U.S. patent application number 12/240541 was filed with the patent office on 2009-05-28 for isolating cells expressing secreted proteins.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. Invention is credited to Gang Chen, James P. Fandl, Neil Stahl, George D. Yancopoulos.
Application Number | 20090137416 12/240541 |
Document ID | / |
Family ID | 40670242 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090137416 |
Kind Code |
A1 |
Fandl; James P. ; et
al. |
May 28, 2009 |
Isolating Cells Expressing Secreted Proteins
Abstract
A method of detecting and isolating cells that produce a
secreted protein of interest (POI) that has a T cell receptor
variable domain, comprising: a) constructing a cell line
transiently or stably expressing a cell surface capture molecule,
which binds the POI, by transfecting the cell line with a nucleic
acid that encodes such cell surface capture molecule; b)
transfecting said cell simultaneously or subsequently with a second
nucleic acid that encodes a POI wherein such POI is secreted; c)
detecting the surface-displayed POI by contacting the cells with a
detection molecule, which binds the POI; and d) isolating cells
based on the detection molecule.
Inventors: |
Fandl; James P.;
(LaGrangeville, NY) ; Chen; Gang; (Yorktown
Heights, NY) ; Stahl; Neil; (Carmel, NY) ;
Yancopoulos; George D.; (Yorktown Heights, NY) |
Correspondence
Address: |
REGENERON PHARMACEUTICALS, INC
777 OLD SAW MILL RIVER ROAD
TARRYTOWN
NY
10591
US
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
40670242 |
Appl. No.: |
12/240541 |
Filed: |
September 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11434403 |
May 15, 2006 |
7435553 |
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12240541 |
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11099158 |
Apr 5, 2005 |
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11434403 |
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10050279 |
Jan 16, 2002 |
6919183 |
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11099158 |
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60261999 |
Jan 16, 2001 |
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Current U.S.
Class: |
506/9 ;
435/7.21 |
Current CPC
Class: |
G01N 33/566 20130101;
C12N 15/65 20130101; G01N 33/6854 20130101; G01N 2333/7051
20130101; G01N 33/56966 20130101 |
Class at
Publication: |
506/9 ;
435/7.21 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method of detecting and isolating a cell that produces a
secreted protein of interest, comprising: (a) constructing a cell
line transiently or stably expressing a cell surface capture
molecule that binds a protein of interest, by transfecting the cell
line with a first nucleic acid that encodes said cell surface
capture molecule; (b) transfecting the cell line constructed in (a)
with a second nucleic acid that encodes a protein of interest that
comprises a T cell receptor variable region, wherein the protein of
interest is capable of being secreted; (c) allowing the cell line
to express the protein of interest; (d) detecting the protein of
interest displayed on the surface of a cell of the transfected cell
line produced in (b) by contacting said transfected cell line
produced in (b) with a detection molecule that binds the protein of
interest, wherein said contacting is carried out in the presence of
a blocking molecule that binds the cell surface capture molecule
and prevents binding of said protein of interest to said cell
surface capture molecule but does not bind to said detection
molecule; (e) isolating the cell bearing the surface displayed
protein of interest detected in step (d).
2. The method of claim 1 wherein the protein of interest is a
soluble T cell receptor or a TCR-Fc.
3. The method of claim 2, wherein the protein of interest is a
TCR-Fc.
4. The method of claim 1, wherein the nucleic acid comprising the T
cell receptor variable region that encodes the protein of interest
is obtained from a DNA library.
5. The method of claim 1, wherein the cell surface capture molecule
is selected from an antibody that binds a T cell receptor variable
region, an Fc receptor, protein A, protein G, protein L, protein H,
and an anti-immunoglobulin ScFv.
6. The method of claim 5 wherein the cell surface capture molecule
is an Fc receptor.
7. The method of claim 5, wherein the Fc receptor is
hFc.gamma.R1.
8. The method of claim 5, further comprising adding a membrane
anchor to a protein such that it remains anchored in a cell
membrane, exposed to the outside of the cell, and functions as a
cell surface capture molecule.
9. The method of claim 8, wherein the membrane anchor is a
transmembrane anchor or a GPI link.
10. The method of claim 1, wherein the detection molecule(s) are
two molecules that bind each other and are differentially
labeled.
11. The method of claim 1, wherein a blocking molecule which binds
the cell surface capture molecule or protein of interest is added
to reduce the binding of the protein of interest to a neighboring
cell.
12. A method of detecting and/or isolating a eukaryotic cell that
produces a secreted protein of interest, comprising: (a) providing
a cell comprising a nucleic acid that encodes a protein of interest
comprising a T cell receptor variable region and a nucleic acid
that encodes a cell surface capture molecule capable of binding the
protein of interest, wherein the nucleic acid encoding the protein
of interest or the nucleic acid encoding the cell surface capture
molecule, or both, are transfected into the cell; (b) culturing the
cell under conditions in which the protein of interest and cell
surface capture molecule are expressed, and a protein of
interest-capture molecule complex is formed intracellularly and
displayed on the cell surface; (c) contacting the cell with a
detection molecule, which binds to the protein of interest
displayed by the cell; and (d) detecting and/or isolating the cell
due to it being bound to the detection molecule.
13. The method of claim 12, wherein the providing step comprises
transfecting the nucleic acid encoding the protein of interest and
the nucleic acid encoding the cell surface capture molecule into
the cell.
14. The method of claim 12, wherein the cell is detected in step
(d) by flow cytometry.
15. The method of claim 12, wherein the detection molecule is
linked to a solid support or particle.
16. The method of claim 12, performed on a population of cells,
wherein the isolating step isolates the cells binding to the
detection molecule from the population.
17. The method claim 16, wherein the cells of the population
express different levels of the POI, and the isolating step
isolates cells based on relative expression level of the protein of
interest.
18. The method of claim 16, further comprising contacting the cells
of the population with a blocking molecule that binds the cell
surface capture molecule or the protein of interest to block
diffusion of protein of interest between cells.
19. The method of claim 12, wherein the protein of interest
comprises an Fc domain.
20. The method of claim 12, wherein the capture molecule comprises
a membrane anchor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 11/434,403 filed 15 May 2006, now U.S. Pat. No. 0,000,000,
which is a continuation of U.S. Ser. No. 11/099,158 filed 5 Apr.
2005, now abandoned, which is a divisional of U.S. Ser. No.
10/050,279 filed 16 Jan. 2002, now U.S. Pat. No. 6,919,183, which
claims the benefit under 35 USC .sctn. 119(e) of U.S. Provisional
Patent Application Ser. No. 60/261,999 filed 16 Jan. 2001, which
applications are each herein specifically incorporated by reference
in their entirety.
BACKGROUND
Field of the Invention
[0002] The field of this invention is a method for identifying and
isolating cells that produce secreted proteins. More specifically,
the method allows rapid isolation of high expression recombinant
antibody-producing cell lines, or may be applied directly to rapid
isolation of specific hybridomas.
[0003] Prior art methods for expressing a gene of interest (GOI) in
a host cell are known. Briefly, an expression vector carrying the
GOI is introduced into the cell. Following stable integration,
standard methods for isolating high expression cells may involve
collection of cell pools, hand-picking colonies from plates,
isolation of single cells by limited dilution, or other methods
known in the art. Pools or individual clones are then expanded and
screened for production of the protein of interest (POI) by direct
measurement of POI activity, by immunological detection of POI, or
by other suitable techniques. These procedures are laborious,
inefficient, expensive, and the number of clones that can be
analyzed is usually limited to a few hundred.
[0004] The large degree of heterogeneity in protein expression by
cells following stable integration requires that many individual
clones be screened in an effort to identify the rare integration
event that results in a stable, high expression production cell
line. This requirement calls for methods that enable rapid
identification and isolation of cells expressing the highest level
of protein production. Moreover, the collection of clone pools, or
hand-picked colonies, risks losing high expression cells, which
often grow more slowly, to faster growing low expression cells.
Therefore, a need exists for methods that allow rapid screening and
isolation of individual cells capable of high level expression of a
secreted POI.
[0005] Incorporation of flow cytometry into methods used for the
isolation of stable expression cell lines has improved the
capability of screening large numbers of individual clones,
however, currently available methods remain inadequate for diverse
reasons. Diffusion of the POI between cells of different
characteristics was also a problem.
BRIEF SUMMARY
[0006] The present invention describes a high-throughput screening
method for the rapid isolation of those cells that secrete protein
by directly screening for the protein of interest (POI). This
invention also allows for the convenient monitoring of POI
expression on a single-cell basis during the manufacturing process.
Furthermore, this technology can be directly applied to screening
of antibody producing cells. The technology can also be directly
applied to screening of cells producing modified T cell receptors,
such as, for example, cells that produce soluble forms of T cell
receptors.
[0007] In one aspect, the invention provides a method of detecting
and isolating cells that produce a secreted protein of interest
(POI), comprising: a) constructing a nucleic acid molecule that
encodes a cell surface capture molecule capable of binding a POI;
b) transfecting a cell expressing the POI with the nucleic acid
molecule of step a); c) detecting the surface-displayed POI by
contacting the cells with a detection molecule, where in the
detection molecule binds the POI; and d) isolating cells based on
the detection molecule.
[0008] In various embodiments, the protein of interest is a ligand,
a soluble receptor protein, a growth factor, an antibody, an Fab, a
single chain antibody (ScFv), or a fragment thereof. When the
protein of interest is an antibody, the antibody is selected from
the group consisting of IgM, IgG, IgA, IgD or IgE, as well as
various subtypes of these. In a specific embodiment, the antibody
is an anti-DII4 or an anti-IL-6 receptor antibody. In more specific
embodiments, the protein of interest is a growth factor selected
from the group consisting of Interleukin (IL)-1, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21,
Ciliary Neurotrophic Factor (CNTF), erythropoietin, Vascular
Endothelial Growth Factor (VEGF), angiopoietin 1 (Ang-1),
angiopoietin 2 (Ang-2), TNF, Interferon-gamma, GM-CSF, TGF.beta.,
and TNF Receptor.
[0009] In various embodiments, the protein of interest comprises a
variable domain of a T cell receptor. In specific embodiments, the
protein of interest is a soluble T cell receptor (sTCR), or a
protein comprising a T cell receptor extracellular domain fused to
an Fc (TCR-Fc), In a specific embodiment, the Fc is a human Fc. In
various embodiments, the protein comprises a variable domain of a T
cell receptor extracellular domain. In various embodiments, the
protein comprises a variable domain and a constant region of a T
cell receptor extracellular domain.
[0010] The nucleic acid that encodes the protein of interest may be
from any source, naturally occurring or constructed through
recombinant technology, and may be selected from a DNA library.
[0011] In various embodiments, the cell surface capture molecule is
a ligand-specific receptor, a receptor-specific ligand, an
antibody-binding protein, an antibody or antibody fragment, such as
an ScFv, or a peptide. When the capture molecule is a peptide, the
peptide may be isolated from a phage display library. In more
specific embodiments, the capture molecule may be Ang1, And2, VEGF,
Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1 or KDR), CNTF,
CNTFR-.alpha., cytokine receptor components, fusions of two or more
cytokine receptor components, or a fragments thereof. When the
capture molecule is an antibody-binding protein, the
antibody-binding protein may be an Fc receptor, an
anti-immunoglobulin antibody, an anti-immunoglobulin ScFv, Protein
A, Protein L, Protein G, Protein H or functional fragments
thereof.
[0012] In various embodiments where the protein of interest
comprises a T cell receptor variable domain, the cell surface
capture molecule comprises an Fc receptor or a membrane-associated
antigen capable of being recognized by the variable domain of the T
cell receptor.
[0013] In several embodiments, the methods of the invention further
comprise a membrane anchor that serves to anchor the POI to the
cell membrane, exposed to the outside of the cell, and thus
functions as a cell surface capture molecule. In specific
embodiments, the membrane anchor is a transmembrane anchor or a GPI
link. The membrane anchor may be native to the cell, recombinant,
or synthetic.
