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WO1995015376A9 - METHODE DE DETECTION IN VIVO D'INHIBITEURS DE REPONSES ALLERGIQUES INDUITES PAR L'IgE - Google Patents

METHODE DE DETECTION IN VIVO D'INHIBITEURS DE REPONSES ALLERGIQUES INDUITES PAR L'IgE

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Publication number
WO1995015376A9
WO1995015376A9 PCT/US1994/013660 US9413660W WO9515376A9 WO 1995015376 A9 WO1995015376 A9 WO 1995015376A9 US 9413660 W US9413660 W US 9413660W WO 9515376 A9 WO9515376 A9 WO 9515376A9
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fceri
ige
mice
gene
mouse
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PCT/US1994/013660
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English (en)
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WO1995015376A3 (fr
WO1995015376A2 (fr
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Priority to AU12136/95A priority Critical patent/AU1213695A/en
Publication of WO1995015376A2 publication Critical patent/WO1995015376A2/fr
Publication of WO1995015376A3 publication Critical patent/WO1995015376A3/fr
Publication of WO1995015376A9 publication Critical patent/WO1995015376A9/fr

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  • a mouse line is established in which murine genes encoding structural proteins that are essential for an allergic response are replaced with their human counterparts, to provide an in vivo biological assay for inhibitors of an IgE- ediated human allergic response.
  • a mammalian allergic response is a complex phenomenon resulting from the action of many genes encoding a plurality of structural and functional proteins.
  • Two classical models of allergic reactions are cutaneous and systemic anaphylaxis (Klein, 1982) .
  • Mouse mast cells express at least three types of IgE receptor: the tetrameric high affinity IgE receptor (FceRI) which binds monomeric IgE (Kinet, 1992a; Metzger, 1992) , and the two low affinity IgG receptors (Fc ⁇ RII and III) , which bind both IgG- and IgE-immune complexes (Takizawa et al . , 1992).
  • FceRI tetrameric high affinity IgE receptor
  • Fc ⁇ RII and III two low affinity IgG receptors
  • FceRII/CD23 Conrad, 1990
  • Mac-2 Cherayil et al.,1989; Frigeri and Liu, 1992; Truong et al . , 1993. Both are found on a large variety of hematopoietic cells.
  • FceRI is not only expressed on mast cells and basophils, but also on Langerhans cells (Wang et al . , 1992; Bieber et al . , 1992) and monocytes(Maurer et al . , 1993) .
  • FceRI initiates the release of cytokines, such as interleukin-3, a mast cell growth factor (Burd et al . , 1989; Plaut et al . , 1989; Wodnar-Filipowicz et al . , 1989; Galli,1993).
  • cytokines such as interleukin-3, a mast cell growth factor (Burd et al . , 1989; Plaut et al . , 1989; Wodnar-Filipowicz et al . , 1989; Galli,1993).
  • FceRI expression occurs early in ontogeny, at stages where mast cells are not yet granulated (Thompson et al . , 1990). In these precursor cells,FceRI cannot mediate degranulation, but it may participate in cytokine secretion and play an as yet undefined role in mast cell development.
  • IgE immunoglobulin E
  • allergen a substance that is activated by immunoglobulin E (IgE) and allergen, play a prominent role in anaphylaxis.
  • these cells express at least three types of IgE receptor,including the high affinity IgE receptor (FceRI) .
  • IgE receptor the high affinity IgE receptor
  • FceRI the high affinity IgE receptor
  • One of the proteins believed to be involved in an allergic response is the FceRI receptor.
  • the relative contribution of this receptor, and possibly other receptors such as CD23/FceRII and Mac-2, to the genesis of in vivo anaphylaxis is still unclear.
  • FceRI or other IgE-binding structures such as FC7RII, Fc ⁇ RIII, CD23/FceRII or Mac-2 are causally involved in IgE-dependent anaphylactic responses.
  • other unknown mechanisms could also play a critical role.
  • Inhibitors of allergic responses may be detected by in vitro assays, or in vivo assays. In Vivo versus In Vitro Assays
  • mice which are models of the human allergic response system are preferable.
  • mice ideally produce a "human ⁇ like response.” This would be facilitated if human transgenes were present.
  • the first transgenic mouse was patented in 1987 (the "Onco MouseTM") .
  • a few other types of transgenic mice have been produced (U.S. Pat. No. 5,175,383 and 5,175,385), but are not suitable for studies of the allergic response.
  • Limitations of transgenic mice include random and unpredictable incorporation of transgenes into a lost genome. Location of incorporation and number of copies have generally not been controlled.
  • Transgenic mice that have been "humanized” that is, had sections of their genome replaced by a human homologue, are not available.
  • an in vivo assay for inhibitors of IgE-mediated allergic response makes use of transgenic mice in which a human gene encoding at least one chain of the FceRI replaces its murine homologue.
  • These are "humanized” mice, not merely mice into which a human transgene is introduced.
  • a "knockout" mouse is produced in which at least one gene encoding an FceRI chain is rendered non-functional.
  • a human gene that is substantially homologous replaces the murine gene in the FceRI-deficient mice.
  • the human gene is incorporated into the genome in the same position as was its murine counterpart.
  • FceRI-deficient mice provide useful in vivo models with which to address questions regarding the physiological role of FceRI so that effective inhibitors of deleterious allergic responses may be developed.
  • Using FceRI deficient mice according to the present invention provides an opportunity to characterize the FceRI -/- phenotype, as well as to address questions about the role of FceRI in initiating anaphylactic responses and in mast cell ontogeny.
