Chimeric IgE Polypeptides and Host Cells
The invention provides methods and materials related to expressing chimeric IgE proteins. Specifically, the invention provides nucleic acid vectors, host cells, and methods for producing chimeric IgE polypeptides.
This application is a continuation of U.S. Ser. No. 10/438,794, filed on May 15, 2003, which claims the benefit of U.S. Provisional Application Ser. No. 60/382,552, filed May 21, 2002.
BACKGROUND1. Technical Field
The invention relates to methods and materials involved in expressing and purifying recombinant chimeric IgE polypeptides.
2. Background Information
Generating a vaccine to treat IgE-induced hypersensitivity conditions such as food, dander, and pollen allergies, asthma, and eczema requires large quantities of a chimeric IgE polypeptide effective to induce the production of anti-self IgE antibodies when administered to a mammal. Recombinant DNA technology is widely used in medicine, diagnostics, agriculture, and related fields to produce large amounts of a desired polypeptide quickly and cost-effectively. Appropriately designed genetic constructs can permit a polypeptide to be produced by a variety of prokaryotic and eukaryotic organisms, including bacteria, yeast, insect larvae, insect cells, and mammalian cells. The organisms and nucleic acid vectors used depend on such factors as structure and function of the desired polypeptide, post-translational modification requirements, purification requirements and restrictions, required yields, and the purified polypeptide's intended application.
SUMMARYThe invention provides methods and materials related to expressing IgE polypeptides such as chimeric IgE polypeptides. Specifically, the invention provides nucleic acid vectors, host cells, and methods for producing chimeric IgE polypeptides. When administered to a mammal, the chimeric IgE polypeptides provided herein can reduce the IgE antibody effects of IgE-related diseases such as asthma, allergies, and eczema.
In one aspect, the invention provides a host cell having a nucleic acid vector, wherein the nucleic acid vector includes a cytomegalovirus promoter, an Ig leader sequence, an insert sequence, and a SV40 late polyadenylation sequence such that the cytomegalovirus promoter is upstream from and operably linked to the insert sequence, the Ig leader sequence is downstream from the cytomegalovirus promoter and upstream from and operably linked to the insert sequence, the SV40 late polyadenylation sequence is downstream from and operably linked to the insert sequence, and the insert sequence encodes a chimeric IgE polypeptide. The insert sequence can have a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17. The chimeric IgE polypeptide encoded by the insert sequence can have a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. The host cell can be a JM109, DH5α, NS0, HeLa, BHK-21, COS-7, Sf9, or CHO cell, or descendants therefrom that include a chimeric IgE polypeptide.
In another aspect, the invention provides a method for producing a chimeric IgE polypeptide by culturing a eukaryotic cell having a vector including a cytomegalovirus promoter, an Ig leader sequence, an insert sequence, and a SV40 late polyadenylation sequence such that the cytomegalovirus promoter is upstream from and operably linked to the insert sequence, the Ig leader sequence is downstream from the cytomegalovirus promoter and upstream from and operably linked to the insert sequence, the SV40 late polyadenylation sequence is downstream from and operably linked to the insert sequence, and the insert sequence encodes a chimeric IgE polypeptide; and recovering the chimeric IgE polypeptide from the culture. Again, the insert sequence can have a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17. Also, the chimeric IgE polypeptide can have a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. Further, the host cell can be a JM109, DH5α, NS0, HeLa, BHK-21, COS-7, Sf9, or CHO cell.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DESCRIPTION OF DRAWINGS
The invention provides methods and materials related to expressing IgE polypeptides such as chimeric IgE polypeptides. Specifically, the invention provides nucleic acid vectors, host cells, and methods for producing chimeric IgE polypeptides.
