U.S. patent application number 11/819917 was filed with the patent office on 2008-08-28 for method for producing monoclonal antibodies.
This patent application is currently assigned to Wyeth. Invention is credited to Randal J. Kaufman, Clive R. Wood.
Application Number | 20080206754 11/819917 |
Document ID | / |
Family ID | 46323542 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080206754 |
Kind Code |
A1 |
Wood; Clive R. ; et
al. |
August 28, 2008 |
Method for producing monoclonal antibodies
Abstract
An improved method for the production of monoclonal antibodies
is disclosed.
Inventors: |
Wood; Clive R.; (Boston,
MA) ; Kaufman; Randal J.; (Boston, MA) |
Correspondence
Address: |
WYETH/FINNEGAN HENDERSON, LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Wyeth
|
Family ID: |
46323542 |
Appl. No.: |
11/819917 |
Filed: |
June 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11324255 |
Jan 4, 2006 |
7247475 |
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11819917 |
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10244894 |
Sep 17, 2002 |
7011974 |
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11324255 |
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07765281 |
Sep 25, 1991 |
6475787 |
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10244894 |
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07386489 |
Jul 28, 1989 |
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07765281 |
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Current U.S.
Class: |
435/6.16 ;
435/91.2 |
Current CPC
Class: |
C07K 16/00 20130101;
C07K 16/44 20130101; C12N 2510/02 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Goverment Interests
[0002] This invention was made with government support under grant
number R44GM37329-01-02-0379428 awarded by the Department of Health
and Human Services. The U.S. government has certain rights in this
invention.
Claims
1-6. (canceled)
7. A method of optimizing the expression level of an antibody or a
fragment thereof, which comprises: (a) producing a eukaryotic host
cell containing and capable of expressing a first DNA sequence
encoding at least an antibody heavy chain or an antigen binding
portion thereof, said first DNA sequence being associated with a
first selectable amplifiable marker gene, and a second DNA sequence
encoding at least an antibody light chain or an antibody binding
portion thereof, said second DNA sequence being associated with
said first selectable amplifiable marker gene; (b) culturing said
host cell in a suitable culture medium; (c) measuring the relative
amounts of said first and second DNA sequences expressed; (d)
introducing into said host cell a third DNA sequence encoding at
least an antibody chain or antigen binding portion thereof that is
limiting, said third DNA sequence being associated with a second
selectable amplifiable marker gene; and (e) differentially
amplifying said third DNA sequence to allow production of the
desired amount of antibody.
8. A method of optimizing the expression level of an antibody or a
fragment thereof, which comprises: (a) producing a eukaryotic host
cell containing and capable of expressing a first DNA sequence
encoding at least an antibody heavy chain or an antigen binding
portion thereof, said first DNA sequence being associated with a
first selectable amplifiable marker gene, and a second DNA sequence
encoding at least an antibody light chain or an antibody binding
portion thereof, said second DNA sequence being associated with
said first selectable amplifiable marker gene; (b) culturing said
host cell in a suitable culture medium; (c) introducing into said
host cell a third DNA sequence selected from: (i) a DNA sequence
encoding at least an antibody heavy chain or antigen binding
portion thereof; and (ii) a DNA sequence encoding at least an
antibody light chain or antigen binding portion thereof, said third
DNA sequence being associated with a second selectable amplifiable
marker gene; and (d) differentially amplifying said third DNA
sequence to allow production of the desired amount of antibody.
9. A method of optimizing the expression level of an antibody or a
fragment thereof, which comprises: (a) producing a eukaryotic host
cell containing and capable of expressing a first DNA sequence
encoding at least an antibody heavy chain or an antigen binding
portion thereof, said first DNA sequence being associated with a
first selectable amplifiable marker gene, and a second DNA sequence
encoding at least an antibody light chain or an antibody binding
portion thereof, said second DNA sequence being associated with
said a second selectable amplifiable marker gene; (b) culturing
said host cell in a suitable culture medium; (c) measuring the
relative amounts of said first and second DNA sequences expressed;
(d) introducing into said host cell a third DNA sequence encoding
at least an antibody chain or antigen binding fragment thereof that
is in limiting amount, said third DNA sequence being associated
with a third selectable amplifiable marker gene; and (e)
differentially amplifying said third DNA sequence to allow
production of the desired amount of antibody.
10. The method of claim 7, wherein said third DNA sequence encodes
an antibody heavy chain or antigen binding portion thereof.
