TRANSGENIC ANIMALS CAPABLE OF PRODUCING HUMANIZED IGE AT MUCH HIGHER LEVELS THAN MOUSE IGE

Abstract

The transgenic non-human animals are constructed, in whose genome the coding sequences of one of the animal's endogenous immunoglobulin C constant regions are replaced by human immunoglobulin C constant region coding sequences. The transgenic animal is mouse, in whose genome the C1 constant regions are replaced by the human immunoglobulin C constant regions and the C constant region is replaced by the human immunoglobulin C constant region. The transgenic mouse yields humanized IgE-secreting B cells and antigen-specific humanized IgE after immunization. The transgenic animals are employed to prepare serum containing humanized IgE, antiserum containing antigen-specific humanized IgE, and monoclonal antigen-specific humanized IgE antibodies by hybridoma and other technologies.

Claims

1. A transgenic animal, in whose genome the gene segment encoding CH1-CH2-CH3-M1-M2 of one of the animal's endogenous immunoglobulins of C is replaced by the gene segment encoding CH1-CH2-CH3-CH4-M1-M2 of human immunoglobulin C.

2. A transgenic animal of claim 1, in which the animal is a mouse, rat, or rabbit.

3. A transgenic animal of claim 1, in which the animal is a mouse and the C is C1.

4. A transgenic mouse of claim 3, in which the mouse is further crossed with a transgenic mouse, in whose genome the mouse's endogenous C constant region coding sequence is replaced by the human immunoglobulin C constant region coding sequences.

5. A method for producing serum or antigen-specific antiserum containing humanized IgE by using a transgenic animal, in whose genome the gene segment encoding CH1-CH2-CH3-M1-M2 of one of the animal's endogenous immunoglobulins of C is replaced by the gene segment encoding CH1-CH2-CH3-CH4-M1-M2 of human immunoglobulin C; for the method of producing antigen-specific antiserum, the animal is immunized with the specific antigen.

6. A method for producing serum or antigen-specific antiserum containing humanized IgE of claim 5, wherein the transgenic animal is a mouse, rat, or rabbit.

7. A method for producing serum or antigen-specific antiserum containing humanized IgE of claim 5, wherein the animal is a mouse and the C is C1.

8. A method for producing serum or antigen-specific antiserum containing humanized IgE of claim 7, wherein the mouse strain is further crossed with a transgenic mouse strain, in whose genome the mouse's endogenous C constant region sequence is replaced by the human immunoglobulin C constant region sequence; the homozygous mouse strain with both transgenic human C and C is used as the host for the production of serum or antigen-specific antiserum.

9. A method of preparing antigen-specific humanized IgE-secreting hybridomas by using the lymphocytes of a transgenic animal, in whose genome the gene segment encoding CH1-CH2-CH3-M1-M2 of one of the animal's endogenous immunoglobulins of C is replaced by the gene segment encoding CH1-CH2-CH3-CH4-M1-M2 of human immunoglobulin C; the animal is immunized with the specific antigen.

10. A method of preparing antigen-specific humanized IgE-secreting hybridomas of claim 9, wherein the transgenic animal is a mouse, rat, or rabbit

11. A method of preparing antigen-specific humanized IgE-secreting hybridomas of claim 9, wherein the animal is a mouse and the C is C1.

12. A method of preparing antigen-specific humanized IgE-secreting hybridomas of claim 11, wherein the mouse strain is further crossed with a transgenic mouse strain, in whose genome the mouse's endogenous

Description

DETAILED DESCRIPTION OF THE INVENTION

1. Altering the Relative Abundance of Immunoglobulin Isotypes

[0009] The immunoglobulin heavy chain gene locus (IGHC) contains in one cluster of the genes encoding the constant regions of all of the classes and subclasses of heavy chains, including chain of IgM, chain of IgD, and chain of IgG, and a chain of IgA, and chain of IgE. In both human and mouse, the class has four subclasses and the class has two subclasses. In human genome, the IGHC is arranged in the order of --3-1-1-2-4--2, and in the mouse genome, IGHC is arranged in the order --3-1-2b-2a (or 2c)--. The gene elements encoding each of the subclasses is separated from the neighboring subclass by the switch (S) regions involved in class switch recombination (CSR).

[0010] The immune-competent resting B lymphocytes bear surface membrane-bound IgM and IgD (mIgM and mIgD). Upon initial antigen stimulation, the first antibodies produced by the lymphocytes are of the IgM class. With continual or repeated antigen stimulation, the activated B lymphocytes expand, differentiate, and secrete antibodies toward the antigens. One important aspect of this antibody response is that the B cells undergo isotype-switching from originally IgM production to the production of another isotype. The regulation and the determination of isotypes are mediated by a network of cytokines, chemokines, transcription activators, and negative regulators. Following antigen stimulation, signaling pathways recruit those factors which regulate the expression of germ line transcripts and the switch regions of the individual genes (Chaudhuri and Alt 2004; Stavnezer and Amemiya 2004; Pan-Hammarstroem et al. 2007). CSR that effectuates the change in antibody class is a deletional recombination where the constant region gene of the heavy chain C is replaced by a downstream C.sub.H gene and the intervening sequences are excised as circular DNA. CSR is initiated by activation-induced deaminase acting within the S region, which is followed with double strand breaks, DNA damage response/repair pathways and nonhomologous end joining (Chaudhuri and Alt 2004). The Ig of different class and subclass is expressed at different levels. In general, IgG, IgA, and IgM are expressed at much higher levels than IgD and IgE. And between IgD and IgE, the latter is still much lower. In addition to the different levels of production among the different classes, the turnover rate of free Ig and the stabilization of each Ig class by its receptor contribute to the overall turnover kinetics, the abundance, and half-life of the Ig class.

