Showing papers by "Ira Pastan published in 1996"

Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers.

Abstract: Monoclonal antibody MAb K1 recognizes a 40-kDa glycoprotein present on the surface of mesothelial cells, mesotheliomas, and ovarian cancers. We have used MAb K1 to isolate a 2138-bp cDNA that encodes this antigen. The cDNA has an 1884-bp open reading frame encoding a 69-kDa protein. When the cDNA was transfected into COS and NIH 3T3 cells, the antigen was found on the cell surface and could be released by treatment with phosphatidylinositol-specific phospholipase C. The 69-kDa precursor is processed to the 40-kDa form. The protein has been named mesothelin because it is made by mesothelial cells. Mesothelin may play a role in cellular adhesion.

Engineering antibody Fv fragments for cancer detection and therapy: disulfide-stabilized Fv fragments.

Abstract: Disulfide-stabilized Fv fragments of antibodies (dsFv) are molecules in which the VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions distant from complementarity-determining regions (CDRs). This method of stabilization is applicable for the stabilization of many antibody Fvs and has also been applied to a T-cell receptor Fv. A summary of the design strategy, and the construction and production of various dsFvs and dsFv-fusion proteins is presented. Included in the discussion are the biochemical features of dsFvs in comparison with scFvs, the effect of disulfide stabilization on Fv binding and activity, and various applications of dsFvs and dsFv-immunotoxins for tumor imaging and the treatment of solid tumors in animal models.

Treatment of advanced solid tumors with immunotoxin LMB–1: An antibody linked to Pseudomonas exotoxin

Abstract: Immunotoxin LMB–1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin. B3 recognizes a carbohydrate antigen (LeY) present on many human solid tumors1. LMB–1 has excellent antitumor activity in nude mice bearing LeY–positive tumors2. We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the LeY antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis. Three patients (two colon, one breast cancer) had minor responses. The maximum tolerated dose of LMB–1 is 75 μg/kg given intravenously three times every other day. The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema. Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display

Abstract: EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Preclinical Development of a Recombinant Toxin Containing Circularly Permuted Interleukin 4 and Truncated Pseudomonas Exotoxin for Therapy of Malignant Astrocytoma

Abstract: Effective treatment is lacking for malignant glioblastoma/astrocytoma We have identified interleukin-4 receptors (IL-4R) on human malignant astrocytoma We demonstrate that 16 of 21 surgical samples of high-grade astrocytoma and glioblastoma but not normal brain tissues expressed IL-4R as assessed by reverse transcriptase PCR We further demonstrate that human malignant astrocytoma cell lines express high-affinity IL-4R Using a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin A, we observed that this toxin IL4(38-37)-PE38KDEL) is highly cytotoxic to IL-4R-bearing glioblastoma cells Compared with a previously reported IL4-PE chimeric protein (IL-PE4E), IL4(38-37)-PE38KDEL bound with higher affinity and was 3-30-fold more cytotoxic to glioblastoma cell lines Upon intrathecal administration in monkeys, high cerebrospinal fluid IL4(38-37)-PE38KDEL levels were achieved using 2- and 6-microg/kg doses without any central nervous system or other abnormalities IL4(38-37)-PE38KDEL levels were not detectable in the serum of any monkey studied When IL4(38-37)-PE38KDEL was injected into the right frontal cortex of rats, localized necrosis was observed at 1000-ng/ml doses but not at < or = 100-ng/ml doses We conclude that by localized administration, nontoxic levels of IL4(38-37)-PE38KDEL can be achieved, which may have significant cytotoxic activity against malignant astrocytoma

Adenovirus-mediated gene transfer to human breast tumor cells: An approach for cancer gene therapy and bone marrow purging

Abstract: To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells. Following infection of breast cancer cells with an adenovirus expressing β-galactosidase gene, high levels of β-galactosidase activity were observed. No β-galactosidase activity was observed in low-density human bone marrow cells. A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing β-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency. In contrast, human bone marrow cells treated under the same conditions expressed very low levels of the transfected β-galactosidase DNA. Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments. A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells. Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells. However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine. On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.

Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins.