  • Preferred mammals in this context are mice. Such detailed knowledge facilitates choice and design of candidate inhibitors of the allergic response.
  • Transgenic mice are used to reconstitute a receptor that is a model for the human receptor.
  • Such mice are prepared by incorporating the human gene encoding the ⁇ chain of the IgE receptor into the strain of mice homozygous for a disrupted ⁇ chain gene.
  • transgenic mice that express only the human and not the murine IgE receptor are produced by incorporating into mice with a disrupted FceRI chain gene, the homologous gene encoding the human chain. Incorporation may be effected by breeding the FceRI-deficient mice to mice having the human transgene.
  • Crossing-over and recombination yields transgenic mice which have the human gene substituted for the murine gene in the mouse genome.
  • the murine promoters are regulating expression of the human counterpart.
  • Other suitable promoters include CMV and HTLV1.
  • a strain of mice is established in which the gene encoding the ⁇ chain of the high affinity IgE receptor is disrupted by homologous recombination.
  • the result of disruption of the ⁇ chain gene in this manner is to abolish the surface expression of the IgE receptor on mast cells.
  • mast cell development proceeds in a normal pattern, despite disruption of one of its cell surface proteins.
  • a targeting plasmid is designed and constructed to disrupt the fourth exon of the FceRI gene.
  • This region is preferred for disruption because it is one of the two exons encoding the two Ig domains.
  • the third exon of the FceRIo gene is also suitable for disruption by homologous recombination.
  • the gene encoding the ⁇ chain also is suitable for disruption. Preferably, both the genes encoding the alpha and the ⁇ chain are disrupted.
  • the targeted plasmid is incorporated into mice via mouse embryonic stem cells by electroporation or calcium phosphate precipitation.
  • mice homozygous for the disrupted alpha gene do not have any overt phenotype. These mice are resistant to both cutaneous and systemic anaphylaxis, demonstrating that the high affinity IgE receptor is necessary for in vivo IgE dependant anaphylaxis. Therefore, interfering with the structure, and thereby the function of the receptor provides an effective means of treating an allergy regardless of the allergen specificity. This generalized treatment is possible because all allergic responses use the IgE receptor as a conduit.
  • transgenic mice are useful as in vivo assays of inhibitors of human allergic reactions, because they express the high affinity IgE receptor only if the human chains of the receptor are produced. Otherwise, because there is no receptor, there is no response.
  • an in vivo assay for an allergic response includes the following steps: (i) providing a transgenic mouse that expresses at least one human gene encoding a chain of the FceRI receptor substituted for the homologous mouse gene (a "humanized” transgenic mouse) and (ii) subjecting said transgenic mouse to conditions conducive to an allergic reaction and then (iii) detecting whether said allergic reaction occurred.
  • the presence of an allergic response in the mice is detectable, for example, by inducing passive cutaneous anaphylaxis or systemic anaphylaxis.
  • the substituted human gene encodes the alpha chain of the FceRI receptor or the beta chain.
  • an in vivo method to screen for an agent that inhibits an allergic response in a human includes the following steps:
  • a method of affecting an allergic response entails treating a patient with an amount of an inhibitor which is determined, via an assay of the present invention, to be sufficient to inhibit said response.
  • a transgenic mouse with a human gene superimposed on a complete murine system is not preferred because (l) unexpected interactions are possible when both murine and human genes are encoding a similar chain; (2) competition between the human and mouse ⁇ chains for ⁇ and chains is likely to decrease receptor expression; this could produce false negative results in an inhibitor assay; (3) human binding of the high affinity receptor in the presence of the mouse high affinity receptor is not known.
  • Candidate substances that may be capable of inhibiting an allergic response are selected for screening. Likely candidates include peptides and small, charged molecules that are not capable of entering a cell. These candidate substances are then screened, generally by in vitro assays, to select candidates for in vivo testing. The in vivo inhibitor assays of the present invention are used after a random in vitro screen has identified potential candidate inhibitors.
  • FIGURE 1 A restriction map of a targeting vector construct and of a wild-type and mutated allele is shown. Exons are indicated by black boxes; neo and thymidine kinase cassettes are represented by hatched boxes. Bg, Bm, E, H, K and X represent restriction sites for Bglll, BamHI, EcoRI, Hindlll Kpnl and Xhol respectively. PCR primers are indicated by arrowheads, and a probe used to detect homologous recombination events is shown.
  • in vivo assays for inhibitors of human allergic responses employ humanized transgenic mice in which at least one human gene encoding a chain of the OFceRI receptor replaces its murine homologue. Such assays are particularly useful for detecting inhibitors of IgE-mediated allergic responses, i . e . , inhibitors of IgE binding to the receptor.
  • FceRI is a tetrameric complex of one a , one ⁇ , and two disulfide-linked ⁇ chains (Kinet, 1992a; Metzger, 1992) . Because only the fully assembled complex is expressed on the cell surface (Blank et al . , 1989), targeting one subunit is a strategy to prevent FceRI expression. Because subunits are part of other Fc receptors and of the T cell antigen receptor (Ravetch and Kinet, 1991) , and play a critical role in signal transduction (Kinet, 1992b; Weiss, 1993), targeting 7 affects the expression and function of receptors other than FceRI.
  • the ⁇ subunit gene is preferably disrupted and replaced with the human homologue.
  • IgE surface receptor and its use in recombinant genetic production of ⁇ subunits, are disclosed in U.S. Pat.
  • the ⁇ chain is also a suitable target for disruption and replacement by the human ⁇ chain encoding gene
  • both the ⁇ and ⁇ chain genes are disrupted and replaced with active human counterparts.
  • Active means the genes are capable of expression under suitable regulatory control.
