U.S. patent application number 10/960169 was filed with the patent office on 2005-04-28 for cell lines and methods for producing proteins.
Invention is credited to Deo, Yashwant M., Parker, Stephen H..
Application Number | 20050090001 10/960169 |
Document ID | / |
Family ID | 34526530 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050090001 |
Kind Code |
A1 |
Parker, Stephen H. ; et
al. |
April 28, 2005 |
Cell lines and methods for producing proteins
Abstract
The present invention relates to the isolation and enrichment of
avian oviduct tumor cells to obtain sustainable cell lines that can
be passaged multiple times in cell culture and can be used for
long-term production of heterologous polypeptides at higher yields.
The methods of the instant invention involve the manipulation
and/or propagation of oviduct tumor cells derived from either
wild-type or transgenic avians.
Inventors: |
Parker, Stephen H.;
(Jefferson, GA) ; Deo, Yashwant M.; (New
Brunswick, NJ) |
Correspondence
Address: |
AVIGENICS, INC.
111 RIVERBEND ROAD
ATHENS
GA
30605
US
|
Family ID: |
34526530 |
Appl. No.: |
10/960169 |
Filed: |
October 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60509353 |
Oct 7, 2003 |
|
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|
Current U.S.
Class: |
435/349 ;
435/456; 800/19 |
Current CPC
Class: |
C12N 5/0682 20130101;
C12N 5/0693 20130101; C12N 2517/02 20130101; C12N 2510/02
20130101 |
Class at
Publication: |
435/349 ;
800/019; 435/456 |
International
Class: |
A01K 067/027; C12P
021/04; C12N 005/06; C12N 015/86 |
Claims
What is claimed is:
1. An isolated avian oviduct tumor cell line comprising a nucleic
acid encoding a heterologous protein, wherein the cell line is
sustainable in culture for at least three or more passages.
2. The cell line of claim 1 wherein the cell line is sustainable in
culture for at least ten or more passages.
3. The cell line of claim 1 wherein the cell line is sustainable in
culture for at least twenty or more passages.
4. The cell line of claim 1 wherein the nucleic acid is operably
linked to a promoter.
5. The cell line of claim 4 wherein the promoter is selected from
the group consisting of a cytomegalovirus (CMV) promoter, a MDOT
promoter and a rous-sarcoma virus (RSV) promoter.
6. The cell line of claim 1 wherein the nucleic acid is operably
linked to an oviduct-specific promoter.
7. The cell line of claim 6 wherein the oviduct-specific promoter
is selected from an ovomucoid promoter, an ovalbumin promoter, and
a lysozyme promoter.
8. The cell line of claim 1 wherein the heterologous protein is a
pharmaceutical composition.
9. The cell line of claim 1 wherein the avian is selected from the
group consisting of a chicken, turkey, duck, goose, quail,
pheasant, parrot, finch, hawk, crow or ratite, ostrich, emu and
cassowary.
10. The cell line of claim 1 wherein the avian is a chicken.
11. The cell line of claim 1 isolated from a transgenic avian that
expresses the heterologous protein in oviduct cells.
12. The cell line of claim 1 wherein the tumor cell line is an
epithelial or mesenchymal tumor.
13. The cell line of claim 1 wherein the tumor is an epithelial
adenoma or adenocarcinoma.
14. The cell line of claim 1 wherein the tumor is an epithelial
adenoma.
15. A composition comprising the cell line of claim 1 and a culture
medium.
16. The composition of claim 15 wherein the culture medium
comprises a heterologous protein expressed by the cell line.
17. An immortal cell line derived from a tubular gland cell
comprising a nucleic acid encoding a heterologous protein.
18. The cell line of claim 17 wherein the nucleic acid is operably
linked to a promoter.
19. The cell line of claim 17 wherein the nucleic acid is operably
linked to an oviduct-specific promoter.
20. The cell line of claim 17 wherein the promoter is selected from
an ovomucoid promoter, an ovalbumin promoter, and a lysozyme
promoter.
21. The cell line of claim 17 wherein the protein is a
pharmaceutical composition.
22. The cell line of claim 17 wherein the protein is a
cytokine.
23. The cell line of claim 17 wherein the protein is an
antibody.
24. The cell line of claim 17 wherein the protein is selected from
the group consisting of a mammalian monoclonal antibody, a fusion
protein, a growth factor, an enzyme, a structural protein,
interferon, granulocyte-colony stimulating factor, and
erythropoietin.
25. The cell line of claim 17 isolated from a transgenic avian that
expresses the heterologous protein in oviduct cells.
26. The cell line of claim 17 wherein the tumor cell line is an
epithelial or mesenchymal tumor.
27. The cell line of claim 17 wherein the tumor is an epithelial
adenoma or adenocarcinoma.
28. The cell line of claim 17 wherein the tumor is an epithelial
adenoma.
29. A composition comprising the cell line of claim 17 and a
culture medium.
30. The composition of claim 29 wherein the culture medium
comprises a heterologous protein expressed by the cell line.
31. A method of producing a heterologous protein comprising,
contacting an avian oviduct tumor cell with a nucleic acid encoding
a heterologous protein under conditions suitable for protein
expression, wherein the tumor cell is sustainable in culture for at
least three or more passages; and isolating the heterologous
protein.
32. The method of claim 31 wherein the nucleic acid is transfected
into the cell in vitro.
33. The method of claim 31 wherein the nucleic acid is contacted
with the cell in vivo.
34. The method of claim 31 wherein the nucleic acid comprises a
selectable marker, and wherein the method further comprises
selecting for tumor cells that express the marker.
35. The method of claim 31 further comprising enriching for tumor
cells that express the heterologous protein.
36. The method of claim 35 wherein the enriching comprises
selecting for cells that express gicerin.
37. The method of claim 36 wherein the selection is performed using
a fluorescently labeled antibody that binds gicerin.
38. The method of claim 31 further comprising enriching for tumor
cells that bind neurite outgrowth factor (NOF).
39. The method of claim 31 wherein the tumor is an epithelial
adenoma.
40. The method of claim 31 wherein the avian is a chicken.
41. The method of claim 31 wherein the transfection is performed by
microinjection, electroporation or lipofection.
42. The method of claim 31 wherein the tumor cell is sustainable in
culture for at least ten or more passages.
43. The method of claim 31 wherein the tumor cell is sustainable in
culture for at least twenty or more passages.
44. The method of claim 31 wherein the protein is a pharmaceutical
composition.
45. The method of claim 31 wherein the protein is a cytokine.
46. The method of claim 31 wherein the protein is an antibody.
47. The method of claim 31 wherein the protein is selected from the
group consisting of a mammalian monoclonal antibody, a fusion
protein, a growth factor, an enzyme, a structural protein,
interferon, granulocyte-colony stimulating factor, and
erythropoietin.
48. A method of producing a heterologous protein comprising:
isolating oviduct tumor cells that expresses the protein, wherein
the cell is sustainable in culture for at least three or more
passages; culturing the tumor cells under conditions suitable for
expression of the protein; and isolating the protein.
49. The method of claim 48 wherein the tumor cell is sustainable in
culture for at least ten or more passages.
50. The method of claim 48 wherein the tumor cell is sustainable in
culture for at least twenty or more passages.
51. The method of claim 48 wherein the avian is a chicken.
52. The method of claim 48 wherein the protein is a pharmaceutical
composition.
Description
RELATED APPLICATION INFORMATION
[0001] This application claims the benefit of U.S. provisional
application 60/509,353, filed Oct. 7, 2003.
BACKGROUND OF THE INVENTION
[0002] Recent advances have allowed for the generation of
transgenic avians that express heterologous proteins in their
oviduct cells. The avian oviduct contains an infundibulum, magnum,
isthmus, shell gland, vagina and cloaca (Etches, Reproduction in
Poultry. 1996, New York, N.Y.: CABI Publishing. 318). An ovulated
ovum enters the tract through the infundibulum and continues to the
magnum where the majority of egg white proteins, such as ovalbumin,
ovomucoid and lysozyme, are produced by tubular gland cells and
deposited on the ova. (Palmiter, J Biol Chem, 1972. 247: 6450-61;
Yu and Marquardt, Biol Reprod, 1973. 8: 283-98). Expression of the
major egg white proteins is controlled by hormone responsive
elements of the respective promoters (Palmiter, J Biol Chem, 1972.
