Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
1999-05-13
2001-12-11
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
C530S300000, C530S350000, C530S827000, C530S829000, C514S002600
Reexamination Certificate
active
06329500
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a therapeutic peptide and compositions thereof, nucleic acid sequences encoding the peptide and a method for inhibiting cytokine activity in a patient by administering a composition comprising the peptide. More particularly the present invention is directed to inhibiting TGF-&bgr; activity by administering a peptide having the TGF-&bgr; binding domain of &agr;
2
-macroglobulin.
BACKGROUND OF THE INVENTION
Human &agr;
2
-macroglobulin (&agr;
2
M) is a 718-kDa glycoprotein that was originally characterized as a broad-spectrum proteinase inhibitor. More recent experiments have demonstrated that (&agr;
2
-Macroglobulin (&agr;
2
M) also binds and regulates the activity of various cytokines, see Gonias, Exp. Hematol. 20:302-311 (1992). The structure of &agr;
2
M consists of four identical subunits, each with 1451 amino acids. The subunits are linked into dimers by disulfide bonds and into intact homotetramers by noncovalent interactions.
Proteinases react with &agr;
2
M by cleaving any of a number of susceptible peptide bonds in the “bait region”, which includes amino acids 666-706. Bait region cleavage causes &agr;
2
M to undergo a major conformational change, which effectively “traps” the attacking proteinase in a complex which is non-dissociable, even when the proteinase and the inhibitor are not covalently linked. Conformational change also reveals binding sites for the &agr;
2
M receptor/low density-lipoprotein receptor-related protein (LRP). These binding sites have been localized to 18-kDa peptides at the C-terminus of each &agr;
2
M subunit; Lys-1370 and Lys-1374 play particularly important roles.
Like the complement components, C3 and C4, each &agr;
2
M subunit contains a novel thiol ester bond, which is formed from the side-chains of Cys-949 and Glu-952, and these thiol esters may be instrumental in determining the conformational state of &agr;
2
M. When &agr;
2
M reacts with a proteinase, the thiol esters emerge from within hydrophobic, solvent-restricted clefts and are cleaved by nucleophiles or H
2
O. Small primary amines, such as methylamine, penetrate the hydrophobic clefts and react with &agr;
2
M thiol esters independently of proteinases, inducing an equivalent or nearly equivalent conformational change.
In addition to its activity as a proteinase inhibitor, &agr;
2
M functions as a major carrier and regulator of certain cytokines, including isoforms of the transforming growth factor-&bgr; (TGF-&bgr;) family. The highest affinity interactions of &agr;
2
M involve members of the transforming growth factor-&bgr; (TGF-&bgr;) and neurotorphin families. O'Connor-McCourt and Wakefield first identified &agr;
2
M as a physiologically significant carrier of TGF-&bgr; in human serum (
J. Biol. Chem
. 262, 14090-14099, 1987). Their studies demonstrated that nearly all of the TGF-&bgr;1 in serum is associated with &agr;
2
M and that the bound TGF-&bgr;1 is inactive. Huang et al. (
J. Biol. Chem
. 263, 1535-1541, 1988) confirmed the role of &agr;
2
M as a TGF-&bgr;-carrier and demonstrated that the TGF-&bgr;-binding activity of &agr;
2
M depends on its conformational state.
More recent studies have demonstrated the function of &agr;
2
M as a TGF-&bgr;-carrier in animal model systems. When radioiodinated TGF-&bgr;1 is injected intravascularly in mice, the cytokine is cleared rapidly at first; however, this is followed by a slow-clearance phase, during which time the TGF-&bgr; is almost entirely &agr;
2
M-associated.
The TGF-&bgr; family of cytokines regulates diverse processes including cellular growth, differentiation, wound healing, and inflammation. At the cellular level, TGF-&bgr; response is mediated by or regulated by a variety of receptors and binding proteins, including the type I and type II receptors, which are serine/threonine kinases, betaglycan, and endoglin. TGF-&bgr; activity is also regulated by processes that alter delivery of the active cytokine to the cell surface. For example, TGF-&bgr; is secreted as a large latent complex that includes the propeptide, latency associated peptide (LAP), and a second gene product, latent TGF-&bgr;-binding protein (LTBP). Conversion of latent TGF-&bgr; into active 25-kDa homodimer requires dissociation of LAP and LTBP in reactions which may be mediated by proteinases, thrombospondin, the mannose 6-phosphate/insulin-like growth factor-II receptor and acidic microenvirornents. Once activated, the 25-kDa form of TGF-&bgr; may bind to &agr;
2
M, once again forming a complex that is unavailable for receptor-binding.