  • mice homozygous for a disrupted ⁇ chain gene have been found not to express cell surface FceRI.
  • FceRI does not play a major role in mast cell development.
  • the presence of intact FceRI is necessary to induce local or systemic
  • IgE-mediated anaphylaxis Therefore, inhibitors of anaphylaxis can be detected by disruptive effects on
  • FceRI is necessary for the induction of IgE-mediated anaphylaxis, with a minor role at most for the other lower affinity IgE-binding proteins.
  • the role of IgG immune complexes also seems to be minimal, because systemic anaphylactic reactions could not be induced in any mice, either FceRI +/+ or -/-, even after intravenous injection of 0.4 mg of IgG.
  • Other IgE-binding proteins may play a role if alternative methods, such as active immunization (Martin et al . , 1993), are used to induce antigen sensitivity. FceRI clearly is necessary, however, for initiating the cascade of events leading to
  • RECTIFIED SHEET (RULE 91) ISA/EP the various symptoms of IgE-mediated anaphylaxis. Accordingly, targeting FceRI is an effective way to prevent or treat allergic reactions.
  • IgE Although the evidence for a protective role of IgE in parasitic infestations is mixed, IgE seems to be protective against chronic infestations of Schistosoma in humans (Hagan et al . , 1991; Rihet et al . , 1991; Dunne et al . , 1992), implicating FceRI in the mechanisms of protection against parasites.
  • the FceRI-deficient mice are useful to determine whether FceRI is necessary for protection against parasites. If it is, compositions enhancing this protection may be developed by use of the humanized transgenic mice of the present invention.
  • FceRI (-/-) is used in this description to denote herein a genotype responsible for a phenotype wherein the IgE high affinity receptor is not expressed. This includes genotypes FceRI ⁇ (-/-) which may be specified to indicate that a gene encoding the ⁇ chain is non-functional (not expressed as an ⁇ chain) .
  • Plasmids used for gene targeting are often designed to inactivate a specific gene by the introduction of a "null mutation,” that is, a mutation which completely destroys the ability of a gene to produce a functional protein.
  • Many of these targeting plasmids are designed to introduce null mutations by inserting exogenous sequences into the center of a gene, thus interrupting the normal protein coding sequence. This strategy has failed in a number of cases, however, because of the ability of the cellular transcriptional machinery to remove the RNA encoded by the exogenous sequences during splicing.
  • a targeting plas id is preferably designed that replaces coding sequences necessary for the function of the encoded protein with exogenous sequences.
  • One obstacle that is encountered and must be overcome in designing a suitable targeting plasmid pertains to the small size of the genes encoding FceRI subunits, notably the ⁇ chain gene, which limits the number of endogenous restriction sites that are suitable for use in constructing a targeting plasmid that would produce the expected null phenotype.
  • a way to circumvent this problem is to use PCR technology to generate the smaller of the two arms of homologous DNA that direct the targeting event. This approach provides maximum flexibility in choosing the length and location of the homologous DNA and the position in which the gene is disrupted.
  • the length of the smaller arm is determined by balancing the need for a reasonable targeting frequency, which increases with the length of homology, with the need to produce the arm by PCR, for which efficiency increases as the length of the reaction product decreases.
  • isogenic DNA is used to construct this arm. This increases the probability that there are no differences between the target locus and the targeting construct.
  • a targeting plasmid is constructed by modifying a known plasmid (see next section) by the addition of a linker sequence. This sequence is preferably one that is believed to ensure a good primer binding site for PCR across the DNA corresponding to the short arm of the targeting plasmid in the targeted cell lines.
  • a linker sequence is preferably one that is believed to ensure a good primer binding site for PCR across the DNA corresponding to the short arm of the targeting plasmid in the targeted cell lines.
  • regions of homology included in the targeting plasmid attention is also paid to the need to isolate genomic probes for verification of targeting events, for example, by Southern analysis. Another uncertainty that is taken into consideration in carrying out the targeting experiments is the relative targeting frequency of individual targeting experiments.
  • a control electroporation is carried out in parallel with each experimental electroporation, where electroporation is used to incorporate the targeting plasmid into a host cell.
  • a construct designed to target a marker such as the hprt locus is suitable. This permits determination of the targeting frequency for a well characterized locus before screening for targeting events at the experimental locus. Thus, it is possible to anticipate whether a targeting event for the FceRI locus is likely to be identified in a given experiment.
  • E14TG2A is derived from the 129/Ola strain, rather than the 129/Sv strain from which most mouse ES cell lines are derived, it carries two recessive mutations, pinkeyed and c ch .
  • a final factor that plays a role in the production of the targeted mice is the breeding scheme used to
  • mice homozygous for the targeted locus.
  • chimeric males that transmit the ES cell genome to large percentages of their offspring in their first litters were bred to their own daughters (e.g. in the illustrative examples herein, mice were selected that transmitted the ES cell genome to at least 75% of their offspring) .
  • the FceRI ⁇ subunit is a type I transmembrane molecule with an extracellular segment containing two immunoglobulin (Ig) related domains, the second of which is necessary for IgE binding (Robertson, 1993) .
  • the FceRI ⁇ gene comprises five exons, with the third and fourth exons encoding the two Ig domains (Tepler et al . 1989; Ye al., 1992).
  • a targeting plasmid is preferably designed to disrupt the fourth exon by insertion of a neomycin gene (an illustrative construction is shown in FIG. 1) .
  • D3 and E14TG2 embryonic stem cells were selected in the presence of G418 and gancyclovir to enrich for those that had integrated the vector by homologous recombination.
