Biomedical subjects

W Debinski

Publications and source records attributed to W Debinski.

At least 37 records · Page 2Linked to original sources

PubMed1997

Reversion of uroepithelial cell tumorigenesis by the ectopic expression of human wnt-5a.

Wnt gene family members are thought to play an important role in cell growth and differentiation. When normal wnt gene expression is disrupted, there is the potential for cell transformation. We have found previously that a strong correlation exists between the loss of normal wnt-5a gene expression and cell transformation (Olson and Papkoff, Cell Growth & Differ., 5: 197-206, 1994). Recently, this has been tested directly using antisense wnt-5a, which, when expressed in mouse mammary cells, results in cell transformation (Olson and Gibo, Antisense wnt-5a transforms C57MG mouse mammary epithelial cells, manuscript in preparation). We hypothesize that wnt-5a is a growth-regulating gene, the disruption of which could result in tumorigenesis. The multistage progression of many human cancers involves the loss of normal tumor suppressor gene(s) activity. Several tumor suppressor genes are thought to map to chromosome 3p11-p25. We have studied the ectopic expression of human wnt-5a (3p14-p21) in a tumorigenic uroepithelial cell line with deletion of chromosome 3p13-p21.2. This results in loss of tumorigenicity in athymic nude mice and suppresses anchorage-independent cell growth in soft agar. This suggests that human wnt-5a is a novel tumor suppressor gene in uroepithelial cell carcinoma and may be one of the suppressor genes deleted or rearranged on chromosome 3p.

Animals↗

PubMed1997

Receptor for interleukin 13 on AIDS-associated Kaposi's sarcoma cells serves as a new target for a potent Pseudomonas exotoxin-based chimeric toxin protein.

AIDS-associated Kaposi's sarcoma (AIDS-KS), the most common malignant complication of human immunodeficiency virus infection, is characterized by neoplastic proliferation of mesenchymal cells. AIDS-KS cells release and respond to an array of cytokines through specific plasma membrane receptors. Specific targeting of potent cytotoxic agents to cell surface receptors/antigens on Kaposi's sarcoma cells may provide effective therapy for this malignancy. We have identified a new target in the form of an interleukin 13 (IL-13) receptor that is overexpressed in the five AIDS-KS cell lines examined. Radiolabeled IL-13 cross-linked to a single protein of about Mr 70,000 in AIDS-KS cells. We utilized a chimeric cytotoxic protein composed of IL-13 and a truncated Pseudomonas exotoxin (IL13-PE38QQR), which was found to be specifically and highly cytotoxic to AIDS-KS cells, as determined by protein synthesis inhibition and clonogenic assays. IL13-PE38QQR demonstrated significant antitumor activity in a human epidermoid carcinoma xenograft model. Normal human umbilical vein-derived endothelial, lymphoid, and bone marrow precursor cells expressed low levels of IL-13 receptors, and IL-13 toxin was not cytotoxic to them. Thus, IL-13 receptor on AIDS-KS cells may represent a novel plasma membrane protein(s) that could be utilized to target therapeutic agents.

ADP Ribose Transferases↗

PubMed1996

Receptor for interleukin (IL) 13 does not interact with IL4 but receptor for IL4 interacts with IL13 on human glioma cells.