Nucleic acid vectors can be designed to express chimeric IgE polypeptides. Such nucleic acid vectors can contain an insert sequence. The term “insert sequence” as used herein refers to a nucleic acid sequence that is inserted into a nucleic acid vector such that that inserted nucleic acid sequence can be expressed. Typically, an insert sequence is a nucleic acid sequence that encodes a chimeric IgE polypeptide. The term “chimeric IgE polypeptide” as used herein refers to a polypeptide having a combination of IgE domains from different species. A chimeric IgE polypeptide typically contains IgE constant heavy (CH) chain domains (e.g., CH1, CH2, CH3, or CH4). For example, an insert sequence having the sequence set forth in SEQ ID NO:2 can encode an opossum CH2-rat CH3-opossum CH4 (ORO) chimeric IgE polypeptide (SEQ ID NO:3). Other examples of insert sequences include, without limitation, an insert sequence having the sequence set forth in SEQ ID NO:5 that encodes an opossum CH2-human CH3-opossum CH4 (OSO) chimeric IgE polypeptide (SEQ ID NO:6), an insert sequence having the sequence set forth in SEQ ID NO:7 that encodes an opossum CH2-rat CH3-opossum CH2-rat CH3-opossum CH4 (ORORO) chimeric IgE polypeptide (SEQ ID NO:8), an insert sequence having the sequence set forth in SEQ ID NO:15 that encodes an opossum CH2-human CH3-opossum CH2-human CH3-opossum CH4 (OSOSO) chimeric IgE polypeptide (SEQ ID NO:16), as well as those chimeric IgE polypeptides disclosed in International Patent Application Serial No. PCT/SE99/01896. In addition to rat and human, IgE domains from other species may be used in chimeric insert sequences. Such species include, without limitation, dog, cat, horse, pig, cow, and monkey. For example, an insert sequence including IgE domains from opossum and monkey (e.g., cynomolgus) can encode an opossum CH2-cynomolgus CH3-opossum CH4 (OCO) chimeric IgE polypeptide. Other insert sequences having IgE domains from opossum and monkey include, without limitation, sequences that encode opossum CH2-cynomolgus CH3-opossum CH4 (OCO-H), where the sequence contains a C-terminal histidine-tag; sequences that encode opossum CH2-cynomolgus CH3-opossum CH2-cynomolgus CH3-opossum CH4 (OCOCO); and sequences that encode opossum CH2-cynomolgus CH3-opossum CH2-cynomolgus CH3-opossum CH4, where the sequence contains a C-terminal histidine-tag (OCOCO-H).
An insert sequence can be modified. Such modifications can include, without limitation, additions, deletions, substitutions, point mutations, and combinations thereof. An insert sequence can be modified to include a C-terminal polyhistidine sequence to aid in the purification of the polypeptide encoded by the insert sequence. Polyhistidine sequences used for this purpose have been described elsewhere (Ford et al., Protein Expr. Purif., 2(2-3):95-107, 1991). For example, an insert sequence having the sequence set forth in SEQ ID NO:13 can encode an OSO chimeric IgE polypeptide including a C-terminal polyhistidine sequence (OSO-H; SEQ ID NO:14). An insert sequence can be modified to contain point mutations. For example, an insert sequence having the sequence set forth in SEQ ID NO:11 can encode an OSOSO chimeric IgE polypeptide containing point mutations in the human CH3 domains that abolish mast cell receptor binding (modOSOSO; SEQ ID NO:12). Other examples of modified insert sequences include, without limitation, an insert sequence having the sequence set forth in SEQ ID NO:17 that encodes an OSOSO chimeric IgE polypeptide including a C-terminal polyhistidine sequence (OSOSO-H; SEQ ID NO:18) and an insert sequence having the sequence set forth in SEQ ID NO:9 that encodes an OSOSO chimeric IgE polypeptide including a C-terminal polyhistidine sequence and containing point mutations in the human CH3 domains that abolish mast cell receptor binding (modOSOSO-H; SEQ ID NO:10).
A nucleic acid vector also can contain components that affect the expression of the insert sequence. Examples of such components include, without limitation, promoters, enhancers, leaders, and polyadenylation sequences. Such components can be operably linked to the insert sequence. The term “operably linked” as used herein refers to an arrangement where components so described are configured so as to perform their usual function. For example, a nucleic acid vector with an insert sequence encoding an OSOSO chimeric IgE polypeptide also can contain a cytomegalovirus (CMV) promoter (see, for example, Thomson et at., Proc. Natl. Acad. Sci. U.S.A., 81(3):659-663, 1984), an immunoglobulin (Ig) leader sequence (see, for example, Neuberger et al., EMBO J., 2(8):1373-1378, 1983), and a bovine growth hormone (bGH) polyadenylation sequence (see, for example, Goodwin et al., J. Biol. Chem., 267:16330-16334, 1992). In this case, the components are operably linked to the insert sequence such that the CMV promoter drives the expression of the insert sequence including the Ig leader sequence and bGH polyadenylation sequence, the Ig leader sequence directs the expressed insert sequence into the lumen of the endoplasmic reticulum in preparation for secretion, and the bGH polyadenylation sequence stabilizes the insert sequence transcript.
