U.S. patent application number 12/122123 was filed with the patent office on 2008-11-13 for methods of treatment with glycosylated antibodies.
This patent application is currently assigned to SmithKline Beecham Corporation. Invention is credited to James S. Crowe, Michael J. Keen, Martin J. Page, Nicholas T. Rapson.
Application Number | 20080279852 12/122123 |
Document ID | / |
Family ID | 10683862 |
Filed Date | 2008-11-13 |
United States Patent
Application |
20080279852 |
Kind Code |
A1 |
Page; Martin J. ; et
al. |
November 13, 2008 |
Methods of treatment with glycosylated antibodies
Abstract
The present invention relates to methods of treating a human
patient suffering from rheumatoid arthritis, comprising: obtaining
a CHO cell comprising antibody heavy and light chain genes which
are constituted of cDNA and in a form that enables balanced
expression of the light and heavy chains of a human or humanized
recombinant antibody; culturing the CHO cell in a serum-free media;
purifying the resulting recombinant antibody; and administering a
therapeutically effective amount of the antibody expressed and
glycosylated in a CHO cell expression system to said human
patient.
Inventors: |
Page; Martin J.; (Oddington,
GB) ; Crowe; James S.; (Stevenage, GB) ;
Rapson; Nicholas T.; (Stevenage, GB) ; Keen; Michael
J.; (Stevenage, GB) |
Correspondence
Address: |
GLAXOSMITHKLINE;Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham
Corporation
|
Family ID: |
10683862 |
Appl. No.: |
12/122123 |
Filed: |
July 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10145992 |
May 16, 2002 |
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12122123 |
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09495861 |
Feb 2, 2000 |
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10145992 |
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08475607 |
Jun 7, 1995 |
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09495861 |
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08155864 |
Nov 23, 1993 |
5545403 |
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08475607 |
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08046893 |
Apr 15, 1993 |
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08155864 |
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07943146 |
Sep 10, 1992 |
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08046893 |
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07777730 |
Oct 16, 1991 |
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07943146 |
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Current U.S.
Class: |
424/133.1 ;
424/141.1; 424/172.1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 2039/505 20130101; C07K 16/00 20130101; C07K 2317/41 20130101;
A61P 3/08 20180101; A61P 35/00 20180101; C07K 16/2893 20130101;
C07K 2317/24 20130101; A61P 17/00 20180101; A61P 11/00 20180101;
C07K 16/2812 20130101; A61P 31/04 20180101; A61P 43/00 20180101;
A61K 38/00 20130101; A61P 37/00 20180101 |
Class at
Publication: |
424/133.1 ;
424/172.1; 424/141.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/00 20060101 A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 1990 |
GB |
9022543.4 |
Claims
1-48. (canceled)
49. A method of treating a human patient suffering from rheumatoid
arthritis, comprising: a) obtaining a CHO cell comprising antibody
heavy and light chain genes which are constituted of cDNA and in a
form that enables balanced expression of the light and heavy chains
of a human or humanized recombinant antibody; b) culturing the CHO
cell in a serum-free media; c) purifying the resulting recombinant
antibody; and d) administering a therapeutically effective amount
of the antibody expressed and glycosylated in a CHO cell expression
system to said human patient.
50. The method of claim 49 wherein the cell is cultured in said
serum-free medium for greater than two months.
51. The method of claim 49 wherein the cell is cultured in said
serum free medium for greater than five months.
52. The method of claim 49 wherein the culture undergoes multiple
passage.
53. The method of claim 49 wherein the serum free medium further
comprises water, an osmolarity regulator, a buffer, an energy
source, L-glutamine and at least one additional amino acid, an
inorganic iron source and a recombinant growth factor wherein each
component of said medium is obtained from a source other than
directly from an animal source.
54. The method of claim 53 wherein the medium is devoid of bovine
serum albumin, pure human transferrin and soybean lecithin.
55. The method of claim 53 wherein the growth factor is recombinant
insulin.
56. The method of claim 49, wherein the recombinant antibody is
able to activate complement.
57. The method of claim 49 wherein the recombinant antibody is a
monoclonal antibody.
58. The method of claim 49 wherein the recombinant antibody is a
humanized antibody.
59. The method of claim 49 wherein the recombinant antibody is a
human antibody.
Description
[0001] This application is a Divisional application Ser. No.
10/145,992, filed on May 16, 2002 which is a Continuation
Application of No. 09/495,861, filed on Feb. 20, 2000, now
abandoned, which is a continuation of application Ser. No.
08/475,607, filed on Jun. 7, 1995, now abandoned, which is a
Continuation of application Ser. No. 08/155,864, filed on Nov. 23,
1993, now U.S. Pat. No. 5,545,403, which is a Continuation of
application Ser. No. 08/046,893 filed on Apr. 15, 1993, now
abandoned, which is a Continuation of application Ser. No.
07/943,146, filed on Sep. 10, 1992, now abandoned, which is a
Continuation of application Ser. No. 07/777,730, filed on Oct. 16,
1991, now abandoned, and for which priority is claimed under 35
U.S.C. .sctn. 120, and this application claims priority of
Application No. 9022543.4 filed in Great Britain on Oct. 17, 1990
under 35 U.S.C. .sctn. 119.
[0002] The present invention relates to Chinese hamster ovary (CHO)
cell lines, to the production of proteins, in particular antibodies
from such cell lines, also to antibodies having CHO
glycosylation.
[0003] Antibodies or immunoglobulins are proteinaceous bifunctional
molecules. One region which is highly variable between different
antibodies is responsible for binding to an antigen (Fab region),
for example the many different infectious agents that the body may
encounter, whilst the second, constant region (or Fc region) is
responsible for binding to the Fc receptors of cells and also
activates complement. In this way, antibodies represent a vital
component of the immune response of mammals in destroying foreign
microorganisms and viruses.
[0004] An antibody molecule is composed of two light chains and two
heavy chains that are held together by interchain disulphide bonds.
Each light chain is linked to a heavy chain by disulphide bonds and
the two heavy chains are linked to each other by disulphide bonds.
Each heavy chain has at one end a variable domain followed by a
number of constant domains, and each light chain has a variable
domain at one end and a constant domain at the other end. The light
chain variable domain is aligned with the variable domain of the
heavy chain. The light chain constant domain is aligned with the
first constant domain of the heavy chain. The remaining constant
domains of the heavy chains are aligned with each other. The
constant domains in the light and heavy chains are not involved
directly in binding the antibody to the antigen.
