ENHANCED PRODUCTION OF IMMUNOGLOBULINS

Abstract

The present invention provides methods and compositions for generating transgenic animals, including transgenic mammals, as well as plasma cells that allow for cell surface capture of secreted immunoglobulin molecules produced endogenously in the plasma cells.

Description

RELATED APPLICATION

[0001] This application claims priority to U.S. Ser. No. 62/291,217, filed Feb. 4, 2016.

FIELD OF THE INVENTION

[0002] This invention relates to production of immunoglobulin molecules, including methods for rapid screening of antigen-specific antibody-secreting cells for the generation of monoclonal antibodies.

BACKGROUND OF THE INVENTION

[0003] In the following discussion certain articles and methods are described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[0004] Monoclonal antibodies are important biologics that have been widely employed in biomedical research, in clinical diagnostics, and as therapeutic agents because of their exquisite ability to bind antigens of diverse molecular forms. In drug development, monoclonal antibodies are often the molecules of choice because they exhibit desirable pharmacokinetics that are associated with powerful immunological functions normally involved in fending off infectious agents. Furthermore, laboratory animals can readily mount a specific antibody response against any target molecule that is not present natively in the body, making antibody generation a relatively low-risk and cost-efficient approach when compared to alternative strategies.

[0005] Although hybridoma technology was developed more than four decades ago, today it is still the most widely employed technique to generate antigen-specific monoclonal antibodies. In this approach, an animal (typically, a rodent or a rabbit) is first immunized with an antigen of interest. B lymphocytes in the immunized animal that have the receptor specificity for the antigen then become activated, clonally expand, and differentiate into antibody-secreting cells (ASCs). The immunized animal is then sacrificed, and because the ASCs isolated from these animals cannot survive indefinitely in culture, these cells are often immortalized by fusion with malignant plasma cells (such as myeloma or plasmacytoma cells) to generate hybrid cells called hybridomas. The hybridoma cells are then screened and selected for their ability to secrete antibodies with reactivity to the antigen of interest, often involving multiple rounds of limiting dilution and propagation in culture.

[0006] Alternatively, the ASCs can be individually sorted, and the genes encoding the heavy chain and light chain variable domains (V.sub.H and V.sub.L, respectively) directly cloned without the need to propagate the ASCs in vitro. The V.sub.H- and V.sub.L-encoding DNA fragments are next subcloned into an expression vector containing exon sequences for the desired heavy chain and light chain constant regions, respectively. Each V.sub.H and V.sub.L pair of expression vectors are then transfected into a cell line to express the monoclonal antibodies, which are subsequently screened for their ability to recognize the antigen of interest.

[0007] Despite the eventual success in producing monoclonal antibodies against the antigen of interest using either hybridoma or single-cell cloning technique, the efficiency of both techniques is hampered by the labor-intensive process of screening and selection. This is because it has not been feasible to pre-select only the antigen-specific ASCs for fusion with myeloma cells or for single-cell cloning. When B lymphocytes differentiate into ASCs in response to an antigenic encounter, the membrane-bound form of antigen receptors is down-regulated in favor of the secreted form. Thus, selection methods based on the cell surface expression of antigen receptors, such as magnetic or flow-cytometric sorting, do not work well as tools to select for antigen-specific ASCs. Due to this lack of ability to pre-select ASCs, only a small fraction of cells screened in both hybridoma and single-cell cloning techniques produces monoclonal antibodies with specificity for the antigen of interest.

[0008] U.S. Pat. No. 7,148,040 B2 provides methods to express the membrane-bound form of antigen receptors on hybridoma cells to improve the efficiency of hybridoma screening by selection techniques based on the cell surface expression of antigen receptors. In this approach, myeloma cells are transfected with expression constructs encoding CD79A and CD79B, also known as Ig.alpha. and Ig.beta., respectively. CD79A and CD79B are expressed as heterodimers that are necessary for both cell surface expression and signaling functions of the antigen receptors on B cells. As B lymphocytes differentiate into ASCs, they down-regulate CD79A and CD79B expression, thus contributing to the loss of antigen receptor expression on the cell surface. Therefore, re-introducing the expression of CD79A and CD79B allows for increased representation of the membrane-bound form of antigen receptors on the hybridomas. Although this strategy helps reduce the labor of hybridoma screening and selection, the efficiency could be greatly improved if it was feasible to pre-select only the antigen-specific ASCs for fusion with myeloma cells. Moreover, the specified methods do not provide a strategy to increase the efficiency of monoclonal antibody generation using direct V.sub.H and V.sub.L cloning technique from sorted single cells.

[0009] Re-introduction of CD79A and CD79B expression by ASCs in vivo may not provide a viable strategy to increase the expression of antigen receptors on the cell surface either. Because CD79A and CD79B expression is tightly regulated during B lymphocyte development, alterations in their expression levels in vivo may have profound consequences on B lymphocyte survival, functions, and/or antigen receptor selection. Moreover, the antigen receptors on ASCs are likely to be internalized at the time of ASC isolation due to their active engagement with the immunogen, since the immune response is still ongoing when the mouse is euthanized. If instead, signaling-deficient mutant CD79A and CD79B are expressed on ASCs to prevent antigen receptor internalization, it remains unexplored whether the mutant forms of these molecules exhibit a dominant-negative effect that negatively impacts ASC survival and functions in vivo. Finally, expressing CD79A and CD79B on ASCs ex vivo to circumvent the aforementioned problems associated with their enforced expression in vivo is not a practical strategy because ASCs are not amenable to gene transfer by most methods currently available.

[0010] Thus, a method for more efficient screening for antigen-specific ASCs is an important unmet need. The methods and compositions provided by the present specification meet this important need.

SUMMARY OF THE INVENTION

[0011] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[0012] The present invention provides methods and compositions for enhanced production of immunoglobulin molecules. Specifically, the invention provides methods and compositions for the capture of secreted immunoglobulin molecules, including those of IgG, IgA, IgE and IgM isotypes, at the surface of ASCs. The present invention also comprises transgenic animals, including transgenic mammals, comprising engineered ASCs that can capture and display on their cell surface immunoglobulin molecules produced endogenously from within the ASCs.

[0013] In one embodiment, the invention provides restricted constitutive expression of an engineered immunoglobulin-capturing molecule comprising one or more immunoglobulin-binding portions or domains derived from bacterial protein(s) such as Protein A and/or Protein G, using an expression system that expresses the immunoglobulin-capturing molecule preferentially on ASCs with minimal expression at the stages of B cell development prior to antigen-induced differentiation.

[0014] In another embodiment, the invention provides restricted constitutive expression of an engineered immunoglobulin-capturing molecule comprising a single-chain antibody with specificity to an immunoglobulin, using an expression system that expresses the immunoglobulin-capturing molecule preferentially on ASCs with minimal expression at the stages of B cell development prior to antigen-induced differentiation.

[0015] The engineered ASCs express immunoglobulin-capturing molecules that are tethered to the cell surface and have the ability to selectively bind immunoglobulin molecules (also, as used herein "immunoglobulins" or "antibodies") with sufficient affinity to immobilize the immunoglobulin molecules at the plasma membrane. Because ASCs secrete thousands of immunoglobulin molecules per second, the immunoglobulin-capturing molecules on a given ASC are saturated primarily with the immunoglobulin molecules secreted by that ASC rather than with immunoglobulins secreted by other ASCs. Expression of genes encoding cell surface immunoglobulin-capturing molecules provides a means for identifying ASCs based on the particular monoclonal immunoglobulin molecules being expressed.

[0016] In certain aspects, the immunoglobulin-capturing molecule is tethered to the membrane by a peptide sequence derived from a transmembrane protein such as but not limited to human Lymphocyte-Activation Gene 3 (LAG3). In other aspects, the immunoglobulin-capturing molecule is tethered to the plasma membrane via a post-translational modification with, e.g., glycosylphosphatidylinositol (GPI). In some of these aspects, the immunoglobulin-capturing molecule further comprises a long stalk for support, flexibility, and extended protrusion into the extracellular space.

[0017] In certain aspects, expression of the immunoglobulin-capturing molecules is driven by a promoter derived from a human or mouse gene that is highly expressed in ASCs developed in vivo or in vitro. In other aspects, the immunoglobulin-capturing molecules are expressed by an inducible system, such as the tetracycline system, in vivo or in vitro. In some aspects, expression of the immunoglobulin-capturing molecule is coupled to the expression of a reporter gene, such as green fluorescent protein (GFP), via an internal ribosomal entry site sequence (IRES) or a picornavirus 2A ribosomal skip sequence in the expression vector.

[0018] The present invention also provides methods for generating a non-human transgenic animal expressing immunoglobulin-capturing molecules on ASCs. The methods comprise introducing an immunoglobulin-capturing molecule-encoding gene into the genome of a non-human vertebrate, wherein the introduced gene provides constitutive or inducible expression of the immunoglobulin-capturing molecule on host ASCs. In some aspects the transgenic animal is a rodent, preferably a mouse. In other aspects, the transgenic animal is avian, preferably a chicken. In particularly preferred aspects, the transgenic animal is a mouse that expresses human genes encoding the variable domains of the heavy and light chains and lacks the mouse versions of these genes; for example, as described in US Pub. No. 2013/0219535, which is incorporated by reference in its entirety.

[0019] The invention additionally provides processes for isolating genes that encode immunoglobulins of a particular specificity from ASCs that display the specific immunoglobulins captured on the surface of the ASCs.

[0020] The present invention also provides libraries for identification of antibodies of interest from the engineered cells of the invention. The antibody libraries produced using the methods and compositions of the invention provide a facilitated means for the screening and production of antibodies that selectively bind to a target of interest. Such libraries thus enhance the isolation of monoclonal antibodies for use in the clinical, diagnostic, and research settings.

[0021] An advantage of the invention is that the determination of immunoglobulin specificity can be made using established techniques such as binding to fluorescently labeled antigen and flow cytometric or microscopic procedures. Such procedures allow for enhanced efficiency in identification and isolation of rare antigen-specific cells and the cloning of the rearranged immunoglobulin genes from the isolated cells.

[0022] These and other aspects, objects and features are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIGS. 1A, 1B and 1C are illustrations of a secreting ASC with no immunoglobulin-capturing molecules on the cell surface (FIG. 1A), an ASC with immunoglobulin-capturing molecules and captured immunoglobulin molecules (i.e., antibodies) on the surface of the ASC (FIG. 1B), and the binding of labeled antigens to the antibodies retained by the immunoglobulin-capturing molecules expressed on an ASC (FIG. 1C).

[0024] FIG. 2A is a schematic diagram depicting part of a DNA vector encoding an embodiment of an immunoglobulin-capturing molecule. FIG. 2B is a simplified illustration of the embodiment of FIG. 2A expressed as an immunoglobulin-capturing molecule on an ASC surface.

[0025] FIG. 3A is a schematic diagram depicting part of a DNA vector encoding an alternative embodiment of an immunoglobulin-capturing molecule. FIG. 3B is a simplified illustration of the embodiment of FIG. 3A expressed as an immunoglobulin-capturing molecule on an ASC surface.

[0026] FIGS. 4A, 4B, and 4C are illustrations of a secreting ASC with no immunoglobulin-capturing molecules on the cell surface (FIG. 4A), an ASC with immunoglobulin-capturing molecules and immunoglobulins (i.e., antibodies) on the surface of the ASC (FIG. 4B, also as depicted in detail in FIG. 3B), and labeled antigens bound to the immunoglobulin-capturing molecules expressed on an ASC (FIG. 4C).

[0027] FIG. 5A is a schematic diagram depicting part of a DNA vector encoding an exemplary embodiment of an immunoglobulin-capturing molecule. FIG. 5B provides two flow cytometry scatter plots showing the results of retention of secreted immunoglobulin molecules on the cell surface of transfected RPMI 8226 (ATCC.RTM. CCL-155.TM.) human cells per the methods of the invention.

DEFINITIONS

[0028] The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.

[0029] An "antibody-secreting cell" or "ASC" refers to a cell that has differentiated from an antigen-experienced B cell and acquired the capacity to express as well as secrete large amounts of immunoglobulin molecules. ASCs include plasmablasts and short-lived or long-lived plasma cells in the animal, as well as plasmablasts and plasma cells developed in vitro from B cell cultures.

[0030] A "capture molecule" is any moiety that contains a region that selectively binds to a part of or a whole molecule of interest.

[0031] "Capture" refers to selective binding and immobilization of a molecule at a cell surface due to a durable interaction between that molecule and a membrane-bound capture molecule.

[0032] "Cell surface" refers to the plasma membrane of a cell, i.e., that part of a cell most directly exposed to extracellular spaces and available for contact both with cells and proteins in the extracellular (including intercellular) space.

[0033] An "immature B cell" refers to a cell at an intermediate phase of B cell differentiation, during which a hematopoietic stem cell undergoes genetic programming to become a mature, yet antigen-inexperienced, B cell. A "mature" B cell refers to an antigen-inexperienced B cell, which is capable of clonal expansion, as well as differentiation into a memory cell or an antibody-secreting cell, upon activation by an antigen.

