WO2007106448A1 - Procédés de production d'anticorps chez des souris génétiquement modifiées - Google Patents

Procédés de production d'anticorps chez des souris génétiquement modifiées Download PDF

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Publication number
WO2007106448A1
WO2007106448A1 PCT/US2007/006239 US2007006239W WO2007106448A1 WO 2007106448 A1 WO2007106448 A1 WO 2007106448A1 US 2007006239 W US2007006239 W US 2007006239W WO 2007106448 A1 WO2007106448 A1 WO 2007106448A1
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antibody
mouse
antibodies
engineered
antigen
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PCT/US2007/006239
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Peter G. Seferian
Gregory M. Landes
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Lexicon Genetics Incorporated
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • A01K67/0276—Knock-out vertebrates
    • 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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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
    • 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
    • A01K2267/02—Animal zootechnically ameliorated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters

Definitions

  • the present invention relates to methods for generating antibodies in genetically engineered mice.
  • Antibodies generated by a method of the invention have particularly desirable specificities and affinities for a desired target.
  • antibodies generated by a method of the invention are particularly suitable for use with a host, patient, or allotype of interest.
  • Antibodies are components of the immune system capable of specifically binding an antigen. In animals, including humans, antibodies play a critical role in the body's defense against substances that are foreign to the body. Due to the specificity with which antibodies can bind their target antigen, they have been used in a variety of applications. For example, antibodies can be obtained and tested for their ability to bind diseased tissue in a patient. Where antibodies are used to target a molecule or tissue in a patient, they have to be sufficiently selective for the desired target, so they recognize the target without cross-reacting with other structures. Antibodies for use in patients preferably have a high and specific affinity for the target of interest.
  • an antibody used in a patient must not be recognized as foreign by the patient's immune system in order to avoid adverse effects.
  • dual goals, high specificity and the ability to avoid an unduly adverse patient immune response, are often difficult to accomplish and typically very large numbers of antibodies must be screened to identify one that sufficiently satisfies these disparate goals.
  • Present techniques for identifying and producing therapeutic or diagnostic antibodies require substantial screening efforts to identify a single antibody with desirable characteristics. Therefore, techniques that facilitate faster and more economical identification of antibodies that have the desired specificities, while avoiding the elicitation of an adverse immune response from patients, would be highly desirable. This is especially true in light of the potential use of antibodies in many therapeutic and diagnostic applications. The current invention provides such techniques.
  • the present invention provides in certain embodiments methods for raising antibodies with desirable characteristics.
  • antibodies generated using the methods of the invention have a high binding affinity for their target antigen, a high binding selectivity for their target antigen, and/or elicit no more than acceptable responses with a patient's immune system, and preferably no or substantially no response.
  • Methods of the current invention use mice to raise antibodies, preferably mice in which the target antigen of interest is absent or substantially absent.
  • the target antigen has been depleted from a mouse used in methods of the invention, preferably by deleting, disrupting, and/or inactivating a gene that results in the production or expression of the target antigen in the mouse.
  • the target antigen is encoded by a gene in a mouse used in the methods of the invention and said gene has been knocked out, for example, by homologous recombination, gene trapping, or some other kind of mutagenesis.
  • a mouse used in the methods of the invention was engineered to have and/or express one or more human genes, gene segments or exons encoding a human antibody, antibody chain (heavy and/or light) and/or antibody fragment.
  • a mouse used in the methods of the invention was engineered to have and/or express one or more human genes, gene segments or exons that encode one or more heavy and/or light chains of an antibody, preferably for one, more than one and/or all allotypes found among human antibodies.
  • mice used in the methods of the invention were engineered to have and/or express antibody genes, gene segments or exons from another species of interest, for example, any mammalian species.
  • a mouse used in the methods of the invention was engineered in both aforementioned ways, i.e., depleting the antigen from it and introducing a human antibody, antibody chain, and/or fragment into the mouse.
  • an antibody obtained using the methods of the invention is matched to the allotype of a patient in which it is to be used.
  • a mouse engineered in accordance with the invention is used to prepare a display library, such as but not limited to phage, bacterial, ribosome and RNA display libraries.