[0005] The variable domains of each pair of light and heavy chains
form the antigen binding site. They have the same general structure
with each domain comprising a framework of four regions, whose
sequences are relatively conserved, connected by three
complementarity determining regions (CDRs). The four framework
regions largely adopt a beta-sheet conformation and the CDRs form
loops connecting, and in some cases comprising part of, the
beta-sheet structure. The CDRs are held in close proximity by the
framework regions and, with the CDRs from the other domain,
contribute to the formation of the antigen binding site.
[0006] The immunisation of an animal with an antigen results in the
production of different antibodies with different specificities and
affinities. An antiserum obtained from the immunised animal will,
therefore, be heterogeneous and contain a pool of antibodies
produced by many different lymphocyte clones. Antibodies thus
obtained are referred to as polyclonal antibodies and this
polyclonal nature has been a major drawback in the use of
antibodies in diagnostic assays and in therapeutic
applications.
[0007] A major step forward occurred in 1975 when Kohler and
Milstein (Nature, 1975, 256, 495-497) reported the successful
fusion of spleen cells from mice immunized with an antigen with
cells of a murine myeloma line. The resulting hybrid cells, termed
hybridomas, have the properties of antibody production derived from
spleen cells and of continuos growth derived from the myeloma
cells. Each hybridoma synthesizes and secretes a single antibody to
a particular determinant of the original antigen. To ensure that
all cells in a culture are identical, i.e. that they contain the
genetic information required for the synthesis of a unique antibody
species, the hybridomas resulting from cell fusion are cloned and
subcloned. In this way, the cloned hybridomas produce homogeneous
or monoclonal antibodies.
[0008] The advantages of hybridoma technology are profound. Because
many hybrids arising from each spleen are screened for their
potential to produce antibodies to the antigen of interest and only
a few are selected, it is possible to immunize with impure antigens
and yet obtain specific antibodies. The immortality of the cell
line assures that an unlimited supply of a homogeneous,
well-characterised antibody is available for use in a variety of
applications including in particular diagnosis and immunotherapy of
pathological disorders. Unfortunately, the usefulness of such
monoclonal antibodies in a clinical setting can be severely
hampered by the development of human anti-mouse antibodies--an
anti-globulin response--which may interfere with therapy or cause
allergic or immune complex hypersensitivity.
[0009] When, for example, murine (or ratine) monoclonal antibodies
are used in human therapy, the induction of an human anti-mouse
antibody response is due to the murine origin of the constant
domains and four framework regions. This problem has therefore been
addressed by the development of antibodies of two basic types. The
first type, referred to as chimeric antibodies, is where the murine
constant domains only are replaced by equivalent domains of human
origin (Morrison et al, P.N.A.S., 1984, 81, 6851-6855; Boulianne et
al, Nature, 1985, 314, 268-270; and Neuberger et al, Nature, 1985,
314, 268-270). The second type is where the murine constant domains
and the murine framework regions are all replaced by equivalent
domains and regions of human origin. This second type of antibody
is referred to as a humanised or CDR-grafted antibody (Jones et al,
Nature, 1986, 321, 522-525; and Riechmann et al, Nature, 1988, 332,
323-327). A human antibody would of course avoid the need for
"humanisation", however cell lines which secrete human antibodies
are very unstable and have generally proven unsuitable for
commercial scale production.
[0010] To generate sufficient quantities of antibody for full
clinical use it is desirable to employ an efficient recombinant
expression system. Since myeloma cells represent a natural host
specialized for antibody production and secretion, cell lines
derived from these have been used for the expression of recombinant
antibodies. Often, complex vector design, based around
immunoglobulin gene regulatory elements, is required, and final
expression levels have been reported which are highly variable
(Winter et al, Nature, 1988, 332, 323-327; Weidle et al, Gene,
1987, 60, 205-216; Nakatani et al, Bio/Technology, 1989, 7,
805-810; and Gillies et al, Bio/Technology, 1989, 7, 799-804).
[0011] An alternative mammalian expression system is that offered
by the use of dihydrofolate reductase (dhfr) deficient Chinese
hamster ovary (CHO) cells. The use of these cells has enabled the
production of large quantities of several therapeutic proteins for
research and clinical use (Kaufman et al, Mol. Cell. Biol, 1985, 5,
1750-1759; and Zettlmeissl et al, Bio/Technology, 1987, 5,
720-725). There are, however, very few instances of the use of
these cells for the expression of antibodies and the levels of
expression of murine antibodies reported to date are low-of the
order of 0.01-0.1 .mu.g/ml (Weidle et al, Gene, 1987, 51, 21-29;
and Feys et al, Int. J. Cancer, 1988, 2, 26-27).
[0012] A process has now been developed that enables the balanced
expression of the light and heavy chains of an antibody from CHO
cells. Balanced expression is desirable given that the light and
heavy chains are linked together in the antibody molecule in
equimolar proportions. This process allows the antibody to be
obtained in functional form and to be secreted in good yields. Thus
the process enables sufficient quantities of functional antibody to
be obtained for use in the immunotherapy of pathological
disorders.
[0013] The invention therefore provides a CHO cell line capable of
producing antibody, the cell line having been co-transfected with a
vector capable of expressing the light chain of the antibody and a
vector capable of expressing the heavy chain of the antibody
wherein the vectors contain independently selectable markers.
[0014] The present invention further provides a CHO cell line
capable of producing a human antibody or an altered antibody, the
cell line having been co-transfected with a vector containing cDNA
encoding the light chain of the antibody and a vector containing
cDNA encoding the heavy chain of the antibody said vectors capable
of expressing the light and heavy chains of the antibody. The
vectors may advantageously contain independently selectable
markers. Hereafter, reference to the markers includes the singular
and vice versa.
[0015] The cell line of the present invention is capable of
producing all kinds of antibodies that generally comprise equimolar
proportions of light and heavy chains. The invention therefore
includes human antibodies wherein the amino acid sequences of the
heavy and light chains are homologous with those sequences of
antibodies produced by human lymphocytes in vivo or in vitro by
hybridomas. Also included in the invention are altered antibodies
such as hybrid antibodies in which the heavy and light chains are
homologous to a natural antibody but are combined in a way that
would not occur naturally. For example, a bispecific antibody has
antigen binding sites specific to more than one antigen. The
constant region of the antibody may relate to one or other of the
antigen binding regions or may be from a further antibody. Altered
antibodies, such as chimaeric antibodies have variable regions from
one antibody and constant regions from another. Thus, chimaeric
antibodies may be species/species chimaeras or class/class
chimaeras. Such chimaeric antibodies may have one or more further
modifications to improve antigen binding ability or to alter
effector functioning. Another form of altered antibody is a
humanised or CDR-grafted antibody including a composite antibody,
wherein parts of the hypervariable regions in addition to the CDRs
are transferred to the human framework. Additional amino acids in
the framework or constant regions of such antibodies may be
altered. Included in the definition of altered antibody are Fab
fragments which are roughly equivalent to the Y branch portions of
the heavy and light chains; these may be included incomplete
fragments or fragments including part of the Fc region. Thus,
within the scope of the invention is included, any altered antibody
in which the amino acid sequence is not one which exists in
nature.
