U.S. patent application number 11/295006 was filed with the patent office on 2006-06-08 for methods and materials for expression of a recombinant protein.
This patent application is currently assigned to XOMA Technology Ltd.. Invention is credited to Eddie Bautista, Robyn Cotter, Masahisa Handa, Arnold Horwitz.
Application Number | 20060121604 11/295006 |
Document ID | / |
Family ID | 36499981 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060121604 |
Kind Code |
A1 |
Handa; Masahisa ; et
al. |
June 8, 2006 |
Methods and materials for expression of a recombinant protein
Abstract
Recombinant expression vectors are provided comprising a 3' UTR
of a light chain and an Epstein-Barr virus origin of replication.
Also provided are host cells comprising such vectors and methods of
producing recombinant protein with such vectors. Additional methods
of producing a recombinant protein involve contacting cells with a
first and second vector, each of which encode a different
polypeptide chain, and wherein the second vector is present in an
amount which is about 1.5 to 2.5 times as much as that of the first
vector. Cells also can be transfected with a recombinant transient
expression vector encoding a protein and are cultured in a medium
in a membrane-enhanced culturing vessel to produce recombinant
protein.
Inventors: |
Handa; Masahisa; (Berkeley,
CA) ; Horwitz; Arnold; (San Leandro, CA) ;
Bautista; Eddie; (San Francisco, CA) ; Cotter;
Robyn; (Oakland, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
XOMA Technology Ltd.
Hamilton
BM
|
Family ID: |
36499981 |
Appl. No.: |
11/295006 |
Filed: |
December 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60633056 |
Dec 3, 2004 |
|
|
|
Current U.S.
Class: |
435/320.1 ;
435/6.14 |
Current CPC
Class: |
C12N 15/85 20130101;
C12N 2820/55 20130101; C12N 2820/60 20130101; C12N 7/00 20130101;
C12N 2710/16243 20130101; C07K 16/00 20130101; C12N 2710/16221
20130101; C12N 2820/10 20130101; C12N 2830/42 20130101; C12N
2800/108 20130101 |
Class at
Publication: |
435/320.1 ;
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/63 20060101 C12N015/63; C12N 15/74 20060101
C12N015/74; C12N 15/00 20060101 C12N015/00; C12N 15/70 20060101
C12N015/70; C12N 15/09 20060101 C12N015/09 |
Claims
1. A recombinant expression vector comprising a 3' UTR of a light
chain gene and an Epstein-Barr virus origin of replication
(oriP).
2. The recombinant expression vector of claim 1, wherein the light
chain gene is a murine light chain gene.
3. The recombinant expression vector of claim 2, wherein the 3' UTR
comprises a nucleotide sequence of nucleotides 1062-2560 of SEQ ID
NO: 1.
4. The recombinant expression vector of claim 1, wherein the
recombinant expression vector is a recombinant transient expression
vector.
5. The recombinant expression vector of claim 1 further comprising
a pUC19 origin of replication, a viral promoter, a 5' UTR intron,
or a combination of any of the foregoing.
6. The recombinant expression vector of claim 5 further comprising
a pUC19 origin of replication, a viral promoter, and a 5' UTR
intron.
7. The recombinant expression vector of claim 5, wherein the viral
promoter is a CMV promoter.
8. The recombinant expression vector of claim 5, wherein the 5' UTR
intron comprises nucleotides 888-974 of SEQ ID NO: 1.
9. The recombinant expression vector of claim 5, wherein the pUC19
origin of replication comprises the nucleotide sequence of
nucleotides 4551-5220 of SEQ ID NO: 1.
10. The recombinant expression vector of claim 1 further comprising
an antibody signal sequence.
11. The recombinant expression vector of claim 1, wherein the
recombinant expression vector does not comprise an antibody signal
sequence.
12. The recombinant expression vector of claim 1 further comprising
a nucleotide sequence encoding a protein or a functional fragment
thereof.
13. The recombinant expression vector of claim 12, wherein the
nucleotide sequence encodes an immunoglobulin chain.
14. The recombinant expression vector of claim 13, wherein the
immunoglobulin chain is selected from the group consisting of a
.gamma..sub.1 heavy chain, a .gamma..sub.2 heavy chain, a
.gamma..sub.4 heavy chain, a .kappa. light chain, and a .lamda.
light chain.
15. A method of producing a recombinant protein, comprising
contacting a cell with the recombinant expression vector of claim
1, whereupon a recombinant protein is produced.
16. A host cell comprising the recombinant expression vector of
claim 1.
17. The host cell of claim 16, wherein the host cell is a mammalian
cell.
18. The host cell of claim 17, wherein the mammalian cell is a
human cell.
19. The host cell of claim 18, wherein the human cell is a human
embryonic kidney cell.
20. The host cell of claim 19, wherein the human embryonic kidney
cell expresses an Epstein Barr virus nuclear antigen-1 (EBNA-1)
protein.
21. The host cell of claim 20, wherein the host cell is a 293E
cell.
22. A method of producing a recombinant protein, comprising
culturing the host cell of claim 16, whereupon a recombinant
protein is produced.
23. A method of producing a recombinant protein, the method
comprising contacting cells in a medium with a first vector and a
second vector, wherein the first vector encodes a first polypeptide
chain and the second vector encodes a second polypeptide chain,
wherein the first polypeptide chain is different from the second
polypeptide chain, and wherein the second vector is present in the
medium in an amount which is about 1.5 to 2.5 times as much as the
amount of the first vector, whereupon a recombinant heterodimeric
or heteromultimeric protein is produced.
24. The method of claim 23, wherein the second vector is present in
the medium in an amount which is about 1.75 to 2.25 times as much
as the amount of the first vector.
25. The method of claim 24, wherein the second vector is present in
the medium in an amount which is twice as much as the amount of the
first vector.
26. The method of claim 23, wherein the recombinant protein is a
heterotetrameric protein.
27. The method of claim 26, wherein the heterotetrameric protein is
an immunoglobulin.
28. The method of claim 27, wherein the first vector encodes a
heavy chain of an immunoglobulin, or a functional fragment thereof,
and the second vector encodes a light chain of an immunoglobulin,
or a functional fragment thereof.
29. The method of claim 28, wherein the heavy chain is a human
.gamma..sub.1 heavy chain, human .gamma..sub.2 heavy chain, or a
human .gamma..sub.4 heavy chain.
30. The method of claim 28, wherein the light chain is a human
.kappa. light chain or a .lamda. light chain.
31. The method of claim 23, wherein each of the first vector and
the second vector is a recombinant transient expression vector.
32. The method of claim 23, wherein each of the first vector and
the second vector comprises a 3' untranslated region (UTR) of a
light chain gene and an oriP.
33. The method of claim 23, wherein each of the first vector and
the second vector comprises a viral promoter, a pUC19 origin of
replication, a 5' UTR intron, or a combination of any of the
foregoing.
34. The method of claim 33, wherein the viral promoter is a CMV
promoter.
35. The method of claim 33, wherein the 5' UTR intron comprises a
nucleotides 888-974 of SEQ ID NO: 1.
36. The method of claim 23, wherein each of the first vector and
second vector comprises an antibody signal sequence.
37. The method of claim 23, wherein the cells are contacted with
the first vector and second vector simultaneously.
38. The method of claim 23, wherein the cells are contacted with
the first vector and second vector in the presence of a cationic
polymer.
39. The method of claim 38, wherein the cationic polymer is
polyethyleneimine (PEI).
40. The method of claim 39, wherein the PEI is a linear PEI.
41. The method of claim 40, wherein the linear PEI is present in an
amount that is about 1.5 to 4.5 times the amount of the first
vector and second vector.
42. The method of claim 41, wherein the linear PEI is present in an
amount that is about 2.5 to 3.5 times the amount of the first
vector and second vector.
43. The method of claim 42, wherein the linear PEI is present in an
amount that is twice the amount of the first vector and second
vector.
44. The method of claim 23, wherein the cells are mammalian
cells.
45. The method of claim 44, wherein the mammalian cells are human
cells.
46. The method of claim 45, wherein the cells are human embryonic
kidney cells.
47. The method of claim 46, wherein the human embryonic kidney
cells express Epstein-Barr virus nuclear antigen-1 protein
(EBNA-1).
48. The method of claim 47, wherein the cells are 293E cells.
49. The method of claim 23 further comprising isolating the cells
from the medium and culturing the cells in a second medium in a
membrane-enhanced culturing vessel, wherein the second medium is
different from the medium.
50. The method of claim 49, wherein the second medium is a
serum-free cell culture medium.
51. The method of claim 50, wherein the second medium is IS293.TM.
medium.
52. The method of claim 49, wherein the membrane-enhanced culturing
vessel is an Integra CL1000.
53. The method of the claim 49 further comprising purifying the
recombinant protein from the second medium.
54. The method of claim 53, wherein the purifying comprises
centrifuging the second medium through a column comprising Protein
A.
55. The method of claim 53, wherein the purifying occurs after 3
days of culturing the cells in the second medium.
56. The method of claim 53, wherein the purifying occurs after 7
days of culturing the cells in the second medium.
57. The method of claim 55, wherein at least 300 .mu.g/ml
recombinant protein is produced in the second medium.
58. The method of claim 57, wherein at least 500 .mu.g/ml
recombinant protein is produced in the second medium.
59. The method of claim 58, wherein at least 700 .mu.g/ml
recombinant protein is produced in the second medium.
60. The method of claim 49, wherein the culturing comprises seeding
cells in the second medium at a cell density between about
1.0.times.10.sup.6 and 2.0.times.10.sup.7 cells/ml.
61. The method of claim 60, wherein the cell density is about
3.0.times.10.sup.6 to about 1.0.times.10.sup.7 cells/ml.
62. A method of producing a recombinant protein comprising
culturing cells, which have been contacted with a recombinant
transient expression vector encoding a recombinant protein, in a
medium in a membrane-enhanced culturing vessel or in a Fembach
flask, whereupon the recombinant protein is produced.
63. The method of claim 62, wherein the medium is a serum-free cell
culture medium.
64. The method of claim 63, wherein the serum-free medium is
IS293.TM. medium.
65. The method of claim 62, wherein the membrane-enhanced culturing
vessel is an Integra CL1000.
66. The method of claim 62, wherein the method further comprises
purifying the recombinant protein from the medium.
67. The method of claim 66, wherein the purifying comprises
centrifuging the medium through a column comprising Protein A.
68. The method of claim 66, wherein the purifying occurs after 3
days of culturing the cells in the medium.
69. The method of claim 66, wherein the purifying occurs after 7
days of culturing the cells in the medium.
70. The method of claim 68, wherein at least 300 .mu.g/ml
recombinant protein is produced in the medium.
71. The method of claim 70, wherein at least 500 .mu.g/ml
recombinant protein is produced in the medium.
72. The method of claim 71, wherein at least 700 .mu.g/ml
recombinant protein is produced in the medium.
73. The method of claim 62, wherein the culturing comprises seeding
cells in the medium at a cell density between about
1.0.times.10.sup.6 and 2.0.times.10.sup.7 cells/ml.
74. The method of claim 73, wherein the cell density is
3.0.times.10.sup.6 to about 1.0.times.10.sup.7 cells/ml.
75. The method of claim 62, wherein the recombinant transient
expression vector comprises a 3' untranslated region (UTR) of a
light chain gene and an oriP.
76. The method of claim 62, wherein the recombinant transient
expression vector comprises a pUC19 origin of replication, a viral
promoter, a 5' UTR intron, or a combination of any of the
foregoing.
77. The method of claim 76, wherein the viral promoter is a CMV
promoter.
78. The method of claim 76, wherein the 5' UTR intron comprises
nucleotides 888-974 of SEQ ID NO: 1.
79. The method of claim 62, wherein the cells have been contacted
with a recombinant transient expression vector in the presence of a
cationic polymer.
80. The method of claim 79, wherein the cationic polymer is a
polyethyleneimine (PEI).
81. The method of claim 80, wherein the PEI is a linear PEI.
82. The method of claim 81, wherein the PEI is present in an amount
that is about 1.5 to 4.5 times the amount of the recombinant
transient expression vector.
83. The method of claim 82, wherein the PEI is present in an amount
that is about 2.5 to 3.5 times the amount of the recombinant
transient expression vector.
84. The method of claim 83, wherein the PEI is present in an amount
that is twice the amount of the recombinant transient expression
vector.
85. The method of claim 62, wherein the cells are mammalian
cells.
86. The method of claim 85, wherein the mammalian cells are human
cells.
87. The method of claim 86, wherein the human cells are human
embryonic kidney cells.
88. The method of claim 87, wherein the human embryonic kidney
cells express Epstein-Barr virus nuclear antigen-1 protein
(EBNA-1).
