Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-08-06
2001-11-13
Swartz, Rodney P. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S133100, C424S136100, C424S141100, C435S070210, C435S326000, C435S328000, C435S344100, C530S387300, C530S388100
Reexamination Certificate
active
06316600
ABSTRACT:
BACKGROUND OF THE INVENTION
The government may own rights in this application pursuant to grants from The National Institutes of Health including grant number R37 CA 48023
I. Field of the Invention
The present invention relates generally to the field of cellular and molecular immunology. In particular, the present invention relates to the production of monoclonal antibodies in an avian system, e.g., chickens.
II. Related Art
Monoclonal antibodies are valuable tools in basic research and have growing importance for medical diagnosis and therapy. The procedures for making monoclonal antibodies routinely use immunized mice, rats, hamsters or rabbits as the source of antibody producing B cells. Isolated B cells are fused to a nonsecreting, immortalized cell line, and then selected and screened for specific antibody production. A hybridoma will secrete a single monoclonal antibody indefinitely (Kohler and Milstein, 1974). Hybridoma technologies have important limitations, however. In particular, it has been difficult to make antibodies to some proteins and other antigens; it is hypothesized that these molecules have highly conserved epitopes that are not recognized as “foreign” making them less immunogenic in closely related species (Cinader, 1960).
Distinctive features of chicken immunology can be used to advantage to address some of these problems. Chickens can mount an effective immune response to highly conserved mammalian proteins and produce high affinity antibodies (Horton et al., 1984; Larsson et al., 1993). Polyclonal antibodies to calf thymus RNA polymerase II (Carroll and Stoller, 1983) and to the (x-subunit of the rat insulin receptor (Song et al., 1985) were easily made in the chicken; the same antigens were not effective at inducing specific antibody production in the rabbit. Chicken antibodies to 1,25-dihydroxyvitamin D were more specific and less cross-reactive than rabbit antibodies (Bauwens et al., 1987), making it likely that the chicken is recognizing different epitopes. Chicken antibodies made by individual investigators and harvested from egg yolk have been used to study human insulin (Lee et al., 1991), human plasma kallikein (Burger et al., 1985), IL-6 (Wooley et al., 1995), prostaglandins (Fertel et al., 1981) and PCNA (Gassman et al., 1990). In addition, chicken serum containing polyclonal antibodies to human complement components, human erthropoietin, fibrinogen and fibronectin are commercially available (American Research Products, Inc. 1995 Catalogue). However, there is not yet a reliable way to produce chicken monoclonal antibodies given the tendency of chicken cell lines to cease antibody production after a short period of time (Nishinaka et al., 1989, 1991).
Another approach to generating unique antibody sequences recently has developed. It now is possible to clone DNA encoding a specific antibody's heavy and light chains, and to express the cloned antibody chains following transfection into other eukaryotic cells (Neuberger et al., 1983; Gilles et al., 1983). Like hybridomas, these transfected cells can be selected, screened and cloned as stable transfectomas that secrete a monoclonal antibody (Sharon et al., 1984; Morrison, 1985). In addition, the ability to clone the DNA of individual leader (L), variable (V), diversity (D) and joining (J) gene segments (Larrick et al., 1989; Orlandi et al., 1989; Heinrichs et al., 1995) and to manipulate these domain segments by specific mutation and random combination has facilitated the engineering of “artificial” antibody combining sites to a variety of epitopes that can be expressed in transfectomas or by phage display (McMafferty et al., 1990; Winter and Milstein, 1991; Huston et al., 1988; Yamanaka et al., 1996)). These techniques have proved successful in producing monoclonal antibody-type reagents with new specificities and with modified effector functions. Even where successful immunization with a given antigen can be achieved, however, it is technically challenging to identify, and then clone, the heavy and light chain sequences encoding the specific antibody (see Antibody Engineering Chapters 2, 3, 6 Borrebaeck).
Thus, it is clear that there remains a need for improved methods for the production of monoclonal antibodies against epitopes conserved in mammalian species. In addition, there is a need for improved methods in the cloning and manipulation of immunoglobulin genes in various animal species, and in particular, avian species such as chicken.
SUMMARY OF THE INVENTION
It is a goal of the present invention to provide methods for producing antibodies against a variety of antigens, including mammalian antigens with highly conserved epitopes. In addition, it is a goal of the present invention to provide improved methods and compositions for the cloning and manipulation of immunoglobulin genes.
Thus the present invention provides methods for generating monoclonal antibodies comprising the steps of immunizing a chicken with an antigen composition, isolating B cells from the chicken; immortalizing the B cells; selecting an immortalized antibody producing B cell and preparing nucleic acids encoding the antigen binding exons of the light and heavy chain genes thereof; cloning the heavy and light chain antigen binding regions, respectively, into vectors encoding the constant and leader regions of an immunoglobulin heavy chain, the constant and leader regions of an immunogloblin light chain; transferring the vectors into a suitable host cell; culturing the host cell; and isolating antibodies produced by the cell.
In preferred embodiments, the immortalizing comprises fusing the B cells to a chicken lymphoblastoid cell line. In other preferred embodiments, the immortalizing comprises infection of the B cells with an avian retrovirus.
In particular aspects of the invention, the preparing nucleic acids comprises isolating nucleic acids from the B cell and conducting PCR amplification of the coding sequences for the heavy and light chain antigen binding regions. In particular embodiments, the isolated nucleic acids primarily comprise genomic DNA. In preferred aspects it is contemplated that the heavy chain PCR primers have the sequence of SEQ ID NO:7 and SEQ ID NO:8. In other embodiments the light chain PCR primers have the sequence of SEQ ID NO:9 and SEQ ID NO: 10.
In certain embodiments the antigen composition comprises an antigen selected from the group consisting of CFTR, transforming growth factor beta, transcription factors, DNA binding molecules; cyclin dependent kinases and RNA binding proteins and a pharmaceutical buffer carrier or diluent. In other preferred embodiments, the antigen composition comprises an antigen selected from the group consisting of lipids, phospholipids and carbohydrates and a pharmaceutical buffer carrier or diluent.
In other embodiments of the present invention, it is contemplated that the selecting comprises screening the B cells for production of an antibody of interest. In preferred embodiments, the screening comprises measuring the binding of the antibodies to a target antigen. In particular embodiments, the measuring is performed by a method selected from the group consisting of ELISA, Western blot and immune precipitation.
In preferred embodiments, it is contemplated that the host cell is a murine myeloma line. In other preferred embodiments, the culturing performed in vitro. In yet another embodiments, the culturing is performed in vivo. In particular embodiments, the in vivo culturing is performed in a mouse. In certain aspects of the present invention the isolating comprises either or both of protein A affinity and ammonium sulfate precipitation.
Other embodiments of the present invention provide an isolated antibody produced by a process comprising the steps of immunizing a chicken with an antigen composition isolating B cells from the chicken, immortalizing the B cells; selecting an immortalized antibody producing B cell and preparing nucleic acids encoding the antigen binding regions of the light and heavy chain genes thereof; cloning the heavy and light chain ant
Accavitti Mary Ann V
Michael Nancy M.
Thompson Craig B.
ARCH Development Corporation
Fulbright & Jaworski LLP
Swartz Rodney P.
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