Abstract: Recombinant immunotoxins are chimeric proteins in which a truncated toxin is fused to a recombinant antigen-binding domain such as a recombinant Fv or Fab. Recombinant immunotoxins target cell surface receptors and other antigens on tumor cells. The antigen-binding and -targeting domains in recombinant immunotoxins are usually single-chain Fvs (scFv), which are the antibody variable regions connected by a flexible peptide linker and fused directly to a bacterial toxin. However, Fabs have also been used. Recombinant immunotoxins have very good activity in vitro on cultured human tumor cell lines and have produced complete regressions and cures of established tumor xenografts in nude mouse models. Problems with the stability and binding of some scFv immunotoxins as well as scFvs not linked to toxin led to the development of a new type of recombinant Fv immunotoxin in which the targeting variable domains of the Fv are stabilized by an interchain disulfide bond located in structurally conserved framework positions of the VH and VL domains. These are termed disulfide-stabilized Fvs (dsFv) or dsFv immunotoxins. dsFvs and dsFv immunotoxins have several advantages over scFv immunotoxins. This review summarizes the design, construction, activities in vitro and in vivo, and biochemical characteristics of dsFv immunotoxins and compares them with scFv immunotoxins.

Role of CAS, a human homologue to the yeast chromosome segregation gene CSE1, in toxin and tumor necrosis factor mediated apoptosis.

Abstract: We have previously isolated by expression/selection cloning plasmids containing human cDNAs that rendered MCF-7 breast cancer cells resistant to immunotoxins, Pseudomonas exotoxin (PE), and diphthe...

L-Lysine Effectively Blocks Renal Uptake of 125I- or 99mTc-labeled Anti-Tac Disulfide-stabilized Fv Fragment

Abstract: In this study, we investigated the ability of L-lysine to block renal uptake of 125I- or 99mTc- labeled Fv fragments. Anti-Tac disulfide-stabilized Fv fragment (dsFv) was derived from a murine monoclonal antibody that recognizes the alpha subunit of the interleukin-2 receptor (IL-2R alpha). The 125I- or 99mTc-labeled dsFv was injected i.v. into non-tumor-bearing nude mice or into nude mice bearing SP2/Tac (IL-2R alpha positive) and SP2/0 (IL-2R alpha negative) tumor. We then evaluated the pharmacokinetics of L-[3H]lysine and the effect of L-lysine dose, timing of administration, and route of delivery on catabolism and biodistribution of i.v. dsFv. Peak renal uptake of i.v. or i.p. injected L-[3H]lysine occurred within 5 and 15 min, respectively. The kidney uptake of L-lysine exhibited a dose-response effect. When L-lysine was coinfused or injected shortly before dsFv, renal uptake of dsFv was blocked to < 5% of the control, but longer intervals were less effective. Aminosyn II and Travasol 10% (parenteral amino acid solutions) also blocked renal uptake of radiolabeled dsFv. Administration of L-lysine did not alter the blood kinetics and slightly increased the tumor uptake of dsFv, but it did prevent catabolism in the kidney and resulted in lower amounts of catabolites in the serum and urine. In conclusion, we have shown that a blocking dose of lysine, injected with or immediately before the injection of radiolabeled dsFv, is most effective in blocking the renal uptake of dsFv. This is consistent with the rapid uptake of L-[3H]lysine by the kidney and is further substantiated by the relative ineffectiveness of lysine injected immediately after the radiolabeled dsFv injection.

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle

Abstract: Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

Abstract: Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

In Vitro and In Vivo Liposome-Mediated Gene Transfer Leads to Human MDR1 Expression in Mouse Bone Marrow Progenitor Cells

Abstract: The ability to select bone marrow cells (BMC) expressing a selectable gene that confers resistance to anti-cancer drugs would be useful to protect bone marrow during chemotherapy. The human multidrug resistance (MDR1) gene encodes a 170-kD glycoprotein (P-gp), an ATP-dependent transmembrane efflux pump for many different cytotoxic drugs. In this work, we demonstrate efficient expression of the human MDR1 gene in mouse BMC after transfection with a liposomal delivery system (DLS-liposomes). The human MDR1 cDNA expression plasmid (pHaMDR1/A) was encapsulated in DLS-liposomes and delivered to mouse BMC using two approaches: (i) in vitro transfection of BMC followed by bone marrow transplantation and (ii) in vivo direct systemic gene transfer. After both the in vitro and the in vivo approaches, polymerase chain reaction (PCR) analysis confirmed that the human MDR1 gene was successfully transfected to bone marrow, spleen, and peripheral blood (PB) cells, with the human MDR1 gene detected in BMC for up...

Expression of the Human Multidrug Resistance and Glucocerebrosidase cDNAs from Adeno-Associated Vectors: Efficient Promoter Activity of AAV Sequences and In Vivo Delivery via Liposomes

Abstract: Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5′ end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analys...

Cytotoxic and antitumor activity of a recombinant tumor necrosis factor-B1(Fv) fusion protein on LeY antigen-expressing human cancer cells.