[0014] In various embodiments, the protein of interest comprises a
T cell receptor variable region, and the cell surface capture
molecule comprises a membrane-associated antigen. In a specific
embodiment, the membrane-associated antigen is a recombinant fusion
protein comprising an antigen capable of being recognized by the T
cell receptor variable region fused to a membrane anchor wherein
the antigen is associated with the cell surface. In a specific
embodiment, the recombinant fusion protein comprises an antigen
fused to a transmembrane anchor or a GPI link. In another specific
embodiment, the cell surface capture molecule comprises a
recombinant fusion protein comprising an membrane anchor and an
antigen that is capable of binding to a major histocompatibility
(MHC) molecule, including but not limited to, for example, tumor
antigens and self proteins of transformed phenotype.
[0015] In further embodiments, a signal sequence is added to the
amino terminus of a POI, such that the protein is transported to
the cell surface, and functions as a cell surface capture molecule.
The signal sequence may be native to the cell, recombinant, or
synthetic.
[0016] In various embodiments, a blocking molecule which binds the
cell surface capture molecule is added to reduce the diffusion of
the POI from the expressing cell to a neighboring cell. In another
embodiment, the diffusion of the POI from the expressing cell to a
neighboring cell and its adherence to that cell is reduced by
increasing the viscosity of the media.
[0017] The cell isolated by the methods of the invention may be an
antibody-producing cell fused to an immortalized cell. In more
specific embodiments, the antibody-producing cell is a B-cell or
derivative thereof. A B-cell derivative may be a plasma cell, a
hybridoma, a myeloma, or a recombinant cell.
[0018] In addition, the methods of the invention are useful for
identification of B-cells and derivatives thereof, or hybridomas
that express secreted antibodies of a desired specificity, affinity
or isotype. The invention can also be used for isolation of cells
that express desired levels of an antibody or antibody
fragments.
[0019] Detection of the cells with the displayed POI may be
accomplished through the use of any molecule capable of directly or
indirectly binding the displayed POI. Such detection molecules may
facilitate the detection and/or isolation of the cells displaying
the POI. In one embodiment, two molecules that bind each other and
are deferentially labeled are utilized. The detection and/or
isolation may be accomplished through standard techniques known in
the art.
[0020] In another aspect, the invention features a method of
detecting and isolating cells that produce a secreted protein of
interest (POI), comprising: a) transfecting a cell with a nucleic
acid that encodes a cell surface capture molecule, wherein the cell
surface capture molecule is capable of binding the POI; b)
transfecting the cell of a) simultaneously or subsequently with a
second nucleic acid that encodes a POI wherein the POI is expressed
and secreted; c) detecting the surface-displayed POI by contacting
the cell with a detection molecule, which binds the POI; and d)
isolating cells based on the detection molecule.
[0021] In another aspect, the invention features a method of
detecting and isolating cells that produce a POI, comprising: a)
detecting a cell that expresses a cell surface capture molecule in
high yield; b) isolating and culturing the cell detected in (a); c)
transfecting the cell in (b) with a nucleic acid that encodes a POI
wherein such POI is secreted; d) detecting the surface-displayed
POI by contacting the cells with a detection molecule which binds
the POI; and e) isolating cells based on the detection
molecule.
[0022] In another aspect, the invention provides a method of
detecting and isolating cells that produce high levels of protein
of interest (POI), comprising: a) transfecting cells with a nucleic
acid that encodes such cell surface capture molecule capable of
binding the POI, wherein the cell expresses the POI; b) detecting a
cell from (a) that expresses said cell surface capture molecule in
high yield; c) isolating and culturing a high yield cell; d)
detecting the surface-displayed POI by contacting the cell with a
detection molecule binds the POI; and e) isolating the detected
cell.
[0023] In another aspect, the invention provides a method of
detecting and isolating cells that produce high levels of an
immunoglobulin, comprising: (a) transfecting cells with a nucleic
acid that encodes a cell surface capture molecule capable of
binding the immunoglobulin, wherein the cell expresses the
immunoglobulin; (b) detecting a cell of (a) that expresses the
surface capture molecule in high yield; (c) isolating and culturing
the cell that expresses the surface capture molecule in high yield;
(d) detecting the immunoglobulin on the surface of the isolated and
cultured cell of step (c) with a detection molecule that binds the
immunoglobulin; and (e) isolating the cell detected in step (d)
that bears the detected immunoglobulin on its surface.
[0024] In another aspect, a method for detecting cells that produce
a desired level of an affinity agent that comprises a T-cell
receptor (TCR) variable region is provided.
[0025] In another aspect, a method for detecting cells that produce
a desired level of a TCR-Fc is provided, comprising: (a)
transfecting cells with a nucleic acid that encodes an Fc receptor
capable of binding a TCR-Fc, wherein the cell expresses an antigen
recognized by the TCR-Fc; (b) detecting a cell of (a) that
expresses the TCR-Fc in high yield; (c) isolating and culturing the
cell that expresses the TCR-Fc in high yield; (d) detecting the
antigen on the surface of the isolated and cultured cell of step
(c) with a detection molecule; and (e) isolating the cell detected
in step (d) that bears the detected antigen on its surface.
[0026] In various embodiments, the TCR is selected from a human TCR
and a rodent TCR such as a rat, mouse, or hamster TCR. In a
specific embodiment the Fc is a human Fc. In another specific
embodiment, the Fc is a human Fc and the Fc receptor is a high
affinity human Fc receptor. In a specific embodiment, the high
affinity human Fc receptor is a human Fc.gamma.RI.
[0027] In various embodiments, the cell surface capture protein is
surface-bound antigen. In a specific embodiment, the antigen is
bound to the surface by fusion to a transmembrane domain or a GPI
linker.
[0028] Other objects and advantages will become apparent from a
review of the ensuing detailed description.
DETAILED DESCRIPTION
[0029] Before the present methods are described, it is to be
understood that this invention is not limited to particular
methods, and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0030] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus for example, a
reference to "a method" includes one or more methods, and/or steps
of the type described herein and/or which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to describe in their entirety.
[0032] General Description
[0033] The method of the invention provides substantial advantages
over current methods for isolation and identification of
protein-secreting cells. For example, cells that secrete antibodies
may be rapidly and conveniently isolated based on desired
specificity, avidity, or isotype. Furthermore, the amount of
secreted protein produced may be directly quantified, unlike many
methods in the prior art wherein production of secreted protein is
indirectly quantified.
[0034] Recently, two additional methods that utilize flow cytometry
have been developed for the high throughput isolation of stable
high expression cell lines. The first method involves modification
of the expression plasmid to include a transcriptional read out for
the GOI mRNA. This is most often accomplished by inserting an
internal ribosomal entry site (IRES) and a gene whose protein
product is easily monitored by flow cytometry, most frequently
green fluorescent protein (GFP), between the stop codon of the GOI
and the terminal poly A site (Meng et al. (2000) Gene 242:201). The
presence of an IRES allows the POI and GFP to be translated from
the same mRNA. Therefore, the expression level of the GFP gene is
indirectly related to the mRNA level for the GOI. Clones that
accumulate the GFP at high levels are isolated by flow cytometry
and then screened for POI production. Because this method depends
on the coupling of GOI expression to the reporter gene by use of an
IRES in a recombinant construction, it is not applicable to the
isolation of hybridomas.
[0035] The use of flow cytometry in the isolation of expression
clones allows for the rapid analysis of large numbers of clones in
a high throughput format. Moreover, use of flow cytometry
significantly reduces the direct handling of cells. Unfortunately,
the level of GFP production is not a direct measure of the
production level of the POI. Various mechanisms may uncouple the
production of secreted POI from accumulation of GFP. Differences in
production of the POI and the GFP reporter may result from
differences in the translation efficiency of the two genes,
secretion efficiency of the POI, or stability of the polycistronic
mRNA.
[0036] Another method that uses flow cytometry to isolate
expression clones involves encapsulation of cells within agarose
microdrops (Weaver et al. (1990) Methods Enzymol. 2:234). In this
method biotinylated antibodies specific for the POI are bound to
the biotinylated agarose through streptavidin such that secreted
POI is captured and retained within the microdrop (Gray et al.,
(1995) J. Immunol. Methods 182:155). The trapped POI is detected by
immuno-staining with an antibody specific for the POI. To reduce
the encapsulating agarose from absorbing POI secreted from adjacent
cells, the cells are placed in a low-permeability medium. Those
cells with the highest antibody staining of the POI in the
embedding agarose are identified and isolated by flow cytometry.
The gel microdrop approach screens cells directly for their ability
to secrete POI, rather than indirectly screening for expression of
GOI mRNA, but requires the availability of suitable antibodies for
trapping and staining the secreted POI and the procedure requires
special equipment to generate the agarose gel microdrops. Moreover,
some cells may be sensitive to the encapsulation process.
[0037] A variation of this method circumvents the requirement for
embedding cells in a matrix by directly binding an antibody,
specific for the POI, to the cell surface (Manz et al. (1995) PNAS
92:1921-1925). In this method, non-specific biotinylation of cell
surface proteins with biotin-hydroxysuccinimide ester is followed
by contact with a streptavidin-conjugated antibody capable of
binding the POI. Cells secreting the POI become decorated with the
POI which is then detected with an appropriately labeled second
antibody. However, diffusion of POI between neighboring cells is
problematic, and this method also requires a high viscosity medium
to reduce diffusion of POI away from expressing cells. Because
these high viscosity media are required for discriminating cells,
the cells must be washed and placed in a medium suitable for cell
sorting if so desired.
[0038] The problems associated with identification and isolation of
high expression recombinant cell lines especially applies to the
isolation of hybridomas that express an antibody of interest.
However, the identification of useful hybridomas includes several
additional problems; they must be screened first for
antigen-binding activity, then for immunoglobulin isotype.
Moreover, GFP-based methods are not applicable to the
identification and isolation of hybridomas because construction of
hybridomas does not include a recombinant construct such that
expression of the antibody genes can be linked to a transcriptional
reporter such as GFP. Hybridoma screening is a slow, laborious
endeavor where the number of clones screened is limited by existing
technologies.
[0039] The instant invention describes a novel and previously
unknown method of identifying and isolating cells that produce
secreted proteins. The invention is based on the production of a
cell line that expresses a molecule, localized to the cell surface,
which binds the POI. The cell surface-displayed POI can then be
detected by labeling with various detection molecules. The amount
of POI displayed on the cell surface, under specific conditions, is
a direct measure of the total amount of POI secreted. POI producers
may then be isolated from non-producers, and levels of production
or POI characteristics may be differentiated. The advantage of the
invention is that it directly quantifies the secreted POI rather
than indirectly measuring the mRNA.
[0040] This invention relates to the construction or use of cells
that express cell surface capture molecules which bind various
secreted POIs in the same cell that produces the POI. As the cell
secretes the POI, these cell surface capture molecules bind it, or
complexes of POI and cell surface capture molecules may form
intracellularly and then get secreted. Binding may occur in an
autocrine manner or while being secreted. The cells that produce
the secreted POI may then be identified and isolated. Such
identification and isolation may be based on characteristics of the
POI, production of the POI or lack thereof, or by specified levels
of production. The cell surface capture molecule and/or the POI may
be produced by the cell in its native state, or the cell surface
capture molecules and/or the POI may be recombinantly produced.
Through the construction or use of such a cell, any secreted
protein may be captured by the cell surface capture molecule
provided there is a corresponding affinity between the two. As
explained further, any molecule may be manipulated such that it can
be used as a cell surface capture molecule. Therefore, this
invention may be utilized to isolate any cell that secretes a
protein.