247: 6450-61; Schimke et al., Basic Life Sci, 1973. 1: 123-35;
Mulvihill and Palmiter, J Biol Chem, 1977. 252: 2060-8; McKnight
and Palmiter, J Biol Chem, 1979. 254: 9050-8; Mulvihill and
Palmiter, J Biol Chem, 1980. 255: 2085-91; Shepherd et al., J Cell
Biol, 1980. 87: 142-51; Palmiter et al., J Biol Chem, 1981. 256:
7910-6; Sanders and McKnight, Endocrinology, 1985. 116:
398-405).
[0003] Chicken oviduct cells, when stimulated by steroid hormones
during egg-laying, secrete three principal polypeptides, ovalbumin,
ovomucoid and lysozyme (Tsai et al., (1978) Biochemistry 17:
5773-5779). The mRNA transcript encoding ovalbumin constitutes
about 50% of the total mRNA of these cells. Ovomucoid and lysozyme
mRNAs contribute about 6.6% and 3.4% respectively of the total mRNA
of the steroid stimulated cells. (Hynes et al. (1977) pp 932). The
ability of the avian oviduct to express large amounts of various
proteins makes it an attractive target for producing heterologous
proteins. To date, the most common method of expressing
heterologous proteins in avian oviducts is through the production
of transgenic hens. In one example, transgenic hens expressing
human Interferon alpha-2b (hIFN .alpha.2b) were generated (Rapp et
al., Transgenic Research, 2003). Also, transgenic hens were
generated that express human monoclonal antibody in their egg
white. U.S. Pat. No. 6,730,822, issued May 5, 2004, and U.S. patent
application Ser. No. 10/463,980, filed Jun. 17, 2003 filed
internationally as PCT/US04/01833, U.S. patent application Ser. No.
09/877,374, filed Jun. 8, 2001 filed internationally as
PCT/US02/02454, U.S. patent application Ser. No. 10/679,034, filed
Oct. 2, 2003 filed internationally as PCT/US02/29878, and U.S.
patent application Ser. No. 10/856,218, filed May 28, 2004 filed
internationally as PCT/US04/16827 disclose methods and compositions
useful for the generation of transgenic avians for production of
heterologous proteins. The disclosure of this issued US patent and
these four pending patent applications and their corresponding PCT
applications are incorporated herein by reference.
[0004] Primary cultures of oviduct tissue can be generated by
removing the magnum section of a sexually mature hen treated with
estrogen (Sanders and McKnight, Endocrinology, 1985. 116: 398-405).
The tissue is digested with collagenase and dispase to liberate
small cell clumps that are cultured for short durations. Cells
collected and cultured in this manner typically die or
differentiate into cells that do not produce egg white protein
within three days making primary cultures of normal oviduct cells
unsuitable for use in large scale in vitro production of
heterologous proteins. Accordingly, improved methods for producing
heterologous proteins in avian oviduct cells are needed.
SUMMARY OF THE INVENTION
[0005] The present invention is based, at least in part, on the
generation of avian cell lines, such as immortal avian cell lines,
that can be passaged multiple times in cell culture and provide
sustained production of heterologous polypeptides. In one
embodiment, the methods of the present invention rely upon the
manipulation and/or propagation of oviduct tumor cells derived from
either wild-type or transgenic avians.
[0006] Accordingly, in one aspect, the present invention features
an isolated avian cell line comprising a nucleic acid encoding a
heterologous protein, wherein the cell line is sustainable in
culture for at least 3 or more passages. Typically, the cell line
is sustainable in culture for at least 5 passages or 10 passages or
15 passages or 20 passages or 25 passages or 30 passages or 35
passages or 40 passages or 45 passages or 50 passages or 500
passages or 1000 passages or 5000 passages. In a particularly
useful embodiment, the cell line is an immortal cell line capable
of an unlimited number of passages. The nucleic acid is typically
operably linked to an oviduct-specific promoter or a non-specific
promoter capable of controlling expression of the nucleic acid.
[0007] Suitable oviduct-specific promoters include, for example,
and without limitation, ovomucoid promoters, ovalbumin promoters,
and lysozyme promoters, conalbumin promoters, ovomucin promoters,
ovotransferrin promoters and functional portions of each of these
promoters. Suitable non-specific promoters include, for example and
without limitation, cytomegalovirus (CMV) promoters, MDOT promoters
and rous-sarcoma virus (RSV) promoters, murine leukemia virus (MLV)
promoters, mouse mammary tumor virus (MMTV) promoters and SV40
promoters and functional portions of each of these promoters.
Nonlimiting examples of other promoters which may be useful in the
present invention include, without limitation, Pol III promoters
(for example, type 1, type 2 and type 3 Pol III promoters) such as
HI promoters, U6 promoters, tRNA promoters, RNase MPR promoters and
functional portions of each of these promoters. Typically,
functional terminator sequences are selected for use in the present
invention in accordance with the promoter that is employed.
[0008] Avian oviduct cell lines as disclosed herein are
contemplated as being derived from any suitable avian source
including, but not limited to, chicken, turkey, duck, goose, quail,
pheasant, parrot, finch, hawk, crow or ratite, for example,
ostrich, emu, cassowary. In one useful embodiment, the cell line is
derived from a chicken. In one embodiment, the cell line is
isolated from a wild type or transgenic avian that expresses the
heterologous protein in oviduct cells. In one embodiment, the tumor
cell line is contemplated as being derived from certain oviduct
tumors, such as an epithelial or mesenchymal tumors, which include,
for example, adenocarcinomas, mesenchymal adenomas, leiomyomas, and
fibromas. In particularly useful embodiment, the cell line is
derived from epithelial adenomas tumors.
[0009] The present invention further features a composition
comprising the isolated avian oviduct cell line of the invention
within a culture medium. In a related embodiment, the culture
medium comprises a heterologous protein expressed by the cell
line.
[0010] The present invention also features a method of producing a
heterologous protein comprising contacting an avian oviduct tumor
cell with a nucleic acid encoding a heterologous protein under
conditions suitable for expression of the protein, wherein the
tumor cell is sustainable in culture for at least 3, for example,
5, 10 passages, 15 passages, 20 passages, 50 passages or more
passages, and then isolating the heterologous protein. The nucleic
acid can be transfected into the cell in vitro, for example, by
microinjection, electroporation or lipofection or may be
transfected into the cell in vivo. The nucleic acid can further
include a selectable marker to facilitate selection of tumor cells
that express the marker. In an additional related embodiment, the
method includes enriching for tumor cells that express the
heterologous protein, by, for example, selecting for tumor cells
that express gicerin (for example, using a fluorescently labeled
antibody that binds gicerin) or selecting for tumor cells that bind
neurite outgrowth factor (NOF).
[0011] The present invention also features a method of producing a
heterologous protein comprising isolating oviduct tumor cells that
express the protein. In one embodiment, the cell is sustainable in
culture for at least 3 or more passages, culturing the tumor cells
under conditions suitable for expression of the protein, and then
isolating the protein.
[0012] The invention also provides heterologous proteins that are
produced by the foregoing methods and cell lines. Such proteins can
include, for example, antibodies, cytokines, fusion proteins,
growth factors, enzymes, structural proteins, interferons,
granulocyte-colony stimulating factor, and erythropoietins.
Pharmaceutical compositions comprising the heterologous proteins
along with a pharmaceutically acceptable carrier also are
provided.
[0013] Any combination of features described herein is included
within the scope of the present invention provided that the
features included in any such combination are not mutually
inconsistent. Such combinations will be apparent based on this
specification and the knowledge of one of ordinary skill in the
art.