  • ES embryonic stem cells
  • PCR polymerase chain reaction
  • Southern Blot analysis demonstrating integration via homologous recombination at the FceRI ⁇ locus. Hybridization of the same blots with a neo probe showed a single copy of the neo gene.
  • ES cells from three of these lines were injected into C57BL/6 blastocysts.
  • the chimeras generated from two of these cell lines transmitted the mutant gene to their offspring. This was demonstrated by Southern Blot analysis of mouse tail DNA which showed hybridization with a 6.1 kb wild type allele fragment and/or a 7.7 kb fragment for the mutated (alpha chain gene deficient) allele. No overt phenotype was observed in animals that were either heterozygous or homozygous for the mutant gene. Heterozygous and homozygous animals were present in litters at the expected ratios. Construction of the Targeted Plasmid
  • the targeting vector is constructed by cloning two fragments of the mouse FceRI ⁇ into a vector such as pJNS2.
  • the pJNS2 vector is derived by inserting a 50 bp polylinker between the Xbal and Ba HI sites of pNT2 (Tybulewicz et al . 1991) .
  • a genomic clone is isolated by screening, for example, a 129/Ola genomic lambda phage library with mFceRI ⁇ cDNA (Ra et al . , 1989).
  • a 1.6 kb Bglll-Kpnl fragment encompassing exons IV and V is generated by PCR of the genomic clone with primers C A G G T A C C C A G T G T T T A T T G A G a n d GAAGATCTGGATCCTTTGACATCAGATGCC, then inserted between the BamHI and Kpnl sites of pJNS2 vector, 3' to the neo gene and 5 1 to the TK gene.
  • a second fragment, a 5.5kb Xhol-Bglll fragment including exons I to III is available from a Balb/c library genomic clone (Ye et al .
  • the final targeting construct is identical to the corresponding region in the mouse genome, except that a 0.3 kb Bglll-BamHI fragment that encompasses the 3 ' end of intron III and the 5* end of exon IV has been replaced by the positively selectable neo gene in pJNS2.
  • the ES cell lines D3 Doetsch an et al . , 1987
  • E14TG2a Hooper et al . , 1987
  • Electroporation is carried out in culture medium containing 3nM of targeting plasmid (linearized with NotI) , using conditions described previously, except that a 1-sec discharge from a 150-250 mF capacitor charged to 250-400 V is used.
  • Transfected colonies are selected by growth in G418 and enriched for targeted colonies by the addition of gancyclovir (a gift from Syntex, Palo Alto, CA) to the growth medium. Surviving colonies are expanded to a level sufficient for PCR and Southern blot analysis.
  • the targeting vector is constructed by cloning two fragments of the FceRI ⁇ gene into the vector pJNS2.
  • the pJNS2 vector is derived by inserting a 50 bp polylinker between the Xba I and Bam HI sites of pNT2 (Tybulewicz et al . , 1991) .
  • a genomic clone including the FceRI ⁇ gene is isolated by screening a 129/Ola genomic lambda library with the mFceRI ⁇ cDNA.
  • a 1.6 kb Bgl II-Kpn I fragment encompassing exons IV and V is generated by PCR of the genomic clone with primers CAGGTACCCAGTGTTTATTGAG and GAAGATCTGGATCCTTTGACATCAGATGCC and then inserted between the Bam HI and Kpn I sites of the pJNS2 vector, 3' to the neo gene and 5 1 to the tk gene.
  • a second fragment, a 5.5 kb Xho I-Bgl II fragment includes exons 1-3 and is available from a BALB/c library genomic clone, subcloned in pSL301 (In Vitrogen) and then excised by digestion with Xho I and Sal I and inserted into the Xho I site 5• of the neo gene of pJNS2.
  • the final targeting construct is identical to the corresponding region in the mouse genome, except that a 300 bp Bgl II-Bam HI fragment that encompasses the 3 ' end of intron 3 and the 5' end of exon 4 is replaced by the positively selectable neo gene in pJNS2.
  • Neomycin resistent embryonic fibroblasts are prepared from embryos from /32(-/-) females that had mated 14 to 17 days earlier. These mice contain a copy of a neomycin gene within the ⁇ 2-microglobulin locus and thus are capable of growth in media containing G418. Electroporation conditions are similar to those that have been described previously.
  • ES cells are trypsinized, resuspended in culture media at a concentration of 4 x 10 7 /ml and electroporated in the presence of the targeting DNA at a concentration of 12 nM DNA in a first experiment and 5 nm in a second.
  • a voltage of 300 V and capacitance of 150- 250 mF are optimal for an electroporation cell of 5 mm length and 100 mm 2 cross section.
  • 5 x 10 6 electroporated cells are plated onto irradiated fibroblasts in 100 mm dishes in the presence of Dulbecco's modified Eagle's media (DMEM) supplemented with 15% fetal bovine serum (FBS) and 0.1 mM 2-mercaptoethanol.
  • DMEM Dulbecco's modified Eagle's media
  • FBS fetal bovine serum
  • 2-mercaptoethanol The media is replaced 24 hr after electroporation with media containing 200 ⁇ g/ml G418 and 1 ⁇ M
  • EP65, EP66 and EP67 were carried out. These experiments were called EP65, EP66 and EP67.
  • 2x 10 7 E14TG2a cells were electroporated in the presence of the FceRI ⁇ targeting plasmid, which was linearized by restriction with Not I. Cells were cultured in G418 and ganciclovir. All experiments were identical, except that in EP672xl0 7
  • ES cell colonies visible 10-14 days after electroporation are picked with drawn out capillary pipettes into 24 well plates that are seeded with irradiated feeders. After sufficient growth (usually two to three days) wells are trypsinized and cells moved to 60mm wells seeded with feeder cells. When plates become confluent, cells are again trypsinized, and one half of the cells are frozen using conditions which have been found to be optimal for recovery of embryonic stem cells.