11. The method of claim 7, wherein said third DNA sequence encodes
an antibody light chain or antigen binding portion thereof.
12. The method of claim 7, wherein the antibody expression level is
about 60 .mu.g/106 cells/48 hrs.
13. The method of claim 7, wherein the first selectable amplifiable
marker gene is an ADA gene or a DHFR gene.
14. The method of claim 7, wherein the second selectable
amplifiable marker gene is an ADA gene or a DHFR gene.
15. The method of claim 9, wherein the third selectable amplifiable
marker gene is an ADA gene or a DHFR gene.
16. The method of claim 7, wherein the first selectable amplifiable
marker gene is a DHFR gene and the second selectable amplifiable
marker gene is an ADA gene.
17. The method of claim 7, wherein the first selectable amplifiable
marker gene is an ADA gene and the second selectable amplifiable
marker gene is a DHFR gene.
18. The method of claim 7, wherein the antibody or fragment thereof
is a monoclonal antibody.
19. The method of claim 7, wherein the antibody or fragment thereof
is a genetically engineered antibody.
20. The method of claim 19, wherein the genetically engineered
antibody or fragment thereof is a chimeric, humanized or a
CDR-swapped antibody.
21. The method of claim 7, wherein the antibody fragment is
selected from the group consisting of Fv, Fab, and F(ab)'2.
22. The method of claim 9, wherein the antibody or fragment thereof
is a chimeric protein.
23. The method of claim 22, wherein the chimeric protein comprises
a Fab linked to a non-antibody sequence.
24. The method of claim 22, wherein the chimeric protein is a
Fab-enzyme or a Fab-toxin.
25. The method of claim 9, wherein the host cell is a mammalian
cell.
26. The method of claim 9, wherein the host cell is a non-lymphoid
cell.
27. The method of claim 26, wherein the non-lymphoid cell is
selected from the group consisting of Chinese Hamster Ovary (CHO)
cell, HeLa cells, human 293 cell, COS monkey cell, Bowes cell,
mouse L-929 cell, 3T3 cell line, BHK hamster cell, and HaK hamster
cell.
28. The method of claim 26, wherein the non-lymphoid cell is a CHO
cell.
29. The method of claim 9, wherein the host cell is a
lymphocyte-derived cell line.
30. The method of claim 29, wherein the lymphocyte-derived cell
line is a murine hybridoma SP2/0-Ag14 or a murine myeloma cell.
31. The method of claim 9, wherein the first DNA sequence is a cDNA
or genomic DNA.
32. The method of claim 9, wherein the second DNA sequence is a
cDNA or genomic DNA.
33. The method of claim 9, wherein the third DNA sequence is a cDNA
or a genomic DNA.
34. The method of claim 9, wherein the first, second, and third DNA
sequences are stably integrated into the eukaryotic host cell
chromosomal DNA.
35. The method of claim 9, wherein the first and second selectable
amplifiable marker gene sequences are stably integrated into the
eukaryotic host cell chromosomal DNA.
36. The method of claim 9, wherein the first and second DNA
sequences are contained in separate vectors.
37. The method of claim 9, wherein the first and second selectable
amplifiable markers are in separate vectors from the first and
second DNA sequences.
38. The method of claim 9, wherein the first selectable amplifiable
marker is in the same vector as the first DNA sequence.
39. The method of claim 9, wherein the second selectable
amplifiable marker is in the same vector as the second DNA
sequence.
40. The method of claim 36, wherein the vectors containing the
first and second DNA sequences are cotransformed into the
eukaryotic host cell.
41. The method of claim 9, wherein said third DNA sequence is
introduced into said host cell by fusing said host cell with a host
cell comprising said third DNA sequence.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
11/324,255, filed Jan. 4, 2006, which is a continuation of U.S.
patent application Ser. No. 10/244,894, filed Sep. 17, 2002, now
U.S. Pat. No. 7,011,974, which is a continuation of U.S. patent
application Ser. No. 07/765,281, filed Sep. 25, 1991, now U.S. Pat.
No. 6,475,787, which is a continuation-in-part of U.S. application
Ser. No. 07/386,489, filed on Jul. 28, 1989 (now abandoned). These
applications are hereby incorporated by reference in their
entirety.