[0034] An "immunoglobulin" refers to an antibody, whether a part of or whole antibody molecule. In most vertebrate animals including humans, antibodies normally exist as dimers of two identical heavy (H) chains that are each paired with an identical light (L) chain. The N-termini of both H and L chains consist of a variable domain (V.sub.H and V.sub.L, respectively) that together provide the H-L pair with its unique antigen-binding specificity. The constant region of the H chain consists of 3 to 4 immunoglobulin domains (referred to as C.sub.H1 to C.sub.H4) with or without a hinge, depending on the isotype (or antibody class). In mice, the isotypes are IgM, IgD, IgG3, IgG1, IgG2b, IgG2a or IgG2c, IgE, and IgA. The light chain constant region consists of either a .kappa. or .lamda. immunoglobulin domain (referred to as C.kappa. or C.lamda.). In both mice and humans, the presence of .kappa. light chains predominates over that of .lamda. light chains in the total pool of immunoglobulins within an individual. In certain mammals, such as camelids or animals made deficient in light chain expression, immunoglobulins may consist of heavy chains only. Despite the lack of light chains, these immunoglobulins are also efficiently retained on the cell surface by immunoglobulin-capturing molecules designed to bind to the immunoglobulin heavy chain described in the present invention. Additionally, an immunoglobulin can refer to an unconventional antibody, whether in part or in whole, such as a bispecific antibody that consists two or more V.sub.H and/or V.sub.L domains, for example, as described in U.S. Ser. No. 15/246,181, filed 24 Aug. 2016, which is incorporated by reference in its entirety. Finally, an immunoglobulin also refers to a hybrid molecule consisting of part of an antibody, particularly the antibody constant region, and part of another protein. The immunoglobulin-capturing molecules described in the present invention also may be designed and engineered to retain hybrid immunoglobulin molecules for display at the cell surface.

[0035] An "immunoglobulin-capturing molecule" refers to a plasma membrane-bound molecule that can bind, retain, and display immunoglobulin molecules (i.e., immunoglobulins or antibodies) at the cell surface.

[0036] An "immunoglobulin superfamily" or "IgSF" molecule refers to a molecule that possesses immunoglobulin folds (Ig folds) that are structurally similar to the immunoglobulin domains found in antibody molecules.

[0037] The term "transgene" is used herein to describe genetic material which has been or is about to be artificially inserted into the genome of a cell.

[0038] "Transgenic animal" refers to a non-human animal, usually a mammal such as a rodent, particularly a mouse or rat although other animals are envisioned, having an exogenous nucleic acid sequence present as a chromosomal or extrachromosomal element in a portion of its cells or stably integrated into its germ-line DNA (i.e., in the genomic sequence of most or all of its cells).

[0039] A "vector" or "expression construct" includes plasmids and viruses and any DNA or RNA molecule, whether self-replicating or not, which can be used to transform, transduce, or transfect a cell.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999) Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2007), PCR Primer: A Laboratory Manual; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Green and Sambrook (2012), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., New York, N.Y.; and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y.; Nagy, et al., Eds. (2003) Manipulating the Mouse Embryo: A Laboratory Manual (3rd Ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Immunology Methods Manual (Lefkovits ed., Academic Press 1997); Gene Therapy Techniques, Applications and Regulations From Laboratory to Clinic (Meager, ed., John Wiley & Sons 1999); M. Giacca, Gene Therapy (Springer 2010); Gene Therapy Protocols (LeDoux, ed., Springer 2008); Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, eds., John Wiley & Sons 1998); and Mammalian Chromosome Engineering--Methods and Protocols (G. Hadlaczky, ed., Humana Press 2011), all of which are herein incorporated in their entirety by reference for all purposes.

[0041] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an immunoglobulin" refers to one or more such immunoglobulins, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0042] 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. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.

[0043] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

The Invention in General

[0045] Antibody-secreting cells (ASCs) normally do not display the immunoglobulins they express and secrete on their plasma membrane, making highly advanced techniques based on cell surface labeling, such as magnetic and flow-cytometric sorting, inapplicable as methods to select for antigen-specific ASCs. The present invention was born out of the need for a system that allows for efficient screening of ASCs based on cell surface presentation of secreted immunoglobulin molecules (also as used herein "immunoglobulins" or "antibodies"). Specifically, the present invention provides a means for expressing immunoglobulin-capturing molecules that can retain and immobilize immunoglobulins at the surface of the secreting cells, such as ASCs or hybridomas, which do not normally express high levels of membrane-bound immunoglobulins or naturally have the ability to retain immunoglobulins on their cell surface. ASCs express and release large amounts of immunoglobulins (thousands of molecules per second) (see, e.g., Mitchell, Advances in Immunology 28:451-511 (1979)). Therefore, the immunoglobulin-capturing molecules expressed on these cells are saturated primarily with immunoglobulins produced from within, rather than with the immunoglobulins secreted by other cells. Thus, the immunoglobulin-capturing molecules must possess a high affinity and a low dissociation rate for the immunoglobulin molecules they capture. The present invention provides methods and compositions for expression of such high-affinity immunoglobulin-capturing molecules with low dissociation rates.

[0046] Engineering ASCs to capture endogenously produced immunoglobulins on their cell surface provides a facile means for discriminating the antigen specificity of the antibodies that each ASC produces, and for separating ASCs secreting desired immunoglobulins from those ASCs that do not. Discrimination can be accomplished by, e.g., using antigens labeled with substances that facilitate identification and purification of cells (e.g., magnetic, biotinylated, fluorescent, radioactive, or enzymatic molecules) by well-established procedures known in the art.

[0047] FIGS. 1A, 1B and 1C illustrate the principles of the present invention in one exemplary embodiment. As depicted in FIG. 1A, an antibody-secreting cell (ASC) (101) does not normally express the membrane-bound form of the immunoglobulin molecule, nor does it retain the secreted form of the immunoglobulin molecule (i.e., antibody) (102) on its cell surface. The present invention provides methods and compositions for the expression of immunoglobulin-capturing molecules (103) on the cell surface of ASCs. According to the invention, during the synthesis of the immunoglobulin molecules in the endoplasmic reticulum and subsequent packaging in the vesicles for secretion--or soon after their secretion-some immunoglobulin molecules (102) are retained on the cell surface by immunoglobulin-capturing molecules (103) as depicted in FIG. 1B. Labeled antigens (104) (e.g., fluorescently-labeled antigens) are then allowed to bind to the immunoglobulin molecules (antibodies) (102) that have bound to an immunoglobulin-binding portion of the immunoglobulin-capturing molecules (103) on the cell surface of the ASC, as depicted in FIG. 1C. Antigen-specific ASCs are then identified by the labeled antigens (104) bound to the immunoglobulin-binding portion of the immunoglobulin-capturing molecules (102) that have been captured on the cell surface. Detection of antigen binding on the ASCs is accomplished by, e.g., using antigens that are directly labeled with a fluorophore or other reporter molecule. The ASCs that bind labeled antigens are then purified by, e.g., cell-sorting techniques known in the art.

[0048] The purified ASCs expressing antibodies specific for a particular antigen may then be immortalized by fusion with myeloma or plasmacytoma cells, or directly used as a source of nucleic acids (DNA or mRNA) for the creation of libraries of sequences encoding immunoglobulins.

[0049] Libraries from purified ASCs contain rearranged immunoglobulin genes encoding antibodies of defined specificity (i.e., specificity for the antigens used in the purification process). The V.sub.H and V.sub.L genes can be identified from the antigen-specific ASCs by deep sequencing coupled with bioinformatics data mining (see, e.g., Haessler and Reddy, Methods in Molecular Biology 1131:191-203 (2014)). Alternatively, the antigen-specific ASCs can be individually sorted. The V.sub.H and V.sub.L domains unique to each ASC are then cloned via established RT-PCR or 5' Rapid Amplification of cDNA Ends (5' RACE) techniques adapted to single-cell cloning (for review, see, e.g., Tiller, et al., New Biotechnology 5:453-7 (2011)). In yet another alternative, the V.sub.H and V.sub.L sequences can be identified using the methods and materials described in U.S. Pat. Nos. 9,328,172; 8,309,035; and 8,309,317.

The Immunoglobulin-Capturing Molecule

[0050] The immunoglobulin-capturing molecules of the present invention that are expressed at the cell surface comprise at least two components, and in preferred embodiments may comprise additional components, as described in detail below. In a simple form, the immunoglobulin-capturing molecules comprise a cell surface tether component, and an immunoglobulin-binding component. The cell surface tether component may comprise a transmembrane peptide domain that tethers or anchors the expressed immunoglobulin-binding component in the cell surface membrane, or the cell surface tether component may comprise a chemical moiety (for example, glycosylphosphatidylinositol) that allows for the immunoglobulin-binding component to be tethered to the cell surface membrane via a chemical bond. In addition to these components, the immunoglobulin-capturing molecules of the present invention may comprise a stalk component, one or more linker components, and/or a reporter peptide.

[0051] In one embodiment, the immunoglobulin-capturing molecule consists of one or more immunoglobulin-binding domains or portions derived from one or more bacterial proteins that naturally have affinity for the constant region of the heavy or light chain of immunoglobulins. Such immunoglobulin-binding proteins include but are not limited to Protein A from Staphylococcus aureus, Protein G from group C and G Streptococci, Protein H from Streptococcus pyogenes, or Protein L from Peptostreptococcus magnus. In some embodiments, the immunoglobulin-capturing molecule is expressed as a hybrid molecule comprising two or more immunoglobulin-binding domains derived from two or more different bacterial proteins. As an example, the capture molecule may be expressed as a fusion protein, which contains two immunoglobulin-binding domains from Protein G and two immunoglobulin-binding domains from Protein A. In some aspects of this embodiment, one or more of the bacterial immunoglobulin-binding protein domains are modified to, e.g., remove potential sites for glycosylation or other post-translational modifications in eukaryotic cells, improve affinity for certain immunoglobulin isotypes, or improve translation efficiency in mammalian cells by codon optimization.

[0052] In another embodiment, the immunoglobulin-capturing molecules consist of single-chain variable fragments (scFv). The scFv is expressed as a fusion protein of the V.sub.H and V.sub.L domains derived from a hybridoma cell line that produces monoclonal antibodies against the heavy chain or light chain constant region of another immunoglobulin molecule (e.g., a common epitope present in all murine IgG isotypes). In some aspects, the scFv capture molecule comprises the V.sub.H domain connected in tandem to the V.sub.L domain by a glycine/serine-rich linker sequence in either order. The glycine/serine-rich linker sequence includes but is not limited to repetitions of (Gly-Gly-Gly-Gly-Ser).sub.n [as in SEQ ID No. 29] or (Gly-Ser).sub.n [as in SEQ ID No. 28].

[0053] In some embodiments, a polypeptide sequence encoding a transmembrane domain is fused to the immunoglobulin-binding domain in order to tether the immunoglobulin-capturing molecule on the cell surface. Preferably in this embodiment, the transmembrane domain is inert (lacking cell signaling functions) and not prone to internalization. Such a transmembrane domain could be an artificial sequence, or a motif derived from Major Histocompatibility Class I (MHC I), an IgSF molecule such as Lymphocyte-Activation Gene 3 (LAG3 or CD223), or any other transmembrane protein of any species--that is naturally inserted into the plasma membrane upon protein translation.

[0054] In other embodiments, the immunoglobulin-capturing molecule contains--in addition to the immunoglobulin-binding domain--a C-terminal peptide sequence for post-translational modification with, e.g., glycosylphosphatidylinositol (GPI), where GPI acts as a tether portion of the immunoglobulin-capturing molecule. GPI is a normal post-translational moiety that comprises a phosphoethanolamine group, a trimannosyl-nonacetylated glucosamine (Man.sub.3-GlcN) core, and a phosphatidylinositol group that tethers the protein to the plasma membrane. The phosphoethanolamine group of GPI is linked to a protein C-terminus via a phosphodiester bond. The GPI tether sequences may consist of the C-termini of proteins that are naturally anchored to the ASC plasma membrane by this post-translational process. Table 3 lists exemplary GPI tether or anchor sequences that may be used to construct the immunoglobulin-capturing molecule.

[0055] In certain embodiments, the immunoglobulin-capturing molecule contains a "stalk" structure for structural flexibility and support, as well as for increased exposure to the extracellular space. Since the cell surface is ubiquitously crowded with various molecules, the immunoglobulins captured on the immunoglobulin-capturing molecules may be occluded from access to their cognate antigen in the extracellular space by other molecules on the ASC surface. Thus, inclusion of a long stalk in the immunoglobulin-capturing molecule can alleviate any steric hindrance that compromises antigen binding by the displayed immunoglobulins. In preferred aspects of the invention, the stalk of the immunoglobulin-capturing molecule comprises one or more immunoglobulin domains derived from one or more IgSF proteins. Examples of these domains include but are not limited to the immunoglobulin domains of CD2, CD4, or CD22. Additionally, the stalk of the immunoglobulin-capturing molecule may be expressed as a macromolecular complex of two or more subunits. For example, the stalk of the ScFv-containing capture molecule may consist of C.sub.H2 and C.sub.H3 domains as well as the hinge region of an IgG molecule; thus, the immunoglobulin-capturing molecule is expressed as a homodimer.

Expression of the Immunoglobulin-Capturing Molecule

[0056] In certain aspects of the invention, expression of the immunoglobulin-capturing molecules is driven by a promoter derived from a gene that is highly expressed in ASCs but not in immature B cells or antigen-inexperienced mature B cells. These genes include but are not limited to B Lymphocyte-Induced Maturation Protein 1 (Blimp1), Syndecan 1 (Sdc1), Tumor Necrosis Factor Receptor Superfamily Member 17 (Tnfrsf17), and Fucosyltransferase 1 (Fut1). The gene chosen for ASC expression may be of mouse origin, or it may be from another species in which the gene shows an appropriately conserved expression pattern.

[0057] In certain other aspects, expression of the immunoglobulin-capturing molecules is driven by an inducible promoter, such as the tetracycline- or tamoxifen-inducible system. The inducible promoter is used to drive the expression of the immunoglobulin-capturing molecule either directly or indirectly via expression of a recombinase such as Cre (see, e.g., Albanese, et al., Seminars in Cell & Developmental Biology, 13:129-141 (2002); Sakai, Methods in Molecular Biology, 1142:33-40 (2014)). Such inducible expression in ASCs is accomplished either in the transgenic animal or in vitro during culture of ASCs as well as at the stage of hybridoma culture.

