Many research and clinical applications require large quantities of full-length antibodies with l... more Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.
Thrombocytopenia is common in advanced-stage liver disease and is partly caused by inadequate thr... more Thrombocytopenia is common in advanced-stage liver disease and is partly caused by inadequate thrombopoietin (TPO) production in the failing liver. Treatment of chronic hepatitis C with interferon alfa (IFN-␣) often induces thrombocytopenia, sometimes even leading to discontinuation of treatment. TPO regulation in response to IFN-␣induced thrombocytopenia was studied in patients with chronic hepatitis C with and without cirrhosis (Child A). An in vitro culture system with HepG2 cells was used to demonstrate any direct effects of IFN-␣ on TPO mRNA expression, TPO synthesis, or TPO secretion from liver cells. Thrombocyte count was lower (U test: P F .05) in patients with hepatitis C cirrhosis compared with patients with chronic hepatitis C without cirrhosis before IFN therapy, and decreased in both patient groups (Wilcoxon matched-pairs test: P F .05) on IFN therapy, the median decrease in both groups being comparable (noncirrhotic patients, 35%; cirrhotic patients, 32%; U test: P ؍ .57). TPO levels rose in noncirrhotic patients (Wilcoxon matchedpairs test: P F .05), but not in patients with cirrhosis (noncirrhotic patients' median increase: 43% vs. cirrhotic patients' median decrease: 5%; U test: P F .001). Even in patients without cirrhosis, the increase in TPO levels was relatively small for the decrease in platelet count. No effect of IFN-␣ could be demonstrated on TPO mRNA expression in vitro, but TPO secretion from liver cells was significantly reduced. Lower platelet counts but similar TPO levels in patients with chronic hepatitis C and cirrhosis compared with noncirrhotic patients and a moderate increase in TPO levels in noncirrhotic patients with a missing increase in cirrhotic patients during IFN-␣-induced thrombocytopenia provide further evidence for an impairment of TPO production in patients with cirrhosis and during IFN therapy. Recombinant human TPO could be of value in patients developing severe thrombocytopenia under IFN-␣ therapy. (HEPATOLOGY 1998;28:1424-1429.) PATIENTS AND METHODS Patients. The 45 patients included participated in a prospective trial of IFN-␣ treatment for chronic hepatitis C. 17 Included in this trial were patients with chronic hepatitis C (n ϭ 30) and with compensated hepatitis C cirrhosis (Child-class A only, n ϭ 15
Little is known about thrombopoietin (Tpo) production in human fetuses and neonates. As a step to... more Little is known about thrombopoietin (Tpo) production in human fetuses and neonates. As a step toward determining whether Tpo is relevant to platelet production in the fetus and neonate, we hypothesized that: (1) like other cytokines, Tpo is present in the cord blood in higher concentrations than in adult plasma; (2) Tpo and its receptor (c-mpl) are expressed in fetuses at, and following, 5-6 weeks post-conception (when platelet production begins); and (3) the sites of Tpo and c-mpl production in the fetus are similar to those of adults. We quantified Tpo, by ELISA, in the plasma of 50 adults, as well as in the umbilical cord plasma of 50 preterm and term infants. We also characterized, by RT-PCR, the organ distribution of Tpo and c-mpl during fetal development (at 8 and 16 weeks). Tpo concentrations were measurable ($ 41 pg / ml) in only two of the 50 adult samples (44 and 46 pg / ml), but in 24 of the 50 cord plasma samples (of the 24 samples, the median was 62 pg / ml; mean6SD, 80639 pg / ml). Tpo levels did not correlate with either gestational age or platelet count at birth. Similarly to adults, in the fetal tissues, Tpo transcripts were found in all organs tested, but the most dense bands were from liver. C-mpl transcripts were also predominantly from liver. We conclude that: (1) Tpo is present in higher concentrations in cord plasma than in venous plasma of adults; (2) Tpo and c-mpl transcripts are detected in human fetuses as early as the onset of platelet appearance; and
Many research and clinical applications require large quantities of full-length antibodies with l... more Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.
