Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1994-10-28
2001-02-06
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S326000
Reexamination Certificate
active
06184342
ABSTRACT:
BACKGROUND OF THE INVENTION
The U.S. Government has a paid-up non-exclusive license in this invention, and may have other rights as stipulated in 35 USC S202(C).
1. Field of the Invention
This invention relates to the fields of protein biochemistry, protein sequences, drugs and therapeutics. More specifically, the present invention relates to peptides and antibodies useful for modulating neutrophil chemotaxis in the immune response and in wound healing.
2. Description of Related Art
An enzyme in blood (thrombin) plays an important role in the inflammatory process and in initiating early stages of wound healing (Carney 1992; Carney et al. 1992b) by stimulating a number of cellular events which increase vascular permeability and recruit inflammatory cells to the site of tissue injury. It activates platelets and stimulates proliferation of fibroblasts (Carney et al. 1978; Perez-Rodriguez et aL 1981), capillary endothelial cells (Belloni et al. 1992), epithelial cells (He et aL 1991), neuronal cells (Gurwitz and Cunningham 1988), monocytes (Bar-Shavit et aL 1986), and T cells (Naldini et al. 1993). Additionally, a thrombin-derived synthetic peptide, TRAP-508, accelerates wound healing and revascularization through mechanisms that mimic normal effects of thrombin on microvascular endothelial cells and the recruitment of inflammatory cells to the wound site in vivo (Carney et al. 1992a; Stiernberg et al. 1993). However, the mechanisms by which this enzyme and related synthetic peptides stimulate these cellular events are quite complex and not generally understood.
It is highly useful for research and clinical purposes to have available the biochemical factors which mediate the various mechanisms that regulate the wound healing and inflammation processes. Neutrophil cell chemotaxis initiated by thrombin is one such mechanism involved in the wound healing/inflammation response process about which more needs to be known.
What is known about these processes is that thrombin and thrombin peptides play a role in chemotactic recruitment of inflammatory cells, including neutrophils (a.k.a. polymorphonuclear leukocytes) to a wound site. Further, it is known that at the injury site, thrombin causes proteolytic cleavage and activation of a G-protein-linked Proteolytically Activated Receptor for Thrombin (PART) that is present on the surface of platelets and endothelial cells (Vu et al. 1991; Rasmussen et al. 1991; Zhong et al. 1992), which results in release of an N-terminal peptide of approximately 15-amino acids.
However, prior to the present invention, the fate of this N-terminal peptide cleavage fragment was not known, nor was there any known function or use for this peptide fragment. Tests on fibroblasts and other cells using the released N-terminal peptide had found no apparent activity of the released peptide Van Obberghen-Schilling and Pouyssegur 1993).
SUMMARY OF THE INVENTION
The present invention embodies synthetic peptides which are neutrophil cell chemotactic agents, and which mimic the activity and role of the cleavage fragment of the Proteolytically Activated Receptor for Thrombin (PART). The benefits of these agents and the antibodies to them is their utility in research and clinical applications for studying and enhancing aspects of the wound healing and inflammatory response processes.
An object of the present invention is a number of peptides that are useful as chemotactic agents for cells having a receptor for the Neutrophil Targeting Peptide (NTP): SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 (see Table 1). This object also embodies peptides comprising a series of at least seven amino acids of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. A further aspect of this object is that these peptides are capable of specific binding to an NTP receptor on neutrophil cells generally, and to an NTP receptor on human neutrophil cells in particular.
Another object of the present invention is a process of stimulating neutrophil cell chemotactic migration by forming a gradient of a peptide of the present invention in an environment in which neutrophil cells are present or otherwise available, e.g., recruitable from the circulation in an in vivo system. The gradient may be accomplished by adding at a site in the environment toward which the neutrophil cells are to migrate an effective amount of the peptide sufficient to establish a gradient of the peptide against which the neutrophil cells migrate. It is a particular aspect of this process where the neutrophil cells are human neutrophil cells. The amount of the peptide to be added at the site can be any amount that one skilled in the art would recognize as establishing the required peptide gradient. However, it is a further aspect of this embodiment that the added amount of peptide is equivalent to a concentration of about 10
−10
to 10
−5
Molar in the area of the addition.