[0016] The cell line of the invention is preferentially employed
for the production of altered antibodies most preferably chimaeric
antibodies or CDR-grafted antibodies. Particular examples of these
include antibodies against T cell markers such as CD2, CD3, CD4,
CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD25, CD45 and CDw52 and
especially CDR grafted antibodies against the CDw52 antigen, such
as CAMPATH-1H_ (Campath is a Trademark of the Wellcome Foundation
Ltd) described in EP 328404. Further examples include CDR-grafted
antibodies against various cancer cell marker antigens such as CD33
and CD38.
[0017] After co-transfection into recipient CHO cells, the
resulting colonies may be selected using both markers. Colonies
exhibiting the dual phenotype are generally capable of
co-expressing both the light and heavy chains. The selectable
markers may or may not be of a dominant nature. Examples of
selectable markers for use in co-transfection include adenosine
deaminase (Kaufman et al, P.N.A.S, 1989, 83, 3136-40) asparagine
synthetase (Cartier et al, Mol. Cell. Biol., 1987, 7, 1623-28), E.
Coli trpB gene and Salmonella hisD gene (Hartman et al, P.N.A.S.,
1988, 85, 8407-51), M2 mouse ribonucleotide reductase (Thelander et
al, EMBO J, 1989, 8, 2475-79), human multidrug resistance gene
(Kate et al, Gene, 1989, 84, 439-446), glutamine synthetase
(Bebbington et al, DNA Cloning, Vol III, 1987, Ed. D. M. Glover,
163-188, IRL Press), xanthine guanine phosphoribosyl transferase
(gpt) (Mulligan et al, Science, 1980, 209, 1422-27), hygromycin B
(Santerre et al, Gene, 1984, 30, 147-156), neomycin gene (Southern
et al, J. Mol. Appl. Genet., 1982, 1, 327-341), and dihydrofolate
reductase (Subramani et al, Mol. Cell. Biol., 1981, 1, 854-864).
One particularly preferred selectable marker is dhfr which is
usually employed with a parental CHO cell line of the dhfr.sup.-
phenotype (Urlaub et al, P.N.A.S. 1980, 77, 4216-4220).
Successfully co-transfected CHO cells will possess the dhfr.sup.+
phenotype and can readily be selected by culturing the colonies on
media devoid of thymidine and hypoxanthine and optionally
containing methotrexate (MTX). A preferred selectable marker for
use with the other of the vectors is a dominant resistance marker,
such as neomycin (neo). CHO cells successfully transfected with
this marker can readily be selected by culturing the colonies on
media containing the antibiotic, G418, otherwise known as
Geneticin.
[0018] A second preferred system of selection and amplification is
provided by the glutamine synthetase selectable marker or (GS
system) which is described in WO87/04462. CHO cells which have been
successfully transfected with the gene encoding the GS enzyme and
the desired antibody gene can be selected by culturing colonies in
media devoid of glutamine as described in PCT published application
number WO87/04462.
[0019] At least one of the selectable markers preferably also
provides the basis upon which the genes encoding the light and
heavy chains may be amplified. In co-transfection of a CHO cell
line, the vector DNAs are often integrated into the chromosome of
the cell at the same locus. Thus, the use of only one of the
selectable markers as the basis for amplification normally results
in a parallel increase in the copy number of both genes. One
particularly preferred selectable marker for use in this way is
dhfr which enables the desired amplification to be obtained through
the use of increasing concentrations of MTX. A second preferred
selectable marker is GS which allows amplification by the addition
of methionine sulphoximine (MSX).
[0020] The selectable markers are of course under the control of
regulatory elements of DNA so as to provide for their expression.
In the case of the use of dhfr as a selectable marker, the
regulatory elements are preferably of a vital source, such as from
DNA tumour viruses. Particularly preferred are the use of an SV40
or adenovirus major late promoter. It is particularly advantageous
in this regard to remove the enhancer element from the promoter
thus effectively "crippling" it. This modification allows for
increased levels of gene amplification at each concentration of
methotrexate selection than would otherwise occur if a strong
promoter was used. In the case of the use of neo as a selectable
marker, an example of a suitable promoter is the mouse
metallothionein promoter.
[0021] The light and heavy chain genes may constitute genomic DNA
or, preferably, cDNA, and are cloned using procedures known in the
art (Molecular Cloning: A Laboratory Manual, Second Edition,
Maniatis et al, Cold Spring Harbor). The genes are also under the
control of regulatory elements of DNA so as to provide for their
expression. The use of the same regulatory elements for both chains
is preferred so that their expression is substantially balanced.
The regulatory elements may be of vital origin and examples include
those mentioned above in conjunction with the expression of dhfr as
a selectable marker. Another example is the use of the .beta.-actin
promoter and cognate .beta.-actin polyadenylation signal.
[0022] One or both of the vectors may also contain an SV40 origin
of replication to allow for the vector constructs to be checked by
rapid transient assay.
[0023] Construction of the expression vectors may be carried out in
accordance with procedures known in the art (Molecular Cloning: A
Laboratory Manual, Second Edition, Maniatis et al, Cold Spring
Harbor).
[0024] Co-transfection of the CHO cell line with the expression
vectors may be carried out simply by using equimolar quantities of
both vectors and standard transfection procedures, such as calcium
phosphate precipitation or lipofectin. Selection of the desired
co-transfected cell line may be carried out in accordance with
standard procedures known for the particular selectable
markers.
[0025] The present invention also provides a process for the
production of an antibody which comprises culturing a CHO cell line
of the present invention. Culture of the CHO cell line may be
carried out in serum-containing or preferably serum and protein
free media. In one preferred instance where the CHO cell line is a
dhfr.sup.+ transformant, the medium preferably lacks hypoxanthine
and/or thymidine and optionally contains MTX. Where a selectable
marker is glutamine synthetase the medium preferably lacks
glutamine and optionally contains MSX. Expression of both chains in
substantially equimolar proportions enables optimum yields of
functional antibody to be obtained. The two chains assemble within
the cell and are then secreted into the culture medium as
functional antibody. The resulting antibody may be purified and
formulated in accordance with standard procedures.