[0058] In order to express the immunoglobulin-capturing molecule on the cell surface, a signal peptide is included for protein translation in the endoplasmic reticulum. The signal peptide may be a consensus sequence or one that naturally exists as part of cell surface or secreted protein. In preferred aspects of the invention, the signal peptide is derived from that of an immunoglobulin heavy chain [as in SEQ ID Nos. 5-7] or light chain protein [as in SEQ ID Nos 1-3].

[0059] In some aspects, in addition to the immunoglobulin-capturing molecule, the expression vector may include an open-reading frame for a reporter protein such as GFP, red fluorescent protein (RFP), or the like. The reporter gene in the expression construct is linked to the immunoglobulin-capturing molecule via, e.g., an IRES sequence or a picornavirus 2A ribosomal skip sequence. Expression of the reporter gene allows for improved purity when used in combination with antigen selection to sort for antigen-specific ASCs.

[0060] Transgenes providing for expression of the immunoglobulin-capturing molecules are generated by inserting the coding sequences for the immunoglobulin-capturing molecules into a large piece of genomic DNA containing the gene that is highly expressed in ASCs (e.g, Blimp1 or Tnfrsf17). The insertion can be accomplished by homologous recombination mediated by sequences appended to the ends of the coding fragments, or by other standard molecular biology approaches. The large pieces of genomic DNA may be contained within bacterial artificial chromosome vectors, e.g., such as the pieces of DNA in these vectors that can be obtained from commercially or publicly available genomic DNA libraries.

[0061] Transgenic mice (or other animals) expressing the immunoglobulin-capturing molecules may be generated by any facility with the requisite skills using known techniques, as will be understood by one skilled in the art upon reading the present disclosure. Analysis of the animals carrying the transgene is performed using standard methodology such as immunofluorescence microscopy, flow cytometry and/or immunoblotting.

[0062] Illustrated in FIGS. 2A and 2B are the transgene (201) and expressed structure (202) of an immunoglobulin-capturing molecule according to one embodiment. The transgene (201) comprises two exons with an intervening intron (203) [SEQ ID Nos. 5-7]. The first exon and the beginning of the second exon encode a leader peptide (e.g., V.sub.H leader peptide). Contiguous with the leader peptide-encoding sequence are sequences encoding the following components: one or more immunoglobulin-binding domains (204) derived from one or more bacterial proteins [e.g., a sequence chosen from SEQ ID Nos. 8-11], a glycine/serine-rich linker (205) [e.g., a sequence chosen from SEQ ID Nos. 12 or 13], a "stalk" structure or region (206) [e.g., a sequence chosen from SEQ ID Nos. 14-16], and a transmembrane domain (207) [e.g., a sequence chosen from SEQ ID No. 17-20]. Following protein translation, the leader peptide is excised from the immunoglobulin-capturing molecule (202), which is expressed as a cell surface protein tethered to the plasma membrane (212). The respective components (208-211) of the immunoglobulin-capturing molecule (202) shown are immunoglobulin-binding domain(s) (208) [e.g., a sequence chosen from SEQ ID Nos. 24-27], glycine/serine-rich linker (209) [e.g., a sequence chosen from SEQ ID Nos. 28 or 29], stalk (210) [e.g., a sequence chosen from SEQ ID Nos. 30-32], and transmembrane domain (211) [e.g., a sequence chosen from SEQ ID Nos. 33-36].

[0063] Exemplary nucleic acid sequences for components of the immunoglobulin-capturing molecule illustrated in FIG. 2A (with the expressed structure illustrated in FIG. 2B) are listed in Table 1. The immunoglobulin-capturing molecule may be assembled by combining together one sequence of the several possible options for each component from Table 1 in the order depicted in FIG. 2A (i.e., from N-terminus to C-terminus). For example, a small immunoglobulin-capturing molecule may consist of only two immunoglobulin-binding domains of Protein G, a (glycine-serine).sub.3 linker, and a transmembrane domain without a stalk; while a larger one may contain five Protein A immunoglobulin-binding domains as well as four Protein G immunoglobulin-binding domains, a (Gly-Gly-Gly-Gly-Ser).sub.3 linker, a human CD22 stalk composed of six immunoglobulin folds, and a long human CD7 transmembrane domain.

[0064] An expression construct and the molecular structure of an alternative embodiment of an immunoglobulin-capturing molecule is illustrated in FIGS. 3A and 3B. In this embodiment of the invention, the transgene (301) similarly comprises a leader sequence encoded by two exons with an intervening intron (303), followed by sequences encoding the components of a scFv with specificity for a part of an immunoglobulin molecule (e.g., a conserved part of the heavy or light chain constant region): V.sub.H (304), glycine/serine-rich linker (305), and V.sub.L (306). For extended protrusion of the immunoglobulin-capturing molecule into the extracellular space, a sequence encoding a stalk comprising a hinge (307) as well as Fc fragment (308) of an immunoglobulin molecule is appended to the scFv-encoding sequence. Finally, one or more exons (309) encoding a transmembrane domain is also included in the expression construct (301). Shown in FIG. 3B is the immunoglobulin-capturing molecule (302) expressed as a homodimer of two subunits, each consisting of a V.sub.L (310) domain, glycine/serine-rich linker (311), and V.sub.H (312) domain of scFv connected to a hinge (313) and Fc (314) of an immunoglobulin molecule. The two subunits of the immunoglobulin-binding portion of the immunoglobulin-capturing molecule are covalently linked via disulfide bonds in the hinge region (313) of each chain. The expressed immunoglobulin-capturing molecule is tethered or anchored into the plasma membrane (316) by a transmembrane domain (315).

[0065] FIG. 4 illustrates the presentation of immunoglobulin-capturing molecules on a cell surface by an scFV embodiment of the immunoglobulin-capturing molecule. As demonstrated previously, an antibody-secreting cell (401) normally does not express the membrane-bound form of antigen receptors and lacks the ability to display on the cell surface the immunoglobulin molecules (402) they secrete. Expression of the scFv version of the immunoglobulin-capturing molecule (403) allows some of the immunoglobulin molecules (402) to be retained on the cell surface as they are being synthesized in the endoplasmic reticulum and subsequently packaged in the vesicles for secretion, or soon after their secretion. Antigen-specific ASCs are then identified by the binding of antigens (404) to the captured immunoglobulin molecules (402) on the cell surface. Detection of antigen binding on the ASCs is accomplished by using antigens that are directly labeled with a fluorophore or any other reporter molecule.

Transgenic Cell Libraries

[0066] The transgenic cells of the invention also are used to produce expression libraries, preferably low complexity libraries, for identification of antibodies of interest on the surface of ASCs. The present invention thus also includes antibody libraries produced using the cell technologies of the invention for identification of antigen-specific antibodies expressed on ASCs.

Transgenic Animals

[0067] The present invention also provides transgenic animals that have been modified to express immunoglobulin-capturing molecules on the cell surface of ASCs.

[0068] In preferred aspects, the transgenic animals of the invention further comprise human immunoglobulin regions. Numerous methods have been developed for replacing endogenous mouse immunoglobulin regions with human immunoglobulin sequences to create partially- or fully-human antibodies for drug discovery purposes. Examples of such mice include those described in, for example, U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669.

[0069] The exons that encode the antibody V.sub.H and V.sub.L domains do not exist in the germ-line DNA. Instead, each V.sub.H or V.sub.L exon is generated, respectively, by the recombination of randomly selected V, D, and J genes present in the H chain locus, or of randomly selected V and J genes in the light chain locus. There are multiple V, D, and J genes in the H chain locus as well as multiple V and J genes in each L chain locus, thus allowing for the generation of a vast antibody diversity repertoire per individual when the permutations of H chain VDJ rearrangements are combined with the permutations of L chain VJ gene rearrangements.

[0070] In particularly preferred aspects, the transgenic animals of the invention are as described in co-pending application US Pub. No. 2013/0219535, which is incorporated by reference in its entirety herein. Such transgenic animals have a genome comprising an introduced partially human immunoglobulin region, wherein the endogenous non-human V, D, and J gene coding sequences have been replaced with those of human origin without altering the endogenous noncoding sequences. Preferably, the transgenic cells and animals of the invention have genomes in which part or all of the endogenous immunoglobulin genes are removed.

[0071] In other aspects, the transgenic animals of the invention are avian, preferably chickens.

Use in Antibody Production

[0072] Culturing cells in vitro has been the basis of the production of numerous therapeutic biotechnology products, and involves the production of protein products in cells and release into the support medium. The quantity and quality of protein production over time from the cells growing in culture depends on a number of factors, such as, for example, cell density, cell cycle phase, cellular biosynthesis rates of the proteins, condition of the medium used to support cell viability and growth, and the longevity of the cells in culture. (See, for example, Fresney, Culture of Animal Cells, Wiley, Blackwell (2010); and Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Ozturk and Ha, Eds., CRC Press, (2006).)

[0073] For certain products, such as monoclonal antibodies, enhancing the presence and protein-expression efficiency of the cells that are actually producing the product is a key aspect of efficient protein production. Capturing antibodies on the surface of ASCs secreting them provides opportunities for discriminating ASCs on the basis of their immunoglobulin specificities, and this in turn provides opportunities for optimizing and enhancing the production of antibodies for various uses.

EXAMPLES

[0074] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0075] Efforts have been made to ensure accuracy with respect to terms and numbers used (e.g., vectors, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Example 1: Expression of a Minimal Protein G-Containing Membrane-Bound Immunoglobulin-Capturing Molecule

[0076] An expression vector encoding a small membrane-bound form of the immunoglobulin-capturing molecule without a stalk is generated by direct DNA synthesis or standard molecular cloning techniques. A diagram of the protein-coding part of this vector (501) is shown in FIG. 5A. The expression vector encodes two immunoglobulin-binding domains of streptococcal Protein G (504) [SEQ ID No. 8] that are tethered to the cell surface by means of a membrane-spanning domain derived the human LAG3 (or CD223) protein (506) [SEQ ID No. 17]. A fragment of DNA encoding a short linker consisting of Gly-Ser-Gly-Ser-Gly-Ser sequence (505) [SEQ ID No. 28] is placed between the DNA fragments encoding the Protein G immunoglobulin-binding domains (504) and the transmembrane domain (506) to provide structural flexibility to the expressed protein. Finally, a sequence encoding a signal peptide (leader peptide) (503) is included in the construct to allow for extrusion of the immunoglobulin-capturing molecule into the lumen of the endoplasmic reticulum during its biosynthesis. The signal peptide sequence in this example is derived from an immunoglobulin light chain variable (V.sub.L) gene segment which includes its native intron (503) [SEQ ID Nos. 1-3]. The promoter is indicated at (502). The nucleotide and amino acid sequences of various components comprising the immunoglobulin-capturing molecule in this example are specified in Table 1 and Table 2, respectively.

[0077] The expression vector is transfected into various myeloma, hybridoma or other cell lines using commonly accessible methodology such as electroporation. The transfected cells are then examined for surface expression of the immunoglobulin-capturing molecule using procedures such as immunofluorescence microscopy, flow cytometry, and/or immunoblotting of the membrane protein fractions. The cells are further analyzed using a subset of these procedures for the capacity of the cell surface immunoglobulin-capturing molecules to retain immunoglobulins produced by the transfected cells or added to them.

[0078] FIG. 5B illustrates the expression of this small immunoglobulin-capturing molecule in a plasmacytoma cell line and its ability to retain immunoglobulin molecules on the cell surface. Human RPMI 8226 (ATCC.RTM. CCL-155.TM.) cells were transfected with DNA plasmids encoding the immunoglobulin-capturing molecule (501) under control of the Blimp1 promoter (502). The cells were also co-transfected with a plasmid encoding mouse IgG. Compared to untransfected cells (top, 507), the transfected cells (bottom, 508) exhibit captured immunoglobulins on the cell surface.

[0079] Transgenic animals are then generated to express the membrane-bound immunoglobulin-capturing molecules containing Protein G on the ASCs, and the capacity of the transgene-encoded molecules to capture immunoglobulins on ASCs is determined directly on the ASCs taken from the transgenic mice by standard flow cytometry.

Example 2: Expression of a Protein G-Containing Membrane-Bound Immunoglobulin-Capturing Molecule Containing a Stalk

[0080] An expression vector encoding a membrane-bound form of the immunoglobulin-capturing molecule containing a long stalk is generated by direct DNA synthesis or standard molecular cloning techniques. The expression vector encodes three immunoglobulin-binding domains derived from the C-terminal half of streptococcal Protein G [SEQ ID No. 9]. DNA fragments encoding a short linker consisting of Gly-Ser-Gly-Ser-Gly-Ser [SEQ ID No. 28] sequence, a stalk consisting of six immunoglobulin domains derived from human CD22 protein [SEQ ID No. 16], and a transmembrane domain derived from human CD58 [SEQ ID No. 18] are appended to the immunoglobulin-binding domain-encoding DNA fragment of the vector. Finally, a sequence encoding a signal peptide (leader peptide) is placed preceding the entire open-reading frame of the immunoglobulin-capturing molecule to allow for extrusion of the translated protein into the lumen of the endoplasmic reticulum during its biosynthesis. The sequences encoding the signal peptide in this example are derived from an immunoglobulin heavy chain variable (V.sub.H) gene segment and include its native intron [SEQ ID Nos. 5-7]. The nucleotide and amino acid sequences of components comprising the immunoglobulin-capturing molecule in this example are specified in Table 1 and Table 2, respectively.