Abstract: We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the scFv region of monoclonal antibody (mAb) B1, an antibody that recognizes LeY antigen present on many human cancer cells. Our rationale for fusing the scFv to the COOH terminus of TNF was to diminish the binding of the fusion protein to TNF receptors because the COOH terminus of TNF is involved in binding, and thus to partially inactivate (detoxify) the molecule. The Fv region should then target and accumulate the fusion protein on cancer cells, which should compensate for the reduced binding affinity of the TNF moiety and lead to selective killing of TNF-sensitive antigen-expressing cancer cells. The fusion protein was expressed in Escherichia coli and found in insoluble inclusion bodies. After refolding and purification by anion exchange, Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to LeY antigen on cancer cells with the same affinity as B1(scFv) and B1(scFv) immunotoxins but with significantly lower affinity to the TNF receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to LeY antigen-expressing cancer cells that are sensitive to TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is antibody targeted and TNF mediated because it can be prevented (as shown on MCF-7 cells) by an antibody competing for LeY antigen binding and by an antibody that neutralizes TNF-alpha. TNF-B1(Fv) kills TNF-alpha-sensitive cells that do not express the target antigen only at much higher doses than TNF trimer, and it does not kill LeY-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regression of MCF-7 tumor xenografts in mice at doses that are not toxic to the mice. Thus, the reduced binding of the TNF moiety to TNF receptors, combined with binding of the B1(Fv) portion to LeY antigen, makes TNF-B1(Fv) an agent for selective killing of LeY-expressing TNF-sensitive cancer cells.

Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.

Abstract: B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation.

Abstract: PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23. In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region. The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L [Reiter et al. (1994) J. Biol. Chem. 269, 18327-18331]. In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain. PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin. The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located. Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C. Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment. PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment. To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit. Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.

Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy.

Abstract: Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 microgram/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene.

The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders.

Abstract: Chemoresistance genes have been identified as an impediment to anticancer drug treatment. In particular, P-glycoprotein, the product of the multidrug-resistance (MDR1) gene, plays a major role in clinical treatment failure. Conversely, expression of an MDR1 cDNA in bone marrow of transgenic animals renders hematopoietic cells chemoresistant. Efficient transfer of drug-resistance genes to normal hematopoietic progenitor cells has been achieved with the use of retroviral vectors. In this article we review approaches which use the multidrug-resistance gene to protect bone marrow from myelosuppression following chemotherapy and as a selectable markerin vivo to increase the expression of nonselectable genes which correct hereditary diseases of the hematopoietic system.

Recombinant single‐chain and disulfide‐stabilized Fv‐immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice

Abstract: Monoclonal antibody (MAb) 55.1 specifically recognizes an antigen on the surface of human colon adenocarcinoma cells. We constructed recombinant immunotoxins composed of the heavy- and light-chain variable regions of MAb 55.1 fused to a recombinant form of Pseudomonas exotoxin (PE). The heavy- and light-chain variable regions are stabilized by 2 means. One is by a flexible peptide linker to form a single-chain antigen binding protein (scFv) and the second by an interchain disulfide bond engineered between structurally conserved framework regions. These are termed disulfide stabilized Fvs (dsFv). The 2 Fv forms are fused to truncated forms of PE lacking the cell binding domain. The recombinant scFv- and dsFv-immunotoxins were expressed in E. coli and purified to near homogeneity. The scFv- and dsFv-immunotoxins were shown to be specifically cytotoxic to human colon adenocarcinoma cell lines. The scFv-immunotoxin containing PE38KDEL was more active than the immunotoxin containing PE38 with the native carboxyl terminus (REDLK). However, the PE38KDEL immunotoxin is about 2-fold more toxic in mice, and therefore it does not appreciably increase the therapeutic window in mice. Intravenous administration of the scFv- and dsFv- recombinant immunotoxins caused complete regression of a human colon carcinoma (Colo205) growing subcutaneously in immunodeficient mice. The dsFv-immunotoxin has better antitumor activity compared with its scFv-immunotoxin counterpart. © 1996 Wiley-Liss, Inc.

Treatment of acute myelocytic leukemia with interleukin-6 Pseudomonas exotoxin fusion protein in a rat leukemia model