89. The method of claim 88, wherein the cells are 293E cells.
90. The method of claim 62, wherein the protein is an
immunoglobulin chain or a functional fragment thereof.
91. The method of claim 90, wherein the immunoglobulin chain is a
heavy chain or a light chain.
92. The method of claim 91, wherein the immunoglobulin chain is a
heavy chain, and the heavy chain is a human .gamma..sub.1 heavy
chain, human .gamma..sub.2 heavy chain, or a human .gamma..sub.4
heavy chain.
93. The method of claim 91, wherein the immunoglobulin chain is a
light chain, and the light chain is a human .kappa. light chain or
a .lamda. light chain.
Description
FIELD OF THE INVENTION
[0001] This invention pertains to methods of producing a
recombinant protein and recombinant expression vectors and host
cells for use therein.
BACKGROUND OF THE INVENTION
[0002] Large-scale transient expression of recombinant proteins has
been an area of rapid development in the past several years as an
alternative or precursor to stable cell line development to
generate multi-milligram quantities of protein (Wurm et al., Curr.
Opn. Biotech. 10: 156-159 (1999)). Human embryonic kidney (HEK293)
cells are one of the most widely used cell lines for transient
expression and have been successfully adapted to suspension-growth
to help facilitate culture scale-up. Recent reports have
successfully demonstrated the usage of transiently expressing
suspension-adapted HEK293 cells in 1-3 L cultures to generate
recombinant proteins including soluble polypeptides, transmembrane
proteins, and human antibodies (Durocher et al., Nucleic Acids Res.
30: 1-9 (2002); Meissner et al., Biotechnol. Bioeng. 75: 197-203
(2000); and Cote et al., Biotechnol. Bioeng. 59: 567-575
(1998)).
[0003] In particular, Durocher et al. has shown that HEK293E cells
expressing the Epstein-Barr virus (EBV) nuclear antigen-1 protein
(EBNA1) were able to routinely generate >10 mg/L of a number of
different recombinant proteins using the cationic polymer
transfection reagent, polyethyleneimine (PEI) (Boussif et al.,
Proc. Natl. Acad. Sci. 92: 7297-7301 (1995); and Mislick et al.,
Proc. Natl. Acad. Sci. 93: 12349-12354 (1996)).
[0004] Despite these advances, there is still a need in the art for
improved expression systems including optimized transient
transfection systems for time- and cost-efficient production of
recombinant proteins. The invention provides such optimized methods
of producing recombinant proteins. These and other advantages of
the invention, as well as additional inventive features, will be
apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides recombinant expression vectors useful
in methods of producing a recombinant protein. One of the inventive
recombinant expression vectors comprises a 3' untranslated region
(UTR) of a light chain gene. Another recombinant expression vector
provided herein comprises a 3' UTR and an Epstein-Barr virus origin
of replication. Host cells comprising any of the inventive
recombinant expression vectors are also provided herein.
[0006] The invention further provides methods of producing a
recombinant protein. In a first method, the recombinant protein is
a heterodimeric or heteromultimeric protein, either of which
comprises a first polypeptide chain and a second polypeptide chain,
wherein the first polypeptide chain is different from the second
polypeptide chain. The method comprises contacting cells in a
medium with a first vector and a second vector, wherein the first
vector encodes the first polypeptide chain and the second vector
encodes the second polypeptide chain, and the second vector is
present in the medium in an amount which is about 1.5 to about 2.5
times as much as the amount of the first vector.
[0007] In a second method of producing a recombinant protein, the
method comprises culturing cells, which have been contacted with a
recombinant transient expression vector encoding the protein, in a
medium in a membrane-enhanced culturing vessel, whereupon a
recombinant protein is produced. Alternatively, the second method
comprises culturing cells, which have been contacted with a
recombinant transient expression vector encoding the protein, in a
medium in a Fembach flask.
[0008] In a third method, the recombinant protein is produced upon
contacting cells with at least one of the inventive recombinant
expression vectors described herein. In a fourth method, the
recombinant protein is produced upon culturing host cells
comprising any of the inventive recombinant expression vectors
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A is an illustration of the pMXT recombinant
expression vector without any recombinant protein coding sequences.
FIG. 1B is an illustration of the pMXT vector encoding a human
.gamma..sub.2 heavy chain, while FIG. 1C is an illustration of the
pMXT vector encoding a human .kappa. light chain. The following
abbreviations are used in FIGS. 1A-1C: Ap, ampicillin resistance
marker; CMV promoter, cytomegalovirus promoter; MCS, multiple
cloning sequence; 5' UT intron, 5' untranslated region intron; SP,
signal peptide; V, variable region; C, constant region; LC 3' UT,
light chain 3' untranslated region, OriP, Epstein-Barr virus origin
of replication; pUC19ori, origin of replication from the pUC19
plasmid.
[0010] FIGS. 2A-2I are flow cytometry data graphs depicting the
levels of fluorescence of green fluorescence protein (GFP) and
propidium iodide (PI) under differing transfection conditions,
specifically differing DNA and polyethyleneimine (PEI)
concentrations. In FIG. 2A, cells were transfected with 1 .mu.g/ml
DNA and 1 .mu.g/ml PEI. In FIG. 2B, cells were transfected with 2
.mu.g/ml DNA and 2 .mu.g/ml PEI. In FIG. 2C, cells were transfected
with 5 .mu.g/ml DNA and 5 .mu.g/ml PEI. In FIG. 2D, cells were
transfected with 1 .mu.g/ml DNA and 2 .mu.g/ml PEI. In FIG. 2E,
cells were transfected with 2 .mu.g/ml DNA and 4 .mu.g/ml PEI. In
FIG. 2F, cells were transfected with 5 .mu.g/ml DNA and 10 .mu.g/ml
PEI. In FIG. 2G, cells were transfected with 1 .mu.g/ml DNA and 5
.mu.g/ml PEI. In FIG. 2H, cells were transfected with 2 .mu.g/ml
DNA and 10 .mu.g/ml PEI. In FIG. 2I, cells were transfected with 5
.mu.g/ml DNA and 25 .mu.g/ml PEI.
[0011] FIG. 3 is a graph showing the % cell viability (X) and % GFP
positive 293E cells (.box-solid.) that were adapted to suspension
growth in different serum-free media and optimized for
transfection. A control set of 293E cells were grown in DMEM.
[0012] FIG. 4 is a graph showing the antibody production by cells
which were co-transfected with different heavy chain (HC):light
chain (LC) ratios of different vector types.
[0013] FIG. 5A is a graph showing antibody production
(.circle-solid.) and cell viability (X) of transiently transfected
293E cells in Integra flasks as a function of time
post-transfection. FIG. 5B is a graph showing the production of
antibodies in shake flasks vs. Integra flasks by transfected cell
cultures at day 7 post-transfection.
[0014] FIG. 6A is a graph of the percentage of viable cells
transfected with DNA encoding Ab#1 as a function of time
post-transfection. FIG. 6B is a graph of the percentage of viable
cells transfected with DNA encoding Ab#2 as a function of time
post-transfection. In both FIGS. 6A and 6B, .box-solid. is I-50;
.tangle-solidup. is I-100; .circle-solid. is I-200; .diamond-solid.
is I-400; and X is E-200.
[0015] FIG. 7A is a graph of the number of viable cells transfected
with DNA encoding Ab#1 as a function of time post-transfection.
FIG. 7B is a graph of the number of viable cells transfected with
DNA encoding Ab#2 as a function of time post-transfection. In both
FIGS. 7A and 7B, .box-solid. is I-50; .tangle-solidup. is I-100;
.circle-solid. is I-200; .diamond-solid. is I-400; and X is
E-200.
[0016] FIG. 8A is a graph of the concentration of antibody produced
by cells transfected with DNA encoding Ab#1 as a function of time
post transfection. FIG. 8B is a graph of the concentration of
antibody produced by cells transfected with DNA encoding Ab#2 as a
function of time post-transfection. In both FIGS. 8A and 8B,
.box-solid. is I-50; .tangle-solidup. is I-100; .circle-solid. is
I-200; .diamond-solid. is I-400; and X is E-200.
[0017] FIG. 9A is a graph of the total antibody produced by cells
tranfected with DNA encoding Ab#1 as a function of time
post-transfection. FIG. 9B is a graph of the total yield of
antibody produced by cells transfected with DNA encoding Ab#2 as a
function of time post-transfection.
[0018] FIG. 10 shows SEQ ID NO: 1, which is the nucleotide sequence
of pMXT5 (FIG. 1A) without any coding sequences. Restriction enzyme
sites are labeled with the name of the enzyme above the position of
the site. CMV promoter comprises nucleotides 1-1037; 5' UTR intron
comprises nucleotides 888-974; MCS comprises nucleotides 1038-1061;
LC 3' UT comprises nucleotides 1062-2560; OriP comprises
nucleotides 2561-4550; pUC19 ori comprises nucleotides 4551-5220;
and Ap comprises nucleotides 5221-6380.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The invention provides recombinant expression vectors useful
in methods of producing a recombinant protein. One of the inventive
recombinant expression vectors comprises a 3' untranslated region
(UTR) of a light chain gene. Another recombinant expression vector
provided herein comprises a 3' UTR and an Epstein-Barr virus origin
of replication (oriP). Inventive recombinant expression vectors
optionally comprise a pUC19 origin of replication (pUC19ori).
[0020] For purposes herein, the term "recombinant expression
vector" means a genetically-modified oligonucleotide (i.e.,
polynucleotide) construct that permits the production of a protein
within a cell, when the construct comprises a nucleotide sequence
encoding the protein, and the construct is contacted with the cell
under conditions sufficient to have the protein expressed within
the cell. As the expression vector is recombinant, the vector of
the invention is not naturally-occurring as a whole. However, parts
of the vectors can be naturally-occurring.
[0021] The recombinant expression vector can comprise any type of
nucleotides, including, but not limited to DNA and RNA, which can
be single-stranded or double-stranded, which can be synthesized or
obtained in part from natural sources, and which can contain
natural or non-natural or altered nucleotides. Examples of
non-natural or altered nucleotides that can be used to generate the
recombinant expression vectors include, but are not limited to,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and
2,6-diaminopurine.
[0022] The recombinant expression vector can comprise
naturally-occurring or non-naturally-occuring internucleotide
linkages, or both types of linkages, such as phosphoroamidate
linkages or phosphorothioate linkages, instead of the
phosphodiester linkages found between the nucleotides of an
unmodified oligonucleotide. Preferably, the non-naturally occurring
or altered nucleotides or internucleotide linkages do not hinder in
any way the transcription or replication of the vector.
[0023] The recombinant expression vector can be any suitable
recombinant expression vector, and can be used to transform or
transfect any suitable host. For example, one of ordinary skill in
the art appreciates that transformation or transfection is a
process by which, for example, exogenous nucleic acids such as DNA
are introduced into cells wherein the transformation or
transfection process involves contacting the cells with the
exogenous nucleic acids such as the recombinant expression vector
as described herein. Suitable vectors include those designed for
propagation and expansion or for expression or both, such as
plasmids and viruses. The vector can be selected from the group
consisting of the pUC series (Fermentas Life Sciences), the
pBluescript series (Stratagene, LaJolla, Calif.), the pET series
(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,
Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).
Bacteriophage vectors, such as .lamda.GT10, .lamda.GT11,
.lamda.ZapII (Stratagene), .lamda.EMBL4, and .lamda.NML149, also
can be used. Examples of plant expression vectors include pBI01,
pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of
animal expression vectors include pEUK-Cl, pMAM, and pMAMneo
(Clontech). A preferred recombinant expression vector includes the
pMXT vector as shown in FIGS. 1A-1C.
[0024] The recombinant expression vector can be prepared using
standard recombinant DNA techniques described in, for example,
Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d
edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
and Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, New York, N.Y.
(1994).
[0025] Desirably, the recombinant expression vector comprises
regulatory sequences, such as transcription and translation
initiation and termination codons, which are specific to the type
of host (e.g., bacterium, fungus, plant, or animal) into which the
vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA- or RNA-based.
[0026] A construct of a recombinant expression vector, which is
circular or linear, can be prepared to contain a replication system
functional in a prokaryotic or eukaryotic host cell. Replication
systems can be derived from ColEl, 2 .mu.plasmid, .lamda., SV40,
bovine papilloma virus, and the like. The recombinant expression
vector of the invention can comprise a replication system, which
comprises an oriP. Preferably, the inventive recombinant expression
vector comprises an oriP, and not an Epstein Barr virus nuclear
antigen (EBVNA), which EBVNA is known to activate an oriP.