[0026] Antibodies are glycoproteins containing between 3 and 12%
carbohydrate. The carbohydrate units are transferred to acceptor
sites on the antibody chains after the heavy and light chains have
combined. The major carbohydrate units are attached to amino acid
residues of the constant region of the antibody. Carbohydrate is
also known to attach to the antigen binding sites of some
antibodies and may affect the antibody-binding characteristics by
limiting access of the antigen to the antibody binding site. There
are a number of roles associated with the carbohydrate units. They
may affect overall solubility and the rate of catabolism of the
antibody. It is also known that carbohydrate is necessary for
cellular secretion of some antibody chains. It has been
demonstrated that glycosylation of the constant region plays a
vital role in the effector functioning of an antibody; without this
glycosylation in its correct configuration, the antibody may be
able to bind to the antigen but may not be able to bind for example
to macrophages, helper and suppressor cells or complement, to carry
out its role of blocking or lysing the cell to which it is
bound.
[0027] It has now been found that antibody glycosylated by CHO
cells maintains antigen binding capability and effector
functionality. This has been demonstrated in vitro complement lysis
assays and in vivo in a human patient.
[0028] The invention therefore provides an antibody having CHO
glycosylation. Such antibodies may be natural, such as human
antibodies, altered antibodies for example hybrid antibodies or
bispecific antibodies, chimaeric or CDR-grafted antibodies,
including Fab fragments.
[0029] The CHO glycosylation may be associated with the antigen
binding site or other parts of the variable domain. It may
alternatively or additionally be associated with the constant
region. The glycosylated antibody is prepared by expression of the
antibody genes in a suitably engineered CHO cell followed by
recovery and if necessary, purification of the antibody from the
cell culture medium.
[0030] CHO glycosylated antibodies are useful in medical therapy
for treating numerous human disorders, generally as
immunosuppressives more particularly for example T-cell mediated
disorders including severe vasculitis, rheumatoid arthritis,
systemic lupis, also autoimmune disorders such as multiple
sclerosis, graft vs host disease, psoriasis, Juvenile onset
diabetes, Sjogrens' disease, thyroid disease, myasthenia gravis,
transplant rejection and asthma. These antibodies are also useful
in treating cancer such as Non-Hodgkins lymphoma, multiple myeloma,
and infectious diseases such as HIV and herpes.
[0031] The invention therefore provides the use of CHO glycosylated
antibodies in the manufacture of a medicament for the treatment of
any of the aforementioned disorders. Also provided is a method of
treating a human being having any such a disorder comprising
administering to said individual a therapeutically effective amount
of a CHO glycosylated antibody.
[0032] The dosages of such antibodies will vary with the condition
being treated and the recipient of the treatment, but will be in
the range 1 to about 100 mg for an adult patient preferably 1-10 mg
usually administered daily for a period between 1 and 30 days. A
two part dosing regime may be preferable wherein 1-5 mg are
administered for 5-10 days followed by 6-15 mg for a further 5-10
days.
[0033] Also included within the invention are formulations
containing CHO glycosylated antibody. Such formulations preferably
include, in addition to antibody, a physiologically acceptable
diluent or carrier possibly in admixture with other agents such as
other antibodies or an antibiotic. Suitable carriers include but
are not limited to physiological saline, phosphate buffered saline,
phosphate buffered saline glucose and buffered saline.
Alternatively, the antibody may be lyophilised (freeze dried) and
reconstituted for use when needed by the addition of an aqueous
buffered solution as described above. Routes of administration are
routinely parenteral including intravenous, intramuscular,
subcutaneous and intraperitoneal injection or delivery.
THE ACCOMPANYING DRAWINGS SHOW
[0034] FIG. 1
(a) the pLD9 construct containing expression cassettes for the
`crippled` dhfr selection/amplification marker and the Campath-1H
light chain cDNA. The small box with the dashed arrow is the
weakened SV40 promoter; the larger dotted box with an arrow is the
.beta.-actin promoter; polyA refers to respectively sourced
polyadenylation and termination signals; the small box with ori
contains the SV40 origin of replication; (b) the pNH316 construct
containing expression cassettes for the neomycin selection marker
and the Campath-1H heavy chain cDNA. The box with an arrow and MT
refers to the mouse metallothionein promoter. Restriction sites
indicated are: --H, HindIII; Bg, BglII; B, BamHI; R1, EcoR1.
[0035] FIG. 2
[0036] Comparative determinations of the rate of Campath-1H
synthesis in confluent A39 cells over 4 consecutive days. Following
the [.sup.35S] methionine pulse period, equal aliquots of cells (C)
and culture medium (M) were Immuno-precipitated and separated by
SDS-PAGE. The position of the Campath-1H heavy and light chains are
indicated (H and L arrows). There was some loss of material for the
day 3 cell sample.
[0037] FIG. 3
[0038] A pulse-chase experiment to determine the rate of secretion
and distribution of radiolabelled Campath-1H in A39 cells.
Confluent cells were pulsed with [.sup.35S] methionine for 6 hours,
then fresh medium containing an excess of unlabelled methionine was
added. Equal aliquots of cells and culture medium were taken at the
indicated time points (in hours following the end of the pulse
period) and treated as described in the legend of FIG. 2. The
samples for the 48 and 72 hour medium time points were run on a
different gel to the 6 and 24 hour points, and the tracks are only
lined up relative to the position of the heavy (H) chain.
[0039] FIG. 4
[0040] Shows growth of C1H 3D11* 44 in WCM5 (protein-free medium)
in a 1 litre fermenter measured as cell count/ml over 90 days.
[0041] FIG. 5
[0042] Shows antibody production from C1H 3D* 44 cells in WCM5 in a
1 litre fermenter measured as micrograms of antibody/ml over 80
days.
[0043] The following Examples are provided purely for illustration
of the present invention.
EXAMPLE 1
Cloning of the Heaving and Light Chain cDNAs for Campath-1H
[0044] The complementarity determining regions from the rat
CAMPATH-1G monoclonal were originally grafted directly into genomic
human heavy and light chain frameworks (Winter et al, Nature, 1988,
322, 323-327). These constructs were engineered for expression in
the myeloma cell line YO and resulted in yields of CAMPATH-1H of up
to 5 .mu.g/ml following 10-14 days in culture (Hale et al, Tissue
Antigens, 1990, 35, 118-127 and Winter et al, Nature, 1988, 325,
323-327). The myeloma cell line TF57 (Hale et al, ibid) was used to
generate size selected cDNA fractions of 0.9-1.2 kb and 1.4-1.7 kb
for the light and heavy chain cDNAs respectively. These were used
to make EcoR1 Tinkered cDNA libraries in .lamda.gt10. All
procedures were as described by Huynh et al (DNA Cloning, Vol I: A
Practical Approach, 1984, Glover, D (Editor), IRL Press, Oxford).
