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WO2008128144A2 - Système de sélection d'anticorps monoclonal, et fabrication et utilisation de celui-ci - Google Patents

Système de sélection d'anticorps monoclonal, et fabrication et utilisation de celui-ci Download PDF

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Publication number
WO2008128144A2
WO2008128144A2 PCT/US2008/060165 US2008060165W WO2008128144A2 WO 2008128144 A2 WO2008128144 A2 WO 2008128144A2 US 2008060165 W US2008060165 W US 2008060165W WO 2008128144 A2 WO2008128144 A2 WO 2008128144A2
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protein
cell
domain
transgenic
antibody
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PCT/US2008/060165
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WO2008128144A3 (fr
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Shuang Zhang
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Shuang Zhang
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the invention is in the field of analysis of cell populations and cell separation and the compositions obtained thereby. More particularly, the invention concerns analysis and separation of antibody-specific B cells based on primary labeling of cells with their secreted products through capture of these products by a specific binding partner for the product anchored or bound to the cell surface.
  • myeloma is a B-cell cancer
  • myeloma is a B-cell cancer
  • This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol, electroporation or, of historical importance, infection with some virus.
  • the fused hybrid cells (called hybridomas) , being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. Cloning and selecting lines with desired binding activities is a laborious procedure that usually relies on limiting dilution microculture .
  • the hybridomas are sufficiently diluted to ensure clonality and grown.
  • the antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA) or immuno-dot blot, and the most sensitive one is picked out.
  • the time frame required for developing a monoclonal antibody using this approach is generally 3 to 9 months .
  • One approach is to place the cell in a medium that inhibits the rate of diffusion from the cell.
  • the typical method has been to immobilize the cell in a gel-like medium (agar) , and then to screen the agar plates for product production using a system reliant upon blotting, for example western blots.
  • the U.S. Pat. 4,510,244 provided a method for isolating specific antibody hybridomas from a hybridoma cell mixture employing antigen-conjugated labeled microspheres and a label activated cell sorter. By selecting for labeled cells which produce light scatter and low red autofluorescence, viable single cells can be isolated and cloned which produce the desired antibodies.
  • Another approach is to couple the cells at their surface to a specific binding partner for the product and allowing the product to be captured by the specific binding partner as it is secreted and released.
  • the product-labeled cells can then be further coupled to suitable labels, if desired, and separated according to the presence, absence, or amount of product.
  • the U.S. Pat. 6,376,170 provided a method for the identification and clonal isolation of antibodies that bind to unique epitopes.
  • the method is based on the use of antibodies as solid phase capture reagents to bind a known capture antibody epitope, thereby precluding the capture antibody epitope from being presented to a population of antibodies to be screened.
  • the method is particularly suited for screening libraries of cloned antibodies, such as phage display combinatorial antibodies .
  • the U.S. Publication 20060263801 disclosed methods of identifying ligands that are internalized into a cell.
  • the methods typically involve i) contacting the cell with a reporter non-covalently coupled to a ligand; ii) dissociating the reporter from the ligand and removing dissociated reporter from the surface of the cell; and iii) detecting the reporter within said cell (if any is present) where the presence of the reporter within said cell indicates that the ligand binds to an internalizing receptor and is internalized.
  • the U.S. Publication 20060073095 disclosed methods and compounds that relate to screening and selection of monoclonal antibodies specific for antigens in heterogeneous antigen mixtures.
  • Antibody-secreting cells such as hybridomas are modified to make them capable of directly binding antigens by capturing their secreted antibody products onto their surface membranes in appropriate binding density and orientation.
  • Selectivity of binding to novel or desired antigens is achieved by first reacting the antigen mixtures affixed to a solid substrate with a polyclonal antibody library that prevents access to the majority of antigens or epitopes other than those that are novel or desired.
  • the present invention provides a DNA that enables a B- cell to bind to its own secreted antibody.
  • the DNA comprises (1) a coding region encoding a chimeric protein that comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of the antibody (usually IgG); and (2) a promoter that drives the expression of the protein.
  • the promoter is an inducible and/or B-cell (especially plasma cell) specific promoter.
  • the binding domain might be derived from a secondary antibody that is generated against the non-variable region of the primary antibody, from Fc-binding domain of Fc receptors (FcRs) , or from the Fc-binding domain of bacterial Fc-receptors, such as Streptococcus Protein G, Staphylococcus aureus Protein A, and Peptostreptococcus magnus Protein L.