[0027] As used herein, the term "oriP" or "Epstein-Barr virus
origin of replication" refers to a nucleotide sequence that is
substantially identical to the Epstein-Barr virus origin of
replication, which has the nucleotide sequence of nucleotides
2561-4550 of SEQ ID NO: 1. It is preferred that no insertions,
deletions, inversion, and/or substitutions are present in this
nucleotide sequence. However, one of ordinary skill in the art
appreciates that the nucleotide sequence of nucleotides 2561-4550
of SEQ ID NO: 1 can have insertions, deletions, inversion, and/or
substitutions that will not negatively affect the function of the
nucleotide sequence, which is to promote high copy episomal plasmid
replication. One of ordinary skill in the art further appreciates
that such high copy episomal plasmid replication occurs in
mammalian cells.
[0028] The recombinant expression vector also preferably comprises
a pUC19 origin of replication. As used herein, the term "pUC19
origin of replication" refers to the nucleotide sequence of the
origin of replication from a pUC 19 vector, which is commercially
available from Fermentas Life Sciences and has the nucleotide
sequence of nucleotides 4551-5220 of SEQ ID NO: 1. It is preferred
that no insertions, deletions, inversion, and/or substitutions are
present in this nucleotide sequence. However, one of ordinary skill
in the art appreciates that nucleotides 4551-5220 of SEQ ID NO: 1
can have insertions, deletions, inversion, and/or substitutions
that will not affect the function of the nucleotide sequence, which
is to promote high copy episomal plasmid replication. One of
ordinary skill in the art further appreciates that such high copy
episomal plasmid replication occurs in bacterial cells.
[0029] The recombinant expression vector can include one or more
marker genes, which allow for selection of transformed or
transfected hosts. Marker genes include biocide resistance, e.g.,
resistance to antibiotics, heavy metals, etc., complementation in
an auxotrophic host to provide prototrophy, and the like. Suitable
marker genes for the inventive recombinant expression vectors
include, for instance, neomycin/G418 resistance genes, hygromycin
resistance genes, histidinol resistance genes, tetracycline
resistance genes, and ampicillin resistance genes.
[0030] The recombinant expression vector can comprise a native or
normative promoter operably linked to the nucleic acid encoding the
protein. The selection of promoters, e.g., strong, weak, inducible,
tissue-specific, and developmental-specific, is within the ordinary
skill in the art. Similarly, the combining of a nucleic acid with a
promoter is also within the skill in the art. The promoter can be a
viral promoter or a non-viral promoter. Preferably, the promoter is
a viral promoter. More preferably, the viral promoter is a strong
viral promoter, such as a cytomegalovirus (CMV) promoter. The CMV
promoter is known in the art and has the nucleotide sequence of
nucleotides 1-1037 of SEQ ID NO: 1. It is preferred that no
insertions, deletions, inversion, and/or substitutions are present
in this nucleotide sequence. However, one of ordinary skill in the
art appreciates that nucleotides 1-1037 of SEQ ID NO: 1 can have
insertions, deletions, inversion, and/or substitutions that will
not affect the function of the nucleotide sequence, which is to
drive the transcription of the recombinant protein coding
sequence.
[0031] The recombinant expression vector comprises a 3' UTR of a
light chain gene. Preferably, the recombinant expression vector
comprises a 3' UTR of a light chain gene in combination with an
Epstein-Barr virus origin of replication (oriP). As used herein,
the term "3' UTR" refers to a nucleotide sequence of a gene that is
untranslated and is located 3' to the stop codon of the coding
sequence of that gene. The phrase "light chain gene" refers to a
gene encoding a light chain of an immunoglobulin. Thus, in regard
to the invention, the 3' UTR of a light chain gene is a nucleotide
sequence that is originally found in a light-chain gene and that is
inserted into the inventive vector. The light chain gene can be a
light chain gene of any mammal, such as a human, mouse, rat, goat,
rabbit, horse, pig, etc. Preferably, the light chain gene is a
mouse (murine) light chain gene. More preferably, the mouse light
chain gene has the nucleotide sequence of nucleotides 1062-2560 of
SEQ ID NO: 1. It is preferred that no insertions, deletions,
inversion, and/or substitutions are present in this nucleotide
sequence. However, one of ordinary skill in the art appreciates
that nucleotides 1062-2560 of SEQ ID NO: 1 can have insertions,
deletions, inversion, and/or substitutions that will not affect the
function of the nucleotide sequence, which is to provide signals
for polyadenylation. With respect to the inventive vectors, the 3'
UTR of a light chain gene is preferably located immediately 3' to
the stop codon of the coding sequence of the vector. If no coding
sequence is present, then the 3' UTR of a light chain gene
preferably is located 3' to the multiple cloning sequence and/or
the CMV promoter. The recombinant expression vector can comprise a
single copy of a 3' UTR or multiple copies of a 3' UTR. Preferably,
the recombinant expression vector comprises a single copy of a 3'
UTR.
[0032] The recombinant expression vector preferably comprises a 5'
UTR intron. As used herein, the term "5' UTR intron" refers to a
nucleotide sequence that is transcribed but is removed by RNA
splicing and thus not retained in the final transcript. It further
is not translated and, thus, is not expressed as part of the
protein, polypeptide, or peptide encoded by the vector. The 5' UTR
intron is preferably located after the promoter in the 5'
untranslated region of the recombinant expression vector. The 5'
UTR intron promotes enhanced expression. The 5' UTR intron can be
from any naturally-occurring source or can be constructed from
portions of different sources, e.g., constructed from splice donor
and acceptor sequences from different sources. For example, the 5'
UTR intron comprises a portion of a CMV intron and a portion of a
SV40 16S intron. Preferably, the splice donor for the 5' UTR intron
is from the sequence downstream of the start of transcription from
the viral promoter, and the splice acceptor is from the SV40 16S
intron and has the nucleotide sequence of nucleotides 888-974 of
SEQ ID NO: 1. It is preferred that no insertions, deletions,
inversion, and/or substitutions are present in this nucleotide
sequence. However, one of ordinary skill in the art appreciates
that nucleotides 888-974 of SEQ ID NO: 1 can have insertions,
deletions, inversion, and/or substitutions that will not affect the
function of the nucleotide sequence, which is to drive the
transcription of the recombinant protein coding sequence.
[0033] In a preferred embodiment, the recombinant expression vector
comprises a 3' UTR, an oriP, a pUC19 origin of replication, a viral
promoter, and a 5' UTR intron. Preferably, the viral promoter is a
CMV promoter and the 5' UTR intron comprises a portion of a CMV
intron and a portion of a SV40 16S intron, e.g., comprises
nucleotides 888-974 of SEQ ID NO: 1. Most preferably, the
recombinant expression vector is the vector plasmid pMXT5, which is
shown pictorially in FIG. 1A (pMXT5), and which has the nucleotide
sequence (without any coding sequences) of SEQ ID NO: 1 (FIG. 10).
For example, as shown in FIG. 10, the CMV promoter comprises
nucleotides 1-1037; 5' UTR intron comprises nucleotides 888-974;
MCS comprises nucleotides 1038-1061; LC 3' UT comprises nucleotides
1062-2560; OriP comprises nucleotides 2561-4550; pUC19 ori
comprises nucleotides 4551-5220; and Ap comprises nucleotides
5221-6380.
[0034] The recombinant expression vector can be designed for either
transient expression or for stable expression. Preferably, the
vector of the invention promotes transient expression, i.e., is a
recombinant transient expression vector, such that the vector is
one that does not integrate into the genome of a host cell. Without
being bound to any particular theory, it is believed that the
recombinant expression vector can be made to be a transient
expression vector by incorporating into the vector an oriP, which
promotes high copy episomal plasmid replication.
[0035] The recombinant expression vector can comprise a nucleic
acid sequence encoding any protein, such as a hormone, growth
factor, antibody, receptor, structural protein, enzyme, etc. The
protein can be, for example, a therapeutic protein, and can be
naturally-occurring or non-naturally occurring, e.g., a genetically
engineered protein including, for example, a fusion protein, a
chimeric protein, etc. Preferably, the recombinant expression
vector comprises such a nucleic acid for the expression of the
protein. It is to be understood that the term "protein" as used
herein includes parts or fragments thereof, and thus, polypeptides
and peptides of any length are included within the meaning of this
term. For example, polypeptides and peptides are included wherein
the polypeptides can comprise, for instance, about 50 or more amino
acids and the peptides can comprise, for instance, about 8-49 amino
acids. The nucleic acid sequence encoding the protein can be
obtained from any source, e.g., isolated from nature, synthetically
generated, isolated from a genetically-engineered organism, and the
like. An ordinarily skilled artisan will appreciate that any type
of nucleic acid sequence (e.g., DNA, RNA, genomic DNA, and cDNA)
that can be inserted into a recombinant expression vector can be
used in connection with the invention. For example, the nucleic
acid sequence encoding a protein can be naturally-occurring, e.g.,
a gene. Alternatively, the nucleic acid sequence encoding a protein
can be non-naturally occurring, e.g., non-native to any organism,
e.g., mammal. For instance, the nucleic acid sequence can be a
codon optimized nucleic acid sequence in which codons within the
nucleic acid sequence, which codons are not generally used by the
host cell translation system, termed "rare codons," are changed by
in vitro mutagenesis to preferred codons without changing the amino
acids of the synthesized protein (Bradel-Tretheway et al., J.
Virol. Meth., 111: 145-156 (2003); Ramakrishna et al., J. Virol.
78: 9174-9189 (2004)). In addition, the nucleic acid sequence
encoding a protein can be further modified, e.g., codon optimized,
to improve the folding of the RNA, such that the folding of the RNA
transcript encoded by the nucleic acid sequence is minimized.
Whatever type of nucleic acid sequence is used, the nucleic acid
sequence preferably encodes a secreted protein. By "secreted" is
meant that the protein is released from the cell into the
extracellular environment, thereby facilitating the purification of
the protein. In this regard, the recombinant expression vector
preferably comprises a signal sequence, which causes the expressed
protein to be secreted from the cell by which it was expressed.
[0036] In a preferred embodiment, the recombinant expression vector
comprises a nucleic acid encoding an immunogloblin chain, e.g.,
light chain or heavy chain. The immunoglobulin chain can be any
immunoglobulin chain derived from any source, genetically-modified,
or synthesized. Preferably, the immunoglobulin chain is a human
immunoglobulin chain selected from the group consisting of a
.gamma..sub.1 heavy chain, a .gamma..sub.2 heavy chain, a
.gamma..sub.4 heavy chain, a .kappa. light chain, and a .lamda.
light chain. Exemplary heavy chain constant region sequences
include: a .gamma..sub.1 heavy chain constant region, which is
encoded by the nucleotide sequence of SEQ ID NO: 4 and comprises
the amino acid sequence of SEQ ID NO: 5; a .gamma..sub.2 heavy
chain constant region, which is encoded by the nucleotide sequence
of SEQ ID NO: 6 and comprises the amino acid sequence of SEQ ID NO:
7; and a .gamma..sub.4 heavy chain constant region, which is
encoded by the nucleotide sequence of SEQ ID NO: 8 and comprises
the amino acid sequence of SEQ ID NO: 9. Exemplary light chain
constant region sequences include: a .kappa. light chain constant
region, which is encoded by the nucleotide sequence of SEQ ID NO:
10 and comprises the amino acid sequence of SEQ ID NO: 11, and a
.lamda. light chain constant region, which is encoded by the
nucleotide sequence of SEQ ID NO: 12 and comprises the amino acid
sequence of SEQ ID NO: 13. Exemplary antibody heavy and light
chains include: an LDP-01 heavy chain, which is encoded by the
nucleotide sequence of SEQ ID NO: 14 and comprises the amino acid
sequence of SEQ ID NO: 15, and an LDP-01 light chain, which is
encoded by the nucleotide sequence of SEQ ID NO: 16 and comprises
the amino acid sequence of SEQ ID NO: 17. The LDP-01 antibody is
referred to herein as Ab#1 and has been described in WO 2004/033693
(PCT/US2003/010154) and U.S. Patent Application Publication No.
2003/0203447 A1.
[0037] In this regard, the recombinant expression vector desirably
comprises an antibody signal sequence, which promotes the secretion
of the antibody into the extracellular environment. Suitable
antibody signal sequences are known in the art. For example, a
preferred signal sequence comprises SEQ ID NO: 2 or SEQ ID NO:
3.
[0038] The recombinant expression vector can alternatively comprise
a nucleic acid sequence encoding a functional fragment of a
protein. The term "functional fragment" which is synonymous with
"functional part" or "functional portion," when used in reference
to a protein, refers to any part or fragment of the protein, which
part or fragment retains a biological activity of the protein of
which it is a part. Functional fragments encompass, for example,
those parts of a protein (the parent protein) that retain a
function of the parent protein to a similar extent, the same
extent, or to a higher extent, as the parent protein. For instance,
if the protein is an immunoglobulin, functional fragments thereof
can include any portion of the immunoglobulin which, for example,
retains the ability to bind to the antigen of the parent
immunoglobulin. Also, for example, if the protein is a cell surface
receptor, functional fragments thereof can include any portion of
the cell surface receptor which, for instance, retains the ability
to bind to the ligand of the parent cell surface receptor. In
reference to the parent protein, the functional fragment can
comprise, for instance, about 10%, 25%, 30%, 50%, 60%, 80%, 90%,
95%, or more of the parent protein. The functional portion can
comprise additional amino acids at the amino or carboxy terminus of
the portion, or at both termini, which additional amino acids are
not found in the amino acid sequence of the parent protein.
