect of IT on endothelial cells, various ITs were incubated with cells for 2 or 20 h, and the incorporation of [3H]leucine into protein was measured. The endothelial cells studied were human umbilical vein endothelial cells, human lung-derived microvascular endothelial cells (HUVECs), human adult dermal microvascular endothelial cells, human pulmonary artery endothelial cells, and human aortic endothelial cells. We found that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with PE38, as well as B3(Fv)-PE38 (LMB-7), a recombinant single chain immunotoxin, inhibited protein synthesis, with 50% inhibitory concentrations between 600 and 1000 ng/ml for 20-h incubation in HUVECs, human lung-derived microvascular endothelial cells, and human adult dermal microvascular endothelial cells but not on human pulmonary artery endothelial cells. The cytotoxic effect was specific since PE38 itself or PE coupled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml. Further evidence that the cytotoxicity of B3-containing ITs is due to specific B3 binding to endothelial cells comes from the fact that the cytotoxicity can be blocked by excess free MAb B3. HUVECs undergo overt morphological changes after treatment with B3-LysPE38 or B3(Fv)PE38. Gaps between the cells are formed after a 20-h exposure but not after 2 h. These studies suggest that VLS in patients is due to capillary damage caused by prolonged exposure to high concentrations of LMB-1.

ADP Ribose Transferases↗

PubMed1995

Coexpression of a multidrug-resistance gene (MDR1) and herpes simplex virus thymidine kinase gene as part of a bicistronic messenger RNA in a retrovirus vector allows selective killing of MDR1-transduced cells

A new retroviral vector, pSXLC/pHa, was constructed to coexpress drug-selectable markers with a second gene of interest as a part of a bicistronic mRNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system was used to develop a new retroviral vector pHa-MDR-IRES-TK which expresses a single mRNA from which translation of the MDR1 gene is cap dependent and translation of the herpes simplex virus thymidine kinase gene is IRES dependent. The pHa-MDR-IRES-TK transfectants showed high levels of P-glycoprotein expression and multidrug resistance. More than 95% of the vincristine-resistant cells transfected or transduced with pHa-MDR-IRES-TK showed hypersensitivity to ganciclovir, which selects against cells expressing herpes simplex virus thymidine kinase. An amphotropic retrovirus titer of 7.8 x 10(4)/ml was obtained with this vector. This safety-modified vector should be useful for introducing the MDR1 gene into bone marrow cells to protect normal cells from the toxic effects of cancer chemotherapy because this vector allows the elimination of cancer cells that have been unintentionally transduced with the MDR1 vector.

Journal Article↗

PubMed1995

Immunotoxins that target an oncogenic mutant epidermal growth factor receptor expressed in human tumors.

Human cancers arise from a series of mutations, many of which direct the expression of mutant proteins with altered functions. These aberrant proteins are attractive targets for new therapeutic agents. One such protein is a mutant epidermal growth factor receptor (EGFRvIII) that has an in-frame deletion near the NH2 terminus of its extracellular domain. This protein was first identified in human gliomas, but has also been shown to be present in lung and breast carcinomas. The deletion results in a receptor with constitutive tyrosine kinase activity that enhances the tumorigenicity of glioblastomas in vivo. The deletion also creates a tumor-specific cell-surface sequence at the deletion junction. Three specific anti-EGFRvIII mAbs have been isolated following immunization with a mixture of a deletion junction synthetic peptide and EGFRvIII as present on cell membranes. We have constructed immunotoxins by conjugating a modified version of Pseudomonas exotoxin A to these mAbs. Immunotoxins were tested on cells that had been transfected with cDNA for the EGFRvIII receptor and expressed receptor protein at 5 x 10(5) receptors/cell. All three immunotoxins were cytotoxic to these cells, with 50% inhibition of protein synthesis occurring in a 15-50 pM range. The immunotoxins specifically targeted EGFRvIII, as their cytotoxicity could be blocked by their respective free antibody. They showed little or no cytotoxicity to cells expressing high levels of normal epidermal growth factor receptors, demonstrating that they are able to discriminate between cells expressing the mutant receptor and those expressing the wild-type receptor. Immunotoxins targeted to mutant epidermal growth factor receptors are promising candidates for further development as tumor cell-specific therapeutic agents.

