Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-03-22
2004-03-09
Bugaisky, Gabrielle (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S350000, C530S380000
Reexamination Certificate
active
06703219
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a mutant protein having at least one of the biological activities of modified-C-reactive protein and to methods and materials for making the mutant protein by recombinant DNA techniques. The invention also relates to methods and materials for using the mutant protein.
BACKGROUND OF THE INVENTION
C-reactive protein was first described by Tillett and Francis [
J. Exp. Med
., 52, 561-71 (1930)] who observed that sera from acutely ill patients precipitated with the C-polysaccharide of the cell wall of
Streptococcus pneumoniae
. Others subsequently identified the reactive serum factor as protein, hence the designation “C-reactive protein.”
C-reactive protein (CRP) is synthesized in the liver, and its concentration in serum may increase as much as 1,000-fold during the acute phase response. See Gewurz et al.,
Adv. Int. Med
., 27, 345-372 (1982); Kushner,
Ann. N.Y. Acad. Sci
., 389, 39-48 (1982); Pepys et al.,
Adv. Immunol
., 34, 141-212 (1983). Although the exact role of CRP in the acute phase response is not known, it is believed to play an important part in host defense. For instance, it has been reported that: (1) CRP binds phosphorylcholine, suggesting a role for CRP as an opsonin for microorganisms and damaged tissue that have exposed phosphorylcholine groups; (2) CRP binds chromatin, suggesting that CRP may act to scavenge chromatin released by cell lysis; (3) CRP neutralizes platelet activating factor, suggesting that CRP may function as a regulator of platelet and neutrophil activities; and (4) CRP complexed to certain other molecules or liposomes activates complement, suggesting that CRP may trigger the complement cascade. See Kaplan et al.,
J. Immunol
., 112, 2135-2147 (1974); Volanakis et al.,
J. Immunol
., 113, 9-17 (1974); Siegel et al.,
J. Exp. Med
., 140, 631-47 (1974); Siegel et al.,
J. Exp. Med
., 142, 709-211(1975); Mold et al.,
J. Exp. Med
., 154, 1703-1708 (1981); Narkates et al.,
Proc. N.Y. Acad. Sci
., 389, 172-182 (1982); Nakayama et al.,
Clin. Exp. Immunol
., 54, 319-326 (1983); Robey et al.,
J. Biol. Chem
., 259, 7311-7316 (1984); Robey et al.,
J. Exp. Med
., 161, 1344-56 (1985); Vigo,
J. Biol. Chem
., 260, 3418-3422 (1985); Shephard et al.,
Clin. Exp. Immunol
., 63, 718-27 (1986); Horowitz et al.,
J. Immunol
., 138, 2598-2603 (1987); Tatsumi et al.,
Clinica Chimica Acta
, 172, 85-92 (1988); DuClos et al.,
J. Immunol
., 141, 4266-4260 (1988); DuClos et al.,
J. Immunol
., 146, 1220-1225 (1991); Xia et al.,
FASEB J
., 6, 1344a (1992).
CRP is a pentamer which consists of five identical subunits, each having a molecular weight of about 23,500. The pentameric form of CRP is sometimes referred to as “native CRP.”
In about 1983, another form of CRP was discovered which is referred to as “modified-CRP” or “mCRP”. Modified-CRP has significantly different charge, size, solubility and antigenicity characteristics as compared to native CRP. Potempa et al.,
Mol. Immunol
., 20, 1165-75 (1983). Modified-CRP also differs from native CRP in binding characteristics; for instance, mCRP does not bind phosphorylcholine. Id.; Chudwin et al.,
J. Allercy Clin. Immunol
., 77, 216a (1986). Finally, mCRP differs from native CRP in its biological activity. See Potempa et al.,
Protides Biol. Fluids
, 34, 287-290 (1986); Potempa et al.,
Inflammation
, 12, 391-405 (1988).
The distinctive antigenicity of mCRP has been referred to as “neo-CRP.” Neo-CRP antigenicity is expressed on:
1) denatured CRP prepared using suitable conditions (described below);
2) the primary translation product of DNA coding for CRP (preCRP); and
3) CRP immobilized on solid surfaces.
Potempa et al.,
Mol Immunol
., 20, 1165-75 (1983); Mantzouranis et al.,
Ped. Res
., 18, 260a (1984); Samols et al.,
Biochem. J
., 227, 759-65 (1985); Chudwin et al.,
J. Allergy Clin. Immunol
., 77, 216a (1986); Potempa et al.,
Inflammation
, 12, 391-405 (1988).