  • a mouse used to prepare a phage-display library is engineered to deplete or substantially deplete the target antigen from said mouse, and/or is engineered to introduce a human antibody, antibody chain, and/or fragment into the mouse.
  • cells from a mouse engineered in accordance with the invention are used to prepare a phage-display library, for example, lymphocytes or any other cell type capable of displaying an antibody, antibody fragment, or antibody chain.
  • a phage-display library according to the invention is used to generate an antibody of interest.
  • the present invention describes methods for raising antibodies.
  • antibodies obtained using the methods of the invention have a high affinity for their target antigen and, in certain other embodiments, do not result in unacceptable side effects due to reactivity of the antibodies with the immune system of a patient in which the antibodies are used, for example, for therapy or diagnostics.
  • Antibodies are typically elicited in animals by introducing the antigen of interest into the animals and identifying an immune response in the animals. Resulting antibodies are then screened for their affinity for the target antigen.
  • the usefulness of antibodies typically increases with their affinity for the antigen.
  • the strength of the animals' immune response is typically indicative of the affinity of antibodies obtained.
  • the closer in structure an antigen is to molecules that naturally occur within an animal the weaker the immune reaction obtained to those antigens, as such molecules are recognized as "self and do not elicit a robust immune response.
  • antibodies are best obtained in animals that have had no developmental exposure to the antigen of interest or any similar structure.
  • Compatibility with a patient's immune system and effective binding of a target antigen are typically distinct qualities of an antibody that must be present to achieve a safe and effective antibody for use in the patient.
  • Antibody therapeutics are typically obtained in a system that is alien to the patient and thus alien to the patient's immune system.
  • antibodies are adapted to the preferences of the patients' species. For example, humanized or human antibodies are a preferred choice for therapeutic and diagnostic applications in humans.
  • antibodies can also be distinguished on the basis of structural features that are related to antigen binding, and features that are unrelated to the antibodies' abilities to recognize their target antigens.
  • antibody constant regions contain allelic markers, genetically inherited structural features that are inherited in a Mendelian fashion and known as allotypes.
  • the immune system of an individual patient typically contains and is compatible with certain antibody allotypes, but not others. Allotypes are antigenic determinants specified by allelic forms of the Ig genes. Such markers have been associated with disease susceptibility and vary between human ethnic groups (Matsumoto, H., 1968, Japanese J of Hum Gen 13 (1) 10-19; Johnson WE, Kohn PH, Steinberg AG 3 1977, CHn Immunol Immunopathol 7(1):97-113; Grubb R, Matsumoto H, Sattar MA, 1988 Arthritis Rheum.
  • IGHG immunoglobulin heavy G-chain
  • Gm haplotypes namely 1/Gm(b,f,n), 2/Gm(b,f,-n), 3/Gm(g,a,n) and 4/Gm(g,a,-n).
  • an antibody may be effective in its ability to recognize an antigen but still be therapeutically and diagnostically useless in a patient, and even dangerous, due to allotype incompatibilities.
  • a given antibody is therefore typically compatible for use in some patients, but not in others, due to varying compatibilities of the antibody's allotype with the immune system of the different patients.
  • the allotype of an antibody made using the methods of the invention may be determined and/or knowing the allotype of an antibody made using the methods of the invention and the allotype found in the immune system of a patient and/or the presence of a preexisting anti-allotype activity, one may identify antibody-patient combinations that are most compatible and least likely to result in unacceptable responses. Furthermore, in some cases while the absence of foreign allotypic constant region determinants may not eliminate the potential for an anti- idiotypic response to develop, its absence may delay that response for at therapeutically significant amount of time.
  • Methods of the current invention in certain embodiments, raise antibodies in animals that do not comprise the antigen of interest and therefore do not recognize the antigen as "self (have not been tolerized).
  • the animals do not comprise the protein, peptide, glycoprotein, nucleic acid, oligonucleotide, polynucleotide, or other molecular entity that comprises the antigen of interest.