DETAILED DESCRIPTION
[0014] The present invention relates to the isolation and
enrichment of avian oviduct cells to obtain sustainable cell lines
that can be passaged multiple times in cell culture and can be used
for long-term production of heterologous polypeptides at higher
yields. The methods of the present invention involve the
manipulation and/or propagation of oviduct tumor cells derived from
either wild-type or transgenic avians. The tumor cells may be
isolated directly from the wild-type or transgenic avians or may be
obtained by genetically altering (for example, by mutagenesis)
cells taken from normal oviduct tissue of wild-type or transgenic
avians. Preferably, the transgenic avians produce heterologous
protein in oviduct cells.
[0015] For purposes of describing the invention, the following
terms and definitions may be used.
[0016] The term "isolated avian oviduct tumor cell" and "avian
oviduct tumor cell" or "oviduct tumor cell" as used herein, refers
to a cell substantially free of other cell types found in tumors or
non-tumorous avian oviduct tissue. An avian oviduct tumor cell line
as disclosed herein, may refer to cell lines obtained from one or
more cells of avian oviduct tumors or cell lines obtained from one
or more cells which have been subjected to in vitro mutagenesis as
disclosed herein.
[0017] The term "sustainable", as used herein, refers to the
ability of the cell or cell line to be passaged in culture for at
least three or more, or ten or more times, preferably twenty or
more or an infinite number of times.
[0018] The term "transfected", as used herein, refers to the uptake
of heterologous DNA or RNA by a cell. Transfection, as used herein,
encompasses all art-recognized procedures for introducing nucleic
acid into cells, including, for example, microinjection,
electroporation, chemical transfection and transduction into a
cell.
[0019] The term "oviduct tumor", as used herein, refers to tumorous
tubular gland cells from an avian oviduct (typically the magnum)
that secretes egg white proteins including, for example, ovalbumin,
ovomucoid, ovoinhibitor, conalbumin, ovomucin and lysozyme. Avian
oviduct tumors are typically of an epithelial or mesenchymal
nature. Epithelial tumors usually consist of adenomas or
adenocarcinomas. Mesenchymal tumors typically consist of adenomas,
leiomyomas or fibromas.
[0020] As used herein the terms "polypeptide" and "protein" refer
to a polymer of amino acids, linked through peptide bonds. The term
"polypeptide" includes proteins, protein fragments, protein
analogues, oligopeptides and the like. The term polypeptide as used
herein can also refer to a peptide.
[0021] The term "heterologous protein", as used herein, refers to a
protein that is not naturally expressed by the cell that produces
the protein.
[0022] The term "avian" as used herein refers to any species,
subspecies or race of organism of the taxonomic class ava, such as
chicken, turkey, duck, goose, quail, pheasants, parrots, finches,
hawks, crows and ratites. The term includes the various known
strains of Gallus gallus, or chickens, for example, White Leghorn,
Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island,
Ausstralorp, Minorca, Amrox, California Gray, Italian
Partidge-colored, as well as strains of turkeys, pheasants, quails,
duck, ostriches and other poultry commonly bred in commercial
quantities.
[0023] The terms "nucleic acid" and "polynucleotide", as used
herein refer to any natural or synthetic array of nucleotides (or
nucleosides), for example cDNA, genomic DNA, mRNA, tRNA,
oligonucleotides, oligonucleosides and derivatives thereof. The
term "gene" as used herein refers to a nucleic acid or
polynucleotide (including both RNA or DNA) that encodes genetic
information for the synthesis of a whole RNA, a whole protein, or
any portion of such whole RNA or whole protein. Genes that are not
naturally part of a particular organism's genome are referred to as
"foreign genes," "heterologous genes" or "exogenous genes" and
genes that are naturally a part of a particular organism's genome
are referred to as "endogenous genes". Such genes may be contained
in an "expression vector" which, as used herein, refers to a
nucleic acid vector that comprises an expression control region
operably linked to a nucleotide sequence coding for at least one
polypeptide. Such expression vectors therefore drive transcription
and expression of the gene.
[0024] As used herein, the term "regulatory sequences" includes
promoters, enhancers, and other elements that may control gene
expression. Standard molecular biology textbooks such as Sambrook
et al. eds "Molecular Cloning: A Laboratory Manual" 3rd ed., Cold
Spring Harbor Press (2001) may be consulted to design suitable
expression vectors that may further include an origin of
replication and selectable gene markers. It should be recognized,
however, that the choice of a suitable expression vector and the
combination of functional elements therein depends upon multiple
factors including the choice of the host cell to be transformed
and/or the type of protein to be expressed. Representative examples
of expression vectors include, for example, bacterial plasmid
vectors including expression, cloning, cosmid and transformation
vectors such as, but not limited to, pBR322, animal viral vectors
such as, but not limited to, modified adenovirus, influenza virus,
polio virus, pox virus, retrovirus, and the like and vectors
derived from bacteriophage nucleic acid, for example, plasmids and
cosmids, artificial chromosomes, such as but not limited to, Yeast
Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes
(BACs), and synthetic oligonucleotides like chemically synthesized
DNA or RNA. Accordingly, the term "nucleic acid vector" or "vector"
as used herein refers to a natural or synthetic single or double
stranded plasmid or viral nucleic acid molecule, or any other
nucleic acid molecule that can be transfected or transformed into
cells and replicate independently of, or within, the host cell
genome. A nucleic acid can be inserted into a vector by cutting the
vector with restriction enzymes and ligating the pieces together.
The nucleic acid molecule can be RNA or DNA.
[0025] The term "heterologous polypeptide", as used herein, refers
to a polypeptide that does not naturally occur in a host cell.
[0026] The term "expressed" or "expression" as used herein refers
to the transcription of a nucleotide sequence into an RNA nucleic
acid molecule at least complementary in part to a region of one of
the two nucleic acid strands of a gene coding sequence and/or to
the translation from an RNA nucleic acid molecule into a protein or
polypeptide.
[0027] The term "operably linked" as used herein refers to the
configuration of coding and control sequences, for example, within
an expression vector, so as to achieve transcription and/or
expression of the coding sequence. Thus, control sequences operably
linked to a coding sequence are capable of effecting the expression
of the coding sequence and regulating in which tissues, at what
developmental time points, or in response to which signals, and the
like, a gene is expressed. A coding sequence is operably linked to
or under the control of transcriptional regulatory regions in a
cell when DNA polymerase will bind the promoter sequence and
transcribe the coding sequence into mRNA that can be translated
into the encoded protein. The control sequences need not be
contiguous with the coding sequence, so long as they function to
direct the expression thereof. Thus, for example, intervening
untranslated or transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding sequence.
Such intervening sequences include but are not limited to enhancer
sequences which are not transcribed or are not bound by
polymerase.
[0028] The terms "gene expression control regions" or "gene
expression controlling regions" as used herein refer to nucleotide
sequences that are associated with a nucleic acid sequence and
which regulate, in whole or in part, the expression of the nucleic
acid sequence, for example, regulate in whole or in part the
transcription of a nucleotide sequence.
[0029] The term "oviduct-specific promoter", as used herein, refers
to a promoter from a gene specifically expressed in avian oviduct
cells including, but not limited to, promoters from ovalbumin,
lysozyme, conalbumin, ovomucoid or ovotransferrin genes.
[0030] The terms "transformation" and "transfection" as used herein
refer to the process of inserting a nucleic acid into a host cell.
Many techniques are well known to those skilled in the art to
facilitate transformation or transfection of a nucleic acid into a
prokaryotic or eukaryotic cell. These methods include a variety of
techniques, which may include, but are not limited to, viral
transduction, microinjection, treating the cells with high
concentrations of salt such as, but not only, a calcium or
magnesium salt, an electric field, detergent, or liposome mediated
transfection, to render the host cell competent for the uptake of
the nucleic acid molecules, and by such methods as sperm-mediated
and restriction-mediated integration. Other transfecting agents
include but are not limited to lipofectin, lipfectamine, DIMRIE C,
Supeffect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA,
DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE
(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP
(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl
dioctadecytammonium bromide), DHDEAB
(N,N-di-n-hexadecyl-N,N-dihydroxyeth- yl ammonium bromide), HDEAB
(N-n-hexadecylN,N-dihydroxyethylammonium bromide), polybrene, or
poly(ethylenimine) (PEI). These non-viral agents have the advantage
that they can facilitate stable integration of xenogeneic DNA
sequences into the vertebrate genome, without size restrictions
commonly associated with virus-derived transfecting agents. Certain
methods useful for the introduction of nucleic acid into a cell are
disclosed in, for example, U.S. patent application Ser. No.