Desirably, the additional amino acids do not interfere with the
biological function of the functional portion.
[0039] The invention further provides a host cell comprising any of
the recombinant expression vectors described herein. As used
herein, the term "host cell" refers to any type of cell that can
contain the inventive recombinant expression vector. The host cell
can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or
can be a prokaryotic cell, e.g., bacteria or protozoa. The cell can
be a cultured cell or a primary call, i.e., isolated directly from
an organism, e.g., a human. The cell can be an adherent cell or a
suspended cell, i.e., a cell that grows in suspension. Suitable
host cells are known in the art and include, for instance, DH5a E.
coli cells, Chinese hamster ovarian (CHO) cells, monkey VERO cells,
COS cells, HEK293 cells, and the like. For purposes of amplifying
or replicating the recombinant expression vector, the host cell is
preferably a prokaryotic cell. More preferably, the host cell is a
DH5.alpha. cell. For purposes of producing a recombinant protein,
the host cell is preferably a mammalian cell. Most preferably, the
host cell is a human cell. While the cell can be any cell of the
human body, it is preferred that the cell is a human embryonic
kidney cell. More preferred is that the human embryonic kidney cell
expresses an Epstein Barr virus nuclear antigen-1 (EBNA-1) protein,
e.g., a 293E cell.
[0040] As used herein, the term "mammal" refers to any mammal,
including, but not limited to, mammals of the order Rodentia, such
as mice and hamsters, and mammals of the order Logomorpha, such as
rabbits. It is preferred that the mammals are from the order
Camivora, including Felines (cats) and Canines (dogs). It is more
preferred that the mammals are from the order Artiodactyla,
including Bovines (cows) and Swines (pigs) or of the order
Perssodactyla, including Equines (horses). It is most preferred
that the mammals are of the order Primates, Ceboids, or Simoids
(monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.
[0041] The invention further provides methods of producing a
recombinant protein. In a first method, the recombinant protein is
a heterodimeric or heteromultimeric protein comprising a first
polypeptide chain and a second polypeptide chain, wherein the first
polypeptide chain is different from the second polypeptide chain.
The first method comprises contacting cells in a medium with a
first vector and a second vector, wherein the first vector encodes
the first polypeptide chain and the second vector encodes the
second polypeptide chain, and the second vector is present in the
medium in an amount which is about 1.5 to about 2.5 times as much
as the amount of the first vector, whereupon a recombinant protein
is produced. The first and second vectors can be any suitable
vector and preferably are inventive recombinant expression vectors
as described herein.
[0042] For purposes of the first inventive method of producing a
protein, the first and second vectors can independently be any type
of vector, i.e., the first and second vectors can have the same
regulatory elements but differ only in the recombinant protein
coding sequence contained therein. By way of example, both the
first vector and second vector can be the pMXT vector as shown in
FIG. 1A. Preferably, each of the first vector and the second vector
is one of the inventive recombinant expression vectors described
herein. Most preferably, the first and second vectors are pMXT
vectors. For example, it is preferred that each of the first and
the second vector is a recombinant transient expression vector. It
is also preferred that each of the first and second vector
comprises a 3' UTR of a light chain gene and an oriP. It is also
preferred that each of the first and second vector comprises a
viral promoter, a pUC19 origin of replication, a 5' UTR intron, or
a combination of any of the foregoing. Preferably, the viral
promoter is a CMV promoter, and the 5' UTR intron comprises
nucleotides 888-974 of SEQ ID NO: 1. Moreover, it is preferred that
each of the first and second vector comprises an antibody signal
sequence.
[0043] Also, with respect to the first inventive method of
producing a protein, the second vector is present in the medium in
an amount which is about 1.5 to about 2.5, e.g., 1.6, 1.7, 1.75,
1.8, 1.9, 2.0, 2.125, 2.25, 2.3, 2.4, and 2.5, times as much as the
amount of the first vector. Preferably, the second vector is
present in the medium in an amount which is about 1.75 to about
2.25 times as much as the amount of the first vector. More
preferably, the second vector is present in the medium in an amount
which is about twice as much as the amount of the first vector.
[0044] The invention further provides a second method of producing
a recombinant protein. The second method comprises culturing cells,
which have been contacted with a recombinant transient expression
vector encoding the protein, in a medium in a membrane-enhanced
culturing vessel, whereupon a recombinant protein is produced. The
second method can alternatively comprise culturing cells, which
have been contacted with a recombinant transient expression vector
encoding the recombinant protein, in a medium in a Fembach flask,
whereupon a recombinant protein is produced. The recombinant
transient expression vector can be any suitable such vector and
preferably is an inventive recombinant expression vector as
described herein.
[0045] In a third method, the recombinant protein is produced upon
contacting cells with at least one of the inventive recombinant
expression vectors described herein. In a fourth method, the
recombinant protein is produced upon culturing any of the inventive
host cells comprising any of the inventive recombinant expression
vectors described herein.
[0046] Any suitable method can be employed to contact cells with a
first vector, a second vector, or a recombinant expression vector,
such that the cells express the protein encoded by the vector.
Methods of contacting cells, such that the cells are modified to
express a particular protein, polypeptide, or peptide, are
well-known in the art. See the references listed in Sambrook et al.
(1989), supra. Suitable methods of contacting cells to this end
include, for instance, infection with a viral vector, transfection
with a lipofection reagent, cationic polymer, DEAE, or calcium
phosphate, and electroporation.
[0047] The cells can be contacted with a first vector, a second
vector, or a recombinant expression vector in the presence of a
suitable cationic polymer. Suitable cationic polymers for
transfecting cells are known in the art, and include, for example,
polylysine and polyethyleneimine (PEI). In a preferred embodiment
of the inventive method, the cationic polymer is PEI. PEI can be
linear or branched and can vary in molecular weight, depending on
the number of base units, which comprise the polymer. Preferably,
the PEI is a linear PEI. More preferably, the linear PEI has a
molecular weight of about 25 kDa. Although the amount of PEI used
in the method can be any amount, it is preferred that the linear
PEI is present in an amount that is about 1.5 to about 4.5, e.g.,
1.5, 1.6, 1.75, 2.0, 2.25, 2.5, 2.6, 2.7, 2.75, 2.8, 2.9, 3.0, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.75, 3.8, 3.9, 4.0, 4.1, 4.25, 4.3, 4.4,
and 4.5, times the amount of the vector(s) contacting the cells.
Preferably, the PEI is present in an amount that is about 2.5 to
3.5 times the amount of the vector(s) contacting the cells. More
preferably, the PEI is present in an amount that is about twice the
amount of the vector(s) contacting the cells.
[0048] For purposes of the inventive method comprising contacting
cells with more than one vector, e.g., a first vector and a second
vector, the cells can be contacted with the first vector and second
vector in a sequential fashion, e.g., first vector contacted with
the cells before the second vector. Alternatively, the cells can be
contacted with the first vector and second vector simultaneously.
Preferably, the cells are contacted with the first vector and
second vector simultaneously. For example, in a method comprising
contacting cells with more than one vector, the cells can be
contacted with a first vector before or simultaneously with a
second or additional vector.
[0049] As used herein, the term "culturing" is synonymous with
"maintaining." Methods of culturing cells are known in the art
(see, e.g., Tissue Engineering Methods and Protocols, Morgan and
Yarmush (eds.), Humana Press, Inc., Totowa, N.J., 1999). As one
ordinarily skilled recognizes, the conditions under which cells are
cultured varies depending on the cell type. The conditions include
temperature of the environment, the culturing vessel containing the
cells, the composition of the various gases, e.g., CO.sub.2, which
comprises the cell culture atmosphere or environment, the medium in
which the cells are maintained, the components and pH of the
medium, the density at which cells are maintained, the schedule by
which the medium needs to be replaced with new medium, etc. These
parameters are often known in the art or can be empirically
determined. For example, with respect to the inventive methods,
wherein cells are cultured in a medium, e.g., a first medium, a
second medium, etc., any method can be employed to culture the
cells in the medium, such that the cells express (and, in some
instances, secrete) the protein encoded by the vector, which was
contacted to the cells.
[0050] The cells are desirably cultured in a membrane-enhanced
culturing vessel or a Fembach flask. For purposes herein, the term
"membrane-enhanced culturing vessel" refers to a container for
holding cell cultures that have been improved upon by the addition
of at least one membrane. Suitable membrane-enhanced culturing
vessels include membrane-based cell culture vessels, dialysis-based
cell culture vessels, membrane-based high density cell culture
vessels, and two-compartment vessels. The term "vessel" as used
herein is synonymous with systems, reactors, bioreactors, flasks,
and devices. Suitable membrane-enhanced culturing vessels include,
for instance, miniPerm.RTM. flasks, OptiCell.RTM. flasks, and the
CELLINE.TM. CL1000 (referred to herein as Integra flasks or Integra
CL1000 flasks), which are commercially available from companies,
such as IBS Integra Biosciences AG (Chur, Switzerland), OptiCell
(Westerville, Ohio), VWR, Fisher Scientific, and Labmate (Asia).
Most preferably, the membrane-enhanced culturing vessel is an
Integra CL1000. For example, one of the ordinary skill in the art
appreciates that a membrane-enhanced culturing vessel such as an
Integra flask may comprise a nutrient chamber and a cultivation
chamber, wherein nutrients from a media reservoir in the nutrient
chamber pass through a semi-permeable membrane into the cultivation
chamber containing cells so as to provide a continuous supply of
nutrients and wherein the membrane also allows for diffusion of
metabolites out of the cultivation chamber and away from contact
with the cells but does not permit diffusion of a recombinant
protein produced by the cells (e.g., an antibody or antibody
fragment) out of the cultivation chamber, and further wherein the
cells also have sufficient gas exchange such as access to oxygen
and carbon dioxide through a separate silicone membrane at the
bottom of the vessel.
[0051] As used herein, the term "Fembach flask" refers to a
commercially available Corning.RTM. polycarbonate Erlenmeyer flask
having the Fembach design. Such flasks are commercially-available
from companies such as Life Sciences.
[0052] Without being bound to any particular theory,
membrane-enhanced flasks (e.g., Integra CL1000, OptiCell.RTM.
flasks, and miniPerm.RTM. flasks) and Fernbach flasks are
particularly suitable for culturing transfected cells, for example,
transiently transfected cells, as these devices permit efficient
gas exchange between the cells and the environment, e.g., the
incubator environment, which permits optimal cell growth and
production of the recombinant protein. Under certain conditions,
shake flasks can also be suitable culturing vessels in which cells
can be cultured for optimal cell growth and production of the
recombinant protein. It should be understood that any flask or
culturing vessel that permits efficient gas exchange between the
cells and the environment are included in the scope of the
invention and are not limited to only the aforementioned flasks and
culturing vessels.
[0053] In the inventive methods comprising culturing cells, the
medium can be any suitable medium for culturing cells known in the
art. The medium can be, for example, a culture medium containing 1%
low immunoglobulin (Ig) fetal bovine serum (FBS). Alternatively,
the medium can be a serum-free cell culture medium, e.g., IS293.TM.
medium. In some instances, the medium is preferably a serum-free
IS293.TM. medium (Irvine Scientific, Irvine, Calif.).
[0054] The cell cultures of the inventive methods can be initiated
or seeded at any suitable cell density. As one of ordinary skill in
the art recognizes, the seeding density depends on a variety of
factors, such as cell type, culturing conditions, and the day which
has been selected for harvesting or purifying the recombinant
protein from the cell culture. Desirably, the cell density is
within the range of about 1.0.times.10.sup.6 to about
2.0.times.10.sup.7 (e.g., about 1.0.times.10.sup.6 to about
1.5.times.10.sup.7). More preferably, the initiating seeding cell
density of the cell culture is about 3.0.times.10.sup.6 to about
1.0.times.10.sup.7. Without being bound to any particular theory,
it is believed that the seeding density of cells, which have been
transiently transfected with a vector encoding a protein, is a
factor in obtaining efficient production of a recombinant
protein.
[0055] For purposes of the inventive methods, the cells that are
cultured or are contacted with a first vector, a second vector, or
a recombinant expression vector can be any cell, such as those
described herein as "host cells." For example, the cells that are
cultured and/or contacted with one or more than one recombinant
expression vector can be any host cells. Preferably, the cells are
mammalian cells, and, more preferably, the cells are human cells.