Thrombocytopenia is common in advanced-stage liver disease and is partly caused by inadequate thr... more Thrombocytopenia is common in advanced-stage liver disease and is partly caused by inadequate thrombopoietin (TPO) production in the failing liver. Treatment of chronic hepatitis C with interferon alfa (IFN-␣) often induces thrombocytopenia, sometimes even leading to discontinuation of treatment. TPO regulation in response to IFN-␣induced thrombocytopenia was studied in patients with chronic hepatitis C with and without cirrhosis (Child A). An in vitro culture system with HepG2 cells was used to demonstrate any direct effects of IFN-␣ on TPO mRNA expression, TPO synthesis, or TPO secretion from liver cells. Thrombocyte count was lower (U test: P F .05) in patients with hepatitis C cirrhosis compared with patients with chronic hepatitis C without cirrhosis before IFN therapy, and decreased in both patient groups (Wilcoxon matched-pairs test: P F .05) on IFN therapy, the median decrease in both groups being comparable (noncirrhotic patients, 35%; cirrhotic patients, 32%; U test: P ؍ .57). TPO levels rose in noncirrhotic patients (Wilcoxon matchedpairs test: P F .05), but not in patients with cirrhosis (noncirrhotic patients' median increase: 43% vs. cirrhotic patients' median decrease: 5%; U test: P F .001). Even in patients without cirrhosis, the increase in TPO levels was relatively small for the decrease in platelet count. No effect of IFN-␣ could be demonstrated on TPO mRNA expression in vitro, but TPO secretion from liver cells was significantly reduced. Lower platelet counts but similar TPO levels in patients with chronic hepatitis C and cirrhosis compared with noncirrhotic patients and a moderate increase in TPO levels in noncirrhotic patients with a missing increase in cirrhotic patients during IFN-␣-induced thrombocytopenia provide further evidence for an impairment of TPO production in patients with cirrhosis and during IFN therapy. Recombinant human TPO could be of value in patients developing severe thrombocytopenia under IFN-␣ therapy. (HEPATOLOGY 1998;28:1424-1429.) PATIENTS AND METHODS Patients. The 45 patients included participated in a prospective trial of IFN-␣ treatment for chronic hepatitis C. 17 Included in this trial were patients with chronic hepatitis C (n ϭ 30) and with compensated hepatitis C cirrhosis (Child-class A only, n ϭ 15
Little is known about thrombopoietin (Tpo) production in human fetuses and neonates. As a step to... more Little is known about thrombopoietin (Tpo) production in human fetuses and neonates. As a step toward determining whether Tpo is relevant to platelet production in the fetus and neonate, we hypothesized that: (1) like other cytokines, Tpo is present in the cord blood in higher concentrations than in adult plasma; (2) Tpo and its receptor (c-mpl) are expressed in fetuses at, and following, 5-6 weeks post-conception (when platelet production begins); and (3) the sites of Tpo and c-mpl production in the fetus are similar to those of adults. We quantified Tpo, by ELISA, in the plasma of 50 adults, as well as in the umbilical cord plasma of 50 preterm and term infants. We also characterized, by RT-PCR, the organ distribution of Tpo and c-mpl during fetal development (at 8 and 16 weeks). Tpo concentrations were measurable ($ 41 pg / ml) in only two of the 50 adult samples (44 and 46 pg / ml), but in 24 of the 50 cord plasma samples (of the 24 samples, the median was 62 pg / ml; mean6SD, 80639 pg / ml). Tpo levels did not correlate with either gestational age or platelet count at birth. Similarly to adults, in the fetal tissues, Tpo transcripts were found in all organs tested, but the most dense bands were from liver. C-mpl transcripts were also predominantly from liver. We conclude that: (1) Tpo is present in higher concentrations in cord plasma than in venous plasma of adults; (2) Tpo and c-mpl transcripts are detected in human fetuses as early as the onset of platelet appearance; and
Uploads
Papers by Paul Sims