In addition, a nucleic acid vector can contain components that aid in the growth, maintenance, or selection of a host cell containing the nucleic acid vector. Such components include, without limitation, origins of replication and antibiotic selection markers. For example, a nucleic acid vector with a CMV promoter, an Ig leader sequence, an SV40 late polyadenylation sequence, and an insert sequence encoding an OSOSO chimeric IgE polypeptide can also contain an fl origin of replication, a sequence that confers ampicillin resistance on a bacterial host cell when expressed, and a sequence that confers neomycin resistance on a mammalian host cell when expressed. Other examples of antibiotic selection markers include, without limitation, sequences that confer resistance to hygromycin B, puromycin, kanamycin, tetracycline, blasticidin S, Geneticin®, and zeocin on a host cell when expressed. Nucleic acid vectors that contain one or more than one component described herein can be obtained commercially from, for example, Invitrogen (Carlsbad, Calif.) and Promega (Madison, Wis.).
In one embodiment, the invention provides a nucleic acid vector (e.g., the pCI-neo vector from Promega, catalogue number E1841) containing at least one of the insert sequences provided herein (e.g., ORO, OSO, ORORO, modORORO-H, modOSOSO, OSO-H, OSOSO, and OSOSO-H). The invention also provides host cells that contain a nucleic acid vector described herein. Such cells can be prokaryotic cells (e.g., JM109 or DH5α) or eukaryotic cells (e.g., NS0, HeLa, BHK-21, COS-7, Sf9, or CHO). Host cells containing a nucleic acid vector provided herein may or may not express a polypeptide. For example, a host cell may function simply to propagate the nucleic acid vector for use in other host cells. In addition, the nucleic acid vector can be integrated into the genome of the host or maintained in an episomal state. Thus, a host cell can be stably or transiently transfected with a nucleic acid vector containing an insert sequence of the invention.
A host cell within the scope of the invention can contain a nucleic acid vector with an insert sequence that encodes a chimeric IgE polypeptide. For example, a host cell can contain a nucleic acid vector with an insert sequence encoding an OSO chimeric IgE polypeptide or any of the chimeric IgE polypeptides provided herein. In addition, a host cell can express the polypeptide encoded by the insert sequence.
Various methods can be used to introduce a nucleic acid vector into a host cell in vivo or in vitro. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods that can be used to introduce a nucleic acid vector into a host cell. In addition, naked DNA can be delivered directly to cells in vivo as described elsewhere (U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466 including continuations thereof). Further, a nucleic acid vector can be introduced into cells in the context of generating transgenic animals.
Transgenic animals can be aquatic animals (such as fish, sharks, dolphin, and the like), farm animals (such as pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (such as baboon, monkeys, and chimpanzees), and domestic animals (such as dogs and cats). Several techniques known in the art can be used to introduce a nucleic acid vector into animals to produce the founder lines of transgenic animals. Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Nat. Acad. Sci., USA, 82:6148 (1985)); gene transfection into embryonic stem cells (Gossler A et al., Proc Natl Acad Sci USA 83:9065-9069 (1986)); gene targeting into embryonic stem cells (Thompson et al., Cell, 56:313 (1989)); nuclear transfer of somatic nuclei (Schnieke A E et al., Science 278:2130-2133 (1997)); and electroporation of embryos (Lo C W, Mol. Cell. Biol., 3:1803-1814 (1983)). Once obtained, transgenic animals can be replicated using traditional breeding or animal cloning.
Various methods can be used to identify a host cell containing a nucleic acid vector of the invention. Such methods include, without limitation, PCR, nucleic acid hybridization techniques such as Northern and Southern analysis, and in situ nucleic acid hybridization. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a nucleic acid vector with a particular insert sequence by detecting the expression of a polypeptide encoded by that particular insert sequence.
