Showing papers by "Ira Pastan published in 1998"

HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter.

Abstract: The FDA approved HIV-1 protease inhibitors, ritonavir, saquinavir, and indinavir, are very effective in inhibiting HIV-1 replication, but their long-term efficacy is unknown. Since in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether these protease inhibitors are recognized by the MDR1 multidrug transporter (P-glycoprotein, or P-gp), thereby reducing their intracellular accumulation. In vitro studies in isolated membrane preparations from insect cells infected with MDR1-expressing recombinant baculovirus showed that these inhibitors significantly stimulated P-gp-specific ATPase activity and that this stimulation was inhibited by SDZ PSC 833, a potent inhibitor of P-gp. Furthermore, photoaffinity labeling of P-gp with the substrate analogue [125I]iodoarylazidoprazosin (IAAP) was inhibited by all three inhibitors. Cell-based approaches to evaluate the ability of these protease inhibitors to compete for transport of known P-gp substrates showe...

Human P-Glycoprotein Exhibits Reduced Affinity for Substrates during a Catalytic Transition State †

Abstract: Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (...

Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity

Abstract: Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37°C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis

Abstract: A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.

Cross-Resistance to Methotrexate and Metals in Human Cisplatin-resistant Cell Lines Results from a Pleiotropic Defect in Accumulation of These Compounds Associated with Reduced Plasma Membrane Binding Proteins

Abstract: Cross-resistance to a wide array of toxic chemicals is a common phenomenon in cisplatin-resistant cell lines. In this study, two independently isolated cisplatin-resistant cell lines derived from a human hepatoma and a cervical adenocarcinoma were shown to be cross-resistant to methotrexate (MTX) and several metal salts, such as sodium arsenite, sodium arsenate, antimony potassium tartrate, and cadmium chloride. A pleiotropic defect resulting in reduced accumulation of cisplatin, 3[H]MTX, 73As3+, and 73As5+ was found in both cisplatin-resistant cell lines. Analysis by immunoblot, indirect immunofluorescence, and Northern hybridization showed dramatically reduced expression of the folate binding protein that mediates MTX uptake in both human cisplatin-resistant cell lines. By photoaffinity labeling with UV irradiation, specific binding proteins of Mr 230,000 and Mr 48,000 for 73As3+ and Mr 190,000 for 73As5+ were found in enriched plasma membrane of both human cisplatin-sensitive parental cell lines. Expression of these specific binding proteins was decreased in cells selected for cisplatin resistance. A protein band at Mr 36,000 that binds to 73As3+ was overexpressed in both human cisplatin-resistant cell lines. The finding of loss of distinct binding proteins for MTX, arsenate, and arsenite in association with decreased accumulation of these agents in cisplatin-resistant cells suggests a pleiotropic, possibly regulatory, alteration in these cells.

Accumulation of a recombinant immunotoxin in a tumor in vivo : Fewer than 1000 molecules per cell are sufficient for complete responses

Abstract: Recombinant immunotoxins have been shown to cure human tumor xenografts in mice, but their biodistribution to both tumors and normal organs has not been reported. Anti-Tac(Fv)-PE38 is a single-chain recombinant immunotoxin composed of the variable heavy and light domains of the anti-Tac monoclonal antibody that reacts with the primate interleukin 2 (IL2) receptor alpha subunit (IL2R alpha or CD25) fused to a truncated form of Pseudomonas exotoxin (PE). 125I-labeled anti-Tac(Fv)-PE38 was given i.v. to immunodeficient mice each bearing two A431 tumors, one that expresses IL2R alpha (ATAC-4) and one that does not (A431, parental). A single i.v. dose of 4 microg/mouse caused complete regression of the IL2R alpha + tumor. At 6 h, over 6% of the injected dose/g was found in the ATAC-4 tumor, and 2% was in the A431 tumor. Uptake in the ATAC-4 tumor was higher than in any other tissue. Sections of tumor examined by autoradiography indicated that anti-Tac(Fv)-PE38 was distributed throughout the entire tumor, with some portions having higher uptake than others. By subtracting uptake in tumors without receptor (A431) from uptake in receptor-containing tumors (ATAC-4), we calculated that at least 400 molecules/cell specifically bound to IL2R alpha-positive tumor cells at 90 min and 750 molecules/cell bound at 360 min. This is similar to the 400-870 molecules/cell required for >99.9% killing of ATAC-4 cells growing as a monolayer. The results show that solid tumors in mice can be eradicated like cells in tissue culture, and that delivery of less than 1000 molecules/cell is sufficient to cause complete tumor regressions.