  • DNA is prepared from the remaining cells and used to screen for targeting events by the polymerase chain reaction (PCR) or by Southern blotting. DNA is prepared following a previously described method (Miller et al . , 1988).
  • cells are resuspended in 300 ⁇ l buffer containing 0.05M Tris(pH8), 0.1M NaCl, 0.1M EDTA, 1 % SDS and 150 ⁇ g of Protinase K. After incubation for 24 hr at 55 degrees, 180 ⁇ l of a saturated NaCl solution is added. Preparations are centrif ⁇ ged and supernates removed. DNA is then precipitated by the addition of two volumes of ethanol. DNA is resuspended in Tris EDTA buffer (10mm Tris pH 7.5, lmm EDTA) at approximately l ⁇ g/ ⁇ l concentration.
  • primers are chosen which hybridize to the pJNS2 polylinker (bases 2-35) and to a region 150 bp 3 1 from exon 5. (GCTTCTAGCAACAGAAGGCAGATTAC) .
  • the length of the expected product is 1.75 kb for cases of homologous recombination. There is no amplification in cases of random integration.
  • Methods suitable to confirm disruption of a gene encoding ⁇ chain of the FCeRI receptor are employed. These methods include Southern analysis and PCR.
  • Southern analysis in an illustrative embodiment, DNA is digested with restriction enzymes as directed by the manufacturers, and fragments are separated on 0.7% agarose gels. DNA is transferred to nylon membranes and probed with a 32 P labelled 350 bp Hind III-Bam HI fragment that is located 150 bp downstream of exon 5.
  • the hybridization pattern obtained with the restriction enzyme Eco RI is expected to be altered by integration of the targeting plasmid into the endogenous FceRI ⁇ locus by homologous recombination.
  • the probe hybridizes to an EcoRI fragment of approximately 6.1kb derived from the endogenous locus.
  • the intensity of the band representing this fragment is expected to be reduced by 50%, and an additional band, generated by homologous recombination of the targeting plasmid with the FceRI ⁇ locus, is expected to be present.
  • the replacement of the 300 bp Bgl II-BamHI fragment of the FceRI ⁇ gene with the 1849 bp neomycin insert in the alpha locus results in an EcoRI fragment that is approximately 1.5kb longer than the corresponding fragment in the native locus.
  • Targeted lines 66-154, 66-161, 66-168, and 66-196 were identified from experiment EP66, while lines 67-327, 67- 490, 67-501, 67-502, and 67-602 were identified from experiment EP67.
  • the transformed cell is used to generate a new mouse strain. This may be accomplished by injecting a mouse blastocyst with the cell.
  • the ES cell lines carrying the inactivated alpha gene are expected to allow the introduction of this mutation into the mouse germ line. Towards this end the following procedures are carried out.
  • Mice may be purchased from Jackson Laboratories (Bar Harbor, ME) .
  • Blastocysts are obtained from 3 to 4 week old superovulated females, in this embodiment, they are from C57BL/6 mice. Uteri are flushed with M2 media 3.5 days after ovulation.
  • Blastocysts are collected, washed several times in fresh M2 media, and placed in a 100 ml droplet of M2 under paraffin oil.
  • ES cells identified as having the inactivated alpha locus are trypsinized, washed once with fresh DMEM media and diluted to approximately 2 x 10 6 cells/ml. 5 ml of cells are added to the droplet containing the blastocysts. 10 to 15 ES cells are injected into the blastocoel of each blastocyst using standard manipulation techniques.
  • blastocysts Following injection, 6 to 9 blastocysts are returned to each uterine horn of pseudopregnant females mated 2.5 days previously with vasectomized males. Development of the blastocysts continued in vivo and pups are born about
  • C57BL/6 x DBA F,mice proved to be excellent foster mothers, because they yielded a pregnancy rate close to 100% and were able to raise small litters
  • the contribution of the ES cells to the offspring is judged visually by examination of the coat color of the pups if coat color genes were in the targeting plasmid.
  • the blastocysts were obtained from C57BL/6 mice, which are solid black in color.
  • the ES cell line E14TG2a was isolated from strain 129/Ola embryos, it and all cell lines derived from it were expected to carry the coat color markers characteristic of this mouse strain. These include the dominant AW allele at the agouti locus, the recessive chinchilla allele at the c-locus, and the recessive p-allele (pink- eyed dilution) at the p-locus.
  • ES cells Contribution of ES cells to the mesoderm-derived portions of hair follicles results in an agouti coat. Hair follicles to which melanocytes of ES cell origin (and therefore carrying the P and c ch mutation) have migrated produce cream colored hairs. Both of these coat colors are easily distinguished from the solid black coat seen in pups derived from non-agouti C57BL/6 host blastocysts.
  • the ES cell line D3 is derived from 129/SV and carries only the dominant Aw mutation. Chimerism is visualized by the presence of agouti coat color.
  • ES cell lines carrying an inactivated alpha gene were injected into C57BL/6 blastocysts. Chimeric animals as judged by coat color were obtained from all three lines. The following lines were injected: 66-154, 66-161, 66-168, 66-196, 67- 357. All cell lines produced chimeras when injected into mouse blastocysts, but germ line transmission of the ES cell genome was only achieved with cell lines 66-154, 66-
  • Chimeric animals are generated as described and bred to suitable mouse lines such as B6D2 or C57BL/6 mice(Koller and Smithies, 1989). Offspring carrying the targeted gene are identified by Southern analysis of tail DNA prepared as described (Miller et al., 1988) . All the manipulations are performed on 4 to 8 week old mice that are FceRI (+/+) and (-/-) littermates.