09/919,143, filed Jul. 31, 2001 and U.S. patent application Ser.
No. 10/811,136, filed Mar. 26, 2004. The disclosure of each of
these two patent applications is incorporated in its entirety
herein by reference.
[0031] The term "enriched", as used herein with respect to avian
oviduct-specific tumor cells, refers to an increase in the number
of such cells relative to the total cell population in which they
are contained (i.e., an increase in the number of tumor cells
relative to the number of non-tumor cells).
[0032] As used herein, a "transgenic avian" includes an avian, for
example, a chicken, turkey, duck, goose, quail, pheasant, parrot,
finch, hawk, crow or ratite, for example, ostrich, emu, cassowary,
in which one or more, for example, essentially all of the cells of
the avian include a transgene. The transgene is introduced into the
cell, directly or indirectly by introduction into a precursor of
the cell, for example, by microinjection, transfection or
infection, for example, by infection with a recombinant virus. The
term genetic manipulation includes the introduction of a
recombinant DNA molecule. This molecule may be integrated within a
chromosome, or it may be extrachromosomally replicating DNA.
[0033] The terms "recombinant nucleic acid" and "recombinant DNA"
as used herein refer a combination of at least two nucleic acids
that is not naturally found in a eukaryotic or prokaryotic cell in
that particular configuration. The nucleic acids may include, but
are not limited to, nucleic acid vectors, gene expression
regulatory elements, origins of replication, suitable gene
sequences that when expressed confer antibiotic resistance,
protein-encoding sequences and the like. The term "recombinant
polypeptide" is meant to include a polypeptide produced by
recombinant DNA techniques such that it is distinct from a
naturally occurring polypeptide either in its location, purity or
structure. Generally, such a recombinant polypeptide will be
present in a cell in an amount different from that normally
observed in nature.
[0034] As used herein, the term "transgenic cell" refers to a cell
containing a transgene.
[0035] As used herein, the term "marker sequence" refers to a
nucleic acid molecule that is used to identify those cells that
have incorporated the targeting construct into their genome. For
example, the marker sequence can be a sequence encoding a protein
which confers a detectable trait on the cell, such as an antibiotic
resistance gene, for example, neomycin resistance gene, or an
assayable enzyme not typically found in the cell, for example,
alkaline phosphatase, horseradish peroxidase, luciferase,
beta-galactosidase and the like.
[0036] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in an organism found in nature.
[0037] As used herein, the term "host cell" is intended to refer to
a cell into which a nucleic acid molecule of the invention, such as
a recombinant expression vector of the invention, has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It should be understood that such
terms refer not only to the particular subject cell but to the
progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein. A host cell may be a
mammalian cell, for example, a human cell. In certain embodiments,
the host cell is an epithelial cell, for example, a pancreatic
epithelial cell.
[0038] As used herein, the term "transgene" means a nucleic acid
sequence (encoding, for example, a human interferon polypeptide)
that is partly or entirely heterologous, i.e., foreign, to the
transgenic animal or cell into which it is introduced, or, is
homologous to an endogenous gene of the transgenic animal or cell
into which it is introduced, but which is designed to be inserted,
or is inserted, into the animal's genome in such a way as to alter
the genome of the cell into which it is inserted (for example, it
is inserted at a location that differs from that of the natural
gene or its insertion results in a knockout). A trangene also
includes a regulatory sequence designed to be inserted into the
genome such that it regulates the expression of an endogenous
coding sequence, for example, to increase expression and/or to
change the timing and or tissue specificity of expression, for
example, to effect "gene activation".
[0039] The term "antibody" as used herein refers to polyclonal and
monoclonal antibodies and fragments thereof, and immunologic
binding equivalents thereof. The term "antibody" refers to a
homogeneous molecular entity, or a mixture such as a polyclonal
serum product made up of a plurality of different molecular
entities, and broadly encompasses naturally-occurring forms of
antibodies (for example, IgG, IgA, IgM, IgE) and recombinant
antibodies such as single-chain antibodies, chimeric and humanized
antibodies and multi-specific antibodies. The term "antibody" also
refers to fragments and derivatives of all of the foregoing, and
may further comprise any modified or derivatised variants thereof
that retains the ability to specifically bind an epitope. Antibody
derivatives may comprise a protein or chemical moiety conjugated to
an antibody. A monoclonal antibody is capable of selectively
binding to a target antigen or epitope. Antibodies may include, but
are not limited to polyclonal antibodies, monoclonal antibodies
(mAbs), humanized or chimeric antibodies, camelized antibodies,
single chain antibodies (scFvs), Fab fragments, F(ab').sub.2
fragments, disulfide-linked Fvs (sdFv) fragments, for example, as
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, intrabodies, synthetic antibodies, and epitope-binding
fragments of any of the above.
[0040] The techniques used to isolate and characterize nucleic
acids and proteins according to the methods of the present
invention are well known to those of skill in the art and standard
molecular biology and biochemical manuals may be consulted to
select suitable protocols without undue experimentation. See, for
example, Sambrook et al. (2001) Molecular Cloning: A Laboratory
Manual, 3rd ed., Cold Spring Harbor Press, the content of which is
herein incorporated by reference in its entirety.
[0041] Some abbreviations used in the present specification include
the following: aa, amino acid(s); bp, base pair(s); cDNA, DNA
complementary to RNA; min, miunte(s); nt, nucleotide(s); SSC,
sodium chloride-sodium citrate; UTR, untranslated region; DMSO,
dimethyl sulfoxide; ul, microliter; and uM; micromolar.
[0042] Various aspects of the invention are described in further
detail in the following.
[0043] The present invention relates to avian oviduct cell lines
which are capable of producing heterologous proteins. In a
particularly useful embodiment, the cell lines are immortal. That
is, the cells are capable of undergoing an indefinite number of
cell divisions.
[0044] Cell lines of the present invention may be obtained using
any useful method. In one embodiment, the tumor cell lines are
derived or obtained from avians having tumors in their reproductive
organs, in particular, the oviduct. It has been determined that
avians, such as chickens, will spontaneously produce tumors in the
oviduct and that such tumors become more prevalent as the avian
ages. For example, it is believed that more than half of the
domestic chickens over the age of five years have such tumors. In
addition, the tumors can be induced in avians using mutagens or
carcinogens as is understood in the field of mutation research. In
one particularly useful embodiment, the avian tumors arise from one
or more tubular gland cells.
[0045] Examples of carcinogens and mutagens contemplated for use in
producing avian oviduct tumors include, without limitation,
acrylonitrile, adriamycin, aflatoxins, arsenic and arsenic
compounds, asbestos, benzene, benzidine n,n-bis
(2-chloroethyl)-2-naphthylamine, benzo (a) pyrene, beryllium and
beryllium compounds, bis (chloromethyl) ether, 1,4-butanediol,
cadmium and cadium compounds, carbon tetrachloride, chloroform,
chromium and chromium compounds, chloromethyl methyl ether,
conjugated estrogens, 2,4-diaminotoluene,
1,2-dibromo-3-chloropropane, dichloroethane, ddt,
diethylstilbestrol, diethylsulfate, 1,4-dioxane, ethylene dibromide
(edb), ethylene oxide, ethylene thiourea, ethyl methanesulfonate,
formaldehye (formalin solutions), hydrazine and hydrazine sulfate,
lead acetate and lead phosphate, 3-methylcholanthrene,
methylhydrazine, methyl iodide, methylene chloride, methyl
methanesulfonate, 1-methyl-3-nitro-1-nitrosogu- anidine,
4,4-methylenebis (2-chloroaniline) (mboca), 2-napthylamine, nickel
and nickel compounds, 4-nitrobiphenyl, nitrogen mustard,
2-nitropropane, n-nitrosodi-n-bytylamine, n-nitrosodiethyl-amine,
n-nitrosodimethyl-amine, n-nitroso-n-ethylurethane, oxymetholone,
phenacetin, phenyloin, polychlorinated byphenyls, procarbazine,
progesterone, 1,3-propane sultone, b-propiolactone, reserpine,
saccharin, safrole, selenium sulfide,
2,3,7,8-tetrachlorodibenzo-p-dioxin (tcdd), thioacetaminde,
thiourea, thoriumdioxide, o-toluidine, m-troluenediamine,
toxaphene, tris (1 aziridinyl) phosphine sulfide, tris
(2,3-dibromopropyl) phosphate, uracil mustard, urethane and vinyl
chloride.