[0041] Most any protein has the capacity to function as a cell
surface capture molecule as described by the invention. What is
necessary is the ability of the desired protein to be anchored to
the cell membrane and exposed to the extracellular space. If the
desired cell has a signal sequence then only a membrane anchor,
including but not limited to a transmembrane anchor or a GPI
linkage signal, need be added to the cell surface capture molecule
such that it remains anchored in the cell membrane exposed to the
outside of the cell. Furthermore, if the desired protein lacks a
signal sequence, a signal sequence may be added to the amino
terminus of the desired protein, such that it is transported to the
cell surface. A signal sequence and a membrane anchor may be native
to the cell, recombinant, or synthetic.
[0042] Cells often secrete a wide variety of proteins, endogenously
or following the introduction of recombinant DNA. Any secreted
protein may be identified and the cell producing it may be isolated
according to the method of this invention. Such secreted proteins
include but are not limited to growth factors, growth factor
receptors, ligands, soluble receptor components, antibodies, sTCRs,
TCR-Fc's, and peptide hormones. Such secreted proteins may or may
not be recombinant. That is, the secretion of some proteins of
interest from the desired cell may not require the introduction of
additional nucleotide sequences. For example, the secretion of
antibodies from B-cells or plasma cells is not the result of
introduction of recombinant nucleotide sequences into the B-cell or
plasma cell. Recombinant secreted proteins may be produced by
standard molecular biology techniques well known to the skilled
artisan (see e.g., Sambrook, J., E. F. Fritsch And T. Maniatis.
Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2,
and 3, 1989; Current Protocols in Molecular Biology, Eds. Ausubel
et al., Greene Publ. Assoc., Wiley Interscience, NY). These
secreted proteins are useful for many commercial and research
purposes. This invention encompasses the production of such
secreted proteins through the methodologies of the invention.
Detection of the cells with the displayed POI may be accomplished
through the use of any molecule capable of directly or indirectly
binding the displayed POI. Such detection molecules may facilitate
the detection and/or isolation of the cells displaying the POI.
[0043] The invention is applicable to the isolation of, inter alia,
a) ligand-producing cells by using the ligand-specific receptor as
the cell surface capture molecule, b) soluble receptor-producing
cells by using a surface bound receptor-specific ligand as the cell
surface capture molecule, c) antibody-producing cells by using an
antibody-binding protein as the cell surface capture molecule, d)
sTCR's by using an s-TCR-binding protein (e.g., and antigen
recognized by the TCR) as the cell surface capture molecule, or e)
TCR-Fc's, by using an Fc-binding protein as a cell surface capture
molecule.
[0044] In accordance with the methodology of this invention, a cell
is first transfected with a vector containing a nucleotide sequence
that encodes a cell surface capture molecule that is capable of
binding the secreted POI, under conditions in which such cell
surface capture molecule is expressed. Transfected cells which are
appropriate producers of such cell surface capture molecules are
then detected and isolated, and such cells are cultured. These
cells may either naturally produce the POI, or the POI may be
recombinantly produced. If the cells naturally produce the POI,
they are ready for detection and isolation. If the POI is to be
recombinantly produced, then the isolated and cultured cells
expressing the specified cell surface capture molecule are
transfected with second nucleotide sequence that encodes the
secreted POI, under conditions in which the secreted POI is
expressed. Upon expression, the secreted POI binds to the cell
surface capture molecules and the cells displaying bound POI are
detected and isolated.
[0045] If the POI is naturally produced by the cell, the cell will
not be transfected with nucleotide sequence encoding the POI.
Therefore, this aspect of the invention is applicable to any and
all cells producing a POI. In addition, if the cell surface capture
molecule is naturally produced by the cell, the cell need not be
transfected with nucleotide sequences encoding the cell surface
capture molecule. Therefore, this aspect of the invention is
applicable to any and all cells producing a cell surface capture
molecule.
[0046] A wide variety of host cells may be transfected. These cells
may be either of eukaryotic or of prokaryotic origin. The cells
will often be immortalized eukaryotic cells, and in particular,
mammalian cells, for example monkey kidney cells (COS), Chinese
hamster ovary cells (CHO), HeLa cells, baby hamster kidney cells
(BHK), human embryonic kidney cells (HEK293), leukocytes, myelomas,
cell lines transfected with adenovirus genes, for example, AD5 E1,
including but not limited to immortalized human retinal cells
transfected with an adenovirus gene, for example, PER.C6.TM. cells,
and embryonic stem cells. The cells may also be non mammalian cells
including bacterial, fungi, yeast and insect cells, including, but
not limited to, for example Escherichia coli, Bacillus subtilus,
Aspergillus species, Saccharomyces cerevisiae, and Pichia pastoris.
All cells may be grown in culture trays medium under appropriate
conditions or in a synergistic host. The most desirable cells will
be mammalian cells capable of culture.
[0047] The secreted POI bound to the cell surface capture molecule
may be detected and isolated by various techniques known in the
art. Cultures cells displaying the secreted POI may be contacted
with (a) molecule(s) capable of directly or indirectly binding the
secreted POI wherein such detection molecule(s) may contain a
detection label, such as, for example, a chromogenic, fluorogenic,
colored, fluorescent, or magnetic label. The label bound to the
detection molecule may be detected and the cell isolated using
various methods. Most preferably, within a cell population the
label will be detected and the cell isolated utilizing flow
cytometry. Alternatively, the detection molecule may be used for
the direct isolation of cells displaying the POI. This may be
accomplished by conjugation of the detection molecule to a culture
plate, paramagnetic molecules, or any other particle or solid
support. In addition, displayed POI may be detected directly by a
property of the detection molecule or the POI.
[0048] In one embodiment, two detection molecules that bind each
other and are differentially labeled are used to detect a displayed
secreted POI that blocks that interaction. If a cell displays a
secreted POI that binds the first detection molecule and blocks the
interaction between the first and second detection molecule, that
cell may be isolated based on the presence of only the first
detection molecule on its surface. On the other hand, if a cell
displays a secreted POI that binds the first detection molecule but
does not block the interaction between the first and second
detection molecule, that cell may be isolated based on the presence
of both detection molecules on its surface. For example, antibody
producing cells expressing antibodies that specifically block, or
do not block, the formation of a receptor-ligand complex may be
identified. If the detection molecules are a receptor and its
ligand which are differentially labeled, then an antibody producing
cell that expresses antibodies that block the receptor-ligand
complex from forming may be detected by the presence of one label
on its surface, whereas an antibody producing cell that expresses
antibodies that do not block the receptor-ligand complex from
forming may be detected by the presence of both labels on its
surface.
[0049] In any of the embodiments and with regards to isolating
expressing cells from non-expressing cells or lesser expressing
cells, one of the principal difficulties, when the POI is a
secreted protein, is diffusion of POI between neighboring cells.
Therefore, it is critical that any system that is designed to
capture the secreted POI on the cell surface must prevent the
diffusion of the POI from the expressing cell to a neighboring cell
and its adherence to that cell. If diffusion is allowed to occur,
and neighboring cells become decorated with the secreted POI, then
separation of cells based upon the degree of POI decoration will
fail to discriminate high expressing cells from cells with low
expression levels, and may fail to effectively isolate expressing
from non-expressing cells.
[0050] Therefore one embodiment of this invention is to block the
diffusion of the secreted POI between neighboring cells. This may
be accomplished by the addition of a blocking molecule that binds
either the cell surface capture molecule or the POI and prevents
the binding of the secreted POI to the cell surface capture
molecule. In this aspect, the detection molecules do not bind the
blocking molecule. For example, if the cell surface receptor is the
hFc.gamma.RI and the secreted POI possesses the human IgG Fc
fragment, then diffusion of the secreted POI between neighboring
cells may be blocked by the addition of exogenous rat IgG to the
culture media. Detection of cells displaying secreted POI, and not
bound rat IgG, is achieved by use of antibodies specific for human
IgG Fc that do not recognize rat IgG. In another embodiment,
binding of the secreted POI between neighboring cells is reduced by
increasing the viscosity of the media.
[0051] In one embodiment of this invention, the secreted POI is not
allowed to accumulate in the media. This may be accomplished by
regulating the expression of the secreted POI and/or the cell
surface capture molecule such that brief expression of the POI
results in sufficient POI to bind the cell surface capture molecule
but insufficient amounts for diffusion. In another embodiment,
cells may be removed from the media containing accumulated POI, the
POI bound to the cells is stripped off, and POI expression is
allowed to continue for a limited period of time such that secreted
POI does not accumulate in the media. Proteins may be stripped by
methods known in the art, for example, washing cells with low pH
buffer.
[0052] According to this invention, those cells in a cell
population that bind the most detection molecules also express the
most secreted POI. In fact, the more POI that an individual cell
secretes, the more POI is displayed on the cell surface. This
correlation between the amount of surface-displayed POI and the
expression level of the POI in that cell allows one to rapidly
identify cells with a desired relative expression level from a
population of cells.
[0053] In one embodiment, a DNA library may be used to express
secreted protein which may be displayed on the cell surface by the
cell surface capture molecule. For example, a library of DNA may
also be generated from the coding regions of the antibody variable
domains from B-cells isolated from immunized animals. The DNA
library may then be expressed in a cell that expresses a cell
surface capture molecule specific for antibodies such that clones
of desired specificity, isotype, or avidity may be identified and
isolated by the method of the invention. In another embodiment, a
library of DNA may be generated from the coding regions of T cell
receptor variable domains from T-cells, and fused to, for example,
an Fc capable of binding to an Fc-binding protein. The DNA library
may them be expressed in a cell that expresses an Fc-binding
protein such that clones of desired specificity, isotype, or
avidity may be identified and isolated as described herein.
[0054] In another embodiment, transgenic mammals may be created
that express a particular cell surface capture molecule in one or
more cell types. The cells from such transgenic mammals may then be
screened directly for the production of a POI. For example, it may
be desirable to express a cell surface capture molecule, specific
for antibodies, in plasma cells. Accordingly, plasma cells from
immunized mice may be harvested and those cells producing
antibodies specific to the desired antigen may be isolated by the
method of the invention.
[0055] In a further embodiment of the invention, antibody
production is measured through the use of a CHO cell line that
expresses the human Fc.gamma.R1 receptor (Fc.gamma.RI) which binds
the particular antibody or TCR-Fc that is the POI.
[0056] In another aspect of the invention, the protein of interest
comprises one or more T cell receptor variable domains or a soluble
T cell receptor. The one or more T cell receptor variable domains
can be covalently linked to a moiety that can bind a cell surface
capture protein. In a specific embodiment, the one or more T cell
receptor variable domains are fused to an Fc sequence, e.g., a
human Fc sequence, and the cell surface capture protein is an Fc
receptor, e.g., an Fc.gamma.R.
[0057] The general structures of TCR variable domains are known
(see, e.g., Lefranc and Lefranc (2001) The T Cell Receptor
FactsBook, Academic Press, incorporated herein by reference; see,
e.g., pp. 17-20; see also, Lefranc et al. (2003) IMGT unique
numbering for immunoglobulin and T cell receptor variable domains
and Ig superfamily V-like domains, Developmental and Comparative
Immunology 27:55-77, and Lefranc et al. (2005) IMGT unique
numbering for immunoglobulin and T cell receptor constant domains
and Ig superfamily C-like domains, Developmental and Comparative
Immunology 29:185-203, each incorporated herein by reference). In
one embodiment, a TCR variable domain of a TCR-Fc comprises an
N-terminal region having a variable domain of 104-125 amino acids.
In another embodiment, the TCR-Fc further comprises a TCR constant
region comprising 91-129 amino acids. In another embodiment, the
TCR-Fc further comprises a connecting peptide comprising 21-62
amino acids.