  • the resulting animals produce sperm that contain the ES cell genome, and thus are capable of passing the targeted mutation on to their offspring.
  • Offspring arising from eggs fertilized by sperm carrying the ES cell genome can be identified by markers such as coat color.
  • the ES cell genomes of all of the targeted lines are homozygous for the dominant color coat marker Aw. If the chimera is mated with an animal that is non-agouti, such as C57BL/6 or B6/D2, offspring that arise from sperm of ES cell origin will have agouti coats, while those derived from sperm of blastocyst origin will not. Using this criterion, it was determined that chimeras generated from two of the three targeted ES cell lines were able to transmit the ES cell genome to their offspring. By random chance, 50% of the resulting agouti offspring would be expected to inherit the mutated alpha gene. Animals carrying the mutant locus were identified by the analysis of DNA isolated from tail biopsies taken from these animals.
  • PCA passive cutaneous anaphylaxis
  • mice 35 between the right and left ears were detected in -/- mice injected with IgE. No effect was seen in either +/+ or -/- mice injected with IgG. Dye extravasation and swelling (13% weight increase) were detected only in the right ears of +/+ mice injected with IgE and antigen.
  • IgE but not IgG is capable of inducing passive cutaneous anaphylaxis, and then only when FceRI is present. 5
  • having the FceRI -/- phenotype appears not to engender any overtly undesirable features.
  • the mice have no obvious propensity to autoimmune diseases, or susceptibility to infection, as would perhaps manifest if their immune systems were overtly altered.
  • IgE is injected intravenously 24h before intravenous injection of antigen.
  • the antigenic challenge is immediately followed by a dramatic increase in vascular permeability, generalized fluid extravasation, and profound shock
  • both +/+ and -/- mice were injected intravenously with 20 mg of anti-DNP IgE or vehicle.
  • mice Another set of mice was injected with 20 mg of mouse anti-DNP IgG or 400 mg of rabbit anti-TNP IgG. All mice were challenged 24h later with 1 mg of DNP 3(M0 -HSA. Evans Blue dye was concomitantly injected with antigen to visualize the fluid extravasation. A significant drop in
  • FceRI (+/+) and 3 FceRI (-/-)animals received 400 ⁇ g of rabbit anti-TNP IgG. All the animals received 1 mg of DNP SMO -HSA in 0.5 % Evans Blue i.v. 24h later. Four additional FceRI(+/+) mice received antigen only. The monitoring of the rectal temperature was started at the time of antigen injection.
  • IgE is iodinated using chloramine T to a specific activity of 4X105 cpm/mg.
  • Twenty or 1 mg of [1251]-IgE in 200 ml PBS with 0.1 % BSA are injected intravenously into +/+ and -/-mice. Blood is collected in heparinized tubes at various intervals of time.
  • [1251]-IgE in blood samples (50 ml) is im uno precipitated with rabbit anti-murine IgE antibodies bound to protein A-sepharose resin (Pharmacia) .
  • a mouse line is created, according to the present invention, in which the crucial structural proteins involved in allergic responses are replaced by their human counterparts. Creation of such a mouse line requires three steps. The first of these is the identification and characterization of proteins that play an important role in allergic responses. Such proteins represent reasonable targets for potential inhibitors of these responses. The second step is the inactivation of genes in the mouse genome that encode the proteins identified in step one. And the final step is the replacement of the genes inactivated in step two with their human homologous counterparts.
  • the FceRI receptor is one of the proteins that plays an important role in allergic responses.
  • the murine gene encoding this receptor is replaced with the equivalent human gene.
  • the first step in this process is to inactivate the FceRI receptor in the mouse genome via homologous recombination in mouse embryonic stem (ES) cells.
  • ES mouse embryonic stem
  • Other inactivation methods are available, such as site-directed mutagenesis.
  • Analysis of animals that are homozygous for the inactivated FceRI gene, and therefore totally unable to produce the FceRI receptor, as related herein has confirmed the importance of this receptor in both passive and active anaphylaxis in mice.
  • the final step in production of mice expressing only human FceRI receptors is the substitution of a human gene for its murine homologue in the genome of FceRI deficient mice.
  • the human FceRI gene is introduced into the mouse genome using one of four different approaches: (1) random integration using conventional transgenic techniques, (2) random integration via ES cells, (3) targeted integration into the hprt locus via ES cells, and (4) targeted replacement of the endogenous FceRI gene via ES cells.
  • random integration using conventional transgenic techniques (2) random integration via ES cells
  • targeted integration into the hprt locus via ES cells targeted replacement of the endogenous FceRI gene via ES cells.
  • These various alternatives are suitable to develop a mouse strain in which both the level and pattern of expression of the FceRI gene mimic those seen in humans. This animal is useful for assessing the efficiency of various inhibitors.
  • mice with transgenes added to the genome are described in U.S. Pat. Nos. 4,736,866, 5,175,383 and 5,175,385.
  • a standard technique for the addition of genes from the human genome to mice is injection of 1 day embryos with a fragment of DNA containing the gene and the necessary regulatory sequences. Using this technique, mice are obtained in which the FceRI gene plus appropriate regulatory sequences have integrated at random sites in the genome. Crossing of these animals with mice homozygous for the disrupted FceRI gene results in animals carrying the transgene and heterozygous for the targeted gene.