Binding of TGF-&bgr; to &agr;
2
M is initially non-covalent and reversible; however, the complex can become covalently stabilized as a result of thiol-disulfide exchange. The latter reaction is observed primarily with conformationally-altered &agr;
2
M, since native &agr;
2
M lacks free thiol groups. A number of complementary methods have been used to determine equilibrium dissociation constants (K
D
) for the interaction of TGF-&bgr; with &agr;
2
M (
Arch. Biochem. Biophys
. 292,487-49,1992
;J. Biol. Chem
. 269,1533-1540, 1994; and
Ann. N. Y. Acad. Sci. USA
737,273-290, 1994). The K
D
s for the binding of TGF-&bgr;1 and TGF-&bgr;2 to native &agr;
2
M are 300 nNM and 10 nM, respectively; the K
D
s for the binding of TGF-&bgr;1 and TGF-&bgr;2 to methylamine-modified &agr;
2
M (&agr;
2
M-MA) are 80 nM and 10 nM, respectively. These binding constants accurately predict the ability of &agr;
2
M to neutralize TGF-&bgr; in cell culture systems.
In cell culture systems, &agr;
2
M neutralizes both exogenously-added and endogenously-synthesized TGF-&bgr;. Neutralization of endogenously-synthesized TGF-&bgr; results in altered gene expression, including greatly increased expression of inducible nitric oxide synthase (iNOS) by murine macrophages and increased expression of platelet-derived growth factor &agr;-receptor by vascular smooth muscle cells (see
J. Biol. Chem
. 270, 21919-21927, 1995 and
J. Biol. Chem
. 270, 30741-30748, 1995). &agr;
2
M gene knock-out mice demonstrate increased tolerance to endotoxin challenge and this characteristic is most likely explained by the enhanced function of TGF-&bgr; as an immunosuppressive, in the absence of &agr;
2
M.
The fate of &agr;
2
M-associated TGF-&bgr; depends on the &agr;
2
M conformation. Native &agr;
2
M, which is the predominant form of &agr;
2
M present in the plasma and probably in most extravascular microenvironments, binds TGF-&bgr; reversibly and noncovalently. Thus, native &agr;
2
M may buffer tissues against rapid changes in TGF-&bgr; levels by binding or slowly releasing the cytokine in response to the free TGF-&bgr; concentration. Based on the K
D
value, it is believed that approximately 95% of the TGF-&bgr;1 in plasma is &agr;
2
M-associated under equilibrium conditions, even though TGF-&bgr;1 binds to native &agr;
2
M with lower affinity than TGF-&bgr;2. Conversion of &agr;
2
M into the transformed conformation, which probably occurs most frequently at sites of inflammation due to the increase in cellular proteinase secretion, alters the mechanisms by which TGF-&bgr; is regulated. First, transformed &agr;
2
M has free Cys residues and thus undergoes thiol-disulfide exchange with TGF-&bgr;, eliminating the potential for release of active cytokine. Second, &agr;
2
M-proteinase complexes bind to the endocytic receptor, LRP; bound TGF-&bgr; is internalized with the &agr;
2
M-proteinase complex and probably delivered to lysosomes.
The mechanism by which &agr;
2
M binds cytokines remains unclear. Early studies, suggesting a prominent role for the thiol ester-derived Cys-residues, were not confirmed for TGF-&bgr;1 and TGF-&bgr;2. When &agr;
2
M-MA was treated with papain to release the 18-kDa receptor binding domains, the TGF-&bgr;-binding activity remained with the residual 600-kDa &agr;
2
M fragment. Thus, the cytokine- and LRP-binding sites are not co-localized. One proposed mechanism for &agr;
2
M binding was that the central cavity in the structure of &agr;
2
M, which serves as the proteinase-trap, als
Gonias Steven L.
Webb Donna J.
Breen John P.
Caputa Anthony C.
Holleran Anne L.
University of Virginia Patent Foundation
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