Abstract: We studied the applicability of interleukin-6 Pseudomonas exotoxin fusion protein (IL-6PE4E) for treatment of acute myelocytic leukemia (AML). Leukemic cells from five out of 10 AML patients studied expressed IL-6 receptor (IL-6R) and proliferation in vitro was inhibited in four of these cases. The potential of this approach in vivo was tested in a pre-clinical model for AML; the Brown Norway acute myelocytic leukemia (BNML). To obtain IL-6R expression levels on BNML cells comparable to the numbers expressed on human AML, human IL-6R gene transfectants of the BNML sub-line LT12 (LT12/IL-6R) were generated. IL-6PE4E is cytotoxic in vitro to LT12/IL-6R expressing 1400 high affinity IL-6R per cell with 50% inhibition of DNA synthesis at 1 ng/ml. In vivo treatment of leukemic rats carrying LT12/IL-6R leukemia indicated that the maximal tolerated dose of IL-6PE4E was 275 +/- 25 microg/kg/day, when continuously administered for 7 days and resulted in a 90% reduction in leukemic cell load. At this dose level of IL-6PE4E no reduction of normal hemopoietic progenitors was seen in non-leukemic rats. At higher dose levels (350-1050 microg/kg/day) severe systemic toxicity was encountered. On the basis of these pre-clinical studies the feasibility of growth factor-toxins for selective in vivo targeting to AML cells is evaluated.

A Novel Strategy for the Negative Selection in Mouse Embryonic Stem Cells Operated with Immunotoxin-Mediated Cell Targeting

Abstract: Immunotoxoin-mediated cell targeting (IMCT) is a technique for conditionally ablating specific cell types based on the cytotoxic activity of a recombinant immunotoxin anti-Tac (Fv)-PE40. To examine the feasibility of this technique for the negative selection in mouse embryonic stem (ES) cells, we investigated the responsiveness of cells expressing human interleukin-2 receptor alpha subunit to anti-Tac(Fv)-PE40. The immunotoxin treatment efficiently eliminated only ES cells bearing the receptor as a consequence of the target specificity of anti-Tac(Fv)-PE40, indicating that IMCT can be used as a novel strategy for positive and negative selection to enrich ES cell clones with a targeted mutation.

Retroviral transfer of the human MDR1 gene confers resistance to bisantrene-specific hematotoxicity.

Abstract: In this work, we demonstrate a protective effect conferred by the human multidrug resistance gene (MDR1) to populations of the murine hematopoietic system against the toxic effects of bisantrene, a novel intercalating cytotoxic agent under investigation as an anticancer agent. In vitro, MDR1-expressing cell lines are highly cross-resistant to bisantrene, and low levels of P-glycoprotein (the MDR1 gene product cell surface protein) confer resistance to the drug. MDR1-positive mice were generated after transplantation of bone marrow cells (BMC) transduced in vitro with a MDR1 retrovirus. Control mice were transplanted with BMC transduced with the neomycin resistance gene. Administration of a single i.v. dose of 50 mg/kg of bisantrene resulted in a decrease of the total WBC count of approximately 40%. In contrast, a decrease of the total WBC count of only 17% was observed in mice transplanted with MDR1-transduced BMC. Immunofluorescence studies with cell lineage-specific monoclonal antibodies showed that bisantrene was specifically toxic for B lymphocytes and macrophages. Double-staining with MRK16 (a monoclonal antibody specific for P-glycoprotein) demonstrated that a single dose of bisantrene increased the relative number of MDR1-transduced positive B cells, macrophages, and (to some extent) granulocytes when compared to the number found in MDR1-untreated mice or the bisantrene-treated neomycin-transduced control mice. These results provide in vivo evidence that bisantrene is a hematotoxic drug capable of selecting for MDR1-transduced hematopoietic cells. Bisantrene might be useful for gene therapy as an in vivo selective agent for cells transduced with MDR1 vectors that also coexpress therapeutic genes of interest.

Cytotoxic effects of TGF-α-Pseudomonas exotoxin A fusion protein in human pancreatic carcinoma cells

Abstract: The epidermal growth factor (EGF) receptor is overexpressed in human pancreatic cancers and cultured cell lines. TP40 is a chimeric protein composed of transforming growth factor-α (TGF-α) linked to a modified Pseudomonas exotoxin A (PE40) that exerts growth inhibitory effects on cells bearing a high number of EGF receptors. Therefore, we compared the effect of TP40 on the growth of Chinese hamster ovary (CHO) cells expressing varying levels of the EGF receptor and on the growth of two human pancreatic cancer cell lines. The growth of CHO cells devoid of endogenous EGF receptors was minimally altered by high concentrations of TP40, even following a 72-h incubation period. In contrast, in CHO cells expressing ∼95,000 and 438,000 EGF receptors per cell, one-half maximal growth inhibition oc- curred at 5 and 3 ng/ml TP40, respectively. Following a 72-h incubation in T3M4 and COLO 357 human pancreatic cancer cells, one-half maximal growth inhibition o curred at 0.2 and 0.4 ng/ml TP40, respectively. This effect was significantly greater than that of native Pseudomonas exotoxin A. These findings indicate that human pancre- atic cancer cells are markedly sensitive to the growth inhibitory effects of TP40 and raise the possibility that TP40 may have a therapeutic role in this disorder.