Immunoglobulin binding protein arrays in eukaryotic cells

Details Immunoglobulin binding protein arrays in eukaryotic cells Immunoglobulin binding protein arrays in eukaryotic cells

2000-05-02

2004-02-24

Wessendorf, T. D. (Department: 1639)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

Nonplant protein is expressed from the polynucleotide

C800S294000, C800S281000, C800S278000, C435S069100, C435S320100, C435S219000, C435S091500, C435S091500

Reexamination Certificate

active

06696620

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to arrays of immunoglobulin binding proteins. The invention is more particularly related to methods for the expression of arrays of foreign immunoglobulin binding proteins in eukaryotic cells, such as plant cells, as well as to transformed eukaryotic cells that express such arrays.
BACKGROUND OF THE INVENTION
Immunoglobulin molecules play key roles in a variety of physiological processes. Such molecules, which include antibodies and portions thereof, are critical for immune system function, and have found numerous therapeutic and diagnostic applications. The discovery of immunoglobulin molecules with desired binding characteristics is the focus of many current drug discovery efforts.
Traditional techniques for immunoglobulin molecule discovery involve the expression of a multitude of immunoglobulin molecule genes in an array of hybridoma cells, other forms of immortalized B-lymphocytes or phage-infected bacteria. For monoclonal antibody expression, individual antibody-producing B-lymphocytes from an immunized animal are generally fused with cells derived from an immortalized B-lymphocyte tumor. Clones of hybrid cells are then screened to identify those that grow indefinitely and secrete the desired immunoglobulin molecule. The polynucleotides encoding the monoclonal antibodies can then be isolated and used to express all or part of the antibody in other organisms, such as bacteria, yeast and plants. The ability to express immunoglobulins on the surface of bacteriophage has enabled the generation of immunoglobulin libraries that could represent all possible combinations of heavy and light chains derived from any population of B-lymphocytes. These libraries have been used successfully to identify high affinity combining sites recognizing a wide variety of antigens. A significant drawback to this technique is the randomization of heavy and light chain combining sites requiring the generation of very large numbers of recombinant phage to identify specific heavy and light chain binding pairs. This combinatorial aspect of random libraries makes expression of these libraries in other organisms unfeasible. Newer technologies involve transgenic mice expressing antibodies from human chromosomal segments which can be used to generate hybridoma arrays expressing human antibodies.
Arrays formed in B-lymphocytes, phage infected bacteria or transgenic animals have been useful within certain immunoglobulin molecule screens, but difficulties have been encountered with producing large quantities of immunoglobulin molecules in these cells. Large-scale production of immunoglobulin molecules from any of the traditional organisms is typically very expensive. Further, phage infected bacteria are incapable of providing the variety of immunoglobulin molecule structures that may be desired. Similarly, the usefulness of transgenic animal cells has been limited by the susceptibility of such cells to infection with viruses or other microorganisms.
For economic and other reasons, it would be desirable to use genetically engineered plants as the primary vehicle for the discovery of immunoglobulin molecules, as well as for the ultimate production of immunoglobulin molecules to be used in industrial, clinical or research applications. The advantages of plants for production of immunoglobulin molecules include a low cost of production, relatively low capital investment compared to fermentation systems, the absence of animal viruses and prions, production of the immunoglobulin molecule in a biochemical background of defined proteins such as seed proteins, ease of storage and transport, and a facile scale-up to unlimited quantities of raw material. It would also be desirable to be able to express a library of binding proteins that is not derived from a combinatorial process of randomly paired heavy and light chains.
It is known that immunoglobulin molecules can be expressed in a variety of eukaryotic hosts including plant cells. A wide variety of structural genes have been isolated from mammalian cells and viruses, joined to transcriptional and translational initiation and termination regulatory signals from a source other than the structural gene, and introduced into plant hosts in which these regulatory signals are functional. Among those host cells that have been transformed with individual immunoglobulin molecule-encoding nucleic acids are monocots (e.g., corn, rice and wheat), dicots (e.g., tobacco, soybean, alfalfa, petunia, and Arabidopsis) and lower plants (e.g., Chlamydomonas). Plants transformed with nucleic acids encoding individual immunoglobulin molecules have been able to produce fully functional and fully assembled immunoglobulins (see Hiatt et al.,
Nature
342:76-78, 1989; Firek et al.,
Plant Molecular Biology
23:861-870, 1993; Van Engelen et al.,
Plant Molecular Biology
26:1701-1710, 1994; Ma et al.,
Science
268:716-719, 1995; Magnuson et al.,
Protein Expression and Purification
7:220-228, 1996; Schouten et al.,
Plant Molecular Biology
30:781-793, 1996; Fiedler et al.,
Immunotechnology
3:205-216, 1997; Verch et al.,
J. Immunol. Meth.
220:69-75, 1998; Zeitlin et al.,
Nature Biotechnology
16:1361-1364, 1998; DeJaeger et al.,
Eur. J. Biochem.
259:426-434, 1999; Fischer et al.,
Biol. Chem.
380:825-839, 1999; Khoudi et al.,
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64(2):135-143, 1999; McCormick et al.,
Proc. Natl. Acad. Sci. USA
96:703-708, 1999; Russell,
Curr. Top. Microbiol. Immunol.
240:119-138, 1999).
In previous plant cell transformations, the transforming nucleic acid introduced a single immunoglobulin molecule. For example, tobacco plants have been transformed with individual gamma or kappa chains to produce individual plants expressing immunoglobulin molecule components. The respective tobacco transformants were then cross-pollinated to produce plants expressing a single antibody, wherein covalent bond formation between the two components resulted in the formation of enhanced binding capacity. In another instance, an antibody molecule was introduced into a single plant using a single vector. The vector encoded two immunoglobulin component chains and resulted in the formation, in the plant, of an immunoglobulin molecule comprising covalently linked heavy and light immunoglobulin chains.
Plant cells have not been used to express a diversity of immunoglobulin molecules in an array. As noted above, the ability to prepare an array of immunoglobulin molecules in plants or plant cells would facilitate identification of useful immunoglobulin molecules and would enable a rapid transition from immunoglobulin molecule discovery to full scale production in a single organism.
Accordingly, there remains a need in the art for methods for generating arrays of immunoglobulin molecules in plants and plant cells, as well as other eukaryotic organisms and cells. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides methods for the production of arrays of biologically or physiologically active immunoglobulin binding proteins in eukaryotic cells. Within certain aspects, methods are provided for preparing an immunoglobulin binding protein array in plant cells, comprising the steps of: (a) transforming a population of plant cells with a library of at least two different polynucleotides encoding different immunoglobulin binding protein (IgBP) polypeptides that: (i) specifically bind to a ligand with a K
D
<10
−6
moles/liter; or (ii) form one or more disulfide bonds with one or more polypeptides in the transfected cell, to generate a binding protein that specifically binds to a ligand with a K
D
<10
−6
moles/liter; wherein the IgBP polypeptides (i) comprise four framework regions (e.g., human or murine) alternating with three complementarity determining regions and (ii) comprise at least one peptide sequence having at least 75%, preferably at least 95%, sequence identity to a framework region of a native IgM, IgG, IgA

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