  • the animals in which antibodies are elicited are genetically engineered to lack, or substantially lack, the antigen of interest, or to comprise the antigen of interest in amounts that are substantially less than in an animal of that species that has not been so genetically engineered.
  • mice Animals so engineered are referred to herein as "depleted animals" (or mice, if mice are used, or any other species).
  • antibodies are obtained in methods of the invention by exposing the immune system of a depleted animal to the antigen of interest.
  • animals used to raise antibodies are engineered to express genes, gene segments, or one or more exons encoding one or more human antibodies (or whichever other species in which the antibodies elicited are to be used), one or more human antibody chains (light chains and/or heavy chains), and/or one or more segments or fragments of a human antibody and/or antibody chain.
  • Animals so engineered are referred to herein as "antibody-engineered animals” (or mice, if mice are used, or any other species), in these embodiments, the antibodies obtained using methods of the invention are meant for use in a human patient.
  • animals are engineered to express human antibody heavy chains of one or more allotypes, and most preferably of a single allotype, of human antibody heavy chains.
  • methods of the invention raise antibodies in animals, for example, mice, which are depleted animals and which are also antibody-engineered animals.
  • Animals so engineered are referred to herein as “dual-engineered animals” (or mice, if mice are used, or any other species).
  • an animal engineered in accordance with the invention is used to prepare a display library, such as but not limited to a phage-display library, a yeast surface display library, a bacterial display library, a ribosome display library and an mRNA display library.
  • a depleted animal, an antibody-engineered animal, and/or a dual-engineered animal is used to prepare a phage-display library.
  • cells from a depleted, an antibody-engineered, and/or dual-engineered animal are used to prepare a phage-display library, for example, lymphocytes or any other cell type capable of displaying an antibody, antibody fragment, or antibody chain.
  • a phase-display library in accord with the invention can be screened to identify an antibody of interest, for example, by contacting said library with an antigen or epitope of interest.
  • the present invention also provides a business method for providing antibodies.
  • a business method of the invention comprises receiving antigenic and/or allotypic information pertinent to an antibody application of interest, designing and/or engineering an animal, e.g.
  • the business method may further comprise recommending a particular antigen and/br allotype for use in generating an antibody using the methods of the invention.
  • Methods of the current invention raise antibodies, in certain embodiments, in mice. More preferably, antibodies are elicited in mice that were genetically engineered. Mice used in methods of the invention may be depleted mice, antibody-engineered mice, and/or, most preferably, dual-engineered mice.
  • a depleted mouse in certain embodiments, comprises a significant reduction in the amount of one or more molecular components comprising said mouse. In certain embodiments, said reduction is at least 50 percent compared to a wild-type mouse, more preferably at least 70 percent, or at least 90 percent, or at least 95 percent, and most preferably 100 percent.
  • a depleted mouse for use in a method of the invention is generated, in certain embodiments, by engineering a genomic mutation into a mouse, for example, by gene targeting or gene trapping or any other method capable of mutating a segment of the genome in a mouse.
  • a depleted mouse in certain embodiments, may be homozygous or heterozygous for the engineered genomic mutation.
  • the genomic mutation engineered into a mouse used in a method of the invention results in a significant reduction in the amount of one or more molecular components comprising said mouse.
  • the mutation may disrupt a gene encoding a protein, or leading to the expression of one or more proteins, and therefore the protein, or proteins, would no longer be expressed or generated, at least in an amount that is significantly reduced when compared to the amount found in a wild-type mouse.
  • One method for generating a depleted mouse is by gene trapping. Background on generating a depleted mouse, including cloning, tissue culture, mutagenesis and generating mice, is also described in U.S. Patent Application Nos.
  • a depleted mouse is analyzed to determine which molecule is significantly reduced in said mouse, for example, a protein encoded by a gene disrupted by a gene trap vector used to generate said mouse.
  • An antibody-engineered mouse in certain embodiments, is capable of generating a human (or another species of interest) antibody, antibody chain (for example, light and/or heavy chain) and/or antibody fragment.
  • An antibody-engineered mouse in certain embodiments, may be generated by replacing parts of the genome of the mouse with sequences encoding human antibody, antibody chains (for example, light and/or heavy chain) and/or antibody fragments.