[0046] In another embodiment, avian oviduct cells, for example,
tubular gland cells, may be immortalized in vitro. Immortalization
of the avian cells may be spontaneous, or may be induced by
mutagens or by transfection using certain oncogenes. Mutagens, such
as those disclosed for use herein for the production of avian
tumors, can be employed for the in vitro production of immortal
avian oviduct tumor cell lines utilizing methodologies well known
to those of ordinary skill in the art. Examples of oncogenes which
may be useful for the production of immortalized cells include,
without limitation, genes for: growth factors, growth factor
receptors, protein kinases, signal transducers, nuclear
phosphoproteins, and transcription factors. Such oncogenes may be
employed by a person of ordinary skill in the field utilizing
methods known in the art to produce immortal avian oviduct tumor
cell lines.
[0047] Oviduct tumor cells can be isolated from tumorous avian
oviducts using any suitable tissue isolation technique known in the
art. For example, tumor cells can be obtained by removing and
dissecting the oviduct of a female avian to expose the luminal
side. The oviduct is examined and any tumors, polyps or abnormal
growths are excised for processing. Avian oviduct tumors are
typically of an epithelial or mesenchymal nature. Epithelial tumors
usually consist of adenomas or adenocarcinomas. Mesenchymal tumors
typically consist of adenomas, leiomyomas or fibromas. Once
collected, the tumor can be placed in a suitable basal salt medium,
such as F12, and stored on ice for up to 4 hours.
[0048] The oviduct tumor can be finely minced, for example, into
1-2 mm pieces, and incubated in a suitable media, such as F12 with
collagenase (for example, 0.08-8.0 mg/ml) and dispase (for example,
0.1-10.0 mg/ml). In one embodiment, the tissue is shaken at
37-42.degree. C. for 1 to 30 minutes, (400 rpm) and triturated to
generate clumps each consisting of 100-1000 cells. Clumps of cells
are allowed to settle out and the supernatant, containing red blood
cells and cellular debris, is discarded. The clumps are suspended
in pre-warmed F12 media, supplemented with collagenase (0.08-8.0
mg/ml) and dispase (0.1-10.0 mg/ml), and incubated at 37-42.degree.
C. with shaking (400 rpm). Clumps are triturated every ten minutes
to generate clumps consisting of 5-100 cells that are subsequently
pelleted with low speed centrifugation (.about.800 rpm). The
resultant pellet is suspended in ice cold F12 and filtered through
a single layer of surgical gauze tape to remove larger clumps.
Cells are washed twice with ice cold F12, pelleted and suspended in
the growth medium at an OD.sub.600 of 0.3-3.0. Cells collected with
this technique are suitable for use in the methods described
below.
[0049] All subsequent culturing typically utilizes a basal salt
media, such as F12, DMEM, MEM, and the like supplemented with fetal
calf serum, fetal bovine serum or chicken serum to 0.1%-15% total
volume, 0.01-1000 nM estrogen, 1-10000 nM corticosterone and
0.01-1000 uM insulin. This medium is hereafter referred to as
"growth media". Cells derived from oviduct tumors can be isolated
through multiple passages in such appropriate growth media.
Initially, cells isolated from the original oviduct samples are
grown in growth media (for example, at 1-5% CO.sub.2 and
37'-42.degree. C.) until the culture reaches confluency or the
diameter of cell clumps reaches approximately one millimeter. Cell
clumps and monolayers are subsequently treated with basal salts
media containing collagenase (0.08-8 mg/ml) and dispase (0.1-10
mg/ml) to produce single cell suspension. Cells are pelleted and
washed twice with basal salt media. After final wash, the cell
pellet is suspended in growth media at a cell density of
1.times.10.sup.4-1.times.10.sup.6 cells per milliliter.
[0050] Single colony isolates of cultured cells can be isolated
using limiting dilution techniques. Cultured cells are dispersed as
described above. Single cells are plated in each well of 96 well
plate and incubated in growth media. Alternatively single cells can
be cultured on a monolayer of cells such as whole embryo
fibroblasts, STO (Martin and Evans, Proc Natl Acad Sci USA, 1975.
72: 1441-5), or LMH (Kawaguchi et al., Cancer Res, 1987. 47:
4460-4). Supernatent from each well can be collected every three
days and the levels of ovalbumin, ovomucoid and lysozyme determined
by ELISA (ovomucoid and ovalbumin) or bioactivity assay (lysozyme).
Clones that express any or all of the defined egg white proteins
are passaged as needed to maintain viability.
[0051] Avian tumor cells isolated as described above can be
transfected with nucleic acids, for example, transgenes, encoding a
variety of heterologous proteins using techniques well known in the
art, such that sustained production of the proteins is achieved.
Typically, the nucleic acid is present within a suitable expression
vector. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced, for example, bacterial
vectors having a bacterial origin of replication and episomal
mammalian vectors. Other vectors, such as non-episomal mammalian
vectors, are integrated into the genome of a host cell upon
introduction into the host cell, and thereby are replicated along
with the host genome. Moreover, certain vectors are capable of
directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "expression
vectors". In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include other forms of expression
vectors, such as viral vectors, for example, replication defective
retroviruses, adenoviruses and adeno-associated viruses.
[0052] Suitable vectors for use in the present invention comprise
nucleic acid sequence encoding heterologous protein(s), that are
operatively linked to one or more regulatory sequences, for
example, promoter sequences. The phrase "operably linked" is
intended to mean that the nucleotide sequence of interest, for
example, the sequence encoding the heterologous protein, is linked
to the regulatory sequence(s) in a manner which allows for
expression of the nucleotide sequence (for example, in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). The term "regulatory sequence"
is intended to include promoters, enhancers and other expression
control elements, for example, polyadenylation signals. Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells, for example,
oviduct-specific regulatory sequences. Other elements included in
the design of a particular expression vector can depend on such
factors as the choice of the host cell to be transformed, the level
of expression of protein desired and the like. The expression
vectors of the invention can be introduced into host cells to
thereby produce proteins or peptides, including fusion proteins or
peptides, encoded by nucleic acids as described herein.
[0053] Vectors as described above can be introduced into isolated
avian tumor cells using any of a variety of well known techniques
well known in the field. Suitable methods may be described, for
example, in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1992), which is
hereby incorporated by reference. See, also, Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md.(1989); Hitt et al., "Construction and propagation of
human adenovirus vectors," in Cell Biology: A Laboratory Handbook,
Ed. J. E. Celis., Academic Press. 2.sup.nd Edition, Volume 1, pp:
500-512, 1998; Hitt et al., "Techniques for human adenovirus vector
construction and characterization," in Methods in Molecular
Genetics, Ed. K. W. Adolph, Academic Press, Orlando, Fla., Volume
7B, pp:12-30, 1995; Hitt, et al., "Construction and propagation of
human adenovirus vectors," in Cell Biology: A Laboratory Handbook,
Ed. J. E. Celis. Academic Press. pp:479-490, 1994, each of which is
also hereby incorporated by reference. The methods include, for
example, stable or transient transfection, lipofection,
electroporation and infection with recombinant viral vectors.