[0011] The present invention pertains to genetically altering an animal, so that the IgE in the altered animal becomes humanized IgE and its production is much higher than the IgE in an unaltered animal host. For achieving this, a mouse, rat, or rabbit is used, because genetic alteration of the antibody genes in these animals can be achieved with existing tools of molecular biology and embryonic stem cell manipulation, and the information concerning the immunoglobulin gene complexes in these animals. Furthermore, among these animals, mouse is a good choice because the time for reproduction is short and the tools for preparing transgenic strains are well established.

[0012] To increase the overall IgE levels, the coding sequences for the constant region of one of C immunoglobulin, such as C1, which is expressed at high levels, is replaced by the coding sequence for the constant region of human C. In doing so, the regulatory sequences in the promoter and the S regions of the mouse own C gene are kept, so that the control of expression of the knock-in human C may also achieve high expression. It is noted that since human IgE is not recognized by mouse FcRI, the transgenic mice should not have adverse conditions even they produce large quantities of humanized IgE.

2. Construction of a Chimeric Transgene Comprising Human C Coding Sequences Replacing the Mouse C1 Coding Sequences in Mouse Immunoglobulin Heavy Chain Gene Locus (mIGHG)

[0013] The replacement is achieved via homologous recombination between a designed construct and a mouse BAC clone containing the mouse IGHG locus (Clone ID RP24-258E20, FIG. 1A). The construct can be generated by PCR amplification incorporating the coding regions of human C CH1-CH2-CH3-CH4-M1-M2, flanked at either end with 2,000 bp each of the mouse sequences upstream and downstream, respectively, of the mouse C1 gene at the recombination sites. The homologous recombination can be performed in E. coli using the Red/ET Recombination methodology (Gene Bridges GmbH, Dresden, Germany). Specifically, the homologous recombination occurs in two steps. First, a counter selection marker rpsL-neo replaces the mouse C1 coding region for CH1-H-CH2-CH3-M1-M2 and is incorporated between the mouse homologous arms (the 2,000 bp sequences described above). H represents the hinge region. Then, the counter selection marker is replaced with the human C region encoding CH1-CH2-CH3-CH4-M1-M2.

3. Construction of a Chimeric Transgene Comprising Human C Coding Sequences Replacing the Mouse C Coding Sequences in Mouse Immunoglobulin Light Chain Locus (IGKC)

[0014] A construct is designed with PCR amplification incorporating human C coding sequences flanked at either end with 50 bp each of the mouse sequences in the noncoding region upstream and downstream, respectively, of the mouse C gene at the recombination sites. The construct is then integrated into a mouse BAC clone containing the IGKC locus (Clone ID RPCI23-59O5, FIG. 1A) via Red/ET Recombination methodology in E. coli (Gene Bridges GmbH, Dresden, Germany). Again, the homologous recombination occurs in two steps. First, a counter selection marker rpsL-neo replaces the mouse C coding region and is incorporated between the mouse homologous arms (the 50 bp sequences described above). Then, the counter selection marker is replaced with the human C coding sequences.

4. Generation of Transgenic Mice Harboring the Chimeric Transgenes

[0015] The method for transgene transfer employs the embryonic stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos. Transgenes can be efficiently introduced into the ES cells by electroporation, retrovirus-mediated transduction or other methods. The preferred method is electroporation. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.

[0016] Homologous recombination can also be used to introduce transgenes. Homologous recombination can be mediated by either RecE/RecT (RecE/T) or Red /. In E. coli, any intact, independently replicating, circular DNA molecule can be altered by RecE/T or Red / mediated homologous recombination with a linear DNA fragment flanked by short regions of DNA sequence identical to regions present in the circular molecule. Integration of the linear DNA fragment into the circular molecule by homologous recombination replaces sequences between its flanking sequences and the corresponding sequences in the circular DNA molecule.

[0017] The homologous recombination described in sections 3 and 4 above yield transgenes comprising modified mouse BAC clones harboring the human C coding sequences and C coding sequences, respectively. Each transgene is then introduced via electroporation into embryonic stem cells of mouse strain C57BL/6 where homologous recombination of the transgene and the corresponding endogenous gene locus takes place. The colonies verified to contain successfully recombined transgenes are then injected into blastocysts of C57BL/6, which are subsequently transferred into the uterus of pseudopregnant mice of the C57BL/6J-c2J strain. The embryos are allowed to develop into chimeric mice, which are then monitored to produce transgenic mice as in the standard procedures listed above.