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport†

Abstract: P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.

Complete regression of established human glioblastoma tumor xenograft by interleukin-4 toxin therapy.

Abstract: No curative therapy is available for malignant gliomas. We have discovered that human glioblastoma cells express high affinity interleukin-4 receptor (IL-4R), which is an attractive target for receptor-directed IL-4 toxin therapy. The IL-4 toxin, IL-4(38-37)-PE38KDEL, is a fusion protein containing translocation and enzymatic domains of Pseudomonas exotoxin and a circularly permuted human IL-4. The IL-4 toxin binds specifically to the IL-4R and is highly cytotoxic to glioblastoma cells, as determined by clonogenic and protein synthesis inhibition assays. Intratumoral administration of the IL-4 toxin given on alternate days for 3-4 doses into U251 glioblastoma flank tumors in nude mice, showed a complete remission of small (approximately 13 mm3) and large (approximately 60 mm3) tumors in all animals, without any evidence of toxicity. A significant antitumor activity was also observed when the IL-4 toxin was administered via i.p. and i.v. routes. These results demonstrate that the IL-4 toxin may be a new therapeutic drug for the treatment of human glioblastoma. Therefore, we have begun a Phase I clinical trial with IL-4(38-37)-PE38KDEL for treatment of human glioblastoma.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

Abstract: We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Role of caspases in immunotoxin-induced apoptosis of cancer cells.

Abstract: Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonas exotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the “death substrate” poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFα. Also the fluorescent substrate, DEVD-AFC, is cleaved 2−4-fold more rapidly by lysates from B3(Fv)-PE38 t...

Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein.

Abstract: P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, ( 125 I)iodoarylazi- doprazosin (IAAP), were abrogated, with little effect on (R- 32 P)-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino- proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12.

Reconsidering targeted toxins to eliminate HIV infection: You gotta have HAART

Abstract: The success of highly active anti-retroviral therapy (HAART) has inspired new concepts for eliminating HIV from infected individuals. A major obstacle is the persistence of long-lived reservoirs of latently infected cells that might become activated at some time after cessation of therapy. We propose that, in the context of treatment strategies to deliberately activate and eliminate these reservoirs, hybrid toxins targeted to kill HIV-infected cells be reconsidered in combination with HAART. Such combinations might also prove valuable in protocols aimed at preventing mother-to-child transmission and establishment of infection immediately after exposure to HIV. We suggest experimental approaches in vitro and in animal models to test various issues related to safety and efficacy of this concept.

Co-Expression of Human Adenosine Deaminase and Multidrug Resistance Using a Bicistronic Retroviral Vector

Abstract: Current gene therapy protocols designed to treat adenosine deaminase (ADA) deficiency and other metabolic disorders suffer from low-efficiency delivery to target cells and a lack of long-term stability in expression of the therapeutic proteins. These problems may be resolved by use of an in vivo dominant selection. The multidrug transporter (MDR1) has been suggested as a useful selective marker for gene therapy. In this work, we co-expressed ADA and MDR1 cDNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system produced a bicistronic mRNA containing both ADA and MDR1, which enables co-expression of ADA and MDR1, and also allows the two proteins to be translated separately. After in vitro selection using a cytotoxic MDR1 substrate, vincristine, we demonstrated that functional ADA was co-expressed with MDR1 in proportion to the expression level of MDR1, whereas MDR1 expression was proportional to the stringency of the vincristine selection...

Evaluation of the in vivo biodistribution of yttrium-labeled isomers of CHX-DTPA-conjugated monoclonal antibodies.

Abstract: 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.

[33] Functional expression of human P-glycoprotein from plasmids using vaccinia virus-bacteriophage T7 RNA polymerase system

Abstract: Publisher Summary This chapter describes the vaccinia-T7 expression system that has proved to be ideal for functional studies of P-glycoprotein (Pgp) and the transporter associated with multidrug resistance (MDR). The heterologous expression systems have been explored but none have been completely satisfying. The baculovirus/insect cell system appears to be most promising for the large-scale synthesis of protein for biochemical and structural analysis. The expression of human Pgp in the yeast Saccharomyces cerevisiae and E. coli have met with limited success owing to low expression levels, toxicity, or intrinsically high ATPase levels, but neither proved as versatile as the vaccinia-T7 system. Higher-level expression in the yeast Pichia pastoris appears to be feasible. The use of this vaccinia-T7 transient system that does not involve drug selection for Pgp expression eliminates the need to consider these possible complications in the interpretation of the observed phenotypes. The major drawback of the vaccinia-T7 system is that the infected transfected cells cannot be used to measure relative resistance to MDR drugs in cell proliferation assays because the infected cells are committed to virus-induced lysis.

