U.S. patent application number 16/392174 was filed with the patent office on 2020-01-16 for early post-transfection isolation of cells (epic) for biologics production.
The applicant listed for this patent is Genzyme Corporation. Invention is credited to Victor R. Cairns, Christine DeMaria, Jason Vitko.
Application Number | 20200018682 16/392174 |
Document ID | / |
Family ID | 57211576 |
Filed Date | 2020-01-16 |
United States Patent
Application |
20200018682 |
Kind Code |
A1 |
Cairns; Victor R. ; et
al. |
January 16, 2020 |
EARLY POST-TRANSFECTION ISOLATION OF CELLS (EPIC) FOR BIOLOGICS
PRODUCTION
Abstract
Provided herein are methods for selecting a population of cells
expressing a target polypeptide. In some aspects, the disclosure
provides methods for sorting and selecting populations of
transfected host cells based on their early expression of a
selectable polypeptide. In certain embodiments, the sorting is
performed using fluorescence-activated cell sorting or
magnetic-activated cell sorting based on the selectable
polypeptide. Such selection methods can be further utilized to
generate clonal populations of producer cells, e.g. for large-scale
manufacturing of a target polypeptide of interest.
Inventors: |
Cairns; Victor R.;
(Bridgewater, NJ) ; DeMaria; Christine;
(Bridgewater, NJ) ; Vitko; Jason; (Bridgewater,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Family ID: |
57211576 |
Appl. No.: |
16/392174 |
Filed: |
April 23, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15288050 |
Oct 7, 2016 |
10317329 |
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16392174 |
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62239515 |
Oct 9, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/10 20130101;
G01N 15/14 20130101; C07K 16/00 20130101; G01N 33/5044 20130101;
G01N 2333/70592 20130101; C12N 15/1086 20130101; G01N 2015/149
20130101; C12N 15/85 20130101; C12N 2510/02 20130101 |
International
Class: |
G01N 15/14 20060101
G01N015/14; C12N 15/85 20060101 C12N015/85; G01N 33/50 20060101
G01N033/50; C07K 16/00 20060101 C07K016/00; C12N 15/10 20060101
C12N015/10 |
Claims
1-30. (canceled)
31. A method of producing a population of producer cells expressing
a target polypeptide, the method comprising: (a) transfecting host
cells with one or more vectors that encode one or more mRNAs,
wherein the one or more mRNAs encode a selectable polypeptide and
the target polypeptide; (b) isolating from the transfected host
cells, within 2 to 15 days of transfection, a sub-population of
early-expressing transfected host cells which express the
selectable polypeptide; and (c) expanding the sub-population of
transfected host cells, thereby producing a population of producer
cells expressing the target polypeptide.
32. The method of claim 31, wherein steps (b) and (c) are each
performed in drug-selection-free medium.
33. The method of claim 31, further comprising isolating the target
polypeptide from the population of producer cells.
34. The method of claim 31, further comprising isolating one or
more single transfected host cells from the expanded sub-population
and culturing the one or more single transfected host cells to
produce clonal populations of the one or more single transfected
host cells.
35. The method of claim 34, wherein at least one of the clonal
populations of the one or more single transfected host cells yields
a 2- to 30-fold improvement in production of the target polypeptide
compared to that of a stable pool of transfected but uncloned host
cells obtained at step (c).
36. The method of claim 31, wherein the transfected host cells
subject to selection in (b) contains at least 80-120.times.10.sup.6
cells.
37. The method of claim 31, wherein the isolating in step (b) is
performed less than 6 days after transfection.
38. The method of claim 31, wherein the sub-population of
transfected host cells contains 0.5-6.0.times.10.sup.6 cells prior
to expansion in step (c).
39. The method of claim 31, wherein the expanding in step (c) is
for between 4-31 days.
40. The method of claim 31, wherein a first of the one or more
vectors encodes the mRNA encoding the target polypeptide and a
second of the one or more vectors encodes the mRNA encoding the
selectable polypeptide.
41. The method of claim 31, wherein the mRNA encoding the target
polypeptide and the mRNA encoding the selectable polypeptide are
both encoded on one vector.
42. The method of claim 31, wherein the target polypeptide and the
selectable polypeptide form a fusion polypeptide.
43. The method of claim 31, wherein the target polypeptide and the
selectable polypeptide are encoded by a single multicistronic
mRNA.
44. The method of claim 43, wherein the multicistronic mRNA
comprises a first open reading frame (ORF) that encodes the
selectable polypeptide and a second ORF that encodes the target
polypeptide, wherein the first ORF is 5' to the second ORF.
45. The method of claim 44, wherein the first ORF has a non-AUG
start codon.
46. The method of claim 44, wherein the second ORF has an AUG start
codon.
47. The method of claim 44, wherein the ORF that encodes the
selectable polypeptide is devoid of any AUG sequences.
48. The method of claim 31, wherein the selectable polypeptide is
CD52 or CD59.
49. A clonal population of transfected host cells that express a
selectable polypeptide and a target polypeptide obtainable by the
method of claim 31.
50. The clonal population of claim 49, wherein the clonal
population yields a 2- to 30-fold improvement in production of the
target polypeptide compared to that of the sub-population of
transfected host cells in (b).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/288,050, filed Oct. 7, 2016, which claims
the benefit of priority to U.S. Provisional Patent Application No.
62/239,515, filed Oct. 9, 2015. The contents of the aforementioned
applications are hereby incorporated by reference in their
entirety.
BACKGROUND
[0002] Methods for selection of producer cell populations and cell
clones are imperative for the manufacturing of biologics, such as
antibodies and fusion proteins. Such methods generally rely on use
of a selection agent, such as methotrexate (MTX) or methionine
sulphoximine (MSX), to bias and amplify the production of
biologics. Selection agent-based methods may affect the viability
or growth rate of selected populations or may have a negative
impact on clonal stability. Such drug-based selections can also be
time consuming, often requiring multiple rounds of selection to
obtain populations which contain clones that are suitable for
biologic manufacturing. There remains a need for rapid and reliable
methods of generating both large cell populations and clones that
produce high titers of biologics with less negative impact to the
host cell.
SUMMARY OF THE INVENTION
[0003] In some aspects, the disclosure provides methods of
selecting a population of cells expressing a target polypeptide. As
described herein, methods for selection were developed that relied
upon sorting of populations shortly following their transfection.
Thus the methods feature the step of isolating a sub-population of
transfected cells for early detectable expression of the
transfected vector. In certain embodiments, the selection is based
on early expression of a selectable polypeptide, which is different
from the target polypeptide and detectable on the surface of the
cell.
[0004] Unexpectedly, the methods described herein were found to be
faster than traditional methods which use two rounds of MTX
selection to generate a pool, and more productive than traditional
MTX amplification, including single-round MTX selection.
[0005] The methods described herein are useful, e.g., for the
generation of pools of cells for screening of polypeptides of
interest (such as in early clinical development and for the
generation of high titer clones, which can be utilized to produce a
polypeptide of interest both for small and large scale
manufacturing.
[0006] Accordingly, in some aspects, the disclosure provides a
method of producing a population of producer cells expressing a
target polypeptide, the method comprising: (a) transfecting host
cells with one or more vectors that encode one or more mRNAs,
wherein the one or more mRNAs encode a selectable polypeptide and
the target polypeptide; (b) isolating from the transfected host
cells, within 2 to 15 days after transfection, a sub-population of
early-expressing transfected host cells which express the
selectable polypeptide; and (c) expanding the sub-population of
early-expressing transfected host cells, thereby producing a
population of producer cells.
[0007] In some embodiments, step (b) is performed in
drug-selection-free medium.
[0008] In some embodiments, step (c) is performed in
drug-selection-free medium.
[0009] In some embodiments, step (b) and step (c) are each
performed in drug-selection-free medium.
[0010] In some embodiments of any one of the methods provided, the
method further comprises isolating the target polypeptide from the
expanded sub-population.
[0011] In some embodiments of any one of the methods provided, the
method further comprises isolating one or more single transfected
host cells from the expanded sub-population and culturing the one
or more single transfected host cells to produce clonal populations
of the one or more single transfected host cells.
[0012] In some embodiments of any one of the methods provided, at
least one of the clonal populations of the one or more single
transfected host cells yields a 2- to 30-fold improvement in
production of the target polypeptide compared to that of a stable
pool of transfected but uncloned host cells obtained at step
(c).
[0013] In some embodiments of any one of the methods provided, the
transfected host cells subject to isolation in step (b) contains
80-120.times.10.sup.6 cells.
[0014] In some embodiments of any one of the methods provided, the
isolation in step (b) is performed less than six days after
transfection. In some embodiments of any one of the methods
provided, the isolation in step (b) is performed between two and
four days after transfection. In some embodiments of any one of the
methods provided, the isolation in step (b) is performed two days
after transfection. In some embodiments of any one of the methods
provided, the isolation in step (b) is performed three days after
transfection.
[0015] In some embodiments of any one of the methods provided, the
sub-population of transfected host cells contains
0.5-6.0.times.10.sup.6 cells prior to expansion in step (c).
[0016] In some embodiments of any one of the methods provided, the
expanding in step (c) is for between 4-31 days.
[0017] In some embodiments of any one of the methods provided, a
first of the one or more vectors encodes the mRNA encoding the
target polypeptide, and a second of the one or more vectors encodes
the selectable polypeptide.
[0018] In some embodiments of any one of the methods provided, the
mRNA encoding the target polypeptide and the mRNA encoding the
selectable polypeptide are both encoded on one vector.
[0019] In some embodiments of any one of the methods provided, a
first of the one or more vectors encodes the mRNA encoding the
target polypeptide, and a second of the one or more vectors encodes
the selectable polypeptide.
[0020] In some embodiments of any one of the methods provided, the
mRNA encoding the plurality of target polypeptides and the mRNA
encoding the plurality of selectable polypeptides are both encoded
on one vector.
[0021] In some embodiments of any one of the methods provided, the
isolation in step (b) employs magnetic-activated cell sorting
(MACS), fluorescence-activated cell sorting (FACS), or
ClonePix.
[0022] In some embodiments of any one of the methods provided, the
selectable polypeptide is a FACS selectable polypeptide and the
isolation in step (b) employs FACS.
[0023] In some embodiments of any one of the methods provided, the
target polypeptide and the selectable polypeptide form a fusion
polypeptide.
[0024] In some embodiments of any one of the methods provided, the
mRNA is a multicistronic mRNA. In some embodiments of any one of
the methods provided, the multicistronic mRNA comprises a first
open reading frame (ORF) that encodes the selectable polypeptide
and a second ORF that encodes the target polypeptide, wherein the
first ORF is 5' to the second ORF. In some embodiments of any one
of the methods provided, the first ORF has a non-AUG start codon.
In some embodiments of any one of the methods provided, the second
ORF has an AUG start codon. In some embodiments of any one of the
methods provided, the non-AUG start codon is a UUG, GUG, or CUG in
a Kozak consensus sequence. In some embodiments of any one of the
methods provided, the ORF that encodes the selectable polypeptide
is devoid of any AUG sequences.
[0025] In some embodiments of any one of the methods provided, the
selectable polypeptide is CD52 or CD59.
[0026] In some embodiments of any one of the methods provided, the
target polypeptide is a therapeutic agent. In some embodiments of
any one of the methods provided, the target polypeptide is a
secreted protein. In some embodiments of any one of the methods
provided, the target polypeptide is an antibody or an Fc fusion
protein.
[0027] In some embodiments of any one of the methods provided, the
host cells are CHO cells, HEK293 cells, or HeLa cells.
[0028] Other aspects of the disclosure relate to a clonal
population of transfected host cells that express a selectable
polypeptide and a target polypeptide obtainable by any one of the
methods described above or otherwise described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A is a schematic depicting comparison between
traditional transfection and selection and EPIC-based transfection
and selection. Early expression refers to expression early after
transfection, prior to significant genomic integration.
[0030] FIG. 1B is a diagram showing reporter expression of a
transfected population of cells from day 3 to 21 in a
nucleotide-deficient selection process (compared to mock
transfected population). Transfected cells exhibited an apparent
early expression shortly after transfection (e.g., day 3-4) and
then transitioned to stable expression upon completion of selection
(day 18-21).
[0031] FIG. 2 is a series of FACS histogram offsets depicting the
early expression of both red fluorescent protein (RFP) and cell
surface reporter CD52 expression from the same vector (pGZ729-RFP).
No selection pressure was applied to the transfected cells. Peak
early expression for RFP and CD52 occurs between days 2 and 3.
[0032] FIG. 3 is a series of FACS histogram offsets depicting the
day 3 early expression of RFP and CD52 in cells transfected with
pGZ729-RFP (encoding both selectable polypeptide CD52 and target
polypeptide RFP) or pGZ700-RFP (encoding only target polypeptide
RFP).
[0033] FIG. 4 is a schematic showing both the methodology of EPIC
to generate a sub-population of cells for selection shortly after
transfection and the beneficial effects to both the reporter
expression and monoclonal antibody (mAb) titers upon
isolation/expansion of the sort-enriched population. Mock refers to
mock transfection.
[0034] FIG. 5 is a graph depicting day 14 unfed batch titers for
EPIC-generated pools as compared to traditional MTX
methodologies.
[0035] FIG. 6 is a graph depicting day 14 unfed batch titers from
EPIC-generated clones which achieved top expression ranging from
1.5-2.0 g/L. Leftmost bar (0.5 g/L) represents titer for
EPIC-sorted pool prior to cloning. All other vertical bars
represent titers for individual clones.
[0036] FIG. 7 is a series of histogram offsets depicting the
comparative benefit of EPIC targeting to generate stable pools
transfected with pGZ729-RFP. EPIC was used to target early RFP
expression at day 2 which yielded a stable pool with improved RFP
(and CD52 reporter expression) as compared to traditional
transfection/selection methodologies (0 nM MTX).
