Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-01-08
2001-01-23
Scheiner, Laurie (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007930, C422S067000, C530S388900, C424S188100
Reexamination Certificate
active
06177253
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed in this application.
Cyclosporine A (CsA) is a cyclic undecapeptide of fun-gal origin which is a immunosuppressive agent useful in preventing organ rejection in transplant patients (1-3).
Because the therapeutic index of CsA is narrow, it is important to measure serum cyclosporine levels in patients treated with CsA (4). This can be accomplished by high performance liquid chromatography or by RIA, with the latter procedure being the more convenient one.
It has reported, and we have confirmed (unpublished), that CSA, itself, is non-immunogenic (5). To obtain antibodies, therefore, it is necessary to link CsA to a protein carrier. The side chains of CsA, however, consist only of aliphatic groups with none of the functional groups customarily used to link a hapten to a carrier. Previous workers have made immunogenic cyclosporine C (CsC)-protein conjugates because the CsC has a threonine residue in position 2 (5). Linkage to a protein was via a hemisucciniate, using a water soluble carbodiimide as a coupling agent. Polyclonal antisera were successfully raised in this way and are routinely used to measure CsA in patients sera (5). More recently, monoclonal antibodies were prepared using an activated ester of a lysyl-CsA derivative (6).
We have chosen to use CsA, itself, as a hapten by converting it to a reactive carboxyl-containing peptide via a photochemical reaction. Coupling of this derivative to proteins has led to the successful raising of CsA-specific rabbit antibodies that can be used to measure CsA levels in sera of transplant patients under treatment with CsA.
Recently, Luban et al. (30) have shown that HIV-1 Gag protein binds to Cyclophilin A and B and that the Gag portion of SIV binds only to Cyclophilin B. We therefore determined that since Gag and CsA bind to cyclophillin, an antibody directed against CsA might bind to Gag and thereby treat HIV infection by inhibition of HIV replication. Further it is believed that the compositions of matter made according to this specification would be useful to treat AIDS.
SUMMARY OF THE INVENTION
The present invention provides a molecule having the structure:
where each R may independently be H or X, provided that at least one R is X, where X is a ligand which is produced as the result of a photochemical reaction between a precursor of X containing a photochemically activatable group and a hydrogen of cyclosporine A and which comprises a reactive group.
The invention further provides that the reactive group may be a group which is reactive with a macromolecule. In a preferred embodiment of this invention, the macro-molecule may be a polypeptide. In a very preferred embodiment, the invention further provides that the. polypeptide may be a protein. In a preferred embodiment, the reactive group may be a carboxyl.
Specific examples of X may include but are not limited to the following:
In a preferred embodiment of the invention, the probability is greater that 0.75 that only one R in the aforementioned molecule is X. In a very preferred embodiment, the probability is about 1.0.
The present invention further provides a molecule which comprises a congener of cyclosporine A characterized by the structural backbone of cyclosporine A in which one or more hydrogen atoms are replaced by one or more ligands, each such ligand both comprising a reactive group and being attached to the structural backbone of cyclosporine A at a location which a hydrogen atom has been replaced as the result of a photochemical reaction between a precursor of the ligand containing a photochemically activatable group and the hydrogen atom being replaced.
The present invention further provides an immunosuppressive agent useful for preventing organ rejection in a transplant subject comprising an amount of the aforementioned molecules effective to inhibit organ rejection in a transplant subject and a pharmaceutically acceptable carrier.
The present invention also provides a composition of matter which comprises a conjugate of a compound and the aforementioned molecule wherein the compound is bound to the molecule through the reactive group of the ligand X.
The invention further provides a composition of matter which comprises a conjugate of a macromolecule and the aforementioned molecule wherein the macromolecule is bound to the molecule through the reactive group of the ligand X.
Similarly, the invention provides a composition of matter which comprises a conjugate of a polypeptide and the aforementioned molecule wherein the polypeptide is bound to the molecule through the reactive group of the ligand X.
Moreover, the invention provides a composition of matter which comprises a conjugate of a protein and the aforementioned molecule wherein the protein is bound to the molecule through the reactive group of the ligand X.
The invention also provides a method for preventing rejection in a transplant subject comprising administering to the subject an amount of the aforementioned molecule effective to inhibit organ rejection in the transplant subject.
The subject invention further provides an antibody directed to the aforementioned composition of matter specific for cyclosporine A or congener of cyclosporine A. In accordance with the teachings of the invention the antibody may further be characterized as polyclonal or monoclonal. In addition, these antibodies may be detectably labeled.
The invention further provides a method of detecting the presence of cyclosporine A or congener of cyclosporine A in a biological tissue sample which comprises treating the biological tissue sample with the aforementioned detectably labeled antibody under conditions permitting the antibody to bind to cyclosporine A or congener and form a complex therewith, removing labeled antibody which is not bound to cyclosporine A or congener, detecting the presence of labeled antibody bound to cyclosporine A or congener and thereby detecting the presence of cyclosporine A or congener in the biological tissue sample.
The invention further provides another method of detecting the presence of cyclosporine A or a congener of cyclosporine A in a biological tissue sample which comprises treating the biological tissue sample with the aforementioned unlabeled antibody under conditions permitting the antibody to bind to cyclosporine A or congener and form a complex therewith, removing antibody which is not bound to cyclosporine A or congener, treating the complex with a labeled antibody directed to the unlabeled antibody under conditions such that the labeled antibody binds to the unlabeled antibody of the complex, removing labeled antibody which is not bound to the complex, detecting the presence of labeled antibody bound to the complex and thereby detecting the presence of cyclosporine A or congener in the biological tissue sample.
Additionally, this invention provides a method of determining the concentration of cyclosporine A or congener of cyclosporine A in a biological fluid sample which comprises, contacting a solid support with an excess of the aforementioned composition of matter under conditions permitting the composition of matter to attach to the surface of the solid support, contracting a predetermined volume of biological fluid sample with a predetermined amount of the aforementioned labeled antibody under conditions such that the cyclosporine A or congener in the sample binds to the labeled antibody and forms a complex therewith, contacting the resulting complex to the solid support to the surface of which the composition of matter is attached under conditions permitting the labeled antibody of the co
Chen Bi-Xing
Erlanger Bernard F.
Cooper & Dunham LLP
Parkin Jeffrey S.
Scheiner Laurie
The Trustees of Columbia University in the City of New York
White John P.
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