[0058] In one embodiment, the Fc sequence is fused directly or
through a linker to the TCR variable domain. In another embodiment,
the TCR-Fc comprises a TCR variable region and a TCR constant
region, and the Fc sequence is fused directly or through a linker
to the TCR constant region. In another embodiment, the TCR-Fc
comprises a TCR variable region, a TCR constant region, and a
connecting peptide, and the Fc sequence is fused directly or
through a linker to the connecting peptide.
[0059] The sTCR, TCR-Fc, or fusion protein comprising one or more T
cell receptor variable regions can be selected so as to
specifically bind an antigen of interest, for example, a substance
produced by a tumor cell, for example, tumor cell substance that is
capable of producing an immune response in a host. In a specific
embodiment, the antigen is an antigen that is present on the
surface of a tumor cell (i.e., a tumor antigen), is recognized by a
T cell, and that produces an immune response in a host. Tumor
antigens include, for example, alphafetoprotein (AFP),
carcinoembryonic antigen (CEA), MUC-1, epithelial tumor antigen
(ETA), tyrosinase (e.g., for malignant melanoma),
melanoma-associated antigen (MAGE), and mutated or abnormal forms
of other proteins such as, for example, ras, p53, etc.
[0060] In one embodiment, the POI is a TCR-Fc, and the TCR-Fc
comprises a TCR .alpha. chain variable region fused to an Fc
sequence and a TCR .beta. chain fused to the Fc sequence (each
directly or through a linker), wherein the TCR .alpha. chain-Fc
fusion and the TCR .beta. chain-Fc fusion associate to form an
.alpha..beta. TCR-Fc. In a specific embodiment, the .alpha..beta.
TCR-Fc comprises the following two polypeptides: (1) a TCR .alpha.
chain variable region fused to a TCR .alpha. chain constant region
fused to an Fc sequence, and (2) a TCR .beta. chain variable region
fused to a TCR .beta. chain constant region fused to an Fc
sequence.
[0061] In another embodiment, the POI is a TCR-Fc having a TCR
.alpha. variable region and a TCR .beta. variable region and,
optionally, a TCR .alpha. constant region and/or a TCR .beta.
constant region. In a specific embodiment, the TCR-Fc is encoded by
a nucleic acid comprising (5' to 3') a TCR .alpha. variable region
sequence, optionally followed by a TCR .alpha. constant region
sequence, a TCR .beta. variable region sequence, optionally
followed by a TCR .beta. constant region sequence, optionally a
linker, then an Fc sequence. In a specific embodiment, the TCR-Fc
is encoded by a nucleic acid comprising (5' to 3') a TCR .beta.
variable region sequence, optionally followed by a TCR .beta.
constant region sequence, a TCR .alpha. variable region sequence,
optionally followed by a TCR .alpha. constant region sequence,
optionally a linker, then an Fc sequence. In various embodiments,
constructs encoding TCR-Fc's are preceded by signal sequences,
e.g., secretion signal sequences, to render them secretable.
[0062] In another embodiment, the POI is a TCR-Fc, and the TCR-Fc
comprises a TCR-Fc comprising a TCR .gamma. chain fused to an Fc
sequence and a TCR .delta. chain variable region fused to an Fc
sequence to form a .gamma..delta. TCR-Fc. In a specific embodiment,
the .gamma..delta. TCR-Fc comprises the following two polypeptides:
a TCR a chain variable region fused to a TCR .gamma. chain constant
region fused to an Fc sequence, and (2) a TCR .delta. chain
variable region fused to a TCR .delta. chain constant region fused
to an Fc sequence.
[0063] T cell receptor variable regions can be identified and/or
cloned by any method known in the art. The T cell receptor variable
regions of the protein of interest are obtainable, for example, by
expressing rearranged T cell receptor variable region DNA in a
cell, for example, fused to a human Fc sequence. Rearranged T cell
receptor variable regions specific for a particular antigen can be
obtained by any suitable method known in the art (see references
below), for example, by exposing a mouse to an antigen and
isolating T cells of the mouse, making hybridomas of the T cells of
the mouse, and screening the hybridomas with the antigen of
interest to obtain a hybridoma of interest. Rearranged T cell
variable regions specific for the antigen of interest can be cloned
from the hybridoma(s) of interest. T cell receptor variable regions
specific for an antigen can also be identified using phage display
technology, for example, as provided in references below. The
variable regions can then be cloned and fused, for example, to a
human Fc to make a protein of interest that can bind to a cell
surface capture molecule that is an Fc.gamma.R.
[0064] Methods for identifying and/or cloning T cell receptor
variable regions are described, for example, in U.S. Pat. No.
5,635,354 (primers and cloning methods); Genevee et al. (1992) An
experimentally validated panel of subfamily-specific
oligonucleotide primers (V.alpha.1-w29/V.beta.1-w24) for the study
of human T cell receptor variable V gene segment usage by
polymerase chain reaction, Eur. J. Immunol. 22:1261-1269 (primers
and cloning methods); Gorski et al. (1994) Circulating T Cell
Repertoire Complexity in Normal Individuals and Bone Marrow
Recipients Analyzed by CDR3 Size Spectratyping, J. Immunol.
152:5109-5119 (primers and cloning methods); Johnston, S. et al.
(1995) A novel method for sequencing members of multi-gene
families, Nucleic Acids Res. 23/15:3074-3075 (primers and cloning
methods); Pannetier et al. (1995) T-cell repertoire diversity and
clonal expansions in normal and clinical samples, Immunology Today
16/4:176-181 (cloning methods); Hinz, T. and Kabelitz, D. (2000)
Identification of the T-cell receptor alpha variable (TRAV) gene(s)
in T-cell malignancies, J. Immunol. Methods 246:145-148 (cloning
methods); van Dongen et al. (2002) Design and standardization of
PCR primers and protocols for detection of clonal immunoglobulin
and T-cell receptor gene recombinations in suspect
lymphoproliferations: U.S. Pat. No. 6,623,957 (cloning methods and
primers); Report of the BIOMED-2 Concerted Action BMH4-CT98-3936,
Leukemia 17:2257-2317 (primers and cloning methods); Hodges et al.
(2002) Diagnostic role of tests for T cell receptor (TCR) genes, J.
Clin. Pathol. 56:1-11 (cloning methods); Moysey, R. et al. (2004)
Amplification and one-step expression cloning of human T cell
receptor genes, Anal. Biochem. 326:284-286 (cloning methods);
Fernandes et al. (2005) Simplified Fluorescent Multiplex PCR Method
for Evaluation of the T-Cell Receptor V.beta.-Chain Repertoire,
Clin. Diag. Lab. Immunol. 12/4:477-483 (primers and cloning
methods); Li, Y. et al. (2005) Directed evolution of human T-cell
receptors with picomolar affinities by phage display, Nature
Biotech. 23/3:349-354 (primers and cloning methods); Wlodarski et
al. (2005) Pathologic clonal cytotoxic T-cell responses: nonrandom
nature of the T-cell receptor restriction in large granular
lymphocyte leukemia, Blood 106/8:2769-2780 (cloning methods);
Wlodarski et al. (2006) Molecular strategies for detection and
quantitation of clonal cytotoxic T-cell responses in aplastic
anemia and myelodysplastic syndrome, Blood 108/8:2632-2641 (primers
and cloning methods); Boria et al. (2008) Primer sets for cloning
the human repertoire of T cell Receptor Variable regions, BMC
Immunology 9:50 (primers and cloning methods); Richman, S, and
Kranz, D. (2007) Display, engineering, and applications of
antigen-specific T cell receptors, Biomolecular Engineering
24:361-373 (cloning methods). Examples of sTCRs are provided in,
for example, U.S. Pat. Nos. 6,080,840 and 7,329,731; and, Laugel, B
et al. (2005) Design of Soluble Recombinant T Cell Receptors for
Antigen Targeting and T Cell Inhibition, J. Biol. Chem.
280:1882-1892; incorporated herein by reference. Fc sequences are
disclosed herein; examples of Fc sequences, and their use in fusion
proteins, are provided, for example, in U.S. Pat. No. 6,927,044 to
Stahl et al. All of the foregoing references are incorporated
herein by reference.
EXAMPLES
[0065] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Construction of pTE084.
[0066] pTE084 was constructed by ligating the 1,436 bp Xba I
fragment from pCAE100 that encodes the human Fc.gamma.RI
(hFc.gamma.RI; GenBank accession number M21091) into the Xba I site
of pRG821. The orientation of hFc.gamma.RI in desirable plasmids
resulting from the ligation was examined by restriction mapping
with Not I, Pst I, Eco RI, and Stu I. pTE084 was designed for the
high level expression of hFc.gamma.RI, the high affinity cell
surface receptor for the Fc domain of human IgG. It contains two
independent expression cassettes. One cassette is a hFc.gamma.RI
gene driven by the CMV-MIE promoter, and the second cassette is the
neomycin phosphotransferase II (npt) gene, which confers resistance
to G418, driven by the SV40 late promoter.
[0067] Construction of a CHO K1 derivative that expresses
hFc.gamma.RI. CHO K1 cells (4.times.10.sup.6) were transfected with
pTE084 using Lipofectamine.TM. (Life Technologies; Rockville, Md.)
following manufacturer's suggestions. The cells were placed in the
culture medium (10% fetal bovine serum, 90% Ham's F-12, 2 mM
L-glutamine; all reagents were from Life Technologies, Rockville,
Md.) containing 500 .mu.g/ml G418 (Life Technologies) for 15 days.
The cells that survived G418 selection were trypsinized, pooled,
and stained with FITC-conjugated human IgG, Fc fragment (FITC-hFc;
Jackson ImmunoResearch Laboratories, West Grove, Pa.). Briefly, the
cells grown on 10 cm culture plates were washed once with
Dulbecco's phosphate-buffered saline (PBS) without calcium chloride
and magnesium chloride (Life Technologies). Three mls of 0.25%
trypsin (Life Technologies) was added to each plate. The plates
were swirled until the cells detached from the plate. Ten ml
culture medium was immediately added to each plate of the detached
cells. The cells were then collected by centrifugation at
1,000.times.g for 4 minutes. After removal of supernatant, the
cells were resuspended in 4 ml of 2 .mu.g/ml FITC-hFc diluted in
culture medium. The cells were then placed on a platform shaker and
stained for one hour at room temperature. To remove unbound
FITC-hFc, the cells were washed twice with 20 ml PBS. The degree of
FITC-hFc label on the cells was measured by flow cytometry on a
MOFLO.TM. cell sorter (Cytomation; Fort Collins, Colo.). The
FITC-hFc did not stain mock-transfected parental CHO K1 cells but
gave rise to a distribution of fluorescence in the G418-resistant,
pTE084-transfected pool. The top 1% most fluorescent cells from the
selected pool were placed into 96-well plates at 1 cell/well by
flow cytometry. Nine days later, 88 cell clones in the 96-well
plates were expanded into 24-well plates. After 3 days, the cells
in individual wells were washed once with 1 ml PBS, stained with
0.5 ml of 2 .mu.g/ml FITC-hFc for 1 hour, washed twice with 1 ml
PBS and examined for cell surface staining under a fluorescent
microscope. The thirty three most fluorescent clones were chosen,
expanded, then screened by flow cytometry.
[0068] Diffusion of secreted protein between expressing cells and
non-expressing cells among cells was blocked by adding IgG: As all
cells in a hFc.gamma.RI clonal cell line express a cell surface
hFc.gamma.RI, they all possess the ability to bind IgG or fusion
proteins consisting of the Fc domain of IgG. Because hFc.gamma.RI
binds IgG from a variety of species (van de Winkel and Anderson,
1991), a panel of animal IgGs was tested for the ability to block
the binding of a protein containing a human IgG1 (hIgG1) Fc tag
(4SC622) to hFc.gamma.RI-expressing cells. 4SC622 is a chimeric
molecule consisting of IL-2R.gamma. extracellular domain fused to
the hIL-4R.gamma. extracellular domain which is then fused to the
hIG-1 Fc domain. In this experiment, cultures of RGC1, a
hFc.gamma.RI-expressing cell line selected from CHO K1 cells that
have been stably transfected with pTE084, were incubated with 1
.mu.g/ml 4SC622 for 18 hours in the presence or absence of 1 mg/ml
IgG from different species in a 37.degree. C. tissue culture
incubator.