An additional object of this invention is a process of generating antibodies using as an antigen a series of amino acids defined by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 and the products of that process. Specifically the polyclonal antibody pAb51-IgG anti-NTP is an embodiment of this object.
A further object of the present invention is a process of modulating neutrophil cell chemotactic migration by adding antibodies of the present invention at a site in the system at which neutrophil migration is to be modulated in an amount effective to modulate the neutrophil cell migration. It is a further aspect of this embodiment that the added amount of peptide is equivalent to a concentration of about 10
−10
to 10
−4
Molar in the area of the addition.
It is also an object of this invention that the compositions and processes be accomplished both in vivo and in vitro.
TABLE 1
PEPTIDE SEQUENCES
Sequence
Source
Amino Acid Sequence
SEQ ID NO:1
Human
+
N-RRPESKATNA TLDPR
SEQ ID NO:2
Hamster
+
N-RQPESEMTDA TVNPR
SEQ ID NO:3
Mouse
+
N-SQPESERTDA TVNPR
SEQ ID NO:4
Rat
+
N-RQPESERMYA TPYATPNPR
SEQ ID NO:5
Hamster
+
N-CRQPESEMTDA TVNPR
REFERENCES:
patent: 5840499 (1998-11-01), Brass et al.
Cooper et al, The Biochemical Basis of Neuropharm. Oxford Univ. Press, NY, 1986, pp. 93-94.
Carney, D.H. 1992. Postclotting cellular effects of thrombin mediated by interaction with high-affinity thrombin receptors. inThrombin Structure and Function. L. Berliner, ed. Plenum, New York. 351.
Carney, D.H., W.R. Redin & L. McCroskey. 1992. Role of high-affinity thrombin receptors in postclotting effects of thrombin.Semin. Thromb. Hemost. 18: 91.
Carney, D.H., K.C. Glenn & D.D. Cunningham. 1978. Conditions which affect initation of animal cell division by trypsin and thrombin.J. Cell, Physiol. 95: 13.
Perez-Rodriguez, R., A. Franchi & J. Pouyssegur. 1981. Growth factor requirements of Chinese hamster lung fibroblasts in serum-free media: High mitogenic reaction of thrombin.Cell Biol. Int. Rep. 5: 347.
Belloni, P.N., D.H. Carney & G.L. Nicolson. 1992. Isolation and partial characterization of murine and human endothelial cells from various organs: Differential responsiveness to thrombin and other growth factors.Microvas. Res. 43: 20.
He, C.-J., E. Rondeau, R.L. Medcalf, R. Lacave, W.-D. Schleuning & J.-D.C. Sraer. 1991. Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.J. Cell. Physiol. 146: 131.
Gurwitz, D. & D.D. Cunningham. 1988. Thrombin modulates and reverses neuroblastoma neurite outgrowth.Proc. Natl. Acad. Sci. USA. 85: 3440.
Bar-Shavit, R., A.J. Kahn, K.G. Mann & G.D. Wilner. 1986. Identification of a thrombin sequence with growth factor activity on macrophages.Proc. Natl. Acad. Sci. USA. 83: 976.
Naldini, A., D.H. Carney, V. Bocci, K.D. Klimpel, M. Asuncion, L.E. Soares & G.R. Klimpel. 1993. Thrombin enhances T cell proliferative responses and cytokine production.Cell. Immunol. 147: 367.
Carney, D.H., R. Mann, W.R. Redin, S.D. Pernia, D. Berry, J.P. Heggers, P.G. Hayward, M.C. Robson, J. Christie, C. Annable, J.W. Fenton II., & K.C. Glen
Carney Darrell H.
Ramakrishnan Shyam
Chrysalis Biotechnology, Inc.
Fulbright & Jaworski
Ungar Susan
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