WO2008128144A2

WO2008128144A2 - Monoclonal antibody selecting system, and making and using thereof

Info

Publication number: WO2008128144A2
Application number: PCT/US2008/060165
Authority: WO
Inventors: Shuang Zhang
Original assignee: Shuang Zhang
Priority date: 2007-04-15
Filing date: 2008-04-14
Publication date: 2008-10-23
Also published as: WO2008128144A3

Abstract

The present invention provides a DNA encoding a chimeric protein that comprises one transmembrane domain and one extracellular Fc-binding domain and provides transgenic myeloma cells and transgenic mice having the DNA integrated into their genome. The invention also provides methods of preparing transgenic myeloma cell and transgenic mice expressing the chimeric protein. The invention further provides methods of using the transgenic myeloma cells and transgenic mice said mice to facilitate isolation of monoclonal antibodies-producing B-cells. Specifically, antibody-producing cells are engineered to express a chimeric protein that comprises a transmembrane domain and an extracellular Protein G' domain, rendering the cells binding to antibodies released by themselves. Such character simplifies the subsequent selection of monoclonal antibody-producing cells.

Description

MONOCLONAL ANTIBODY SELECTING SYSTEM, AND MAKING AND USING THEREOF

BACKGROUND OF THE INVENTION

1. Field of Invention

The invention is in the field of analysis of cell populations and cell separation and the compositions obtained thereby. More particularly, the invention concerns analysis and separation of antibody-specific B cells based on primary labeling of cells with their secreted products through capture of these products by a specific binding partner for the product anchored or bound to the cell surface.

2. Background of the Invention

Since Kohler and Milstein first developed somatic fusions of B-lymphocytes with myeloma cells to produce monoclonal antibody-making cell lines in 1975, hybridoma technology has greatly advanced our understanding in basic biological research and modern medical science. Their unique recognizing ability makes monoclonal antibodies (mAbs) the perfect and sensitive tools for detecting and localizing specific biological molecules. Monoclonal antibodies can be made against almost any cell molecule, enabling that molecule to be identified, localized and purified. Monoclonal antibodies have become the key player in disease diagnostics and therapeutics. The monoclonal antibody market has reached sales of $14bn in 2005. During the next 5 years, antibodies for oncology and arthritis, immune and inflammatory disorders (AIID) are forecast to continue to lead the market, with sales of $14bn and $llbn, respectively in 2011. Antibodies are also being developed in respiratory, cardiovascular and ophthalmology indications.

However, despite the central role of monoclonal antibodies in these developments in medicine and molecular biology, the process for producing and screening monoclonal antibodies has changed little since the mid-1970s. The approach most often used to produce a monoclonal antibody against a specific antigen requires a series of immunizations of mice or rats with an antigen over the course of several weeks to enhance the activation and proliferation of mature B cells producing antigen-specific antibodies. Multiple mice are generally immunized and are tested periodically for the presence of relevant serum immunoglobulin titers prior to sacrificing the animals. To produce monoclonal antibodies, one removes B-cells from the spleen or lymph nodes of the animal that has been challenged several times with the antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer) and that have lost the ability to produce antibodies. This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol, electroporation or, of historical importance, infection with some virus. The fused hybrid cells (called hybridomas) , being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. Cloning and selecting lines with desired binding activities is a laborious procedure that usually relies on limiting dilution microculture . The hybridomas are sufficiently diluted to ensure clonality and grown. The antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA) or immuno-dot blot, and the most sensitive one is picked out. The time frame required for developing a monoclonal antibody using this approach is generally 3 to 9 months .

The development of RIMMS (repetitive, multiple site immunization strategy) has enabled somatic fusions to take place just 8-14 days after the initiation of immunization. The supernatants of the hybridomas produced can then be screened using standard immunoassays, allowing a monoclonal antibody against a specific antigen to be isolated much more quickly. However, even using RIMMS, the production and screening of monoclonal antibodies against large numbers of different antigens requires considerable time and resources. As more and more novel proteins are discovered, there is a need for faster and more efficient methods for producing and screening monoclonal antibodies against these proteins, in order to allow their further characterization.

