Patent Grant
11053288
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.
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.
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.
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.
Alternatively, the ASCs can be individually sorted, and the genes encoding the heavy chain and light chain variable domains (VH and VL, respectively) directly cloned without the need to propagate the ASCs in vitro. The VH- and VL-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 VH and VL 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.
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.
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 Igα and Igβ, 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 VH and VL cloning technique from sorted single cells.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
These and other aspects, objects and features are described in more detail below.
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.
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.
A “capture molecule” is any moiety that contains a region that selectively binds to a part of or a whole molecule of interest.
“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.
“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.
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.
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 (VH and VL, 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 CH1 to CH4) 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 κ or λ immunoglobulin domain (referred to as Cκ or Cλ). In both mice and humans, the presence of κ light chains predominates over that of λ 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 VH and/or VL 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.
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.
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.
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.
“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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 VH and VL 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 VH and VL 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 VH and VL 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
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.
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.
In another embodiment, the immunoglobulin-capturing molecules consist of single-chain variable fragments (scFv). The scFv is expressed as a fusion protein of the VH and VL 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 VH domain connected in tandem to the VL 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)n [as in SEQ ID No. 29] or (Gly-Ser)n [as in SEQ ID No. 28].
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.
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 (Man3-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.
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 CH2 and CH3 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
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.
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.
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].
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.
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.
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.
Illustrated in
Exemplary nucleic acid sequences for components of the immunoglobulin-capturing molecule illustrated in
An expression construct and the molecular structure of an alternative embodiment of an immunoglobulin-capturing molecule is illustrated in
Transgenic Cell Libraries
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
The present invention also provides transgenic animals that have been modified to express immunoglobulin-capturing molecules on the cell surface of ASCs.
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.