  • FcRs Fc receptors
  • bacterial Fc-receptors such as Streptococcus Protein G, Staphylococcus aureus Protein A, and Peptostreptococcus magnus Protein L.
  • the chimeric protein can further have a detectable domain.
  • the preferred detectable domain is a fluorescent protein, such as the monomeric red fluorescent protein (mRFP) .
  • the detectable domain can be either extracellular or intracellular.
  • the present invention also provides a method for associating a B-cell with its own secreted antibody, comprising the steps of: a) expressing a protein in the B-cell, wherein the protein comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of the antibody; and b) incubating said B-cell from step a) with antigen labeled with a label moiety.
  • the present invention also provides a transgenic myeloma cell which expresses a transgene integrated into its genome, wherein the transgene comprises a DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to the non-variable region of an antibody.
  • the present invention further provides a transgenic animal which expresses a transgene integrated into its genome, wherein the transgene comprises DNA encoding a protein having at least one transmembrane domain and an extracellular domain, wherein said extracellular domain comprises at least one binding domain that binds to the non-variable region of the animal antibody.
  • the transgenic animal is preferably mouse or rat.
  • the present invention also provides methods to isolate B- cells expressing antibodies against specific antigen by using the transgenic myeloma cell or the transgenic animal disclosed here.
  • Antigen might be either labeled in vitro with materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles, or expressed as a fusion protein with a detectable domain, such as a fluorescent protein.
  • the labeled cells may then be separated or detected using standard cell sorting techniques based on the particular properties of these labels. Such techniques include flow cytometry, magnetic separation, high gradient magnetic separation, centrifugation, and the like.
  • Fig. IA is a schematic diagram showing the plasmids used in the present invention
  • Fig. IA is a schematic diagram showing two inducer plasmids with different promoters
  • Fig. IB is a schematic diagram showing two different reporter plasmids
  • Fig.2 is a schematic diagram showing the standard procedure to generate transgenic myeloma cell line with both inducer and reporter plasmids integrated in its genome, as disclosed in the present invention
  • Fig.3 is a schematic diagram showing one preferred embodiment of using the transgenic myeloma cells to isolate antibody-producing B-cells as disclosed in the present invention
  • Fig.4 is a schematic diagram showing the standard procedure to produce transgenic mice with both inducer and reporter plasmids integrated in their genome, as disclosed in the present invention.
  • Fig.5 is a schematic diagram showing one preferred embodiment of using the transgenic mouse to isolate antibody-producing B-cells as disclosed in the present invention .
  • One plasma cell produces one specific mAb at very high rate, up to 10000 molecules/second.
  • a cell X produces a mAb Y. It is conceivable that the Y is at its highest concentration surrounding X. If an antibody- binding protein Z is on the surface of X, Z is most likely to bind Y, but not to antibodies made by other cells, as long as the amount of Z is far less than that of Y surrounding X. In addition, Z might bind to Y when they co-exist in ER and/or Golgi, before they reach to the EM.
  • a transgene encoding a chimeric protein is integrated into the genome of a myeloma cell or an animal.
  • the chimeric protein comprises at least one transmembrane domain and an extracellular domain, wherein the extracellular domain comprises at least one binding domain that binds to non-variable region of an antibody.
  • an inducible promoter is preferred.
  • One of the inducible expression systems is the tetracycline (Tet) regulatory system, which is based on the unusual specificity of interaction between the Tet repressor (TetR) and its specific DNA binding site, the tet operator (tetO) and between Tet repressor and its inducer, particularly Doxycycline (Dox) .
  • Tet Tetracycline
  • Tet-Off Tet control systems.
  • Dox prevents binding of tTA to the tetO sequence within P tet/ and thus abolishes transcription.
  • the rtTA reverse tetracycline controlled transactivator
  • Tet-On requires Dox for binding to and activation of P te t-
  • Tet control system has been widely and successfully applied in many cell systems and animal systems, including mouse. Transgene expression in these animals is exclusively dependent on the administration/absence of tetracycline or tetracycline derivatives (such as Dox) .
  • Tet-Off system or other inducible systems might be used as well .
  • Tet-On systems in which some use two plasmids and some use one plasmid, the commercial Tet-On system from Clontech is used and described here.