  • an antibody-engineered mouse is only capable of generating antibodies resulting from the engineered mutation but not mouse antibodies of the type corresponding to those of the engineered mutation. Background on how to prepare a mouse capable of raising human antibodies and on raising human antibodies in mice is also described in U.S. Patent Nos.
  • a mouse is used in the methods of the invention that is engineered to have both types of mutations, i.e., a mutation engineered into a depleted mouse and a mutation engineered into an antibody-engineered mouse.
  • a dual-engineered mouse is most preferable for use in a method of the invention to raise antibodies because it is more likely than a wild-type or solely antibody-engineered mouse to yield high affinity antibodies because the antigen is present in the mouse in significantly reduced amounts and therefore the mouse is more likely to recognize the antigen as foreign.
  • Another reason for preferring a dual-engineered mouse in the methods of the invention is that it is capable of generating antibodies that are more suitable for applications, including therapeutics and diagnostics, in a species of interest, for example, in humans, because the mouse was engineered to be capable of generating antibodies specific to the species of interest, for example, human.
  • the present invention describes the generation of human or chimeric-human antibodies in knockout animals.
  • the described knockout alleles are engineered in embryonic stem cells that have been derived from animals that have been previously engineered to functionally replace the endogenous antibody heavy and/or light (kappa and/or lambda) chain encoding genes with human antibody heavy and/or light chain genes or portions thereof, (e.g., CDR regions, see generally Lonberg, 2005, Nat. Biotech. 23(9): 1117-1125).
  • a genetically engineered mutation found in a depleted mouse is generated in embryonic stem (ES) cells that are homozygous for human antibody heavy and/or light chain genes.
  • ES embryonic stem
  • the resulting ES cells can be used, in certain embodiments, to generate a mouse that is dual-engineered.
  • dual-engineered mice when bred to the homozygous state, will be able to mount a "naive” or “non-tolerized” immunologic response to introduced “human” antigens that directly results in the production of "human” antibodies.
  • This feature would remove the need to "humanize” antibodies produced in the described dual-engineered animals (e.g., mice) (which typically have an endogenous repertoire of antibody heavy and light chain genes that often require further engineering to increase their suitability for human therapeutic use).
  • the ES cells contain human antibody heavy and/or light chain genes that are derived from specific isotypes (IgA(I & 2), IgD, IgE, IgG(l-4), and IgM) and/or allotypes.
  • ES cells can be prepared from genetically engineered animals comprising human IgGl genes from Glm(f), Glm(x), Glm(z), or Glm(a); human IgG2 genes from G2m(n); human IgG3 genes from G3m(bO); G3m(bl), G3m(b4) 3 G3m(b5); G3m(c3), G3m(c5); G3m(g), G3m(g5), G3m(s); G3m(t), G3m(u), G3m(v); human IgA2 genes A2m(l), A2m(2) and human IgE genes Em(I) and/or human kappa light chain genes Km(I) , Km(2) or Km(3) allotypes and any and all practical combinations and permutations of allotypes (some allotypic combinations are the result from the diploid nature of the genome and because multiple allotypes can be present on
  • the described human allotypes can be present in a homozygous or heterozygous state or otherwise engineered to provide for any desired combination of human heavy and human light chain allotypes in the antibodies produced in depleted animals that have been genetically engineered using these antibody- engineered ES cells.
  • a dual-engineered mouse of the invention is further engineered to replace other components of the mouse immune system with an analogous component of the human immune system to further assist in the generation of human or humanized antibodies in the mouse.
  • replacements include, but are not limited to, a T cell receptor, a cytokine gene, a gene encoding a monocyte-specific function, a gene encoding a function involved in antigen presentation, and any other gene involved in the immune response that leads to the generation of antibodies.
  • a dual-engineered mouse is generated by first preparing a depleted mouse and then introducing the mutation or mutations rendering the mouse also antibody-engineered, and thus a dual-engineered mouse.
  • a dual-engineered mouse is generated by breeding a depleted mouse with an antibody-engineered mouse to eventually obtain a dual-engineered mouse.