[0069] Cell surface binding of 4SC622 was determined by flow
cytometry after washed cells were stained with
phycoerythrin-conjugated mouse IgG1 monoclonal AG184 (PE-AG184)
specific for the hIL-2R.gamma. component of 4SC622 (BD Pharmingen;
San Diego, Calif.), following procedures outlined for cell staining
with FITC-hFc.
[0070] It was found that hIgG completely blocked 4SC622 from
binding to the hFc.gamma.R1 expressed on the surface of RGC1. Rat,
rabbit and canine-derived IgG also effectively blocked binding
whereas bovine and ovine-derived IgG did not block. The ability of
exogenously added rat IgG to block the binding of an exogenously
added hIgG1 Fc-tagged protein (4SC622) to cell surface hFc.gamma.RI
suggests that rat IgG can also block transfer between cells
expressing a hlgG1 Fc-tagged protein at different levels. To test
this, two cell lines that can be distinguished by the presence or
absence of the green fluorescent protein (EGFP) were generated from
RGC1. Briefly, to mark RGC1 cells with EGFP, 2.times.10.sup.6 RGC1
cells were co-transfected with 0.5 mg PTE073 which encodes a
hygromycin B phosphotransferase gene driven by phosphoglycerate
kinase promoter, and 5 mg pRG816-EGFP which encodes EGFP gene
driven by CMV-MIE promoter. The transfected cells were selected
with 200 .mu.g/ml hygromycin B (Sigma; St. Louis, Mo.) for two
weeks. Green fluorescent cells were isolated by flow cytometry. One
EGFP and hFc.gamma.RI-expressing clone, RGC2, was used in cell
mixing experiments. The other cell line used in these experiments,
RGC4, was generated by stable transfection of RGC1 with plasmid
pEE14.1-622. pEE14.1-622 is a plasmid in which expression of 4SC622
is driven by the CMV-MIE promoter and includes a glutamine
synthetase minigene, which confers resistance to the analog
methionine sulfoximine (MSX), and allows for selection of stable
integration events. RGC4 cells express hFc.gamma.RI on the cell
surface and secrete the hlgG1 Fc-tagged protein 4SC622. One plate
of mixed cells comprising 50% RGC2 and 50% RGC4 cells was incubated
with 1 mg/ml rat IgG for 18 hours prior to staining with PE-AG184
then examined by flow cytometry. EGFP fluorescence of RGC2 cells
shows that RGC2 cells also bind exogenously added 4SC622 (1
.mu.g/ml) as indicated by an increase in PE-AG184 fluorescence.
RGC4 did not fluoresce in the EGFP gate. Significantly, exogenously
added rat IgG did not reduce the percentage of RGC4 cells that
stained positive for cell surface 4SC622, suggesting that the
binding of 4SC622 to hFc.gamma.RI occurred while the proteins were
in transit to the cell surface. When RGC2 and RGC4 cells were
mixed, the 4SC622 protein secreted from RGC4 cells accumulated in
the medium and bound most of the RGC2 cells. However, the addition
of 1 mg/ml rat IgG significantly reduced the percentage of RGC2
cells that bound 4SC622, demonstrating that rat IgG blocked the
transfer of secreted hIgG1 Fc-tagged protein from expressing cells
to non-expressing cells.
Example 2
Cell Surface Fluorescence Correlates with the Expression Level of
4SC622
[0071] RGC1 cells (4.times.10.sup.6) were transfected with
pEE14.1-622 and a pool of stable transfectants was obtained after
selection for 2 weeks in medium comprised of 10% dialyzed fetal
bovine serum, 90% glutamine-free Dulbecco's Modified Eagle's Medium
(DMEM), 1.times.GS supplement, and 25 .mu.M MSX (All reagents were
from JRH Biosciences, Lenexa, Kans.). Rat IgG was added to the
culture medium to 1 mg/ml 18 hours prior to immunostaining. The
cells were trypsinized, washed with PBS, and stained with 1.5
.mu.g/ml of a polyclonal FITC-conjugated anti-human IgG (H+ L)
F(ab').sub.2 fragment (Jackson ImmunoResearch Laboratories) for one
hour at room temperature following procedures as described for
FITC-hFc staining in Example 1. Cell staining was then analyzed by
flow cytometry. The distribution of fluorescence suggested that the
selected pool contained cells with a wide range of 4SC622
expression levels. Cells in the top 3% (R3 bracket), 7-11% (R5
bracket), and 15-19% (R7 bracket) with respect to their
immunofluorescence were sorted into three distinct pools and
expanded for 9 days. Average 4SC622 production per cell for the
pools was determined by measuring cell numbers and 4SC622 levels in
the media after 3 days growth by an immuno-based Pandex assay
(Idexx; Westbrook, Me.) following the manufacturer's
recommendations. In the Pandex assay, fluoricon polystyrene assay
particles coated with goat anti-human IgG, g-chain specific
antibody (Sigma) were used to capture 4SC622 from the medium, and a
FITC-conjugated goat anti-human IgG, Fc specific (Sigma) was used
to detect bead-bound 4SC622. Known amounts of purified 4SC622 were
included in the assay for calibration. Cells in the top 3%, 7-11%,
and 15-19% pool were found to produce 4SC622 at 1.42, 0.36, and
0.22 pg/cell/day, respectively. Thus, there was a correlation
between cell surface 4SC622 staining and specific protein
production. This result suggests that individual cells that express
4SC622 at high levels may be obtained by isolating cells that were
stained brightest by the polyclonal FITC-conjugated anti-human IgG
(H+ L) F(ab').sub.2 fragment.
Example 3
Isolation of Expression Clones in RGC1: IL-4 Trap
[0072] To directly demonstrate the efficiency in generating clonal
cell lines with high level secreted protein production by our
methodology, clonal 4SC622 producing cell lines were generated from
RGC1. RGC1 cells (4.times.10.sup.6) were transfected with
pEE14.1-622, and selected for two weeks with 25 .mu.M MSX to obtain
a pool of stable transfectants. MSX-resistant cells were pooled and
incubated with 1 mg/ml human IgG for 18 hours, prior to staining
with PE-AG184. Six cells from the top 5% gate, as determined by
flow cytometry analysis of cell surface 4SC622 staining, were
isolated and expanded. 4SC622 production from the six clonal lines
was determined and compared to 4SC622 production from clones
obtained by hand-picking selected colonies followed by dilution
cloning and amplification. One RGC1-derived clone, RGC4, produced
4SC622 at 12 pg/cell/day. This level is similar to that of the best
4SC622 producer isolated by hand-picking and analyzing 2,700
clones. Thus, compared with hand-picking colonies, the methodology
outlined in this invention proves to be far more efficient in the
screening and cloning of high producers.
[0073] VEGF Trap. Plasmids pTE080 and pTE081 encode the genes for
VEGF Traps, hVEGFR1R2 and hVEGF-R1R3. hVEGF-R1R2 is a chimeric
molecule consisting of the first Ig domain of hVEGFR1 fused to the
second Ig domain of hVEGFR2 which is then fused to the hIg1FC
domain. hVEGFR1R3 is a chimeric molecule consisting of the first Ig
domain of hVEGFR1 fused to the second Ig domain of hVEGFR3 which is
then fused to the hIg1FC domain. In these plasmids, the gene for
the VEGF Trap is driven by the CMV-MIE promoter and a glutamine
synthetase minigene, which confers resistance to MSX, is expressed
for selection of stable integration events. RGC1 cells were
transfected with either of these plasmids and grown in medium
containing 25 .mu.M MSX for 2 weeks to select for cells in which
the plasmid has stably integrated. MSX-resistant cells were
incubated with 0.1 .mu.g/ml Ig2a and mouse IgG3 for 18 hours prior
to staining with 1.5 .mu.g/ml polyclonal FITC-conjugated anti-human
IgG (H+ L) F(ab').sub.2 fragment. Cell were stained for 1 hour then
washed twice with PBS prior to flow cytometry. Single cells were
sorted into 96-well tissue culture plates from the pool of cells
whose fluorescence was among the highest 1%. The cells in
individual wells were expanded and their productivities were
determined by Pandex assays. RGC-derived clones expressing both
hVEGFR1R2 and hVEGFR1R3 had higher specific productivities and were
isolated by screening fewer clones as compared to the
highest-expressing hand-picked MSX-resistant colonies. See Table
1.
TABLE-US-00001 TABLE I SPECIFIC PRODUCTIVITY COMPARISON Hand-picked
CHO K1 RGC1-derived Stable Cell Lines Stable Cell Lines Sp. Prod.
Sp. Prod. Transient (pg/cell/ # clones (pg/cell/ # clones Protein
(.mu.g/ml) day) screened day) screened 4SC622 1.1 12 2700 12 6
hVEGF- 33 68 190 77 62 R1R2 hVEGF- 27 5 100 22.6 42 R1R3
Example 4
Cell Surface-Bound hIgG1 Fc-Tagged Protein is Internalized by
RGC1
[0074] hFc.gamma.RI is known to induce internalization of its cell
surface-bound ligand. To analyze whether RGC1 cells could
internalize cell surface-bound 4SC622, 1 .mu.g/ml 4SC622 was added
to RGC1 cells for 1 hour and then the cells were immediately
processed for 4SC622 immunostaining with PE-AG184 and flow
cytometry analysis. Ninety-three percent of the cells stained
positive for cell surface 4SC622. Alternatively, 1 .mu.g/ml 4SC622
was added to RGC1 cells for 1 hour, then the cells were washed and
incubated in culture medium without 4SC622 with PE-AG184 for 18
hours. Flow cytometry analysis following immunostaining for 4SC622
showed that 9% of the cells retained 4SC622 on the cell surface. To
further characterize the loss of surface-bound 4SC622, purified
4SC622 protein was added to the media of RGC1 and parental CHO K1
cells, then levels of 4SC622 in the media were measured over time.
4SC622, added to 2 .mu.g/ml to the culture media in a 10 cm plate,
was significantly lower in RGC1 conditioned medium after 3 days
incubation as compared to the CHO K1 control. These results show
that the concentration of 4SC622 in the culture medium is reduced
by the presence of hFc.gamma.RI on the cell surface. The results
suggest that the depletion of 4SC622 from the media was the result
of hFc.gamma.RI-4SC622 complex internalization. This
internalization of receptor-ligand complexes may facilitate the
effective removal of all 4SC622 from non-expressing cells in the
presence of blocking IgG during the 18-hour blocking step.
Example 5
Construction of CHO K1 Cell Lines with Inducible hFc.gamma.RI
Expression
[0075] Flow cytometry-based autologous secretion trap (FASTR)
methods that utilize the hFc.gamma.RI allow rapid isolation of high
expression clones. However, if hFc.gamma.RI mediates turnover of
Fc-tagged proteins, then the realized production of the secreted
protein by engineered hFc.gamma.RI expressing cells would be higher
if hFc.gamma.RI expression could be inhibited during the production
period. To this end, a CHO K1 cell line in which the expression of
hFc.gamma.RI is induced by tetracycline, or the analog doxycycline,
was constructed. In this system, CHO K1 cells were first engineered
to express the tetracycline repressor protein (TetR) and
hFc.gamma.RI was placed under transcriptional control of a promoter
whose activity was regulated by TetR. Two tandem TetR operators
(TetO) were placed immediately downstream of the CMV-MIE
promoter/enhancer in pTE084 to generate pTE158. Transcription of
hFc.gamma.RI from the CMV-MIE promoter in pTE158 was blocked by
TetR in the absence of tetracycline or some other suitable inducer.