  • the inducer plasmid pTet-ON is the first half of the Clontech Tet-On system, in which the reverse tetracycline transactivator (rtTA) is expressed under the CMV promoter.
  • the CMV promoter originated from human cytomegalovirus immediate-early gene, is one of the most widely used promoters in mammalian expression systems.
  • the CMV promoter induces high-level constitutive expression in a variety of mammalian cell lines, including mouse plasma cells.
  • a B-cell (especially plasma-cell) specific promoter could be used instead of the CMV promoter.
  • One of the plasma-cell specific promoters is from the Blimp-1 gene (Prdml), whose expression is high in plasma cells but either low or absent in other B cells.
  • Prdml Blimp-1 gene
  • a mouse Blimp-1 promoter fragment spanning -3500 to +1 is generated by PCR and is used to replace the CMV promoter in the pTet-ON plasmid.
  • pTet-ON is used in the following description unless stated otherwise.
  • Fig. IB shows two different reporter plasmids, derived from the second half of the Clontech Tet-on system.
  • the chimeric protein consists of an N- terminal signal peptide, a Protein G' domain and a transmembrane domain.
  • a signal peptide is a short peptide chain that directs the post-translational transport of a protein.
  • An ER signal peptide is the best characterized signal peptide. It exists at the amino terminal of a protein. The protein is guided to the ER by a signal-recognition particle (SRP) .
  • SRP signal-recognition particle
  • a typical signal peptides for transport to the ER i s H2N-Met-Met- Ser- Phe-Val - Ser-Leu-Leu-Leu-Val -Gly- I le-
  • an N-terminal signal peptide initiates translocation, but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is translocated cross the ER membrane.
  • This stop-transfer- signal anchors the protein in the membrane after the ER signal sequence has been released from the translocator and has been cleaved off.
  • the transmembrane (TM) domain can function as the stop-transfer signal.
  • Proteins that bind to the constant (Fc) region of IgG have been found on the surface of a variety of staphylococci and streptococci bacteria. Among them, Protein A from Staphylococcus aureus, Protein G from Streptococcus and Protein L from Peptostreptococcus magnus are the best known and well studied.
  • Protein G binds to mammalian IgGs mainly through Fc regions.
  • Native Protein G has 3 IgG binding domains and also domains for albumin and cell-surface binding.
  • Protein G' (NCBI ACCESSION: CAA37410), a part of Streptococcus protein G, contains only three Fc-binding domains.
  • Protein G has greater affinity than Protein A for most mammalian IgGs, especially for certain subclasses including human IgG3, mouse IgGl and rat IgG2a.
  • Protein G does not bind to human IgM, IgD and IgA. Both modified Protein G and Protein A have been used for purification of mammalian monoclonal and polyclonal IgGs.
  • Protein L is an immunoglobulin-binding protein that binds to immunoglobulin kappa light chains without interfering with the antigen-binding site and binds a wider range of Ig classes and subclasses than other antibody-binding proteins such as Protein A or Protein G. Protein L binds to all classes of Ig (i.e., IgG, IgM, IgA, IgE and IgD) . Protein L also binds single chain variable fragments (Scfv) and Fab fragments.
  • pTRE-SpGTMR has an additional detectable domain.
  • the detectable domain is a monomeric red fluorescent protein (mRFP) .
  • the detectable domain can be either extracellular or intracellular.
  • the mRFP locates at the intracellular region of the chimeric protein.
  • pTRE-SpGTM is used in the following description unless stated otherwise.
  • an artificial intron is added to stimulate the transport of mRNA out of the nucleus.
  • the artificial intron could be placed at either end of the protein-coding region, preferably at the 3' end.
  • myeloma cell line that has both inducer and reporter plasmids integrated into its genome is well documented (see Clontech Tet-On® Advanced Inducible Gene Expression System User Manual PT3898-1) .
  • myeloma cells are first transfected with the inducer plasmid, which also contains a Neo r selectable maker.
  • G418-resistant transfectants are further screened by transient transfections with pTRE- Tight-Luc for clones with low background and high Dox- dependent induction of luciferase activity. Selected cells are then co-transfected with the reporter plasmid and a Linear Hygromycin Marker.
  • Hygromycin-resistant transfectants are further screened for clones with low background and high Dox-dependent induction of the chimeric protein.
  • the resulting clones are double-stable transgenic myeloma cell line, which has both inducer and reporter plasmids integrated into its genome.
