Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-09-20
2003-12-09
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S252300, C435S320100, C435S325000, C435S375000, C530S300000, C530S399000, C536S023100, C536S023500
Reexamination Certificate
active
06660467
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to CNTF variants with enhanced neuronal receptor selectivity, useful for the treatment of neurological or other diseases or disorders.
Ciliary neurotrophic factor (CNTF) is a 23-kDa neuro-cytokine, which is expressed in both the peripheral and central nervous system beginning in the late embryonic period (reviewed by Manthorpe et al., 1993; Ip and Yancopoulos 1996). Initially identified by its ability to promote the in vitro survival of embryonic chick parasympathetic neurons, CNTF was subsequently shown to exert potent growth-promoting and/or differentiating actions on a variety of neuronal and glial cells, including motoneurons, sensory neurons, sympathetic neurons, hippocampal neurons, and oligodendrocytes (reviewed by Manthorpe et al., 1993; Ip and Yancopoulos 1996). In vivo administration of CNTF prevents degeneration of chick spinal motoneurons during development of axotomized rat facial motoneurons and of motoneurons in mutant progressive motor, neuronopathy mice. The neuroprotective effects of CNTF make it a candidate for the treatment of human motoneuron disease and possibly other neurodegenerative diseases (Manthorpe et al., 1993; Ip and Yancopoulos 1996).
In addition to its neuronal actions, CNTF can also elicit biological effects in non-neuronal cells, such as glia (Hughes et al. 1988; Louis et al. 1993), hepatocytes (Schooltnik et al. 1992), skeletal muscle cells (Helgren et al. 1994), embryonic stem cells (Conover et al.1993), bone marrow stromal cells (Gimble et al.1994), and tumor plasma cells (Zhang et al.1994).
The functional pleiotropy of CNTF is one of the likely reasons for the problems associated with the therapeutic use of this protein. CNTF has a short half-life in vivo (Davies et al., 1994), and needs to be administered at high doses in order to achieve pharmacologically useful concentrations in target tissues. At high dosages CNTF produces side-effects, such as weight loss and acute-phase response (Dittrich et al., 1995). There is therefore a need for agents that are able to mimic the neurotrophic effects of CNTF without eliciting all or part of its side effects.
CNTF exerts its biological actions through the binding, sequential assembly, and activation of a multisubunit receptor complex composed of a ligand-specific a-receptor (CNTFR) and the signal transducing subunits gp130 and leukemia inhibitory factor receptor-b (LIFR) (Ip and Yancopoulos, 1996). Binding of CNTF to CNTFR triggers the subsequent association and heterodimerization of gp130 and LIFR, leading to the activation of a signaling cascade mediated by protein tyrosine kinases of the Jak family and STAT transcription activators. Similar to gp80, the a-receptor for IL-6, which mediates homodimerization of gp130, CNTFR can function in either membrane-bound or soluble forms (Ip and Yancopoulos, 1996). The membrane-bound form of CNTFR (m-CNTFR), which is anchored to the cell surface via a glycosyl-phosphatidylinositol linkage, is expressed predominantly in neuronal and skeletal muscle cells (Davies et al., 1991; Ip et al., 1993). The soluble form of CNTFR (s-CNTFR), which can be produced by phospholipase C-mediated cleavage of m-CNTFR, serves as a cofactor in potentiating CNTF actions on cells that express gp130 and LIFR (Davis et al., 1993). Soluble CNTFR has been detected in cerebrospinal fluid and serum (Helgren et al., 1994; Davis et al., 1993), suggesting that it may be involved in mediating some of the non-neuronal actions of CNTF, such as acute-phase response (Dittrich et al., 1994).
Since m-CNTFR is required for neuronal action of CNTF, while s-CNTFR is thought to mediate non-neuronal effects, modified CNTF proteins with increased selectivity for m-CNTFR are expected to produce a more neuron-specific spectrum of pharmacological activities.