  • a subsequent round of breeding is necessary to yield animals homozygous for the targeted mutation and therefore defective in the production of mouse FceRI.
  • mice Because of the presence of the transgene, however, these mice express a humanized IgE receptor.
  • 1-day embryos are obtained that are from FceRI deficient animals, and test breeding is carried out by mating founders to FceRI deficient animals.
  • the fragment introduced into the 1-day embryos is of two typas. The first contains a large segment of upstream DNA 5' to the human alpha gene. This fragment is sufficient to direct the cell type specific synthesis of this gene.
  • a construct in which the human alpha gene is driven by a suitable promotor, either the murine promoter for the alpha chain gene, CMV, HTVL1, or the like, is introduced into 1-day embryos. While expression in a large number of cell types is expected, this should include those in which binding of IgE triggers anaphylaxis.
  • ES cells to obtain random integration of foreign DNA into the mouse genome is described in more detail in the following section.
  • the human FceRI gene is introduced into ES cells via electroporation or calcium phosphate precipitation, either in the presence of DNA carrying a selectable marker gene or as part of a construct that contains the marker gene.
  • ES cells that have incorporated DNA as demonstrated by their growth in selectable media are then screened for the presence of the human FceRI gene.
  • These ES cells are then introduced into mouse blastocysts. Resulting female chimeras are sacrificed and analyzed for the expression of the human FceRI, taking into account the relative contribution of ES cells to various tissues. Males from those lines which display appropriate levels and patterns of expression levels are then tested for germ line transmission.
  • neomycin resistance gene Two different marker genes, the neomycin resistance gene and the hprt gene, are suitable for initial selection of the ES cells carrying the transgene in this scheme.
  • the neomycin gene is small and can easily be added to a construct carrying the human FceRI gene. This ensures that virtually all of the ES cell colonies that are resistant to neomycin carry the alpha gene.
  • the hprt gene has the advantage of allowing use as a starting population of the ES cell line carrying one disrupted mouse FceRI locus.
  • this line has become resistant to neomycin as a result of manipulation to target this locus, it originates from an ES cell line that carries a spontaneous mutation in the endogenous hprt locus, thus allowing the hprt gene to be used to identify cells that have integrated DNA into their genome. This means one generation of breeding time can be eliminated in producing animals that express only the human FceRI gene.
  • Another suitable strategy is to use homologous recombination to replace the coding sequences of the mouse FceRI gene, with the coding sequences from the corresponding human gene. This places the human gene under the same regulatory constraints as the endogenous gene. The high targeting frequency that has been demonstrated for this locus in the present invention makes such an approach desirable.
  • the present invention concerns a method for identifying an inhibitory compound which interferes with an IgE-mediated allergic reaction.
  • These "candidate substances” are first screened in vitro . It is contemplated that inhibitors will be small, charged molecules, although the invention is not limited to detecting these compounds.
  • the substance After determining a candidate substance by in vitro screening, the substance is introduced into the system of a transgenic mouse in which a human gene encoding at least one chain of the FceRI replaces its murine homologue.
  • the substance is generally introduced by intravenous injection of the substance in a suitable carrier or diluent.
  • mice are preferred, including a transgenic mouse of the present invention injected with a control substance, for example, only the carrier or diluent.
  • Other controls include mice transgenic for other human genes.
  • An identical environment is provided to control and treated mice to induce an allergic reaction. Suitable methods are required to determine whether an allergic reaction occurred. Absence of an allergic reaction in the treated, but not the control mice, provides evidence that the candidate substance is inhibitory.
  • the substance to be tested should be in a relatively purified form, to assay for the effect of the substance to be tested not effects caused by imp ⁇ rities.
  • a range of concentrations of the candidate substance is tested to avoid a false negative result arising from concentrations that are too low, and to estimate effective concentrations to test in clinical trials of candidate substances that are inhibitory in the transgenic mice tests.
  • FceRI plays a role in mast cell development. This speculation was based on the early appearance of FceRI on precursors of mast cells (Thompson et al . , 1990; Ashman et al . , 1991) and on the capacity of FceRI to mediate synthesis and release of mast cell growth factors such as interleukin-3. Investigations using FceRI deficient mice shows that FceRI does not play an important role in mast cell development. The absence of FceRI did not affect the number or appearance of mast cells in any of the tissues and organs examined. In addition, mast cells appeared to function normally when stimulated by non-specific mast cell activators.
  • BMMC bone marrow derived mast cells
  • Spleen B cells constitutively expressing FceRII/CD23 were also analyzed by double staining with monomeric IgE and an antibody against surface immunoglobulins. In both +/+ and -/- mice, the population of B cells appeared normal and virtually all B cells showed IgE binding. This binding was due to CD23 because, unlike the binding of monomeric IgE to BMMC, it was completely inhibited by anti-CD23. Thymocytes were also analyzed for various markers, including TCR ⁇ , TCR7S, Fc ⁇ RII/III, CD5, CD3, CD4 and CD8 and for their capacity to bind monomeric IgE. The thymocytes from both +/+ and -/- mice did not bind IgE and their various markers of differentiation were qualitatively and quantitatively normal.
  • FC7RII and FC7RIII react equivalently with the monoclonal antibody 2.4G2 (Ravetch and Kinet, 1991). Therefore, BMMC were stained with 2.4G2 to detect expression of FC7RII/III. Binding of IgE-immune complexes and oligomeric IgG to FC7RII and FC7RIII on BMMC of FceRI (+/+) and FceRI (-/-) animals. Expression of FC7RII/III was exhibited using 2.4G2-FITC. The binding of oligomeric IgG was revealed by using goat F(ab') 2 anti-rabbit IgG-FITC in the absence or presence of 2.4G2.