[0081] The expression vector is transfected into various myeloma, hybridoma or other cell lines using commonly accessible methodology such as electroporation. The transfected cells are then examined for surface expression of the Protein G molecule using procedures such as immunofluorescence microscopy, flow cytometry and immunoblotting of the cell membrane protein fractions. The cells are further analyzed using a subset of these procedures for the capacity of the cell surface Protein G to capture immunoglobulins produced by the transfected cells or added to them.

[0082] Transgenic animals are then generated to express the membrane-bound immunoglobulin-capturing molecules consisting of Protein G, CD22 and CD58 fusion in the ASCs, and the capacity of the transgene-encoded molecules to capture immunoglobulins on ASCs is determined directly on the ASCs taken from the transgenic mice by standard flow cytometry.

Example 3: Expression of a Protein G-Containing Immunoglobulin-Capturing Molecule Anchored to the Membrane by a GPI Post-Translational Modification

[0083] An expression vector encoding two immunoglobulin-binding domains derived from the streptococcal Protein G is synthesized. Included in this expression vector downstream of the Protein G-encoding sequence are DNA fragments that encode the following: a Gly/Ser-rich linker sequence, a stalk consisting of two immunoglobulin domains of human CD4, and a GPI anchor sequence. Finally, a signal peptide sequence (leader sequence) is included in the construct to allow for extrusion of the translated protein into the lumen of the endoplasmic reticulum during its biosynthesis. The sequences encoding the signal peptide in this example are derived from an immunoglobulin light chain variable (V.sub.L) gene segment and include its native intron. The nucleotide and amino acid sequences of components comprising the immunoglobulin-capturing molecule in this example are specified in Table 1 and Table 2, respectively. The GPI anchor sequences are specified in Table 3.

[0084] The expression vector is transfected into various myeloma, hybridoma or other cell lines using commonly accessible methodology such as electroporation. The transfected cells are then examined for surface expression of the Protein G molecule using procedures such as immunofluorescence microscopy, flow cytometry and immunoblotting of the cell membrane protein fractions. The cells are further analyzed using a subset of these procedures for the capacity of the cell surface Protein G to capture immunoglobulins produced by the transfected cells or added to them.

[0085] Transgenic animals are then generated to express the GPI-anchored immunoglobulin-capturing molecules in ASCs, and the capacity of the transgene-encoded molecules to capture immunoglobulins on the surface of ASCs is determined directly on the ASCs taken from the transgenic mice by standard flow cytometry.

Example 4: Expression of a Membrane-Bound scFv Derived from an Antibody Specific for an Immunoglobulin Constant Region

[0086] An expression vector encoding a scFv specific for the constant domain of an immunoglobulin is generated by standard molecular cloning or direct DNA synthesis. In this example, the single-chain antibody is specific for the constant domain of the mouse kappa light chain, which is present in more than 90% of antibodies found in normal mice. The exon encoding the scFv comprises V.sub.L, linker, and V.sub.H sequences specified at [SEQ ID Nos. 43-48, respectively]. Included in this expression vector downstream of the ScFv-encoding sequence is a contiguous sequence that encodes the Fc part of rat IgG1 consisting of the following: a hinge region, C.sub.H2 domain, and C.sub.H3 domain of the secreted form. The rat IgG1 Fc-encoding sequence is specified at [SEQ ID Nos. 49]. The vector also includes sequences encoding the transmembrane domain of a mouse Major Histocompatibility Complex Class I protein (the mouse K molecule from the b haplotype), specified at [SEQ ID Nos. 50-54].

[0087] The expression vector is transfected into various myeloma, hybridoma and other cell lines using commonly accessible methodology such as electroporation. The transfected cells are examined for surface expression of the single chain antibody molecule using procedures such as immunofluorescence microscopy, flow cytometry and immunoblotting of the cell membrane protein fractions. The cells are further analyzed using a subset of these procedures for the capacity of the cell surface single chain antibody molecule to capture immunoglobulins produced by the transfected cells or added to them.

[0088] Transgenic animals are generated to express the scFv-containing immunoglobulin-capturing molecules on ASCs, and the capacity of the transgene-encoded molecules to capture immunoglobulins on the surface of ASCs is determined by standard flow cytometry directly on the ASCs taken from the mice.

Example 5: Use of Transgenic Animal Expressing Immunoglobulin-Capturing Molecules to Isolate ASCs Producing Monoclonal Antibodies Against Antigen of Interest

[0089] Transgenic mice are generated using a bacterial artificial chromosome vector containing the promoter of human TNFRSF17 gene, the coding sequence of an immunoglobulin-capturing molecule, for example as in Examples 1-4, an IRES sequence, and GFP. Spleen, lymph nodes, and bone marrow from several transgenic founder lines are harvested, processed, and analyzed for the expression of GFP as well as the immunoglobulin-capturing molecule by standard flow cytometry. GFP-positive cells from the transgenic mice are then pooled, sorted, and verified by enzyme-linked immunospot (ELISPOT) for their ability to secrete immunoglobulins. A transgenic line that stably expresses detectable levels of GFP and the immunoglobulin-capturing molecule is selected for propagation.

[0090] Adult transgenic mice are immunized with an antigen of interest. Spleens as well as the relevant lymph nodes are isolated from the immunized mice, processed, and stained for flow cytometric analyses. Additionally, the isolated cells are subjected to antigen binding during the flow cytometric staining. The antigen is either directly labeled with a fluorophore or with biotin for use with a labeled avidin, streptavidin, or similar system. ASCs are sorted on the basis of GFP-positive staining as well as antigen-positive staining.

[0091] The purified ASCs are then fused to myeloma cells to generate hybridoma cells using established methodologies familiar to those with ordinary skill in the art. In this invention, the provided methods to express the immunoglobulin-capturing molecules also allow for the screening of hybridoma cells based on GFP expression as well as positive staining of antigens captured on the cell surfaces.

[0092] Alternatively, the purified ASCs are individually sorted, and genes encoding their V.sub.H and V.sub.L domains are cloned via RT-PCR or 5' RACE techniques adapted for single cells. The cloned V.sub.H- and V.sub.L-coding sequences are subcloned into an expression vector containing a sequence encoding the desired constant regions of heavy chain and light chain, respectively. The V.sub.H- and V.sub.L-expression vectors are transfected into a HEK-293T or CHO cell lines, and the secreted monoclonal antibodies are further tested for antigen binding and other functions.

