The present invention provides methods for preparing, and compositions comprising, stabilized protein-polymer conjugates. More particularly, the present invention relates to the stabilization of individual and complexed subunits of multisubunit protein complexes by conjugation to polymers. Such conjugation acts to stabilize the specific subunit complexes in their native conformation in liquid medium.

BACKGROUND OF THE INVENTION

The present invention relates to the conjugation of individual and complexed subunits of multisubunit protein complexes with polymers to stabilize their conformation. More specifically, the present invention describes a method of stabilizing individual and complexed subunits via covalent conjugation to a natural or synthetic polymer. The present invention also relates to stabilized conjugates of cardiac troponin I and C, and methods for its preparation.

SUMMARY OF THE INVENTION

Proteins are composed of long chains of amino acids. The structure of proteins can be considered on four different levels. The primary structure refers to the specific order of amino acids in the polymer chain. The secondary structure refers to the interactions among and between the amino acids in the chain to form such structures as α-helices and β-pleated sheets. The tertiary structure refers to the three-dimensional structure of the protein, which is also referred to as a protein's conformation. The quaternary structure refers to the spatial arrangement of individual polypeptides or subunits of multisubunit proteins.

The native conformation of a protein is only marginally stable. Thus, many proteins which are removed from their native environment and purified undergo conformational changes which can cause a loss of biological activity, such as enzyme activity or antibody binding capacity. In particular, the individual (uncomplexed) subunits of multisubunit protein complexes may undergo dramatic conformational changes when separated from the other subunits of the complex and stored in a liquid medium.

It is often desirable to separate the individual subunits of a multisubunit protein complex, for example to study or exploit the biological activity of each individual subunit. However, this may not be possible if the individual subunits undergo conformational changes in their uncomplexed state that alter their biological activity. Therefore, it is often desirable to stabilize specific subunit complexes of more than one, but less than all, subunits of a multisubunit protein. Accordingly, it is an object of the present invention to provide a method for stabilizing specific subunit complexes of multisubunit proteins.

Troponin is an example of a multisubunit protein complex which consists of three individual subunits; troponin T, troponin C, and troponin I. The troponin complex is involved in the calcium-sensitive switch that regulates the interaction of actin and myosin in striated muscles. Troponin T binds the troponin complex to tropomyosin, while troponin I is the inhibitory subunit of the complex, because it inhibits the actomyosin Mg²⁺-ATPase. Whereas troponin (TnC), which binds Ca²⁺, from skeletal muscle and cardiac muscle is identical, troponin I and T (TnI and TnT) from these two sources exist as different isoforms, each having a different amino acid sequences and thus a unique structure. Thus, cardiac troponin I (cTnI), cardiac troponin T (cTnT), and subunit complexes, such as cardiac troponin I and C (cTnIC) are of particular interest as cardiospecific markers.

The majority of the research into the troponin complex has centered around the regulatory function and structure of the troponin complex in skeletal muscle. The troponin complex assists in muscle contraction. The TnC molecule has four binding domains to bind divalent metal ions. The Ca²⁺/Mg²⁺ binding sites are in the C-terminal region and the Ca²⁺ binding sites are in the N-terminal region. In studies of skeletal muscle, in the absence of Ca²⁺, the N-terminus of TnI binds to the C-terminal region of TnC and to the globular C-terminal region of TnT. Thus, research indicates that TnI and TnC are anti-parallel and TnI and TnT are anti-parallel. The presence of calcium ion increases the C-terminal domain's affinity for the inhibitory and C-terminal regions of TnI. In addition, there is a hydrophobic surface in the N-terminal domain of TnC that represents a Ca²⁺ dependent binding site for TnI and TnT. It has been proposed that the Ca²⁺ dependent reactions relate to the regulatory mechanism and Ca²⁺ independent interactions maintain the structural integrity of the complex. In order to study structure and function of the troponin complex in its regulation of skeletal muscle, cross-linking studies have been accomplished. See Farah, C. and Reinach, F. Review: The Troponin complex and regulation of muscle contraction. FASEB Journal 9 pp. 755-767 (1995). Covalent binding between TnC and skeletal muscle TnI has been formed between the carboxyl groups in the TnC and lysine groups in TnI using EDC. See Kobayoshi et al. (1994), Structure of the troponin complex: implications of photocross-linking of troponin I to troponin C thiol mutants. J. Biol. Chem. 269, 5725-5729. In addition, Leszyk et al. (1987) Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-malemidobenzophenone; identification of residues in troponin I that are close to cystein-98 of troponin C. Biochemistry 26, 7042-7047, reported that the main product of cross-linking between TnC and skeletal muscle TnI comprises segments derived from the N-terminal regulatory domain of TnC (residues 46 to 78) and the inhibitory region of skeletal TnI (residues 96-116).

U.S. patent application Ser. No.08/865,468, filed on May 29, 1997, discloses that the majority of native cTnI in human serum after myocardial infarction (MI) is associated with TnC and TnT. The presence of TnI in a complex with other troponin subunits in MI patient serum increases its stability and protects it from further degradation. In addition, the troponin complex protects the sites where cardiac-specific antibodies bind. U.S. patent application Ser. No. 08/865,468, filed on May 29, 1997, also discloses methods to isolate the complex from MI patient serum.