The libraries were screened using [.sup.32P] nick translated probes
specific for the variable regions to isolate full length cDNA
clones. For the light chain cDNA, the 5' untranslated leader was
removed up to position -32 using Bal-31 exonuclease and a HindIII
linker added. For the 3' end, use was made of a unique SacI site 47
bp upstream of the stop codon. A SacI-HindIII oligonucleotide pair
was used to regenerate this sequence and position the HindIII site
immediately after the stop codon. For the 5' end of the heavy chain
cDNA, the unique NcoI site overlapping the ATG start codon was used
to re-build a 29 bp untranslated leader, identical to that of the
light chain, using a HindIII-NcoI oligonucleotide pair. At the 3'
end, the unique NaeI site 12 bp downstream of the stop codon was
converted into a HindIII site using linkers.
EXAMPLE 2
Construction of Vectors
[0045] The human .beta.-actin promoter was excised from
pH.beta.APr-3-neo (which corresponds to pH.beta.APr-1-neo (Gunning
et al, P.N.A.S., 1987, 84, 483-35) except that the SV40
polyadenylation/termination signal has been replaced with the
respective human .beta.-actin signals) as a 2860 bp PvuII-HindIII
fragment, in which the PvuII site was subsequently converted to a
BglII site using linkers. To isolate the human .beta.-actin
polyadenylation and termination signals from pH.beta.APr-3-neo, an
SphI site 1.4 kb downstream of the unique HindIII site was
converted to a BamHI site using linkers. The basal dhfr vector
called p104, was constructed as follows The SDhI site at position
-128 in the SV40 promoter in pSV2dhfr (Subramani et al, Mol. Cell.
Biol., 1981, 1, 54-864) was converted into a SalI site to remove
all enhancer elements from the promoter. The weakened dhfr
expression unit was then subcloned as a SalI-BamHI fragment into
the homologous sites in pSVOd (Mellon et al., Cell, 1981, 27,
279-288).
[0046] To construct pLD9, the p104 vector was digested with BamHI,
phosphatased, and ligated with three other fragments consisting of
the BglII-HindIII .beta.-actin promoter, the HindIII Campath-1H
light chain cDNA and the HindIII-BamHI .beta.-actin
polyA/termination signals. To construct pNH316, the construct
pdBPV-MMTneo (Law et al, Mol. Cell. Biol., 1993, 3, 2110-2115) was
digested with BamHI, phosphatased, and the fragment containing the
neomycin gene isolated following separation on an agarose gel. This
was ligated to the two .beta.-actin fragments and the Campath-1H
heavy chain cDNA. The constructs, pLD9 and pNH316 are depicted in
FIG. 1.
EXAMPLE 3
Expression of Campath-1H in CHO Cells
[0047] The dhfr.sup.- CHO cell line DUK-B11 (Urlaub et al,
P.N.A.S., 1980, 77, 4216-4220) was grown in Iscove's MEM
supplemented with 10% fetal bovine serum, and 4 .mu.g/ml each of
hypoxanthine and thymidine. 10 .mu.g of pLD9 and pNH316 was
co-precipitated onto cells using the calcium phosphate method,
(Gorman et al, DNA Cloning, 1985, Vol II, 143-190, Academic Press,
N.Y.) and selected for the double phenotype of dhfr.sup.+/neo
resistance by using the medium above except that 10% dialysed serum
was used, the hypoxanthine/thymidine were omitted, and G418 (Gibco)
was included at 500 .mu.g/ml. In some experiments MTX was included
directly in the first round selection for dhfr.sup.+ transformants.
Several hundred resistant colonies were pooled and assayed for the
production of Campath-1H antibody in the culture medium. The
average yield was 0.5 .mu.g/ml for non-amplified first round
transformants.
[0048] Each pooled cell population was then cultured in the
presence of 10.sup.-7 M MTX, and after two weeks, resistant
colonies were again pooled and titred for Campath-1H production.
There was a considerable increase in yield of up to 80-fold (Table
1). These cells were dilution cloned, screened for Campath-1H
yield, and two high producer lines isolated, called A37 and 3D9
(Table 1). These were both amplified further in the presence of
10.sup.-6 M MTX, then dilution cloned and screened as above. The
increase in expression at this second, and final, amplification
stage was not so dramatic as seen previously; nevertheless, when
re-fed at confluence and left for a further 4 days, the cell lines
A39 and 3D11 were capable of producing up to 200 .mu.g/ml of
Campath-1H.
TABLE-US-00001 TABLE 1 Expression Levels of Campath-1H using
Stepwise Amplification Accumulated Construct Selection stage
Campath-1H (.mu.g/ml) pLD9 + pNH316 dhfr.sup.+/neo basal pool 0.5
10.sup.-7 M MTX amplified pool 18-40 Cell lines A37 and 3D9 40
10.sup.-6 M MTX amplified pool 60-90 Cell line A39 100 Cell line
3D11 150-200 Legend to Table Cells were allowed to reach confluence
in a T175 tissue culture flask, then refed with fresh 50 ml of
tissue culture medium and left for a further 4 days. The Campath1H
antibody that had accumulated in the medium during this period was
measured by ELISA. Total cell counts on the day of assay were
usually 2.5 .times. 10.sup.7. The yield from the 3D11 cell line
reflects a productivity of 100 .mu.g/10.sup.6 cells/day.
[0049] The co-transfection vectors pLD9 and pNH316 were further
employed to evaluate an alternative amplification strategy to the
one described above. The dhfr.sup.- CHO cells were co-transfected
as usual, and two days later split directly into a series of flasks
containing G418 (for neomycin selection) and increasing
concentrations of MTX ranging from 3.times.10.sup.-9 M to 10.sup.-7
M. Following two weeks of this selection, the number of resistant
colonies were counted and pooled for each flask. When the cell
populations had stabilized, they were assayed for Campath-1H
antibody titres and the results are shown in Table 2. As the MTX
level was increased, there was a marked decrease in the number of
surviving dhfr.sup.+ colonies, but they expressed proportionately
more Campath-1H. Thus, in a one step direct selection at high
concentrations of MTX, it is possible to isolate cell populations
which produce up to 60-fold increase in antibody yield compared to
cell populations selected for basal dhfr levels.