DETAILED DESCRIPTION
[0037] Before the present invention is described, it is to be
understood that this invention is not limited to particular methods
and experimental conditions disclosed herein; 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.
[0038] Furthermore, the practice of the invention employs, unless
otherwise indicated, conventional molecular and cellular biological
and immunological techniques within the skill of the art. Such
techniques are well known to the skilled worker, and are explained
fully in the literature. See, e.g., Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,
N.Y. (1987-2008), including all supplements; M. R. Green and J.
Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition),
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2012); and
Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2013, 2.sup.nd
edition).
I. Definitions
[0039] Unless otherwise defined herein, scientific and technical
terms used herein have the meanings that are commonly understood by
those of ordinary skill in the art. In the event of any latent
ambiguity, definitions provided herein take precedent over any
dictionary or extrinsic definition. Unless otherwise required by
context, singular terms shall include pluralities and plural terms
shall include the singular. The use of "or" means "and/or" unless
stated otherwise. The use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting.
[0040] Generally, nomenclatures used in connection with cell and
tissue culture, molecular biology, immunology, microbiology,
genetics and protein and nucleic acid chemistry and hybridization
described herein are those well-known and commonly used in the art.
The methods and techniques provided herein are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification unless
otherwise indicated. Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0041] That the disclosure may be more readily understood, select
terms are defined below.
[0042] As used herein, the term "polynucleotide" intends a
polymeric form of nucleotides of any length, examples of which
include, but are not limited to, a gene or gene fragment, exons,
introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,
ribozymes, complementary DNA (cDNA), recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and
primers. A polynucleotide may comprise modified nucleotides, such
as methylated nucleotides and nucleotide analogs.
[0043] As used herein, the term "polypeptide" intends a polymeric
form of amino acids of any length, examples of which include, but
are not limited to, a protein, a protein fragment, a multimeric
protein, a fusion protein, an antibody (including fragments
thereof), and a peptide.
[0044] As used herein, a "selectable polypeptide" is a polypeptide
that can be detected, directly or indirectly, by any suitable
method including, for example and without limitation,
fluorescence-activated cell sorting (FACS), magnetic-activated cell
sorting (MACS), ClonePix, and affinity chromatography. In certain
embodiments, the selectable polypeptide is expressed on the surface
of a cell, i.e., is a cell surface polypeptide. Examples of
selectable polypeptides include polypeptides that include an
extracellular domain (e.g., CD52 or CD59) that are capable of being
bound to or by a detectable binding partner (e.g., a
fluorescently-labeled antibody). Other examples of selectable
polypeptides include fluorescent proteins such as green fluorescent
protein (GFP), red fluorescent protein (RFP), yellow fluorescent
protein (YFP), blue fluorescent protein (BFP), and variants thereof
including eGFP, Venus, mCherry, mTomato, and the like. In certain
embodiments, the selectable polypeptide may be conveniently
detected, directly or indirectly, by flow cytometry.
[0045] As used herein, "fluorescence-activated cell sorting" or
"FACS" refers to a method of separating a population of cells into
one or more sub-populations based on the presence, absence, or
level of one or more FACS-selectable polypeptides expressed by the
cells. FACS relies on optical properties, including fluorescence,
of individual cells in order to sort the cells into
sub-populations. FACS cell sorters suitable for carrying out a
method described herein are well-known in the art and commercially
available. Exemplary FACS cell sorters include BD Influx.TM. (BD
Biosciences) and other equivalent cell sorters produced by other
commercial vendors such as Sony, Bio-Rad, and Beckman Coulter.
[0046] As used herein, a "FACS selectable polypeptide" is a
polypeptide that can be detected, directly or indirectly, by flow
cytometry. Examples of FACS selectable polypeptides include
polypeptides that include an extracellular domain (e.g., CD52 or
CD59) that are capable of being bound to a detectable binding
partner (e.g., a fluorescently-labeled antibody) for indirect
detection of the polypeptide by flow cytometry. Other examples of
FACS selectable polypeptides include fluorescent proteins such as
green fluorescent protein (GFP), red fluorescent protein (RFP),
yellow fluorescent protein (YFP), blue fluorescent protein (BFP),
and variants thereof including eGFP, Venus, mCherry, mTomato, and
the like, which may be detected directly by flow cytometry.
[0047] As used herein, magnetic-activated cell sorting, or "MACS"
refers to a method of separating a population of cells into one or
more sub-populations based on the presence, absence, or level of
one or more MACS-selectable polypeptides expressed by the cells.
MACS relies on magnetic susceptibility properties of tagged
individual cells in order to sort the cells into sub-populations.
MACS cell sorters suitable for carrying out a method described
herein are well-known in the art and commercially available.
Exemplary MACS cell sorters include MACSQuant.RTM. flow cytometer
(Miltenyi Biotec).
[0048] As used herein, a "MACS selectable polypeptide" is a
polypeptide that can be detected, directly or indirectly, by
magnetic-activated cell sorting. Examples of MACS selectable
polypeptides include polypeptides that include an extracellular
domain (e.g., CD52 or CD59) that are capable of being bound to a
magnetically susceptible binding partner (e.g., an iron-, nickel-,
or cobalt-labeled bead coupled to an antibody) for direct or
indirect detection of the polypeptide. In certain embodiments, the
selectable polypeptide may be conveniently detected, directly or
indirectly, by flow cytometry.
[0049] As used herein, "ClonePix" refers to a method of, and device
for, separating a population of cells into one or more
sub-populations based on the presence, absence, or level of one or
more selectable polypeptides expressed by the cells. ClonePix
relies on optical properties, including white light and
fluorescence detection, of individual cells or colonies of cells in
order to sort the cells into sub-populations. ClonePix is described
in U.S. Pat. Nos. 7,776,584; 8,034,612; 8,034,625; 8,293,520;
8,293,525; 8,293,526; and 8,293,527, each to Richmond et al., and
is commercially available from Molecular Devices (Sunnyvale,
Calif.).
[0050] As used herein, "target polypeptide" refers to a protein, a
protein fragment, a multimeric protein, a fusion protein, an
antibody (including fragments thereof), or a peptide that can be
produced in host cells and in the aspects exemplified herein, the
target polypeptide is selected because of its potential as a
therapeutic agent, e.g., an antibody (including a fragment
thereof), a Fc fusion protein, a hormone or an enzyme. In some
embodiments, the target polypeptide is a secreted protein. However,
the methods described herein are not limited for the selection and
scale-up of therapeutic polypeptides. For example, diagnostic
polypeptides or polypeptides for use in the environment are also
contemplated for use as a target polypeptide in a method disclosed
herein.
[0051] In certain embodiments, the selectable polypeptide is a cell
surface polypeptide, and the target polypeptide is a secreted
polypeptide.
[0052] As used herein, the term "antibody" refers to such
assemblies (e.g., intact antibody molecules, antibody fragments, or
variants thereof) which have significant known specific
immunoreactive activity to an antigen of interest. Antibodies and
immunoglobulins comprise light and heavy chains, with or without an
interchain covalent linkage between them. Basic immunoglobulin
structures in vertebrate systems are relatively well
understood.
[0053] As used herein, the term "antibody" includes entire
antibodies as well as antigen-binding fragments and variants of
such antibodies. Antibodies may be of any class, such as IgG, IgA
or IgM; and of any subclass, such as IgG1 or IgG4. The antibody can
be a polyclonal or a monoclonal antibody, or it can be fragments of
the polyclonal or monoclonal antibody. The antibody can be
chimeric, humanized, totally human, bi-specific, or bi-functional.
Any antigen-binding fragment or variant of an antibody is also
contemplated, such as Fab, Fab', F(ab').sub.2, single-chain
variable regions (scFv) and variations of the same.
[0054] As used herein, an "Fc fusion protein" refers to a protein
comprising an immunoglobulin Fc domain that is linked, directly or
indirectly, to a polypeptide, such as a protein or peptide. The
linked polypeptide can be any proteinaceous molecule of interest,
such as a ligand, a receptor, or an antigenic peptide.
[0055] As used herein, the term "producer cell" refers to a cell
expressing a polypeptide of interest. In certain embodiments, a
producer cell is a cell expressing a target polypeptide as
disclosed herein. In certain embodiments, a producer cell is a cell
expressing both a selectable polypeptide and a target polypeptide
as disclosed herein.
[0056] In certain embodiments, the term "producer cells" refers to
cells that are suitable for production of proteins, e.g., in a
small- or large-scale manufacturing method for producing biologics.
In some embodiments, producer cells are mammalian or insect cells.
Producer cells are further discussed herein.
[0057] As used herein, a "population of producer cells" is a
population of cells that expresses an enhanced level of one or more
polypeptides, e.g., a FACS selectable polypeptide and a target
polypeptide that are encoded by the same multicistronic mRNA. In
certain embodiments, a "population of producer cells" is a
population of cells that expresses an enhanced level of a target
polypeptide. In some embodiments, the enhanced level is at least
10-fold, at least 100-fold, at least 1,000-fold, or at least
10,000-fold of the one or more polypeptides in an unselected
population. In some embodiments, the enhanced level is at least
10-fold, at least 100-fold, at least 1,000-fold, or at least
10,000-fold of a FACS-selectable polypeptide in an unselected
population as detected by flow cytometry (e.g., on a BD Influx.TM.
cell sorter). In some embodiments, the enhanced level is at least
10-fold, at least 100-fold, at least 1,000-fold, or at least
10,000-fold of a MACS-selectable polypeptide in an unselected
population as detected by flow cytometry (e.g., on a MACSQuant.RTM.
flow cytometer (Miltenyi Biotec)). Methods for generating
populations of producer cells are described herein.
[0058] As used herein, a "population of producer cells" is a
population of cells that expresses detectable levels of one or more
polypeptides, e.g., a FACS selectable polypeptide and a target
polypeptide that are encoded by the same multicistronic mRNA.
Methods for generating populations of producer cells are described
herein.
[0059] As used herein, a "multicistronic mRNA" is an mRNA that
contains at least two open reading frames (ORFs) that are capable
of encoding two or more polypeptides.
[0060] As used herein, a "drug-selection-free medium" is a culture
medium that is devoid of a drug (e.g., methotrexate (MTX)) that is
used to select a population or sub-populations of cells that
express a protein that confers drug resistance (e.g., dihydrofolate
reductase) to the population or sub-population.
[0061] As used herein, "medium-based selection" is a selection
process by which the culture medium is altered to include a
selection agent (e.g., MTX) or to exclude a component of medium,
which results in selection of a sub-population that is resistant to
the selection agent or can survive in the absence of the excluded
medium component.
[0062] As used herein, "nucleotide-deficient medium" is culture
medium that is devoid of or contains low levels (e.g., less than 10
micrograms/mL) of nucleotides having one or more of the nucleobases
adenine (A), cytosine (C), guanine (G), thymine (T), hypoxanthine,
or thymidine. In some embodiments, nucleotide-deficient medium is
medium that is devoid of hypoxanthine and thymidine. Exemplary
nucleotide-deficient medium includes CD CHO Medium (Gibco, Life
Technologies, Catalogue numbers 10743 (liquid) and 12490
(granulated)).
[0063] As used herein, a "viability marker" is a cell
characteristic that is indicative of cell viability and is
detectable by FACS. Exemplary viability markers include forward
scatter, side scatter, propidium iodide stain, or combinations
thereof.
[0064] As used herein, the term "non-AUG start codon" is intended
to include any non-AUG polynucleotide (typically a triplet) that
functions as a start site for translation initiation with reduced
efficiency relative to that of an AUG start codon. Naturally
occurring alternate start codon usage is known in the art and
described for example in Kozak (1991) J Cell Biol. 115(4): 887-903;
Mehdi et al. (1990) Gene 91:173-178; Kozak (1989) Mol. Cell. Biol.
9(11): 5073-5080. In general, non-AUG start codons have decreased
translation efficiencies compared to that of an AUG; for example,
the alternate start codon GUG may have 3-5% translation efficiency
compared to that of an AUG (100%). The translation efficiency of a
non-AUG start codon can also be affected by its sequence context;
for example, an optimal Kozak consensus sequence is reported to
have a positive effect on translation initiation at non-AUG start
codons (Mehdi et al. (1990) Gene 91:173-178; Kozak (1989) Mol.
Cell. Biol. 9(11): 5073-5080). The complete Kozak DNA consensus
sequence is GCCRCCATGG (SEQ ID NO:1), where the start codon ATG
(AUG in RNA) is underlined, the A of the ATG start codon is
designated as the +1 position, and "R" at position -3 is a purine
(A or G). The two most highly conserved positions are a purine,
preferably an A, at -3 and a G at +4 (Kozak (1991) J Cell Biol
115(4): 887-903). Alternate start codon usage is described for
attenuated expression of a selectable marker in U.S. Patent
Publication 2006/0172382 and U.S. Patent Publication 2006/0141577,
the entire contents of which are incorporated herein by reference.
One of skill in the art will recognize that the sequences described
herein as DNA will have correlative sequences as RNA molecules,
e.g., DNA sequence ATG would correspond to RNA sequence AUG, and
vice versa.
[0065] As used herein, the term "EPIC" refers to Early
Post-transfection Isolation of Cells, as described in more detail
herein.
[0066] As used herein, the term "FLARE" refers to "FLow Cytometry
Attenuated Reporter Expression." FLARE is an expression system
utilizing a multicistronic mRNA that contains at least two open
reading frames (ORFs), an upstream ORF containing a non-AUG start
codon and encoding a FACS selectable polypeptide, and a downstream
ORF containing an AUG start codon and encoding a target
polypeptide. See U.S. patent application Ser. No. 12/441,806 which
is incorporated by reference herein in its entirety.
[0067] As used herein, the term "about" shall refer to a range of
tolerance of 10% around a stated value. Therefore, when the term
"about" is used to modify a stated value, the range indicated will
encompass any number within .+-.0.01%, 0.02%, 0.05%, 0.1%, 0.2%,
0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the stated
value.