The invention also provides methods for producing recombinant chimeric IgE polypeptides. Such methods involve culturing a host cell that expresses a chimeric IgE polypeptide and recovering the expressed chimeric IgE polypeptides. Any method can be used to recover a recombinant chimeric IgE polypeptide. For example, recombinant chimeric IgE polypeptides that are present in a host cell homogenate can be recovered using ion exchange chromatography. In another example, recombinant chimeric IgE polypeptides with polyhistidine sequences can be recovered from a host cell homogenate by passing the homogenate over a nickel column and eluating the polyhistidine-containing polypeptides with imidazole. A particular recombinant chimeric IgE polypeptide with a leader sequence that directs that polypeptide's secretion can be recovered from the growth medium of a host cell expressing that polypeptide. For example, the growth medium from a culture of mammalian host cells expressing and secreting ORO polypeptides can be collected, and the ORO polypeptides can be recovered using chromatography. It is understood that a leader sequence that directs the secretion of a polypeptide typically is removed from that polypeptide in the host cell by proteolysis. Thus, the recovered secreted polypeptide, in many cases, is free of any translated leader sequence.
In one embodiment, the cell medium from a clonal CHO cell line expressing and secreting ORO polypeptides is collected and centrifuged to remove cell debris. After centrifuging, the supernatant is dialyzed and passed over an ion exchange column allowing the ORO polypeptides to bind. The bound ORO polypeptides are eluted using a sodium chloride/sodium acetate gradient, and the eluated fractions are screened for recombinant ORO polypeptides using an ELISA technique. The eluated fractions with the best ELISA reactivity can be pooled and dialyzed again, and the dialyzed pooled fractions can be passed over a hydrophobic interaction column allowing the ORO polypeptides to bind. The bound ORO polypeptides are eluted using a sodium phosphate gradient, and the eluated fractions are again screened for recombinant ORO polypeptides using an ELISA technique. The eluated fractions with the best ELISA reactivity can be further analyzed by silver stained SDS-PAGE to estimate the purity of the ORO polypeptides.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES Example 1 Nucleic Acid Vectors for Expressing Chimeric IgE PolypeptidesPlasmids containing cDNA sequences encoding polyhistidine-tagged ORO or OSO are used in PCRs with primers designed to amplify ORO or OSO without a polyhistidine tag (5′-AACTCGAGTACTTTATCTCTCCCAGAA-3′ (SEQ ID NO:19) and 5′-ATCTCGAGATTAGTTACCAGCTGAGTAATGCA-3′ (SEQ ID NO:20)). XhoI restriction enzyme recognition sites are included in each PCR primer to allow PCR amplification products to be ligated into an XhoI-digested and phosphatase-treated plasmid. Each PCR amplification cycle consists of a denaturing step at 94° C. for 30 seconds, an annealing step at 52° C. for 30 seconds, and an elongating step at 68° C. for 3 minutes. This amplification cycle was repeated 25 times. Each reaction begins with a single denaturing step at 94° C. for 2 minutes and ends with a single elongating step at 68° C. for 10 minutes. The resulting PCR amplification products are purified and digested with XhoI. The digested products are then purified. The resulting purified amplification products are ligated into an XhoI-digested and phosphatase-treated plasmid, producing ORO and OSO nucleic acid vectors. DNA sequencing and transient expression in CHO cells is used to verify the orientation, reading frame, and fidelity of each nucleic acid vector.
Example 2 Isolating Stable CHO Clones Expressing Chimeric IgE PolypeptidesThe nucleic acid vectors described in Example 1 are linearized by digestion with PvuI. CHO cells are transfected with the linearized nucleic acid vectors using LipofectAMINE™ 2000 (Life Technologies, Rockville, Md.). After 24 hours, the transfected cells are seeded into three 96-well plates with selective medium consisting of DMEM containing 10% FBS and 600 μg/mL G418. After 21 days, clones are screened for recombinant chimeric IgE polypeptide expression by ELISA. Briefly, the wells of ELISA plates are coated with rabbit anti-OOO immunoglobulin diluted 1:5000 in coating buffer (0.1 M sodium carbonate, pH 9.5). The coated plates are incubated overnight at 4° C. After washing the coated plates four times with PBS containing 0.05% Tween20, each well of the washed ELISA plates is treated with 100 μL of PBS containing 3% BSA for one hour at room temperature to block any subsequent non-specific binding interactions. After blocking, 100 μL of a recombinant IgE chimera protein sample is loaded into each well, and the loaded ELISA plate is incubated. Following the incubation, the plates are washed four times in PBS containing 0.05% Tween20. The washed plates are treated with 100 μL of either an HRP-conjugated goat anti human IgE antibody (KPL 074-1004, 1:2000; Merck Eurolab, Stockholm, Sweden) when detecting OSO (SEQ ID NO:6), or a monoclonal mouse anti-rat IgE antibody (MAS 314, Harlan Seralab, Leicestershire, England) when detecting ORO (SEQ ID NO:3), and then incubated for 1 hour at room temperature. Detecting ORO requires an additional incubation with an HRP-conjugated rabbit anti-mouse Ig antibody (P0260, Dako, Carpinteria, Calif.) for 1 hour at room temperature. Following the incubations, the treated plates are washed 8 times with PBS containing 0.05% Tween20 and developed according to the manufacturer's instructions. The protein concentrations are determined by measuring the absorbance at 490-650 nm.