The cells are desirably human embryonic kidney cells. In a most
preferred embodiment, the human embryonic kidney cells express
Epstein-Barr virus nuclear antigen-1 protein (EBNA-1), e.g., 293E
cells.
[0056] Cells, which have been contacted with a recombinant
transient expression vector, can be obtained by transiently
transfecting cells by any method known in the art, including those
described herein. Recombinant transient expression vectors are
known in the art and include, for instance, pCEP4, pcDNA3, and any
of the recombinant expression vectors described herein which
comprise an oriP. Preferably, the recombinant transient expression
vectors are pMXT vectors. For example, the vectors can be any of
the inventive recombinant expression vectors as described
herein.
[0057] With respect to the first method of producing a recombinant
protein (e.g., comprising contacting cells with a first vector and
a second vector), the method can further comprise the second
inventive method of producing a recombinant protein. That is, the
method of producing a recombinant protein can further comprise the
step of culturing the cells, which have been contacted with a first
vector and a second vector, in a second medium in a
membrane-enhanced culturing vessel (e.g., an Integra CL1000, an
OptiCell.RTM. flask, a miniPerm.RTM. flask), a Fernbach flask, or
like flask. In such an embodiment, the second medium can be
different from the medium in which the first and second vectors are
present. For purposes of the methods, which comprise culturing
cells in a membrane-enhanced culturing vessel, a Fembach flask, or
like flask, the suitable medium for use in such a vessel or flask
can be a serum-free cell culture medium, e.g., IS293 medium.
Preferably, the medium is serum-free IS293 medium (Irvine
Scientific, Irvine, Calif.).
[0058] With respect to the second inventive method of producing a
recombinant protein, the method can comprise the first inventive
method of producing a recombinant protein. One of ordinary skill in
the art recognizes that the methods described herein can be
combined in such a way, such that all of the limitations of the
methods are met. Such a combined method is within the scope of the
invention.
[0059] With respect to any of the inventive methods comprising
culturing cells, e.g., in a membrane-enhanced culturing vessel, a
Fernbach flask, or like flask, the method can further comprise
purifying or isolating the recombinant protein from the medium,
e.g., the serum-free medium. As used herein, the terms "purifying"
and "isolating" do not necessarily refer to absolute purity or
isolation, as one of ordinary skill in the art appreciates that a
partially purified or partially isolated protein can be useful or
of value.
[0060] Methods of purifying proteins from mixtures are known in the
art. Suitable purification methods include, for example,
chromatography, electrophoresis, and the like. Suitable
chromatographic methods of purifying polypeptides include, for
example, HPLC, ion-exchange chromatography, affinity
chromatography, etc. Preferably, the purifying comprises
chromatographing the medium through a resin, such as a cationic
resin, an anionic resin, and an affinity resin. If the polypeptide
is an immunoglobulin chain, the purifying preferably comprises the
use of resin comprising Staphylococcus aureus Protein A, which is a
bacterially-produced protein that binds to the Fc regions of IgG
antibodies. More preferably, the purifying comprises centrifuging
the medium through a column comprising Protein A, e.g.,
centrifuging the medium through a Protein A spin column (which is
commercially available from Pro-Chem).
[0061] The purifying can occur at any point in time after culturing
the cells, which have been contacted with a vector. In some
instances, it is preferable for the purifying to occur after about
3 days of culturing, e.g., after about 3, 4, 5, 6 or more days. In
other instances, it is preferable for the purifying to occur after
about 7 days of culturing, e.g., after about 7, 8, 9, 10, 11, 12,
13, 14, 15 or more days.
[0062] The invention provides fast and efficient methods of
producing high levels of recombinant proteins. In some instances,
at least 300 .mu.g/ml recombinant protein is produced after 3 days
of culturing. In other instances, at least 500 .mu.g/ml recombinant
protein is produced after 3 days of culturing. In some preferred
instances, at least 700 .mu.g/ml recombinant protein is produced
after 3 days of culturing.
[0063] The term "recombinant protein" as used herein, refers to any
protein or part thereof that is produced by a
genetically-engineered organism. For example, the recombinant
protein can be any of the proteins described herein.
[0064] For purposes of the first method of producing a recombinant
protein, the recombinant protein is a heterodimeric protein or a
heteromultermeric protein, such as a tetramer, which comprises two
copies of two different polypeptide chains. Such proteins are known
in the art, and include, for instance, hemoglobin, immunoglobulins,
T cell receptors, and B cell receptors, etc. In a preferred
embodiment of the first inventive method, the recombinant protein
is a heterotetrameric protein. Desirably, the heterotetrameric
protein is an immunoglobulin. In this instance, it is preferred
that the first vector encodes a heavy chain of an immunoglobulin,
or a part thereof, and the second vector encodes a light chain of
an immunoglobulin, or a part thereof. The heavy chain can be any
heavy chain of any immunoglobulin, as described herein. The light
chain can be any light chain of any immunoglobulin, as described
herein. Exemplary antibody heavy and light chains: an LDP-01 heavy
chain, which is encoded by the nucleotide sequence of SEQ ID NO: 14
and comprises the amino acid sequence of SEQ ID NO: 15, and an
LDP-01 light chain, which is encoded by the nucleotide sequence of
SEQ ID NO: 16 and comprises the amino acid sequence of SEQ ID NO:
17. The LDP-01 antibody is referred to herein as Ab#1 and has been
described in WO 2004/033693 (PCT/US2003/010154) and U.S. Patent
Application Publication No. 2003/0203447 A1.
EXAMPLES
[0065] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0066] This example demonstrates the construction of recombinant
expression vectors of the invention.
[0067] Transient expression vectors for expression of any gene were
constructed with a multilinker site containing unique restriction
sites positioned between the 3' end of the CMV promoter and the 5'
end of the mouse light chain 3' untranslated region. Transient
expression vectors containing cDNAs, which encode light chain
.kappa. or .lamda. genes or heavy chain .gamma..sub.1,
.gamma..sub.2, or .gamma..sub.4 genes, under the control of a CMV
promoter (Boshart et al., Cell 41: 521-530 (1985)) and mouse light
chain 3' untranslated region (Xu et al., J. Biol. Chem. 261:
3838-3845 (1986)) were contructed. Unique restriction sites were
positioned at the 5' end of the V region (e.g., SalI) and in the
junction regions between the V and constant regions (BlpI for heavy
chain, BsiWI for .kappa. light chain and AvrII for lambda) for the
cloning of any new V region adjacent to the desired cognate
constant region. The vectors also contained the Epstein Barr virus
oriP sequence (Reisman et al., Mol. Cell. Biol. 5: 1822-1832
(1985)) for episomal plasmid replication in 293E cells, the origin
of replication from the vector pUC19, and the gene encoding
resistance to ampicillin for selection of tranformants in E. coli.
The transient expression vectors containing the multilinker sites,
the heavy chain, and the light chain are shown in FIGS. 1A-1C.
Example 2
[0068] This example demonstrates a method of transiently
transfecting cells for producing recombinant proteins.
[0069] 293E cells (Invitrogen, R620-07) were maintained as adherent
cultures in Dulbecco's Modified Eagle Medium (DMEM)
(Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS,
Hyclone), 2 mM glutamine, and 250 .mu.g/ml G418 antibiotic
(Gibco-Invitrogen). For growth in suspension culture, the cells
were adapted to the following serum-free media formulations:
IS293.TM. (Irvine Scientific), IS293-V.TM. (Irvine Scientific), 293
SFM II (Gibco-Invitrogen), H-SFM (Gibco-Invitrogen), and
HYQ.RTM.PF293 (HyClone). The cells were originally supplemented
with 10% low IgG FBS (HyClone) and 2 mM glutamine and gradually
weaned down to 1% low IgG FBS over a period of several weeks. Once
in 1% low IgG FBS, the cells were transferred to shake flasks for
continued adaptation to suspension growth. Growth and viability
were monitored using the VICELL.TM. XR Cell Viability Analyzer
(Beckman-Coulter).
[0070] All plasmids were transformed into DH5a cells (Invitrogen)
and purified using endotoxin-free plasmid purification kits
(QIAGEN.RTM.). For transfections in 6-well plates, 2 ml of cells at
5.times.10.sup.5 cells/ml were seeded per well. For transfections
in shake flask cultures, cells were seeded at 8.times.10.sup.5
cells/ml at the appropriate volumes prior to transfection. DNA (2
.mu.g/ml) was pre-incubated with linear polyethyleneimine (PEI, 25
kDa MW, Polysciences) at a concentration of 4 .mu.g/ml for 10 min
at room temperature prior to addition to cells. The DNA/PEI mixture
was then added to cells, and the cells with the DNA/PEI were either
maintained in shake flasks or transferred to Integra flasks.
Example 3
[0071] This example demonstrates the determination of the optimal
PEI:DNA ratio for transient transfections.
[0072] Adherent 293E cells grown in DMEM supplemented with 10% FBS
in 6-well plates were transfected with pQBI-pGK (GFP expressing
plasmid, Q-biogene) using linear polyethyleneimine (PEI) as
described in Example 2. DNA (1 .mu.g/ml, 2 .mu.g/ml, or 5 .mu.g/ml)
was pre-incubated with linear PEI (1, 2, 4, 5, 10, or 25 .mu.g/ml)
for 10 min at room temperature prior to the addition to cells, then
the PEI/DNA mixture was added to cells, and the cells were
maintained in shake flasks or Integra flasks.
[0073] GFP expression was monitored 24 hours post-transfection
using a Becton Dickinson FACScan flow cytometer equipped with the
Cytek Automated Microsampler System (AMS) 96-well plate reader.
Flow data was analyzed using FlowJo (Tree Star, Inc.). Cells also
were counterstained with 1 .mu.g/ml propidium iodide (PI) to
determine cell viability. Growth and viability of the cells
post-transfection were monitored using the VICELL.TM. XR Cell
Viability Analyzer (Beckman-Coulter). The cells transfected with 1
.mu.g/ml DNA and 1 .mu.g/ml PEI (FIG. 2A); 2 .mu.g/ml DNA and 2
.mu.g/ml PEI (FIG. 2B); 5 .mu.g/ml DNA and 5 .mu.g/ml PEI (FIG.
2C); 1 .mu.g/ml DNA and 2 .mu.g/ml PEI (FIG. 2D); 2 .mu.g/ml DNA
and 4 .mu.g/ml PEI (FIG. 2E); 5 .mu.g/ml DNA and 10 .mu.g/ml PEI
(FIG. 2F); 1 .mu.g/ml DNA and 5 .mu.g/ml PEI (FIG. 2G); 2 .mu.g/ml
DNA and 10 .mu.g/ml PEI (FIG. 2H); and 5 .mu.g/ml DNA and 25
.mu.g/ml PEI (FIG. 2I) were measured for GFP expression
.alpha.-axis) and PI staining (y-axis) by flow cytometry, and the
resulting data was plotted in the series of graphs of FIGS.
2A-2I.
[0074] As shown in FIGS. 2A-2I, the DNA concentration of 1 .mu.g/ml
at a PEI:DNA ratio of 2:1 gave the highest percentage of cells
expressing GFP with relatively low cellular cytotoxicity 24 hours
post-transfection.
[0075] The results of this example demonstrated the production of
recombinant protein and confirmed that the optimal PEI:DNA ratio
for transient transfection is 2:1.
Example 4
[0076] This example demonstrates the determination of the optimal
medium for culturing transiently transfected cells.
[0077] 293E cells were grown and transfected in the presence of 1%
low-IgG serum in 6-well plates and shake flasks, as described in
Example 2. Twenty-four hours after transfection, 293E cells were
adapted to suspension growth in one of 5 different serum-free media
formulations (IS293.TM., H-SFM, IS293V.TM., SFMII, or
HYQ.RTM.PF293) or one serum-containing media formulation (DMEM) as
in Example 2.
[0078] Twenty-four to forty-eight hours later, GFP expression by
transfected cells were determined as described in Example 3. Cells
also were counterstained with 1 .mu.g/ml PI to determine cell
viability. Growth and viability of the cells post-transfection were
monitored using the VICELL.TM. XR Cell Viability Analyzer
(Beckman-Coulter). The resulting data from GFP expression (bars)
and for PI staining (X) was plotted to form the graph of FIG.
3.
[0079] As shown in FIG. 3, IS293.TM. medium (Irvine Scientific)
gave the highest percentage of GFP-expressing cells with minimal
cytotoxicity in shake flasks; values were comparable to those
obtained with adherent 293E cells cultured in DMEM supplemented
with 10% FBS.
[0080] The results of this example demonstrated that IS293.TM.
medium is the optimal serum-free medium to be used with transiently
transfected cells for producing recombinant proteins.