  • in vitro protein evolution, selection and screening can be accomplished using display libraries, such as phage, bacterial, ribosome, RNA or on-bead display libraries, in which proteins are physically linked to their encoding sequence.
  • a phage-display library may be generated from an animal (for example, a mouse) of the invention.
  • a phage-display library of the invention is generated using cells from a mouse of the invention, for example, lymphocytes or any cell type capable of displaying an antibody, antibody fragment or antibody chain (preferably a human or humanized sequence).
  • Any method known in the art can be used to generate a phage-display library of the invention. Examples of generating a phage-display library of the invention are taught in U.S. Patent Nos. 5,679,526; 6,555,310, 6,794,132, and in U.S. Patent Application Nos. 20030104477; 20060068421, all of which are incorporated herein by reference in their entirety for all purposes.
  • display libraries may be generated from an animal (for example, a mouse) of the invention.
  • Additional embodiments include but are not limited to, yeast surface display libraries (as taught in U.S. Patent Nos. 6,300,065; 6,423,538; 6,509,893; 6,699,658; 6,696,251, all of which are incorporated herein by reference in their entirety for all purposes), bacterial surface display libraries (as taught in U.S. Patent Nos. 5,348,867; 5,866,344, both of which are incorporated herein by reference in their entirety for all purposes), ribosome display libraries (as taught in U.S.
  • kits used to determine one or more allotypes present in a given patient for example, by using standard antibody assays or polynucleotide based screening of the patient's genome at sites of allotypic variation (via PCR, sequencing, RFLP analysis, high-stringency hybridization, etc.)
  • assays include, but are not limited to, those described in WO 2003062825 Al and U.S. Patent Nos: 05,665,356 and 6,589,756, which are herein incorporated by reference in their entirety for all purposes).
  • Certain other embodiments of the invention relate to methods for determining one, two, three, four, five, or more allotypes in one or more patients or a patient population (allotyping of a patient). Certain other embodiments relate to methods for detecting one or more anti-allotypic antibodies in a patient or patient population, preferably to lower the risk of anaphylaxis in said patient during antibody therapy by selecting a therapeutic antibody allotype that is compatible with (in other words, that does not react with) anti-allotypic antibody present in said patient.
  • Certain other embodiments of the invention relate to selecting therapeutic antibodies that have a compatible or suitably matching allotype or allotypes for a patient or patient population, for example, by selecting an antibody that has been generated using animals (bearing in mind that the actual therapeutic monoclonal antibodies are typically obtained by manipulating antibody producing cells taken from the described animals for subsequent monoclonal generation, identification, and production) that have been engineered to express human antibodies of an allotype compatible with the natural repertoire of allotypes in a patient or patient population and avoiding any preexisting anti-allotypic responses.
  • the "allotype-compatible" animals are animals that have been engineered to knockout the endogenous ortholog of the protein or molecule being targeted for antibody generation.
  • an antibody of the present invention is specific to an antigen or epitope which is significantly reduced or absent in a depleted mouse and/or dual-engineered mouse of the invention (depleted antigen).
  • a depleted antigen is absent in a homozygote of a depleted mouse of the invention, but not absent in a wild-type of the same mouse line.
  • a depleted antigen is synthesized in a cell of a wild-type mouse, but not in a cell of a homozygote of a depleted mouse of the invention.
  • a depleted antigen is expressed by a gene in a wild-type mouse, but is not expressed by a gene in a homozygote of a depleted mouse of the invention.
  • a depleted antigen is encoded by a gene in a wild-type mouse, but not a gene in a homozygote of a depleted mouse of the invention.
  • an antibody of the invention is identified or obtained by using a display library, such as but not limited to phage, bacterial, ribosome and RNA display libraries, of the invention.
  • a phage-display library of the invention can be screened with an antigen or epitope of interest to identify an antibody, antibody fragment or antibody chain.
  • an antibody of the current invention is specific for a target antigen that is known or suspected to be relevant to a disease condition. Examples of such antigens are described in Lonberg, 2005, Nat. Biotech. 2S(9): ⁇ ⁇ 17-1125, which is incorporated herein by reference for any purpose.