II. Methods for Early Selection of Producer Cells
[0068] In some aspects, the disclosure relates to a method of
producing a population of producer cells expressing a target
polypeptide. In some embodiments, the method comprises:
[0069] (a) transfecting host cells with one or more vectors that
encode one or more mRNAs, wherein the one or more mRNAs encode a
selectable polypeptide and the target polypeptide;
[0070] (b) isolating from the transfected host cells, within 2 to
15 days of transfection, a sub-population of early-expressing
transfected host cells which express the selectable polypeptide;
and
[0071] (c) expanding the sub-population of transfected host cells,
thereby producing a population of producer cells expressing the
target polypeptide.
[0072] Early-expressing transfected cells can comprise different
classes of exogenous DNA, part of which has not integrated into the
cells' genomic DNA, and part of which has integrated into the
cells' genomic DNA. Both these types of DNA have the potential to
lead to expression of the polypeptide or polypeptides they
encode.
[0073] Host cells are transfected with one or more vectors that
encode one or more mRNAs, wherein the one or more mRNAs encode a
selectable polypeptide and the target polypeptide. A producer cell
can be generated using any cell type suitable for production of a
target polypeptide from a multicistronic mRNA. In some embodiments,
the host cell is a eukaryotic cell. Examples of suitable eukaryotic
cells to produce a target polypeptide include, but are not limited
to, a Chinese Hamster Ovary (CHO) cell line, including those
designated CHO-DBX11, CHO-DG44, CHO-S, CHO-K1, and the hamster cell
line BHK-21; the murine cell lines designated NIH3T3, NSO, C127,
the simian cell lines COS, Vero; and the human cell lines HeLa,
HEK293 (also called 293), NIH-3T3, U-937 and Hep G2. Additional
examples of suitable host cells include yeast cells, insect cells
(e.g., Drosophila Schnieder S2 cells, Sf9 insect cells (WO
94/26087), BTI-TN-5B1-4 (High Five.TM.) insect cells (Invitrogen)),
plant cells, avian cells, and bovine cells. Examples of yeast
useful for expression include, but are not limited to
Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis,
Yarrowia, and Pichia. See e.g., U.S. Pat. Nos. 4,812,405;
4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and
6,258,559. Other examples of producer cells can be prokaryotic,
including bacterial cells such as E. coli (e.g., strain
DH5.alpha..TM.) (Invitrogen, Carlsbad, Calif.), PerC6 (Crucell,
Leiden, NL), B. subtilis and/or other suitable bacteria. The cells
can be purchased from a commercial vendor such as the American Type
Culture Collection (ATCC, Rockville, Md.) or cultured from an
isolate using methods known in the art.
[0074] To make a producer cell, recombinant or exogenous
polynucleotide(s) can be inserted into the host cell using any
suitable transfer technique (e.g., by transfection, transformation,
electroporation or transduction). Vectors that encode one or more
mRNAs include DNA vectors. Vectors that may be used include
plasmids, viruses, phage, transposons, and minichromosomes of which
plasmids are a typical embodiment. Generally such vectors further
include a signal sequence, origin of replication, one or more
marker genes, a promoter and transcription termination sequences
operably linked to the gene encoding the multicistronic mRNA so as
to facilitate expression. Examples of suitable DNA viral vectors
include adenovirus (Ad) and adeno-associated virus (AAV).
Adenovirus-based vectors for the delivery of polynucleotides are
known in the art and may be obtained commercially or constructed by
standard molecular biological methods. Adenoviruses (Ads) are a
group of viruses, including over 50 serotypes. See, e.g.,
International Patent Application No. WO 95/27071. Other viral
vectors for use in the present disclosure include vectors derived
from vaccinia, herpesvirus (e.g., herpes simplex virus (HSV)), and
retroviruses. Gene delivery vehicles also include several non-viral
vectors, including DNA/liposome complexes, and targeted viral
protein-DNA complexes.
[0075] For use in transfection, in certain embodiments circular
vectors may be pre-linearized, i.e., linearized prior to
introduction into the host cell, for example by restriction at one
or more restriction endonuclease sites. Linearization is believed
to be necessary for integration into the genome, and this can be
effected by pre-linearization or in a random fashion by
endonucleases naturally present within the host cell.
Pre-linearization has the potential advantage of introducing a
degree of control into the site of restriction. Thus, in certain
embodiments, circular vectors, including supercoiled circular
vectors, may be introduced into the host cell. In certain
embodiments in accordance with the instant invention, the one or
more vectors are linear at the time of transfection.
[0076] Vectors that contain both a promoter and a cloning site into
which a polynucleotide can be operatively linked are known in the
art and available from commercial vendors. Such vectors are capable
of transcribing RNA in vitro or in vivo, and are commercially
available from sources such as Agilent Technologies and Promega
Corporation. In order to optimize expression and/or in vitro
transcription, it may be necessary to remove, add or alter 5'
and/or 3' untranslated portions to eliminate extra, potentially
inappropriate alternative translation initiation codons or other
sequences that may interfere with or reduce expression, either at
the level of transcription or translation. Alternatively, consensus
ribosome binding sites can be inserted immediately 5' of the start
codon to enhance expression.
[0077] A promoter can be provided for expression in the producer
cell. Promoters can be constitutive or inducible. For example, a
promoter can be operably linked to a nucleic acid encoding a
multicistronic mRNA such that it directs expression of the encoded
polypeptides. A variety of suitable promoters for prokaryotic and
eukaryotic hosts are available. Prokaryotic promoters include lac,
tac, T3, T7 promoters for E. coli; 3-phosphoglycerate kinase or
other glycolytic enzymes e.g., enolase, glyceraldehyde 3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose 6 phosphate isomerase,
3-phosphoglycerate mutase, and glucokinase. Eukaryotic promoters
include inducible yeast promoters such as alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, metallothionein, and enzymes
responsible for nitrogen metabolism or maltose/galactose
utilization; RNA polymerase II promoters including viral promoters
such as polyoma, fowlpox and adenoviruses (e.g., adenovirus 2),
bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV,
in particular, the immediate early gene promoter), retrovirus,
hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter, and
the early or late Simian virus 40 (SV40) and non-viral promoters
such as EF-1 alpha (Mizushima and Nagata (1990) Nucleic Acids Res.
18(17):5322). Those of skill in the art will be able to select the
appropriate promoter for expressing any given polypeptide in a
given host cell.
[0078] Where appropriate, e.g., for expression in cells of higher
eukaryotes, additional enhancer elements can be included instead of
or as well as those found located in the promoters described above.
Suitable mammalian enhancer sequences include enhancer elements
from globin, elastase, albumin, fetoprotein, metallothionein, and
insulin. Alternatively, one may use an enhancer element from a
eukaryotic cell virus such as SV40 enhancer, cytomegalovirus early
promoter enhancer, polyoma enhancer, baculoviral enhancer or murine
IgG2a locus (see, WO 2004/009823). Whilst such enhancers are often
located on the vector at a site upstream to the promoter, they can
also be located elsewhere, e.g., within the untranslated region or
downstream of the polyadenylation signal. The choice and
positioning of enhancer may be based upon compatibility with the
host cell used for expression.
[0079] In addition, the vectors (e.g., expression vectors) may
comprise a selectable marker for selection of host cells carrying
the vector, and, in the case of a replicable vector, an origin of
replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in
prokaryotic (e.g., .beta.-lactamase gene (ampicillin resistance),
tet gene (tetracycline resistance) and eukaryotic cells (e.g.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin,
or hygromycin B resistance genes). The dihydrofolate reductase
(DHFR) gene permits selection with methotrexate or
nucleotide-deficient medium in a variety of hosts. Similarly, the
glutamine synthetase (GS) gene permits selection with methionine
sulphoximine. Genes encoding the gene product of auxotrophic
markers of the host (e.g., LEU2, URA3, HIS3) are often used as
selectable markers in yeast. Use of viral (e.g., baculovirus) or
phage vectors, and vectors which are capable of integrating into
the genome of the host cell, such as retroviral vectors, are also
contemplated.
[0080] In eukaryotic systems, polyadenylation and termination
signals may be operably linked to a polynucleotide encoding the
multicistronic mRNA as described herein. Such signals are typically
placed 3' of an open reading frame. In mammalian systems,
non-limiting examples of polyadenylation/termination signals
include those derived from growth hormones, elongation factor-1
alpha and viral (e.g., SV40) genes or retroviral long terminal
repeats. In yeast systems, non-limiting examples of
polyadenylation/termination signals include those derived from the
phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH)
genes. In prokaryotic systems polyadenylation signals are typically
not required and it is instead usual to employ shorter and more
defined terminator sequences. The choice of
polyadenylation/termination sequences may be based upon
compatibility with the host cell used for expression. In addition
to the above, other features that can be employed to enhance yields
include chromatin remodeling elements, introns and host cell
specific codon modification.
[0081] The producer cells of the disclosure contain a recombinant
polynucleotide (e.g., a recombinant cDNA) that encodes a
multicistronic mRNA molecule from which the target and selectable
polypeptides are separately translated from different ORFs. In some
embodiments, the selectable polypeptide is a cell surface
polypeptide. In certain embodiments, the producer cells of the
disclosure contain a plurality of recombinant polynucleotides, each
of which encodes a multicistronic mRNA molecule from which a target
polypeptide and a selectable polypeptide are separately translated
from different ORFs. Each target polypeptide can thus be associated
with a particular selectable polypeptide. In some embodiments, the
selectable polypeptide is a cell surface polypeptide.
[0082] Examples of cell surface polypeptides include, but are not
limited to CD2, CD20, CD52, and CD59. Exemplary, non-limiting,
amino acid sequences for CD52 and CD59 cell surface polypeptides
are provided below.
TABLE-US-00001 Amino Acid Sequence for Exemplary Human CD52
polypeptide: (SEQ ID NO: 2)
LERFLFLLLTISLLVLVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFV ANAIIHLFCFS
Amino Acid Sequence for Exemplary Human CD59 polypeptide (splice
acceptor mutant): (SEQ ID NO: 3)
LGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDA
CLITKAGLQVYNNCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQL
ENGGTSLSEKTVLLLVTPFLAAAWSLHP Amino Acid Sequence for Exemplary
Mouse CD52 polypeptide: (SEQ ID NO: 4)
LKSFLLFLTIILLVVIQIQTGSLGQATTAASGTNKNSTSTKKTPLKSGAS
SIIDAGACSFLFFANTLICLFYLS
[0083] In some embodiments, a first ORF is provided which encodes a
selectable polypeptide, such as CD52 or CD59. Exemplary,
non-limiting ORF sequences for CD52 and CD59 are provided
below.
TABLE-US-00002 Nucleotide Sequence for Exemplary Human CD52 ORF:
(SEQ ID NO: 6) ttggagcgcttcctcttcctcctactcaccatcagcctcctcgttttggt
acaaatacaaaccggactctccggacaaaacgacaccagccaaaccagca
gcccctcagcatccagcaacataagcggaggcattttccttttcttcgtc
gccaacgccataatccacctcttctgcttcagttga Nucleotide Sequence for
Exemplary Human CD59 ORF: (SEQ ID NO: 7)
ttgggaatccaaggagggtctgtcctgttcgggctgctgctcgtcctcgc
tgtcttctgccattccggtcatagcctgcagtgctacaactgtcctaacc
caactgctgactgcaaaacagccgtcaattgttcatctgattttgacgcg
tgtctcattaccaaagctgggttacaagtgtataacaactgttggaagtt
tgagcattgcaatttcaacgacgtcacaacccgcttgagggaaaacgagc
taacgtactactgctgcaagaaggacctgtgtaactttaacgaacagett
gaaaacggagggacatccttatcagagaaaacagttettctgctggtgac
tccatttctggcagctgcttggagccttcatccctaa Nucleotide Sequence for
Exemplary Mouse CD52 ORF: (SEQ ID NO: 8)
ttgaagagettectcctcttcctcactatcattcttctcgtagtcattca
gatacaaacaggatccttaggacaagccactacggccgcttcaggtacta
acaaaaacagcacctccaccaaaaaaaccccettaaagagcggggcctca
tccatcatcgacgcgggcgcttgcagtttcctcttcttcgccaataccct
tatttgcctcttctacctcagctaactgagtaa
[0084] As discussed below, each the foregoing exemplary ORFs has
been modified to eliminate all internal ATG triplets.
[0085] In some embodiments, a second ORF is provided which encodes
a target polypeptide, such as an antibody, enzyme, or Fc fusion
protein. In some embodiments, separate translation is accomplished
by use of a non-AUG start codon for translation initiation of the
selectable polypeptide and the use of an AUG start codon for
translation initiation of the target polypeptide.
[0086] In this embodiment, generally the polynucleotide encoding
the target polypeptide is located downstream of the polynucleotide
encoding the selectable polypeptide. Separate translation can also
be achieved using an internal ribosome entry site (IRES). In some
embodiments, the IRES element is located upstream of the
polynucleotide encoding the target polypeptide and downstream of
the polynucleotide encoding the selectable polypeptide. In some
embodiments, the IRES element is located upstream of the
polynucleotide encoding the selectable polypeptide and downstream
of the polynucleotide encoding the target polypeptide.
[0087] In some embodiments, a non-AUG start codon is located within
the DNA encoding the selectable polypeptide in such a way that
translation of the selectable polypeptide is less efficient than
translation of the target polypeptide. To achieve decreased
translation efficiency, the AUG start codon of the selectable
polypeptide may be changed to an alternate non-AUG start codon,
examples of which include but are not limited to: CUG, GUG, UUG,
AUU, AUA, and ACG.