TABLE-US-00001 TABLE 1 exemplary nucleic acid sequences SEQ Corresponding ID No. Description Structure Sequence 1 Leader FIG. 2A, 203 ATGGACATCAGGGCTCCTGCTCAGTTTCTTGGCATC Exon 1/Intron/ FIG. 3A, 303 TTGTTGCTCTGGTTTCCAG Exon 2/Tags FIG. 5A, 503 V.sub.L Leader Exon 1 2 Leader FIG. 2A, 203 GTAAAATGAACTAAAATGGGAATTTCACTGTAAGTG Exon1/Intron/ FIG. 3A, 303 TTGACAGGCATTTGGGGACTGTGTTCTTTTATCATG Exon 2/Tags FIG. 5A, 503 CTTACCTTTGTAGATATTCATTATGTCTCCACTCCT V.sub.L intron AG 3 Leader FIG. 2A, 203 GTGCCAGATGTGACATCCAGATG Exon1/Intron/ FIG. 3A, 303 Exon 2/Tags FIG. 5A, 503 V.sub.L Leader Exon 2 4 Leader FIG. 2A, 203 GACTACAAGGATGACGACGACAAGGGCAGCGGCGAA Exon1/Intron/ FIG. 3A, 303 CAGAAGCTGATTTCGGAGGAGGACCTG Exon 2/Tags FIG. 5A, 503 FLAG + Myc Tags 5 Leader FIG. 2A, 203 ATGGGATGGAGCTGTATCATGCTCTTCTTGGCAGCA Exon 1/Intron/ FIG. 3A, 303 ACAGCTACAG Exon 2/Tags FIG. 5A, 503 V.sub.H Leader Exon 1 6 Leader FIG. 2A, 203 GTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGAC Exon 1/Intron/ FIG. 3A, 303 ATATACATGGGTGACAATGACATCCACTTTGCCTTT Exon 2/Tags FIG. 5A, 503 CTCTCCACAG V.sub.H Intron 7 Leader FIG. 2A, 203 GTGTCCACTCCCAGGTCCAACTG Exon 1/Intron/ FIG. 3A, 303 Exon 2/Tags FIG. 5A, 503 V.sub.H 8 Ig-Binding FIG. 2A, 204 GGTACCCCAGCCGTGACCACCTACAAGCTCGTCATC Domain FIG. 5A, 504 AACGGAAAGACGCTCAAGGGCGAAACCACTACCAAG 2-Domain GCGGTGGATGCCGAAACCGCCGAAAAGGCCTTCAAG Protein G CAGTACGCTAACGACAATGGGGTGGACGGAGTCTGG ACGTACGATGATGCCACCAAGACTTTCACCGTGACC GAAGTGAACACTCCGGCCGTCACCACTTATAAGCTC GTGATCAACGGGAAAACCCTGAAGGGAGAGACTACC ACAAAGGCCGTGGATGCTGAGACTGCAGAGAAGGCG TTCAAACAGTACGCCAACGACAACGGCGTGGACGGC GTCTGGACCTACGATGACGCCACTAAGACCTTCACT GTGACCGAA 9 Ig-Binding FIG. 2A, 204 ATAGATGAAATTTTAGCTGCATTACCTAAGACTGAC Domain FIG. 5A, 504 ACTTACAAATTAATCCTTAATGGTAAAACATTGAAA 3-Domain GGCGAAACAACTACTGAAGCTGTTGATGCTGCTACT Protein G GCAGAAAAAGTCTTCAAACAATACGCTAACGACAAC GGTGTTGACGGTGAATGGACTTACGACGATGCGACT AAGACCTTTACAGTTACTGAAAAACCAGAAGTGATC GATGCGTCTGAATTAACACCAGCCGTGACAACTTAC AAACTTGTTATTAATGGTAAAACATTGAAAGGCGAA ACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAA AAAGTCTTCAAACAATACGCTAACGACAACGGTGTT GACGGTGAATGGACTTACGACGATGCGACTAAGACC TTTACAGTTACTGAAAAACCAGAAGTGATCGATGCG TCTGAATTAACACCAGCCGTGACAACTTACAAACTT GTTATTAATGGTAAAACATTGAAAGGCGAAACAACT ACTAAAGCAGTAGACGCAGAAACTGCAGAAAAAGCC TTCAAACAATACGCTAACGACAACGGTGTTGATGGT GTTTGGACTTATGATGATGCGACTAAGACCTTTACG GTAACTGAA 10 Ig-Binding FIG. 2A, 204 GTGGATAACAAGTTCAACAAGGAACAGCAGAACGCC Domain FIG. 5A, 504 TTTTACGAGATTCTGCATCTGCCCAACCTGAATGAG 2-Domain GAACAGCGGAACGCATTCATTCAGTCTCTGAAGGAT Protein A + 2- GATCCTAGCCAGTCGGCCAACCTCCTGGCTGAAGCA Domain Protein AAGAAGCTGAACGATGCCCAAGCGCCCAAAGTGGAC G AACAAGTTTAACAAGGAGCAGCAGAATGCTTTCTAC GAGATCCTGCACCTCCCGAATCTGAACGAGGAGCAG AGAAACGCCTTCATCCAATCACTGAAGGACGACCCG TCACAGTCCGCCAACCTTCTGGCGGAAGCCAAGAAA CTGAACGACGCCCAGGCGCCAAAGGTGGACGGATCC GGGTCCGGCAGCGGTACCCCAGCCGTGACCACCTAC AAGCTCGTCATCAACGGAAAGACGCTCAAGGGCGAA ACCACTACCAAGGCGGTGGATGCCGAAACCGCCGAA AAGGCCTTCAAGCAGTACGCTAACGACAATGGGGTG GACGGAGTCTGGACGTACGATGATGCCACCAAGACT TTCACCGTGACCGAAGTGAACACTCCGGCCGTCACC ACTTATAAGCTCGTGATCAACGGGAAAACCCTGAAG GGAGAGACTACCACAAAGGCCGTGGATGCTGAGACT GCAGAGAAGGCGTTCAAACAGTACGCCAACGACAAC GGCGTGGACGGCGTCTGGACCTACGATGACGCCACT AAGACCTTCACTGTGACCGAA 11 Ig-Binding FIG. 2A, 204 GCCAATGCCGCCCAGCACGACGAGGCTCAGCAGAAC Domain FIG. 5A, 504 GCATTCTACCAGGTGCTGAACATGCCAAACCTCAAC 5-Domain GCCGATCAGCGCAATGGTTTCATTCAGTCCCTGAAG Protein A + 4- GACGATCCGAGCCAGTCAGCTAACGTGCTCGGGGAG Domain Protein GCCCAAAAGCTGAATGACTCCCAGGCGCCGAAGGCC G GACGCCCAGCAAAACAACTTCAACAAGGATCAGCAA TCCGCCTTCTATGAAATCCTGAATATGCCTAACCTG AACGAAGCTCAGCGGAACGGGTTCATCCAGAGCCTT AAGGACGACCCTAGCCAGTCCACCAACGTGCTGGGG GAGGCCAAGAAACTTAACGAATCCCAGGCCCCGAAG GCGGACAACAACTTTAACAAGGAACAGCAGAACGCC TTTTACGAGATCCTCAACATGCCGAACCTCAACGAG GAACAGCGCAACGGTTTCATCCAGTCCCTGAAGGAC GATCCATCCCAGTCCGCCAACCTGTTGAGCGAGGCG AAGAAGCTGAATGAGTCCCAAGCCCCCAAGGCTGAC AACAAGTTCAATAAGGAACAACAGAATGCCTTCTAC GAAATTCTGCACTTGCCCAATCTGAACGAGGAGCAG CGCAACGGCTTCATCCAATCTCTGAAAGACGACCCG TCGCAGTCGGCCAACTTGCTGGCCGAAGCCAAGAAG CTCAACGACGCTCAGGCCCCTAAGGCCGACAACAAG TTCAACAAAGAGCAACAGAACGCGTTCTACGAGATT CTCCACTTGCCGAACCTGACCGAAGAACAACGGAAC GGATTCATTCAGAGCCTGAAGGATGACCCTTCGGTG TCAAAGGAGATCCTGGCAGAAGCCAAAAAGCTGAAC GATGCCCAGGCACCAAAGGAAGAGGACAACAACAAG CCGGGCGACCCGAGGATCTCCGAAGCCACTGATGGG CTGTCCGATTTTCTGAAGTCACAGACTCCTGCTGAG GACACCGTGAAGTCCATCGAGCTCGCCGAGGCCAAG GTGCTGGCCAACCGGGAGCTGGATAAGTACGGAGTG TCCGACTACTACAAAAACCTGATTAACAACGCCAAG ACTGTGGAAGGAGTGAAGGCATTGATCGATGAAATC CTGGCGGCGCTCCCAAAAACCGACACCTACAAACTG ATTCTCAACGGAAAGACGCTGAAGGGGGAAACTACC ACCGAAGCGGTGGACGCCGCCACCGCCGAAAAGGTG TTTAAGCAGTATGCTAACGACAACGGTGTCGACGGA GAGTGGACCTACGACGACGCCACTAAGACTTTCACC GTGACCGAGAAGCCCGAGGTCATCGACGCGAGCGAG CTCACTCCCGCCGTGACCACCTACAAGCTGGTCATC AATGGAAAGACTCTGAAGGGCGAAACTACTACTGAA GCCGTGGATGCGGCAACCGCCGAGAAAGTGTTCAAG CAATACGCAAACGATAACGGGGTGGACGGAGAGTGG ACCTACGACGATGCCACAAAGACCTTCACCGTCACC GAAAAGCCCGAAGTGATCGACGCTTCCGAACTGACG CCGGCCGTGACAACTTACAAGCTCGTCATTAACGGA AAGACCCTTAAGGGCGAAACCACGACCAAGGCAGTG GACGCCGAAACTGCCGAGAAGGCGTTCAAGCAGTAC GCCAACGACAACGGCGTGGACGGAGTGTGGACTTAC GATGATGCGACCAAGACGTTCACTGTGACCGAGATG GTCACCGAAGTGCCG 12 Gly/Ser Linker FIG. 2A, 205 GGATCCGGCTCCGGATCC FIG. 3A, 305 FIG. 5A, 505 13 Gly/Ser Linker FIG. 2A, 205 GGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGC FIG. 3A, 305 GGAGGCTCG FIG. 5A, 505 14 Stalk FIG. 2A, 206 GAGATGGTGTCCAAGCCGATGATCTACTGGGAGTGT Rat CD2 FIG. 5A, 506 TCCAACGCGACTCTGACCTGTGAAGTGCTGGAGGGA ACCGACGTGGAACTGAAGCTGTACCAGGGTAAAGAA CATCTGCGGTCGTTGCGCCAAAAGACCATGAGCTAC CAGTGGACCAACTTGCGGGCGCCTTTCAAGTGCAAA GCCGTCAATAGAGTGTCCCAGGAGAGCGAAATGGAG GTCGTGAACTGCCCCGAAAAGGGACTG 15 Stalk FIG. 2A, 206 TCAACTTCCATCACCGCCTACAAGAGCGAGGGAGAG Rat CD4 FIG. 5A, 506 AGCGCCGAGTTTTCCTTCCCCCTGAACCTGGGCGAA GAAAGCCTCCAGGGAGAACTGCGCTGGAAGGCAGAA AAGGCCCCAAGCTCTCAGTCCTGGATCACCTTCAGC CTGAAGAACCAGAAGGTGTCCGTGCAGAAGTCCACT TCAAACCCGAAGTTCCAGCTCTCCGAAACCCTCCCT CTGACCCTGCAAATCCCTCAAGTGTCGCTGCAATTC GCGGGGAGCGGAAATCTGACTCTGACTCTTGACCGG GGCATCTTGTACCAGGAGGTGAACCTGGTGGTCATG AAGGTGACCCAGCCCGATAGCAACACCCTGACCTGT GAAGTGATGGGACCCACGTCCCCGAAGATGCGGCTC ATTCTGAAGCAGGAGAACCAGGAGGCTCGGGTGTCC AGACAGGAAAAGGTCATCCAAGTGCAGGCCCCGGAA GCCGGCGTGTGGCAGTGCCTGCTGTCCGAGGGAGAG GAAGTCAAGATGGACTCGAAAATCCAGGTGCTGTCC AAAGGGCTGAACCAGACTATG 16 Stalk FIG. 2A, 206 GAAAGGCCTTTTCCACCTCATATCCAGCTCCCTCCA Human CD22 FIG. 5A, 506 GAAATTCAAGAGTCCCAGGAAGTCACTCTGACCTGC TTGCTGAATTTCTCCTGCTATGGGTATCCGATCCAA TTGCAGTGGCTCCTAGAGGGGGTTCCAATGAGGCAG GCTGCTGTCACCTCGACCTCCTTGACCATCAAGTCT GTCTTCACCCGGAGCGAGCTCAAGTTCTCCCCACAG TGGAGTCACCATGGGAAGATTGTGACCTGCCAGCTT CAGGATGCAGATGGGAAGTTCCTCTCCAATGACACG GTGCAGCTGAACGTGAAGCACACCCCGAAGTTGGAG ATCAAGGTCACTCCCAGTGATGCCATAGTGAGGGAG GGGGACTCTGTGACCATGACCTGCGAGGTCAGCAGC AGCAACCCGGAGTACACGACGGTATCCTGGCTCAAG GATGGGACCTCGCTGAAGAAGCAGAATACATTCACG CTAAACCTGCGCGAAGTGACCAAGGACCAGAGTGGG AAGTACTGCTGTCAGGTCTCCAATGACGTGGGCCCG GGAAGGTCGGAAGAAGTGTTCCTGCAAGTGCAGTAT GCCCCGGAACCTTCCACGGTTCAGATCCTCCACTCA CCGGCTGTGGAGGGAAGTCAAGTCGAGTTTCTTTGC ATGTCACTGGCCAATCCTCTTCCAACAAATTACACG TGGTACCACAATGGGAAAGAAATGCAGGGAAGGACA GAGGAGAAAGTCCACATCCCAAAGATCCTCCCCTGG CACGCTGGGACTTATTCCTGTGTGGCAGAAAACATT CTTGGTACTGGACAGAGGGGCCCGGGAGCTGAGCTG GATGTCCAGTATCCTCCCAAGAAGGTGACCACAGTG ATTCAAAACCCCATGCCGATCGAGAAGGAGACACAG TGACCCTTTCCTGTAACTACAATTCCAGTAACCCCA GTGTTACCCGGTATGAATGGAAACCCCATGGCGCCT GGGAGGAGCCATCGCTTGGGGTGCTGAAGATCCAAA ACGTTGGCTGGGACAACACAACCATCGCCTGCGCAG CTTGTAATAGTTGGTGCTCGTGGGCCTCCCCTGTCG CCCTGAATGTCCAGTATGCCCCCCGAGACGTGAGGG TCCGGAAAATCAAGCCCCTTTCCGAGATTCACTCTG GAAACTCGGTCAGCCTCCAATGTGACTTCTCAAGCA GCCACCCCAAAGAAGTCCAGTTCTTCTGGGAGAAAA ATGGCAGGCTTCTGGGGAAAGAAAGCCAGCTGAATT TTGACTCCATCTCCCCAGAAGATGCTGGGAGTTACA GCTGCTGGGTGAACAACTCCATAGGACAGACAGCGT CCAAGGCCTGGACACTTGAAGTGCTGTATGCACCCA GGAGGCTGCGTGTGTCCATGAGCCCGGGGGACCAAG TGATGGAGGGGAAGAGTGCAACCCTGACCTGTGAGA GCGACGCCAACCCTCCCGTCTCCCACTACACCTGGT TTGACTGGAATAACCAAAGCCTCCCCTACCACAGCC AGAAGCTGAGATTGGAGCCGGTGAAGGTCCAGCACT CGGGTGCCTACTGGTGCCAGGGGACCAACAGTGTGG GCAAGGGCCGTTCGCCTCTCAGCACCCTCACCGTCT ACTATAGCCCGGAGACC 17 Transmembrane FIG. 2A, 207 GCGCCTGGAGCGCTGCCGGCCGGTCATCTGTTGTTG Domain FIG. 5A, 507 TTCCTGACCCTGGGGGTGCTGTCACTGCTGCTGCTC Human LAG3 GTGACCGGGGCATTCGGTTTCCACCTGTGGAGAAGG CAGTGGCGGTAG 18 Transmembrane FIG. 2A, 207 CATTCCCGGCACCGCTACGCGCTGATTCCGATTCCT Domain FIG. 5A, 507 CTGGCCGTGATCACCACCTGTATCGTGCTCTACATG Human CD58 AACGGTATCCTGAAATGCGACAGAAAGCCCGACAGG ACTAACAGCAATTAG 19 Transmembrane FIG. 2A, 207 CCGCTGTACCTGATCGTGGGGGTGTCAGCCGGCGGT Domain FIG. 5A, 507 CTGCTGCTCGTGTTCTTCGGGGCACTGTTCATCTTC Rat CD2 TGCATTTGCAAGAGGAAGAAGCGGTAG 20 Transmembrane FIG. 2A, 207 CCACCCCGGGCGTCCGCACTGCCGGCGCCCCCTACC Domain FIG. 5A, 507 GGAAGCGCGCTGCCCGATCCGCAAACCGCCAGCGCC Human CD7 CTGCCTGACCCGCCCGCGGCTAGCGCCTTGCCTGCC GCACTGGCCGTGATTTCATTCCTGCTGGGTCTGGGG CTCGGGGTGGCCTGCGTGTTGGCACGGACTCAGATC AAGAAGCTGTGCTCCTGGAGAGACAAAAACTCCGCC GCCTGTGTGGTGTACGAGGACATGTCACACTCGAGG TGCAATACCCTGTCCTCGCCGAACCAGTACCAGTAG

TABLE-US-00002 TABLE 2 exemplary peptide sequences SEQ Corresponding ID No. Description Structure Sequence 21 Leader MGWSCIMLFLAATATGVHSQVQL Exon1/Intron/ Exon 2/Tags V.sub.L Leader Exon 22 Leader DYKDDDDKGSGEQKLISEEDL Exon1/Intron/ Exon 2/Tags FLAG + Myc Tags 23 Leader MDIRAPAQFLGILLLWFPGARCDIQM Exon1/Intron/ Exon 2/Tags V.sub.H Leader Exon 24 Ig-Binding FIG. 2B, 208 GTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFK Domain QYANDNGVDGVWTYDDATKTFTVTEVNTPAVTTYKL 2-Domain VINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDG Protein G VWTYDDATKTFTVTE 25 Ig-Binding FIG. 2B, 208 IDEILAALPKTDTYKLILNGKTLKGETTTEAVDAAT Domain AEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVI 3-Domain DASELTPAVTTYKLVINGKTLKGETTTEAVDAATAE Protein G KVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDA SELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKA FKQYANDNGVDGVWTYDDATKTFTVTE 26 Ig-Binding FIG. 2B, 208 VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD Domain DPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFY 2-Domain EILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKK Protein A + 2- LNDAQAPKVDGSGSGSGTPAVTTYKLVINGKTLKGE Domain Protein TTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKT G FTVTEVNTPAVTTYKLVINGKTLKGETTTKAVDAET AEKAFKQYANDNGVDGVWTYDDATKTFTVTE 27 Ig-Binding FIG. 2B, 208 ANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLK Domain DDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQ 5-Domain SAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLG Protein A + 4- EAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNE Domain Protein EQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKAD G NKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDP SQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEI LHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLN DAQAPKEEDNNKPGDPRISEATDGLSDFLKSQTPAE DTVKSIELAEAKVLANRELDKYGVSDYYKNLINNAK TVEGVKALIDEILAALPKTDTYKLILNGKTLKGETT TEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT VTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTE AVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVT EKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAV DAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEM VTEVP 28 Gly/Ser Linker FIG. 2B, 209 GSGSGS 29 Gly/Ser Linker FIG. 2B, 209 GGGGSGGGGSGGGGS 30 Stalk FIG. 2B, 210 EMVSKPMIYWECSNATLTCEVLEGTDVELKLYQGKE Rat CD2 HLRSLRQKTMSYQWTNLRAPFKCKAVNRVSQESEME VVNCPEKGL 31 Stalk FIG. 2B, 210 STSITAYKSEGESAEFSFPLNLGEESLQGELRWKAE Rat CD4 KAPSSQSWITFSLKNQKVSVQKSTSNPKFQLSETLP LTLQIPQVSLQFAGSGNLTLTLDRGILYQEVNLVVM KVTQPDSNTLTCEVMGPTSPKMRLILKQENQEARVS RQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVLS KGLNQTM 32 Stalk FIG. 2B, 210 MKVTQPDSNTLTCEVMGPTSPKMRLILKQENQEARV Human CD22 SRQEKVIQVQAPEAGVWQCLLSEGEEVKMDSKIQVL SKGLNQTM 33 Transmembrane FIG. 2B, 211 APGALPAGHLLLFLTLGVLSLLLLVTGAFGFHLWRR Domain QWR Human LAG3 34 Transmembrane FIG. 2B, 211 HSRHRYALIPIPLAVITTCIVLYMNGILKCDRKPDR Domain TNSN Human CD58 35 Transmembrane FIG. 2B, 211 PLYLIVGVSAGGLLLVFFGALFIFCICKRKKR Domain Rat CD2 36 Transmembrane FIG. 2B, 211 PPRASALPAPPTGSALPDPQTASALPDPPAASALPA Domain ALAVISFLLGLGLGVACVLARTQIKKLCSWRDKNSA Human CD7 ACVVYEDMSHSRCNTLSSPNQYQ