  • Antibody types contemplated under the current invention include, but are not limited to, polyclonal, monoclonal, multispecif ⁇ c, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • an antibody of the current invention binds its antigen with a binding affinity (K 3 ) of 10 6 M “ ' or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, more preferably 10 9 M “1 or greater, more preferably 10 10 M “ 1 or greater, more preferably 10 1 ' M “1 or greater, and most preferably 10 12 M '1 or greater.
  • K 3 binding affinity
  • Therapeutic Utility AB antibody of the present invention may be used for therapeutic applications.
  • Therapeutic applications, for example, of antibodies of the invention include treatment of disease symptoms and phenotypic traits as presaged by the observation and study of a homozygous KO mouse line of the invention.
  • the present invention provides, in certain embodiments, diagnostic tools.
  • An antibody of the present invention may be used, for example, to diagnose the presence or quantity of an antigen or epitope that the antibody is specific for, or a disease condition associated with a phenotype found in the animal in which the antibody is obtained. Such antibodies may also have prognostic value.
  • Retroviral vectors such as those exemplified and described in detail in U.S. Patents Nos. 6,080,576, 6,136,566, 6,139,833 were used to generate a collection of gene trapped ES cell clones. Plasmids containing various VICTR cassettes described above were constructed by conventional cloning techniques. Usually, the cassettes contained a PGK promoter directing transcription of an exon that ends in a canonical splice donor sequence. The transcript encoding the exon was engineered to contain sequences that allow for the annealing of two nested PCR and sequencing primers. The vector backbone was based on pBluescript KS+ from Stratagene Corporation.
  • This primer has unique sequences (for subsequent PCR) on its 5' end and nine random nucleotides or nine T (thymidine) residues on it's 3' end.
  • Reaction products from the first strand synthesis were added directly to a PCR with outer primers specific for the engineered sequences of puromycin and the "RS" primer. After amplification, aliquots of reaction products were subjected to a second round of amplification using primers internal, or nested, relative to the first set of PCR primers. This second amplification provided more reaction product for sequencing and also provided increased specificity for the specifically gene trapped DNA.
  • the products of the nested PCR were visualized by agarose gel electrophoresis, and seventeen of the eighteen clones provided at least one band that was visible on the gel with ethidium bromide staining. Most gave only a single band, which is an advantage in that a single band is generally easier to sequence.
  • the PCR products were sequenced directly after excess PCR primers and nucleotides were removed by filtration in a spin column (Centricon-100, Amicon). DNA was added directly to dye terminator sequencing reactions (purchased from ABI) using the standard Ml 3 forward primer, a region for which was built into the end of the puro exon in all of the PCR fragments.
  • VICTR 20 is exemplary of a broader family of vectors that incorporate two main functional units: a sequence acquisition component having a strong promoter element (phosphoglycerate kinase 1) active in ES cells that is fused to the puromycin resistance gene coding sequence that lacks a polyadenylation sequence but is followed by a synthetic consensus splice donor sequence (PGKpuroSD); and 2) a mutagenic component that incorporates a splice acceptor sequence fused to a selectable, colorimetric marker gene and followed by a polyadenylation sequence (for example, SA ⁇ geopA or SAIRES ⁇ geop A).
  • stop codons have been engineered into all three reading frames in the region between the 3 1 end of the selectable marker and the splice donor site.
  • VICTRs 3, 20, and various variations and modifications thereof were used in the commercial scale application of the presently disclosed invention; many mutagenized ES cell clones were rapidly engineered and obtained. Sequence analysis obtained from these clones has identified a wide variety of both previously identified and novel sequences.
  • the cloned 3' RACE products resulting after the target ES cells were infected with VICTR 20 were purified using conventional column chromatography (e.g., S300 and G- 50 columns), and the products were recovered by centrifugation. Purified PCR products were quantified by fluorescence using PicoGreen (Molecular Probes, Inc., Eugene Oregon) as per the manufacturer's instructions.