Example 5
[0081] This example demonstrates the determination of optimum heavy
and light chain plasmid ratios for maximum antibody
productivity.
[0082] Various ratios of pMXT (heavy chain (HC)):pMXT (light chain
(LC)) (see Example 1) or pCEP4 (HC):pCEP4 (LC) were tested for
effects on antibody productivity in cells grown in IS293.TM. medium
supplemented with 1% low-IgG serum in shake flasks. The pCEP4
vector containing the nucleotide sequence encoding the Ab# 1 heavy
chain (SEQ ID NO: 14) was constructed by cloning the coding
sequence into KpnI and Xho sites. The pCEP4 vector containing the
nucleotide sequence encoding the Ab#1 light chain (SEQ ID NO: 16)
was constructed by cloning the coding sequences into Nhe and Xho
sites. The encoded heavy chain and light chain of Ab#1 is set forth
as SEQ ID NOs: 15 and 17, respectively. All plasmids were amplified
by transformation into DH5 .alpha. cells and purified as described
in Example 2. 293E cells were transiently transfected as described
in Example 2. Transfected cells were transferred to IS293.TM.
medium in shake flasks for 7-10 days. Antibody expression by the
cells transfected with a 1:1, 1:2, or 2:1 ratio of vector encoding
HC:vector encoding LC, wherein the vectors were either pMXT or
pCEP4 was determined by sandwich ELISA and the data analyzed in
PRISM.TM. (GraphPad). The resulting data was plotted to form the
graph of FIG. 4.
[0083] As shown in FIG. 4, a 1:2 ratio of HC:LC generated the
highest antibody productivity with Ab#1 achieving levels of 60-70
.mu.g/ml after 7-10 days. The highest productivity for Ab#1
(LDP-01) in pMXT was .about.3.times. greater than the best output
achieved using pCEP4.
[0084] The results of this example demonstrated that the pMXT
vector is optimal for co-transfecting cells with vectors encoding
different polypeptide chains at a ratio of 1:2.
Example 6
[0085] This example demonstrates that the level of antibody
production by transiently transfected cells cultured
post-transfection in membrane-enhanced culturing vessels are
comparable to the level of antibody production achieved by
transfected cells cultured post-transfection in shake flasks.
[0086] 293E cells were transiently transfected in shake flasks as
described in Example 2. Cells were either maintained in the shake
flasks or transferred to 15 ml of medium and placed in Integra
CL1000 flasks. After 7-10 days, cell culture supernatant was
harvested, clarified, and purified for antibodies using a standard
Protein A column, if cells were cultured in shake flasks, or a
Protein A spin column, if cells were cultured in Integra flasks.
Cell viability and antibody production of both sets of cells 0, 4,
7, and 14 days post-transfection were assayed as described in
Examples 4 and 5, respectively. For antibody expression using the
Integra CL1000 flask, 200 ml of transfected 293E cells were
resuspended in 15 ml of IS293.TM. medium supplemented with 1% low
IgG FBS and 250 .mu.g/ml G418 antibiotic and transferred into the
membrane compartment. One liter of IS293.TM. medium was added to
the upper media chamber.
[0087] The cell viability (X) and antibody production
(.circle-solid.) of the transfected cells maintained in Integra
flasks are shown in FIG. 5A, whereas the levels of antibody
production for Ab#1 by cells maintained in either shake flasks or
by Integra CL1000 flasks are shown in FIG. 5B.
[0088] As shown in FIG. 5A, antibody production of cells cultured
in Integra flasks peaked at 7 days, producing over 1 mg/ml
antibody. This level is comparable to the level of antibody
production of transiently transfected cells cultured in shake
flasks as shown in FIG. 5B.
[0089] The results of this example demonstrated that Integra flasks
are suitable culturing vessels for maintaining small volumes of
transiently transfected cells. The small volume permits the use of
Protein A spin columns, which facilitates the purification of
antibodies from the cell culture supernatant.
Example 7
[0090] This example demonstrates a method of producing antibodies
in membrane-enhanced culturing vessels at optimized seeding
densities.
[0091] Suspension-adapted HEK 293E cells were maintained in
IS293.TM. medium (Irvine Scientific) supplemented with 1% low IgG
FBS (HyClone), 2 mM glutamine (Gibco-Invitrogen), and 250 .mu.g/ml
G418 antibiotic (Gibco-Invitrogen). For transfection, cells were
seeded at 8.times.10.sup.5 cells/ml in shake flasks at the
appropriate volumes prior to transfection. DNA encoding Ab#1 or
Ab#2 (which differed from Ab#1) was pre-incubated with linear
polyethyleneimine (PEI, 25 kDa MW, Polysciences) at optimized
conditions (see, e.g., Example 3; see also, e.g., Handa et al.,
American Society for Cell Biology, poster presentation #1937
(2004)) prior to addition to cells. For antibody expression using
the Integra CL1000 flask, cells at the following seeding densities
were resuspended in 30 ml of IS293.TM. medium supplemented with 1%
low IgG FBS, 2 mM glutamine, and 250 .mu.g/ml G418 antibiotic and
transferred into the cultivation chamber: 1.3.times.10.sup.6
(I-50), 2.7.times.10.sup.6 (I-100), 5.3.times.10.sup.6 (I-200), and
1.1.times.10.sup.7 (I-400). For comparison, 8.times.10.sup.5 cells
(E-200) were seeded in Erlenmeyer flasks. All flasks were incubated
for 3, 5, 7, or 10 days post-transfection. One ml samples from the
nutrient chambers and cultivation chambers of the Integra CL 1000
flasks were removed and analyzed at 3, 5, 7, or 10 days
post-transfection.
[0092] Growth and viability were monitored using the VICELL.TM. XR
Cell Viability Analyzer (Beckman-Coulter). The percentage of viable
cells 1, 3, 5, 7, and 10 days after transfection for cells
tranfected at different seeding densities is shown in FIGS. 6A and
6B. The viable cell count of cells 0, 1, 3, 5, 7, and 10 days after
transfection for cells transfected at different seeding densities
is shown in FIGS. 7A and 7B.
[0093] As shown in FIGS. 6A (Ab#1), 6B (Ab#2), 7A (Ab#1) and 7B
(Ab#2), cell viability did not vary between flasks, but viable cell
growth was improved in the Integra flasks for all seeding densities
tested. Maximum densities of 3-5.times.10.sup.7 cells/ml were
achieved for all conditions over the 10 day analysis period.
[0094] Analytes, gases, and pH of the samples were determined 3, 5,
7, and 10 days post-transfection using a BIOPROFILE.TM. Chemistry
Analyzer (Nova Biomedical). The data for selected nutrients and
metabolites of the media containing cells producing Ab#1 or the
media containing no cells (Media Only) are set forth in Table 1.
TABLE-US-00001 TABLE 1 Flask Media Day Day Day Day Nutrient Sample
Chamber Only 3 5 7 10 Glucose I-50 cultivation 5.37 3.35 2.85 2.57
2.76 (g/L) I-50 nutrient 5.36 5.11 4.49 4.12 3.47 I-100 cultivation
5.37 3.86 2.93 2.99 2.41 I-100 nutrient 5.36 5.05 4.39 3.97 3.85
I-200 cultivation 5.37 3.27 3.09 3.01 2.84 I-200 nutrient 5.36 4.62
4.12 3.68 3.10 I-400 cultivation 5.37 2.86 3.11 2.98 2.74 I-400
nutrient 5.36 4.52 3.94 3.67 3.23 E-200 N/A 5.36 4.42 2.51 2.36
2.06 Glutamine I-50 cultivation 6.96 6.07 4.77 4.80 4.32 (mmol/L)
I-50 nutrient 6.82 6.30 5.89 5.54 4.79 I-100 cultivation 6.96 5.84
4.57 4.65 4.03 I-100 nutrient 6.82 6.37 5.88 5.41 5.10 I-200
cultivation 6.96 5.36 4.64 4.50 4.68 I-200 nutrient 6.82 6.07 5.62
5.19 4.63 I-400 cultivation 6.96 5.47 5.25 5.02 4.21 I-400 nutrient
6.82 6.37 5.91 5.68 5.12 E-200 N/A 6.82 6.31 5.82 5.53 5.08 Lactate
I-50 cultivation 0.26 1.92 2.42 3.17 2.82 (g/L) I-50 nutrient 0.34
0.74 1.36 1.82 2.27 I-100 cultivation 0.26 1.85 2.43 2.74 2.82
I-100 nutrient 0.34 0.99 1.62 1.94 2.52 I-200 cultivation 0.26 2.47
2.32 2.84 2.72 I-200 nutrient 0.34 1.25 1.86 2.09 2.33 I-400
cultivation 0.26 2.57 2.31 2.78 2.66 I-400 nutrient 0.34 1.63 2.17
2.35 2.38 E-200 N/A 0.34 1.53 2.50 2.59 2.48
[0095] As shown by the resulting data, transiently transfected
cells maintained in 30 ml media in the cultivation chamber of an
Integra CL1000 flask can reach cell densities of up to
3-5.times.10.sup.7 viable cells/ml (e.g., 4.5.times.10.sup.7).
Nutrients from the media reservoir in the nutrient chamber pass
through a semi-permeable membrane into the cultivation chamber
providing a continuous supply of essential nutrients. The membrane
also allows for diffusion of metabolites out of the cultivation
chamber and away from contact with cells. Cells also have efficient
access to oxygen and carbon dioxide through a separate silicone
membrane at the bottom of the flask.
[0096] The Integra supernatant from the cultivation chamber had
higher glucose levels than shake flasks but lower glutamine levels.
The levels of lactate appeared similar between the two cultures.
The higher relative levels of glucose to lactate in the Integra
cultures could indicate that the cells are generating more ATP by
promoting efficient entry of pyruvate from glycolysis into the TCA
cycle.
[0097] Antibody titers of transfected cells placed in Integra
flasks or shake flasks at different seeding densities were
determined using the EASY-TITER.TM. Human IgG Assay Kit (Pierce) 0,
3, 5, 7, and 10 days post-tranfection. The data expressed as the
concentration of antibody titers (.mu.g/ml) is shown in FIGS. 8A
(Ab#1) and 8B (Ab#2), whereas the data expressed as the total
antibody yield (mg) is shown in Table 2. TABLE-US-00002 TABLE 2 Day
3 Day 5 Day 7 Day 10 % of E-200 % of E-200 % of E-200 % of E-200
Sample Yield (mg) Max Yield Yield (mg) Max Yield Yield (mg) Max
Yield Yield (mg) Max Yield Ab#1 E-200 7.6 52% 14.5 100% 7.4 51% 7.6
52% I-50 0.8 6% 5.2 36% 8.5 59% 13.3 92% I-100 5.2 36% 8.3 57% 14.7
101% 24.0 166% I-200 9.4 65% 13.8 95% 13.8 95% 19.0 131% I-400
11.00 76% 15.7 108% 14.3 99% 11.7 81% Ab#2 E-200 4.1 50% 6.3 77%
7.5 92% 8.2 100% I-50 1.2 15% 4.3 52% 7.6 93% 6.4 78% I-100 3.5 43%
8.3 101% 10.4 127% 12.9 157% I-200 6.1 74% 13.0 159% 17.0 207% 22.0
268% I-400 10.8 132% 17.8 217% 21.0 256% 22.0 268%
[0098] As shown in FIGS. 8A and 8B, antibody productivity for the
two antibodies tested, Ab#1 and Ab#2, were different in Erlenmeyer
flasks. Ab#1 peaked early at day 5 (.about.70 .mu.g/ml), followed
by a decrease in antibody concentration. Ab#2 showed slower and
steadier productivity over the full 10 days, achieving a maximal
antibody output of .about.40 .mu.g/ml. For both Ab#1 and Ab#2,
antibody productivity in the Integra flasks achieved steady levels
of increasing Ab productivity over the 10 day period. An exception
was the 1-400 sample for Ab# 1, which showed slight decreases in
productivity at days 7 and 10; however, the decrease was
substantially less as compared to that in the E-200 samples.
[0099] As shown in Table 2, the maximal yield for Ab#1 in the E-200
culture was .about.15 mg at day 5. Comparable levels (.gtoreq.90%
of E-200 maximum) were obtained in the I-200 and I-400 cultures at
day 5 as well, and higher total yields were obtained with I-100 and
I-200 after day 10, namely 166% (24 mg) and 131% (19 mg),
respectively.
[0100] As also shown in Table 2, the maximal yield for Ab#2 in the
E-200 culture was .about.8 mg at day 10. Comparable levels
(.gtoreq.90% of E-200 maximum) were obtained as early as day 3 with
I-400 (.about.11 mg), at day 5 with I-100 and I-200 (.about.8 mg
and .about.13 mg, respectively), and day 7 with I-50 (.about.8 mg).