[0003] The production of monoclonal antibodies using hybridoma
cells is now well known in the art. Briefly, isolated
antibody-producing lymphocytes from an immunized animal, typically
a mouse, are fused with an immortalized cell line, and the
resultant hybridomas are screened for the production of the desired
monoclonal antibody. Such methods have been successfully used to
produce a wide array of antibodies.
[0004] However, several inherent shortcomings limit the utility of
such methods and the resultant monoclonal antibodies (MAbs).
Foremost of those limitations is that the Mabs so produced are
essentially murine in nature and reactivity. Use of murine MAbs in
human patients, whether for diagnostic or perhaps especially for
therapeutic or prophylactic use, incurs a risk of untoward
antigenic response by the patient.
[0005] In order to avoid such antigenicity, genetically engineered
antibodies have been produced which retain the specific
antigen-binding domains of the parent murine antibody, while
substituting corresponding human antibody domains for part or all
of the remaining murine polypeptide regions. It is hoped that such
antibodies will not prove antigenic in humans because of their
greater resemblance to human antibodies.
[0006] Briefly, chimeric antibodies may be produced by isolating
the MAb-encoding DNA sequences from a desired hybridoma, excising
the portion of the murine DNA which is not required to encode the
antigen-binding domains, and replacing such DNA sequences with
corresponding human DNA sequences. This has been done in two
alternative ways. Firstly, the complete murine variable or V region
DNA of each chain can be appropriately joined to human constant or
C region DNA sequences. The resultant DNAs encode polypeptides with
a murine V and human C domains. Examples are provided by Morrison
et al, 1984, Proc. Natl. Acad, Sci. USA 81:6851 and Liu et al,
1987, J. immunol. 139:3521. The antibody V regions are known to
encode the antigen-binding portions of the antibody, and the C
regions encode the biological effector functions, such as
complement fixation. In the second approach, the portions of the
murine V regions thought to encode the `antigen-binding`
specificity , or complementarily-determining regions (CDRs) are
identified, and the same CDRs are used to replace the human CDRs of
human V regions linked to human c regions. These are `CDR-swap`
antibodies, and examples are provided by Jones et al, 1986, Nature
321:522: Verhoeyen et al, 1988, Nature 332:323; and Reichmann et
al, 1988, Nature 332:323. The resultant DNAs obtained by either
approach thus encode "humanized" heavy and light chains.
[0007] While such genetically engineered antibodies may overcome
limitations on the use of murine MAbs, expression of the chimeric
DNAs encoding such MAbs or even of cloned murine MAb genes is still
problematic. In one approach the DNAs are introduced into murine
hybridoma or myeloma cells for heterologous expression. However,
such methods have net with only limited success, in large part
because of the disappointingly low expression levels achieved thus
far. Thus, a continuing need exists for a method for heterologous
expression of antibody-encoding DNAs. One object of this invention
is to provide an improved heterologous expression system for such
DNAs which affords high levels of expression of antibodies,
preferably chimeric antibodies.
[0008] Heterologous gene expression is typically accomplished by
introducing the desired gene (or DNA encoding the desired protein)
into a host cell in association with an amplifiable marker such as
a gene encoding dihydrofolate reductase (DHFR) .The transfected or
transformed host cells are then iteratively subjected to increasing
selective pressure such that the number of copies of the marker
gene and the associated desired gene are increased. Where the
marker is a DHFR gene, the selective agent is methotrexate (MTX),
as is well known in the art. However, where heavy and light chain
antibody genes are so introduced into a host cell, no practical
method exists to ensure that both genes are appropriately
amplified. It should be noted that if expression of one chain
predominates, then the expression level of the other chain can
limit the amount of antibody actually produced. Additionally, heavy
chain expression, in the absence of light chain expression may be
deleterious to the producing cells. Heavy chain toxicity is
discussed in Kohler, G, 1980, Proc. Natl. Acad. Sci. USA 77:2197
and Haas and Wabl, 1984, ibid. 81:7185.
[0009] We have found that high expression levels for antibodies
depends in part on differentially amplifying the heavy and light
chain DNAs to optimize the relative gene copy numbers of the heavy
and light chain DNAs. In the practice of this invention, such
optimization of relative gene copy number and thus the relative
expression levels may be conveniently achieved by introducing the
heavy chain and light chain DNAs respectively associated with
different amplifiable markers, presumably into different
chromosomal locations when the introduced DNA is chromosomally
integrated. The heavy chain DNA and the light chain DNA are then
separately amplified by application of selective conditions for the
respective markers until appropriate optimization of gene
expression is achieved.
