Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2002-02-14
2004-10-05
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C424S184100, C424S192100, C436S547000, C436S548000, C514S04400A, C800S004000, C800S005000
Reexamination Certificate
active
06800462
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods and compositions for producing proteins and generating antibodies.
BACKGROUND OF THE INVENTION
Antibodies are valuable tools for both basic research and the diagnosis and treatment of various diseases. Protein-specific binding antibodies allow for the precise identification and quantitation of a particular species of protein in a biological sample. In addition, the interaction between a protein and its binding antibody can modulate the activity or function of a protein. Modulation of the activity or function of a protein is particularly useful in treating a disease condition characterized by either excessive or insufficient activity of a given protein.
The generation of protein-specific antibodies frequently involves immunization using a purified form of a protein against which antibodies are desired. Providing sufficient quantities for immunization with a specific, properly folded protein is a common challenge in the field of antibody generation.
SUMMARY OF THE INVENTION
The invention is directed to methods of producing a protein by administering a nucleic acid encoding the protein to an animal. According to these methods, any nucleic acid sequence of interest can be administered to an animal. Following the administration of the nucleic acid to the animal, the protein is produced in vivo and can be isolated following the removal of a biological sample from the animal.
A protein produced according to these methods can be used to generate in a second animal an antibody that binds to a protein sequence of interest. In some instances, the methods include an initial isolation of the protein from the biological sample. This isolated protein is then used to immunize an animal. This invention thus permits the efficient generation of antibodies directed against a target amino acid sequence.
In one aspect, the invention includes a method of generating an antibody by performing the following steps: (1) administering to a first mammal a nucleic acid encoding a fusion protein and expressing the fusion protein in the first mammal, wherein the fusion protein contains a first amino acid sequence and a second amino acid sequence, and wherein the second amino acid sequence contains a first member of a specific binding pair; (2) removing from the first mammal a biological sample that contains the fusion protein; (3) binding a second member of the specific binding pair to the fusion protein via the first member of the specific binding pair to thereby isolate the fusion protein; and (4) administering the fusion protein to a second mammal, to thereby generate an antibody response in the second mammal against the first amino acid sequence of the fusion protein.
In one embodiment, the method also includes a step of cleaving the first amino acid sequence from the second amino acid sequence.
In one embodiment, the first member of the specific binding pair is a peptide of at least five amino acids in length. For example, the first member of the specific binding pair can be an Fc domain of an immunoglobulin.
The biological sample used in the method can include, for example, serum or a tissue lysate. The method can include an additional step of generating a lysate of the biological sample.
In one embodiment, the second member of the specific binding pair is an antibody, e.g., a monoclonal antibody.
The method can include an additional step of administering a protease inhibitor to the animal before removing the biological sample from the animal.
The method can also include an additional step of immobilizing the fusion protein.
In one embodiment, the first amino acid sequence of the fusion protein is identical to all or a portion of a naturally occurring human protein.
The method can include an additional step of isolating from the second mammal antisera containing an antibody that specifically binds to the first amino acid sequence of the fusion protein, wherein the antibody is produced in the second mammal following the administration of the fusion protein.
The method can include an additional step of removing a B lymphocyte from the second mammal and fusing the B lymphocyte in vitro with a second cell to form a hybridoma, wherein the hybridoma produces a monoclonal antibody that specifically binds to the first amino acid sequence of the fusion protein.
The method can include an additional step of removing components of the biological sample that are not bound to the second member of the specific binding pair, to thereby provide a purified fusion protein.
In another aspect, the invention features a method of generating an antibody by performing the following steps: (1) administering to a first mammal an isolated nucleic acid encoding a protein and expressing the protein in the first mammal; (2) removing from the first mammal a biological sample that contains the protein; and (3) administering the protein to a second mammal, to thereby generate an antibody response in the second mammal against the protein.
The protein used in the method can optionally be a fusion protein. The fusion protein can contain a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence contains a first member of a specific binding pair. In one embodiment, the first member of the specific binding pair is a peptide of at least five amino acids in length. For example, the first member of the specific binding pair can be an Fc domain of an immunoglobulin.
The biological sample used in the method can include, for example, serum or a tissue lysate. The method can include an additional step of generating a lysate of the biological sample.
The method can include an additional step of isolating from the second mammal antisera containing an antibody that specifically binds to the protein, wherein the antibody is produced in the second mammal following the administration of the protein.
The method can include an additional step of removing a B lymphocyte from the second mammal and fusing the B lymphocyte in vitro with a second cell to form a hybridoma, wherein the hybridoma produces a monoclonal antibody that specifically binds to the protein.
An advantage of the invention is that it provides rapid and efficient methods for the production of large amounts of a protein or portion thereof in its native conformation. The purified protein can then be used in generating antibodies that bind to the corresponding native protein sequence. These production methods avoid the time-consuming, expensive, and laborious tasks associated with the in vitro production and purification of proteins.
Another advantage of the invention is that it avoids several of the difficulties associated with recombinant protein production in bacteria. Mammalian proteins expressed in bacteria lack post-translational modification and frequently do not possess their native conformation. In addition, some proteins cannot even be translated in some bacteria because some nucleic acid constructs contain codons that are rarely used in bacteria.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification will control. In addition, the described materials and methods are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and the claims.
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patent: 5541087 (1996-07-01), Lo et al.
patent: 5698679 (1997-12-01), Nemazee
patent: 5703055 (1997-12-01), Felgner et al.
patent: 5721121 (1998-02-01), Etcheverry et al.
patent: 5726044 (1998-03-01), Lo et al.
patent: 5843678 (1
Hsu Pei-Ling
Lin Rong-Hwa
Wu Chung-Hsiun
AbGenomics Corporation
Fish & Richardson P.C.
Saunders David
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