3T3 Cells↗

PubMed1995

Recombinant F(ab') C242-Pseudomonas exotoxin, but not the whole antibody-based immunotoxin, causes regression of a human colorectal tumor xenograft.

We have previously made a Mr 195,000 immunotoxin (IT) composed of mAb C242 coupled to Pseudomonas exotoxin A. This IT inhibited growth but did not cause regression of a human colorectal cancer xenograft growing in nude mice (W. Debinski et al., J. Clin. Invest., 90: 405-411, 1992). Since smaller proteins penetrate into tissues and tumors better than larger proteins, we have made a smaller recombinant (r) IT to overcome the hypothesized poor tumor penetration of the Mr 195,000 conjugate. This was accomplished by making a C242rF(ab')-based Mr 86,000 IT. To make rF(ab')-IT, the Fd and kappa chains of mAb C242 were cloned, and kappa was fused at the gene level to PE38QQR, a mutant form of Pseudomonas exotoxin. Both C242Fd and C242kappa-PE38QQR were expressed in a bacterial expression system, and large amounts of the C242Fd attached via a disulfide bond to the C242kappa-PE38QQR were obtained. The C242rF(ab')-IT covalently linked heterodimer has a 50% inhibitory concentration of 0.2-2.0 ng/ml (2-20 pm, respectively) on human colon adenocarcinoma cell lines that express the C242 antigen. When injected into mice bearing Colo205 tumors, the C242rF(ab')-PE38QQR caused an immediate regression of the tumors, while the C242-PE38QQR conjugate had only a growth inhibitory effect. In addition, several cures were obtained. Our results indicate that rIT C242F(ab')-PE38QQR is a much more potent antitumor agent than an IgG conjugate.

ADP Ribose Transferases↗

PubMed1995

Characterization of B1(Fv)PE38 and B1(dsFv)PE38: single-chain and disulfide-stabilized Fv immunotoxins with increased activity that cause complete remissions of established human carcinoma xenografts in nude mice.

The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Pastan et al., Cancer Res., 51: 3781-3787, 1991). We have generated plasmids encoding immunotoxins in which the Fv domain of B1, either as a single-chain Fv or as a disulfide-stabilized Fv (dsFv), was fused to PE38, a truncated form of Pseudomonas exotoxin A. To compare the activities of the two types of recombinant immunotoxins, the proteins were prepared from cytoplasmic inclusion bodies produced in Escherichia coli. The immunotoxins were evaluated for stability, antigen binding, specific cytotoxicity, pharmacokinetics, and antitumor activity in a nude mouse model. Although the single-chain immunotoxin is relatively stable when incubated at 37 degreesC (t(1/2) approximately 4 h), the dsFv immunotoxin is much more stable, with no loss of activity after 8 h at 37 degreesC. The single-chain immunotoxin has a 2-fold better binding affinity and cytotoxicity toward antigen-positive cultured cells than the dsFv immunotoxin. The half-lives in the blood of mice of B1(Fv)PE38 (single-chain) and B1(dsFv)PE38 (disulfide-stabilized) are 23 and 27 min, respectively. Their therapeutic potential was evaluated in athymic nude mice bearing human epidermoid carcinoma xenografts. Both immunotoxins caused complete regressions of the s.c. (30-40 mm3) tumors when given i.v. in three doses of 0.025 mg/kg every other day. This is one-twentieth of the mouse LD50. Recombinant immunotoxins containing the B1(Fv) are 2-3-fold more potent antitumor agents than previously described immunotoxins containing the B3(Fv) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620, 1991), which also target LeY and related carbohydrates in human tumors, but have a similar toxicity in mice. Thus, their therapeutic window is 2-3-fold larger. In addition, B1(dsFv)PE38 has only a 50% decrease in the apparent binding affinity of B1(Fv)PE38, whereas B3(dsFv)PE38 has a much greater loss in antigen binding.

ADP Ribose Transferases↗