[0010] By way of example; the heavy chain-encoding DNA may be
linked to an adenosine deaminase (ADA) gene and the light
chain-encoding DNA linked to a DHFR gene. Each of the antibody
genes with its respective marker gene is then introduced into the
host cells, preferably Chinese Hamster Ovary (CHO) cells by
conventional methods. For example, each set of DNA may be
introduced into separate CHO cells, e.g. by electroporation, and
the resultant transformants fused. The ADA.sup.+, DHFR.sup.+ CHO
cells so obtained contain the heavy chain DNA associated with an
ADA gene and the light chain DNA associated with a DHFR gene, each
of which DNAs is then specifically amplified by treatment with
iteratively increasing amounts of MTX (amplifies light chain DNA,
but not heavy chain DNA) and 2'-deoxycoformycin (dCF, amplifies
heavy chain DNA but not light chain DNA). During the course of
amplification the host cells are analyzed for antibody production
(by ELISA). Cells so amplified for optimized antibody production
were found to produce MAbs which retained the specific hapten
binding characteristics of the parental MAb and which bind
complement. Expression levels of about 60 .mu.g/l 10.sup.6 cells/48
hrs have been obtained, which may be ever further improved by
additional rounds of amplification. So far as we are aware,
efficient production of antibodies in non-lymphoid cells has never
been demonstrated heretofore.
[0011] It should be noted that the DNAs encoding the respective
chains may be cDNA or genomic DNA. It should also be noted that
this invention should be useful for the production not just of
cloned antibodies, but also of genetically engineered antibodies
such as CDR-swapped antibodies as previously mentioned, and in
addition, genetically engineered antibody fragments or derivatives
such as Fv, Fab, F(ab)'.sub.2 fragments using truncated DNAs and
chimeric proteins such as Fab-enzyme and Fab-toxin fusion proteins.
Thus, this approach will also be of general value in the production
of hetero-dimeric molecules, other than complete antibodies.
Examples include other forms of genetically-engineered antibodies,
such as Fab and F(ab).sub.2' forms, and antigen-binding portions,
such as a Fab, linked to non-antibody peptide sequences. Examples
of the genetic engineering of such molecules are found in Newberger
et al, 1984, Nature 312:604; Skerra and Pluckthun, 1988, Science
240:1038; Better et al, 1988, Science 240:1041 and Reichmann et al,
1988, J. Mol. Biol. 203:825.
DETAILED DESCRIPTION OF THE INVENTION
[0012] I. Production of Hybridoma Cells
[0013] Hybridoma cell lines producing a desired antibody may be
produced by conventional methods such as the well known methods of
Kohler and Milstein. Briefly, an animal, preferably a rodent such
as a Balb/C mouse is immunized and later re-immunized (boosted)
with the desired immunogen, with an adjuvant as desired, as is well
known in the art. Assaying the serum of the animal by conventional
methods such as a specific ELISA reveals whether the animal is
producing an antibody of the desired affinity and avidity. An
immunized animal having an appropriate titer of the desired
antibody is sacrificed and its spleen removed. The spleen cells are
then carefully separated and fused with a suitable myeloma cell
line by conventional procedures or otherwise immortalized, as is
also well known in the art. The immortalized cells producing the
desired antibody are then identified by routine, conventional
screening and are then subcloned as desired.
[0014] II. Cloning heavy and light chain-encoding DNAs
[0015] Methods for cloning immunoglobulin heavy and light chains is
well known in the art. See e.g. Beidler et al, 1988, J. Immunol.
141:4053 (genomic) and Liu et al, 1987, Proc. Natl. Acad. Sci. USA
84:3439 (CDNA). Briefly, CDNA or genomic libraries are constructed
for the RNA or genomic DNA, respectively, from hybridomas producing
a specific antibody of interest, as is known in the art. The
immunoglobulin clones from such libraries can be identified by
hybridization to DNA or oligonucleotide probes specific for J.sub.H
or C.sub.H sequences for the heavy chain clones, or J.sub.L or
C.sub.L sequences for the light chain clones. The positive clones
are then further characterized by conventional restriction
endonuclease site mapping and nucleotide sequencing.