Higher yields were obtained for I-100, I-200, and I-400 at day 10,
157% (13 mg), 268% (22 mg), and 268% (22 mg), respectively.
[0101] The results of this example demonstrated high levels of
antibody production in Integra flasks within short periods of time.
Cell densities of 1.0.times.10.sup.6 and 1.5.times.10.sup.7 were
examples of optimal densities for producing high levels of
antibodies. As shown herein, transiently transfected cells, for
example, 293E cells, in membrane-enhancing culturing vessels such
as Integra flasks, generated higher total antibody yields over
cells cultured in Erlenmeyer flasks, irrespective of antibody
productivity levels in shake cultures. Transiently expressing
antibodies in membrane-enhancing culturing vessels such as Integra
flasks also appeared to better retain antibody stability upon
exhaustion of the culture.
[0102] As shown by the results obtained herein, total antibody
yields from transiently transfected 293E cells are significantly
increased when cultured in Integra flasks vs. standard Erlenmeyer
flasks. Increasing the number of transfected cells seeded in the
Integra flask can substantially decrease the time to reach maximum
antibody yield, while decreasing the seeding density allows for
multi-mg production of antibodies using a fraction of the cells
under normal conditions in an Erlenmeyer flask. Generating
transiently expressed antibodies in Integra flasks also better
maintains the antibody titer for longer periods of time thus
allowing for greater confidence to allow cultures to proceed to
extinction without significant loss of antibody. Advantageously,
usage of membrane-enhancing culturing vessels, such as Integra
flasks, for transient protein production, such as antibody
production, allows for increased total yield, faster production by
using more cells, and/or conservation of cells by using fewer cells
while maintaining productivity comparable to non-membrane culturing
vessels, such as Erlenmeyer flasks.
[0103] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0104] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0105] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
17 1 7006 DNA Epstein Barr virus 1 aagcttgagt tttatgggtg gcagtcactg
gctggctagg cacatagcca ggccaaacct 60 aggcctccaa gggctcccca
aaatctgaat ttctgagtag tcttcatccc ctctcctgct 120 ctaaggtcag
gtccatcctc tctggtcctt accttgatga caaggatcga cattgattat 180
tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
240 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
gacccccgcc 300 cattgacgtc aataatgacg tatgttccca tagtaacgcc
aatagggact ttccattgac 360 gtcaatgggt ggagtattta cggtaaactg
cccacttggc agtacatcaa gtgtatcata 420 tgccaagtac gccccctatt
gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 480 agtacatgac
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 540
ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
600 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
caccaaaatc 660 aacgggactt tccaaaatgt cgtaacaact ccgccccatt
gacgcaaatg ggcggtaggc 720 gtgtacggtg ggaggtctat ataagcagag
ctcgtttagt gaaccgtcag atcgcctgga 780 gacgccatcc acgctgtttt
gacctccata gaagacaccg ggaccgatcc agcctccgcg 840 gccgggaacg
gtgcattgga acgcggattc cccgtgccaa gagtgacgta agtaccgcct 900
atagagtcta taggcccacc cccttggctt cttatggatc cggtggtggt gcaaatcaaa
960 gaactgctcc tcagtggatg ttgcctttac ttctaggcct gtacggaagt
gttacttctg 1020 ctctaaaagc tgctgcaggt cgacgaattc atcgatgata
tctcgagccc gcccgtcaca 1080 aagagcttca acaggggaga gtgttagagg
gagaagtgcc cccacctgct cctcagttcc 1140 agcctgaccc cctcccatcc
tttggcctct gacccttttt ccacagggga cctaccccta 1200 ttgcggtcct
ccagctcatc tttcacctca cccccctcct cctccttggc tttaattatg 1260
ctaatgttgg aggagaatga ataaataaag tgaatctttg cacctgtggt ttctctcttt
1320 cctcactaga ggatctctgt ctttcttact aaatggtagt aatcagttgt
ttttccagtt 1380 acctgggttt ctcttctaaa gaagttaaat gtttagttgc
cctgaaatcc accacactta 1440 aaggataaat aaaaccctcc acttgccctg
gttggctgtc cactacatgg cagtcctttc 1500 taaggttcac gagtactatt
catggcttat ttctctgggc catggtaggt ttgaggaggc 1560 atacttccta
gttttcttcc cctaagtcgt caaagtcctg aagggggaca gtctttacaa 1620
gcacatgttc tgtaatctga ttcaacctac ccagtaaact tggcgaagca gtagaatcat
1680 tatcacagga agcaaaggca acctaaatgt gcaagcaata ggaaaatgtg
gaagcccatc 1740 atagtacttg gacttcatct gcttttgtgc cttcactaag
tttttaaaca tgagctggct 1800 cctatctgcc attggcaagg ctgggcacta
cccacaacct acttcaagga cctctatacc 1860 gtgagattac acacatacat
caaaatttgg gaaaagttct accaagctga gagctgatca 1920 ccccactctt
aggtgcttat ctctgtacac cagaaacctt aagaagcaac cagtattgag 1980
agactcattt atgaaagtct aaaactggat acaaccaaaa tgtccaccaa cagttaaatt
2040 atgacatgtt cacaattgag ctattactta ataaggagaa ttaataaaat
aaaacttaag 2100 agcatagttt aatctcataa acaagataat aagcaaaaca
aaacattttt tcatccatgt 2160 aagtttaaaa gcaggtaaaa tttaaaatta
agagagacat aagttttgag gtagcaagat 2220 ggaaactctg gggcttgggg
aatgttctgt ctctctgtat gggatgtgaa agttactatt 2280 gtggaattgg
gatctatgtt cttcctgtat atattgtata cttcataata acttcaccta 2340
aagaaatatc taatacccag tgcatacata aaagaggata caaggaatga atcatacgtc
2400 aaggccagaa agacaataaa gtaggggatc cagacatgat aagatacatt
gatgagtttg 2460 gacaaaccac aactagaatg cagtgaaaaa aatgctttat
ttgtgaaatt tgtgatgcta 2520 ttgctttatt tgtaaccatt ataagctgca
ataaacaagt tctctagatg tgtaactctt 2580 ggctgaagct cttacaccaa
tgctggggga catgtacctc ccaggggccc aggaagacta 2640 cgggaggcta
caccaacgtc aatcagaggg gcctgtgtag ctaccgataa gcggaccctc 2700
aagagggcat tagcaatagt gtttataagg cccccttgtt aaccctaaac gggtagcata
2760 tgcttcccgg gtagtagtat atactatcca gactaaccct aattcaatag
catatgttac 2820 ccaacgggaa gcatatgcta tcgaattagg gttagtaaaa
gggtcctaag gaacagcgat 2880 atctcccacc ccatgagctg tcacggtttt
atttacatgg ggtcaggatt ccacgagggt 2940 agtgaaccat tttagtcaca
agggcagtgg ctgaagatca aggagcgggc agtgaactct 3000 cctgaatctt
cgcctgcttc ttcattctcc ttcgtttagc taatagaata actgctgagt 3060
tgtgaacagt aaggtgtatg tgaggtgctc gaaaacaagg tttcaggtga cgcccccaga
3120 ataaaatttg gacggggggt tcagtggtgg cattgtgcta tgacaccaat
ataaccctca 3180 caaacccctt gggcaataaa tactagtgta ggaatgaaac
attctgaata tctttaacaa 3240 tagaaatcca tggggtgggg acaagccgta
aagactggat gtccatctca cacgaattta 3300 tggctatggg caacacataa
tcctagtgca atatgatact ggggttatta agatgtgtcc 3360 caggcaggga
ccaagacagg tgaaccatgt tgttacactc tatttgtaac aaggggaaag 3420
agagtggacg ccgacagcag cggactccac tggttgtctc taacaccccc gaaaattaaa
3480 cggggctcca cgccaatggg gcccataaac aaagacaagt ggccactctt
ttttttgaaa 3540 ttgtggagtg ggggcacgcg tcagccccca cacgccgccc
tgcggttttg gactgtaaaa 3600 taagggtgta ataacttggc tgattgtaac
cccgctaacc actgcggtca aaccacttgc 3660 ccacaaaacc actaatggca
ccccggggaa tacctgcata agtaggtggg cgggccaaga 3720 taggggcgcg
attgctgcga tctggaggac aaattacaca cacttgcgcc tgagcgccaa 3780
gcacagggtt gttggtcctc atattcacga ggtcgctgag agcacggtgg gctaatgttg
3840 ccatgggtag catatactac ccaaatatct ggatagcata tgctatccta
atctatatct 3900 gggtagcata ggctatccta atctatatct gggtagcata
tgctatccta atctatatct 3960 gggtagtata tgctatccta atttatatct
gggtagcata ggctatccta atctatatct 4020 gggtagcata tgctatccta
atctatatct gggtagtata tgctatccta atctgtatcc 4080 gggtagcata
tgctatccta atagagatta gggtagtata tgctatccta atttatatct 4140
gggtagcata tactacccaa atatctggat agcatatgct atcctaatct atatctgggt
4200 agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
atatctgggt 4260 agcatatgct atcctaatct atatctgggt agtatatgct
atcctaattt atatctgggt 4320 agcataggct atcctaatct atatctgggt
agcatatgct atcctaatct atatctgggt 4380 agtatatgct atcctaatct
gtatccgggt agcatatgct atcctcatgc atatacagtc 4440 agcatatgat
acccagtagt agagtgggag tgctatcctt tgcatatgcc gccacctccc 4500
aagggggcgt gaattttcgc tgcttgtcct tttcctgctg gttggcatgc cggggagagg
4560 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
gctcggtcgt 4620 tcggctgcgg cgagcggtat cagctcactc aaaggcggta
atacggttat ccacagaatc 4680 aggggataac gcaggaaaga acatgtgagc
aaaaggccag caaaaggcca ggaaccgtaa 4740 aaaggccgcg ttgctggcgt
ttttccatag gctccgcccc cctgacgagc atcacaaaaa 4800 tcgacgctca
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4860
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
4920 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
ggtatctcag 4980 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
gaaccccccg ttcagcccga 5040 ccgctgcgcc ttatccggta actatcgtct
tgagtccaac ccggtaagac acgacttatc 5100 gccactggca gcagccactg
gtaacaggat tagcagagcg aggtatgtag gcggtgctac 5160 agagttcttg
aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 5220
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
5280 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
gcagaaaaaa 5340 aggatctcaa gaagatcctt tgatcttttc tacggggtct
gacgctcagt ggaacgaaaa 5400 ctcacgttaa gggattttgg tcatgagatt
atcaaaaagg atcttcacct agatcctttt 5460 aaattaaaaa tgaagtttta
aatcaatcta aagtatatat gagtaaactt ggtctgacag 5520 ttaccaatgc
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 5580
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
5640 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
cagcaataaa 5700 ccagccagcc ggaagggccg agcgcagaag tggtcctgca
actttatccg cctccatcca 5760 gtctattaat tgttgccggg aagctagagt
aagtagttcg ccagttaata gtttgcgcaa 5820 cgttgttgcc attgctacag
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 5880 cagctccggt
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 5940
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
6000 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
gatgcttttc 6060 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
tgtatgcggc gaccgagttg 6120 ctcttgcccg gcgtcaatac gggataatac
cgcgccacat agcagaactt taaaagtgct 6180 catcattgga aaacgttctt
cggggcgaaa actctcaagg atcttaccgc tgttgagatc 6240 cagttcgatg
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 6300
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
6360 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
tttatcaggg 6420 ttattgtctc atgagcggat acatatttga atgtatttag
aaaaataaac aaataggggt 6480 tccgcgcaca tttccccgaa aagtgccacc
tgacgtctaa gaaaccgcgg ccgcaacaga 6540 cgtctaagaa accattatta
tcatgacatt aacctataaa aataggcgta tcacgaggcc 6600 ctttcgtctc
gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 6660
gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
6720 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
agattgtact 6780 gagagtgcac catatgcggt gtgaaatacc gcacagatgc
gtaaggagaa aataccgcat 6840 caggcgccat tcgccattca ggctgcgcaa
ctgttgggaa gggcgatcgg tgcgggcctc 6900 ttcgctatta cgccagctgg
cgaaaggggg atgtgctgca aggcgattaa gttgggtaac 6960 gccagggttt
tcccagtcac gacgttgtaa aacgacggcc agtgcc 7006 2 57 DNA Homo Sapiens
misc_feature Signal Sequence 2 atg gga tgg agc tgt atc atc ctc ttc
ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtc cac tcc 57 Val His Ser 3
19 PRT Homo Sapiens 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser 4 993 DNA Homo Sapiens
misc_feature Heavy Chain Gamma 1 4 gcc agc aca aag ggc cca tcg gtc
ttc ccc ctg gca ccc tcc tcc aag 48 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 agc acc tct ggg ggc aca
gcg gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg
gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg
cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc 240
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg
gac aag 288 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 aga gtt gag ccc aaa tct tgt gac aaa act cac aca
tgt cca ccg tgc 336 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 cca gca cct gaa ctc ctg ggg gga ccg tca
gtc ttc ctc ttc ccc cca 384 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 aaa ccc aag gac acc ctc atg atc
tcc cgg acc cct gag gtc aca tgc 432 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 gtg gtg gtg gac gtg agc
cac gaa gac cct gag gtc aag ttc aac tgg 480 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 tac gtg gac
ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 528 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 gag
cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 576 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac
624 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc
aaa ggg 672 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 cag ccc cga gaa cca cag gtg tac acc ctg ccc cca
tcc cgg gag gag 720 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 atg acc aag aac cag gtc agc ctg acc
tgc ctg gtc aaa ggc ttc tat 768 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 ccc agc gac atc gcc gtg gag
tgg gag agc aat ggg cag ccg gag aac 816 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 aac tac aag acc acg
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 864 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 ctc tat agc
aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 912 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 gtc
ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg 960 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 cag aag agc ctc tcc ctg tcc ccg ggt aaa tga 993 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 5 330 PRT Homo Sapiens 5 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 6 981 DNA Homo Sapiens misc_feature Heavy Chain Gamma 2
6 gcc agc aca aag ggc cca tcg gtc ttc ccc ctg gcg ccc tgc tcc agg
48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15 agc acc tcc gag agc aca gcg gcc ctg ggc tgc ctg gtc aag
gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc
gct ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag
tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg acc
tcc agc aac ttc ggc acc cag acc 240 Leu Ser Ser Val Val Thr Val Thr
Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 tac acc tgc aac gta gat
cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Thr Cys Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aca gtt gag cgc
aaa tgt tgt gtc gag tgc cca ccg tgc cca gca cca 336 Thr Val Glu Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 cct gtg
gca gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 384 Pro Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
acc ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg gac 432