The exons that encode the antibody VH and VL domains do not exist in the germ-line DNA. Instead, each VH or VL 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.
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.
In other aspects, the transgenic animals of the invention are avian, preferably chickens.
Use in Antibody Production
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).)
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.
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.
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.
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
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.
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.
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 (VH) 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.
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.
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.
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 (VL) 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.
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.
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.
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 VL, linker, and VH 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, CH2 domain, and CH3 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].
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.
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.
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.
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.
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.
Alternatively, the purified ASCs are individually sorted, and genes encoding their VH and VL domains are cloned via RT-PCR or 5′ RACE techniques adapted for single cells. The cloned VH- and VL-coding sequences are subcloned into an expression vector containing a sequence encoding the desired constant regions of heavy chain and light chain, respectively. The VH- and VL-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.
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. § 112,¶6.
This application claims priority to U.S. Ser. No. 62/291,217, filed Feb. 4, 2016.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4892824 | Skaletsky | Jan 1990 | A |
| 5591669 | Krimpenfort et al. | Jan 1997 | A |
| 5593598 | McGinness et al. | Jan 1997 | A |
| 5612205 | Kay et al. | Mar 1997 | A |
| 5633425 | Lonberg et al. | May 1997 | A |
| 5661016 | Lonberg et al. | Aug 1997 | A |
| 5789215 | Berns et al. | Aug 1998 | A |
| 5789650 | Lonberg et al. | Aug 1998 | A |
| 5814318 | Lonberg et al. | Sep 1998 | A |
| 5874299 | Lonberg et al. | Feb 1999 | A |
| 5877397 | Lonberg et al. | Mar 1999 | A |
| 5939598 | Kucherlapati et al. | Aug 1999 | A |
| 5955300 | Faure | Sep 1999 | A |
| 5985615 | Jakobovits et al. | Nov 1999 | A |
| 6023010 | Krimpenfort et al. | Feb 2000 | A |
| 6091001 | Jakobovits et al. | Jul 2000 | A |
| 6114598 | Kucherlapati et al. | Sep 2000 | A |
| 6130364 | Jakobovits et al. | Oct 2000 | A |
| 6162963 | Kucherlapati et al. | Dec 2000 | A |
| 6492575 | Wagner et al. | Dec 2002 | B1 |
| 6570061 | Rajewsky et al. | May 2003 | B1 |
| 6586251 | Economides | Jul 2003 | B2 |
| 6596541 | Murphy | Jul 2003 | B2 |
| 6653113 | Berns et al. | Nov 2003 | B1 |
| 6673986 | Kucherlapati et al. | Jan 2004 | B1 |
| 6998514 | Bruggeman | Feb 2006 | B2 |
| 7041870 | Tomizuka et al. | May 2006 | B2 |
| 7041871 | Lonberg | May 2006 | B1 |
| 7064244 | Jakobovits et al. | Jun 2006 | B2 |
| 7105348 | Murphy | Sep 2006 | B2 |
| 7129084 | Buelow | Oct 2006 | B2 |
| 7145056 | Jakobovits | Dec 2006 | B2 |
| 7148040 | Meagher et al. | Dec 2006 | B2 |
| 7205148 | Economides et al. | Apr 2007 | B2 |
| 7294754 | Poueymirou et al. | Nov 2007 | B2 |
| 7473557 | Economides et al. | Jan 2009 | B2 |
| 7476536 | Kuroiwa et al. | Jan 2009 | B2 |
| 7501552 | Lonberg | Mar 2009 | B2 |