TABLE-US-00003 TABLE 3 GPI anchor sequences SEQ ID No. Description Sequence 37 Human CD59 GAATTCCTTGAAAATGGTGGGACATCCTTATCAGAGAAAACAGTT CTTCTGCTGGTGACTCCATTTCTGGCAGCAGCCTGGAGCCTTCAT CCC 38 Human CD59 EFLENGGTSLSEKTVLLLVTPFLAAAWSLHP 39 Human CD24 ACCAATGCCACAACAAAGGCAGCAGGGGGAGCACTCCAGTCAACA GCAAGTTTGTTTGTCGTGTCACTGAGTCTCTTGCATCTTTATTCA 40 Human CD24 TNATTKAAGGALQSTASLFVVSLSLLHLYS 41 Human CNTN1 GTCTCCCAGGTGAAAATTTCAGGAGCCCCTACCCTCTCCCCATCC (Contactin 1) CTCCTGGGTTTGCTGCTGCCCGCCTTTGGCATTCTCGTGTATCTG GAGTTC 42 Human CNTN1 VSQVKISGAPTLSPSLLGLLLPAFGILVYLEF (Contactin 1)

TABLE-US-00004 TABLE 4 Example 4 sequences SEQ ID No. Description Sequence 43 V.sub.L Leader exon 1 ATGGAATCACAGACCCAGGTCCTCATGTTTCTTCTGCTCTGGGT ATCTG 44 V.sub.L intron GTAAGAAATTTAAAGTATTAAAACCTTTTCAAAGTTTCATCTTT GTGGTAAGAAATTTGCAATATGTGCCAGTGTGTAATATTTCTTA CATAATAAATTTGTGACAGTATGATAAGGACATTTAAATGAAAA ATTTCGACTGTTGTTATAATCTATGTCTGTGTATCTATGAATTT TCACTGCCTATTAATTATTACAG 45 V.sub.L exon 2 end GTGCCTGTGCA of VL leader sequence 46 VL exon 2 GACATTCAGATGACCCAGTCTCCATCCTCCATGTCTGTGTCTCT GGGAGACACAGTCACTATTACTTGCCGGGCAAGTCAGGACGTTG GGATTTATGTAAACTGGTTCCAGCAGAAACCAGGGAAATCTCCT AGGCGTATGATTTATCGTGCAACGAACTTGGCAGATGGGGTCCC ATCAAGGTTCAGCGGCAGTAGGTCTGGATCAGATTATTCTCTCA CCATCAGCAGCCTGGAGTCTGAAGATGTGGCAGACTATCACTGT CTACAGTATGATGAGTATCCATTCACGTTCGGATCCGGGACGAA GTTGGAAATAAAACGG 47 V.sub.L exon 2 linker GGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTC G 48 V.sub.H exon 2 CAGGTACAGCTGAAAGAGTCAGGACCTGGTCTGGTGCAGCCCTC ACAGACCCTGTCTCTCACCTGCACTGTCTCTGGACTCTCATTAA TCAGTTATGGTGTAAGTTGGGCTCGCCAGCCTCCAGGGAAGGGT CTGGAGTGGATTGCAGCAATATCAAGTGGTGGAAGCACATATTA TAATTCAGTTCTCACATCTCGACTGAGCATCAGCAGGGACACCT CCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAA GACACAGCCATTTACTTCTGTACCAGAGAACTCTGGGACTACTA TGATTACTGGGGCCAAGGAGTCATGGTCACAGTCTCCTCA 49 Exon 2-Rat GCTGAAACAACAGCCCCCAGAAACCCGGGAGGTGATTGCAAGCC IgG1 Fc TTGTATATGTACAGGCTCAGAAGTATCATCTGTCTTCATCTTCC CCCCAAAGCCCAAAGATGTGCTCACCATCACTCTGACTCCTAAG GTCACGTGTGTTGTGGTAGACATTAGCCAGGACGATCCCGAGGT CCATTTCAGCTGGTTTGTAGATGACGTGGAAGTCCACACAGCTC AGACTCGACCACCAGAGGAGCAGTTCAACAGCACTTTCCGCTCA GTCAGTGAACTCCCCATCCTGCACCAGGACTGGCTCAATGGCAG GACGTTCAGATGCAAGGTCACCAGTGCAGCTTTCCCATCCCCCA TCGAGAAAACCATCTCCAAACCCGAAGGCAGAACACAAGTTCCG CATGTATACACCATGTCACCTACCAAGGAAGAGATGACCCAGAA TGAAGTCAGTATCACCTGCATGGTAAAAGGCTTCTATCCCCCAG ACATTTATGTGGAGTGGCAGATGAACGGGCAGCCACAGGAAAAC TACAAGAACACTCCACCTACGATGGACACAGATGGGAGTTACTT CCTCTACAGCAAGCTCAATGTGAAGAAGGAAAAATGGCAGCAGG GAAACACGTTCACGTGTTCTGTGCTGCATGAAGGCCTGCACAAC CACCATACTGAGAAGAGTCTCTCCCACTCCCCCGGT 50 Exon 2 part of AAAGAGCCTCCTCCATCCACTGTCTCCAACATGGCGACCGTTGC mouse MHC I TGTTCTGGTTGTCCTTGGAGCTGCAATAGTCACTGGAGCTGTGG (H2K.sup.b) TGGCTTTTGTGATGAAGATGAGAAGGAGAAACACAG transmembrane domain 51 Intron GTAGGAAAGGGCAGAGTCTGAGTTTTCTCTCAGCCTCCTTTAGA GTGTGCTCTGCTCATCAATGGGGAACACAGGCACACCCCACATT GCTACTGTCTCTAACTGGGTCTGCTGTCAGTTCTGGGAACTTCC TAGTGTCAAGATCTTCCTGGAACTCTCACAGCTTTTCTTCTCAC AG 52 Exon 3-part of GTGGAAAAGGAGGGGACTATGCTCTGGCTCCAG mouse MHC I (H2K.sup.b) transmembrane domain 53 Intron GTTAGTGTGGGGACAGAGTTGTCCTGGGGACATTGGAGTGAAGT TGGAGATGATGGGAGCTCTGGGAATCCATAATAGCTCCTCCAGA GAAATCTTCTAGGTGCCTGAGTTGTGCCATGAAATGAATATGTA CATGTACATATGCATATACATTTGTTTTGTTTTACCCTAG 54 Exon 4-end of GCTCCCAGACCTCTGATCTGTCTCTCCCAGATTGTAAAGGTGAC mouse MHC I ACTCTAGGGTCTGATTGGGGAGGGGCAATGTGGACATGA (H2K.sup.b) transmembrane domain 55 V.sub.L leader MESQTQVLMFLLLWVSGACA 56 V.sub.L DIQMTQSPSSMSVSLGDTVTITCRASQDVGIYVNWFQQKPGKSP RRMIYRATNLADGVPSRFSGSRSGSDYSLTISSLESEDVADYHC LQYDEYPFTFGSGTKLEIKR 29 Linker GGGGSGGGGSGGGGS 57 V.sub.H QVQLKESGPGLVQPSQTLSLTCTVSGLSLISYGVSWARQPPGKG LEWIAAISSGGSTYYNSVLTSRLSISRDTSKSQVFLKMNSLQTE DTAIYFCTRELWDYYDYWGQGVMVTVSS 58 Rat IgG1 Fc AETTAPRNPGGDCKPCICTGSEVSSVFIFPPKPKDVLTITLTPK VTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNSTFRS VSELPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPEGRTQVP HVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQEN YKNTPPTMDTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHN FIHTEKSLSHSPG 59 Mouse MHC I KEPPPSTVSNMATVAVLVVLGAAIVTGAVVAFVMKMRRRNTGGK (H2K.sup.b) GGDYALAPGSQTSDLSLPDCKGDTLGSDWGGAMWT transmembrane domain

[0093] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims that follow, unless the term "means" is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. .sctn. 112, 6.