Recently, we have demonstrated that human (h) glioma cell lines express large number of receptors (R) for interleukin 13 (IL13) (Debinski, W., Obiri, N. I., Powers, S. K., Pastan, I., and Puri, R. K. (1995) Clin. Cancer Res. 1, 1253-1258). These cells are extremely sensitive to a chimeric protein composed of hIL13 and a derivative of Pseudomonas exotoxin (PE), PE38QQR. We have found that the cytotoxicity of hIL13-PE38QQR was blocked by hIL13 but not by hIL4 on the U-251 MG and U-373 MG cells, contrary to what was observed on several adenocarcinoma cell lines. In the present study, we further explored interactions between receptor for IL13 and IL4 on glioma cells. Established human glioma cell lines, such as DBTRG MG, Hs 683, U-87 MG, SNB-19, and A-172, are very susceptible to hIL13-PE38QQR, and the action of the chimeric toxin is not blocked by hIL4 on all these cells either. Also, hIL4 is not a competitor for 125I-hIL13 binding sites on glioma cells. Of interest, a corresponding hIL4-based chimeric toxin, hIL4-PE38QQR, is poorly active or not active on all the tested glioma cell lines. When active, however, hIL4 toxin action was blocked by hIL13. hIL13 is a competitor for 125I-hIL14 binding in a competitive binding assay on glioma cells. hIL13 and hIL4 did not affect the growth of the tested glioma cell lines. Human glioblastoma multiforme explant cells exhibited similar responses to the chimeric toxins and interleukins when compared with that found in established glioma cultures. Our results suggest that the hIL13R on glioma cells is expressed in one predominant form, the form that does not interact with IL4. Thus, this type of hIL13R is apparently different from the one demonstrated previously on several adenocarcinoma cell lines.

ADP Ribose Transferases↗

PubMed1996

Targeting of interleukin-13 receptor on human renal cell carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR).

We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor. To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR. We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines. IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow-derived cells were not susceptible to the cytotoxic effect of IL 13-PE38QQR. The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R. The cytotoxic activity of IL13-PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay. Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC. IL13-PE38QQR competes for [125I]-IL-13 binding sites on RCC cells, although at a lower affinity than the wild-type recombinant cytokine. Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR. Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells. IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

ADP Ribose Transferases↗

PubMed1996

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain.

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.

Antibodies↗

PubMed1995

A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4.

Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies.

ADP Ribose Transferases↗

PubMed1995

Receptor for interleukin 13. Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins 2, 4, 7, 9, and 15.

Interleukin 13 (IL-13) shares many biological properties with IL-4, and although the receptor for IL-4 (IL-4R) has been characterized, the expression and structure of IL-13 receptor are unknown. We report here that human renal cell carcinoma (RCC) cells express large numbers of functional IL-13R. Human B lymphocytes and monocytes expressed a very small number of IL-13R, while resting or activated human T cells expressed little or no IL-13R. IL-4 did not compete for IL-13 binding, while IL-13 competed for IL-4 binding, even though IL-4R and IL-13R are structurally distinct on human RCC cells. IL-13 cross-linked with one major protein that is similar in size to the gamma c subunit of IL-2, -4, -7, -9, and -15 receptors but was not recognized by anti-gamma c or anti-IL-4R antibodies. IL-4, on the other hand, cross-linked with two major proteins, the smaller of which appears to be similar in size to IL-13R and gamma c, but (like the IL-13R) it did not react with anti-gamma c antibody. Although as shown in this study and in previous studies, gamma c is a functional component of IL-4R in lymphoid cells, it does not appear to be associated with IL-4R on RCC cells. Even in the absence of common gamma chain IL-4 and IL-13 were able to up-regulate intracellular adhesion molecule-1 antigen on RCC cells. These data suggest that the interaction of IL-13 with IL-4R does not involve gamma c and IL-13R itself may be a novel subunit of the IL-4R.

Cross Reactions↗

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

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↗

PubMed1994

Interleukin-4 receptors expressed on tumor cells may serve as a target for anticancer therapy using chimeric Pseudomonas exotoxin.

Interleukin-4 receptors (IL4R) are present on a wide variety of human cancer cells derived from both hematopoietic and epithelial malignancies. We have targeted IL4R on a human solid tumor xenograft with chimeric proteins composed of human IL4 (hIL4) and 2 different mutant forms of a powerful bacterial toxin, Pseudomonas exotoxin A (PE). The 2 chimeric toxins, termed hIL4-PE4E and hIL4-PE38QQR, showed specific, hIL4R-dependent and dose-dependent antitumor activities. Neither of the chimeric toxins showed antitumor potency when the ADP-ribosylation activity of the toxin was inactivated by mutagenesis. One of the chimeras, hIL4-PE38QQR, caused a complete although transient regression of established solid tumors. These observations indicate that hIL4-PE chimeric proteins should be further evaluated for the treatment of human malignancies.