| 7541513 | Bruggeman | Jun 2009 | B2 |
| 7659442 | Poueymirou et al. | Feb 2010 | B2 |
| 7868223 | Tomizuka et al. | Jan 2011 | B2 |
| 8158419 | Lonberg | Apr 2012 | B2 |
| 8163546 | Akamatsu et al. | Apr 2012 | B2 |
| 8232449 | Tanamachi | Jul 2012 | B2 |
| 8293480 | Lonberg | Oct 2012 | B2 |
| 8367888 | Bruggeman | Feb 2013 | B2 |
| 8502018 | Murphy | Aug 2013 | B2 |
| 8754287 | MacDonald et al. | Jun 2014 | B2 |
| 9012717 | MacDonald et al. | Apr 2015 | B2 |
| 9580491 | Green et al. | Feb 2017 | B2 |
| 10494445 | Green et al. | Dec 2019 | B2 |
| 10526420 | Green et al. | Jan 2020 | B2 |
| 10575504 | Green et al. | Mar 2020 | B2 |
| 10604587 | Green et al. | Mar 2020 | B2 |
| 10618977 | Green et al. | Apr 2020 | B2 |
| 10626188 | Green et al. | Apr 2020 | B2 |
| 10662255 | Green et al. | May 2020 | B2 |
| 20030017534 | Buelow | Jan 2003 | A1 |
| 20060015957 | Lonberg | Jan 2006 | A1 |
| 20070061900 | Murphy | Mar 2007 | A1 |
| 20090055943 | Economides | Feb 2009 | A1 |
| 20090111126 | Akamatsu | Apr 2009 | A1 |
| 20090136950 | Dubridge | May 2009 | A1 |
| 20100317539 | Yu | Dec 2010 | A1 |
| 20110145937 | MacDonald et al. | Jun 2011 | A1 |
| 20110236378 | Green | Sep 2011 | A1 |
| 20110258710 | Murphy | Oct 2011 | A1 |
| 20110283376 | Murphy | Nov 2011 | A1 |
| 20120047585 | Rohrer et al. | Feb 2012 | A1 |
| 20120073004 | MacDonald | Mar 2012 | A1 |
| 20120090041 | Buelow | Apr 2012 | A1 |
| 20120096572 | MacDonald et al. | Apr 2012 | A1 |
| 20130137101 | Economides | May 2013 | A1 |
| 20130219535 | Wabl et al. | Aug 2013 | A1 |
| 20130263292 | Liang | Oct 2013 | A1 |
| 20130333057 | MacDonald et al. | Dec 2013 | A1 |
| 20140283153 | Trianni | Sep 2014 | A1 |
| 20150183820 | Honda et al. | Jul 2015 | A1 |
| 20170058052 | Wabl et al. | Mar 2017 | A1 |
| 20170188557 | Green et al. | Jul 2017 | A1 |
| 20170218090 | Green et al. | Aug 2017 | A1 |
| 20170303517 | Wabl | Oct 2017 | A1 |
| 20170306352 | Wabl | Oct 2017 | A1 |
| 20180230238 | Wabl et al. | Aug 2018 | A1 |
| 20200181285 | Green et al. | Jun 2020 | A1 |
| 20200181286 | Green et al. | Jun 2020 | A1 |
| 20200407464 | Green et al. | Dec 2020 | A1 |
| Number | Date | Country |
|---|---|---|
| 2089661 | Mar 1992 | CA |
| 1399575 | Jul 2006 | EP |
| 1399559 | Apr 2008 | EP |
| 0817835 | Oct 2008 | EP |
| 2264163 | Dec 2010 | EP |
| 2517556 | Oct 2012 | EP |
| 2517557 | Oct 2012 | EP |
| 2398784 | Sep 2004 | GB |
| 2561352 | Oct 2018 | GB |
| 9203918 | Mar 1992 | WO |
| 9425585 | Nov 1994 | WO |
| WO-9530750 | Nov 1995 | WO |
| 9640915 | Dec 1996 | WO |
| 9945962 | Jun 1999 | WO |
| WO 11004192 | Jan 2001 | WO |
| 0109187 | Feb 2001 | WO |
| WO 0212437 | Feb 2002 | WO |
| 2002057423 | Jul 2002 | WO |
| 02066618 | Aug 2002 | WO |
| WO 02066630 | Aug 2002 | WO |
| 2008070367 | Jun 2008 | WO |
| 2008081197 | Jul 2008 | WO |
| 2009013620 | Jan 2009 | WO |
| WO-2009033743 | Mar 2009 | WO |
| 2009157771 | Dec 2009 | WO |
| 2010005863 | Jan 2010 | WO |
| 2010039900 | Apr 2010 | WO |
| 2011123708 | Oct 2011 | WO |
| WO 11158009 | Dec 2011 | WO |
| WO 11163311 | Dec 2011 | WO |
| WO 12018610 | Feb 2012 | WO |
| 2012123949 | Sep 2012 | WO |
| 2013022782 | Feb 2013 | WO |
| 2013092720 | Jun 2013 | WO |
| 2013138681 | Sep 2013 | WO |
| 2013171505 | Nov 2013 | WO |
| 2014013075 | Jan 2014 | WO |
| 2015112790 | Jul 2015 | WO |
| 2015188141 | Dec 2015 | WO |
| 2017035252 | Mar 2017 | WO |
| 2017095939 | Jun 2017 | WO |
| 2018189520 | Oct 2018 | WO |
| 2020074874 | Apr 2020 | WO |
| Entry |
|---|
| Van Keuren et al., 2009, Transgenic Res., vol. 18(5), pp. 769-785 (Year: 2009). |
| Ivies et al. (2014, Nature Protocols, vol. 9(4), pp. 810-827) (Year: 2014). |
| West et al., 2016, J. Equine Vet. Sci., vol. 41, pp. 1-12 (Year: 2016). |
| Meng et al. (2015, J. Animal Sci. and Biotech., pp. 1-7 (Year: 2015). |
| Brevini et al., 2010, Theriogenology, vol. 74, pp. 544-550 (Year: 2010). |
| Paris et al. (2010, Theriogenology, vol. 74, pp. 516-524) (Year: 2010). |