In the presence of inducer TetR protein was incapable of binding
TetO and transcription of hFc.gamma.RI occurs.
CHO K1 cells were transfected with pcDNA6/TR, a plasmid that
confers resistance to blasticidin in which expression of TetR
originates from the CMV-MIE promoter (Invitrogen; Carlsbad,
Calif.). After two weeks of selection with 2.5 .mu.g/ml blasticidin
(Invitrogen), the stable transfectants were pooled. This pool was
then transfected with pTE158, a plasmid that confers resistance to
G418 in which the expression of hFc.gamma.RI is dependent on a
CMV-MIE/TetO hybrid promoter. The cells consecutively transfected
with pcDNA6/TR and pTE158 were selected with 400 .mu.g/ml G418 and
2.5 .mu.g/ml blasticidin for 12 days then pooled. The pool was
induced for two days by the addition of 1 .mu.g/ml doxycycline then
stained with FITC-hFc to identify cells that express hFc.gamma.RI.
The top 5% of cells expressing hFc.gamma.RI were collected as a
pool, expanded for 6 days in the absence of doxycycline, and were
again stained with FITC-hFc for the presence of hFc.gamma.RI. Cells
that did not stain for hFc.gamma.RI were collected and expanded in
culture medium containing 1 .mu.g/ml of doxycycline for three days.
The pool was then stained for the presence of hFc.gamma.RI and were
isolated by flow cytometry. Cells that expressed the highest levels
of hFc.gamma.RI (top 1%) were sorted onto 96 well plates at one
cell per well. These cells presumably contained cell that had low
non-induced expression levels of Fc.gamma.R1 and high inducible
levels of Fc.gamma.R1. After expansion, the induction of
hFc.gamma.RI by doxycycline in 20 clones was confirmed by
immunostaining with FITC-hFc and flow cytometry. One clone was
chosen for further characterization and was named RGC10.
[0076] In the absence of doxycycline, RGC10 did not express
detectable levels of hFc.gamma.RI, whereas high levels of
hFc.gamma.RI were observed in cells that were induced with 1
.mu.g/ml of doxycycline for three days. The mean fluorescence of
RGC10 cells increased by more than 1,000 fold after induction by
doxycycline.
Example 6
Isolation of 4SC622-Producing Cell Lines from RGC10
[0077] RGC10 cells were transfected with pEE14.1-622, and
MSX-resistant cells were pooled after selection with 25 mM MSX for
two weeks. Expression of hFc.gamma.RI was induced by the addition
of 1 .mu.g/ml of doxycycline to the culture medium for three days.
One mg/ml rat IgG was added to the culture medium containing
doxycycline 18 hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+ L) F(ab').sub.2 fragment and
analysis by flow cytometry. Cells that expressed the highest levels
of 4SC622 (top 1%) were sorted into 96 well plates at 1 cell per
well. Without induction of hFc.gamma.RI expression by doxycycline,
staining with polyclonal FITC-conjugated anti-human IgG (H+ L)
F(ab').sub.2 fragment fails to detect cell surface bound 4SC622.
Sixty clones were expanded in the absence of doxycycline. The
specific productivity of the 13 highest producers was determined by
Pandex assay. The specific productivity of clone 1C2 was 17.8
pg/cell/day, significantly better than the 12 pg/cell/day observed
for the best 4SC622 cell line previously isolated using the
unregulated hFc.gamma.RI cell line RGC1.
Example 7
Sp2/0 Myeloma Cells can be Engineered to Express a Cell Surface
Capture Protein
[0078] In this example, the Sp2/0-Ag14 myeloma cell line was
engineered to stably express hFc.gamma.RI to demonstrate that the
autologous secretion trap method was applicable to cell lines other
than CHO. The gene for hFc.gamma.RI was introduced into the myeloma
cell by retroviral infection. The plasmid pLXRN (Clontech; Palo
Alto, Calif.), a retroviral DNA vector wherein a gene of interest
may be expressed from the upstream Moloney murine sarcoma virus
long terminal repeat (MoMuSV LTR) promoter, was used to generate
retrovirus encoding the hFc.gamma.RI gene. The 1,363 bp Xho I
fragment from pTE084, encoding the human Fc.gamma.RI gene, was
cloned into the Xho I site of pLXRN. A plasmid in which
hFc.gamma.RI cDNA expression was dependent on the MoMuSV LTR was
chosen and named pTE255.
[0079] Pantropic retrovirus for the expression of hFc.gamma.RI was
generated essentially following the manufacturer's guidelines. The
packaging cell line GP-293, a HEK 293-based cell line that stably
expresses the viral gag and pol proteins (Clontech; Palo Alto,
Calif.), was co-transfected with 10 mg each of pVSV-G and pTE255.
The plasmid pVSV-G allows expression of the viral envelope protein
VSV-G that confers broad host range upon the infective
particles.
[0080] Construction of Sp2-hFc.gamma.RI-4. The pantropic
hFc.gamma.RI retrovirus was used to infect 1.times.10.sup.7
Sp2/0-Ag14 myeloma cells (American Type Culture Collection;
Manassas, Va.) at a multiplicity of about 10 infective particles
per cell. Three days after infection, cells were stained for 1 hour
then washed twice with PBS prior to analysis by flow cytometry.
Those cells expressing hFc.gamma.RI, as indicated by bound
FITC-hFc, were collected as a pool by flow cytometry. The pool was
expanded for 13 days then again stained with FITC-hFc and cells
expressing hFc.gamma.RI were collected as a pool by flow cytometry.
These sorted cells were cultured in 10% fetal bovine serum 90%
Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/l glucose and
4 mM glutamine for 3 weeks, stained with FITC-hFc, and the cells
with mean fluorescence in the top 1% of the population were cloned
by single cell sorting. After expansion, 24 clones were examined by
flow cytometry for expression of hFc.gamma.RI, as described above,
and one clone, Sp2-hFc.gamma.RI-4, was chosen for additional
characterization.
[0081] Isolation of Sp2-hFc.gamma.RI-4 cells expressing 4SC622
protein. Sp2-hFc.gamma.RI-4 cells (1.times.10.sup.7) were
transfected with pTE209, a plasmid that allows constitutive
expression of 4SC622 from the CMV-MIE promoter and confers
resistance to hygromycin. The transfected cells were placed in
medium containing 10% FCS, 90% D-MEM and 400 .mu.g/ml hygromycin
for 14 days. Hygromycin-resistant cells were incubated with 1 mg/ml
rabbit IgG for eighteen hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+ L) F (ab').sub.2 fragment. Cells
were stained for 1 hour then washed twice with PBS prior to
analysis by flow cytometry. Labeled cells were collected as a pool
by flow cytometry then cultured for 5 days and sorted as described
above. Cells from the expanded pool that bound the most polyclonal
FITC-conjugated anti-human IgG (H+ L) F (ab').sub.2 fragment, top
1% population, were then cloned by single cell sorting (FIG. 17).
Production of 4SC622 from ten clones was analyzed by ELISA and all
10 clones were found to express 4SC622; clone 5H11 produced 4SC622
at 0.5 pg per cell per day. These data showed that clones secreting
4SC622 were efficiently isolated by the autologous secretion trap
method from a heterogeneous pool of cells derived from stable
transfection of Sp2-hFc.gamma.RI-4 cells with pTE209.
[0082] To confirm that 4SC622 was autologously displayed on the
surface of myeloma cells expressing both 4SC622 and hFc.gamma.RI,
clone 5H11 was incubated with 1 mg/ml rabbit IgG for 18 hours then
stained with FITC-conjugated anti-human IgG (H+ L) F(ab').sub.2
fragment and found to display cell surface 4SC622. Secreted protein
was displayed under conditions in which cross-feeding was blocked
by rabbit IgG, demonstrating the autologous display of 4SC622.
These data indicated that the autologous secretion trap method
described above was not limited to CHO cells and may be extended to
myeloma and other cell types as well.
Example 8
Protein G Chimeric Protein can Function as a Cell Surface Capture
Protein
[0083] To demonstrate the application of the autologous secretion
trap method to a cell surface capture protein other than
hFc.gamma.RI, a cell line expressing Protein G was constructed.
Protein G, from the Streptococcus strain G148, binds to all human
and mouse IgG subclasses, and as such has utility for the isolation
of recombinant cells expressing antibodies or IgG Fc fusion
proteins. To demonstrate that the Protein G IgG Fc binding domain
could be used as a cell surface capture protein capable of binding
to all human and mouse IgG subclasses, we constructed a CHO line
expressing a chimeric protein comprised of the Fc binding domain of
Protein G fused to the hFc.gamma.RI transmembrane and intracellular
domain. The Fc binding domain of Protein G contains three
homologous repeats of 55 amino acids long (Guss et al., (1986) EMBO
5:1567 and Sjobring et al., (1991) J. Biol. Chem. 266:399) and each
repeat is capable of binding one IgG Fc. To improve the expression
of this chimeric protein in CHO cells, we constructed a synthetic
DNA in which the signal sequence from the mouse ROR1 gene was fused
to the Fc binding domain, amino acids 303 to 497 of Protein G
(accession # X06173) (SEQ ID NO:1). This synthetic DNA was
generated by a combination of oligonucleotide annealing, gap
filling, and PCR amplification. The synthetic DNA was then fused,
by PCR, to DNA encoding the transmembrane and intracellular
domains, amino acids 279 to 374 (SEQ ID NO:2), of hFc.gamma.RI
(accession M21091). The resultant DNA encoding the Protein
G/hFc.gamma.RI chimeric protein was cloned into pTE158 downstream
of the CMV-MIE promoter, replacing the gene encoding hFc.gamma.RI,
to yield the plasmid pTE300.
A CHO K1 cell line adapted to grow in serum-free medium, RGC14, was
transfected with pTE300, and after three days 400 .mu.g/ml G418 was
added to the culture medium to select for stable integration of
pTE300. Two weeks after the start of selection, the cells were
stained with FITC-hFc to identify cells that expressed
hFc.gamma.RI. These cells were analyzed by flow cytometry and cells
expressing hFc.gamma.RI were collected as a pool. The cells were
expanded for 10 days and the population of cells expressing
hFc.gamma.RI was again isolated by flow cytometry. The cells were
again expanded, stained with FITC-hFc, and single cells expressing
high levels of the Protein G/hFc.gamma.RI chimeric protein were
isolated by flow cytometry. Single cells that stained positive for
FITC-hFc binding were sorted into medium composed of 10% fetal
bovine serum, 90% Ham's F12, and 400 .mu.g/ml G418. After two weeks
incubation, 48 clones were examined for binding to bovine IgG
present in the culture medium by staining with FITC-conjugated
anti-bovine IgG F(ab').sub.2 fragment (Jackson ImmunoResearch
Laboratories, West Grove, Pa.). One clone, RGC18 that stained
positive with this antibody was chosen for further
characterization.
[0084] Isolation of expression clones in RGC18: RGC18 cells
(6.times.10.sup.6) were transfected with pTE209 and selected for
integration of the plasmid by growth in 400 .mu.g/ml hygromycin for
18 days. Hygromycin-resistant cells were incubated with 1 mg/ml
rabbit IgG for eighteen hours prior to staining with polyclonal
FITC-conjugated anti-human IgG (H+ L) F (ab').sub.2 fragment. Cells
were stained for 1 hour then washed twice with PBS prior to
analysis by flow cytometry. The most fluorescent cells (top 5%)
were isolated by single cell sorting and expanded for 3 weeks. Ten
clones were examined for 4SC622 secretion. All clones tested
secreted 4SC622 at high level, and the best clone, RGC19, had a
specific productivity of 6.4 pg/cell day. This result demonstrated
that 4SC622-expressing cells were efficiently isolated from a
heterogeneous pool of cells derived from stable transfection of
RGC18 with pTE209 by the autologous secretion trap method.