[0088] Thus, when using an alternate non-AUG start codon,
expression of a selectable polypeptide can be attenuated relative
to that of a co-expressed target polypeptide. In addition to
alteration of the start codon, the DNA encoding the selectable
polypeptide may be modified at all internal ATG triplets to prevent
internal initiation of translation. In some embodiments, the
selectable polypeptide has a short amino acid sequence (<200
amino acids) and is encoded by a polynucleotide with few (<10)
ATG triplets.
[0089] Without wishing to be bound by theory, to initiate
translation of the mRNA encoding both the selectable polypeptide
and the target polypeptide, ribosomes begin scanning at the 5' cap
structure of the mRNA with the majority scanning past the alternate
start codon (for example, UUG) and instead initiating translation
at the downstream AUG start codon. However, translation initiation
can occur at the alternate start codon, albeit with very low
frequency, so that a low level of the selectable polypeptide is
also expressed.
[0090] From the transfected host cells is selected a sub-population
of early-expressing transfected host cells which express detectable
levels of the selectable polypeptide. During transfection
individual host cells take up different amounts of exogenous
polynucleotide, e.g., DNA, in an essentially random manner. Some
cells will take up many copies of the exogenous polynucleotide,
others will take up fewer copies, and some will take up none. The
amount of DNA taken up into a given cell affects the fate of the
DNA, including its early expression and its integration into the
genome.
[0091] Following transfection with DNA, at least some of the
polynucleotide that has been introduced into the cell is
translocated into the nucleus where it is transcribed into mRNA. In
the first few days, expression of the introduced DNA may be driven
off one or more classes of DNA, some of which has not yet been
integrated into the genome of the host cell, and some of which has
been integrated into the genome of the host cell. At this point the
extent of expression is believed to be principally proportional to
the "dose" of DNA introduced into the host cell and its nucleus.
The greater the amount of exogenous DNA taken up by the host cell
the greater the degree of early expression. However, a small amount
of DNA that has been introduced into the host cell, particularly
once it is linear, can become integrated into the genome of the
host cell. Thus, in the first several days following transfection,
there is a competition between degradation and loss of the
exogenous DNA on the one hand, and stochastic integration of the
exogenous DNA into the genome on the other hand. Integration can
include single or multiple copies of introduced DNA. The greater
the amount of exogenous DNA taken up by the host cell, the greater
the chance (and degree) of its integration. Ultimately, it is the
integrated DNA that is responsible for long-term productive
expression, i.e., expression after all nonintegrated DNA (e.g.,
plasmid or episomal DNA) is degraded to the point of being
incapable of meaningful expression.
[0092] Thus in the first 2 to about 15 days (e.g., 2 days, 3 days,
4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,
12 days, 13 days, 14 days, 15 days) following transfection, there
are early-expressing transfected host cells which express
detectable amounts of the selectable polypeptide. Particularly in
the first 2-6 days, more particularly in the first 2-4 days, and
even more particularly in the first 2-3 days, this early expression
is believed to be largely, but not necessarily exclusively, driven
off exogenous DNA that has not yet been integrated into the genome
of the host cell. During this early period following transfection,
there may be some degree of integration of exogenous DNA into the
host cell genome. Because this early expression depends on the
"dose" of DNA taken up by the host cell and its nucleus, and the
dose is essentially random among transfected cells, during this
early period the transfected host cells include sub-populations of
cells expressing different amounts of polypeptide encoded by the
exogenous DNA. Also during this early period the sub-populations of
cells expressing greater amounts of polypeptide encoded by the
exogenous DNA presumably took up greater amounts of exogenous DNA
and therefore have a greater chance of incorporating the DNA into
their genome.
[0093] Accordingly, the term "early-expressing" or "early
expression", as used herein, refers to detectable expression in the
first 2 to about 15 days (e.g., 2-15 days, 2-14 days, 2-13 days,
2-12 days, 2-11 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6
days, 2-5 days, 2-4 days, 2-3 days, 3-15 days, 3-14 days, 3-13
days, 3-12 days, 3-11 days, 3-10 days, 3-9 days, 3-8 days, 3-7
days, 3-6 days, 3-5 days, 3-4 days, 4-15 days, 4-14 days, 4-13
days, 4-12 days, 4-11 days, 4-10 days, 4-9 days, 4-8 days, 4-7
days, 4-6 days, 4-5 days, 5-15 days, 5-14 days, 5-13 days, 5-12
days, 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-6 days,
6-15 days, 6-14 days, 6-13 days, 6-12 days, 6-11 days, 6-10 days,
6-9 days, 6-8 days, 6-7 days, 7-15 days, 7-14 days, 7-13 days, 7-12
days, 7-11 days, 7-10 days, 7-9 days, 7-8 days, 8-15 days, 8-14
days, 8-13 days, 8-12 days, 8-11 days, 8-10 days, 8-9 days, 9-15
days, 9-14 days, 9-13 days, 9-12 days, 9-11 days, 9-10 days)
following transfection. In certain embodiments, the term
"early-expressing" or "early expression" refers to detectable
expression in the first 2 to about 10 days following transfection.
In certain embodiments, the term "early-expressing" or "early
expression" refers to detectable expression in the first 2 to about
6 days following transfection. In certain embodiments, the term
"early-expressing" or "early expression" refers to detectable
expression in the first 2 to about 5 days following transfection.
In certain embodiments, the term "early-expressing" or "early
expression" refers to detectable expression in the first 2 to about
4 days following transfection. In certain embodiments, the term
"early-expressing" or "early expression" refers to detectable
expression in the first 2 to about 3 days following
transfection.
[0094] Any method known in the art useful for detecting a cell
surface marker may be used in connection with the methods of the
disclosure. For example, an antibody or other cell surface
marker-specific binding agent is contacted directly or indirectly
with the transfected host cells under conditions that permit or
favor binding of antibody to the selectable polypeptide and thereby
select a sub-population of early-expressing transfected host cells.
The selection of the antibody or other binding agent is determined
by: 1) its ability to selectively bind the selectable polypeptide
that is expressed on the host cell; and 2) its ability to be
labeled with a detectable label or bind to a detectable label, for
example, for use in flow cytometry or FACS.
[0095] In an alternate embodiment, a first agent can be a protein
or peptide that binds to the selectable polypeptide, which first
agent also in turn binds to a second agent that is capable of being
detectably labeled (e.g., incorporating a fluorescent, enzymatic,
colorimetric, magnetically susceptible, or other detectable label).
It is intended, although not always explicitly stated that
"indirect" binding to the selectable polypeptide includes the use
of any number of intermediate partners. In certain embodiments,
"indirect" binding to the selectable polypeptide includes the use
of one intermediate partner, e.g., one unlabeled antibody or other
binding agent.
[0096] In some embodiments, the antibody or other binding agent
binds directly to the cell surface marker and comprises a
fluorescent label. Suitable fluorescent labels include, but are not
limited to, fluorescein isothiocyanate (FITC), rhodamine,
tetramethylrhodamine, eosin, phycoerythrin (PE), erythrosin,
allophycocyanin (APE), coumarin, methyl-coumarins, pyrene,
Malachite green, stilbene, Lucifer Yellow, Cascade Blue, and Texas
Red. Other suitable optical dyes are described in the Molecular
Probes.RTM. Handbook, 11.sup.th Edition, 2010.
[0097] In some embodiments, the fluorescent label is functionalized
to facilitate covalent attachment to the antibody or other agent.
Suitable functional groups, include, but are not limited to,
isothiocyanate groups, amino groups, haloacetyl groups, maleimides,
succinimidyl esters, and sulfonyl halides, all of which may be used
to attach the fluorescent label to a second molecule. The choice of
the functional group of the fluorescent label will depend on the
site of attachment to the antibody or other binding agent, the
selectable polypeptide, or the second labeling agent.
[0098] Attachment of the fluorescent label may be either direct or
via a linker to the antibody or other binding agent. In one aspect,
the linker is a relatively short coupling moiety that generally is
used to attach molecules. In this embodiment, attachment of the
first labeling moiety to the candidate agents will be done as is
generally appreciated by those in the art, and may include
techniques outlined above for the incorporation of fluorescent
labels.
[0099] Materials and techniques for design and construction of
labeled antibodies and other agents for use in cytometry are known
in the art and described for example, in Bailey et al. (2002)
Biotechnol. Bioeng. 80(6); 670-676; Carroll and Al-Rubeai (2004)
Expt. Opin. Biol. Therapy 4:1821-1829; Yoshikawa et al. (2001)
Biotechnol. Bioeng. 74:435-442; Meng et al. (2000) Gene
242:201-207; Borth et al. (2001) Biotechnol. Bioeng. 71
(4):266-273; Zeyda et al. (1999) Biotechnol. Prog. 15:953-957;
Klucher et al. (1997) Nucleic Acids Res. 25(23):4853-4860; and
Brezinsky et al. (2003) J. Imumunol. Methods 277:141-155.
[0100] Suitable binding pairs for use in indirectly linking the
label to the agent (which in turn, binds the selectable
polypeptide) include, but are not limited to, antigens/antibodies,
including digoxigenin/antibody, dinitrophenol (DNP)/anti-DNP,
dansyl-X/anti-dansyl, fluorescein/anti-fluorescein, lucifer
yellow/anti-lucifer yellow, rhodamine/anti-rhodamine; and
biotin/avidin (or biotin/strepavidin). The binding pairs should
have high affinities for each other, sufficient to withstand the
shear forces during cell sorting or other detection system used in
connection with the disclosure.
[0101] Thus, in some aspects, first labeling moieties (when second
labeling moieties are used), include, but are not limited to,
haptens such as biotin. Biotinylation of target molecules is well
known, for example, a large number of biotinylation agents are
known, including amine-reactive and thiol-reactive agents, for the
biotinylation of proteins, nucleic acids, carbohydrates, and
carboxylic acids. Similarly, a large number of other haptenylation
reagents are also known.
[0102] The antibodies used in a method described herein can be
produced in cell culture, in phage, or in various animals,
including but not limited to cows, mice, rats, hamsters, guinea
pigs, rabbits, sheep, goats, horses, cows, camelids, monkeys,
chimpanzees, etc., so long as the antibodies retain specificity of
binding for the selectable polypeptide. Antibodies can be tested
for specificity of binding by comparing binding to appropriate
antigen to binding to irrelevant antigen or antigen mixture under a
given set of conditions.
[0103] In embodiments in which the antibody or other binding agent
for the selectable polypeptide is not directly labeled, the
antibody or binding agent preferably also contains and retains the
ability to bind a secondary agent which is detectable after binding
to the cell via the selectable polypeptide.
[0104] In some embodiments, when the selectable polypeptide is
CD52, the selectable polypeptide may be detected using an anti-CD52
antibody. "Anti-CD52 antibody" refers to an antibody that
specifically recognizes and binds CD52. Anti-CD52 antibodies can be
generated by methods well known in the art. See for example,
Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
1987 to present versions) and Antibodies: A Laboratory Manual,
Second edition (Greenfield, ed. 2013). Additionally, several
anti-CD52 antibodies are commercially available (e.g., antibodies
conjugated to a fluorescent label, such as those sold by the
commercial vendors AbCam, SeroTec, and BioLegend).
[0105] In some embodiments, when the selectable polypeptide is
CD59, the selectable polypeptide may be detected using an anti-CD59
antibody. "Anti-CD59 antibody" refers to an antibody that
specifically recognizes and binds CD59. Anti-CD59 antibodies can be
generated by methods well known in the art. Additionally, several
anti-CD59 antibodies are commercially available (e.g., antibodies
conjugated to a fluorescent label, such as those sold by the
commercial vendors AbCam, SeroTec, and BioLegend).
[0106] In a particular embodiment, when the selectable polypeptide
is CD20, the FACS selectable polypeptide may be detected using an
anti-CD20 antibody. "Anti-CD20 antibody" refers to an antibody that
specifically recognizes and binds CD20. Anti-CD20 antibodies can be
generated by methods well known in the art. Additionally, several
anti-CD20 antibodies are commercially available from vendors such
as BD Pharmingen; Beckman Coulter, Inc. (Fullerton, Calif.,
numerous clones including Catalog No. 6604106 Clone H299 (B1);
Isotype IgG2a and Catalog No. IM1565 Clone L26, Isotype IgG2a);
Invitrogen (Carlsbad, Calif., Clone: BH-20, Isotype: IgG2a and
Clone: B-H20, Isotype: IgG2a); BioLegend (San Diego, Calif.,
Catalog. No. 302301, Clone: 21-7, Isotype: IgG2b, .kappa.); EMD
Biosciences, Inc., CALBIOCHEM.RTM. Brand (San Diego, Calif.,
Catalog No. 217670 Clone 2H7, Isotype: IgG2b); and Anaspec (San
Jose, Calif., Catalog No. 29587).
[0107] For use in MACS, where there is a more limited number of
antigen-specific magnetic beads, a labeled or unlabeled primary
antibody or other binding agent (e.g., Fc fusion protein) can be
used to bind to the selectable polypeptide, followed by binding by,
for example, an isotype-specific magnetic bead. For example,
Miltenyi Biotec sells CD20 microbeads and anti-mouse IgG
microbeads, but neither CD52 nor CD59 microbeads; anti-mouse IgG
microbeads could be used to label primary mouse IgG anti-human CD52
or mouse IgG anti-human CD59.
[0108] In an exemplary, non-limiting method, a population of
transfected host cells as described herein is contacted with an
agent that recognizes and directly or indirectly binds the
selectable polypeptide, if present, on the surface of the cells.
The contacting is performed under conditions that favor or are
suitable for specific binding (directly or indirectly) of the agent
or antibody with the selectable polypeptide. The cells that are
bound to the agent or antibody are then selected for using a
suitable method such as FACS (e.g., by gating for cells that
express the FACS-selectable polypeptide at a high level such as a
level that is at least 80% of the level of the population) and used
to select a sub-population of early-expressing transfected host
cells. Alternatively, the cells that are bound to the agent or
antibody are then selected for using a suitable method such as MACS
(e.g., by gating for cells that express the MACS-selectable
polypeptide at a high level such as a level that is at least 80% of
the level of the population) and used to select a sub-population of
early-expressing transfected host cells.