Many methods have been developed to associate secreted antibodies with the cells where these antibodies were produced. Since such associationn will greatly facilitate the identification and isolation of the B-cells that produce specific antibodies.

One approach is to place the cell in a medium that inhibits the rate of diffusion from the cell. The typical method has been to immobilize the cell in a gel-like medium (agar) , and then to screen the agar plates for product production using a system reliant upon blotting, for example western blots. The U.S. Pat. 4,510,244 provided a method for isolating specific antibody hybridomas from a hybridoma cell mixture employing antigen-conjugated labeled microspheres and a label activated cell sorter. By selecting for labeled cells which produce light scatter and low red autofluorescence, viable single cells can be isolated and cloned which produce the desired antibodies.

Another approach, as disclosed in U.S. Pat. 7166423, is to couple the cells at their surface to a specific binding partner for the product and allowing the product to be captured by the specific binding partner as it is secreted and released. The product-labeled cells can then be further coupled to suitable labels, if desired, and separated according to the presence, absence, or amount of product.

The U.S. Pat. 6,376,170 provided a method for the identification and clonal isolation of antibodies that bind to unique epitopes. The method is based on the use of antibodies as solid phase capture reagents to bind a known capture antibody epitope, thereby precluding the capture antibody epitope from being presented to a population of antibodies to be screened. The method is particularly suited for screening libraries of cloned antibodies, such as phage display combinatorial antibodies .

The U.S. Publication 20060263801 disclosed methods of identifying ligands that are internalized into a cell. The methods typically involve i) contacting the cell with a reporter non-covalently coupled to a ligand; ii) dissociating the reporter from the ligand and removing dissociated reporter from the surface of the cell; and iii) detecting the reporter within said cell (if any is present) where the presence of the reporter within said cell indicates that the ligand binds to an internalizing receptor and is internalized.

The U.S. Publication 20060073095 disclosed methods and compounds that relate to screening and selection of monoclonal antibodies specific for antigens in heterogeneous antigen mixtures. Antibody-secreting cells such as hybridomas are modified to make them capable of directly binding antigens by capturing their secreted antibody products onto their surface membranes in appropriate binding density and orientation. Selectivity of binding to novel or desired antigens is achieved by first reacting the antigen mixtures affixed to a solid substrate with a polyclonal antibody library that prevents access to the majority of antigens or epitopes other than those that are novel or desired.

SUMMARY OF THE INVENTION

The present invention provides a DNA that enables a B- cell to bind to its own secreted antibody. The DNA comprises (1) a coding region encoding a chimeric protein that comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of the antibody (usually IgG); and (2) a promoter that drives the expression of the protein. In preferred embodiments, the promoter is an inducible and/or B-cell (especially plasma cell) specific promoter.

The binding domain might be derived from a secondary antibody that is generated against the non-variable region of the primary antibody, from Fc-binding domain of Fc receptors (FcRs) , or from the Fc-binding domain of bacterial Fc-receptors, such as Streptococcus Protein G, Staphylococcus aureus Protein A, and Peptostreptococcus magnus Protein L.

The chimeric protein can further have a detectable domain. The preferred detectable domain is a fluorescent protein, such as the monomeric red fluorescent protein (mRFP) . The detectable domain can be either extracellular or intracellular.

The present invention also provides a method for associating a B-cell with its own secreted antibody, comprising the steps of: a) expressing a protein in the B-cell, wherein the protein comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of the antibody; and b) incubating said B-cell from step a) with antigen labeled with a label moiety.

The present invention also provides a transgenic myeloma cell which expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of an antibody.

The present invention further provides a transgenic animal which expresses a transgene integrated into its genome, wherein the transgene comprises DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of the animal antibody. The transgenic animal is preferably mouse or rat.

The present invention also provides methods to isolate B- cells expressing antibodies against specific antigen by using the transgenic myeloma cell or the transgenic animal disclosed here.