Specific Killing of HIV-infected Lymphocytes by a Recombinant Immunotoxin Directed against the HIV-1 Envelope Glycoprotein

Abstract: 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 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 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°C, retaining 80% of its original activity after 24 hr 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

Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo.

Abstract: 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.

Photoaffinity labeling of human P-glycoprotein: effect of modulator interaction and ATP hydrolysis on substrate binding.

Abstract: Publisher Summary This chapter discusses the methods to evaluate the differential effect of adenosine triphosphate (ATP) hydrolysis and modulator interaction on substrate binding to these two sites. The results presented in the chapter deal with P-glycoprotein (Pgp) in isolated membranes from baculovirus-infected insect cells. These assays can also be successfully used for studies with Pgp expressed in human and murine cells as well as with the purified protein on its reconstitution into proteoliposomes. Pgp shows internal homology of amino acid sequence between its N- and C-terminal halves. Each half of the protein contains six putative transmembrane regions (TMs) followed by a consensus nucleotide-binding domain (NBD). Four major steps are involved in drug transport: (1) substrate recognition, (2) ATP binding, (3) ATP hydrolysis, and (4) coupling of ATP hydrolysis to drug translocation. Photoaffinity labeling of Pgp with analogs of substrates and modulators has revealed two major areas, one on each homologous half of the protein, as primary sites of drug interaction.

Construction and characterization of bicistronic retroviral vectors encoding the multidrug transporter and beta-galactosidase or green fluorescent protein.

Abstract: Multidrug resistance (MDR) can be conferred by overexpression of the adenosine triphosphate-driven multidrug transporter P-glycoprotein (Pgp) known as MDR1 Thus, two potential applications of the MDR1 gene that may be useful in gene therapy are the protection of bone marrow cells from the cytotoxic effects of chemotherapy regimens in cancer patients and its possible use as an in vivo selectable gene when linked to a therapeutic gene In this study, we have designed two retroviral bicistronic expression vectors by linking the MDR1 gene to the reporters known as beta-galactosidase and the red-shifted green fluorescent protein (GFP) We report the creation of stable producer cell lines that synthesize virus particles carrying the MDR-internal ribosomal entry site (IRES)-lacZ and the MDR-IRES-GFP transgenes These transcriptional fusions allow coordinate expression of Pgp and the reporter gene product to easily mark the MDR phenotype Using the MDR-IRES-lacZ retrovirus, we demonstrate that periodic pulses of cytotoxic drug selection with a Pgp substrate enable sustained, long-term expression of the reporter beta-galactosidase in otherwise unstable transductants We have also incorporated the improved features of GFP as a reporter gene into our MDR-IRES-GFP retrovirus This vector allows rapid and specific identification of MDR1 gene transfer and expression in living cells either by fluorescence microscopy or by fluorescence-activated cell sorter analysis These two MDR/reporter gene systems should be useful for in vivo studies, the evaluation of the potential of the MDR1 gene in gene therapy applications, and as a monitor of the selective efficacy of its MDR phenotype

Clinical Trials with Pseudomonas Exotoxin Immunotoxins

Abstract: Pseudomonas exotoxin (PE) has been used to make immunotoxins for cancer therapy for more than a decade (FitzGerald et al. 1983). The function of PE is described in detail elsewhere in this book. In summary, PE is a 613 amino acid (66kDa) single-chain protein secreted by Pseudomonas aeruginosa. X-ray crystallography (Allured et al. 1986) and mutational studies (Gray et al. 1984; Hwang et al. 1987) have shown that PE is composed of three major structural and functional domains: an NH2-terminal cell binding domain (domain Ia, composed of amino acids 1–252), a central translocation domain (domain II, amino acids 253–364), and a COOH-terminal domain (III, amino acids 399–613). The latter catalyzes the ADP-ribosylation and inactivation of elongation factor 2 and thereby inhibits protein synthesis and leads to cell death. Domain III contain a COOH-terminal sequence (REDLK) that directs the endocytosed and processed toxin into the endoplasmic reticulum (see below). Substitution of REDLK with a KDEL sequence, which is known to retain newly synthesized proteins in the endoplasmic reticulum (Seetharam et al. 1991), results in a PE molecule that is more toxic to cells probably because it is more efficiently brought to the endoplasm reticulum where translocation seems to occur. Domain Ib is composed of amino acids 365–399 and has no known function; deletion of all of this domain results in no loss of activity (Fig. 1).