  • hybridoma cells are produced by fusing the transgenic myeloma cells with B-cells isolated from spleen and lymph nodes of an immunized animal. The hybridoma cells are then grown in a culture medium containing Dox at the concentration between 10-lOOOng/ml for 24-48 hours to induce the chimeric protein expression. After induction, the hybridoma cells are incubated with antigen-GFP fusion protein. Fluorescent-activated cell sorting (FACS) is performed to isolate cells with labeling intensity above a given threshold.
  • FACS Fluorescent-activated cell sorting
  • the reporter plasmid pTRE-SpGTMR is favored over pTRE-SpGTM since the sorting criteria could be set to base upon the ratio of GFP/mRFP.
  • the endogenous mRFP provides a base for comparing the binding affinity of different antibodies to the antigen.
  • antigen-GFP fusion protein is used in the above description, antigen might also be labeled in vitro with materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles. In the case that the antigen is not a protein, in vitro labeling becomes necessary. Such labeling techniques are well documented in the field to which this invention belongs.
  • FACS cell sorting/detecting techniques
  • Such techniques including flow cytometry, magnetic separation, high gradient magnetic separation, centrifugation, and the like, are all well documented in the field to which this invention belongs.
  • Transgenic animals are genetically modified animals into which cloned genetic material has been experimentally introduced.
  • the cloned genetic material is referred to as a transgene.
  • the nucleic acid sequence of the transgene is integrated at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the recitations in U.S. Pat. Nos. 5,489,743 and 5,602,307.
  • mice and rats are preferred.
  • linearized inducer or reporter plasmid is injected into the pronuclei of fertilized mouse eggs.
  • the plasmid is incorporated into random loci, usually in head-to-tail concatemers consisting of varying numbers of copies.
  • the eggs are transferred to the oviducts of pseudopregnant foster mothers, generated by mating females with vasectomized males so the females do not produce any fertilized embryos of their own.
  • the offspring resulting from injected eggs are genotyped using DNA extracted from a small piece of tissue cut from the tip of the tail.
  • the mice that do carry the transgene are called founders. Offspring from each founder are tested for transmission and expression of the transgene.
  • mice that have the transgene on one chromosome are heterozygous because they do not have a corresponding allele on the other chromosome. These heterozygous can further cross to produce homozygous.
  • Heterozygous or homozygous inducer plasmid transgenic mice were mated with heterozygous or homozygous reporter plasmid transgenic mice to generate double-transgenic progeny. Double transgenic mice are confirmed by southern blotting or PCR analysis with genomic DNA isolated from tail biopsies.
  • a double transgenic mouse is first immunized with a target antigen. Once the titer of the antibody is satisfied, the mouse is administered with Dox (2g/l in 2.5% sucrose) in light-protected bottles in drinking water every 24 hours for 48-72 hours. The mouse is then sacrificed and B-cells from its spleen and lymph nodes are isolated. The B-cells are incubated with labeled antigen and are sorted accordingly as described above. Sorted B-cells can be further characterized as described above .
  • Dox induction can be performed after B- cells are isolated from the spleen and lymph nodes of the immunized mouse.
  • Dox concentration should be between 10-lOOOng/ml and the induction lasts 24-48 hours .
  • the Fc region (Fragment, crystallizable) , is derived from the stem of the "Y, " and is composed of two heavy chains that each contributes two to three constant domains (depending on the class of the antibody) . Fc binds to various cell receptors and complement proteins. In this way, it mediates different physiological effects of antibodies (opsonization, cell lysis, degranulation of mast cells, basophils and eosinophils and other processes) .
  • Each end of the forked portion of the "Y" on the antibody is called the Fab region (Fragment, antigen binding) . It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope—the antigen-binding site—at the amino terminal end of the monomer. The two variable domains bind the epitope on their specific antigens.
  • Fc receptor is a protein found on the surface of certain cells; including natural killer cells, macrophages, neutrophils and mast cells; that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (Fragment, crystallizable) region. FcRs bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity.
  • FcR Fc receptor-gamma receptors
  • Fc ⁇ R Fc-gamma receptors
  • Fc ⁇ R Fc-alpha receptors
  • Fc ⁇ R Fc-epsilon receptors
  • the 780bp fragment mIL2/pG ' (Fc) /TM (hCD2) (SEQ ID NO. 1) was first in vitro synthesized and assembled, which consists of optimized DNA sequence encoding the IL2 signal peptide, the Protein G' Fc binding domain, the hCD2 transmembrane domain, and a C-terminal short end.