Sequence CWU 1

1

59155DNAHomo sapiens 1atggacatca gggctcctgc tcagtttctt ggcatcttgt tgctctggtt tccag 552110DNAHomo sapiens 2gtaaaatgaa ctaaaatggg aatttcactg taagtgttga caggcatttg gggactgtgt 60tcttttatca tgcttacctt tgtagatatt cattatgtct ccactcctag 110323DNAHomo sapiens 3gtgccagatg tgacatccag atg 23463DNAArtificial SequenceLeader Exon1/Intron/Exon 2/TagsFLAG+Myc Tags 4gactacaagg atgacgacga caagggcagc ggcgaacaga agctgatttc ggaggaggac 60ctg 63546DNAHomo sapiens 5atgggatgga gctgtatcat gctcttcttg gcagcaacag ctacag 46682DNAHomo sapiens 6gtaaggggct cacagtagca ggcttgaggt ctggacatat acatgggtga caatgacatc 60cactttgcct ttctctccac ag 82723DNAHomo sapiens 7gtgtccactc ccaggtccaa ctg 238369DNAEscherichia coli 8ggtaccccag ccgtgaccac ctacaagctc gtcatcaacg gaaagacgct caagggcgaa 60accactacca aggcggtgga tgccgaaacc gccgaaaagg ccttcaagca gtacgctaac 120gacaatgggg tggacggagt ctggacgtac gatgatgcca ccaagacttt caccgtgacc 180gaagtgaaca ctccggccgt caccacttat aagctcgtga tcaacgggaa aaccctgaag 240ggagagacta ccacaaaggc cgtggatgct gagactgcag agaaggcgtt caaacagtac 300gccaacgaca acggcgtgga cggcgtctgg acctacgatg acgccactaa gaccttcact 360gtgaccgaa 3699621DNAEscherichia coli 9atagatgaaa ttttagctgc attacctaag actgacactt acaaattaat ccttaatggt 60aaaacattga aaggcgaaac aactactgaa gctgttgatg ctgctactgc agaaaaagtc 120ttcaaacaat acgctaacga caacggtgtt gacggtgaat ggacttacga cgatgcgact 180aagaccttta cagttactga aaaaccagaa gtgatcgatg cgtctgaatt aacaccagcc 240gtgacaactt acaaacttgt tattaatggt aaaacattga aaggcgaaac aactactgaa 300gctgttgatg ctgctactgc agaaaaagtc ttcaaacaat acgctaacga caacggtgtt 360gacggtgaat ggacttacga cgatgcgact aagaccttta cagttactga aaaaccagaa 420gtgatcgatg cgtctgaatt aacaccagcc gtgacaactt acaaacttgt tattaatggt 480aaaacattga aaggcgaaac aactactaaa gcagtagacg cagaaactgc agaaaaagcc 540ttcaaacaat acgctaacga caacggtgtt gatggtgttt ggacttatga tgatgcgact 600aagaccttta cggtaactga a 62110741DNAEscherichia coli 10gtggataaca agttcaacaa ggaacagcag aacgcctttt acgagattct gcatctgccc 60aacctgaatg aggaacagcg gaacgcattc attcagtctc tgaaggatga tcctagccag 120tcggccaacc tcctggctga agcaaagaag ctgaacgatg cccaagcgcc caaagtggac 180aacaagttta acaaggagca gcagaatgct ttctacgaga tcctgcacct cccgaatctg 240aacgaggagc agagaaacgc cttcatccaa tcactgaagg acgacccgtc acagtccgcc 300aaccttctgg cggaagccaa gaaactgaac gacgcccagg cgccaaaggt ggacggatcc 360gggtccggca gcggtacccc agccgtgacc acctacaagc tcgtcatcaa cggaaagacg 420ctcaagggcg aaaccactac caaggcggtg gatgccgaaa ccgccgaaaa ggccttcaag 480cagtacgcta acgacaatgg ggtggacgga gtctggacgt acgatgatgc caccaagact 540ttcaccgtga ccgaagtgaa cactccggcc gtcaccactt ataagctcgt gatcaacggg 600aaaaccctga agggagagac taccacaaag gccgtggatg ctgagactgc agagaaggcg 660ttcaaacagt acgccaacga caacggcgtg gacggcgtct ggacctacga tgacgccact 720aagaccttca ctgtgaccga a 741111681DNAEscherichia coli 11aaacctcaac gccgatcagc gcaatggttt cattcagtcc ctgaaggacg atccgagcca 60gtcagctaac gtgctcgggg aggcccaaaa gctgaatgac tcccaggcgc cgaaggccga 120cgcccagcaa aacaacttca acaaggatca gcaatccgcc ttctatgaaa tcctgaatat 180gcctaacctg aacgaagctc agcggaacgg gttcatccag agccttaagg acgaccctag 240ccagtccacc aacgtgctgg gggaggccaa gaaacttaac gaatcccagg ccccgaaggc 300ggacaacaac tttaacaagg aacagcagaa cgccttttac gagatcctca acatgccgaa 360cctcaacgag gaacagcgca acggtttcat ccagtccctg aaggacgatc catcccagtc 420cgccaacctg ttgagcgagg cgaagaagct gaatgagtcc caagccccca aggctgacaa 480caagttcaat aaggaacaac agaatgcctt ctacgaaatt ctgcacttgc ccaatctgaa 540cgaggagcag cgcaacggct tcatccaatc tctgaaagac gacccgtcgc agtcggccaa 600cttgctggcc gaagccaaga agctcaacga cgctcaggcc cctaaggccg acaacaagtt 660caacaaagag caacagaacg cgttctacga gattctccac ttgccgaacc tgaccgaaga 720acaacggaac ggattcattc agagcctgaa ggatgaccct tcggtgtcaa aggagatcct 780ggcagaagcc aaaaagctga acgatgccca ggcaccaaag gaagaggaca acaacaagcc 840gggcgacccg aggatctccg aagccactga tgggctgtcc gattttctga agtcacagac 900tcctgctgag gacaccgtga agtccatcga gctcgccgag gccaaggtgc tggccaaccg 960ggagctggat aagtacggag tgtccgacta ctacaaaaac ctgattaaca acgccaagac 1020tgtggaagga gtgaaggcat tgatcgatga aatcctggcg gcgctcccaa aaaccgacac 1080ctacaaactg attctcaacg gaaagacgct gaagggggaa actaccaccg aagcggtgga 1140cgccgccacc gccgaaaagg tgtttaagca gtatgctaac gacaacggtg tcgacggaga 1200gtggacctac gacgacgcca ctaagacttt caccgtgacc gagaagcccg aggtcatcga 1260cgcgagcgag ctcactcccg ccgtgaccac ctacaagctg gtcatcaatg gaaagactct 1320gaagggcgaa actactactg aagccgtgga tgcggcaacc gccgagaaag tgttcaagca 1380atacgcaaac gataacgggg tggacggaga gtggacctac gacgatgcca caaagacctt 1440caccgtcacc gaaaagcccg aagtgatcga cgcttccgaa ctgacgccgg ccgtgacaac 1500ttacaagctc gtcattaacg gaaagaccct taagggcgaa accacgacca aggcagtgga 1560cgccgaaact gccgagaagg cgttcaagca gtacgccaac gacaacggcg tggacggagt 1620gtggacttac gatgatgcga ccaagacgtt cactgtgacc gagatggtca ccgaagtgcc 1680g 16811218DNAArtificial SequenceGly/Ser linker 12ggatccggct ccggatcc 181345DNAArtificial SequenceGly/Ser linker 13ggaggcggag gcagcggagg cggtggctcg ggaggcggag gctcg 4514243DNARattus rattus 14gagatggtgt ccaagccgat gatctactgg gagtgttcca acgcgactct gacctgtgaa 60gtgctggagg gaaccgacgt ggaactgaag ctgtaccagg gtaaagaaca tctgcggtcg 120ttgcgccaaa agaccatgag ctaccagtgg accaacttgc gggcgccttt caagtgcaaa 180gccgtcaata gagtgtccca ggagagcgaa atggaggtcg tgaactgccc cgaaaaggga 240ctg 24315561DNARattus rattus 15tcaacttcca tcaccgccta caagagcgag ggagagagcg ccgagttttc cttccccctg 60aacctgggcg aagaaagcct ccagggagaa ctgcgctgga aggcagaaaa ggccccaagc 120tctcagtcct ggatcacctt cagcctgaag aaccagaagg tgtccgtgca gaagtccact 180tcaaacccga agttccagct ctccgaaacc ctccctctga ccctgcaaat ccctcaagtg 240tcgctgcaat tcgcggggag cggaaatctg actctgactc ttgaccgggg catcttgtac 300caggaggtga acctggtggt catgaaggtg acccagcccg atagcaacac cctgacctgt 360gaagtgatgg gacccacgtc cccgaagatg cggctcattc tgaagcagga gaaccaggag 420gctcgggtgt ccagacagga aaaggtcatc caagtgcagg ccccggaagc cggcgtgtgg 480cagtgcctgc tgtccgaggg agaggaagtc aagatggact cgaaaatcca ggtgctgtcc 540aaagggctga accagactat g 561161638DNAHomo sapiens 16gaaaggcctt ttccacctca tatccagctc cctccagaaa ttcaagagtc ccaggaagtc 60actctgacct gcttgctgaa tttctcctgc tatgggtatc cgatccaatt gcagtggctc 120ctagaggggg ttccaatgag gcaggctgct gtcacctcga cctccttgac catcaagtct 180gtcttcaccc ggagcgagct caagttctcc ccacagtgga gtcaccatgg gaagattgtg 240acctgccagc ttcaggatgc agatgggaag ttcctctcca atgacacggt gcagctgaac 300gtgaagcaca ccccgaagtt ggagatcaag gtcactccca gtgatgccat agtgagggag 360ggggactctg tgaccatgac ctgcgaggtc agcagcagca acccggagta cacgacggta 420tcctggctca aggatgggac ctcgctgaag aagcagaata cattcacgct aaacctgcgc 480gaagtgacca aggaccagag tgggaagtac tgctgtcagg tctccaatga cgtgggcccg 540ggaaggtcgg aagaagtgtt cctgcaagtg cagtatgccc cggaaccttc cacggttcag 600atcctccact caccggctgt ggagggaagt caagtcgagt ttctttgcat gtcactggcc 660aatcctcttc caacaaatta cacgtggtac cacaatggga aagaaatgca gggaaggaca 720gaggagaaag tccacatccc aaagatcctc ccctggcacg ctgggactta ttcctgtgtg 780gcagaaaaca ttcttggtac tggacagagg ggcccgggag ctgagctgga tgtccagtat 840cctcccaaga aggtgaccac agtgattcaa aaccccatgc cgattcgaga aggagacaca 900gtgacccttt cctgtaacta caattccagt aaccccagtg ttacccggta tgaatggaaa 960ccccatggcg cctgggagga gccatcgctt ggggtgctga agatccaaaa cgttggctgg 1020gacaacacaa ccatcgcctg cgcagcttgt aatagttggt gctcgtgggc ctcccctgtc 1080gccctgaatg tccagtatgc cccccgagac gtgagggtcc ggaaaatcaa gcccctttcc 1140gagattcact ctggaaactc ggtcagcctc caatgtgact tctcaagcag ccaccccaaa 1200gaagtccagt tcttctggga gaaaaatggc aggcttctgg ggaaagaaag ccagctgaat 1260tttgactcca tctccccaga agatgctggg agttacagct gctgggtgaa caactccata 1320ggacagacag cgtccaaggc ctggacactt gaagtgctgt atgcacccag gaggctgcgt 1380gtgtccatga gcccggggga ccaagtgatg gaggggaaga gtgcaaccct gacctgtgag 1440agcgacgcca accctcccgt ctcccactac acctggtttg actggaataa ccaaagcctc 1500ccctaccaca gccagaagct gagattggag ccggtgaagg tccagcactc gggtgcctac 1560tggtgccagg ggaccaacag tgtgggcaag ggccgttcgc ctctcagcac cctcaccgtc 1620tactatagcc cggagacc 163817120DNAHomo sapiens 17gcgcctggag cgctgccggc cggtcatctg ttgttgttcc tgaccctggg ggtgctgtca 60ctgctgctgc tcgtgaccgg ggcattcggt ttccacctgt ggagaaggca gtggcggtag 12018123DNAHomo sapiens 18cattcccggc accgctacgc gctgattccg attcctctgg ccgtgatcac cacctgtatc 60gtgctctaca tgaacggtat cctgaaatgc gacagaaagc ccgacaggac taacagcaat 120tag 1231999DNARattus rattus 19ccgctgtacc tgatcgtggg ggtgtcagcc ggcggtctgc tgctcgtgtt cttcggggca 60ctgttcatct tctgcatttg caagaggaag aagcggtag 9920288DNAHomo sapiens 20ccaccccggg cgtccgcact gccggcgccc cctaccggaa gcgcgctgcc cgatccgcaa 60accgccagcg ccctgcctga cccgcccgcg gctagcgcct tgcctgccgc actggccgtg 120atttcattcc tgctgggtct ggggctcggg gtggcctgcg tgttggcacg gactcagatc 180aagaagctgt gctcctggag agacaaaaac tccgccgcct gtgtggtgta cgaggacatg 240tcacactcga ggtgcaatac cctgtcctcg ccgaaccagt accagtag 2882123PRTHomo sapiens 21Met Gly Trp Ser Cys Ile Met Leu Phe Leu Ala Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu 202221PRTArtificial SequenceLeader Exon1/Intron/Exon 2/TagsFLAG+Myc Tags 22Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gly Glu Gln Lys Leu Ile1 5 10 15Ser Glu Glu Asp Leu 202326PRTHomo sapiens 23Met Asp Ile Arg Ala Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala Arg Cys Asp Ile Gln Met 20 2524123PRTEscherichia coli 24Gly Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr1 5 10 15Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu 20 25 30Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp 35 40 45Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Val Asn Thr 50 55 60Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys65 70 75 80Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala 85 90 95Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr 100 105 110Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu 115 12025207PRTEscherichia coli 25Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys Leu1 5 10 15Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val 20 25 30Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn 35 40 45Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr 50 55 60Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro Ala65 70 75 80Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu 85 90 95Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 100 105 110Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 115 120 125Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala 130 135 140Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly145 150 155 160Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr 165 170 175Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly 180 185 190Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu 195 200 20526247PRTEscherichia coli 26Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile1 5 10 15Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn 50 55 60Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu65 70 75 80Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro 85 90 95Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala 100 105 110Gln Ala Pro Lys Val Asp Gly Ser Gly Ser Gly Ser Gly Thr Pro Ala 115 120 125Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu 130 135 140Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys145 150 155 160Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Asp 165 170 175Ala Thr Lys Thr Phe Thr Val Thr Glu Val Asn Thr Pro Ala Val Thr 180 185 190Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr 195 200 205Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr 210 215 220Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr225 230 235 240Lys Thr Phe Thr Val Thr Glu 24527581PRTEscherichia coli 27Ala Asn Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln1 5 10 15Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile 20 25 30Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu 35 40 45Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln 50 55 60Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn65 70 75 80Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu 85 90 95Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys 100 105 110Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys Glu 115 120 125Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu 130 135 140Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln145 150 155 160Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala 165 170 175Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr 180 185 190Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe 195 200 205Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala 210 215 220Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys225 230 235 240Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro 245 250 255Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp 260 265 270Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn 275 280 285Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Asp Pro 290 295 300Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln305 310 315 320Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys 325 330 335Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr 340 345 350Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu 355 360 365Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys Leu 370 375 380Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val385 390 395 400Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn 405 410 415Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr 420 425 430Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro Ala 435 440 445Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu 450 455 460Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala

Glu Lys Val Phe Lys465 470 475 480Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 485 490 495Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala 500 505 510Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly 515 520 525Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr 530 535 540Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly545 550 555 560Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Met 565 570 575Val Thr Glu Val Pro 580286PRTArtificial SequenceGly/Ser linker 28Gly Ser Gly Ser Gly Ser1 52915PRTArtificial SequenceGly/Ser Linker 29Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 153081PRTRattus rattus 30Glu Met Val Ser Lys Pro Met Ile Tyr Trp Glu Cys Ser Asn Ala Thr1 5 10 15Leu Thr Cys Glu Val Leu Glu Gly Thr Asp Val Glu Leu Lys Leu Tyr 20 25 30Gln Gly Lys Glu His Leu Arg Ser Leu Arg Gln Lys Thr Met Ser Tyr 35 40 45Gln Trp Thr Asn Leu Arg Ala Pro Phe Lys Cys Lys Ala Val Asn Arg 50 55 60Val Ser Gln Glu Ser Glu Met Glu Val Val Asn Cys Pro Glu Lys Gly65 70 75 80Leu31187PRTRattus rattus 31Ser Thr Ser Ile Thr Ala Tyr Lys Ser Glu Gly Glu Ser Ala Glu Phe1 5 10 15Ser Phe Pro Leu Asn Leu Gly Glu Glu Ser Leu Gln Gly Glu Leu Arg 20 25 30Trp Lys Ala Glu Lys Ala Pro Ser Ser Gln Ser Trp Ile Thr Phe Ser 35 40 45Leu Lys Asn Gln Lys Val Ser Val Gln Lys Ser Thr Ser Asn Pro Lys 50 55 60Phe Gln Leu Ser Glu Thr Leu Pro Leu Thr Leu Gln Ile Pro Gln Val65 70 75 80Ser Leu Gln Phe Ala Gly Ser Gly Asn Leu Thr Leu Thr Leu Asp Arg 85 90 95Gly Ile Leu Tyr Gln Glu Val Asn Leu Val Val Met Lys Val Thr Gln 100 105 110Pro Asp Ser Asn Thr Leu Thr Cys Glu Val Met Gly Pro Thr Ser Pro 115 120 125Lys Met Arg Leu Ile Leu Lys Gln Glu Asn Gln Glu Ala Arg Val Ser 130 135 140Arg Gln Glu Lys Val Ile Gln Val Gln Ala Pro Glu Ala Gly Val Trp145 150 155 160Gln Cys Leu Leu Ser Glu Gly Glu Glu Val Lys Met Asp Ser Lys Ile 165 170 175Gln Val Leu Ser Lys Gly Leu Asn Gln Thr Met 180 1853280PRTHomo sapiens 32Met Lys Val Thr Gln Pro Asp Ser Asn Thr Leu Thr Cys Glu Val Met1 5 10 15Gly Pro Thr Ser Pro Lys Met Arg Leu Ile Leu Lys Gln Glu Asn Gln 20 25 30Glu Ala Arg Val Ser Arg Gln Glu Lys Val Ile Gln Val Gln Ala Pro 35 40 45Glu Ala Gly Val Trp Gln Cys Leu Leu Ser Glu Gly Glu Glu Val Lys 50 55 60Met Asp Ser Lys Ile Gln Val Leu Ser Lys Gly Leu Asn Gln Thr Met65 70 75 803339PRTHomo sapiens 33Ala Pro Gly Ala Leu Pro Ala Gly His Leu Leu Leu Phe Leu Thr Leu1 5 10 15Gly Val Leu Ser Leu Leu Leu Leu Val Thr Gly Ala Phe Gly Phe His 20 25 30Leu Trp Arg Arg Gln Trp Arg 353440PRTHomo sapiens 34His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala Val Ile1 5 10 15Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Ile Leu Lys Cys Asp Arg 20 25 30Lys Pro Asp Arg Thr Asn Ser Asn 35 403532PRTRattus rattus 35Pro Leu Tyr Leu Ile Val Gly Val Ser Ala Gly Gly Leu Leu Leu Val1 5 10 15Phe Phe Gly Ala Leu Phe Ile Phe Cys Ile Cys Lys Arg Lys Lys Arg 20 25 303695PRTHomo sapiens 36Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly Ser Ala Leu1 5 10 15Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro Ala Ala Ser 20 25 30Ala Leu Pro Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly 35 40 45Leu Gly Val Ala Cys Val Leu Ala Arg Thr Gln Ile Lys Lys Leu Cys 50 55 60Ser Trp Arg Asp Lys Asn Ser Ala Ala Cys Val Val Tyr Glu Asp Met65 70 75 80Ser His Ser Arg Cys Asn Thr Leu Ser Ser Pro Asn Gln Tyr Gln 85 90 953793DNAHomo sapiens 37gaattccttg aaaatggtgg gacatcctta tcagagaaaa cagttcttct gctggtgact 60ccatttctgg cagcagcctg gagccttcat ccc 933831PRTHomo sapiens 38Glu Phe Leu Glu Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val Leu1 5 10 15Leu Leu Val Thr Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro 20 25 303990DNAHomo sapiens 39accaatgcca caacaaaggc agcaggggga gcactccagt caacagcaag tttgtttgtc 60gtgtcactga gtctcttgca tctttattca 904030PRTHomo sapiens 40Thr Asn Ala Thr Thr Lys Ala Ala Gly Gly Ala Leu Gln Ser Thr Ala1 5 10 15Ser Leu Phe Val Val Ser Leu Ser Leu Leu His Leu Tyr Ser 20 25 304196DNAHomo sapiens 41gtctcccagg tgaaaatttc aggagcccct accctctccc catccctcct gggtttgctg 60ctgcccgcct ttggcattct cgtgtatctg gagttc 964232PRTHomo sapiens 42Val Ser Gln Val Lys Ile Ser Gly Ala Pro Thr Leu Ser Pro Ser Leu1 5 10 15Leu Gly Leu Leu Leu Pro Ala Phe Gly Ile Leu Val Tyr Leu Glu Phe 20 25 304349DNAHomo sapiens 43atggaatcac agacccaggt cctcatgttt cttctgctct gggtatctg 4944199DNAHomo sapiens 44gtaagaaatt taaagtatta aaaccttttc aaagtttcat ctttgtggta agaaatttgc 60aatatgtgcc agtgtgtaat atttcttaca taataaattt gtgacagtat gataaggaca 120tttaaatgaa aaatttcgac tgttgttata atctatgtct gtgtatctat gaattttcac 180tgcctattaa ttattacag 1994511DNAHomo sapiens 45gtgcctgtgc a 1146324DNAHomo sapiens 46gacattcaga tgacccagtc tccatcctcc atgtctgtgt ctctgggaga cacagtcact 60attacttgcc gggcaagtca ggacgttggg atttatgtaa actggttcca gcagaaacca 120gggaaatctc ctaggcgtat gatttatcgt gcaacgaact tggcagatgg ggtcccatca 180aggttcagcg gcagtaggtc tggatcagat tattctctca ccatcagcag cctggagtct 240gaagatgtgg cagactatca ctgtctacag tatgatgagt atccattcac gttcggatcc 300gggacgaagt tggaaataaa acgg 3244745DNAHomo sapiens 47ggaggcggag gcagcggagg cggtggctcg ggaggcggag gctcg 4548348DNAHomo sapiens 48caggtacagc tgaaagagtc aggacctggt ctggtgcagc cctcacagac cctgtctctc 60acctgcactg tctctggact ctcattaatc agttatggtg taagttgggc tcgccagcct 120ccagggaagg gtctggagtg gattgcagca atatcaagtg gtggaagcac atattataat 180tcagttctca catctcgact gagcatcagc agggacacct ccaagagcca agttttctta 240aaaatgaaca gtctgcaaac tgaagacaca gccatttact tctgtaccag agaactctgg 300gactactatg attactgggg ccaaggagtc atggtcacag tctcctca 34849696DNARattus rattus 49gctgaaacaa cagcccccag aaacccggga ggtgattgca agccttgtat atgtacaggc 60tcagaagtat catctgtctt catcttcccc ccaaagccca aagatgtgct caccatcact 120ctgactccta aggtcacgtg tgttgtggta gacattagcc aggacgatcc cgaggtccat 180ttcagctggt ttgtagatga cgtggaagtc cacacagctc agactcgacc accagaggag 240cagttcaaca gcactttccg ctcagtcagt gaactcccca tcctgcacca ggactggctc 300aatggcagga cgttcagatg caaggtcacc agtgcagctt tcccatcccc catcgagaaa 360accatctcca aacccgaagg cagaacacaa gttccgcatg tatacaccat gtcacctacc 420aaggaagaga tgacccagaa tgaagtcagt atcacctgca tggtaaaagg cttctatccc 480ccagacattt atgtggagtg gcagatgaac gggcagccac aggaaaacta caagaacact 540ccacctacga tggacacaga tgggagttac ttcctctaca gcaagctcaa tgtgaagaag 600gaaaaatggc agcagggaaa cacgttcacg tgttctgtgc tgcatgaagg cctgcacaac 660caccatactg agaagagtct ctcccactcc cccggt 69650124DNAMus musculus 50aaagagcctc ctccatccac tgtctccaac atggcgaccg ttgctgttct ggttgtcctt 60ggagctgcaa tagtcactgg agctgtggtg gcttttgtga tgaagatgag aaggagaaac 120acag 12451178DNAMus musculus 51gtaggaaagg gcagagtctg agttttctct cagcctcctt tagagtgtgc tctgctcatc 60aatggggaac acaggcacac cccacattgc tactgtctct aactgggtct gctgtcagtt 120ctgggaactt cctagtgtca agatcttcct ggaactctca cagcttttct tctcacag 1785233DNAMus musculus 52gtggaaaagg aggggactat gctctggctc cag 3353172DNAMus musculus 53gttagtgtgg ggacagagtt gtcctgggga cattggagtg aagttggaga tgatgggagc 60tctgggaatc cataatagct cctccagaga aatcttctag gtgcctgagt tgtgccatga 120aatgaatatg tacatgtaca tatgcatata catttgtttt gttttaccct ag 1725483DNAMus musculus 54gctcccagac ctctgatctg tctctcccag attgtaaagg tgacactcta gggtctgatt 60ggggaggggc aatgtggaca tga 835520PRTHomo sapiens 55Met Glu Ser Gln Thr Gln Val Leu Met Phe Leu Leu Leu Trp Val Ser1 5 10 15Gly Ala Cys Ala 2056108PRTHomo sapiens 56Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly1 5 10 15Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ile Tyr 20 25 30Val Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Arg Met Ile 35 40 45Tyr Arg Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Val Ala Asp Tyr His Cys Leu Gln Tyr Asp Glu Tyr Pro Phe 85 90 95Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 10557116PRTHomo sapiens 57Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ile Ser Tyr 20 25 30Gly Val Ser Trp Ala Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ala Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Asn Ser Val Leu Thr 50 55 60Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr 85 90 95Arg Glu Leu Trp Asp Tyr Tyr Asp Tyr Trp Gly Gln Gly Val Met Val 100 105 110Thr Val Ser Ser 11558232PRTRattus rattus 58Ala Glu Thr Thr Ala Pro Arg Asn Pro Gly Gly Asp Cys Lys Pro Cys1 5 10 15Ile Cys Thr Gly Ser Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 20 25 30Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 35 40 45Val Val Asp Ile Ser Gln Asp Asp Pro Glu Val His Phe Ser Trp Phe 50 55 60Val Asp Asp Val Glu Val His Thr Ala Gln Thr Arg Pro Pro Glu Glu65 70 75 80Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Leu His 85 90 95Gln Asp Trp Leu Asn Gly Arg Thr Phe Arg Cys Lys Val Thr Ser Ala 100 105 110Ala Phe Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Glu Gly Arg 115 120 125Thr Gln Val Pro His Val Tyr Thr Met Ser Pro Thr Lys Glu Glu Met 130 135 140Thr Gln Asn Glu Val Ser Ile Thr Cys Met Val Lys Gly Phe Tyr Pro145 150 155 160Pro Asp Ile Tyr Val Glu Trp Gln Met Asn Gly Gln Pro Gln Glu Asn 165 170 175Tyr Lys Asn Thr Pro Pro Thr Met Asp Thr Asp Gly Ser Tyr Phe Leu 180 185 190Tyr Ser Lys Leu Asn Val Lys Lys Glu Lys Trp Gln Gln Gly Asn Thr 195 200 205Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 210 215 220Lys Ser Leu Ser His Ser Pro Gly225 2305979PRTMus musculus 59Lys Glu Pro Pro Pro Ser Thr Val Ser Asn Met Ala Thr Val Ala Val1 5 10 15Leu Val Val Leu Gly Ala Ala Ile Val Thr Gly Ala Val Val Ala Phe 20 25 30Val Met Lys Met Arg Arg Arg Asn Thr Gly Gly Lys Gly Gly Asp Tyr 35 40 45Ala Leu Ala Pro Gly Ser Gln Thr Ser Asp Leu Ser Leu Pro Asp Cys 50 55 60Lys Gly Asp Thr Leu Gly Ser Asp Trp Gly Gly Ala Met Trp Thr65 70 75

Claims

1. A method for generating an antibody-secreting cell capable of expressing a membrane-bound immunoglobulin-capturing molecule that can bind, retain, and display endogenously produced immunoglobulin molecules at its cell surface, comprising the step of introducing into the antibody-secreting cell, or the progenitor of an antibody-secreting cell, a nucleic acid vector comprising a promoter, nucleic acid sequence coding for an immunoglobulin-binding peptide, and nucleic acid sequence coding for a cell surface tether peptide.

2. The method of claim 1, wherein the immunoglobulin-binding peptide is derived from: a. one or more bacterial proteins that naturally have affinity for an immunoglobulin; or b. one or more variable domains of an immunoglobulin that has affinity for any part of another immunoglobulin.

3. The method of claim 2, wherein the bacterial protein is Protein A or Protein G.

4. The method of claim 1, wherein the antibody-secreting cell is a hybridoma cell or a cell of B lymphocyte lineage.

5. The method of claim 1, wherein the promoter is a constitutive promoter or an inducible promoter.

6. The method of claim 5, wherein the promoter expresses the immunoglobulin-capturing molecule preferentially in antibody-secreting cells with minimal expression during B cell development prior to antigen encounter.

7. The method of claim 6, wherein the promoter is selected from B Lymphocyte-Induced Maturation Protein 1, Syndecan 1, Tumor Necrosis Factor Receptor Superfamily Member 17, or Fucosyltransferase 1 genes.

8. The method of claim 5, where in the inducible promoter is a tetracycline-responsive promoter or a tamoxifen-responsive promoter.

9. The method of claim 1, wherein the cell surface tether peptide is: a. a transmembrane peptide; or b. a peptide sequence that can be post-translationally modified to tether the immunoglobulin-binding peptide to the cell surface of the antibody-secreting cell.

10. The method of claim 9, wherein the transmembrane peptide is derived from human Lymphocyte Activation Gene 3, human CD58, rat CD2, or human CD7.

11. The method of claim 10, wherein the C-terminal peptide sequence mediates glycosylphosphatidylinositol linkage to the plasma membrane.

12. The method of claim 1, wherein the nucleic acid vector further comprises: a. a nucleic acid sequence coding for a stalk structure; b. a nucleic acid sequence coding for a reporter peptide; c. an IRES sequence or a picornavirus 2A ribosomal skip sequence; d. a nucleic acid sequence coding for a signal peptide; or e. a combination thereof.

13. The method of claim 12, wherein the reporter peptide is a fluorescent peptide.

14. The method of claim 12, wherein the nucleic acid vector further comprises nucleic acid sequences coding for a stalk structure and a reporter peptide linked to an IRES sequence or picornavirus 2A ribosomal skip sequence.

15. An antibody-secreting cell produced by the method of claim 1.

16. Part of or whole immunoglobulin molecules derived from the antibody-secreting cell of claim 24.

17. The method of claim 1, wherein the antibody-secreting cell is used for large-scale production of antibodies.

18. A genetically modified animal comprising antibody-secreting cells comprising a gene encoding an immunoglobulin-capturing molecule comprising a cell surface tether portion and an immunoglobulin-binding portion, wherein the immunoglobulin-capturing molecule can bind, retain, and display endogenously produced immunoglobulin molecules at a cell surface of the antibody-secreting cells.

19. A vector for expressing a membrane-bound immunoglobulin-capturing molecule that can bind, retain, and display immunoglobulin molecules at a cell surface of an antibody-secreting cell, comprising a promoter, nucleic acid sequence coding for an immunoglobulin-binding peptide, and nucleic acid sequence coding for a cell surface tether peptide.

20. The vector of claim 19, further comprising nucleic acid sequences coding for a signal peptide, a stalk structure, and a reporter peptide linked to an IRES sequence or picornavirus 2A ribosomal skip sequence.