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130
135 140 gtg agc cac gaa gac ccc gag gtc cag ttc aac tgg tac gtg gac
ggc 480 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly 145 150 155 160 atg gag gtg cat aat gcc aag aca aag cca cgg gag
gag cag ttc aac 528 Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn 165 170 175 agc acg ttc cgt gtg gtc agc gtc ctc acc
gtc gtg cac cag gac tgg 576 Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp 180 185 190 ctg aac ggc aag gag tac aag tgc
aag gtc tcc aac aaa ggc ctc cca 624 Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 gcc ccc atc gag aaa acc
atc tcc aaa acc aaa ggg cag ccc cga gaa 672 Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys
Gly Gln Pro Arg Glu 210 215 220 cca cag gtg tac acc ctg ccc cca tcc
cgg gag gag atg acc aag aac 720 Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn 225 230 235 240 cag gtc agc ctg acc tgc
ctg gtc aaa ggc ttc tac ccc agc gac atc 768 Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 gcc gtg gag tgg
gag agc aat ggg cag ccg gag aac aac tac aag acc 816 Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 aca cct
ccc atg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 864 Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285
ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 912
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290
295 300 tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc
ctc 960 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 305 310 315 320 tcc ctg tct ccg ggt aaa tga 981 Ser Leu Ser Pro
Gly Lys 325 7 326 PRT Homo Sapiens 7 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Met Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185
190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310
315 320 Ser Leu Ser Pro Gly Lys 325 8 984 DNA Homo Sapiens
misc_feature Heavy Chain Gamma 4 8 gcc agc aca aag ggc cca tcc gtc
ttc ccc ctg gcg ccc tgc tcc agg 48 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 agc acc tcc gag agc aca
gcc gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg
gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg
cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65
70 75 80 tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg
gac aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 aga gtt gag tcc aaa tat ggt ccc cca tgc cca tca
tgc cca gca cct 336 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 gag ttc ctg ggg gga cca tca gtc ttc ctg
ttc ccc cca aaa ccc aag 384 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 gac act ctc atg atc tcc cgg acc
cct gag gtc acg tgc gtg gtg gtg 432 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 gac gtg agc cag gaa gac
ccc gag gtc cag ttc aac tgg tac gtg gat 480 Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 ggc gtg gag
gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 528 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 aac
agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 576 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc
624 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205 ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag
ccc cga 672 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 gag cca cag gtg tac acc ctg ccc cca tcc cag gag
gag atg acc aag 720 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys 225 230 235 240 aac cag gtc agc ctg acc tgc ctg gtc
aaa ggc ttc tac ccc agc gac 768 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 atc gcc gtg gag tgg gag agc
aat ggg cag ccg gag aac aac tac aag 816 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 acc acg cct ccc gtg
ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 864 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 agg cta acc
gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 912 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 tgc
tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 960 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 ctc tcc ctg tct ctg ggt aaa tga 984 Leu Ser Leu Ser Leu Gly
Lys 325 9 327 PRT Homo Sapiens 9 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Leu Gly Lys 325 10 318 DNA Homo Sapiens
misc_feature Light Chain Kappa 10 gtg gct gca cca tct gtc ttc atc
ttc ccg cca tct gat gag cag ttg 48 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 aaa tct gga act gcc tct
gtt gtg tgc ctg ctg aat aac ttc tat ccc 96 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30 aga gag gcc aaa
gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt 144 Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35 40 45 aac tcc
cag gag agt gtc aca gag cag gac agc aag gac agc acc tac 192 Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60
agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac 240
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65
70 75 80 aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg
ccc gtc 288 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 85 90 95 aca aag agc ttc aac agg gga gag tgt tag 318 Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 105 11 105 PRT Homo Sapiens 11
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5
10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 12 312 DNA Homo Sapiens misc_feature Light
Chain Lambda 12 aag gcc aac ccc act gtc act ctg ttc ccg ccc tcc tct
gag gag ctc 48 Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu Leu 1 5 10 15 caa gcc aac aag gcc aca cta gtg tgt ctg atc
agt gac ttc tac ccg 96 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe Tyr Pro 20 25 30 gga gct gtg aca gtg gcc tgg aag gca
gat ggc agc ccc gtc aag gcg 144 Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Gly Ser Pro Val Lys Ala 35 40 45 gga gtg gag acc acc aaa ccc
tcc aaa cag agc aac aac aag tac gcg 192 Gly Val Glu Thr Thr Lys Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala 50 55 60 gcc agc agc tac ctg
agc ctg acg ccc gag cag tgg aag tcc cac aga 240 Ala Ser Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80 agc tac agc
tgc cag gtc acg cat gaa ggg agc acc gtg gag aag aca 288 Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 85 90 95 gtg
gcc cct aca gaa tgt tca tag 312 Val Ala Pro Thr Glu Cys Ser 100 13
103 PRT Homo Sapiens 13 Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro
Ser Ser Glu Glu Leu 1 5 10 15 Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe Tyr Pro 20 25 30 Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Gly Ser Pro Val Lys Ala 35 40 45 Gly Val Glu Thr Thr
Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 50 55 60 Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80 Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 85 90
95 Val Ala Pro Thr Glu Cys Ser 100 14 1410 DNA HomoSapiens
misc_feature LDP-01 Heavy Chain 14 atg gga tgg agc tgt atc atc ctc
ttc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtc cac tcc cag gtc caa
ctg cag gag agc ggt cca ggt ctt gtg aga 96 Val His Ser Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Arg 20 25 30 cct agc cag acc
ctg agc ctg acc tgc acc gtg tct ggc ttc acc ttc 144 Pro Ser Gln Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe 35 40 45 acc gat
tac ctt ctg cac tgg gtg aga cag cca cct gga cga ggt ctt 192 Thr Asp
Tyr Leu Leu His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu 50 55 60
gag tgg att gga tgg att gat cct gag gat ggt gaa aca aag tat ggt 240
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Gly 65
70 75 80 cag aag ttt caa agc aga gtg aca atg ctg gta gac acc agc
aag aac 288 Gln Lys Phe Gln Ser Arg Val Thr Met Leu Val Asp Thr Ser
Lys Asn 85 90 95 cag ttc agc ctg aga ctc agc agc gtg aca gcc gcc
gac acc gcg gtc 336 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val 100 105 110 tat tat tgt gca aga ggc gaa tat aga tac
aac tcg tgg ttt gat tac 384 Tyr Tyr Cys Ala Arg Gly Glu Tyr Arg Tyr
Asn Ser Trp Phe Asp Tyr 115 120 125 tgg ggt caa ggc tca cta gtc aca
gtc tcc tca gcc tcc acc aag ggc 432 Trp Gly Gln Gly Ser Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg gtc ttc ccc ctg
gca ccc tcc tcc aag agc acc tct ggg ggc 480 Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 aca gcg gcc
ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 528 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 acg
gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576 Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190 ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg
624 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205 acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc
aac gtg 672 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220 aat cac aag ccc agc aac acc aag gtg gac aag aaa
gtt gag ccc aaa 720 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 225 230 235 240 tct tgt gac aaa act cac aca tgc cca
ccg tgc cca gca cct gaa ctc 768 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255 gcg ggg gca ccg tca gtc ttc
ctc ttc ccc cca aaa ccc aag cac acc 816 Ala Gly Ala Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys His Thr 260 265 270 ctc atg atc tcc cgg
acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864 Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 agc cac gaa
gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 912 Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300
gag
gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 960 Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310
315 320 acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg
ctg 1008 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 325 330 335 aat ggc aag gag tac aag tgc aag gtc tcc aac aaa
gcc ctc cca gcc 1056 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala 340 345 350 ccc atc gag aaa acc atc tcc aaa gcc
aaa ggg cag ccc cga gaa cca 1104 Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 cag gtg tac acc ctg ccc
cca tcc cgg gat gag ctg acc aag aac cag 1152 Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 gtc agc ctg
acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1200 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395
400 gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg
1248 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 405 410 415 cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac
agc aag ctc 1296 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu 420 425 430 acc gtg gac aag agc agg tgg cag cag ggg
aac gtc ttc tca tgc tcc 1344 Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 435 440 445 gtg atg cat gag gct ctg cac
aac cac tac acg cag aag agc ctc tcc 1392 Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 ctg tct ccg ggt
aaa tga 1410 Leu Ser Pro Gly Lys 465 15 469 PRT HomoSapiens 15 Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10
15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe
Thr Phe 35 40 45 Thr Asp Tyr Leu Leu His Trp Val Arg Gln Pro Pro
Gly Arg Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asp
Gly Glu Thr Lys Tyr Gly 65 70 75 80 Gln Lys Phe Gln Ser Arg Val Thr
Met Leu Val Asp Thr Ser Lys Asn 85 90 95 Gln Phe Ser Leu Arg Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala
Arg Gly Glu Tyr Arg Tyr Asn Ser Trp Phe Asp Tyr 115 120 125 Trp Gly
Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145
150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Ala
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys His Thr 260 265
270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390
395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Lys 465 16
702 DNA HomoSapiens misc_feature LDP-01 Light Chain 16 atg gga tgg
agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48 Met Gly Trp
Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtc
cac tcc gac atc cag atg acc cag agc cca agc agc ctg agc gcc 96 Val
His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25
30 agc gtg ggt gac aga gtg acc atc acc tgt aaa gca agt aag agc att
144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile
35 40 45 agc aat tat tta gcc tgg tac cag cag aag cca ggt aag gct
cca aag 192 Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys 50 55 60 ctg ctg atc tac tat ggg tca act ttg cga tct ggt
gtg cca agc aga 240 Leu Leu Ile Tyr Tyr Gly Ser Thr Leu Arg Ser Gly
Val Pro Ser Arg 65 70 75 80 ttc agc ggt agc ggt agc ggt acc gac ttc
acc ttc acc atc agc agc 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser 85 90 95 ctc cag cca gag gac atc gcc acc
tac tac tgc caa cag tat tat gaa 336 Leu Gln Pro Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Glu 100 105 110 aga ccg ctc acg ttc ggc
caa ggg acc aag gtg gaa atc aaa cga act 384 Arg Pro Leu Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 gtg gct gca cca
tct gtc ttc atc ttc ccg cca tct gat gag cag ttg 432 Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 aaa tct
gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc 480 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155
160 aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt
528 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
165 170 175 aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc
acc tac 576 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr 180 185 190 agc ctc agc agc acc ctg acg ctg agc aaa gca gac
tac gag aaa cac 624 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 195 200 205 aaa gtc tac gcc tgc gaa gtc acc cat cag
ggc ctg agc tcg ccc gtc 672 Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val 210 215 220 aca aag agc ttc aac agg gga gag
tgt tag 702 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 17 233 PRT
HomoSapiens 17 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr
Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Lys Ser Ile 35 40 45 Ser Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60 Leu Leu Ile Tyr Tyr
Gly Ser Thr Leu Arg Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95 Leu
Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Glu 100 105
110 Arg Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr
Lys Ser Phe Asn Arg Gly Glu Cys 225 230
* * * * *