Antigen might be either labeled in vitro with materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles, or expressed as a fusion protein with a detectable domain, such as a fluorescent protein. The labeled cells may then be separated or detected using standard cell sorting techniques based on the particular properties of these labels. Such techniques include flow cytometry, magnetic separation, high gradient magnetic separation, centrifugation, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. IA is a schematic diagram showing the plasmids used in the present invention; Fig. IA is a schematic diagram showing two inducer plasmids with different promoters; Fig. IB is a schematic diagram showing two different reporter plasmids;

Fig.2 is a schematic diagram showing the standard procedure to generate transgenic myeloma cell line with both inducer and reporter plasmids integrated in its genome, as disclosed in the present invention; Fig.3 is a schematic diagram showing one preferred embodiment of using the transgenic myeloma cells to isolate antibody-producing B-cells as disclosed in the present invention;

Fig.4 is a schematic diagram showing the standard procedure to produce transgenic mice with both inducer and reporter plasmids integrated in their genome, as disclosed in the present invention; and

Fig.5 is a schematic diagram showing one preferred embodiment of using the transgenic mouse to isolate antibody-producing B-cells as disclosed in the present invention .

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

Principle

Although the pathway of antibody synthesis and secretion is a complex series of steps and the cellular regulation of these steps is still not fully understood, it is generally believing that intracellular transport of antibodies are followed conventional secreted protein pathway, from endoplasmic reticulum (ER) to Golgi apparatus to extracellular medium (EM) . The heavy and light chains are first assembled in the ER, where posttranslational processes occur, such as disulfide bond formation. Then the assembled antibodies are transported from the ER to the Golgi, the primary glycosylation site in the cell. And finally matured antibodies are transported and released to the EM.

One plasma cell produces one specific mAb at very high rate, up to 10000 molecules/second. Let us say a cell X produces a mAb Y. It is conceivable that the Y is at its highest concentration surrounding X. If an antibody- binding protein Z is on the surface of X, Z is most likely to bind Y, but not to antibodies made by other cells, as long as the amount of Z is far less than that of Y surrounding X. In addition, Z might bind to Y when they co-exist in ER and/or Golgi, before they reach to the EM.

In the present invention, a transgene encoding a chimeric protein is integrated into the genome of a myeloma cell or an animal. The chimeric protein comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to non-variable region of an antibody.

To have better control on when and how much the chimeric protein should be made, an inducible promoter is preferred. One of the inducible expression systems is the tetracycline (Tet) regulatory system, which is based on the unusual specificity of interaction between the Tet repressor (TetR) and its specific DNA binding site, the tet operator (tetO) and between Tet repressor and its inducer, particularly Doxycycline (Dox) . There are two complementary Tet control systems. In the tTA (tetracycline controlled transactivator) or Tet-Off system, Dox prevents binding of tTA to the tetO sequence within P_tet/ and thus abolishes transcription. By contrast, the rtTA (reverse tetracycline controlled transactivator) or Tet-On system requires Dox for binding to and activation of P_tet-

The Tet control system has been widely and successfully applied in many cell systems and animal systems, including mouse. Transgene expression in these animals is exclusively dependent on the administration/absence of tetracycline or tetracycline derivatives (such as Dox) .

Although a Tet-on system is used in the preferred embodiments described here, it is worthy of note that Tet-Off system or other inducible systems might be used as well .

Although there are many versions of Tet-on systems, in which some use two plasmids and some use one plasmid, the commercial Tet-On system from Clontech is used and described here.

DNA Constructs

In Fig. IA, the inducer plasmid pTet-ON is the first half of the Clontech Tet-On system, in which the reverse tetracycline transactivator (rtTA) is expressed under the CMV promoter. The CMV promoter, originated from human cytomegalovirus immediate-early gene, is one of the most widely used promoters in mammalian expression systems. The CMV promoter induces high-level constitutive expression in a variety of mammalian cell lines, including mouse plasma cells.

Alternatively, a B-cell (especially plasma-cell) specific promoter could be used instead of the CMV promoter. One of the plasma-cell specific promoters is from the Blimp-1 gene (Prdml), whose expression is high in plasma cells but either low or absent in other B cells. To construct the P_Biimp-rtTA transgene plasmid, a mouse Blimp-1 promoter fragment spanning -3500 to +1 is generated by PCR and is used to replace the CMV promoter in the pTet-ON plasmid. To be simplified, pTet-ON is used in the following description unless stated otherwise.

