Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
2000-05-02
2002-12-10
Ponnaluri, Padmashri (Department: 1627)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C514S970000, C435S015000, C530S412000, C530S418000, C530S421000, C530S810000, C530S821000
Reexamination Certificate
active
06491923
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the conjugation of uncomplexed subunits of multisubunit protein complexes with polymers to stabilize their conformation. More specifically, the present invention describes a method of stabilizing these individual subunits via covalent conjugation to a natural or synthetic polymer. The present invention also relates to stabilized conjugates of cardiac troponin I and stabilized conjugates of cardiac troponin T, and methods for their preparation.
BACKGROUND ART
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 a helices and &bgr; 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. Accordingly, it is an object of the present invention to provide a method for stabilizing individual subunits of multisubunit protein complexes.
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. Whereas troponin C from skeletal muscle and cardiac muscle is identical, troponin I and T 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) and cardiac troponin T (cTnT) are of particular interest as cardiospecific markers.
After myocardial infarction, cTnT levels increase and remain elevated for an extended period. However, it has been reported that in a variety of disease states, cTnT is also expressed in skeletal muscle, which contributes to a lack of cardiospecificity of this protein. Furthermore, uremia, a condition associated with cardiomyopathy, is associated with elevated cTnT. Thus, a lack of absolute cardiospecificity makes this marker less than optimal for use in the early diagnosis of acute myocardial infarction (AMI).
Cardiac TnI is also released after acute myocardial infarction. In contrast to cTnT, cTnI has never been found in a healthy population, which includes marathon runners, in people with skeletal disease, or in patients undergoing non-cardiac operations. Thus, cTnI is a more specific marker for the diagnosis of AMI than other serum proteins.
A variety of immunoassays have been developed that utilize antibodies that can distinguish between the three troponins, 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 PCT 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 &mgr;g/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 Corp., Indianapolis, lnd.
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 control reagents of known concentration. One limitation in the development of immunoassays for the troponins involves the instability of the troponins in their uncomplexed state. Accordingly, there is a need for stabilized compositions of cTnI and cTnT that can be stored for extended periods of time, while retaining antibody binding capacity for use as control reagents in cTnI and cTnT immunoassays.
Stabilization of proteins via covalent conjugation to various polymers has been described. See, for example, U.S. Pat. No. 4,902,502; U.S. Pat. No. 5,468,478; U.S. Pat. No. 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 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 individual protein subunits of multisubunit proteins to polymers stabilizes the protein subunit, i.e. it maintains its native conformation in a liquid medium for a longer period of time than the equivalent unconjugated individual subunit. In a preferred embodiment, the present invention also relates to cTnI-polymer conjugates and cTnT-polymer conjugates which exhibit stabilized antibody binding capacity.
DISCLOSURE OF THE INVENTION
The present invention concerns stabilization of individual subunits of multisubunit protein complexes. Stabilization is accomplished by conjugating the individual subunits to a polymer. The stabilizing effect of conjugation allows the individual subunit to be stored in liquid medium for longer periods of time than an equivalent unconjugated or “free” individual subunit. This greatly enhances the shelf life of the composition. In a preferred embodiment, the stabilized individual subunits are either cTnI or cTnT, which in their unconjugated form are highly unstable in liquid medium.
Polymers which are useful in the present invention can be naturally occurring or synthetic. Whereas certain synthetic polymers may be preferred for stabilization of free cTnI, as will be discussed below, natural polymers such as serum proteins are preferred for stabilization of cTnT. A particularly preferred class of synthetic polymer is PEG. Other suitable polymers include, but are not limited to polyalkylene glycols, polyoxyethylated polyols, polyvinylpyrrolidone, polyhydroxyethyl methacrylate, polyvinyl alcohols, and polyurethane.
The polymers which are useful in the present invention may vary in molecular weight, and must have a molecular weight which is sufficient to stabilize the indi
Botyanszki Janos
Dave Kirti I.
Sintar Eva
Modrovich Ivan E.
Morrison & Foerster / LLP
Ponnaluri Padmashri
LandOfFree
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