Cytotactin derivatives that stimulate attachment and neurite...

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...

Reexamination Certificate

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C530S350000, C424S198100, C424S192100, C424S195110, C424S425000, C424S426000

Reexamination Certificate

active

06482410

ABSTRACT:

TECHNICAL FIELD
The present invention relates to cytotactin proteins, polypeptides, antibodies and other cytotactin derivatives useful in the mediation of neuronal attachment and enhancement of the outgrowth of neurites, as well as to methods of using same. Methods of making the disclosed proteins, polypeptides, antibodies, derivatives and related compositions, which have a variety of diagnostic and therapeutic applications, are also disclosed.
BACKGROUND
Cytotactin (CT) is a multidomain extracellular matrix (ECM) protein which plays a role in cell migration, proliferation, and differentiation during development (Crossin, et al.,
J. Cell Biol
. 102: 1917-1930(1986); Prieto, et al.,
J. Cell Biol
. 111: 685-698 (1990)), which may be controlled by other developmentally important genes. The restricted spatiotemporal expression of cytotactin that results from its developmental regulation is tightly linked to a number of cellular primary processes, including adhesion (Grumet, et al.,
Proc. Natl. Acad. Sci. USA
82: 8075-8079 (1985)), migration (Chuong et al.,
J. Cell Biol
. 104: 331-342 (1987); Halfter, et al.,
Dev. Biol
. 132: 14-25 (1989); Tan, et al.,
PNAS USA
84: 7977-7981 (1987)), proliferation (Chiquet-Ehrismann, et al.,
Cell
53: 383-390 (1988); Crossin,
PNAS USA
88: 11403-11407 (1991)), differentiation (Mackie, et al.,
J. Cell Biol
. 105: 2569-2579 (1987)), epithelial-mesenchymal interactions (Aufderheide, et al.,
J. Cell Biol
. 105: 2341-2349 (1988); Aufderheide, et al.,
J. Cell Biol
. 105: 599-608 (1987)), and cell death (Williamson, et al.,
Embryonic Develop. Morphol
. 209: 189-202 (1991)).
Cytotactin, which is also known as tenascin (TN) (Chiquet-Ehrismann, et al.,
Cell
47: 131-139 (1986)), J1 2201200 (Kruse, et al.,
Nature
316: 146-148 (1985)), hexabrachion (Erickson, et al.,
Nature
311: 267-269 (1984); Gulcher, et al.,
PNAS USA
86: 1588-1592 (1989)), the glioma-mesenchymal extracellular matrix protein (Bourdon, et al.,
Cancer Res
. 43: 2796-2805 (1983)), and myotendinous antigen (Chiquet et al.,
J. Cell Biol
. 98: 1926-1936 (1984)), exists in at least three isoforms generated by alternative splicing (Zisch, et al.,
J. Cell Biol
. 119: 203 (1992)). The three known chicken CT isoforms, which are composed of polypeptides having molecular weights of 190, 200, and 220 kD have been isolated from chicken brain (Grumet, et al.,
PNAS USA
82: 8075-8079 (1985)); relative to the 190 kD isoform, the 200 kD form contains one, and the 220 kD form contains three, additional fn type III domains (Zisch, Id, (1992)). The CT found in other species, including human and murine CT, for example, exists in a variety of isoforms as well.
As noted, variation in the polypeptide structure arises from alternative splicing of transcripts from a single gene (Jones, et al.,
PNAS USA
85: 2186-2190 (1988); Jones, et al.,
PNAS USA
86: 1905-1909 (1989); Spring, et al.,
Cell
59: 325-334 (1989)). The polypeptides are disulfide-linked to form a multimeric structure (Grumet, et al.,
PNAS USA
82: 8075-8079 (1985); Hoffman, et al.,
J. Cell Biol
. 106: 519-532 (1988)). Electron microscopy of the rotary-shadowed molecule has revealed a characteristic six-armed structure, called a hexabrachion (Erickson, et al.,
Nature
311: 267-269 (1984); Erickson, et al.,
Adv. Cell Biol
. 2: 55-90 (1988)), in which six polypeptides are linked through disulfide bonds at their aminotermini.
The sequence of cytotactin reveals a multidomain structure (Jones, et al.,
PNAS USA
86: 1905-1909 (1989); Spring, et al.,
Cell
59: 325-334 (1989)) with homologies to three other protein families. The amino-terminal portion contains the cysteine involved in interchain disulfide bonding, followed by an array of 13 repeats of 31 amino acids in length that resemble those found in epidermal growth factor (EGF). These EGF-like repeats are followed by a variable number of repeats similar to fibronectin type III repeats. In the chicken, cytotactin polypeptides contain between 8 and 11 type III repeats as a consequence of alternative RNA splicing. Different variants have been shown to be expressed preferentially at certain times and anatomical sites during development (Prieto, et al.,