[0054] Isolated avian oviduct cell lines of the inventions
transfected or transformed as described above are also referred to
herein as "recombinant host cells". The term "recombinant host
cell" refers not only to the initial cell transfected, but also to
the progeny or potential progeny of the cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0055] Selection vectors that contain, for example,
oviduct-specific promoters, such as ovalbumin, ovomucoid or
lysozyme promoters or other suitable non-specific promoters that
drive expression of an antibiotic resistance gene such as neomycin,
hydromycin or puromycin, can be transfected into avian tumor cells
using the art-recognized techniques such as those described herein.
Presence of the selection vector in the cell can be transient or
clones that contain integrated copies of the selection vector can
be selected for.
[0056] In one embodiment, the presence of the appropriate
antibiotic, cells will survive if they are able to initiate
transcription from the oviduct-specific promoter located on the
selection vector provided. This technique therefore selects for
those cells with active endogenous oviduct-specific promoters. In
one useful embodiment, cells will survive if they are able to
initiate transcription of an antibiotic resistance gene from the
oviduct-specific promoter located on a selection vector. This
technique therefore selects for those cells with active cloned
oviduct-specific promoters. In one aspect of the invention, an
oviduct-specific promoter drives transcription of an RNA molecule
encoding both an antibiotic resistance protein product and a
protein of interest such as a pharmaceutical composition. In such a
case, an internal ribosome entry site may be employed between the
two coding sequences.
[0057] The foregoing selection methods can be used with the initial
primary cell culture or cells from subsequent passages. The cells
can be continually passaged in suitable growth medium for as long
as desired.
[0058] Alternatively, the selection vectors described above can
drive expression of a fluorescent protein, such as Enhanced Green
Fluorescence Protein (EGFP). Cells that transcribe the EGFP can
then be sorted and collected using standard Florescence Assisted
Cell sorting (FACS) techniques. Cells collected in this manner can
be cultured and additional rounds of selection used to isolate
cells with the desired egg white producing phenotype.
[0059] Metastasis of epithelial tumors often involves cell adhesion
molecules (CAMs) and epithelial-extracellular matrix (ECM) proteins
(Takeichi, Curr Opin Cell Biol, 1993. 5: 806-11; Evans, Cell Biol
Int Rep, 1992. 16: 1-10). Tsukamoto demonstrated that oviductal
tumor cells bind to neurite outgrowth factor (NOF) through
interactions with gicerin, a CAM expressed by cells of oviduct
tumors. This interaction is evidenced by the fact that oviductal
tumor cells pre-incubated with an anti-gicerin antibody exhibited
decreased binding to purified and cell associated NOF. While NOF is
expressed by mesentery cells of oviduct tumors, it is also
expressed on chicken gizzard, skeletal muscle, heart, liver and
ciliary ganglion cells (Hayashi and Miki, J Biol Chem, 1985.260:
14269-78).
[0060] The ability of oviductal tumor cells to bind NOF can be used
as a tool to isolate and enrich for metastatic cells of avian
oviduct tumors. Tumor cells can be collected as described earlier
and placed on tissue culture plates coated with purified NOF or on
a monolayer culture of avian cells prepared from the gizzard,
skeletal muscle, heart, liver or ciliary ganglion, or mesenchym of
an oviductal tumor. This can be done, for example, by incubating
tumor cells for approx. 1 hour at 37'-41.degree. C. at 5% CO.sub.2
to allow attachment of gicerin positive cells to the purified or
cell associated NOF. The cultures are washed twice with warm
(37.degree.-41.degree. C.) DMEM to dislodge any unbound cells.
Seeded cells are allowed to grow until colonies consisting of
10-1000 cells form. Colonies that arise from these cells are
removed by placing a cloning cylinder around the colony and
removing the culture media. The colony is then digested and
liberated from the underlying NOF coated plate or NOF expressing
cell monolayer using F12 containing collagenase (0.08-8 mg/ml) and
dispase (0.1-10 mg/ml). The colony and its' cells are placed in an
Ependorff tube and further digested with fresh F12
collagenase/dispase media. Once digestion is completed the cells
are placed back into culture. This process can be repeated with the
isolated colonies additional times to enrich for oviduct tumor
cells. After several rounds of enrichment the cultures can be
digested and single colonies from these cultures screened for
production of ovalbumin, ovomucoid or lysozyme and viability after
multiple passages.
[0061] Alternatively, oviduct tumor cells that express gicerin can
be isolated using an anti-gicerin antibody and FACS. Cells are
initially incubated with an anti-gicerin antibody. Cells are then
washed to remove unbound antibody and then incubated with a
detection antibody specific for the isotype of the anti-gicerin
antibody. After removing unbound detection antibody, the cells that
stain positive are sorted and collected using standard FACS
techniques. Cells collected in this manner can be cultured and
additional rounds of selection used to isolate cells with the
desired heterologous protein producing phenotype. Cells and their
subsequent lines should remain viable for multiple passages in a
defined growth medium. The growth medium can contain, for example,
supplements of fetal calf serum, fetal bovine serum or chicken
serum to 0.1%-15% total volume, and 0.01-1000 nM estrogen, 1-10000
nM corticosterone and 0.01-1000 uM insulin.
[0062] In one useful embodiment of the invention, the avian cell
lines originate with one or more tubular gland cells. Typically,
such cell lines of the invention will extrude or secrete the
heterologous protein along with, or in place of, egg white protein.
The avian oviduct tumor cell lines can be assessed for the ability
to express heterologous proteins using a variety of art-recognized
techniques. Expression of the heterologous protein can be driven by
either oviduct-specific or non-specific promoters. The expressed
protein can be detected, for example, by ELISA or bioactivity
assay. Such heterologous proteins can be any expressible protein
and may include, for example, therapeutic proteins such as
cytokines, growth factors, enzymes, structural proteins,
immunoglobulins, granulocyte-colony stimulating factor, or any
other polypeptide of interest that is capable of being expressed by
an avian cell or tissue. In one embodiment, the heterologous
protein is mammalian, for example, a human protein or is derived
from or is a portion of a mammalian, or human protein.
[0063] Expression of heterologous proteins may be assessed by any
of a wide variety of well known methods for detecting expression of
a transcribed nucleic acid or polypeptide. Non-limiting examples of
such methods include immunological methods for detection of
secreted, cell-surface, cytoplasmic, or nuclear polypeptides,
protein purification methods, protein function or activity assays,
nucleic acid hybridization methods, nucleic acid reverse
transcription methods, and nucleic acid amplification methods.
[0064] In one embodiment, expression of the protein is assessed
using an antibody, for example a radio-labeled,
chromophore-labeled, fluorophore-labeled, or enzyme-labeled
antibody, an antibody derivative, such as an antibody conjugated
with a substrate or with the protein or ligand of a protein-ligand
pair, for example biotin-streptavidin, or an antibody fragment, for
example a single-chain antibody, an isolated antibody hypervariable
domain, which binds specifically with a polypeptide or fragment
thereof, including a polypeptide which has undergone all or a
portion of its normal post-translational modification.
[0065] In another embodiment, expression of the protein is assessed
by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from
cells, and by hybridizing the mRNA/cDNA with a reference
polynucleotide which is a complement of a polypeptide nucleic acid,
or a fragment thereof. cDNA can, optionally, be amplified using any
of a variety of polymerase chain reaction methods prior to
hybridization with the reference polynucleotide. Expression of one
or more proteins can likewise be detected using quantitative PCR to
assess the level of expression of the protein(s).
[0066] The invention provides methods for producing multimeric
proteins, for example, immunoglobulins, such as antibodies, and
antigen binding fragments thereof.