TABLE-US-00002 TABLE 2 Expression Levels of Campath-1H using Direct
Selection Accumulated Selection (M MTX) dhfr.sup.+colonies
Campath-1H (.mu.g/ml) No MTX 500 0.5 3 .times. 10.sup.-9 40 2
10.sup.-8 5 7 3 .times. 10.sup.-8 5 30 10.sup.-7 -- -- Legend to
Table Colonies at each MTX selection stage were pooled and assayed
as described in the legend of Table 1.
[0050] This selection procedure was repeated following another
co-transfection of cells, and in this instance, the entire
population was selected in medium containing G418 and
3.times.10.sup.-8 M MTX. This generated a larger pool of resistant
colonies which were subsequently pooled and re-amplified twice more
using MTX concentrations of 6.times.10.sup.-7 M, then
3.times.10.sup.-6 M. At this stage, the cells were dilution cloned
and screened for Campath-1H levels. The two highest producer cell
lines isolated were capable of producing antibody levels up to
100-150 .mu.g/ml and were designated as lines 4F11 and 5E10.
[0051] The growth rates of these cell lines, and the A39/3D11 lines
described above, were considerably slower than the parental
non-transformed dhfr.sup.- CHO cells. This is usually a common
feature of these cells once they have been engineered to express
high quantities of a product gene. The yields from the 5E10 and
4F11 cell lines proved to be quite variable over time, and the
latter appeared to have only a limited passage life lasting about 3
weeks before entering crisis and death. This instability was not
evident at all in the other cell lines, although in general, the
lines isolated from the second amplification procedure, including
5E10, were usually more fickle to culture. Of all the lines, the
3D11 coupled good growth and stability with high Campath-1H yields.
To ensure the propagation of these features, the 3D11 cell line was
dilution cloned once more to generate the 3D11* line and this
similarly produced Campath-1H yields up to 200 .mu.g/ml.
EXAMPLE 4
Growth of and Production from C1H 3D11* 44 in WCM4
[0052] a) C1H 3D11* cells growing as a monolayer in Iscoves +10%
FBS Flow, non-essential amino acids, 10.sup.-6 M Methotrexate and
antibiotics were approximately 90% confluent. These cells were
removed from the plastic with trypsin/versene, washed in Iscoves
medium without supplements, centrifuged and resuspended at
5.times.10.sup.4/ml in WCM4 medium Table 3+0.25% peptone+0.1%
polyethylene glycol (PEG) 10,000+0.5% fetal boine serum (FBS)
without methotrexate (MTX).
TABLE-US-00003 TABLE 3 Formulation for medium WCM4. Iscoves
modification of DMEM without BSA, transferrin and lecithin.
Available from GIBCO Ltd., Unit 4, Cowley Mill Td. Est., Uxbridge
UBS 27G. Similar to published medium (Iscoves and Melcher (1978) J.
Exp. Med. 1. 47, 923) without the bovine serum albumin, pure human
tranferrin, or soyabean lecithin. +5 ml/litre 200 mM L glutamine
+50 mg/litre L proline +50 mg/litre L threonine +50 mg/litre L
methionine +50 mg/litre L cysteine +50 mg/litre L tyrosine +25
mg/litre ascorbic acid +0.062 mg/litre vitamin B6 +1.36 mg/litre
vitamin B12 +0.2 mg/litre lipoic acid +0.088 mg/litre methyl
linoleate +1 .mu.M methotrexate +1 mg/litre FeSO.sub.4 +1 mg/litre
ZnSO.sub.4 +0.0025 mg/litre CuSO.sub.4 +5 mg/litre recombinant
insulin (Nucellin) +50,000 Iu/litre polymyxin +20,000 Iu/litre
neomycin +0.16 mg/litre putrescine-2 HCL.
[0053] Three 25 cm.sup.2 flasks were set up with 10 ml of cell
suspension+hypoxanthine (H), thymidine (T) or HT. These flasks were
incubated at 36.5.degree. C. in 5% CO.sub.2 incubator.
[0054] After six days, the flasks were pooled and added to an equal
volume of WCM4+MTX without peptone or PEG, and were transferred to
a 75 cm.sup.2 flask.
[0055] These cells were used to seed a 500 ml Techner spinner,
incubated at 36.5.degree. C. spinning at 40 rpm. Cells continued
growing serum free for a period of over five months and although it
was found that the cells needed a period of adaptation, the growth
rate and viability steadily improved. The population doubling time
was calculated to be 73.1 hours over approximately 7 weeks; this
decreased to 47.4 hours over the subsequent 20 days then
stabilised. Antibody secretion remained high at levels in excess of
60 .mu.g/ml. It was determined that the gene copy number in these
cells did not decrease according to band intensity using Northern
blot analysis.
[0056] In fermenters, these cells produced antibody in excess of 70
.mu.g/ml and regularly achieve levels of 100 .mu.g/ml or more.
These cells are denoted C1H 3D11* 44.
b) Cells from a) above which had been growing serum-free for over 2
months were transferred to a SGi 1 liter fermenter with a stainless
steel angled paddle turning at 70 rpm. The temperature was set at
37.degree. C., dO.sub.2 at 10% and pH control to 7-7.2. The
fermenter was seeded on day 0 with 0.22.times.10.sup.6 cells/ml in
WCM4 (Table 3) with 0.1% polyethylene glycol (PEG) 10,000 and 0.25%
soy peptone, and was top gassed with O.sub.2. The cells were
routinely passaged using fresh medium and a split rate typically
between 1 to 2 and 1 to 4.
[0057] On day 33 the top gassing was replaced with deep sparging
which is can be expected to cause more physical damage to the
cells.
[0058] On day 50 onwards WCM5 (Table 4) was used together with
peptone and PEG instead of WCM4.
TABLE-US-00004 TABLE 4 Formulation for Medium WCM5 Iscoves
modification of DMEM without BSA, transferrin or lecithin (see
Table 3). +5 ml/litre 200 mM L glutamine +50 mg/litre L proline +50
mg/litre L threonine +50 mg/litre L methionine +50 mg/litre L
cysteine +50 mg/litre L tyrosine +25 mg/litre L ascorbic acid
+0.062 mg/litre Vitamin B6 +1.36 mg/litre Vitamin B12 +2 mg/litre
Ferric citrate +1 mg/litre Zinc sulphate +0.0025 mg/litre Copper
sulphate +50,000 IU/litre Polymyxin +20,000 IU/litre Neomycin +3
.mu.l/litre Ethanolamine +0.16 mg/litre Putrescine +5 mg/litre
Recombinant Insulin (Nucellin)
[0059] On day 53 the PEG was replaced with 0.1% pluronic F68. The
resulting growth and antibody levels achieved are shown the
attached graphs (FIGS. 4 and 5), and demonstrate the capacity of
the invention to allow protein-free production of antibody in
excess of 100 .mu.g/ml in fermenters.