J. Cell Biol
. 111: 685-698 (1990)) and they may have different binding or morphogenic functions (Kaptony, et al.,
Development
(
Camb
.) 112: 605-614 (1991); Matsuoka, et al.,
Cell Differ
. 32: 417-424 (1990); Murphy-Ullrich, et al.,
J. Cell Biol
. 115: 1127-1136 (1991)).
More recently, it has been shown that the third fibronectin type III (CTfn3) repeat can mediate RGD-dependent cell attachment via integrins &agr;
v
&bgr;
3
and &agr;
v
&bgr;
6
and that the whole molecule bound to a &bgr;
1
integrin but the binding site was not determined. The carboxy-terminal portion of cytotactin is homologous to the distal domain of the &bgr; and &ggr; chains of fibrinogen and contains a putative Ca
2+
binding site.
Early studies of cell attachment to cytotactin-coated surfaces suggested that multiple modes of binding to the molecule existed. For example, fibroblasts bind both to intact cytotactin and to a chymotryptic fragment derived from the carboxy-terminal end of the protein (Friedlander, et al.,
J. Cell Biol
. 107: 2329-2340 (1988)). These binding activities are inhibitable by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and by antibodies to specific regions of the cytotactin protein. In contrast to their rounded cell morphology on intact cytotactin, cells exhibit a spread morphology on the chymotryptic fragment. Using a variety of recombinant fragments of cytotactin, a smaller region of the molecule has been identified as a cell binding site, but no spreading was observed (Spring, et al.,
Cell
59: 325-334 (1989)).
In these studies, a fragment in the amino-terminal region containing the EGF domains appeared to prevent cell binding to other substrates. Together, these observations suggested that at least two binding activities are present in intact cytotactin, one in the carboxy-terminal half of the protein, mediating cell attachment and flattening, and one in the amino-terminal portion, responsible for so-called anti-adhesive effects (Spring, et al.,
Cell
59: 325-334 (1989)) and rounding of cells exposed to the molecule (Chiquet-Ehrismann, et al.,
Cell
47: 131-139 (1986); Friedlander, et al.,
J. Cell Biol
. 107: 2329-2340 (1988)). Studies on the effects of cytotactin on neural attachment and neurite outgrowth have suggested at least one additional interactive site on the molecule based on antibody inhibition studies (Crossin, et al.,
Exp. Neurol
. 109: 6-18 (1990); Faissner, et al.,
Neuron
5: 627-637 (1990); Grierson, et al.,
Dev. Brain Res
. 55: 11-19 (1990); Husmann, et al.,
J. Cell Biol
. 116: 1475-1486(1992); Lochter, et al.,
J. Cell Biol
. 113: 1159-1171 (1991); Wehrle, et al.,
Development
(
Camb
.) 110: 401-415 (1990)).
BRIEF SUMMARY OF THE INVENTION
We have now unambiguously identified the regions of CT responsible for its ability to promote or to inhibit neurite outgrowth, as well as the regions primarily responsible for cell attachment and spreading. Understanding which regions of this complex protein are responsible for these various functions is essential to determine how the protein may affect neural development and regeneration. One working hypothesis is that the inhibition and promotion of neurite outgrowth may be mapped to specific domains of the protein and may be modulated by other CT binding proteins in the ECM. Fusion proteins have now been generated in the pGEX expression system comprising almost the entire linear structure of the protein and have now been expressed in bacteria. Other new constructs comprising portions of CT, some in unique combinations, are also disclosed herein.
Using these bacterially-generated fusion proteins, smaller domains within the CT protein (e.g., CTfn3) have now been identified that have the ability to promote neurite outgrowth. Another major contribution of the within-disclosed invention is the contribution to the understand

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