Furthermore, these data clearly demonstrated that a fragment of
Protein G could be engineered to include a signal sequence and
transmembrane domain, and function as a cell surface capture
protein.
[0085] To confirm that 4SC622 was autologously displayed on the
surface of RGC19 cells expressing both Protein G/hFc.gamma.RI
chimeric protein and 4SC622, RGC19 was incubated with 1 mg/ml
rabbit IgG for 18 hours then stained with FITC-conjugated
anti-human IgG (H+ L) F(ab').sub.2 fragment and analyzed by flow
cytometry. RGC19 cells were found to possess cell surface 4SC622
under these conditions in which cross-feeding was blocked by rabbit
IgG, suggesting autologous display of 4SC622. Rabbit IgG
effectively blocked binding of exogenous 4SC622 protein to RGC18
cells, but did not block display of 4SC622 on the cell surface of
cells expressing 4SC622. These data demonstrated that the
properties of the Protein G/hFc.gamma.RI chimeric protein were
similar to those of hFc.gamma.RI as a cell surface capture protein,
and suggested that the autologous secretion trap method can employ
other proteins as cell surface capture proteins.
Example 9
Isolation of Antibody-Producing Cells from RGC10
[0086] To demonstrate the utility of the autologous secretion trap
method for the isolation of CHO cell lines that express recombinant
antibodies we cloned the DNA encoding variable light and variable
heavy genes from the KD5 hybridoma. KD5 is a hybridoma that
expresses a monoclonal antibody specific for the human Tie-2
receptor.
[0087] The mouse IgG constant region gene sequences were cloned
from 500 ng of mouse spleen polyA+ RNA (Clontech, Palo Alto,
Calif.). Single stranded cDNA was synthesized using SuperScript
First-Strand Synthesis System for RT-PCR, primed with 50 ng of
random hexamers (Invitrogen Life Technologies, Carlsbad, Calif.).
The mouse kappa light constant DNA sequence (accession # Z37499)
was amplified from this cDNA by PCR using the primers 5' mCLK1
(Z37499) (5'-CGGGCTGATG CTGCACCAAC TGTATCCATC TTC-3') (SEQ ID NO:3)
and 3' mCLK1 (Z37499) (5'-ACACTCTCCC CTGTTGAAGC TCTTGACAAT GGG-3')
(SEQ ID NO:4). The mouse IgG2a constant region DNA sequence
(accession # AJ294738) was also amplified from this cDNA by PCR
using the primers 5' mCH2a (AJ294738) (5'-GCCAAAACAA CAGCCCCATC
GGTCTATCCA C-3') (SEQ ID NO:5) and 3' mCH2a (AJ294738)
(5'-TCATTTACCC GGAGTCCGGG AGAAGCTCTT AGTCG-3') (SEQ ID NO:6). The
PCR products were cloned into pCR2.1-TOPO using TOPO TA Cloning kit
(Invitrogen Life Technologies, Carlsbad, Calif.) and the sequence
of the constant regions were verified.
[0088] The KD5 variable region genes were amplified by RT-PCR from
KD5 hybridoma mRNA and cloned into pCR2.1-TOPO using the heavy and
light chain variable region primer mixes from Amersham-Pharmacia
Biotech (Piscataway, N.J.). The variable heavy chain gene was PCR
amplified using the pCR2.1-TOPO cloned variable region as template
with the primers 5' BspMI/KD5VH N-term (5'-GAGAGTACCT GCGTCATGCA
GATGTGAAAC TGCAGGAGTC TGGCCCT-3') (SEQ ID NO:7) and 3' BspMI/KD5VH
C-term (5'-GAGAGACCTG CGTCAGCTGA GGAGACGGTG ACCGTGGT-3') (SEQ ID
NO:8), digested with BspMI and ligated to the Bsal-digested IgG2a
constant heavy gene PCR fragment amplified with the primers 5'
Bsal/CH2a N-term (5'-GAGAGGGTCT CACAGCCAAA ACAACAGCCC CATCG-3')
(SEQ ID NO:9) and 3' Bsal/CH.sub.2a C-term (5'-GAGAGGGTCT
CCGGCCGCTC ATTTACCCGG AGTCCGGG AGAA-3') (SEQ ID NO:10). This
fragment was then ligated into the BspMI and NotI sites of pRG882.
The resulting plasmid, pTE317, was capable of expressing the KD5
recombinant heavy chain gene, fused to the mROR1 signal sequence,
from the CMV-MIE promoter. The variable light chain gene was PCR
amplified using the pCR2.1-TOPO cloned variable region as template
with the primers 5' BsmBI/KD5VL N-term (5'-GAGAGCGTCT CATGCAGACA
TCCAGATGAC CCAGTCTCCA-3') (SEQ ID NO:11) and 3' BsmBI/KD5VL C--
term (5'-GAGAGCGTCT CACAGCCCGT TTTATTTCCA GCTTGGTCCC-3') (SEQ ID
NO:12), digested with BsmBI and ligated to the Bsal-digested kappa
constant light gene PCR fragment amplified with the primers 5'
Bsal/CLK N-term (5'-GAGAGGGTCT CAGCTGATGC TGCACCAACT GTATCC-3')
(SEQ ID NO:13) and 3' Bsal/CLK C-term (5'-GAGAGGGTCT CAGGCCGCTC
AACACTCTCC CCTGTTGAAG CTCTTGAC-3') (SEQ ID NO:14). This fragment
was then ligated into the BspMI and NotI sites of pRG882. The
resulting plasmid, pTE316, was capable of expressing the KD5
recombinant light chain gene, fused to the mROR1 signal sequence,
from the CMV-MIE promoter.
[0089] The 1450 bp EcoRI-NotI fragment from pTE317, encoding the
KD5 heavy chain gene, was cloned into the EcoRI and NotI sites of
pRG980, a vector that confers resistance to hygromycin and allows
expression of recombinant genes for the UbC promoter, to yield
plasmid pTE322. Similarly, the 750 bp EcoRI-NotI fragment from
pTE316, encoding the KD5 light chain gene, was cloned into the
EcoRI and NotI sites of pRG985, a vector that confers resistance to
puromycin and allows expression of recombinant genes for the UbC
promoter, to yield plasmid pTE324. RGC10 cells (5.times.10.sup.6)
were transfected with 3 .mu.g pTE322 and 3 .mu.g pTE322 and
selected for integration of the plasmids by growth in F12 medium
supplemented with 10% fetal calf serum with 20 .mu.g puromycin and
400 .mu.g/ml hygromycin for 14 days. Expression of hFc.gamma.RI was
induced by the addition of 1 .mu.g/ml of doxycycline to the culture
medium for three days. Double-resistant cells were incubated with 1
mg/ml rabbit IgG for eighteen hours prior to staining with goat
polyclonal FITC-conjugated anti-mouse IgG (Fc.gamma.) F (ab').sub.2
fragment (Jackson ImmunoResearch Laboratories, West Grove, Pa.).
Cells were stained for 1 hour then washed twice with PBS prior to
analysis by flow cytometry. The most fluorescent cells (top 5%)
were isolated as a pool and expanded for 10 days, after which the
protocol was repeated but the top 1% most fluorescent cells were
isolated as a pool. This pool was expanded for 10 days then the top
0.1% most fluorescent cells were isolated as single cells into
96-well plates. Clones were analyzed by ELISA for expression of
antibody and seven clones were chosen from 53 clones analyzed. The
average specific productivity of these clones was 35 pg/cell/day
and the best clone expressed the recombinant KD5 monoclonal
antibody at 54 pg/cell/day.
Example 10
FASTR Screens Unaffected by CSCP Expression Level
[0090] To demonstrate that the expression level of the CSCP does
not significantly affect the ability to isolate cells expressing an
associated sPOI, FASTR screens for the same sPOI in two different
host cell lines that each express the same CSCP but at either a
high level or a low level were compared.
[0091] The FASTR host cell line RGC10 was selected for high-level
expression of hFc.gamma.RI protein by stable integration of pTE158
and was found to contain 40 hFc.gamma.RI integrated gene copies. A
new cell line, RS527, that expressed hFc.gamma.RI protein at a
lower level, was generated from CHO K1 after stable transfection
and selection for single copy gene integration. RS527 cells
expressed significantly less hFc.gamma.RI protein than RGC10 cells
as determined by Western blot analysis of whole cell lysates of the
FASTR cell lines.
[0092] Briefly, RGC10 and RS527 cells were transfected with pTE462,
a plasmid capable of expressing a secreted hFc-fusion protein
Rc1-hFc and conferring resistance to hygromycin. The transfected
cultures were selected with hygromycin for two weeks. The
hygromycin-resistant cells were induced with 1 .mu.g/ml doxycycline
(Dox) and blocked with rabbit IgG overnight, following the FASTR
method described herein. The next day, the RGC10/pTE462 and
RS527/pTE462 cultures were stained by a FITC-conjugated antibody
specific for hFc and then analyzed by flow cytometry. Three cell
bins R4, R5, and R6 marking cells with low, medium, and high
fluorescence respectively were sorted from each host line and
expanded in tissue culture.
[0093] To compare Rc1-hFc protein production level from the six
cell bins, six cultures were set up using equal number of cells for
each bin. Three days later, conditioned media were collected. The
Rc1-hFc protein titers in the conditioned media were determined by
ELISA and were plotted against mean fluorescence of the respective
cell bins. For both RGC10 and RS527 host lines, there was a similar
correlation between mean fluorescence (amount of Rc1-hFc displayed
on the cell surface) and sPOI protein production levels of the
isolated cell pools. Most significantly, the sPOI titers in the two
high fluorescence R6 bins derived from RGC10 and RS527 were
similar. These data demonstrate that the expression level of the
CSCP in a FASTR host cell line does not significantly affect the
use of that host to isolate transfected cells based on expression
level of a sPOI.
Example 11
Tie2 Receptor as a Cell Surface Capture Protein
[0094] Cell surface capture proteins (CSCP's) other than
Fc.gamma.R1 can be used in the methods described herein. In this
example, the Tie2 receptor functions as a CSCP and is used to
isolate cells expressing a Tie-specific ScFv.sub.C1b-Fc fusion
protein made from the C1b monoclonal antibody that specifically
binds the extracellular domain of Tie2 receptor. Although the CSCP
for ScFv.sub.C1b-Fc can be hFcgRI, this example demonstrates that
Tie2 can also be used as the CSCP for ScFv.sub.C1b-Fc.
[0095] To construct an inducible Tie2 CSCP cell line, CHO K1 was
first stably transfected with the TetR plasmid pcDNA6/TR. The
blasticidin-resistant cell pool was then stably transfected with
pTE259, a plasmid that allows inducible expression of a protein
comprised of the extracellular domain and transmembrane domain of
Tie2. Inducible cell clones were isolated by flow cytometry after
staining with an antibody specific for Tie2 (FIG. 3). The RGC54
clone was chosen to study the feasibility of FASTR for the
expression of ScFv.sub.C1b-Fc.
[0096] RGC54 cells were stably transfected with pTE988, a plasmid
capable of expressing the secreted hFc-fusion protein
ScFv.sub.C1b-Fc and conferring resistance to hygromycin. The
transfected culture was selected with hygromycin for two weeks. The
hygromycin-resistant cells were induced with Dox and blocked with 1
mg/ml of purified C1b mAb. The C1b monoclonal antibody was the
source of the variable regions in ScFv.sub.C1b-Fc. The next day,
the cell pool was stained by a FITC-conjugated antibody specific
for hFc and then analyzed by flow cytometry. Three cell bins R6,
R7, and R8 marking cells with high, medium, and low fluorescence
respectively were sorted and expanded in tissue culture. Three
cultures were set up using an equal number of cells for each bin to
determine ScFv.sub.C1b-Fc protein production as determined by
ELISA. A correlation existed between mean fluorescence (amount of
ScFv.sub.C1b-Fc binding to Tie2 on the cell surface) and
ScFv.sub.C1b-Fc protein production levels of the isolated cell
pools.