[0016] III. Expression Vector Construction
[0017] Any conventional eukaryotic, preferably mammalian,
expression vectors designed for high expression levels, of which
many are known in the art, may be used in the practice of this
invention. However, in the practice of this invention the
expression vector for the light chain antibody DNA contains or is
cotransfected with a first selectable, amplifiable marker gene
while the expression vector for the heavy chain antibody DNA
contains or is cotransfected with a second selectable, amplifiable
marker. The two selectable, amplifiable markers must be
differentially amplifiable, i.e. must each be susceptible to
amplification under conditions which do not result in amplification
of the other.
[0018] The eukaryotic cell expression vectors described herein may
be synthesized by techniques well known to those skilled in this
art. The components of the vectors such as the bacterial replicons,
selection genes, enhancers, promoters, and the like may be obtained
from natural sources or synthesized by known procedures. See
Kaufman et al., J. Mol. Biol., 159:601-621 (1982); Kaufman, Proc
Natl. Acad. Sci. 82:689-693 (1985). Eukaryotic expression vectors
useful in practicing this invention may also contain inducible
promoters or comprise inducible expression systems as are known in
the art.
[0019] pMT2 and PMT3SVA are exemplary expression vectors which are
described below. Both vectors contain an SV40 origin of replication
and enhancer, adenovirus major late promoter and tripartite leader
sequence, a cloning site followed by an SV40 polyadenylation site,
the adenovirus VA I gene, E coli origin of replication and an
ampicillin resistance gene for bacterial selection. PMT2 further
contains a DHFR gene between the cloning site and the
polyadenylation signal, while pMT3SVA contains an adenosine
deaminase (ADA) gene under the expression control of the SV40 early
promoter. While both of these vectors contain appropriate
selectable, amplifiable markers, it should be understood that
separate vectors containing the markers may be cotransfected or
cotransformed by conventional means with the respective heavy and
light chain DNAs.
[0020] IV. Production of transformed cell lines
[0021] Established cell lines, including transformed cell lines,
are suitable as hosts. Normal diploid cells, cell strains derived
from in vitro culture of primary tissue, as well as primary
explants (including relatively undifferentiated cells such as
hematopoietic stem cells) are also suitable. Candidate cells need
not be genotypically deficient in the selection gene so long as the
selection gene is dominantly acting.
[0022] The host cells preferably will be established mammalian cell
lines. For stable integration of the vector DNA into chromosomal
DNA, and for subsequent amplification of the integrated vector DNA,
both by conventional methods, CHO (Chinese Hamster Ovary) cells are
currently preferred. Other usable mammalian cell lines include
HeLa, human 293 cells, COS-1 monkey cells, melanoma cell lines such
as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss,
Balb/c or NIH Mice, BHK or HaK hamster cell lines and the like, as
well as lymphocyte derived cell lines such as the murine hybridoma
SP2/0-Ag14 or murine myeloma cells such as P3.653 and J558L or
Abelson murine leukemia virus transformed pre-B lymphocytes.
[0023] The expression vectors may be introduced into the host cells
by purely conventional methods, of which several are known in the
art. Electroporation has been found to be particularly useful.
[0024] Stable transformants may then be screened for the presence
and relative amount of incorporated antibody DNA and corresponding
mRNA and polypeptide synthesis by standard methods. For example,
the presence of the DNA encoding the desired antibody chain may be
detected by standard procedures such as Southern blotting, the
corresponding nRNA by Northern blotting and the protein thereby
encoded by Western blotting.
[0025] It should be appreciated that the two antibody genes may be
introduced serially into the same host cells, or may be introduced
in parallel into separate host cells. In the former case, the
antibody genes would be transfected separately, and the
transfectants after the first of the two transfections, may or may
not be selected in iteratively increasing amounts of the
appropriate selective agent, prior to the second transfection. In
the latter case, the two transfectants may be fused by conventional
means to produce a cell containing and capable of expressing both
antibody chains, as well as both selectable markers to facilitate
isolation of hybrid cells, as exemplified in the Examples which
follow one of the parental cells of a fusion may be exposed to
ionizing radiation before the fusion event. In addition, both heavy
and light chain DNAs may be co-transfected with a single
selectable, amplifiable marker, and the transfectants then passaged
in iteratively increasing amounts of the selective agent. Once the
relative levels of the heavy and light chains expressed in such a
transfectant has been determined, a DNA encoding the chain found in
limiting amounts can then be transfected into the cell, linked to a
different selectable, amplifiable marker. The expression level for
that chain can then be increased by iterative amplification as
previously described.