Clones exhibiting the highest levels of recombinant chimeric IgE polypeptide expression for each nucleic acid vector are expanded and further selected by limiting dilution into 96-well plates. After selection, 48 clones are screened for recombinant chimeric IgE polypeptide expression by ELISA. Two clones exhibiting the highest levels of recombinant chimeric IgE polypeptide expression for each nucleic acid vector are expanded and further selected by limiting dilution into 96-well plates. After selection, the number of clones retaining the expression of recombinant chimeric IgE polypeptides after limiting dilution is determined by ELISA. One clone for each nucleic acid vector is expanded for large-scale recombinant chimeric IgE polypeptide production. A portion of the expanded clone is frozen and preserved as a stock. Clonal cell lines expressing ORO and OSO are generated in this manner.
Example 3 Purifying Recombinant Chimeric IgE PolypeptidesThe clonal cell lines described in Example 2 are grown and maintained at 37° C., 5% CO2 in cell medium. The cell medium containing secreted chimeric IgE polypeptides is collected every few days. The collected medium is centrifuged to remove cell debris. After centrifuging, the supernatant is dialyzed against 50 volumes of 25 mM sodium acetate buffer (pH 5.9) for 24 hours. The dialyzed supernatant is passed over a SP-Sepharose ion exchange column allowing the chimeric IgE polypeptides to bind. The bound chimeric IgE polypeptides are eluted in fractions using a gradient of 2 M sodium chloride in 25 mM sodium acetate buffer (pH 5.9). The eluated fractions are screened for recombinant chimeric IgE polypeptides by ELISA. The eluated fractions with the best ELISA reactivity are pooled and dialyzed against 10 mM sodium phosphate buffer containing 1.6 M ammonium sulphate (pH 6.5). The pooled fractions are passed over a phenyl- or butyl-Sepharose hydrophobic interaction column allowing the chimeric IgE polypeptides to bind. The bound chimeric IgE polypeptides are eluted in fractions using a gradient of 10 mM sodium phosphate buffer without ammonium sulphate (pH 6.5). The eluated fractions are screened for recombinant chimeric IgE polypeptides by ELISA. The eluated fractions with the best ELISA reactivity are further analyzed by silver stained SDS-PAGE to estimate the purity of the chimeric IgE polypeptides.
OTHER EMBODIMENTSIt is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1. A clonal cell line expressing a chimeric IgE polypeptide.
2. The clonal cell line of claim 1, wherein said cell line is a JM109, DH5α, NS0, HeLa, BHK-21, COS-7, Sf9, or CHO cell line.
3. The clonal cell line of claim 1, wherein said cell line is a CHO cell line.
4. The clonal cell line of claim 1, wherein said chimeric IgE polypeptide comprises a human IgE CH3 domain.
5. The clonal cell line of claim 1, wherein said cell line comprises a nucleic acid comprising a cytomegalovirus promoter, an immunoglobulin (Ig) leader sequence, an insert sequence, and a SV40 late polyadenylation sequence, wherein said cytomegalovirus promoter is upstream from and operably linked to said insert sequence, said Ig leader sequence is downstream from said cytomegalovirus promoter and upstream from and operably linked to said insert sequence, said SV40 late polyadenylation sequence is downstream from and operably linked to said insert sequence, and said insert sequence encodes said chimeric IgE polypeptide.
6. The clonal cell line of claim 1, wherein said chimeric IgE polypeptide has the amino acid sequence set forth in SEQ ID NO:13.
Type: Application
Filed: May 9, 2007
Publication Date: Jan 10, 2008
Inventors: Mats Lundgren (Stockholm), Alexis Fuentes (Uppsala), Ann-Christin Magnusson (Uppsala)
Application Number: 11/746,382
International Classification: C12N 5/06 (20060101);