[0067] In one embodiment of the present invention, the multimeric
protein is an immunoglobulin, wherein the first and second
heterologous polypeptides are an immunoglobulin heavy and light
chains respectively. Illustrative examples of this and other
aspects and embodiments of the present invention for the production
of heterologous multimeric polypeptides in avian cells are fully
disclosed in U.S. patent application Ser. No. 09/877,374, filed
Jun. 8, 2001, published as US-2002-0108132-A1 on Aug. 8, 2002, and
U.S. patent application Ser. No. 10/251,364, filed Sep. 18, 2002,
each of which are incorporated herein by reference in their
entirety. In one embodiment of the present invention, therefore,
the multimeric protein is an immunoglobulin wherein the first and
second heterologous polypeptides are an immunoglobulin heavy and
light chain respectively. Accordingly, the invention provides
immunoglobulin and other multimeric proteins that have been
produced by transgenic avians of the invention.
[0068] In the various embodiments of this aspect of the present
invention, an immunoglobulin polypeptide encoded by the
transcriptional unit of at least one expression vector may be an
immunoglobulin heavy chain polypeptide comprising a variable region
or a variant thereof, and may further comprise a D region, a J
region, a C region, or a combination thereof. An immunoglobulin
polypeptide encoded by the transcriptional unit of an expression
vector may also be an immunoglobulin light chain polypeptide
comprising a variable region or a variant thereof, and may further
comprise a J region and a C region. It is also contemplated to be
within the scope of the present invention for the immunoglobulin
regions to be derived from the same animal species, or a mixture of
species including, but not only, human, mouse, rat, rabbit and
chicken. In certain embodiments, the antibodies are human or
humanized.
[0069] In other embodiments of the present invention, the
immunoglobulin polypeptide encoded by the transcriptional unit of
at least one expression vector comprises an immunoglobulin heavy
chain variable region, an immunoglobulin light chain variable
region, and a linker peptide thereby forming a single-chain
antibody capable of selectively binding an antigen.
[0070] Another aspect of the present invention provides a method
for the production in an avian of an heterologous protein capable
of forming an antibody suitable for selectively binding an antigen
including producing a transgenic avian incorporating at least one
transgene, wherein the transgene encodes at least one heterologous
polypeptide selected from an immunoglobulin heavy chain variable
region, an immunoglobulin heavy chain comprising a variable region
and a constant region, an immunoglobulin light chain variable
region, an immunoglobulin light chain comprising a variable region
and a constant region, and a single-chain antibody comprising two
peptide-linked immunoglobulin variable regions.
[0071] In an embodiment of this method of the present invention,
the isolated heterologous protein is an antibody capable of
selectively binding to an antigen. In one embodiment, the antibody
may be generated by combining at least one immunoglobulin heavy
chain variable region and at least one immunoglobulin light chain
variable region, for example, cross-linked by at least one
di-sulfide bridge. The combination of the two variable regions will
generate a binding site capable of binding an antigen using methods
for antibody reconstitution that are well known in the art.
[0072] It is, however, contemplated to be within the scope of the
present invention for immunoglobulin heavy and light chains, or
variants or derivatives thereof, to be expressed in separate
transgenic avians, and therefore isolated from separate media
including serum or eggs, each isolate comprising a single species
of immunoglobulin polypeptide. The method may include combining
certain isolated heterologous immunoglobulin polypeptides, thereby
producing an antibody capable of selectively binding to an antigen.
In this embodiment, two individual transgenic avians may be
generated wherein one transgenic produces serum or eggs having an
immunoglobulin heavy chain variable region, or a polypeptide
comprising such, expressed therein. A second transgenic animal,
having a second transgene, produces serum or eggs having an
immunoglobulin light chain variable region, or a polypeptide
comprising such, expressed therein. The polypeptides may be
isolated from their respective sera and eggs and combined in vitro
to generate a binding site capable of binding an antigen.
[0073] The present invention is useful for the production of many
biological products such as, pharmaceutical compositions. For
example, the present invention can be useful for the production of
biological molecules such as hormones including cytokines (i.e.,
secreted polypeptides that affect a function of cells and modulates
an interaction between cells in an immune, inflammatory or
hematopoietic response), antibodies and other useful pharmaceutical
molecules which include polypeptides. Cytokines includes, but are
not limited to, monokines and lymphokines. Examples of cytokines
include, but are not limited to, interferon .alpha.2b,
Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),
Tumor Necrosis Factor-.alpha. (TNF-.alpha..) and Tumor Necrosis
Factor .beta. (TNF-.beta.), antibodies such as polyclonal and
monoclonal antibodies and fragments thereof, and immunologic
binding equivalents thereof. Antibodies may include, but are not
limited to polyclonal antibodies, monoclonal antibodies (MAbs),
humanized or chimeric antibodies, single chain antibodies, FAb
fragments, F(Ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments thereof. Also contemplated is the
production of antibody fusion proteins, for example, Fc fusion
proteins in accordance with the present methods. The methods of the
present invention can also be useful for producing immunoglobulin
polypeptides which are constituent polypeptides of an antibody or a
polypeptide derived therefrom. An "immunological polypeptide" may
be, but is not limited to, an immunological heavy or light chain
and may include a variable region, a diversity region, joining
region and a constant region or any combination, variant or
truncated form thereof. Immunological polypeptides also include
single-chain antibodies comprised of, but not limited to, an
immunoglobulin heavy chain variable region, an immunoglobulin light
chain variable region and optionally a peptide linker.
[0074] Examples of certain antibodies that can be produced in
methods of the invention may include but are not limited to
HERCEPTIN.RTM. (Trastuzumab) (Genentech, CA) which is a humanized
anti-HER2 monoclonal antibody for the treatment of patients with
metastatic breast cancer; REOPRO.RTM. (abciximab) (Centocor) which
is an anti-glycoprotein IIb/IIIa receptor on the platelets for the
prevention of clot formation; ZENAPAX.RTM. (daclizumab) (Roche
Pharmaceuticals, Switzerland) which is an immunosuppressive,
humanized anti-CD25 monoclonal antibody for the prevention of acute
renal allograft rejection; PANOREX.TM. which is a murine anti-17-IA
cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2
which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone
System); IMC-C225 which is a chimeric anti-EGFR IgG antibody
(ImClone System); VITAXIN.TM. which is a humanized
anti-.alpha.V.beta.3 integrin antibody (Applied Molecular
Evolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti
CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized
anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN.TM.
which is a chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech,
Roche/Zettyaku); LYMPHOCIDE.TM. which is a humanized anti-CD22 IgG
antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody
(ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDEC
Pharm/Mitsubishi); ZEVALIN.TM. is a radiolabelled murine anti-CD20
antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L
antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody
(IDEC); IDEC-152 is a primatized anti-CD23 antibody
(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG
(Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5
(C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-.alpha.
antibody (CAT/BASF); CDP870 is a humanized anti-TNF-.alpha. Fab
fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1
antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human
anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized
anti-TNF-.alpha. IgG4 antibody (Celltech); LDP-02 is a humanized
anti-.alpha.4.beta.7 antibody (LeukoSite/Genentech); OrthoClone
OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech);
ANTOVA.TM. is a humanized anti-CD40L IgG antibody (Biogen);
ANTEGREN.TM. is a humanized anti-VLA-4 IgG antibody (Elan); and
CAT-152 is a human anti-TGF-.beta..sub.2 antibody (Cambridge Ab
Tech).
[0075] Other possible pharmaceutical compositions contemplated for
production in accordance with the present invention, but are not
limited to, albumin, .alpha.-i antitrypsin, antithrombin III,
collagen, factors VIII, IX, X (and the like), fibrinogen,
hyaluronic acid, insulin, lactoferrin, protein C, erythropoietin
(EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte
macrophage colony-stimulating factor (GM-CSF), tissue-type
plasminogen activator (tPA), feed additive enzymes, somatotropin,
and chymotrypsin. Other pharmaceutical compositions which may be
produced as disclosed herein are disclosed in, for example, patents
or pending patent applications which have incorporated herein by
reference elsewhere in the application.