ADP Ribose Transferases↗

PubMed1994

Human renal cell carcinoma cells are sensitive to the cytotoxic effect of a chimeric protein composed of human interleukin-4 and Pseudomonas exotoxin.

We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5' end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hIL4-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from < 1 ng/ml (12 pM) to 10 ng/ml (120 pM) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hIL4-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hIL4-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID50 = 1000 ng/ml) and a human fibroblast cell line (ID50 approximately 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID50, approximately 500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID50, approximately 4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

ADP Ribose Transferases↗

PubMed1993

Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

ADP Ribose Transferases↗

PubMed1993

A wide range of human cancers express interleukin 4 (IL4) receptors that can be targeted with chimeric toxin composed of IL4 and Pseudomonas exotoxin.

A chimeric toxin has been constructed by fusion of a gene encoding human interleukin 4 (hIL4) to a gene encoding a mutant form of Pseudomonas exotoxin A (PE) which cannot bind to its receptors (PE4E). The chimeric gene was expressed in Escherichia coli where large amounts of the chimeric toxin, hIL4-PE4E, was produced. Purified hIL4-PE4E was very cytotoxic to cancer cell lines of both hematopoietic and solid tumor origin. In the HUT 102 T cell leukemia and Daudi B cell lymphoma cell lines, protein synthesis was inhibited by 50% (ID50) at a hIL4-PE4E concentration of 2 and 7 ng/ml (25 and 86 pM, respectively). hIL4-PE4E was also very cytotoxic to cell lines derived from carcinomas of the colon, breast, stomach, liver, adrenals, and prostate, as well as melanoma and epidermoid carcinoma, indicating that hIL4 receptors are widely expressed on human malignancies. We also found that human phytohemagglutinin-activated peripheral blood lymphocytes were extremely sensitive to hIL4-PE4E with an ID50 of 0.2 ng/ml (2.5 pM). The cytotoxic action of hIL4-PE4E was specific because it was blocked by an excess of hIL4 and not of human interleukin 2. In addition, hIL4-PE4ED553, an enzymatically inactive form of the chimeric toxin, was not cytotoxic. These results suggest that the hIL4 receptor may be a target for therapy in malignant and immunologic disorders using hIL4 chimeric toxin.

ADP Ribose Transferases↗

PubMed1993

Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our data suggest that in hypertension, genetic overexpression of the ANP A-receptor subtype is related to the exaggerated biological response to ANP in this disease.

Affinity Labels↗

PubMed1992

Monovalent immunotoxin containing truncated form of Pseudomonas exotoxin as potent antitumor agent.

Recombinant truncated forms of Pseudomonas exotoxin A that lack the cell binding domain of Pseudomonas exotoxin A were coupled to an F(ab') fragment of a monoclonal antibody HB21 directed against the human transferrin receptor. One of these was NlysPE40. The other, NlysPE38QQR, has two amino groups on residues near the NH2-terminus and has no amino groups near the COOH-terminus. The proteins were linked by a stable thioether bond that connected the sulfhydryl group present in the hinge region of the antibody fragment to an amino group on the toxin. The F(ab')-PE40 immunotoxin, containing NlysPE40, exhibited potent cytotoxic activity on human carcinoma cell lines with a concentration of immunotoxin at which isotope incorporation falls by 50% when compared to nontreated cells (ID50) of 5.3 pM (0.5 ng/ml) on both the epidermoid carcinoma A431 and on the colon carcinoma Colo205. Immunotoxins made with whole antibody were considerably less active, with an ID50 of 15.9 pM (3.1 ng/ml) on these cell lines. F(ab')-PE38QQR, the immunotoxin containing NlysPE38QQR, was found to be the most active agent with an ID50 of 1.05 pM (0.1 ng/ml) on A431 cells. The greater cytotoxicity of immunotoxins containing fragmented antibody was probably due to the higher binding affinity of F(ab') conjugates in comparison to whole antibody conjugates to the transferrin receptor. The increase in cytotoxic activity of the immunotoxin made with NlysPE38QQR than that with NlysPE40 may reflect selective coupling of the toxin through NH2-terminal amino groups. The monovalent and divalent immunotoxins had dose-dependent antitumor effects on human epidermoid carcinoma xenografts in nude mice. A431 tumors completely regressed in all animals at a total dose of 105 pmol (10 micrograms) of F(ab')-PE38QQR and of 154 pmol (30 micrograms) of IgG-PE38QQR. Furthermore, the F(ab') immunotoxin was less toxic to mice than the conjugate containing IgG (840 pmol or 80 micrograms of total dose causing measurable adverse effects versus 208 pmol or 40 micrograms, respectively). Thus, a truncated Pseudomonas exotoxin A molecule coupled to the F(ab') fragment of an antibody is more active and less toxic in mice than an immunotoxin made with a whole antibody. Therefore, the therapeutic index for the monovalent immunotoxin is about four times better than that for the divalent immunotoxin.