[0018] The transgenic mice harboring the human C coding region substituting mouse C1 coding region and those harboring the human C coding region substituting mouse C coding region are then crossed to produce mice harboring both transgenes in place of the respective endogenous coding sequences. The resulted mouse strain that harbors both transgenes is used for the production of antigen-specific humaninzed IgE and hybridomas secreting antigen-specific humanized IgE.

5. Production of Antiserum Containing Antigen-Specific Humanized IgE and Hybridomas Secreting Antigen-Specific Humanized IgE

[0019] The transgenic mice resulted from the crosses as described in section 4 are used to generate antigen-specific humanized IgE and hybridomas secreting antigen-specific humanized IgE. Two examples of specific IgE production are: (i) antigens, such as dust mites, and weed, grass or tree pollens, and (ii) Geohelminth parasites, such as Necator americanus (human hookworm) and Trichurls suis (pig whipworm).

Examples

1. Preparation of Recombination-Potent Bacterial Artificial Chromosome (BAC)-Bearing Bacteria and Replacing Mouse C1-Encoding Gene with a Prokaryotic Selection DNA Cassette

[0020] The bacterial clone carrying BAC RP24-258E20, which contains gene exons encoding mouse four C heavy chains (FIG. 1A and FIG. 2, sequence a), was purchased from BACPAC Resources Center. The gene replacement was accomplished by using the Red/ET-based recombination system.

[0021] To prepare recombination-potent BAC-bearing bacteria, the pRed/ET plasmid DNA which encodes enzymatic proteins essential for mediating homologous recombination was delivered into the BAC-bearing bacteria. A single colony of BAC-bearing bacteria grown on LB agar with chloramphenicol and streptomycin was inoculated in 1 ml LB medium with antibiotics. After culturing at 37 C. overnight, the bacteria (30 l) were added into 1.4 ml of LB medium with antibiotics and cultured at 37 C. for 2 hours. The bacteria were placed on ice followed by centrifugation at 11,000 rpm for 30 s and the supernatant was removed. The pellet was washed with 1 ml of chilled 10% glycerol and centrifuged to remove the supernatant. The pellet was resuspended in 20-30 l of chilled 10% glycerol and placed on ice. The pRed/ET plasmid DNA (20 ng) was added into the bacteria and mixed briefly. The mixture was transferred into a chilled 1-mm electroporation cuvette and shocked at 1.8 kV, 200 ohms, and 25 F for 4.55.0 ms. The electroporation condition was used in the following examples. LB medium (1 ml) was added to resuspend the bacteria and then transferred into a culture vessel. The bacteria were cultured at 30 C. for 70 mins and 100 l of cultured bacteria was spread onto an LB agar plate with chloramphenicol and tetracycline. The plate was incubated at 30 C. overnight for growth of pRed/ET plasmid DNA-carrying bacteria which were recombination-potent.

[0022] The mouse C1-encoding gene in the recombination-potent BAC-bearing bacteria was replaced by a prokaryotic selection DNA cassette which contains a hybrid rpsL-neo gene that confers streptomycin-sensitive and kanamycin-resistant selection for transfected bacteria. A single colony of the recombination-potent BAC-bearing bacteria was inoculated in 1 ml of LB with chloramphenicol and tetracycline. After culturing at 30 C. overnight, 30 l of cultured bacteria were added into 1.4 ml of LB medium with antibiotics followed by culturing at 30 C. for 2 hours. L-arabinose at final 10% was added into the culture bacteria with culturing at 37 C. for another 1 hour. The bacteria were placed on ice and then centrifuged at 11,000 rpm for 30 s to remove the supernatant. The pellet was then washed with 1 ml of chilled 10% glycerol and centrifuged to remove the supernatant. The pellet was then resuspended in 20-30 l of chilled 10% glycerol and placed on ice. The DNA stretch containing the hybrid rpsL-neo gene flanked with two 50-bp DNA sequences corresponding to intronic sequences of the overhangs of mouse C1-encoding gene (SEQ ID NO:1) was prepared by polymerase chain reaction (PCR) with specific primers (TABLE 1, primers G1_CH1-rpsL-neo+ and G1_M2-rpsL-neo-). The purified DNA product (100-200 ng) was added into the resuspended bacteria with brief mix. The mixture was transferred into a chilled 1 mm cuvette for electroporation. LB medium (1 ml) without antibiotics was added to resuspend the shocked bacteria and transferred into a culture vessel. The bacteria were cultured at 37 C. for 70 mins and 100 l of the cultured medium was spread onto an LB agar plate containing chloramphenicol, kanamycin, and tetracycline. The plate was incubated at 30 C. overnight and the grown colonies were screened for identifying bacteria carrying rpsL-neo knock-in BAC by colony PCR with specific primers (TABLE 2, primers G1_CH1-up-sc+ and rpsL_sc). Identified clones were grown onto an LB agar plate with antibiotics at 30 C. overnight.