  • Dye terminator cycle sequencing reactions with AmpliTaq® FS DNA polymerase were carried out using approximately 7 pmoles of sequencing primer, and approximately 30-120 ng of 3' template. Unincorporated dye terminators were removed from the completed sequencing reactions using G-50 columns as described above. The reactions were dried under vacuum, resuspended in loading buffer, and electrophoresed through a 6% Long Ranger acrylamide gel (FMC BioProducts, Rockland, ME) on an ABI Prism® 377 with XL upgrade as per the manufacturer's instructions. The sequences of the resulting amplicons, or GTSs, were recorded.
  • AmpliTaq® FS DNA polymerase Perkin Elmer Applied Biosystems, Foster City, CA
  • the promoter from the mouse phosphoglycerate kinase (PGK) gene was placed upstream from the first exon of the naturally occurring murine btk gene (nucleotides 40,043 to 40,250 of the murine btk gene).
  • the first exon of the btk gene does not contain a translational start site and initiation codon marking the 5' region of the coding sequence; however, these features could be engineered into the exon if desired.
  • the 3' end of the coding region of the first exon is marked by a splice donor sequence. Given that splice donor recognition sequences can extend into intronic sequence, 103 bases of intron DNA was retained after the end of the btk first exon.
  • the PGKbtkSD cassette lacks a 3' polyadenylation signal. Accordingly, any transcript produced by the cassette cannot be properly processed, and therefore identified by 3 r RACE 5 unless the transcript is spliced to a 3 1 exon that can be polyadenylated.
  • the btk vector was introduced into the embryonic stem cells using standard techniques. In brief, supernatant from GP +E packaging cells was added to approximately 2 x IQ 6 embryonic stem cells (at an input ratio of approximately 0.1 virus/target cell) for 16 hours and the cells were subsequently selected with G418 for 10 days. G418 resistant cells were subsequently isolated, grown up on 96-well plates and subjected to automated RNA isolation, reverse transcription, PCR and sequencing protocols to obtain the gene trapped sequences.
  • RNA isolation was carried out on DNA bind plates (Corning/Costar) treated with 5'-amino (dT) 42 (GenoSys Biotechnologies) in a 50 mM Sodium Phosphate buffer, pH 8.6, and allowed to sit at room temperature overnight. Immediately prior to use the plates were rinsed three times with PBS and twice with TE. Cells were rinsed with PBS 5 lysed with a solution containing 100 mM Tris-HCl, 500 mM LiCl, 10 mM EDTA, 1% LiDS 5 and 5 mM DTT in DEPC water, and transferred to the DNA binding plate where the mRNA was captured.
  • RNA was washed twice with a solution containing 10 mM Tris-HCl, 150 mM LiCl, 1 mM EDTA, and 0.1% LiDS in DEPC water. The RNA was then ⁇ nsed three times with the same solution minus LiDS. Elution buffer containing 2 mM EDTA in DEPC water was added and the plate was heated at 70 0 C. for five minutes.
  • RT premix containing 2x First Strand buffer, 100 mM Tris-HCl, pH 8.3, 150 mM KCl, 6 mM MgCl 2 , 2 mM dNTPs, RNAGuard (1.5 units/reaction, Pharmacia), 20 mM DTT, QT primer (3 pmol/rxn, GenoSys Biotechnologies) and Superscript II enzyme (200 units/rxn, Life Technologies) was added. The plate was transferred to a thermal cycler for the RT reaction (37 0 C. for 5 min. 42°C. for 30 min. and 55 0 C. for 10 min).
  • ES cells of the current invention are used to generate a mouse of the current invention. Any technique known in the art can be employed to generate a mouse with an ES cell.
  • an ES cell can be used to generate a mouse by injecting the cell into a blastocyst as described, for example, in Bradley, 1987. In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. IRL, Oxford, pp. 113-152. Blastocysts can be isolated from a pregnant mouse on day 3.5 of pregnancy. About 20-25 ES cells are typically injected into a blastocyst.
  • the blastocyst is implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny of the foster animal are used to breed more animals. If an ES cell harboring the engineered genomic mutation entered the germline of the foster animal's off-spring, the mutation will be represented in the off-spring's gene pool. Animals are bred to obtain homozygous, heterozygous or chimeric off-spring and/or wild-type animals. One may determine if an ES cell carrying an engineered genomic mutation of interest has entered the germline by observing the coat color of off-spring mice.