The neo-CRP antigenicity may be detected with antibodies. For instance, an antiserum made specific for neo-CRP can be used. See Potempa et al.,
Mol Immunol
., 24, 531-41 (1987). Alternatively, the unique antigenic determinants of mCRP can be detected with monoclonal antibodies. Suitable monoclonal antibodies are described in U.S. Pat. No. 5,272,257, published PCT application WO 91/00872 (published Jan. 24, 1991; corresponding to U.S. Pat. No. 5,272,257), Ying et al.,
J. Immunol
., 143, 221-228 (1989), Ying et al.,
Immunol
., 76, 324-330 (1992), and Ying et al.,
Molec. Immunol
., 29, 677-687 (1992).
A molecule reactive with antiserum specific for neo-CRP has been identified on the surface of 10-25% of peripheral blood lymphocytes (predominantly NK and B cells), 80% of monocytes and 60% of neutrophils, and at sites of tissue injury. Potempa et al.,
FASEB J
., 2, 731a (1988); Bray et al.,
Clin. Immunol. Newsletter
, 8, 137-140 (1987); Rees et al.,
Fed. Proc
., 45, 263a (1986). In addition, it has been reported that mCRP can influence the development of monocyte cytotoxicity, improve the accessory cell function of monocytes, potentiate aggregated-IgG-ihduced phagocytic cell oxidative metabolism, and increase the production of interleukin-1, prostaglandin E and lipoxygenase products by monocytes. Potempa et al.,
Protides Biol. Fluids
, 34, 287-290 (1987); Chu et al.,
Proc. Amer. Acad. Cancer Res
., 28, 344a (1987); Potempa et al.,
Proc. Amer. Acad. Cancer Res
., 28, 344a (1987); Zeller et al.,
Fed. Proc
., 46, 1033a (1987); Potempa et al.,
Inflammation
, 12, 391-405 (1988); Chu et al.,
Proc. Amer. Acad. Cancer Res
., 29, 371a (1988). Chudwin et al.,
J. Allergy Clin. Immunol
., 77, 216a (1986) teaches that mCRP can have a protective effect in mice challenged with gram-positive type 7F
Streptococcus pneumoniae.
Other activities of mCRP have been discovered and are described in certain issued U.S. patents, co-pending U.S. applications and published PCT applications. In particular, it has been discovered that mCRP binds immune complexes and aggregated immunbglobulin and can, therefore, be used to remove immune complexes and aggregated immunoglobulin from fluids and to quantitate immune complexes. See published PCT application WO 89/09628 (published Oct. 19, 1989), which corresponds to co-pending U.S. application Ser. No. 08/271,137, filed Jul. 6, 1994 (which was a continuation of application Ser. No. 07/582,884, filed Oct. 3, 1990, now abandoned, which was a continuation-in-part of application Ser. No. 07/176,923, filed Apr. 4, 1988, now abandoned). Modified-CRP has also been found to be effective in treating viral infections (see co-pending U.S. application Ser. No. 08/117,874, filed Sep. 7, 1993, a continuation of application Ser. No. 07/799,448, filed Nov. 27, 1991, now abandoned), non-Strentococcal bacterial infections and endotoxic shock (see allowed U.S. application Ser. No. 07/800,508, filed Nov. 27, 1991), and cancer (see issued U.S. Pat. No. 5,283,238 and co-pending U.S. application Ser. No. 08/149,663, filed Nov. 9, 1993).
For a brief review of CRP and mCRP, see Gotschlich,
Ann. N.Y. Acad. Sci
., 557, 9-18 (1989). Kilpatrick and Volanakis,
Immunol. Res
., 10, 43-53 (1991) provides a recent review of CRP.
Prior to the present invention, mCRP was preferably made using purified CRP as a starting material. Generally, mCRP was prepared from CRP by denaturing the CRP. For instance, CRP could be denatured by: (1) treatment with an effective amount of urea (preferably 8M) in the presence of a conventional chelator (preferably ethylenediamine tetraacetic acid (EDTA) or citric acid); (2) adjusting the pH of the CRP to below about 3 or above about 11-12; or (3) heating CRP above 50° C. for a time sufficient to cause denaturation (preferably at 639° C. for 2 minutes) in the absence of calcium or in the presence of a chelator such as those listed above. Urea treatment has been the preferred method. In addition, mCRP can be prepared from CRP by adsorbing the CRP onto solid surfaces. It is believed that mCRP prepared from CRP is formed by the dissociation of the five CRP subunits, each of
Crump Becky L.
Liao Hans H.
Potempa Lawrence A.
Bugaisky Gabrielle
Immtech International Inc.
Mayes Laurie
Sheridan & Ross P.C.
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