| Munoz et al. (2008, Theriogenology, vol. 69, pp. 1159-1164) (Year: 2008). |
| Gomez et al. (2010, Theriogenology, vol. 74, pp. 498-515) (Year: 2010). |
| Buta et al. (2013, Stem Cell Res., vol. 11, pp. 552-562) (Year: 2013). |
| Tong et al. (2010, Nature, vol. 467(7312), pp. 211-213) (Year: 2010). |
| Hong et al. (2012, Stem Cells and Development, vol. 21(9), pp. 1571-1586) (Year: 2012). |
| 2004, Barthold S., Genetica, vol. 122, pp. 75-88, see p. 85 col. 2 parag. 2 lines 1-13). (Year: 2004). |
| Choe et al., 2016, Materials, vol. 9, pp. 1-17 (Year: 2016). |
| Garcia-Arocena D. (2014, The Jackson Laboratory, Same Mutation, Different Phenotype?) (Year: 2014). |
| Heimain-Patterson et al. (2011, Amyotrophic Lateral Schlerosis, vol. 00, pp. 1-8) (Year: 2011). |
| Ait-Azzouzene et al., Journal of Experimental Medicine, vol. 201, No. 5, Mar. 7, 2005 817-828. |
| Cheng TL, Roffler S. Membrane-tethered proteins for basic research, imaging, and therapy. Med Res Rev. Nov. 2008;28(6):885-928. |
| Young et al., Biotechnol. J., 7: 620-634, 2012. |
| Burckstrummer et al., Nature Methods, 3(12): 1013-1019, 2006. |
| Guss et al., The EMBO Journal vol. 5 No. 7 pp. 1567-1575, 1986. |
| Lee et al., Cancer Biol. Ther. 8 (2), 161-166 (2009). |
| Tunyaplin et al., Nucleic Acids Research, 2000, vol. 28, No. 24, 4846-4855. |
| Melidoni et al., PNAS, 110(44): 17802-17807, 2013. |
| Lanitis, Cancer Immunol Res; 1(1); 43-53, 2013. |
| Debono, et al., “Vh Gene Segments in the Mouse and Human Genomes”, J. Mol. Biol., 342:131-34 (2004). |
| Wallace, et al., “Manipulating the Mouse Genome to Engineer Precise Functional Syntenic Replacements with Human Sequence”, Cell, 128:197-209 (2007). |
| Zhang, et al., “:A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments”, PLOS One, 8(5):e63735 (2013). |
| Altschul et al., “Basic local alignment search tool,” J Mol Biol 215:403-410 (1990). |
| Avitahl et al., “A 125 bp region of the Ig VH1 promoter is sufficient to confer lymphocyte-specific expression in transgenic mice,” Int Immunol 8(9):1359-1366 (1996). |
| Bentley et al., “Unrearranged immunoglobulin variable region genes have a functional promoter,” Nucleic Acids Res 10:1841-1856 (1982). |
| Berman et al., “Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus,” EMBO J 7(3):727-738 (1988). |
| Blankenstein et al., “Immunoglobulin VH region genes of the mouse are organized in overlapping cluster,” Eur J Immunol 17:1351-1357 (1987). |
| Brekke et al., “Assembly and analysis of the mouse immunoglobulin kappa gene sequence,” Immunogenetics 56:490-505 (2004). |
| Bruggemann, “The Preparation of Human Antibodies from Mice Harbouring Human Immunoglobulin Loci,” Transgenic Animals: Generation and Use, pp. 397-402, Ed. L.M. Houdebine, CRC Press (1997). |
| Casellas et al., “Igk allelic inclusion is a consequence of receptor editing,” J Exp Med 204(1):153-160 (2007). |
| Cesari et al, “Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange,” Genesis 38:87-92 (2004). |
| Church et al., “Lineage-specific biology revealed by a finished genome assembly of the mouse,” PLoS Biol 7:e1000112 (2009). |
| Clarke et al., “An immunoglobulin promoter region is unaltered by DNA rearrangement and somatic mutation during B-cell development,” Nucleic Acids Res 10:7731-7749 (1982). |
| Decaire et al., “A Publicly Available PCR Methods Laboratory Manual and Supporting Material,” J Microbiol Biol Educ 16:269-270 (2015). |
| Downing et al., “Technical assessment of the first 20 years of research using mouse embryonic stem cell lines,” Stem Cells 22:1168-1180 (2004). |
| Doyen et al., “Analysis of promoter and enhancer cell type specificities and the regulation of immunoglobulin gene expression,” Gene 50:321-331 (1986). |
| Featherstone et al., “The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination,” J Biol Chem 285:9327-9338 (2010). |
| Fishwild et al., “High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice,” Nat Biotechnol, 14:845-851 (1996). |