EXAMPLE 5
Analysis of the Rate of Campath-1H Synthesis and Secretion from CHO
Cells
[0060] During the course of culturing the Campath-1H producing CHO
cells of Example 3, it became clear that even when they reached
confluence, antibody levels continued to accumulate, with time, in
the culture medium. To determine whether this was possibly a
consequence of intracellular accumulation coupled to slow
secretion, the rates of Campath-1H synthesis and secretion were
measured using A39 cells. These analyses were performed over 3-4
consecutive days on cells which were either in growth phase, or
confluent stationary phase. For the cells in either growth state,
the results were identical, and data is presented only for the
immuno-precipitated radiolabelled Campath-1H produced from
stationary cells.
[0061] The rate of antibody synthesis was measured by pulsing the
cells for a short period with [S.sup.35]-methionine on each of four
consecutive days, and then examining the quantity, and
distribution, of immuno-precipitated material. In FIG. 2, it is
clear that the rate of synthesis is equally high at all time points
measured. Furthermore, even by the end of this short pulse, in each
case, more than half of the newly synthesized Campath-1H is already
present in the medium suggesting rapid secretion. This was
confirmed by the data shown in FIG. 3, in which parallel cells were
similarly pulsed, and the distribution of the radiolabelled
Campath-1H chased over a three day period. Within 24 hours,
virtually all of the cellular radiolabelled antibody has been
chased into the medium, where it remained stable for the duration
of the experiment. This demonstrates that even when the recombinant
CHO cells remain stationary for long periods, the rates of
Campath-1H synthesis and secretion are not diminished.
[0062] Campath-1H ELISA assay. Microtiter plates were coated with
anti-human IgG and incubated with the assay sample (in culture
medium). Antibody detection was visualized by using an anti-human
gamma chain specific peroxidase conjugate.
[0063] Analysis of rates of Campath-1H synthesis and secretion.
Cells from Example 3 were grown to confluence in 3 cm tissue
culture wells, then incubated for 30 minutes in methionine-free
Dulbeccos's MEM containing 10% fetal calf serum. Following this,
the cells were labelled in the presence of 120 .mu.Ci/ml [.sup.35S]
methionine (>800 Ci/mmol: Amersham) for the appropriate time
period, then either harvested and lysed in 500 .mu.l of NP-40 lysis
buffer, or incubated further in normal growth medium. Then 125
.mu.l aliquots of cell lysate or culture medium were
immuno-precipitated using goat anti-human IgG (heavy chain
specific; Sigma) and 10% protein-A SEPHAROSE.RTM. (Pharmacia).
Samples were then separated on 10% SDS-PAGE reducing gels according
to Laemmli and the signals amplified with ENHANCE.RTM.
(NEN-Dupont). The dried gels were then autoradiographed
overnight.
Biological Assays for Functional CHO-Glycosylated CAMPATH-1H
Complement Lysis Assay for CAMPATH-1H
[0064] The complement lysis assay is a measure of antibody function
expressed as specific activity, determined by the ability of a
CHO-glycosylated antibody of known concentration to bind to a
pre-determined number of cells and effect cell lysis.
[0065] The assay is carried out on CAMPATH-1H from Example 4 using
Karpas 422 cells (established from B-cell non-Hodgkin lymphoma cell
line--Dyer et al., (1990) Blood, 75 704-714) expressing Campath
antigen on the cell surface. 1.2.times.10.sup.7 cells were loaded
with radiolabel by incubating for 2 hours at 37.degree. C. in a
CO.sub.2 incubator in the presence of 600 .mu.Ci of 51 Cr (sodium
chromate).
[0066] 5.3 ml of the loaded cells in medium (total volume 23.5 ml),
were added to 12.5 ml of normal human serum and 150 .mu.l of the
mixture were pipetted into the wells of a microtitre plate.
[0067] 50 .mu.l samples of the final eluate from three purification
runs were mixed with the cells and incubated for 30 minutes at
4.degree. C. followed by 90 minutes at 37.degree. C. The culture
was centrifuged at 2000 rpm for 5 minutes and the radioactivity in
100 .mu.l of cell supernatant was counted on a gamma counter.
Complement lysis activity in Kilo Units/ml was calculated from a
standard curve of a reference preparation (1000 Units/ml).
[0068] The results are set out in Table 5.
[0069] The concentration of CAMPATH-1H in the 50 .mu.l samples of
final eluate was estimated using samples in PBS pH 7.2 read on a
spectrophotometer at 280 hm. The results are expressed in Table 3
as optical density in mg/ml.
[0070] From this data the specific activity: KU/ml/OD is
determined.
TABLE-US-00005 TABLE 5 Complement lysis Protein Conc Specific
Sample Kilo Units/ml mg/ml Activity A 11.2 11.1 1.0 B 14.8 14.2 1.0
C 13.7 13.6 1.0
[0071] The results indicate that CHO-glycosylated CAMPATH-1H is
functional.
Treatment of an Individual with CHO-Glycosylated CAMPATH-1H
[0072] An individual diagnosed as having severe T-cell mediated
inflammation of the joints (immobilising polyarthritis, pleuritis,
abdominal pains) over five years requiring long periods of
hospitalisation was treated with CHO derived CAMPATH-1H from
Example 4 using the following regime:
2 mg per day over 6 days by intravenous injection 10 mg per day
over subsequent 6 days by intravenous injection. During the second
6 day treatment there was a significant symptomatic improvement. By
the end of the second period the joint inflammation was much
improved and a skin abscess had cleared with antibiotic treatment.
Thirty days after the end of the treatment the individual was
discharged. Approximately 9 months after the initial treatment, the
individual suffered a relapse with multiple joint involvement.
After initial testing for sensitivity with a low dose, the
individual was given a further course of treatment with 10 mg/day
CAMPATH-1H for 10 days with significant improvement.
EXAMPLE 6
Expression of Humanised Anti-CD4 Antibody from CHO Cells
[0073] Construction of the Expression Vector pBan1: Modification of
p342-12
[0074] The complementarily determining regions from a rat IgG2b
raised against human CD4 (The New England Journal of Medicine 1990
323: 250-254) were grafted onto human heavy and light chain
frameworks (Winter et al, Nature, 1988, 322 323-327).