[0109] The selected sub-population of early-expressing transfected
host cells is then grown under conditions that result in expansion
of the sub-population to produce a population of producer cells
expressing the target polypeptide.
[0110] In certain embodiments, the step of isolating from the
transfected host cells, within 2 to 15 days of transfection, a
sub-population of early-expressing transfected host cells which
express the selectable polypeptide is performed in
drug-selection-free medium. For example, in certain embodiments,
the step of isolating from the transfected host cells, within 2 to
15 days of transfection, a sub-population of early-expressing
transfected host cells which express the selectable polypeptide is
performed in 0 nM MTX (i.e., MTX-free) medium.
[0111] In certain embodiments, the step of expanding the selected
sub-population of transfected host cells is performed in
drug-selection-free medium. For example, in certain embodiments,
the step of expanding the selected sub-population of transfected
host cells is performed in 0 nM MTX (i.e., MTX-free) medium.
[0112] In certain embodiments, both the step of (b) isolating from
the transfected host cells, within 2 to 15 days of transfection, a
sub-population of early-expressing transfected host cells which
express the selectable polypeptide, and the step of (c) expanding
the isolated sub-population of transfected host cells are performed
in drug-selection-free medium. For example, in certain embodiments,
both the step of (b) isolating from the transfected host cells,
within 2 to 15 days of transfection, a sub-population of
early-expressing transfected host cells which express the
selectable polypeptide, and the step of (c) expanding the isolated
sub-population of transfected host cells are performed in 0 nM MTX
(i.e., MTX-free) medium.
[0113] Cells, including producer cells, may be cultured in spinner
flasks, shake flasks, roller bottles, wave reactors (e.g., System
1000 from wavebiotech.com) or hollow fiber systems, or for large
scale production, stirred tank reactors or bag reactors (e.g., Wave
Biotech, Somerset, N.J. USA) are used particularly for suspension
cultures. Stirred tank reactors can be adapted for aeration using
e.g., spargers, baffles or low shear impellers. For bubble columns
and airlift reactors, direct aeration with air or oxygen bubbles
may be used. Where the host cells are cultured in a serum-free
culture medium, the medium can be supplemented with a cell
protective agent such as poloxamer 188 (Pluronic.RTM. F-68) to help
prevent cell damage as a result of the aeration process. Depending
on the host cell characteristics, microcarriers may be used as
growth substrates for anchorage-dependent cell lines, or the cells
may be adapted to suspension culture. The culturing of host cells,
particularly vertebrate host cells, may utilize a variety of
operational modes such as batch, fed-batch, repeated batch
processing (see, Drapeau et al. (1994) Cytotechnology 15:103-109),
extended batch process or perfusion culture. Although recombinantly
transformed producer cells may be cultured in serum-containing
media such media comprising fetal calf serum (FCS), in some
embodiments, such host cells are cultured in serum-free media such
as disclosed in Keen et al. (1995) Cytotechnology 17:153-163, or
commercially available media such as ProCHO-CDM or UltraCHO.TM.
(Cambrex, N.J., USA), supplemented where necessary with an energy
source such as glucose and synthetic growth factors such as
recombinant insulin. The serum-free culturing of host cells may
require that those cells are adapted to grow in serum-free
conditions. One adaptation approach is to culture such host cells
in serum containing media and repeatedly exchange 80% of the
culture medium for the serum-free media so that the host cells
adapt to serum-free conditions (see, e.g., Scharfenberg, K. et al.
(1995) In: Animal Cell Technology: Developments Towards the 21st
Century (Beuvery, E. C. et al., eds), pp. 619-623, Kluwer Academic
publishers).
[0114] In certain embodiments, the method further comprises
isolating the target polypeptide from the population of producer
cells. The target polypeptide can be isolated using any method
known in the art and may be further purified, e.g., according to
Current Good Manufacturing Practice (CGMP) for recombinant proteins
and antibodies, to a purity level of at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or more. A target polypeptide according to the described
embodiments may be secreted into the medium and recovered and
purified therefrom using any of a variety of techniques to provide
a degree of purification suitable for the intended use. For
example, the use of a target polypeptide (e.g., an antibody or
Fc-fusion protein) for the treatment of human subjects typically
mandates at least 95% purity as determined by reducing SDS-PAGE,
more typically 98% or 99% purity, when compared to the culture
media comprising the target polypeptide. In the first instance,
cell debris from the culture media can be removed using
centrifugation followed by a clarification step of the supernatant
using e.g., microfiltration, ultrafiltration and/or depth
filtration. Alternatively, a target polypeptide can be harvested by
microfiltration, ultrafiltration or depth filtration without prior
centrifugation. A variety of other techniques such as dialysis and
gel electrophoresis and chromatographic techniques such as
hydroxyapatite (HA), affinity chromatography (optionally involving
an affinity tagging system such as polyhistidine) and/or
hydrophobic interaction chromatography (HIC) (see, U.S. Pat. No.
5,429,746) are available. In one embodiment, a target polypeptide
such as an antibody or Fc-fusion protein, following various
clarification steps, is captured using Protein A or G affinity
chromatography followed by further chromatography steps such as ion
exchange and/or HA chromatography, anion or cation exchange, size
exclusion chromatography and ammonium sulphate precipitation.
Various virus removal steps may also be employed (e.g.,
nanofiltration using, e.g., a DV-20 filter). Following these
various steps, a purified preparation comprising at least 10 mg/mL
or greater, e.g., 100 mg/mL or greater of the target polypeptide
described herein is provided.
[0115] In certain embodiments the methods of the invention further
include the step of isolating one or more single transfected host
cells from the expanded sub-population and culturing the one or
more single transfected host cells to produce clonal populations of
the one or more single transfected host cells. In certain
embodiments the methods of the invention further include the step
of isolating one or more single transfected host cells from the
expanded sub-population and culturing the one or more single
transfected host cells to produce one or more clonal populations of
producer cells expressing the target polypeptide. Preparation of a
clonal population can be performed by any method known in the art.
For example, in one embodiment, the selected cells may be plated
into 96-well (or other size) plates at a density of one cell per
well and permitted to grow for a period of time (e.g., typically
7-28 days) which permits the single cell to grow into a multi-cell
colony of daughter cells (i.e., a clonal population). The method
may next comprise analyzing one or more of the clonal populations
by detecting the level of the selectable polypeptide and/or target
polypeptide expression on said clonal population and selecting one
or more clonal populations with a high expression level of the
selectable polypeptide and/or target polypeptide, thereby selecting
one or more clonal populations stably expressing the target
polypeptide. In certain embodiments, the clonal population is
cultured for 7-28 days after plating at a single cell density
before the clonal populations are analyzed. The method may further
include contacting the clonal population with a detectable antibody
or other binding agent that recognizes and directly or indirectly
binds the selectable polypeptide, if present, on the surface of the
clonal cell under conditions that permit or favor binding of the
antibody or other binding agent with the selectable polypeptide;
and selecting or detecting one or more cells that are directly or
indirectly bound to the antibody or other binding agent. These
cells so selected also can be isolated and cultured. The method may
further include analyzing target polypeptide expression of the one
or more clones, e.g., using protein A screening (such as when the
target polypeptide is an antibody or Fc-fusion protein), Western
blot, SDS polyacrylamide gel electrophoresis (PAGE) with Coomassie
Blue or silver stain, or an enzyme activity assay.
[0116] In certain embodiments, the sub-population of transfected
host cells subject to isolation in step (b) comprises at least
80-120.times.10.sup.6 cells. For example, in certain embodiments,
the sub-population of transfected host cells subject to isolation
in step (b) comprises at least about 80.times.10.sup.6 cells; in
certain embodiments, the sub-population of transfected host cells
subject to isolation in step (b) comprises at least about
90.times.10.sup.6 cells; in certain embodiments, the sub-population
of transfected host cells subject to isolation in step (b)
comprises at least about 100.times.10.sup.6 cells; in certain
embodiments, the sub-population of transfected host cells subject
to isolation in step (b) comprises at least about
110.times.10.sup.6 cells; and in certain embodiments, the
sub-population of transfected host cells subject to isolation in
step (b) comprises at least about 120.times.10.sup.6 cells. For
example, in certain embodiments, the sub-population of transfected
host cells subject to isolation in step (b) comprises about
80.times.10.sup.6 to about 800.times.10.sup.6 cells, about
100.times.10.sup.6 to about 800.times.10.sup.6 cells, about
200.times.10.sup.6 to about 800.times.10.sup.6 cells, about
300.times.10.sup.6 to about 800.times.10.sup.6 cells, about
400.times.10.sup.6 to about 800.times.10.sup.6 cells, about
500.times.10.sup.6 to about 800.times.10.sup.6 cells, about
80.times.10.sup.6 to about 600.times.10.sup.6 cells, about
100.times.10.sup.6 to about 600.times.10.sup.6 cells, about
200.times.10.sup.6 to about 600.times.10.sup.6 cells, about
300.times.10.sup.6 to about 600.times.10.sup.6 cells, about
400.times.10.sup.6 to about 600.times.10.sup.6 cells, about
500.times.10.sup.6 to about 600.times.10.sup.6 cells, about
80.times.10.sup.6 to about 500.times.10.sup.6 cells, about
100.times.10.sup.6 to about 500.times.10.sup.6 cells, about
200.times.10.sup.6 to about 500.times.10.sup.6 cells, about
300.times.10.sup.6 to about 500.times.10.sup.6 cells, about
400.times.10.sup.6 to about 500.times.10.sup.6 cells, about
80.times.10.sup.6 to about 400.times.10.sup.6 cells, about
100.times.10.sup.6 to about 400.times.10.sup.6 cells, about
200.times.10.sup.6 to about 400.times.10.sup.6 cells, about
300.times.10.sup.6 to about 400.times.10.sup.6 cells, about
80.times.10.sup.6 to about 300.times.10.sup.6 cells, about
100.times.10.sup.6 to about 300.times.10.sup.6 cells, about
200.times.10.sup.6 to about 300.times.10.sup.6 cells, about
80.times.10.sup.6 to about 250.times.10.sup.6 cells, about
100.times.10.sup.6 to about 250.times.10.sup.6 cells, about
200.times.10.sup.6 to about 250.times.10.sup.6 cells, about
80.times.10.sup.6 to about 200.times.10.sup.6 cells, or about
100.times.10.sup.6 to about 200.times.10.sup.6 cells.
[0117] In certain embodiments, the isolation in step (b) is
performed less than 6 days after transfection. For example, in
certain embodiments, the isolation in step (b) is performed between
two and four days after transfection. In certain embodiments, the
isolation in step (b) is performed two days after transfection. In
certain embodiments, the isolation in step (b) is performed three
days after transfection.
[0118] In certain embodiments, the sub-population of transfected
host cells comprises about 0.5-6.0.times.10.sup.6 cells prior to
expansion in step (c). For example, in certain embodiments, the
sub-population of transfected host cells comprises about
0.5.times.10.sup.6 cells, about 1.0.times.10.sup.6 cells, about
2.0.times.10.sup.6 cells, about 3.0.times.10.sup.6 cells, about
4.0.times.10.sup.6 cells, about 5.0.times.10.sup.6 cells, or about
6.0.times.10.sup.6 cells prior to expansion in step (c). For
example, in certain embodiments, the sub-population of transfected
host cells comprises about 0.5.times.10.sup.6 to about
1.0.times.10.sup.6 cells, about 0.5.times.10.sup.6 to about
2.0.times.10.sup.6 cells, about 0.5.times.10.sup.6 to about
3.0.times.10.sup.6 cells, about 0.5.times.10.sup.6 to about
4.0.times.10.sup.6 cells, about 0.5.times.10.sup.6 to about
5.0.times.10.sup.6 cells, about 0.5.times.10.sup.6 to about
6.0.times.10.sup.6 cells, about 1.0.times.10.sup.6 to about
2.0.times.10.sup.6 cells, about 1.0.times.10.sup.6 to about
3.0.times.10.sup.6 cells, about 1.0.times.10.sup.6 to about
4.0.times.10.sup.6 cells, about 1.0.times.10.sup.6 to about
5.0.times.10.sup.6 cells, about 1.0.times.10.sup.6 to about
6.0.times.10.sup.6 cells, about 2.0.times.10.sup.6 to about
3.0.times.10.sup.6 cells, about 2.0.times.10.sup.6 to about
4.0.times.10.sup.6 cells, about 2.0.times.10.sup.6 to about
5.0.times.10.sup.6 cells, about 2.0.times.10.sup.6 to about
6.0.times.10.sup.6 cells, about 3.0.times.10.sup.6 to about
4.0.times.10.sup.6 cells, about 3.0.times.10.sup.6 to about
5.0.times.10.sup.6 cells, about 3.0.times.10.sup.6 to about
6.0.times.10.sup.6 cells, about 4.0.times.10.sup.6 to about
5.0.times.10.sup.6 cells, about 4.0.times.10.sup.6 to about
6.0.times.10.sup.6 cells, or about 5.0.times.10.sup.6 to about
6.0.times.10.sup.6 cells, prior to expansion in step (c). In
certain embodiments, the sub-population of transfected host cells
contains greater than 6.0.times.10.sup.6 cells prior to expansion
in step (c). For example, in certain embodiments, the
sub-population of transfected host cells comprises about
7.0.times.10.sup.6 cells, about 8.0.times.10.sup.6 cells, about
9.0.times.10.sup.6 cells, or about 10.0.times.10.sup.6 cells, prior
to expansion in step (c).
[0119] In certain embodiments, the expanding in step (c) is for
between 4-31 days. For example, in various embodiments, the
expanding is for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.