  • FC7RII/III The capacity of FC7RII/III to bind IgE-immune complexes (IgE-IC) was analyzed. Binding of IgE-immune complexes and oligomeric IgG to FC7RII and FC7RIII was detected on BMMC of FceRI (+/+) and FceRI (-/-) animals. Expression of FC7RII/III was exhibited using 2.4G2-FITC. The binding of oligomeric IgG was revealed by using goat F(ab') 2 anti-rabbit IgG-FITC in the absence or presence of 2.4G2. Binding of IgE-immune complexes was displayed in the absence or presence of monomeric IgE or of both IgE and 2.4G2.
  • mice The cells analyzed were from a pool of cultured bone marrow mast cells obtained from two mice.
  • IgE-IC bound principally to FceRI as shown by inhibition with unlabelled monomeric IgE.
  • 2.4G2 further inhibited the binding of IgE-IC.
  • Evidence for this was more striking in -/- mice.
  • Monomeric IgE competed poorly with the binding of IgE-IC. Only 2.4G2 was able to compete efficiently with this binding.
  • BMMC of the -/- mice bind IgE-IC via Example No. 5: Serotonin Release by Activated Mast Cells (BMMC)
  • BMMC were loaded with [ 3 H]serotonin, incubated with monomeric anti-DNP IgE, and then challenged with various amounts of the multivalent antigen dinitrophenol [30-40]-human serum albumin (DNP 3ft40 -HSA) .
  • DNP 3ft40 -HSA multivalent antigen dinitrophenol
  • a dose-dependent release of up to 40% of the total cellular serotonin was observed with the BMMC from +/+ but not -/- mice.
  • -/- BMMC were capable of releasing serotonin after stimulation with ionomycin and therefore were functionally intact. Engagement of FC7RII/III by cross-linking with 2.4G2 on +/+ or -/-BMMC did not lead to detectable serotonin release.
  • Mast cells were stained with acidic toluidine blue (pH 1.5), Giemsa or alcian blue-safranin. The number of mast cell per square millimeter was determined. For each tissue, twenty-five microscopic fields were counted on five non-serial sections.
  • Flow Cytometry Bone marrow derived mast cells were isolated from 4 to 8 week old mice according to Rottem et al. (1992) . Samples (2 to 5X105 cells) of bone marrow derived mast cells,thymocytes or splenocytes were analyzed by flow cytometry.
  • FceRI was detected using biotinylated mouse monoclonal anti-DNP IgE (20 mg/mL) and streptavidin-phycoerythrin(10 mg/mL) (Molecular Probes Inc.) after preincubation with a rat anti-murine FC7RII/III(2.4G2) monoclonal antibody (10 mg/mL). Unstained cells, cells stained with streptavidin-phycoerythrin alone, or cells preincubated with a 10-fold excess of unlabeled IgE were used as controls.
  • Interaction of IgE with FceRII was assessed by inhibition of binding of biotinylated IgE with a rat anti-murine CD23 monoclonal antibody (B3B4) (10mg/mL) (Pharmingen) after preincubation with 2.4G2 (10 mg/mL) . Presence of FC7RII and FC7RIII was detected using 2.4G2-FITC (10 mg/mL). Binding of IgE-antigen immune complexes and of oligomeric IgG to FC7RII/III were performed as described before (Takizawa et al., 1992). Mast cells were identified using rabbit polyclonal anti-murine c-kit receptor antibody (gift from Dr.
  • the Mac-2 antigen is a galactose-specific lectin that binds IgE. J. Exp. Med. 170, 1959-1972.
  • FceRII/CD23 the low affinity receptor for IgE. Ann. Rev. Immunol. 8,623-645. Doetschman, T. C, Eistetter, H. , Katz # ⁇ M. , Schmidt, W. , and Kemler, R. (1987) .
  • Kinet J. P. (1992a) .
  • Kinet, J. P. (1992b) The g-z dimers of Fc receptors as connectors to signal transduction.Curr. Opin. Immunol. 4, 43-48.
  • Human peripheral blood monocytes express the high-affinity IgE receptor, FceRI. J.Immunol. 150, 222A, abstract. 1271.
  • Mast cell lines produce lymphokines in response to cross-linkage of FceRI or to calcium ionophores. Nature 339, 64-67.
  • T cell antigen receptor signal transduction a tale of tails and cytoplasmic protein tyrosine kinases. Cell 73, 209-212.

Abstract

Dans les méthodes de détection in vivo d'inhibiteurs de réponses allergiques humaines on utilise des souris transgéniques humanisées dans lesquelles au moins un gène humain codant une chaîne du récepteur de Fc⊂RI remplace l'expression de son homologue murin. Ces méthodes sont particulièrement utiles pour détecter les inhibiteurs des réponses allergiques induites par l'IgE.
PCT/US1994/013660 1993-12-01 1994-12-01 METHODE DE DETECTION IN VIVO D'INHIBITEURS DE REPONSES ALLERGIQUES INDUITES PAR L'IgE WO1995015376A2 (fr)

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US6111166A (en) * 1994-09-19 2000-08-29 Medarex, Incorporated Transgenic mice expressing human Fcα and β receptors
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US20060179501A1 (en) 2002-12-16 2006-08-10 Chan Andrew C Transgenic mice expressing human cd20
SI24066A (sl) * 2012-05-07 2013-11-29 Univerzitetna klinika za pljuÄŤne bolezni in alergijo Golnik Materiali in metode na nivoju bazofilcev in visokoafinitetnega IgE receptorja povezane z diagnosticiranjem anafilaksije

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