[0026] V. Specific Amplification
[0027] Specific and independent amplification of the two DNAs may
be readily accomplished using conventional amplification procedures
appropriate for each of the respective markers. See e.g. published
international Application WO 88/08035 for an exemplary description
of independently amplifying a first gene linked to a DHFR gene and
a second gene linked to an ADA gene. Other selectable, amplifiable
markers can also be used, and examples are reviewed in Kaufman, R.
J., Genetic Engineering, 9:155, J. K. Setlow, ed. (Plenum
Publishing Corp.) 1987.
[0028] VI. Characterization of MAbs
[0029] The MAbs so produced by the amplified cell lines can be
characterized by standard immunochemical techniques, including
SDS-PAGE, western blotting and immunoprecipitation of intrinsically
.sup.35S-methionine-labeled proteins. The levels of heavy and light
chains produced can be quantitated by ELISAs, and binding to
solid-phase antigens can be demonstrated by ELISA. The binding
characteristics of the antibodies can also be studied in similar
antigen-binding ELISAs in the presence of varying concentrations of
free antigen. The effector functions of the antibodies can be
characterized by standard techniques, e.g. for complement fixation
and antibody- dependent cellular cytotoxicity.
EXAMPLES
Example 1
B1-8 hybridoma, its .alpha.NP MAb and DNAs encoding the heavy
(.mu.) and light (.lamda.) chains of the .alpha.NP MAb
[0030] The B1-8 hybridoma cell line is a fusion of a mouse
splenocyte and a murine myeloma cell line which produces an IgM
antibody directed to the hapten, 4-hydroxy-3-nitrophenyl acetate
(NIP). Those MAbs have been found to bind to
4-hydroxy-5-iodo-3-nitrophenyl acetate (NIP) with greater affinity
than to the immunogen, NP, a characteristic generally termed
"heterocliticity".
[0031] The heavy and light chain cDNAs have been cloned from the
B1-8 hybridoma cell line and are publicly available from Dr. A.
Bothwell of Yale University. The .mu. chain DNA and the .lamda.
chain DNA can each be conveniently isolated as restriction
fragments, as described below.
Example 2
Expression Vector Construction
[0032] The .mu. chain cDNA can be cloned into plasmid pMT3SVA as
follows to produce pMT3A.mu., in which expression of the .mu. gene
is controlled by the adenovirus major late promoter and in which
the .mu. gene is linked to an ADA transcription unit wherein ADA
expression is controlled by the SV40 early promoter and
enhancer.
[0033] The heavy chain expression plasmid can be constructed with
the i heavy chain CDNA of pAB.mu.-11 (Bothwell et al, 1981, Cell
24:625). The .mu. cDNA may be isolated and prepared for cloning
into the Eco RI site of the expression vector pMT3SVA as follows.
pAB.mu.-11 is digested to completion with Bgl II, and then a
partial Pst I digestion is performed. One resulting Bgl II-Pst I
fragment of approximately 1 kb should contain the complete 3' end
of the cDNA and can be purified from a low-melt agarose gel. This
fragment can then be ligated into Bam HI and Pst I digested
Bluescript plasmid (Stratagene, La Jolla, Calif.), and transformed
into E. coli DH5. The resultant transformants can be screened by
restriction enzyme digestion of individual DNA preparations. The
desired clone, with the 3' end of the .mu. cDNA cloned into
Bluescript is called pB.mu.3'. A complete Pst I and Bam HI
digestion of pAB.mu.-11 will generate a Pst I-Bam HI fragment of
approximately 870 bp, that can be purified by elution from a
low-melt agarose gel. This fragment, called .mu.5', contains the 5'
end of the .mu.cDNA, with the exception of the leader sequence.
Another fragment, called .mu.3', can be prepared from pB.mu.3', by
digestion with Bam HI and Eco RI, and elution from a low-melt
agarose gel. This fragment of approximately 1 kb contains the
3'.mu. sequence derived from pAB.mu.-11, with an Eco RI site at the
3' end of the Bluescript polylinker sequence. Fragments .mu.5' and
.mu.3' can be ligated with Eco RI-digested pMT3SVA, and two
synthetic oligodeoxribo-nucleotides, to reconstruct the leader
sequence. The sequences of exemplary synthetic
oligodeoxyribonucleotides are as follows:
TABLE-US-00001 (SEQ ID NO. 1)
5'-AATTCGTAATGGGATGGAGCTGTATCATGCTCTTCTTGGC-AGCAAC
AGCTACAGGTGTCCACTCCCAGGTCCAACTGCA-3' and (SEQ ID NO. 2)
5'-GTTGACCTGGGAGTGGACACCTGTAGCTGTTGCTGCCAAGAAGA-GC
ATGATACAGCTCCATCCCATTAG-3'
[0034] The ligation products can be transformed into E. coli DH5,
and transformants screened by colony hybridization to one of these
two oligodeoxyribonucleotides labeled with .sup.32P, using standard
procedures. Positive colonies can be characterized further with
restriction enzyme digestion analysis of DNA preparations.