ADP Ribose Transferases↗

PubMed1992

Monoclonal antibody C242-Pseudomonas exotoxin A. A specific and potent immunotoxin with antitumor activity on a human colon cancer xenograft in nude mice.

Two immunotoxins were constructed by chemically coupling the monoclonal antibody C242 to Pseudomonas exotoxin A (PE) or a modified form, NlysPE40, that lacks the cell binding domain of PE. Monoclonal antibody C242 recognizes a specific sialylated carbohydrate epitope on a high molecular weight membrane glycoprotein present on cells of human colon, pancreatic, and cervical cancers. C242-PE and C242-NlysPE40 were very cytotoxic for cells expressing this antigen with 50% inhibition of protein synthesis occurring on Colo205 cells at 0.2 ng/ml (0.9 pM) for C242-PE and 6.0 ng/ml (31 pM) for C242-NlysPE40. The two immunotoxins also exhibited a strong antitumor effect on a human colon cancer xenograft grown in nude mice. The specificity and potency of these two C242 immunotoxins warrant their further development for the treatment of cancer.

ADP Ribose Transferases↗

PubMed1991

Substitution of foreign protein sequences into a chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

TGF alpha-PE40 is a chimeric toxin made by replacing domain Ia of Pseudomonas exotoxin (PE) with transforming growth factor alpha (TGF alpha). We have now replaced a portion of domain Ib of PE with different polypeptides or an extra domain III of PE in transforming growth factor alpha-PE40 and maintained cell killing. Thus, TGF alpha-PE40 can be used to transport foreign protein sequences into the cytosol of cells.

ADP Ribose Transferases↗

PubMed1991

High salt intake-induced changes in atrial natriuretic factor kinetics are mediated by clearance receptors.

We have reported a paradoxical plasma atrial natriuretic factor (ANF) decline following prolonged high salt intake that was attributed to an increased tissue uptake of circulating ANF, leading to its augmented distribution volume (Vas) and metabolic clearance rate (MCR) as compared with control rats on a standard diet. To explore this phenomenon further, we evaluated possible chronic salt-loading-induced changes in ANF clearance (C-ANF) receptors, which appear to play a major role in ANF removal from the circulation. We studied changes in plasma [125I]ANF(1-28) and its pharmacokinetics after preoccupation of C-ANF receptors by its specific ligand, C-ANF(4-23), in high-salt-treated rats and their controls. Following C-ANF(4-23) administration, we detected significantly higher circulating [125I]ANF levels throughout the study period (8 min) in high-salt-fed rats compared with the controls (280-470% vs 100-215% increase of basal values, P less than 0.05). C-ANF(4-23) infusion caused a significantly greater decrease of the metabolic clearance rate and distribution volume of [125I]ANF in high-salt-fed rats than in control animals (74 +/- 6% vs 41 +/- 6% and 75 +/- 4% vs 50 +/- 5% of basal values, respectively; P less than 0.05). These data suggest that a prolonged high salt diet may increase the availability of C-ANF receptors and, through this mechanism, may negatively modulate plasma ANF concentrations. C-ANF receptors may thus fulfill a regulatory function on circulating ANF during prolonged salt loading in rats.

Analysis of Variance↗