[0097] These data show that CSCP other than hFc.gamma.RI can serve
as a CSCP, and also suggest that any receptor may be converted into
a CSCP by removal of its cytoplasmic domain. These data also
demonstrate that an antigen can be made into a CSCP and used for
FASTR screening cells expressing an antigen-specific
antibody-related molecule.
Example 12
Effective FASTR Screens with CSCP:sPOI Pairs Having Low
Affinity
[0098] Angiopoetin-1 is a ligand for the Tie2 receptor. A chimeric
protein comprising angiopoetin-1 receptor binding domain and hFc
(FD1-hFc) binds to Tie2 with an affinity constant of 174 nM as
determined by BIAcore.TM.. FD1-hFc and Tie2 were chosen as sPOI and
CSCP, respectively, to determine if a minimum affinity between CSCP
and sPOI is required for FASTR screens
[0099] In cell decoration experiments, exogenously added FD1-hFc
bound specifically to RGC54 cells through Tie2. To determine if the
affinity between Tie2 and FD1-hFc is sufficient to allow FASTR
screening, RGC54 cells were stably transfected with pTE942, a
plasmid capable of expressing the secreted hFc-fusion protein
FD1-hFc and conferring resistance to hygromycin. The transfected
culture was selected with hygromycin for two weeks. The
hygromycin-resistant cells were induced with Dox and blocked with 1
mg/ml of purified FD1-mFc comprising mouse IgG1 Fc. The next day,
the cell pool was stained by a FITC-conjugated antibody specific
for hFc and then analyzed by flow cytometry. Three cell bins R6,
R7, and R8 marking cells with high, medium, and low fluorescence,
respectively, were collected. Cultures were set up using equal
number of cells for each bin to determine FD1-hFc protein
production levels in the conditioned media as determined by ELISA.
There was a correlation between mean fluorescence (FD1-Fc binding
to cell surface-bound Tie2) and FD1-hFc protein production levels
of the isolated cell pools. The bin with the highest fluorescence
produced the most FD1-hFc.
[0100] These data demonstrate that a CSCP:sPOI pair with low
affinity (174 nM KD) can be used for effective FASTR screens.
Importantly, the dissociation t.sub.1/2 for FD1-Fc: Tie2 binding is
less than 2 minutes, suggesting that any CSCP:sPOI pair with a
measurable affinity can work in FASTR screens. In addition, this
experiment also shows that a nonFc.gamma.RI receptor may be used as
the CSCP to isolate cells expressing its ligand.
Example 12
Fusing a Transmembrane Domain onto an ScFv Makes a Functional
CSCP
[0101] An CSCP can be any cell surface-bound protein that has a
measurable affinity to the sPOI. To demonstrate this, a totally
synthetic CSCP was constructed by fusing the transmembrane domain
from the PDGF receptor to an ScFv containing the variable regions
from the murine kappa chain-specific monoclonal antibody HB58. A
FASTR host was constructed that expresses this chimeric protein
(ScFV.sub.HB58-TM.sub.PDGFR) and was used to isolate cells
expressing the angiopoeitin-2 FD domain-specific P12 antibody.
[0102] The RS655 cell line, derived from CHO K1, constitutively
expresses ScFV.sub.HB58-TM.sub.PDGFR. Cells expressing
ScFv.sub.HB58-TM.sub.PDGFR can be stained by sequential incubation
with P12 mAb, FD2-hFc, and FITC-conjugated anti-hIgG-P12 captured
on the cell surface by the HB58 ScFv was detected by its affinity
for FD2, which in turn was detected by recognition of the hFc tag.
RS656 cells were derived from RS655 cells after stable transfection
with a plasmid encoding the gene for eYFP. Nearly 100% of RS656
cells were eYFP-positive, and most (76%) maintained expression of
ScFv.sub.HB58-TM.sub.PDGFR as detected by binding to FD2-hFc.
[0103] RS655 cells were stably transfected with pTE693, a plasmid
capable of expressing the heavy and light chains of the P12
antibody, and conferring resistance to puromycin. The transfected
culture was selected with puromycin for two weeks to yield a pool
of cells that were heterogeneous with regard to P12 mAb expression
(RS655/pTE693).
[0104] To determine if ScFv.sub.HB58-TM.sub.PDGFR could function as
a CSCP and facilitate isolation of antibody-producing cells from
non-producers, equal numbers of RS656 cells and RS655/pTE693 cells
were mixed and co-cultured. When P12 expressed from RS655/pTE693
cells was allowed to diffuse and bind to ScFv.sub.HB58 on the
surface of RS656 cells a large population of yellow cells were also
positive for binding FD2-hFc. However, if the ScFv.sub.HB58 on the
surface of RS656 was bound with excess murine IgG, then only
non-yellow cells were positive for binding FD2-hFc, demonstrating
that expressing cells were effectively separated from
non-expressing cells.
[0105] These data demonstrate that an ScFv can be made into a
functional CSCP by targeting it to the cell membrane. The data also
show that FASTR allows cells expressing a secreted antibody to be
detected with the antibody's antigen.
Example 13
A Protein of Interest Comprising a T Cell Receptor Variable
Region
[0106] A flow cytometry-based autologous secretion trap (FASTR)
method for isolating high expression clones of a cell line that
expresses a protein of interest that is a TCR-Fc is prepared in a
manner analogous to preparing a cell line that expresses an
antibody of interest. High expression clones are identified by
screening cells that display on their surface the TCR-Fc of
interest bound to hFc.gamma.R.
[0107] In these examples, the CHO K1 cell line RGC10, comprising an
inducible Fc.gamma.R1 as a cell surface capture molecule, is
employed. RGC10 is made to express recombinant TCR-Fc's by cloning
TCR variable regions, in frame, to a human Fc region either
directly in frame or with a linker sequence between the TCR
variable regions and the human Fc region.
[0108] To make a protein of interest that is a dimer comprising an
Fc-linked TCR .alpha. variable domain and an Fc-linked TCR .beta.
variable domain, RGC10 is transfected with two vectors: a first
vector capable of expressing a TCR .alpha. variable domain fusion
protein with a human Fc sequence, and a second vector capable of
expressing a TCR .beta. domain fusion protein with the same human
Fc sequence. Each vector includes leader sequence (e.g., a
secretion signal sequence) 5' with respect to the TCR variable
region. and a selectable marker that is a drug resistance gene.
Following each vector transfection, cells containing the vector are
selected by an appropriate drug selection. The selection results in
an RGC10 cell line having both the first and the second vectors.
Cells expressing proteins of interest can be detected by one or
more of an antibody to the .beta. variable domain, an antibody to
the .alpha. variable domain, and an antibody to the Fc domain.
[0109] To make a protein of interest that is a dimer comprising
both an .alpha. and a .beta. TCR variable domain fused to an Fc,
RGC10 is transfected with a single vector encoding a protein of
interest that is constructed as follows: a leader sequence (e.g., a
secretion signal sequence), followed by a TCR variable .beta.
domain fused to a linker, where the linker is, in turn, fused to a
TCR variable a domain, which in turn is fused to an Fc sequence.
Alternatively, the single vector can be constructed as follows: a
leader sequence (e.g., a secretion signal sequence), followed by a
TCR variable .alpha. domain fused to a linker, where the linker is,
in turn, fused to a TCR variable .beta. domain, which in turn is
fused to an Fc sequence. Cells expressing proteins of interest can
be detected by one or more of an antibody to the .beta. variable
domain, an antibody to the .alpha. variable domain, and an antibody
to the Fc domain.
[0110] To make proteins of interest, as above, which also comprise
a TCR .alpha. and/or TCR .beta. constant domain, the TCR variable
domain (.alpha. or .beta.) is fused to a TCR constant domain (e.g.,
TCR variable domain a is fused to TCR constant domain .alpha., and
TCR variable domain .beta. is fused to TCR constant domain p), and
the TCR variable+constant domain is fused directly or through a
linker to the Fc domain. Cells expressing proteins of interest can
be detected by one or more of an antibody to the .beta. variable
domain, an antibody to the .alpha. variable domain, and an antibody
to the Fc domain.
[0111] Cells expressing desired amounts of the TCR-Fc are isolated
using the same procedure as used in isolating 4SC622-producing cell
lines described herein, using one or more of an antibody to the
.alpha. variable domain, an antibody to the .beta. variable domain,
an antibody to the .alpha. constant domain, and antibody to the
.beta. constant domain, and an antibody to the Fc domain. Cells
expressing the highest levels of the TCR-Fc are selected as
TCR-Fc-producing cell lines.
[0112] Although the foregoing invention has been described in some
detail by way of illustration and example, it will be readily
apparent to those of ordinary skill in the art that certain changes
and modifications may be made to the teachings of the invention
without departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
141195PRTStreptococcus sp 1Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr
Leu Lys Gly Glu Thr Thr1 5 10 15Thr Glu Ala Val Asp Ala Ala Thr Ala
Glu Lys Val Phe Lys Gln Tyr20 25 30Ala Asn Asp Asn Gly Val Asp Gly
Glu Trp Thr Tyr Asp Asp Ala Thr35 40 45Lys Thr Phe Thr Val Thr Glu
Lys Pro Glu Val Ile Asp Ala Ser Glu50 55 60Leu Thr Pro Ala Val Thr
Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr65 70 75 80Leu Lys Gly Glu
Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu85 90 95Lys Val Phe
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp100 105 110Thr
Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu115 120
125Val Ile Asp Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys
Leu130 135 140Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr
Lys Ala Val145 150 155 160Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys
Gln Tyr Ala Asn Asp Asn165 170 175Gly Val Asp Gly Val Trp Thr Tyr
Asp Asp Ala Thr Lys Thr Phe Thr180 185 190Val Thr Glu195296PRThomo
sapiens 2Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val Trp Phe His
Val Leu1 5 10 15Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr
Val Leu Trp20 25 30Val Thr Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys
Trp Asp Leu Glu35 40 45Ile Ser Leu Asp Ser Gly His Glu Lys Lys Val
Thr Ser Ser Leu Gln50 55 60Glu Asp Arg His Leu Glu Glu Glu Leu Lys
Cys Gln Glu Gln Lys Glu65 70 75 80Glu Gln Leu Gln Glu Gly Val His
Arg Lys Glu Pro Gln Gly Ala Thr85 90 95333DNAArtificial
SequenceSynthetic 3cgggctgatg ctgcaccaac tgtatccatc ttc
33433DNAArtificial SequenceSynthetic 4acactctccc ctgttgaagc
tcttgacaat ggg 33531DNAArtificial SequenceSynthetic 5gccaaaacaa
cagccccatc ggtctatcca c 31635DNAArtificial SequenceSynthetic
6tcatttaccc ggagtccggg agaagctctt agtcg 35747DNAArtificial
SequenceSynthetic 7gagagtacct gcgtcatgca gatgtgaaac tgcaggagtc
tggccct 47838DNAArtificial SequenceSynthetic 8gagagacctg cgtcagctga
ggagacggtg accgtggt 38935DNAArtificial SequenceSynthetic
9gagagggtct cacagccaaa acaacagccc catcg 351042DNAArtificial
SequenceSynthetic 10gagagggtct ccggccgctc atttacccgg agtccgggag aa
421140DNAArtificial SequenceSynthetic 11gagagcgtct catgcagaca
tccagatgac ccagtctcca 401240DNAArtificial SequenceSynthetic
12gagagcgtct cacagcccgt tttatttcca gcttggtccc 401336DNAArtificial
SequenceSynthetic 13gagagggtct cagctgatgc tgcaccaact gtatcc
361448DNAArtificial SequenceSynthetic 14gagagggtct caggccgctc
aacactctcc cctgttgaag ctcttgac 48
* * * * *