[0120] In certain embodiments, a first of the one or more vectors
encodes the mRNA encoding the target polypeptide, and a second of
the one or more vectors encodes the mRNA encoding the selectable
polypeptide. Where the one or more vectors encoding the mRNA
encoding the target polypeptide and the mRNA encoding the
selectable polypeptide are separate vectors, in certain embodiments
the vectors are independently selected from plasmids, viruses,
phage, transposons, and minichromosomes.
[0121] In certain embodiments, the mRNA encoding the target
polypeptide and the mRNA encoding the selectable polypeptide are
both encoded on one vector. In accordance with these embodiments, a
single vector encodes a polycistronic mRNA encoding both the target
polypeptide and the selectable polypeptide. Also in accordance with
these embodiments, in certain embodiments the mRNA encoding the
selectable polypeptide can be upstream (i.e., 5') of the mRNA
encoding the target polypeptide. Alternatively, in certain
embodiments the mRNA encoding the target polypeptide can be
upstream (i.e., 5') of the mRNA encoding the selectable
polypeptide.
[0122] Thus in certain embodiments, the target polypeptide and the
selectable polypeptide are encoded by a single multicistronic mRNA.
In certain embodiments, the multicistronic mRNA comprises a first
open reading frame (ORF) that encodes the selectable polypeptide
and a second ORF that encodes the target polypeptide, wherein the
first ORF is 5' to the second ORF.
[0123] In certain embodiments, the first ORF has a non-AUG start
codon. In certain embodiments, the non-AUG start codon is a UUG,
GUG, or CUG in a Kozak consensus sequence. A non-AUG start codon
can be installed using standard molecular biology techniques such
as are well known in the art.
[0124] In certain embodiments, the second ORF has an AUG start
codon.
[0125] In certain embodiments, the first ORF has a non-AUG start
codon, and the second ORF has an AUG start codon.
[0126] In certain embodiments, the ORF that encodes the selectable
polypeptide is devoid of any AUG sequences. AUG sequences can be
converted to other triplet sequences, other than stop codons, using
standard molecular biology techniques such as are well known in the
art; for example, and without limitation, AUG sequences can be
converted independently to CUG (L), GUG (V), UUG (L), AAG (K), ACG
(T), AGG (R), AUA (I), AUC (I), AUU (I), GCA (A), GCC (A), GCG (A),
or GCU (A).
[0127] In certain embodiments, the target polypeptide and the
selectable polypeptide form a fusion protein. In certain
embodiments, the fusion protein is membrane-bound. When the fusion
protein is membrane-bound, in certain embodiments the selectable
polypeptide is present in a detectable form, i.e., the target
polypeptide portion of the fusion protein does not prohibit
detection of the selectable polypeptide portion of the fusion
protein. Also when the fusion protein is membrane-bound, in certain
embodiments the target polypeptide is present in a functional form,
i.e., the selectable polypeptide portion of the fusion protein does
not prohibit function of the target polypeptide portion of the
fusion protein. In certain embodiments, the fusion protein is
released from the host cell as a soluble protein. In certain
embodiments, the fusion protein is expressed as a surface protein
but can be cleaved to release the target polypeptide in a soluble,
functional form.
[0128] In certain embodiments, the target polypeptide is a
therapeutic agent, e.g., an antibody, an antigen-binding fragment
of an antibody, an Fc fusion protein, a hormone, or an enzyme.
Polypeptide hormones include, without limitation,
adrenocorticotropic hormone (ACTH), antidiuretic hormone
(vasopressin), atrial natriuretic peptide (ANP), cholecystokinin,
follicle stimulating hormone (FSH), gastrin, glucagon, growth
hormone, insulin, leptin, leuteinizing hormone (LH), oxytocin,
prolactin, somatostatin, and thyroid stimulating hormone (TSH).
Enzymes include, without limitation, acid alpha-glucosidase,
adenosine deaminase, alpha-galactosidase, alpha-L-iduronidase,
arylsulfatase B, beta-galactosidase, beta-glucuronidase,
galactose-6-sulfate sulfatase, glucocerebrosidase, heparan
sulfamidase, heparan-alpha-glucosaminide N-acetyltransferase,
hyaluronidase, iduronate-2-sulfatase,
N-acetylgalactosamine-4-sulfatase, N-acetylglucosamine 6-sulfatase,
and N-acetylglucosaminidase.
[0129] In some embodiments, the target polypeptide is a secreted
protein.
[0130] In certain embodiments, the host cells are mammalian cells.
In certain embodiments, the host cells are selected from the group
consisting of CHO cells, BHK-21 cells, NIH/3T3 cells, HEK293 cells,
HeLa cells, SP2/0 cells, NSO cells, C127 cells, COS cells, Vero
cells, and U937 cells. All of these cells (cell lines) are
commercially available from sources such as American Type Culture
Collection (ATCC, Manassas, Va.). In certain embodiments, the host
cells are selected from the group consisting of CHO cells, HEK293
cells, and HeLa cells.
[0131] An aspect of the invention is a clonal population of
transfected host cells that express a selectable polypeptide and a
target polypeptide obtainable by the method the invention. In
certain embodiments, the clonal population of transfected host
cells expresses a FACS-selectable polypeptide and a target
polypeptide obtainable by the method the invention.
[0132] In certain embodiments, the clonal population yields a 2- to
30-fold improvement in production of the target polypeptide
compared to that of a stable pool of transfected but uncloned host
cells obtained at step (c).
[0133] For example, in some embodiments, the clonal population
yields a 2- to 30-fold, 3- to 30-fold, 5- to 30-fold, 10- to
30-fold, 15- to 30-fold, 20- to 30-fold, 25- to 30-fold, 2- to
25-fold, 3- to 25-fold, 5- to 25-fold, 10- to 25-fold, 15- to
25-fold, 20- to 25-fold, 2- to 20-fold, 3- to 20-fold, 5- to
20-fold, 10- to 20-fold, 15- to 20-fold, 2- to 15-fold, 3- to
15-fold, 5- to 15-fold, 10- to 15-fold, 2- to 10-fold, 3- to
10-fold, 5- to 10-fold, 2- to 5-fold, 3- to 5-fold, or 2- to 3-fold
improvement in production of the target polypeptide compared to
that of a stable pool of transfected but uncloned host cells
obtained at step (c). In certain embodiments, the clonal population
yields a greater than 30-fold improvement in production of the
target polypeptide compared to that of a stable pool of transfected
but uncloned host cells obtained at step (c). For example, in
certain embodiments, the clonal population yields an up to 40-fold,
up to 50-fold, up to 60-fold, up to 70-fold, up to 80-fold, up to
90-fold, or up to 100-fold improvement in production of the target
polypeptide compared to a stable pool of transfected but uncloned
host cells obtained at step (c).
III. Producer Cells and Methods of Production Thereof
[0134] In some embodiments a heterogeneous population of producer
cells is provided. The heterogeneous population of producer cells
can be produced using any method known in the art or described
herein. In some embodiments of any one of the methods provided, the
heterogeneous population of producer cells is produced by
transfecting cells with a vector that encodes the multicistronic
mRNA and subjecting the transfected cells to less than or equal to
one round of medium-based selection to select cells expressing
varying levels (e.g., a variation of at least 10-, 100-, 1,000-, or
10,000-fold) of the multicistronic mRNA. In some embodiments, the
vector further contains a drug-selectable marker, e.g., a
dihydrofolate reductase (DHFR) gene, and the medium-based selection
is methotrexate (MTX, e.g., 1 nM-100 nM MTX), nucleotide-deficient
medium, or a combination thereof. In some embodiments, the vector
further contains a glutamine synthetase (GS) gene and the
medium-based selection is methionine sulphoximine (MSX, e.g.,
25-100 .mu.M MSX). In some embodiments, the vector lacks a
drug-selectable marker, e.g., lacks a DHFR gene or GS gene.
[0135] In some embodiments, FACS is used to select cells expressing
varying levels of the multicistronic mRNA, e.g., by using the FACS
selectable polypeptide level to select the cells.
[0136] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods
described herein may be made using suitable equivalents without
departing from the scope of the embodiments disclosed herein.
Having now described certain embodiments in detail, the same will
be more clearly understood by reference to the following examples,
which are included for purposes of illustration only and are not
intended to be limiting.
EXAMPLES
[0137] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
Example 1: Sorting for Early Post-Transfection Isolation of Cells
(EPIC)--Proof of Concept
[0138] This example demonstrates the feasibility of a method of
sorting to target an unselected transfected early-expressing
population for bulk enrichment prior to selection. This method of
sorting is called "short sorting" or "EPIC" (Early
Post-transfection Isolation of Cells) and is designed to
sort-isolate, or bulk enrich, early reporter expression shortly
after transfection. EPIC may significantly reduce selection
timelines and/or improve productivity of the resulting
heterogeneous population. Experiments have been performed to
investigate the reporter expression profile of a transfected
population throughout the course of a nucleotide-deficient
selection process. FIG. 1A depicts a general scheme for EPIC. FIG.
1B shows the early expression of the reporter gene during the
nucleotide-deficient selection process. These offset histograms
demonstrate that early expression (e.g. day 3-4) is positive and
sortable; making isolating a sub-population of transfected cells
for an EPIC process feasible.
[0139] As shown in FIG. 1A, EPIC can be executed by transfecting a
population and allowing early expression to develop, which can be
targeted for isolation using flow cytometry or other means of cell
sorting. These sort-isolated early-expression sub-populations can
then be placed in a selection media to establish a stable
expression pool. Isolation of these post-transfection early
expression sub-populations prior to selection yields improved
productivity over standard transfection/selection methodologies
alone (e.g., as shown in FIG. 1A).
[0140] To demonstrate proof of concept that detected CD52 signal is
in fact early CD52 reporter expression, vectors directing
expression of both red fluorescent protein (RFP) and CD52
(pGZ729-RFP) or RFP alone (pGZ700-RFP) were constructed and
transfected for early expression evaluation. In this system CD52
corresponds to the detectable polypeptide, and RFP corresponds to
the target polypeptide.
[0141] The pGZ729 vector backbone sequence (including sequence
encoding CD52 but not RFP) is shown below, followed by annotations
of the sequence.