DESCRIPTION OF THE INVENTION
The present invention relates to CNTF variants that, as an effect of specific amino acid substitutions in accordance with the invention, have a reduced ability of binding CNTFR, as compared to the natural CNTF, and a decrease of the biological activity mediated through soluble CNTFR, with an unchanged biological activity mediated through membrane-bound CNTFR.
These variants are on the basis of a method for the treatment of neuronal diseases and disorders, in human and animals. In one embodiment, the biological activities of CNTF variants is compared between human hepatoma cells plus soluble CNTFR and human hepatoma cells stably expressing CNTFR, which provides a method for assessing selectivity for membrane-bound receptor.
In a preferred embodiment, the variant according to the invention is obtained by replacing in the hCNTF (SEQ ID NO: 1) the amino acid threonine in position 169 with isoleucine, and the amino acid histidine in position 174 with alanine (variant which hereinafter is referred as Thr169Ile/His174Ala/hCNTF; IA-CNTF, or SEQ ID NO: 2). This variant is characterized by a reduced ability to bind soluble CNTFR.
The ability of the modified hCNTF to stimulate production of the acute-phase protein haptoglobin is measured in human hepatoma cells in presence of soluble CNTFR. As described hereinafter, the modified CNTF exibits decreased potency as compared to the wild-type CNTF.
In another embodiment, the ability of the modified human CNTF protein to stimulate production of choline acetyltransferase in a human neuroblastoma cell line is measured. As described hereinafter, the modified CNTF protein is equipotent with the wild-type CNTF protein in this assay.
In a preferred embodiment, human hepatoma cells, which do not express CNTFR are engineered to express the full-length human CNTFR, and these cells are used to assay the ability of modified CNTF proteins to stimulate haptoglobin production. Biological activity in this assay is compared to that obtained in parent hepatoma cells assayed in the presence of soluble CNTFR. This procedure provides a measure of selective activation of biological responses through membrane-bound versus soluble CNTFR. As described herein, the modified CNTF protein is equipotent with wild-type human CNTF in this-assay, showing that it maintains high biological activity through membrane-bound CNTFR, while displaying specifically reduced activity through soluble CNTFR. As also described herein, a CNTF variant that was previously shown (Italian patent patent application RM96A000492) to have increased neuronal receptor selectivity (Phe152Ala/Ser166Asp/Gln167His/human CNTF or AKDH-CNTF; a human CNTF variant containing, from amino acids 152 to 167, the sequence reported as SEQ ID NO:3 in the Italian patent application RM96A000492), is also equipotent with wild-type CNTF in hepatoma cells expressing CNTFR. These results shows that this assay system can be used to identify CNTF variants that display different biological activities through soluble and membrane-bound CNTFR.
The ligand retention hypothesis (Baumann et al. 1994) provides the most plausible explanation for the pharmacological behavior of cytokine variants with membrane-bound and soluble receptor isoforms. Baumann and coworkers (see Baumann et al., 1994) calculated that concentrations of cytokine receptors at the cell surface are in the micromolar range (which is far in excess of cytokine-receptor equilibrium dissociation constants, and proposed that this can lead to near unidirectional ligand capture. High membrane concentrations of cytokine receptors would explain why cytokine variants with altered receptor binding affinity can display unchanged agonistic potencies through membrane-bound receptors. The equipotency of CNTF and variants with altered CNTFR affinity in neuronal cells would thus be due to quasi-irreversible ligand capture by in-CNTFR, analogous to the situation in non-neuronal cells in the presence of saturating concentrations of s-CNTFR (Italian patent application RM96A000492).
Thus, according to the invention, certain amino acid substitutions in the human CNTF wild type protein result in modified human CNTF protein that exhibit increased selectivity for membrane-boun
De Martis Anna
Di Marco Annalise
Gloaguen Isabelle
Laufer Ralph
Saggio Isabella
Bunner Bridget E.
Heber Sheldon O.
Instituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A.
Kemmerer Elizabeth
Tribble Jack L.
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