[0075] The cDNA encoding the humanised CD4 light chain was cloned
into pLD9 [Page and Sydenham, M. A. 1991 Biotechnology 9 64-68].
The resulting plasmid was designated p2110. The humanised CD4 heavy
chain was sequenced and cloned into a modified version of plasmid
p342-12 [Law M-F., Byrne, J. C. and Hinley, P. M. 1983 Mol. Cell.
Biol. 3 2110-2115). Plasmid p342-12 was digested with BamH1 to
remove the 7.4 kbp fragment containing part of the BPV-1 genome.
The backbone containing the .beta.-lactamase gene and the neomycin
resistance gene under the control of the mouse metallothionine
promoter was purified and religated at the BamH1 site. This plasmid
was digested with HinDIII, incubated with the large fragment of DNA
polymerase I to remove the HinDIII site and then religated. The
.beta.-actin expression cassette, containing the .beta.-actin
promoter immediately upstream of a unique HinDIII site followed by
the polyadenylation signal, was cloned into the BamHI site of the
modified p342-12 plasmid to generate pBan1.
[0076] Plasmid pBan1, therefore, consisted of the neomycin
resistance gene, the .beta.-lactamase gene and the .beta.-actin
expression cassette containing the unique HinDIII site. The cDNA
encoding the humanised heavy chain was cloned into this site and
the resulting plasmid containing the correctly orientated insert
was designated pBanCD4H. Thus, p2110 and pBanCD4H contained a
different selectable marker and co-transfection into recipient
dhfr-CHO cells would permit the direct selection and isolation of
dhfr.sup.+/neo.sup.r colonies. Cells exhibiting this phenotype
should express functional antiCD4 antibody and could be amplified
to elevate the antibody titres.
Expression of Anti-CD4 Antibody in CHO Cells
a) Cell Culture Methods.
[0077] The dhfr.sup.- CHO line DUK-B11 [Urlaub, G. and Chasin, L.
A. 1980 Proc. Natl. Acad. Sci. USA 77 4216-4220] was propagated in
Iscoves MEM medium supplemented with 10% foetal bovine serum and 4
.mu.g each of hypoxanthine and thymidine (all Flow). After
transfection, transformants were selected in the medium described
above except that the hypoxanthine/thymidine were omitted and
dialysed foetal bovine serum was used. In addition, G418 was
included at 500 .mu.g/ml. To induce spontaneous amplification of
sequences containing and flanking the dhfr gene, MTX was added to a
concentration of 0.1 .mu.m.
b) Transfection and Amplification
[0078] The dhfr.sup.- CHO cell line DUK-B11 was co-transfected with
5 .mu.g of p2110 and 5 .mu.g of pBanCD4H using the transfectant
reagent under the conditions recommended by the manufacturer.
Transformants were selected for the dhfr.sup.+/neo.sup.r phenotype
as described above. Several hundreds of transformants were observed
and pooled. Initital titres indicated that the first round basal
transformants were secreting about 0.1 .mu.g/ml/day. This pooled
population was then cultured in the presence of 0.1 .mu.M MTX for
about 14 days. Resistant colonies were again pooled and assayed.
Expression had increased some 100 fold, the pooled, amplified
colonies producing about 10-12 .mu.g/ml/day. In order to obtain
stable, clonal cell lines giving high antibody titres, the
resistant pools were cloned by limiting dilution in 96-well plates.
Fifty single colonies were identified and assayed and the four
lines giving the highest titres propagated. This process of
identifying highly expressing clones within the resistant
population produced a line designated D419 which expressed the
anti-CD4 antibody at about 20 .mu.g/ml/day.
Characterisation of dhfr.sup.+/neo.sup.r cell lines i)
Determination of copy number and steady state transcription levels
by slot blot analysis of DNA and RNA.
[0079] Whole cell RNA and DNA was prepared from the various stages
of amplification as described by Maniatis et al. [1982 Molecular
Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory, Cold
Spring Harbour, N.Y.]. After fixing onto nitrocellulose filters,
the nucleic acids were probed with [.sup.32-P]-.alpha.ATP labelled
DNA sequences of the heavy chain, the dhfr gene and the
.beta.-actin gene as a control "housekeeping" gene to eliminate
artifacts due to loading errors.
[0080] Inititally, the uncloned 0.1 .mu.M MTX amplified pool was
compared to the first round unamplified transformants and the
untransformed parental B11 cells, with the probes described.
Accordingly, no DNA signal was detected in the parental line when
probed with the heavy chain but a weak signal was detected for
dhfr. This is due to the single, non-functional dhfr allele in the
B11 cell line. As a result, no RNA signal was detected with either
probe. In contrast, a strong signal was detected with both probes
on RNA and DNA in the primary transformants which reflects the
start of expression. A very significant increase in copy number and
steady state levels of RNA of heavy chain and dhfr is observed in
the uncloned amplified pool. This accurately correlates with the
observed increase in expression. Steady state levels of
.beta.3-actin RNA were consistent in all three lines examined.
[0081] A similar comparison was made between the four highest
expressing cloned cell lines. A strong signal was detected on both
the RNA and the DNA blots. However, although the DA19 line was
expressing twice as much antibody as a line designated D423, this
difference was not in either the copy number or steady state levels
of RNA. There are two possible explanations for this observation;
the first is that the DNA in the DA19 line has integrated at a site
in the genome at which it is under the influence of an enhancer.
However, this presumably would be reflected in elevated levels of
RNA. The more likely explanation is that in the replication and
duplication of the tandem arrays in the line D423, some of the
copies of the dhfr/antibody cassette have undergone re-arrangement
and are non-functional and truncated. This is not uncommon since
the site of integration of heterologous genes is often at
breakpoints in the chromosomes such as telomeres which are known to
be "hot spots" for such re-arrangements. This could be resolved by
Northern and Southern analysis.
ii) Protein synthesis and secretion of anti-CD4 antibody in the
D419 line
[0082] The clonal D419 line was labelled with .sup.35S-methionine
and cysteine and the intracellular and secreted antibody extracted
by immunoprecipitation with appropriate antibodies. Following
electrophoresis on reducing SDS-PAGE gels, the gels were dried and
the signal detected by autoradiography.
[0083] It was clear from the result that both heavy and light chain
are efficiently synthesised. Intracellularly, there need not be
stochiometry between heavy and light chains since the two associate
as they pass through the secretory organelles. However, close
stochiometry is observed in the secreted material.
* * * * *