| Gellert, “Molecular analysis of V(D)J recombination,” Annu Rev Genet 26:425-446 (1992). |
| Gopal et al., “Contribution of promoter to tissue-specific expression of the mouse immunoglobulin kappa gene,” Science 229:1102-1104 (1985). |
| Hengartner et al., “Assignment of genes for immunoglobulin kappa and heavy chains to chromosomes 6 and 12 in mouse,” Proc Natl Acad Sci USA 75:4494-4498 (1978). |
| Honjo et al., ed. Immunoglobulin Genes. San Diego, CA: Academic Press Inc., 1989; Chapters 4-6 and 17. |
| Ichihara et al., “Organization of human immunoglobulin heavy chain diversity gene loci,” EMBO J 7(13):4141-4150 (1988). |
| International Human Genome Sequencing Consortium, “Finishing the euchromatic sequence of the human genome,” Nature 431:931-945 (2004). |
| Johnston et al., “Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region,” J Immunol 176:4221-4234 (2006). |
| Jones et al., “Replacing the complementarity-determining regions in a human antibody with those from a amouse,” Nature 321:522-525 (1986). |
| Jung et al., “Unraveling V(D)J Recombination: Insights into Gene Regulation,” Cell 116:299-311 (2004). |
| Kabat et al., “Variable region genes for the immunoglobulin framework are assembled from small segments of DNA—A hypothesis,” Proc Natl Acad Sci USA 75:2429-2433 (1978). |
| Kabat et al., “Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions,” J Exp Med 149:1299-1313 (1979). |
| Kawasaki et al., “One-Megabase Sequence Analysis of the Human Immunoglobulin λ Gene Locus,” Genome Res 7:250-261 (1997). |
| Kitamura et al., “Targeted disruption of μ chain membrane exon causes loss of heavy-chain allelic exclusion,” Nature 356:154-156 (1992). |
| Kozak, “An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs,” Nucleic Acids Res 15:8125-8148 (1987). |
| Kurosawa et al., “Organization, Structure, and Assembly of Immunoglobulin Heavy Chain Diversity DNA Segments,” J Exp Med 155:201-218 (1982). |
| Lander et al., “Initial sequencing and analysis of the human genome,” Nature 2001, 409:860-921 (2001). |
| Landsteiner et al., “On the Specificity of Serological Reactions with Simple Chemical Compounds (Inhibition Reactions),” J Exp Med 54:295-305 (1931). |
| Lee et al., “Genome data mining for everyone,” BMB Reports 41(11):757-764 (2008). |
| Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol 27:55-77 (2003). |
| Lutz et al., “Pro-B cells sense productive immunoglobulin heavy chain rearrangement irrespective of polypeptide production,” Proc Nat Acad Sci USA 108(26):10644-10649 (2011). |
| Mason et al., “Transcription cell type specificity is conferred by an immunoglobulin VH gene promoter that includes a functional consensus sequence,” Cell 41:479-487 (1985). |
| Matsuda et al., “The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus,” J Exp Med 188(11):2151-2162 (1998). |
| Misra et al., “Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination,” Endocrine 19:229-238 (2002). |
| Mouse Genome Sequencing Consortium, “Initial sequencing and comparative analysis of the mouse genome,” Nature 420:520-562 (2002). |
| Ristevski, “Making better transgenic models: conditional, temporal, and spatial approaches,” Mol Biotechnol 29:153-163 (2005). |
| Roebroek et al., “Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development,” Mol Cell Biol 26:605-616 (2006). |
| Saiki et al., “Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes,” Nature 324:163-166 (1986). |
| Sakano et al., “Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes,” Nature 290:562-565 (1981). |
| Schellenberg et al., “Pre-mRNA splicing: a complex picture in higher definition,” Trends Biochem Sci 33:243-246 (2008). |
| Sharon, “The invariant tryptophan in an H chain V region is not essential to antibody binding,” J Immunol 140:2666-2669 (1988). |
| Tonegawa, “Somatic generation of antibody diversity,” Nature 302:575-581 (1983). |