Fig. IB shows two different reporter plasmids, derived from the second half of the Clontech Tet-on system. In pTRE-SpGTM, the chimeric protein consists of an N- terminal signal peptide, a Protein G' domain and a transmembrane domain.

How a protein is integrated into the ER membrane and becomes associated with the plasma membranes has been well documented (see U.S. Publication 20040049010). There are many pathways and signals to anchor a protein on plasma membrane. For illustration purpose, a single-pass transmembrane protein with a cleaved ER signal peptide is used here.

A signal peptide is a short peptide chain that directs the post-translational transport of a protein. An ER signal peptide is the best characterized signal peptide. It exists at the amino terminal of a protein. The protein is guided to the ER by a signal-recognition particle (SRP) . A typical signal peptides for transport to the ER i s H2N-Met-Met- Ser- Phe-Val - Ser-Leu-Leu-Leu-Val -Gly- I le-

Leu- Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys -Cys -Glu-

Val - Phe-Gln .

In the simplest case, an N-terminal signal peptide initiates translocation, but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is translocated cross the ER membrane. This stop-transfer- signal anchors the protein in the membrane after the ER signal sequence has been released from the translocator and has been cleaved off. The transmembrane (TM) domain can function as the stop-transfer signal. Through Golgi apparatus, the protein ends up at plasma membrane with its N-terminus outside and its C-terminus inside of the cell.

Proteins that bind to the constant (Fc) region of IgG have been found on the surface of a variety of staphylococci and streptococci bacteria. Among them, Protein A from Staphylococcus aureus, Protein G from Streptococcus and Protein L from Peptostreptococcus magnus are the best known and well studied.

Protein G binds to mammalian IgGs mainly through Fc regions. Native Protein G has 3 IgG binding domains and also domains for albumin and cell-surface binding. Protein G' (NCBI ACCESSION: CAA37410), a part of Streptococcus protein G, contains only three Fc-binding domains. Although the tertiary structures of Protein A and Protein G are very similar, their amino acid compositions differ significantly, resulting in different binding characteristics. Protein G has greater affinity than Protein A for most mammalian IgGs, especially for certain subclasses including human IgG3, mouse IgGl and rat IgG2a. Unlike Protein A, Protein G does not bind to human IgM, IgD and IgA. Both modified Protein G and Protein A have been used for purification of mammalian monoclonal and polyclonal IgGs.

Protein L is an immunoglobulin-binding protein that binds to immunoglobulin kappa light chains without interfering with the antigen-binding site and binds a wider range of Ig classes and subclasses than other antibody-binding proteins such as Protein A or Protein G. Protein L binds to all classes of Ig (i.e., IgG, IgM, IgA, IgE and IgD) . Protein L also binds single chain variable fragments (Scfv) and Fab fragments.

Compared to pTRE-SpGTM, pTRE-SpGTMR has an additional detectable domain. In the preferred embodiment, the detectable domain is a monomeric red fluorescent protein (mRFP) . The detectable domain can be either extracellular or intracellular. In the preferred embodiment, the mRFP locates at the intracellular region of the chimeric protein. To be simplified, pTRE-SpGTM is used in the following description unless stated otherwise.

As a practical manner, an artificial intron is added to stimulate the transport of mRNA out of the nucleus. The artificial intron could be placed at either end of the protein-coding region, preferably at the 3' end.

Transgenic Myeloma Cells

The technique to generate a myeloma cell line that has both inducer and reporter plasmids integrated into its genome is well documented (see Clontech Tet-On® Advanced Inducible Gene Expression System User Manual PT3898-1) . In brief (see Fig.2), myeloma cells are first transfected with the inducer plasmid, which also contains a Neo^r selectable maker. G418-resistant transfectants are further screened by transient transfections with pTRE- Tight-Luc for clones with low background and high Dox- dependent induction of luciferase activity. Selected cells are then co-transfected with the reporter plasmid and a Linear Hygromycin Marker. Hygromycin-resistant transfectants are further screened for clones with low background and high Dox-dependent induction of the chimeric protein. The resulting clones are double-stable transgenic myeloma cell line, which has both inducer and reporter plasmids integrated into its genome.