Selection and maintenance of multidrug-resistant cells.

Abstract: Publisher Summary This chapter discusses the selection and maintenance of multidrug-resistant cells, including specialized techniques, such as transfection and transduction of multidrug resistance genes and the introduction of multidrug resistance genes into bone marrow to form drug-resistant bone marrow colony-forming units (cfus). In the former approach, a cell line is carefully cloned so that all the cells in the population derive from a single cell; these cloned cells are exposed to a single cytotoxic agent in doses that kill the great majority of the cells in the population. This approach results in single-step resistance to the agent and individual surviving clones may be screened for cross-resistance to other drugs. The second approach is to expose an uncloned cell population to a selective drug in gradually increasing doses, allowing the population to die back and reexpand after each increase in the stringency of selection. This approach may result in highly multidrug-resistant populations in which the mechanisms of multidrug resistance may be relatively unique, particularly if a potent mechanism is responsible for resistance at an early stage of selection and the derivatives of the original resistant cell have overgrown the population. The advantage of this approach is that it casts a wide net for many different possible kinds of drug resistance.

Aminosyn II Effectively Blocks Renal Uptake of 18F-labeled Anti-Tac Disulfide-stabilized Fv

Abstract: Because intact IgG has limitations as a tumor-imaging agent, radiolabeled Fv fragments are being evaluated. Due to the high renal accumulation of Fv fragments, methods to block renal uptake are being sought. This study evaluated how well Aminosyn II, a Food and Drug Administration-approved 15% amino acid solution, would block the renal accumulation of 18F anti-Tac disulfide-stabilized Fv (dsFv) fragments (small fragments with high renal uptake). The anti-Tac dsFv is directed against the α subunit of the interleukin 2 receptor. It was labeled at specific activities of 1.1–2.7 mCi/mg using N -succinimidyl 4-[18F]fluoromethyl benzoate. Four adult baboons were injected i.v. with 0.7–1.9 mCi and 150 µg of dsFv. Each baboon was preinjected with Aminosyn II i.v. and, on a separate occasion, with a control solution. Thirty min before injection of 18F-labeled anti-Tac dsFv, a bolus of either solution was given, followed by a constant infusion of 13.3 ml/kg/h. Quantitative positron emission tomography imaging was performed. The amino acid levels in serum were measured serially. The baseline levels of lysine (and other amino acids) in plasma were not significantly different in either the Aminosyn II or control infusion group and did not change during the control infusion. In the Aminosyn II group, lysine levels in plasma 5 min before anti-Tac dsFv infusion were 5–15 times higher than the baseline value and continued to rise during the infusion. The areas under the curve in blood of the 18F-labeled anti-Tac dsFv, from time of injection to end of imaging, expressed as percentage injected dose (%ID), were 28.94 ± 4.05%ID × h/liter (mean ± SD) for the control group and 32.09 ± 11.15%ID × h/liter for the Aminosyn II group ( P = 0.54). The peak concentration of 18F-labeled anti-Tac dsFv in the kidney of the controls was 24.53 ± 4.34%ID; the value in the Aminosyn II group was 5.39 ± 1.89%ID, representing a mean decrease of 78.5%. The times to reach 90% of the peak levels of 18F in the kidney were 5.6 ± 3.0 min for the Aminosyn II group and 33.8 ± 4.8 min for the control group. The amounts excreted in urine by 90 min were 47.7 ± 8.55%ID and 78.5 ± 12.8%ID ( P = 0.01) for the controls and Aminosyn II group, respectively. In conclusion, Aminosyn II effectively blocks the renal accumulation of 18F-labeled anti-Tac dsFv. Use of Aminosyn II should allow much higher tracer administration for the same radiation exposure to the target organ (kidney).