  • the fragment was cut with Kpnl/Sall and was then inserted into the vector pTRE-Tight (Clontech Laboratories, Inc., Mountain View, California, USA) at the same cloning sites.
  • the junctions of the resulting pTRE-SpGTM plasmid sequence were verified by automated sequencing.
  • mRFP SEQ ID NO. 2
  • the PCR fragment was cut with NgoMIV/Sall and was inserted into the NgoMIV/Sall sites within the pTRE-SpGTM.
  • the junctions of the resulting pTRE-SpGTMR plasmid sequence were verified by automated sequencing.
  • the vector pTet-On-Advanced (Clontech Laboratories, Inc., Mountain View, California, USA) was cut with BsrGI/SacI to remove the CMV promoter and was then ligated with the mouse Blimp-1 promoter fragment spanning -3500 to +1 (SEQ ID NO. 3) , which was generated from mouse genomic DNA by PCR.
  • the junctions of the resulting P B i imp -rtTA plasmid were verified by automated sequencing.
  • the pTet-ON (or P B i imp -rtTA) transgene was generated by excising a BsrGI/Hindlll fragment from the pTet-ON (or P B i imp -rtTA) plasmid, and the P tight -SpG' TM (or P tight -SpG' TMR) transgene was generated by excising an Xhol fragment from the P t ig ht -SpG' TM (or P tight -SpG' TMR) plasmid.
  • Transgenic mice were produced by microinjection of gel-purified transgene DNA into single-cell preimplantation FVB mouse embryos using standard methods.
  • transgenic mice genomic DNA was isolated from tail biopsies, and transgenes were amplified by PCR. Transgene expression was evaluated and confirmed by western blotting analysis. Heterozygous or homozygous pTet-ON (or P Blimp -rtTA) transgenic mice were mated with heterozygous or homozygous P t i ght ⁇ SpG' TM (or P tight -SpG' TMR) transgenic mice to generate double-transgenic progeny.
  • mice were immunized using standard protocol.
  • Dox (2g/l in 2.5% sucrose) was administered to mice 3 days before sacrifice in light-protected bottles in drinking water with sucrose supplementation to combat taste aversion from Dox.
  • B-cells were isolated and prepared from their spleen according to the standard protocol.
  • Bibilalr TA Flickinger MC. A Model of Interorganelle Monoclonal Antibody Transport and Secretion in Mouse Hybridoma Cells. Biotechnology and Bioengineering 38:767- 80.
  • Houdebine LM The Production Of Pharmaceutical Proteins From The Milk Of Transgenic Animals. Reprod Nutr Dev. 35:609-17.

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Abstract

L'invention concerne un ADN codant une protéine chimère qui comprend un domaine transmembranaire et un domaine de liaison Fc extracellulaire et fournit des cellules de myélome transgéniques et des souris transgéniques ayant l'ADN intégré dans leur génome. L'invention propose également des procédés de préparation de cellules de myélome transgéniques et de souris transgéniques exprimant la protéine chimère. L'invention propose en outre des procédés d'utilisation des cellules de myélome transgéniques et des souris transgéniques pour faciliter l'isolation des cellules B produisant des anticorps monoclonaux. De manière spécifique, les cellules produisant des anticorps sont conçues de sorte à exprimer une protéine chimère qui comprend un domaine transmembranaire et un domaine G' de protéine extracellulaire, libérant les cellules liées aux anticorps. Un tel caractère simplifie la sélection ultérieure de cellules produisant des anticorps monoclonaux.
PCT/US2008/060165 2007-04-15 2008-04-14 Système de sélection d'anticorps monoclonal, et fabrication et utilisation de celui-ci WO2008128144A2 (fr)

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Cited By (3)

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US20110041197A1 (en) * 2009-08-14 2011-02-17 Regeneron Pharmaceuticals, Inc. Promoter-Regulated Differentiation-Dependent Self-Deleting Cassette
WO2016079739A3 (fr) * 2014-11-20 2016-07-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Compositions et procédés de production de polypeptides présentant un schéma de glycosylation modifié dans des cellules végétales
CN114134163A (zh) * 2021-11-05 2022-03-04 中国农业大学 单个抗原特异性转基因杂交瘤细胞筛选方法

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Cited By (15)

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