Use of Transgenic Myeloma Cells

In Fig.3, hybridoma cells are produced by fusing the transgenic myeloma cells with B-cells isolated from spleen and lymph nodes of an immunized animal. The hybridoma cells are then grown in a culture medium containing Dox at the concentration between 10-lOOOng/ml for 24-48 hours to induce the chimeric protein expression. After induction, the hybridoma cells are incubated with antigen-GFP fusion protein. Fluorescent-activated cell sorting (FACS) is performed to isolate cells with labeling intensity above a given threshold. In this situation, the reporter plasmid pTRE-SpGTMR is favored over pTRE-SpGTM since the sorting criteria could be set to base upon the ratio of GFP/mRFP. The endogenous mRFP provides a base for comparing the binding affinity of different antibodies to the antigen. Although antigen-GFP fusion protein is used in the above description, antigen might also be labeled in vitro with materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles. In the case that the antigen is not a protein, in vitro labeling becomes necessary. Such labeling techniques are well documented in the field to which this invention belongs.

Although FACS is used in the above description, the cell sorting/detecting techniques have to be chosen based on the particular properties of a cell label. Such techniques, including flow cytometry, magnetic separation, high gradient magnetic separation, centrifugation, and the like, are all well documented in the field to which this invention belongs.

Multiple rounds of separation based on same or different properties of cell labels might be performed to improve the sorting result. Once a label is chosen, separation/ sorting methods based upon such label are generally obvious to a person with ordinary skill in the field to which this invention belongs.

Transgenic Animals

Transgenic animals are genetically modified animals into which cloned genetic material has been experimentally introduced. The cloned genetic material is referred to as a transgene. The nucleic acid sequence of the transgene is integrated at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found. Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the recitations in U.S. Pat. Nos. 5,489,743 and 5,602,307.

Even though murines, especially mice and rats, remain the animals of choice for most transgenic experimentation, transgenic procedures have been successfully performed with a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs. In the present invention, mice and rats are preferred.

As shown in Fig.4, to generate transgenic mice, linearized inducer or reporter plasmid is injected into the pronuclei of fertilized mouse eggs. The plasmid is incorporated into random loci, usually in head-to-tail concatemers consisting of varying numbers of copies. After injection, the eggs are transferred to the oviducts of pseudopregnant foster mothers, generated by mating females with vasectomized males so the females do not produce any fertilized embryos of their own. The offspring resulting from injected eggs are genotyped using DNA extracted from a small piece of tissue cut from the tip of the tail. The mice that do carry the transgene are called founders. Offspring from each founder are tested for transmission and expression of the transgene.

Mice that have the transgene on one chromosome are heterozygous because they do not have a corresponding allele on the other chromosome. These heterozygous can further cross to produce homozygous.

Heterozygous or homozygous inducer plasmid transgenic mice were mated with heterozygous or homozygous reporter plasmid transgenic mice to generate double-transgenic progeny. Double transgenic mice are confirmed by southern blotting or PCR analysis with genomic DNA isolated from tail biopsies.

Use of Transgenic Mice

In Fig.5, a double transgenic mouse is first immunized with a target antigen. Once the titer of the antibody is satisfied, the mouse is administered with Dox (2g/l in 2.5% sucrose) in light-protected bottles in drinking water every 24 hours for 48-72 hours. The mouse is then sacrificed and B-cells from its spleen and lymph nodes are isolated. The B-cells are incubated with labeled antigen and are sorted accordingly as described above. Sorted B-cells can be further characterized as described above .

Alternatively, Dox induction can be performed after B- cells are isolated from the spleen and lymph nodes of the immunized mouse. In this case, Dox concentration should be between 10-lOOOng/ml and the induction lasts 24-48 hours .

To assist a person to better understand the field of art where the present invention belongs to, some additional terms are listed as follows. The Fc region (Fragment, crystallizable) , is derived from the stem of the "Y, " and is composed of two heavy chains that each contributes two to three constant domains (depending on the class of the antibody) . Fc binds to various cell receptors and complement proteins. In this way, it mediates different physiological effects of antibodies (opsonization, cell lysis, degranulation of mast cells, basophils and eosinophils and other processes) .