The determination of the presence or amount of certain constituents or analytes is useful in the diagnosis of disease and physical well-being. Compositions which behave similarly to how constituents present in human bodily fluids behave, e.g., blood, blood serum, plasma, spinal fluid, and urine, are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount or presence of the constituent in a sample. These compositions will be referred to hereinafter as control compositions or controls.

Rapid and simple tests that can be used to accurately diagnose the occurrence of myocardial infarction or distinguish other ischemic events such as unstable angina are extremely important. Cardiac troponin I (cTnI) and troponin T (cTnT) have recently become established as the markers of choice in evaluating cardiac distress. See for example, New England Journal of Medicine Volume 335 No. 18, pages 1342-1349, Antman et al. and pages 1333-1341, Ohman et al.

A variety of immunoassays have been developed that utilize antibodies that can distinguish between the three troponin subunits, and also between their different isoforms. Monoclonal and polyclonal antibodies have been designed and used in immunoassays which can detect the cardiac-specific epitopes formed by the unique amino acid sequence of cTnI. See for example, International Patent Application No. WO 96/10076; European Patent No. 394,819 B1; and Adams et al., Circulation 88:101-106 (1993). Larue et al., (Clin. Chem. 39:972-979 (1993)) describe an immunoenzyme assay that is capable of detecting cTnI in the concentration range of 0.2 to 20 μg/L in 30 minutes.

Immunoassays have also been described which are specific for TnT. See for example, Katus, et al., Circulation, 83(3):902-912 (1991). An immunoassay for TnT is also commercially available from Boehringer Mannheim Corporation (Indianapolis, Ind.).

Most immunoassays are designed to determine the concentration of a given marker in a patient's serum by comparing immunoassay results with the patient's serum to those obtained with controls of known concentration. One limitation in the development of immunoassays for individual troponin subunits involves the instability of the troponins in their uncomplexed state. Accordingly, there is a need for stabilized compositions of tronponin subunit complexes that can be stored for extended periods of time, while retaining antibody binding capacity for use as control reagents in cTnI and/or cardiac troponin IC complex (cTnIC) immunoassays.

In vitro stabilized solutions for cardiac markers have been disclosed. U.S. Pat. No. 5,583,200 and Bodor et al., (1992) Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-I and Preliminary Results in Suspected Cases of Myocardial Infarction, Clin. Chemistry 38, (11) 2203-2214 at 2204 disclose stabilized troponin T and/or troponin I using troponin C and calcium ion. U.S. Pat. No. 5,583,200 discloses that serum may be added. U.S. patent application Ser. No. 08/874,566, filed Jun. 13, 1997, discloses improvements in stabilizing the troponin T or troponin IC complex and discloses solutions useful as calibrators or controls for diagnostic assays measuring troponin. U.S. patent application Ser. No. 08/564,526 and U.S. patent application Ser. No. 08/865,468, filed May 29, 1997, also disclose the effect of TnC upon the immunological and biological activity and non-specific binding of the CNBr-cTnI isoform and other fragments. U.S. patent application Ser. No. 08/564,526 discloses the activity of the complex formed by the CNBr-cTnI isoform, TnC and TnT as useful in immunoassays.

The calibrators and controls in Behring's OPUS® assay are a lyophilized preparation of human cardiac troponin I in processed bovine calf serum with stabilizers. The reconstituted products are stable for seven days when stored at 2 to 8° C. The calibrators and controls in Sanofi Pasteur's troponin I assay are a lyophilized preparation in a buffered human serum matrix. The reconstituted calibrators must be used within fifteen minutes after complete reconstitution, but may be aliquoted and stored frozed at -20° C. for up to about six months. The calibrators and controls in the Dade troponin I assay are provided frozen. When thawed the product is stable for thirty days when stored at 2 to 8° C.

Stabilization of proteins via covalent conjugation to various polymers has been described. See, for example, U.S. Pat. Nos. 4,902,502; 5,468,478; 4,806,524; Katre et al., J. Immunol. 144:209-213 (1990); Abuchowski et al., J. Biol. Chem. 252:3582-3586 (1977). The properties conferred on the conjugated protein have been cited as increased in vivo half life, increased stability in solution, increased solubility, decreased susceptibility to proteases and decreased immunogenicity and antigenicity. For example, Nitecki et al. (U.S. Pat. No. 5,089,261) describe conjugating interleukin-2 to polyethylene glycol (PEG) to reduce immunogenicity.

In addition to stabilization of proteins via covalent conjugations to polymers, it has also been demonstrated that synthetic polymers are capable of providing a stabilizing effect via an ionic interaction with proteins. For example, Marsh and Danielson (Analyst 120:1091-1096 (1995)) have described that the addition of PEG to an aqueous solutions of the multisubunit enzyme lactate dehydrogenase enhances the ability of the enzyme subunits to remain complexed.

The present invention relates to the finding that covalent conjugation of protein individual and complexed subunits of multisubunit proteins to polymers stabilizes the protein subunits, i.e., the subunit complex maintains its native conformation in a liquid medium for a longer period of time than the equivalent unconjugated subunit complex. In a preferred embodiment, the present invention also relates to cTnIC polymer conjugates which exhibit stabilized antibody binding capacity.
 

Claim 1 of 16 Claims

1. A method of stabilizing an individual subunit of a multisubunit protein complex, wherein the individual subunit is cardiac troponin I (cTnI) comprising mixing a solution of cTnI with an active polymer in a molar ratio of CTnI to polymer ranging from about 1:700 to about 1:11550 to form a stabilized cTnI-polymer conjugate.

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