| Toor et al., “Structural insights into RNA splicing,” Curr Opin Struct Biol 19:260-266 (2009). |
| Venter et al., “The sequence of the human genome,” Science 291:1304-1351 (2001). |
| Von Heijne, “Protein targeting signals,” Curr Opin Cell Biol 2:604-608 (1990). |
| Xiong et al., “Chemical gene synthesis: strategies, softwares, error corrections, and applications,” FEMS Microbiol Rev 32:522-540 (2008). |
| International Preliminary Report on Patentability dated Aug. 7, 2018 in corresponding International Patent Application No. PCT/US2017/016521. |
| Gunasekaran et al., “Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects,” J Biol Chem 285:19637-19646 (2010). |
| Liu et al., “A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism,” J Biol Chem 290:7535-7362 (2015). |
| Ma et al., “DNA Synthesis, Assembly and Applications in Synthetic Biology,” Curr Opin Chem Biol 16:260-267 (2012). |
| McLenachan et al., “Flow-cytometric analysis of mouse embryonic stem cell lipofection using small and large DNA constructs,” Genomics 89:708-720 (2007). |
| Rajewsky et al., “Allelic exclusion model questioned,” Scientific Correspondence, Nature 359:371-372 (1992). |
| Sonoda et al, “B Cell Development under the Condition of Allelic Inclusion,” Immunity 6:225-233 (1997). |
| Vetterman et al., “Allelic exclusion of immunoglobulin genes: models and mechanisms,” Immunol Rev 237:22-42 (2010). |
| Wabl et al., “Allelic exclusion model questioned,” Scientific Correspondence, Nature 359:370-371 (1992). |
| Clargo et al., “The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method,” mAbs 6(1): 143-159 (2013). |
| Kontermann et al., “Bispecific antibodies,” Drug Discov Today 20(7):838-847 (2015). |
| Li et al., “Biochemical Analysis of the Regulatory T Cell Protein Lymphocyte Activation Gene-3 (LAG-3; CD223),” Journal of Immunology 173: 6806-6812 (2004). |
| Manz et al., “Analysis and sorting of live cells according to secreted molecules relocated to a cell-surface affinity matrix,” Proceedings of the National Academy of Science USA 92: 1921-1925 (1995). |
| Pinder et al., “Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Ig Capture Assay,” Journal of Immunology 199(12): 4180-4188 (2017). |
| Price et al., “Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas,” Journal of Immunological Methods 343: 28-41 (2009). |
| Verkoczy et al., “Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies,” Immunol. Rev. 275:89-107 (2017). |
| Zhou et al., “Generation of Monoclonal Antibodies against Highly Conserved Antigens,” PLoS One 4(6):e6087 (2009). |
| Notice of Reason for Rejection dated Apr. 7, 2020 in corresponding Japanese Patent Application No. 2018-560448. |
| U.S. Appl. No. 61/361,302, filed Jul. 2, 2010 in the name of ABLEXIS, LLC. |
| U.S. Appl. No. 61/319,690, filed Mar. 31, 2010 in the name of ABLEXIS, LLC. |
| Martin, Jolyon et al., “Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci”, Immunogenetics, 2018, vol. 70, pp. 223-236. |
| Third-Party Submission dated Mar. 31, 2021 in U.S. Appl. No. 16/849,347, filed Apr. 15, 2020. |
| Communication dated Apr. 2, 2021 regarding Third Party Submission. |
| Proudhon, Charlotte et al., “Long Range Regulation of V(D)J Recombination”, Adv Immunol., 2015, vol. 128, pp. 123-182. |
| Ramsden, Dale A. et al., “Conservation of sequence in recombination signal sequence spacers”, Nucleic Acids Research, 1994, vol. 22, No. 10, pp. 1785-1796. |
| Office Action issued Aug. 22, 2019 in U.S. Appl. No. 15/603,334. |
| Amendment/Reply under 37 C.F.R § 1.111 filed Sep. 19, 2019 in response to the Non-Final Office Action dated Aug. 22, 2019 in U.S. Appl. No. 15/603,334. |
| Amendrnent/Reply under 37 C.F.R § 1.114 filed Jul. 10, 2017 in response to the Final Office Action: dated Feb. 24, 2017 in U.S. Appl. No. 13/818,184. |
| Number | Date | Country | |
|---|---|---|---|
| 20170226162 A1 | Aug 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62291217 | Feb 2016 | US |