Each end of the forked portion of the "Y" on the antibody is called the Fab region (Fragment, antigen binding) . It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope—the antigen-binding site—at the amino terminal end of the monomer. The two variable domains bind the epitope on their specific antigens.

An Fc receptor (FcR) is a protein found on the surface of certain cells; including natural killer cells, macrophages, neutrophils and mast cells; that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (Fragment, crystallizable) region. FcRs bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. There are several different types of FcR, which are classified based on the type of antibody that they recognize; those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcγR) , those that bind IgA are called Fc-alpha receptors (FcαR) and those that bind IgE are called Fc-epsilon receptors (FcεR) .

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, applications and publications referred to in the application are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1

To construct the pTRE-SpGTM transgene plasmid, the 780bp fragment mIL2/pG ' (Fc) /TM (hCD2) (SEQ ID NO. 1) was first in vitro synthesized and assembled, which consists of optimized DNA sequence encoding the IL2 signal peptide, the Protein G' Fc binding domain, the hCD2 transmembrane domain, and a C-terminal short end. The fragment was cut with Kpnl/Sall and was then inserted into the vector pTRE-Tight (Clontech Laboratories, Inc., Mountain View, California, USA) at the same cloning sites. The junctions of the resulting pTRE-SpGTM plasmid sequence were verified by automated sequencing.

Example 2

To construct the pTRE-SpGTMR transgene plasmid, mRFP (SEQ ID NO. 2) was first generated by PCR. The PCR fragment was cut with NgoMIV/Sall and was inserted into the NgoMIV/Sall sites within the pTRE-SpGTM. The junctions of the resulting pTRE-SpGTMR plasmid sequence were verified by automated sequencing. Example 3

To construct the P_Bi_imp-rtTA transgene plasmid, the vector pTet-On-Advanced (Clontech Laboratories, Inc., Mountain View, California, USA) was cut with BsrGI/SacI to remove the CMV promoter and was then ligated with the mouse Blimp-1 promoter fragment spanning -3500 to +1 (SEQ ID NO. 3) , which was generated from mouse genomic DNA by PCR. The junctions of the resulting P_Bi_imp-rtTA plasmid were verified by automated sequencing.

Example 4

To generate transgenic Myeloma Cells, the standard procedure, described in Tet-On® Advanced Inducible Gene Expression System User Manual (Clontech) , was strictly followed. The pTRE-SpGTM or pTRE-SpGTMR was linearized with Xhol restriction enzyme before the 2^nd step transfection .

Example 5

The pTet-ON (or P_Bi_imp-rtTA) transgene was generated by excising a BsrGI/Hindlll fragment from the pTet-ON (or P_Bi_imp-rtTA) plasmid, and the P_tight-SpG' TM (or P_tight-SpG' TMR) transgene was generated by excising an Xhol fragment from the P_tig_ht-SpG' TM (or P_tight-SpG' TMR) plasmid. Transgenic mice were produced by microinjection of gel-purified transgene DNA into single-cell preimplantation FVB mouse embryos using standard methods. For genotyping of transgenic mice, genomic DNA was isolated from tail biopsies, and transgenes were amplified by PCR. Transgene expression was evaluated and confirmed by western blotting analysis. Heterozygous or homozygous pTet-ON (or P_Blimp-rtTA) transgenic mice were mated with heterozygous or homozygous P_ti_ght~SpG' TM (or P_tight-SpG' TMR) transgenic mice to generate double-transgenic progeny.

Example 6

The double-transgenic mice were immunized using standard protocol. To induce SpG' TM (or SpG' TMR) expression, Dox (2g/l in 2.5% sucrose) was administered to mice 3 days before sacrifice in light-protected bottles in drinking water with sucrose supplementation to combat taste aversion from Dox. B-cells were isolated and prepared from their spleen according to the standard protocol.

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Claims

CLAIMS I claim:

1. A DNA that enables a B-cell to bind to its own secreted antibody, comprising: a coding region encoding a protein that comprises at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non- variable region of said antibody; and a promoter that drives the expression of said protein.