TABLE-US-00003 Sequence of pGZ729 expression vector (SEQ ID NO: 6):
ggatccgctgtggaatgtgtgtcagttagggtgtggaaagtccccaggct
ccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacc
aggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcat
gcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcc
cgcccctaactccgcccagttccgcccattctccgccccatggctgacta
attttttttatttatgcagaggccgaggccgcctcggcctctgagctatt
ccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagc
ttggggggggggacagctcagggctgcgatttcgcgccaaacttgacggc
aatcctagcgtgaaggctggtaggattttatccccgctgccatcatggtt
cgaccattgaactgcatcgtcgccgtgtcccaaaatatggggattggcaa
gaacggagacctaccctggcctccgctcaggaacgagttcaagtacttcc
aaagaatgaccacaacctcttcagtggaaggtaaacagaatctggtgatt
atgggtaggaaaacctggttctccattcctgagaagaatcgacctttaaa
ggacagaattaatatagttctcagtagagaactcaaagaaccaccacgag
gagctcattttcttgccaaaagtttggatgatgccttaagacttattgaa
caaccggaattggcaagtaaagtagacatggtttggatagtcggaggcag
ttctgtttaccaggaagccatgaatcaaccaggccacctcagactctttg
tgacaaggatcatgcaggaatttgaaagtgacacgtttttcccagaaatt
gatttggggaaatataaacttctcccagaatacccaggcgtcctctctga
ggtccaggaggaaaaaggcatcaagtataagtttgaagtctacgagaaga
aagactaacaggaagatgctttcaagttctctgctcccctcctaaagcta
tgcatttttataagaccatgggacttttgctggctttagatctttgtgaa
ggaaccttacttctgtggtgtgacataattggacaaactacctacagaga
tttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaa
ctactgattctaattgtttgtgtattttagattccaacctatggaactga
tgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgct
cagaagaaatgccatctagtgatgatgaggctactgctgactctcaacat
tctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttcc
ttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactc
ttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatac
aagaaaattatggaaaaatattctgtaacctttataagtaggcataacag
ttataatcataacatactgttttttcttactccacacaggcatagagtgt
ctgctattaataactatgctcaaaaattgtgtacctttagctttttaatt
tgtaaaggggttaataaggaatatttgatgtatagtgccttgactagaga
tcataatcagccataccacatttgtagaggttttacttgctttaaaaaac
ctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgt
tgttaacttgtttattgcagcttataatggttacaaataaagcaatagca
tcacaaatttcacaaataaagcatttttttcactgcattctagttgtggt
ttgtccaaactcatcaatgtatcttatcatgtctggatcctctacgccgg
acgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcct
atatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctc
atgagcgcttgtttcggcgtgggtatggtggcaggccgtggccgggggac
tgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctc
aacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataa
gggagagcgtcgaccgatgcccttgagagccttcaacccagtcagctcct
tccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttc
tttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcatttt
cggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttg
cggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtccc
gccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggc
cgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatgg
ccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcg
ttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagct
tcaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt
ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca
agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc
ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg
gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagc
tcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctggg
ctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggta
actatcgtcttgagtccaacccggtaagacacgacttatcgccactggca
gcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctac
agagttcttgaagtggtggcctaactacggctacactagaaggacagtat
ttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggt
agctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgt
ttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt
tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaa
gggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt
aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaactt
ggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatc
tgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataac
tacgatacgggagggcttaccatctggccccagtgctgcaatgataccgc
gagacccacgctcaccggctccagatttatcagcaataaaccagccagcc
ggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca
gtctattaattgttgccgggaagctagagtaagtagttcgccagttaata
gtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcg
tcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagt
tacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc
cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatg
gcagcactgcataattctcttactgtcatgccatccgtaagatgcttttc
tgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggc
gaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacat
agcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaa
actctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc
gtgcacccaactgatcttcagcatcttttactttcaccagcgtttctggg
tgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgac
acggaaatgttgaatactcatactcttcctttttcaatattattgaagca
tttatcagggttattgtctcatgagcggatacatatttgaatgtatttag
aaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacc
tgacgtctaagaaaccattattatcatgacattaacctataaaaataggc
gtatcacgaggccctttcgtcttcaagaattggggaccaagacagaacca
taagccagtgggatagatcagaaatgttccagaggtgggatggggccaga
gtgcctgccccttgaaccgtcccagggaccagaggtgacaaagtggcaac
acaggtcctgcctgggaatctggtctgctcctacttagtaaagctgcctg
gtgtcacacaagaggcccccacttattcctgcacccctggtggtaggtgg
cgtcttctcccctgcagccaccaggctcccctgagaacactgccggcagt
cctcattgacaggcagtattcgctctgccccacccccacctgtgaattgc
agggctggcaggtcctcaggcagctggcaaaccgcctgaacaactgagag
atacagggccagggccagggcagtcccgtcccccggaggcagggagggga
cgtgctgggaaagttctctctctcaggcccaggttggtgactgcagaagg
cttctgtcaaatctcttttgtgggaaccacagagtagccctgaacgtggg
ggtgtgcttccagtatactctggggtcaccctttccatactggaggcctc
tgcaacttcaaaatgctctgctaccaacctagcacaaggaagttggtcca
gcctccccacgcagggccactgctgcagtccatatatggactaagccttc
cttggtttcaacacctacactcactgagcccctactatgtgtatgcagag
ccgagacaggccctgagcatctcatctgaagcacccttcttgcctaaatt
cagttttctgtcactttctcccaggaggtgtgtgtccctctaagctaagc
caggggtccctcacccctgccccactcccatccctagtgtaggtatcagc
tgaagagcttcctgagcagaacactcttgggtgctgacattttgataaat
aggcccatgtttaggagagcaggggtccgggggcgggagatcttctctgg
tggattgagggctccaagaactactctttgagcacgctgcccctcccaga
gtccccacagcctccagatggactagaacacagttcggctgtggctgcac
ataactaacagaggatagatggtgggtcccagcccaacagtgcctggcaa
tcacccagagccaccagctaacggccttggcttagttttttgcctgggtg
tgatcaggcagccctccaaaactgcccggactccatgacaagttttgctt
gttctatagagcacagttcctttctaggtctggggcaagggacatcggga
gacatcttcctgcaacagctccagtcactggaccaccaggctcgccctgt
ctttggtgtgtggccctgagtctcctaagtggcccaaacctgtgaagacc
cctccaaccacagttttgcttctaaattgtaccccaacacacctagcaaa
ttgaaaccccaccagaagtcccccagatctggctttccggctattgctgg
caagggggagtgactcccggcccattcaatccaggccccgcgtgttcctc
aaacaagaagccacgtaaacataaaccgagcctccatgctgacccttgcc
catcgaggtactcaatgttcacgtgatatccacacccagagggtcctggg
gtgggtgcatgagccccagaatgcaggcttgataaccgagaccctgaatc
gggcagtgtccacaagggcggaggccagtcatgcatgttcgggcctatgg
ggccagcacccaacgccaaaactctccatcctcttcctcaatctcgcttt
ctctctctctctctttttttttttttattttttttttttgcaaaaggagg
ggagagggggtaaaaaaatgctgcactgtgcggctaggccggtgagtgag
cggcgcggagccaatcagcgctcgccgttccgaaagttgccttttatggc
tcgagtggccgctgtggcgtcctataaaacccggcggcgcaacgcgcagc
cactgtcgagtccgcgtccacccgcgagcacaggcctttcgcagctcttt
cttcgccgctccacacccgccaccaggtaagcagggacaacaggcccagc
cggccacagccctcccgtgggcagtgaccgcgctgcagggtcgcggggga
cactcggcgcggacaccggggaaggctggagggtggtgccgggccgcgga
gcggacactttcagatccaactttcagtccagggtgtagaccctttacag
ccgcattgccacggtgtagacaccggtggacccgctctggctcagagcac
gcggcttgggggaacccattagggtcgcagtgtgggcgctatgagagccg
atgcagctttcgggtgttgaaccgtatctgcccaccttggggggaggaca
caaggtcgggagccaaacgccacgatcatgccttggtggcccatgggtct
ttgtctaaaccggtttgcccatttggcttgccgggcgggcgggcgcggcg
ggcccggctcggccgggtgggggctgggttgccactgcgcttgcgcgctc
tatggctgggtattggggcgcgtgcacgctggggagggagcccttcctct
tccccctctcccaagttaaacttgcgcgtgcgtattgagacttggagcgc
ggccaccggggttgggcgagggcggggccgttgtccggaaggggcggggt
cgcagcggcttcggggcgcctgctcgcgcttcctgctgggtgtggtcgcc
tcccgcgcgcgcactagccgcccgccggcggggcgaaggcggggcttgcg
cccgtttggggagggggcggaggcctggcttcctgccgtggggccgcctc
cggaccagcgtttgcctcttatggtaataacgcggccggcctgggcttcc
tttgtcccctgagtttgggcgcgcgccccctggcggcccgaggccgcggc
ttgccggaagtgggcagggcggcagcggctgcgcctagtggcccgctagt
gaccgcgaccctcttttgtgccctgatatagttcgccggatcctaccgcg
gtagcggccgcgccaccttggagcgcttcctcttcctcctactcaccatc
agcctcctcgttttggtacaaatacaaaccggactctccggacaaaacga
caccagccaaaccagcagcccctcagcatccagcaacataagcggaggca
ttttccttttcttcgtcgccaacgccataatccacctcttctgcttcagt
tgaaggccggccaatacgtaggcgcgccattgagtgagtgatttggcgcg
ccaagatatcacacccgggattaattaaaggtacctacgcgtagaattcc
acgtagtggtttaaactctagatactcgagggatctggatcataatcagc
cataccacatttgtagaggttttacttgctttaaaaaacctcccacacct
ccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgt
ttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc
acaaataaagcatttttttcactgcattctagttgtggtttgtccaaact
catcaatgtatcttatcatgtct
Elements of pGZ729 Expression Vector (and Nucleotide Locations):
Nucleotides 1-325--SV40 early promoter (for DHFR transcription)
Nucleotides 347-1089--Dihydrofolate reductase (DHFR) open reading
frame Nucleotides 1090-1934--SV40 early intron and polyA
Nucleotides 2684-3366 --E. coli ColE1 origin Nucleotides
3464-4123--Ampicillin resistance gene Nucleotides
4528-7539--Hamster .beta.-actin promoter (for transcription of gene
of interest) Nucleotides 7568-7753--CD52 open reading frame
(containing TTG start codon) Nucleotides 7781-7791--Stop codons in
each of 3 reading frames Nucleotides 7813-7872--Multiple cloning
site (for insertion of target polypeptide with ATG start codon)
Nucleotides 7882-8123--SV40 early polyA
[0142] As shown in FIG. 2, CHO cells transfected with pGZ729-RFP
produced early expression of both CD52 and RFP that peaked around
days 2 and 3, with signal deteriorating out to day 7
post-transfection. Therefore, EPIC targeting on or near days 2-3 is
suitable for isolation of early-expressing sub-populations of
transfected host cells. In order to demonstrate that these
relatively low degree of fluorescence intensity signals were in
fact CD52 expression, CHO cells transfected with either pGZ729-RFP
or pGZ700-RFP were analyzed for RFP and CD52 expression. As shown
in FIG. 3, both CHO cells transfected with pGZ729-RFP and CHO cells
transfected with pGZ700-RFP robustly expressed RFP (top left and
bottom left, respectively), whereas while CHO cells transfected
with pGZ729-RFP had modest expression of CD52 (top right), CHO
cells transfected with pGZ700-RFP expressed essentially no
detectable CD52 (bottom right). These findings support the notion
that these relatively low degree of fluorescence intensity signals
were in fact CD52 expression from the alternate start expression
cassette, suitable as a target for sorting isolation (EPIC).
Example 2: Sorting for Early Post-Transfection Isolation of Cells
(EPIC)--Producer Cell Pool Generation
[0143] EPIC was initially attempted using mAb #1 in which CHO cells
were transfected and given 2 days to recover, after which 0 nM MTX
selection was initiated to establish early expression. Four days
after transfection, early expression of CD52 cell surface reporter
was targeted for sort isolation (EPIC). Sorting targeted only
positive expression which was collected as a bulk enriched
population of about 1 million cells which was then allowed to
continue selection in nucleotide-deficient media (0 nM MTX). As a
control, a non-sorted transfection was allowed to continue
selection via standard selection procedure. As shown in FIG. 4, by
day 8 sorting for EPIC yielded a slightly enriched population as
seen by CD52 reporter expression as compared to standard selection.
As selection of both populations continued, however, this small
EPIC sub-population became more prominent over time. In fact, the
CD52-negative sub-population was all but eliminated upon selection
completion. Comparatively, the standard selection method
demonstrated a slight improvement in CD52 reporter expression which
is historically typical. Sorting for EPIC, or isolating early
expression, yielded a sub-population of positive expression that
had a preferential survivability over the less expressive cells,
which in turn yielded a more productive stable pool.
[0144] The EPIC and standard selected pools were both used to
establish unfed batch cultures to determine mAb #1 titers. As shown
in FIGS. 4 and 5, the EPIC-generated pool yielded a titer of 502
mg/L, far outpacing any pools generated by MTX amplification, again
using no MTX throughout the processes. Comparatively, the pool
generated by standard selection yielded a titer of 150 mg/L, which
was 3-fold lower than that of the EPIC-generated pool.
[0145] While these initial sorts targeting EPIC took 35 days
(transfection/sort/isolation) to achieve completion to a stable
pool, this was directly related to the small number of sorted cells
collected (1 million) which then had to endure both the expansion
and selection to a stable population. Such timelines could be
greatly reduced by either simply collecting more cells and/or
targeting a purer sort. Many of the cells sorted had high levels of
impurities (cells with little to no expression) and had to be
selected (killed) out, prolonging the selection/expansion
times.
Example 3: Sorting for Early Post-Transfection Isolation of Cells
(EPIC)--Clone Generation
[0146] The EPIC-generated pool of Example 2 was next used to
generate clones using FLARE as previously described (see, e.g.,
Cairns, V. et al. (2011) Utilization of Non-AUG Initiation Codons
in a Flow Cytometric Method for Efficient Selection of Recombinant
Cell Lines. Biotechnol Bioeng 108(11):2611-2622). Briefly, FLARE
was used to isolate and single cell plate the top 3-5% of
reporter-expressing cells from each pool using FACS. Expanded
clones were then screened (taking top 30% positive expressers),
again using FLARE, to identify only the top tier clones to expand
for target polypeptide titer evaluation. As shown in FIG. 6, top
expressing EPIC-generated clones achieved similar titers to those
of best clones from traditional methods, e.g., using MTX-amplified
pools (near 2.0 g/L). Results demonstrated that using EPIC to
isolate early expression populations prior to selection is a viable
alternative to traditional transfection and selection
methodologies. EPIC offers a MTX-independent methodology to achieve
clone titers similar to those from traditional MTX methodologies,
resulting in potentially more robust and stable clones.
Alternatively, EPIC is also amenable to MTX introduction during
selection/expansion of EPIC-generated sub-populations, with the
potential to drive even higher expression in these enriched
populations.