Digestions with Sal I and enzymes that cut in the cDNA, such as Bg1
II and Bam HI can be used to orientate the insert cloned into the
vector, for a unique Sal I site is positioned 3' to the Eco RI site
in pMT3SVA.
[0035] The .mu.cDNA insert used in these studies is also derived
from pAB.mu.-11, and closely resembles the example above. It was
called pMT3A.mu.f.
[0036] The .lamda. chain is introduced into an expression vector to
produce pAd.lamda., in which expression of the .lamda. gene is
present in a bicistronic transcription unit followed by a DHFR
gene, both under the expression control of the adenovirus major
late promoter and SV40 enhancer.
[0037] The mouse immunoglobin .lamda., light chain cDNA used was
derived from pAB.lamda..sub.1-15 (Bothwell et al., 1982, Nature
298:360). Initially the Pst I fragment from this plasmid bearing
the .lamda..sub.1 cDNA was cloned into the Pst I site of pSP65N, to
give p.lamda..sub.1-3. This vector, pSP65N, is derived from the
pSP65 by digestion with Hind III, enzymatic `filling-in` of the
Hind III cohesive ends, and ligation with Not I linkers. The
ligation products were digested with Not I, and religated to
generate pSP65N. pSP65 can be purchased from Promega Biotec. The
orientation of the .lamda..sub.1 cDNA insert in p.lamda..sub.1-3
was found to be such that the vector polylinker Sal I site is at
the end of the insert.
[0038] p.lamda..sub.1-3 was digested with Fok and Sal I and the two
novel bands of approximately 307 bp (I) and 550 bp (II) were
excised frog a low-melt agarose gel, and purified. (I) represents
the 5' Fok I--Fok I fragment consisting of codon--15 to codon 87
(numbering as in Bothwell et al., 1982, Nature 298:380). (II)
represents codon 87 to the 3' end of the coding region, the
remainder of the 3' end of the insert, and extending to the vector
Sal I site.
[0039] The expression vector used was derived from pMT2DGR. This
plasmid was digested with Sal I and Xho I, and the desired vector
fragment was distinguished from the other fragment bearing factor
VIII-related sequences on a low-melt agarose gel, and the vector
fragment was excised and purified. To create pAD.lamda..sub.1, the
pMT2DGR-derived vector fragment was ligated with fragments (I) and
(II), and two synthetic oligodeoxyribonucleotides of the following
sequence:
TABLE-US-00002 5'-TCGACGCCATGGCCTGGATT-3', (SEQ ID NO. 3) and
5'-GTGAAATCCAGGCCATGGCCG-3'. (SEQ ID NO. 4)
[0040] These synthetic sequences annealed to each other, and to the
Fok I cohesive end at the 5' end of (I). Their nucleotide sequence
reconstructs the 5' end of the coding region and creates a small,
5' untranslated region. The ligation products were transformed into
E. coli DH5, and the desired recombinants identified by restriction
enzyme digestion of small-scale DNA preparations from individual
transformants. In addition, pAd.lamda..sub.1 was later transfected
by the DEAE-dextran procedure, into COS-1 cells, and shown to
produce a polypeptide of the correct molecular weight and
immunoreactive with goat anti-mouse .lamda. antisera (from Southern
Biotechnology Associates) on western blot analysis of transfected
cell extracts.