2. The DNA of claim 1, wherein said promoter is inducible.

3. The DNA of claim 1, wherein said binding domain comprises a Fc-binding domain, selected from the group consisting of Streptococcus protein G, Staphylococcus aureus protein A, and Peptostreptococcus magnus protein L.

4. The DNA of claim 3, wherein said promoter is inducible.

5. The DNA of claim 1, wherein said protein further comprises a detectable domain independent of said binding domain.

6. The DNA of claim 5, wherein said detectable domain is a fluorescent protein.

7. A method for associating a B-cell with its own secret antibody, comprising the steps of: a) expressing a protein in said B-cell, wherein said protein comprises at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of said antibody; and b) incubating said B-cell from step a) with antigen labeled with a label moiety.

8. The method of claim 7, wherein said label moiety is fluorochromated .

9. The method of claim 7, wherein said label moiety is magnetizable .

10. The method of claim 7, wherein said binding domain comprises a Fc-binding domain, selected from the group consisting of Streptococcus protein G, Staphylococcus aureus protein A, and Peptostreptococcus magnus protein L.

11. The method of claim 7, wherein said protein further comprises a detectable domain independent of said binding domain.

12. The method of claim 11, wherein said detectable domain is a fluorescent protein.

13. A transgenic myeloma cell which expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of an antibody.

14. The transgenic myeloma cell of claim 13, wherein the expression of said protein is inducible.

15. The transgenic myeloma cell of claim 13, wherein said extracellular domain comprises a Fc-binding domain, selected from the group consisting of Streptococcus protein G, Staphylococcus aureus protein A, and Peptostreptococcus magnus protein L.

16. The method for associating a B-cell with its own secret antibody, comprising the steps of: a) generating a hybridoma cell by fusing said B-cell with a transgenic myeloma cell, wherein said transgenic myeloma cell expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of said antibody; b) incubating said hybridoma cell from step a) with antigen labeled with a label moiety.

17. The method for associating a B-cell with its own secret antibody, comprising the steps of: a) generating a hybridoma cell by fusing said B-cell with a transgenic myeloma cell, wherein said transgenic myeloma cell expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of said antibody, and wherein the expression of said protein is inducible; b) inducing the expression of said protein in said hybridoma cell from step a) ; and c) incubating said hybridoma cell from step b) with antigen labeled with a label moiety.

18. A transgenic animal which expresses a transgene integrated into its genome, wherein the transgene comprises DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of the animal antibody.

19. The transgenic animal of claim 18, wherein the expression of said protein is inducible.

20. The transgenic animal of claim 18, wherein said binding domain comprises a Fc-binding domain, selected from the group consisting of Streptococcus protein G, Staphylococcus aureus protein A, and Peptostreptococcus magnus protein L.

21. The transgenic animal of claim 18, wherein said protein further comprises a detectable domain independent of said binding domain.

22. The transgenic animal of claim 21, wherein said detectable domain is a fluorescent protein.

23. The method for associating a B-cell with its own secret antibody, comprising the steps of: a) isolating said B-cell from a transgenic animal, wherein said transgenic animal expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of said antibody, and wherein the expression of said protein is inducible; b) inducing the expression of said protein in said B- cell from step a) ; and c) incubating said B-cell from step b) with antigen labeled with a label moiety.

24. The method for associating a B-cell with its own secret antibody, comprising the steps of: a) challenging a transgenic animal with an antigen, wherein said transgenic animal expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of said antibody, and wherein the expression of said protein is inducible; b) administrating said transgenic animal a substance to induce the expression of said protein; c) isolating said B-cell from said transgenic animal of step b) and incubating said B-cell with said antigen labeled with a label moiety.

25.A method for generating a transgenic mouse heterozygous for the DNA in claim 2, comprising: a) injecting said DNA into of fertilized eggs; b) transferring the fertilized eggs from step a) to a surrogate mother to produce offspring; c) screening said offspring to identify transgenic founders for expressing said protein.

26. The method according to claim 9 further comprising: e) mating the transgenic mouse; and f) selecting a mouse homozygous for said DNA.