[0076] Heterologous protein(s) produced from avian oviduct tumor
cells in accordance with the present invention can be isolated from
the medium in which the tumor cells are cultured using any of a
variety of art-recognized techniques. Dialysis of the medium
against dilute buffer or a superabsorbant material, followed by
lyophilization, can be employed to remove the bulk of the low
molecular weight components of the medium and to concentrate the
heterologous protein. Alternatively, ultrafiltration or
precipitation by saturation with salts such as sodium or ammonium
sulfate can be used.
[0077] Once obtained in concentrated form, any standard technique,
such as preparative disc gel electrophoresis, ion-exchange
chromatography, gel filtration, size separation chromatography,
isoelectric focusing and the like may be used to purify, isolate,
and/or to identify the heterologous protein. Those skilled in the
art may also readily devise affinity chromatographic means of
heterologous protein purification, especially for those instances
in which a binding partner of the heterologous protein is known,
for example, antibodies.
[0078] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0079] The present invention is further illustrated by the
following examples, which are provided by way of illustration and
should not be construed to limit the invention. The contents of all
references, published patents and patents cited throughout the
present application are hereby incorporated by reference in their
entireties.
EXAMPLE 1
Production of an Immortal Cell Line from Avian Oviduct Tissue
[0080] Epithelial adenoma tumors are isolated from a chicken
oviduct and are finely minced into 1 to 2 mm pieces which are
incubated in F12 medium with 0.8 mg/ml collagenase. The tissue is
shaken at 37.degree. C. for 30 minutes and triturated to generate
cell clumps consisting of approximately 100 to 1000 cells. The
clumps of cells are separated from red blood cells and cellular
debris which is discarded. The clumps are suspended in pre-warmed
F12 media, supplemented with 0.8 mg/ml collagenase and 1.0 mg/ml
dispase, and incubated at 37.degree. C. with shaking. Cell clumps
are triturated every ten minutes to generate clumps consisting of
about 5 to 100 cells. The cells are pelleted with low speed
centrifugation and the resultant pellet is suspended in ice cold
F12 which is filtered through a single layer of sterile surgical
gauze tape to remove larger cell clumps. The cells are washed twice
with ice cold F12, pelleted and resuspended in DMEM 15% FCS growth
medium at an OD.sub.600 of approximately 1.0.
[0081] The cells are grown in growth media at 2% CO.sub.2 and
42.degree. C. until the culture reaches confluency or until the
diameter of cell clumps reach approximately one millimeter. Cell
clumps and monolayers are subsequently treated with basal salts
media which includes 0.8 mg/ml collagenase and 11.0 mg/ml dispase
to produce a single cell suspension. Cells are pelleted and washed
twice with basal salt media. After final wash, the cell pellet is
suspended in growth media at a cell density of
1.times.10.sup.4-1.times.10.sup.6 cells per milliliter.
[0082] Single cells are plated in each well of 96 well plate and
incubated in growth media. Immortal cell lines are identified which
produce ovalbumin as determined by ELISA. Cells that express
ovalbumin are passaged as needed to maintain viability. Each cell
line is passaged a minimum of 20 times.
EXAMPLE 2
Production of an Immortal Cell Line from Avian Oviduct Tissue of a
Transgenic Avian and Production of Heterologous Protein
[0083] Transgenic chickens which produce eggs with interferon
present in the egg white are disclosed in U.S. Pat. No. 6,730,822.
An interferon producing transgenic chicken is matured to five years
of age. Oviduct tissue is removed from the chicken and several
tumors are isolated. An immortal cell line is obtained from the
tumors as described in Example 1.
[0084] Growth medium in which the immortal ovalbumin producing cell
lines are passaged tests positive for interferon.
EXAMPLE 3
Production of an Immortal Cell Line from Avian Tubular Gland
Cells
[0085] Freshly harvested chicken oviduct tissue is isolated from a
healthy 40 week old chicken. The tissue is finely minced into small
pieces approximately 1 mm in diameter. The tissue is shaken at
42.degree. C. for 20 min and triturated to generate clumps each
consisting of about 100 to 1000 cells. The clumps of cells are
separated from red blood cells and cellular debris which is
discarded and are then suspended in pre-warmed McCoy's 5A medium,
supplemented with 0.8 mg/ml collagenase and 1.0 mg/ml dispase, and
incubated at 42.degree. C. with shaking. The cell clumps are
triturated every ten minutes to generate clumps consisting of about
5 to 100 cells. The cells are then collected by low speed
centrifugation. The resultant pellet is suspended in ice cold
McCoy's 5A medium and filtered through a single layer of sterile
surgical gauze tape to remove large clumps. The cells are then
washed 2 times in McCoy's 5A medium.
[0086] Cells are centrifuged, counted, and resuspended in
serum-free RPMI medium 1640 containing 0.4 mg/ml Aroclor
1254-induced S9 rat liver extracts (Moltox, Boone, NC), 0.23 mM
NADP, 0.28 mM glucose 6-phosphate, 0.45 mM MgCl.sub.2, 0.45 mM KCl,
and 200 mM Tris.HCl (pH 7.5) plus 50 uM
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
[0087] The cells are treated for 2 hours at 42.degree. C. After
treatment, the cells are resuspended in complete McCoy's 5A medium
and reseeded. MNNG-resistant cell lines are established by two
successive treatments with 5 uM MNNG and 25 uM
O.sup.6-benzylguanine. Each round of treatment is performed after
cells surviving the prior round of treatment have recovered
exponential growth. Single-cell clones are obtained by limiting
dilution. Each clone is grown to confluency in a well of a six well
plate. Each cell line is passaged a minimum of 20 times.
EXAMPLE 4
Transformation of an Avian Tubular Gland Cell Immortal Cell Line
and Production of Heterologous Protein
[0088] An immortal cell line of Example 1 and Example 3 is
transformed with an OMC24-attB-IRES-G-CSF vector with a neomycin
resistance coding sequence inserted downstream of the ovomucoid
transcription initiation start site such that both the G-CSF and
neomycin resistance are expressed. OMC24-attB-IRES-G-CSF is
disclosed in U.S. patent application Ser. No. 10/940,315, filed
Sep. 14, 2004 and U.S. patent application Ser. No. 10/856,218,
filed May 28, 2004. The disclosure of each of these two patent
applications is incorporated herein in its entirety by
reference.
[0089] OMC24-attB-IRES-G-CSF-Neo is transfected into actively
growing cell lines produced as described in Example 1 and Example
3. For each cell line, 0.2 ml of cells, which have been washed in
OptiMem medium, and 0.6 ml of OptiMem medium plus 10 nm estrogen,
100 nm corticosterone and 1 um insulin (OptiMem) are added to each
of six wells in a six well multiwell plate.
[0090] For each transfection, 5 ul of Dmrie C (Invitrogen, Inc.)
and 95 ul of OptiMem is added to a polycarbonate tube.
Approximately 20 ug of the OMC24attB-IRES-G-CSF-Neo vector in 100
ul of OptiMen is mixed into the Dmrie solution by pipetting. The
DNA-lipid mixture is let to stand for about 15 min at room
temperature in the hood.
[0091] 200 ul of the lipid/DNA/OptiMem mix is added to the 0.8 mls
of cells in each well for a total of 1 ml volume. Incubate at
42.degree. C., 5% CO.sub.2 for at least 5 to 6 hours.
[0092] 2 mls of 15% FCS DMEM plus 10 nm estrogen, 100 nm
corticosterone and 1 urn insulin high glucose with no phenol red
with 2.times.neomycin is added to the transfection medium. The
cells are incubated for 24 hours followed by a change medium to
fresh 10% FCS DMEM hi glucose plus neomycin with no phenol red
followed by 24 hours of incubation. The cells are diluted and grown
for an additional 24 hour at which time individual colonies are
picked, transferred to individual wells of a six well plate and
grown to confluency. For each cell line which is passaged a minimum
of 20 times, the growth medium tests positive for G-CSF.
[0093] While this invention has been described with respect to
various specific examples and embodiments, it is to be understood
that the invention is not limited thereto and that it can be
variously practiced with the scope of the following claims.
* * * * *