  • ES cells are derived from an agouti coat color strain and the blastocyst from a black coat color strain, second or higher generation off-spring carrying agouti coat color would be indicative that an ES cell carrying the engineered genomic mutation has entered the germline.
  • off-Spring animals are examined genetically to detect the presence of the genetrap vector and to characterize the engineered genomic mutation.
  • Transgenic mice may also be generated, for example, as described in Thomas et al. (1999) Immunol., 163:978-84; Kanakaraj et al. (1998) J. Exp. Med., 187:2073-9; or Yeh et al. (1997) Immunity 7:715-725;. Jaenisch (1988) Science, 240:1468-1474.

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Abstract

La présente invention concerne des procédés de production d'anticorps chez un animal, par exemple une souris. Les procédés de l'invention permettent d'obtenir des anticorps chez une souris modifiée de façon qu'elle présente une déplétion de l'antigène d'intérêt. Les procédés de l'invention permettent également d'obtenir des anticorps chez une souris modifiée de façon qu'elle puisse exprimer des anticorps humains ou humanisés. L'invention concerne également des bibliothèques de présentation phagique dérivées d'une souris modifiée de façon qu'elle présente une déplétion de l'antigène d'intérêt et/ou modifiée de façon qu'elle puisse exprimer des anticorps humains ou humanisés, ainsi que des procédés d'obtention d'anticorps en utilisant ces bibliothèques.
PCT/US2007/006239 2006-03-10 2007-03-09 Procédés de production d'anticorps chez des souris génétiquement modifiées WO2007106448A1 (fr)

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WO1996033735A1 (fr) * 1995-04-27 1996-10-31 Abgenix, Inc. Anticorps humains derives d'une xenosouris immunisee
WO2004001035A1 (fr) * 2002-06-21 2003-12-31 Centocor, Inc. Procede de generation d'anticorps monoclonaux
EP0677533B1 (fr) * 1994-04-12 2005-01-12 MILTENYI, Stefan Anticorps contre des épitopes homologues aux auto-antigènes, méthodes de préparation et applications

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EP0677533B1 (fr) * 1994-04-12 2005-01-12 MILTENYI, Stefan Anticorps contre des épitopes homologues aux auto-antigènes, méthodes de préparation et applications
WO1996033735A1 (fr) * 1995-04-27 1996-10-31 Abgenix, Inc. Anticorps humains derives d'une xenosouris immunisee
WO2004001035A1 (fr) * 2002-06-21 2003-12-31 Centocor, Inc. Procede de generation d'anticorps monoclonaux

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CASTROP J ET AL: "CIRCUMVENTION OF TOLERANCE FOR THE NUCLEAR T CELL PROTEIN TCF-1 BY IMMUNIZATION OF TCF-1 KNOCK-OUT MICE", IMMUNOBIOLOGY, FISCHER, STUTTGART, DE, vol. 193, no. 2-4, July 1995 (1995-07-01), pages 281 - 287, XP001013453, ISSN: 0171-2985 *
CLAESSON M H ET AL: "ANTIBODIES DIRECTED AGAINST MONOMORPHIC AND EVOLUTIONARY CONSERVED SELF EPITOPES MAY BE GENERATED IN 'KNOCK-OUT' MICE. DEVELOPMENT OF MONOCLONAL ANTIBODIES DIRECTED AGAINST MONOMORPHIC MHC CLASS I DETERMINANTS", SCANDINAVIAN JOURNAL OF IMMUNOLOGY, BLACKWELL SCIENCE PUBL., OXFORD, GB, vol. 40, no. 2, August 1994 (1994-08-01), pages 257 - 264, XP000867013, ISSN: 0300-9475 *
DECLERCK PAUL J ET AL: "Generation of monoclonal antibodies against autologous proteins in gene-inactivated mice", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 270, no. 15, 14 April 1995 (1995-04-14), pages 8397 - 8400, XP002174630, ISSN: 0021-9258 *
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