Example 3
Transformation and Amplification of CHO Host Cells
[0041] pMT3A.mu.f and pAd.lamda..sub.1 were separately
electroporated into separate pools of CHO DUKX cells (which are
dhfr Pools of transfected clones were made and selected in
increasing concentrations of dCF or MTX, respectively. Two pools
selected at 3 .mu.M dCF (.mu.) or 50 nM MTX (.lamda.) were fused by
conventional means in polyethylene glycol, and ADA.sup.+ DHFR.sup.+
cells were selected up to 3 .mu.M dCF and 50 nM MTX. The cells were
then further selected up to 3 .mu.M dCF and 200 nM MTX and 10 .mu.M
dCF and 50 nM MTX. It was found that only the increased
concentration of dCF led to an increase in the amount of functional
Ab as determined by a hapten-binding ELISA. This correlated with an
increase in the amount of heavy chain produced, and therefore it is
concluded that the amount of heavy chain was limiting the amount of
functional antibody produced. The 10 .mu.M dCF and 50 nM MTX pool
was then further selected at up to 40 .mu.M dCF and 50 nM MTX. At
this stage, clones were obtained by plating the cells at low
density, and after an appropriate period of growth, macroscopic
colonies were cloned out using cloning cylinders as is well known
in the art.
[0042] The levels of .mu., .lamda. and NP-binding MAb produced at
different levels of selection were measured by ELISAs based upon
standard procedures, as described in Voller, A. et al. (1979), the
Enzyme Linked Immunosorbent Assay (ELISA), Dynatech Europe, Borough
House, Rue de Pre, Guernsey, UK; Bos, et al., 1981, J. Immunoassay
2:187; Wood et al., 1984, Nucleic Acids Research 12:3937; and Boss
et al., 1984, Nucleic Acids Research 12:3791.
Example 4
Characterization of the CHO MAb So Produced
[0043] The CHO cells were cultured in alpha medium containing 10%
(by volume) heat-inactivated, dialyzed fetal calf serum and 10
.mu.g/ml penicillin, 10 .mu.g/ml streptomycin and 1 mM L-glutamine
with the selective agents. Selection for DHrR+cells was initially,
carried out in this medium, and then selection was increased with
iteratively increasing concentrations of methotrexate.
[0044] When using ADA section, the cells were cultured in media
supplemented with 0.05 mM L-adenosine, 1 mM uridine and 1.1 mM
adenosine, in addition to dCF. Descriptions of the types of culture
and selection procedures employed are given in Kaufman et al.,
1987, Proc. Nati. Acad. Sci. USA 83:3136; Kaufman et al., 1987,
EMBO J. 6:187: and Kaufman et al., 1985, Mol. Cell Biol. 5:1750.
The medium for selection of ADA.sup.+ DHFR.sup.+ cells contained
10% (v/v) dialyzed fetal calf serum (heat inactivated), 10 .mu.g/ml
each of penicillin and streptomycin, 1 mM L-glutamine, 0.05 mM
L-alanosine, 1 mM uridine, 1.1 mM adenosine, dCF and
methotrexate.
[0045] The CHO MAbs were found to bind immobilized NP, and this
binding could be competed out with 30 .mu.M free NP. The CHO MAb
was found to have a greater affinity for NIP than for NP,
demonstrating the retention of the parental MAb's heterocliticity.
Furthermore, the CHO MAbs were found to be polymeric IgMs and to
produce plaques in an NP-plaque assay--a qualitative measure of
complement fixation. Dresser, D. W., and Greaves, M. F., (1983) in
Handbook of Experimental Immunology, D. M. Weir, ed. (Blackwell
Scientific Publications, Oxford), p271; O'Hara, R. M., Jr., et al.,
(1988) Cell. Immunol. 116:423. Thus, in each of the parameters
measured (hapten binding, complement fixation, and
heterocliticity), the CHO MAbs were found to be strikingly similar
to the parental B1-8 MAbs. The synthesis of these immunoglobin
light and heavy chains were also studied by western blotting, and
pulse-chase labelling with L-.sup.35S-methionine and
immunoprecipitation. The heterologously expressed polypeptides were
found to resemble closely the hybridoma-produced antibody
polypeptides.
Sequence CWU 1
1
4179DNAArtificialleader sequence 1aattcgtaat gggatggagc tgtatcatgc
tcttcttggc agcaacagct acaggtgtcc 60actcccaggt ccaactgca
79269DNAArtificialleader sequence 2gttgacctgg gagtggacac ctgtagctgt
tgctgccaag aagagcatga tacagctcca 60tcccattag
69320DNAArtificialligating oligodeoxyribonucleotide 3tcgacgccat
ggcctggatt 20421DNAArtificialligating oligodeoxyribonucleotide
4gtgaaatcca ggccatggcc g 21
* * * * *