Sequence CWU 1
1
8110DNAHomo sapiens 1gccrccatgg 10261PRTHomo sapiens 2Leu Glu Arg
Phe Leu Phe Leu Leu Leu Thr Ile Ser Leu Leu Val Leu1 5 10 15Val Gln
Ile Gln Thr Gly Leu Ser Gly Gln Asn Asp Thr Ser Gln Thr 20 25 30Ser
Ser Pro Ser Ala Ser Ser Asn Ile Ser Gly Gly Ile Phe Leu Phe 35 40
45Phe Val Ala Asn Ala Ile Ile His Leu Phe Cys Phe Ser 50 55
603128PRTHomo sapiens 3Leu Gly Ile Gln Gly Gly Ser Val Leu Phe Gly
Leu Leu Leu Val Leu1 5 10 15Ala Val Phe Cys His Ser Gly His Ser Leu
Gln Cys Tyr Asn Cys Pro 20 25 30Asn Pro Thr Ala Asp Cys Lys Thr Ala
Val Asn Cys Ser Ser Asp Phe 35 40 45Asp Ala Cys Leu Ile Thr Lys Ala
Gly Leu Gln Val Tyr Asn Asn Cys 50 55 60Trp Lys Phe Glu His Cys Asn
Phe Asn Asp Val Thr Thr Arg Leu Arg65 70 75 80Glu Asn Glu Leu Thr
Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe 85 90 95Asn Glu Gln Leu
Glu Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val 100 105 110Leu Leu
Leu Val Thr Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro 115 120
125474PRTMus musculus 4Leu Lys Ser Phe Leu Leu Phe Leu Thr Ile Ile
Leu Leu Val Val Ile1 5 10 15Gln Ile Gln Thr Gly Ser Leu Gly Gln Ala
Thr Thr Ala Ala Ser Gly 20 25 30Thr Asn Lys Asn Ser Thr Ser Thr Lys
Lys Thr Pro Leu Lys Ser Gly 35 40 45Ala Ser Ser Ile Ile Asp Ala Gly
Ala Cys Ser Phe Leu Phe Phe Ala 50 55 60Asn Thr Leu Ile Cys Leu Phe
Tyr Leu Ser65 7058123DNAArtificial Sequencevector 5ggatccgctg
tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 60cagaagtatg
caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
120ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
ccatagtccc 180gcccctaact ccgcccatcc cgcccctaac tccgcccagt
tccgcccatt ctccgcccca 240tggctgacta atttttttta tttatgcaga
ggccgaggcc gcctcggcct ctgagctatt 300ccagaagtag tgaggaggct
tttttggagg cctaggcttt tgcaaaaagc ttgggggggg 360ggacagctca
gggctgcgat ttcgcgccaa acttgacggc aatcctagcg tgaaggctgg
420taggatttta tccccgctgc catcatggtt cgaccattga actgcatcgt
cgccgtgtcc 480caaaatatgg ggattggcaa gaacggagac ctaccctggc
ctccgctcag gaacgagttc 540aagtacttcc aaagaatgac cacaacctct
tcagtggaag gtaaacagaa tctggtgatt 600atgggtagga aaacctggtt
ctccattcct gagaagaatc gacctttaaa ggacagaatt 660aatatagttc
tcagtagaga actcaaagaa ccaccacgag gagctcattt tcttgccaaa
720agtttggatg atgccttaag acttattgaa caaccggaat tggcaagtaa
agtagacatg 780gtttggatag tcggaggcag ttctgtttac caggaagcca
tgaatcaacc aggccacctc 840agactctttg tgacaaggat catgcaggaa
tttgaaagtg acacgttttt cccagaaatt 900gatttgggga aatataaact
tctcccagaa tacccaggcg tcctctctga ggtccaggag 960gaaaaaggca
tcaagtataa gtttgaagtc tacgagaaga aagactaaca ggaagatgct
1020ttcaagttct ctgctcccct cctaaagcta tgcattttta taagaccatg
ggacttttgc 1080tggctttaga tctttgtgaa ggaaccttac ttctgtggtg
tgacataatt ggacaaacta 1140cctacagaga tttaaagctc taaggtaaat
ataaaatttt taagtgtata atgtgttaaa 1200ctactgattc taattgtttg
tgtattttag attccaacct atggaactga tgaatgggag 1260cagtggtgga
atgcctttaa tgaggaaaac ctgttttgct cagaagaaat gccatctagt
1320gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa
gagaaaggta 1380gaagacccca aggactttcc ttcagaattg ctaagttttt
tgagtcatgc tgtgtttagt 1440aatagaactc ttgcttgctt tgctatttac
accacaaagg aaaaagctgc actgctatac 1500aagaaaatta tggaaaaata
ttctgtaacc tttataagta ggcataacag ttataatcat 1560aacatactgt
tttttcttac tccacacagg catagagtgt ctgctattaa taactatgct
1620caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga
atatttgatg 1680tatagtgcct tgactagaga tcataatcag ccataccaca
tttgtagagg ttttacttgc 1740tttaaaaaac ctcccacacc tccccctgaa
cctgaaacat aaaatgaatg caattgttgt 1800tgttaacttg tttattgcag
cttataatgg ttacaaataa agcaatagca tcacaaattt 1860cacaaataaa
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
1920atcttatcat gtctggatcc tctacgccgg acgcatcgtg gccggcatca
ccggcgccac 1980aggtgcggtt gctggcgcct atatcgccga catcaccgat
ggggaagatc gggctcgcca 2040cttcgggctc atgagcgctt gtttcggcgt
gggtatggtg gcaggccgtg gccgggggac 2100tgttgggcgc catctccttg
catgcaccat tccttgcggc ggcggtgctc aacggcctca 2160acctactact
gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc
2220ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
actatcgtcg 2280ccgcacttat gactgtcttc tttatcatgc aactcgtagg
acaggtgccg gcagcgctct 2340gggtcatttt cggcgaggac cgctttcgct
ggagcgcgac gatgatcggc ctgtcgcttg 2400cggtattcgg aatcttgcac
gccctcgctc aagccttcgt cactggtccc gccaccaaac 2460gtttcggcga
gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct
2520tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt
ctcgcttccg 2580gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag
gcaggtagat gacgaccatc 2640agggacagct tcaaggccag caaaaggcca
ggaaccgtaa aaaggccgcg ttgctggcgt 2700ttttccatag gctccgcccc
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 2760ggcgaaaccc
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
2820gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
ccttcgggaa 2880gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
ttcggtgtag gtcgttcgct 2940ccaagctggg ctgtgtgcac gaaccccccg
ttcagcccga ccgctgcgcc ttatccggta 3000actatcgtct tgagtccaac
ccggtaagac acgacttatc gccactggca gcagccactg 3060gtaacaggat
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
3120ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
aagccagtta 3180ccttcggaaa aagagttggt agctcttgat ccggcaaaca
aaccaccgct ggtagcggtg 3240gtttttttgt ttgcaagcag cagattacgc
gcagaaaaaa aggatctcaa gaagatcctt 3300tgatcttttc tacggggtct
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 3360tcatgagatt
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
3420aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
ttaatcagtg 3480aggcacctat ctcagcgatc tgtctatttc gttcatccat
agttgcctga ctccccgtcg 3540tgtagataac tacgatacgg gagggcttac
catctggccc cagtgctgca atgataccgc 3600gagacccacg ctcaccggct
ccagatttat cagcaataaa ccagccagcc ggaagggccg 3660agcgcagaag
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
3720aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
attgctgcag 3780gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
cagctccggt tcccaacgat 3840caaggcgagt tacatgatcc cccatgttgt
gcaaaaaagc ggttagctcc ttcggtcctc 3900cgatcgttgt cagaagtaag
ttggccgcag tgttatcact catggttatg gcagcactgc 3960ataattctct
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
4020ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
gcgtcaacac 4080gggataatac cgcgccacat agcagaactt taaaagtgct
catcattgga aaacgttctt 4140cggggcgaaa actctcaagg atcttaccgc
tgttgagatc cagttcgatg taacccactc 4200gtgcacccaa ctgatcttca
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 4260caggaaggca
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
4320tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
atgagcggat 4380acatatttga atgtatttag aaaaataaac aaataggggt
tccgcgcaca tttccccgaa 4440aagtgccacc tgacgtctaa gaaaccatta
ttatcatgac attaacctat aaaaataggc 4500gtatcacgag gccctttcgt
cttcaagaat tggggaccaa gacagaacca taagccagtg 4560ggatagatca
gaaatgttcc agaggtggga tggggccaga gtgcctgccc cttgaaccgt
4620cccagggacc agaggtgaca aagtggcaac acaggtcctg cctgggaatc
tggtctgctc 4680ctacttagta aagctgcctg gtgtcacaca agaggccccc
acttattcct gcacccctgg 4740tggtaggtgg cgtcttctcc cctgcagcca
ccaggctccc ctgagaacac tgccggcagt 4800cctcattgac aggcagtatt
cgctctgccc cacccccacc tgtgaattgc agggctggca 4860ggtcctcagg
cagctggcaa accgcctgaa caactgagag atacagggcc agggccaggg
4920cagtcccgtc ccccggaggc agggagggga cgtgctggga aagttctctc
tctcaggccc 4980aggttggtga ctgcagaagg cttctgtcaa atctcttttg
tgggaaccac agagtagccc 5040tgaacgtggg ggtgtgcttc cagtatactc
tggggtcacc ctttccatac tggaggcctc 5100tgcaacttca aaatgctctg
ctaccaacct agcacaagga agttggtcca gcctccccac 5160gcagggccac
tgctgcagtc catatatgga ctaagccttc cttggtttca acacctacac
5220tcactgagcc cctactatgt gtatgcagag ccgagacagg ccctgagcat
ctcatctgaa 5280gcacccttct tgcctaaatt cagttttctg tcactttctc
ccaggaggtg tgtgtccctc 5340taagctaagc caggggtccc tcacccctgc
cccactccca tccctagtgt aggtatcagc 5400tgaagagctt cctgagcaga
acactcttgg gtgctgacat tttgataaat aggcccatgt 5460ttaggagagc
aggggtccgg gggcgggaga tcttctctgg tggattgagg gctccaagaa
5520ctactctttg agcacgctgc ccctcccaga gtccccacag cctccagatg
gactagaaca 5580cagttcggct gtggctgcac ataactaaca gaggatagat
ggtgggtccc agcccaacag 5640tgcctggcaa tcacccagag ccaccagcta
acggccttgg cttagttttt tgcctgggtg 5700tgatcaggca gccctccaaa
actgcccgga ctccatgaca agttttgctt gttctataga 5760gcacagttcc
tttctaggtc tggggcaagg gacatcggga gacatcttcc tgcaacagct
5820ccagtcactg gaccaccagg ctcgccctgt ctttggtgtg tggccctgag
tctcctaagt 5880ggcccaaacc tgtgaagacc cctccaacca cagttttgct
tctaaattgt accccaacac 5940acctagcaaa ttgaaacccc accagaagtc
ccccagatct ggctttccgg ctattgctgg 6000caagggggag tgactcccgg
cccattcaat ccaggccccg cgtgttcctc aaacaagaag 6060ccacgtaaac
ataaaccgag cctccatgct gacccttgcc catcgaggta ctcaatgttc
6120acgtgatatc cacacccaga gggtcctggg gtgggtgcat gagccccaga
atgcaggctt 6180gataaccgag accctgaatc gggcagtgtc cacaagggcg
gaggccagtc atgcatgttc 6240gggcctatgg ggccagcacc caacgccaaa
actctccatc ctcttcctca atctcgcttt 6300ctctctctct ctcttttttt
ttttttattt tttttttttg caaaaggagg ggagaggggg 6360taaaaaaatg
ctgcactgtg cggctaggcc ggtgagtgag cggcgcggag ccaatcagcg
6420ctcgccgttc cgaaagttgc cttttatggc tcgagtggcc gctgtggcgt
cctataaaac 6480ccggcggcgc aacgcgcagc cactgtcgag tccgcgtcca
cccgcgagca caggcctttc 6540gcagctcttt cttcgccgct ccacacccgc
caccaggtaa gcagggacaa caggcccagc 6600cggccacagc cctcccgtgg
gcagtgaccg cgctgcaggg tcgcggggga cactcggcgc 6660ggacaccggg
gaaggctgga gggtggtgcc gggccgcgga gcggacactt tcagatccaa
6720ctttcagtcc agggtgtaga ccctttacag ccgcattgcc acggtgtaga
caccggtgga 6780cccgctctgg ctcagagcac gcggcttggg ggaacccatt
agggtcgcag tgtgggcgct 6840atgagagccg atgcagcttt cgggtgttga
accgtatctg cccaccttgg ggggaggaca 6900caaggtcggg agccaaacgc
cacgatcatg ccttggtggc ccatgggtct ttgtctaaac 6960cggtttgccc
atttggcttg ccgggcgggc gggcgcggcg ggcccggctc ggccgggtgg
7020gggctgggtt gccactgcgc ttgcgcgctc tatggctggg tattggggcg
cgtgcacgct 7080ggggagggag cccttcctct tccccctctc ccaagttaaa
cttgcgcgtg cgtattgaga 7140cttggagcgc ggccaccggg gttgggcgag
ggcggggccg ttgtccggaa ggggcggggt 7200cgcagcggct tcggggcgcc
tgctcgcgct tcctgctggg tgtggtcgcc tcccgcgcgc 7260gcactagccg
cccgccggcg gggcgaaggc ggggcttgcg cccgtttggg gagggggcgg
7320aggcctggct tcctgccgtg gggccgcctc cggaccagcg tttgcctctt
atggtaataa 7380cgcggccggc ctgggcttcc tttgtcccct gagtttgggc
gcgcgccccc tggcggcccg 7440aggccgcggc ttgccggaag tgggcagggc
ggcagcggct gcgcctagtg gcccgctagt 7500gaccgcgacc ctcttttgtg
ccctgatata gttcgccgga tcctaccgcg gtagcggccg 7560cgccaccttg
gagcgcttcc tcttcctcct actcaccatc agcctcctcg ttttggtaca
7620aatacaaacc ggactctccg gacaaaacga caccagccaa accagcagcc
cctcagcatc 7680cagcaacata agcggaggca ttttcctttt cttcgtcgcc
aacgccataa tccacctctt 7740ctgcttcagt tgaaggccgg ccaatacgta
ggcgcgccat tgagtgagtg atttggcgcg 7800ccaagatatc acacccggga
ttaattaaag gtacctacgc gtagaattcc acgtagtggt 7860ttaaactcta
gatactcgag ggatctggat cataatcagc cataccacat ttgtagaggt
7920tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
aaatgaatgc 7980aattgttgtt gttaacttgt ttattgcagc ttataatggt
tacaaataaa gcaatagcat 8040cacaaatttc acaaataaag catttttttc
actgcattct agttgtggtt tgtccaaact 8100catcaatgta tcttatcatg tct
81236186DNAHomo sapiens 6ttggagcgct tcctcttcct cctactcacc
atcagcctcc tcgttttggt acaaatacaa 60accggactct ccggacaaaa cgacaccagc
caaaccagca gcccctcagc atccagcaac 120ataagcggag gcattttcct
tttcttcgtc gccaacgcca taatccacct cttctgcttc 180agttga
1867387DNAHomo sapiens 7ttgggaatcc aaggagggtc tgtcctgttc gggctgctgc
tcgtcctcgc tgtcttctgc 60cattccggtc atagcctgca gtgctacaac tgtcctaacc
caactgctga ctgcaaaaca 120gccgtcaatt gttcatctga ttttgacgcg
tgtctcatta ccaaagctgg gttacaagtg 180tataacaact gttggaagtt
tgagcattgc aatttcaacg acgtcacaac ccgcttgagg 240gaaaacgagc
taacgtacta ctgctgcaag aaggacctgt gtaactttaa cgaacagctt
300gaaaacggag ggacatcctt atcagagaaa acagttcttc tgctggtgac
tccatttctg 360gcagctgctt ggagccttca tccctaa 3878233DNAMus musculus
8ttgaagagct tcctcctctt cctcactatc attcttctcg tagtcattca gatacaaaca
60ggatccttag gacaagccac tacggccgct tcaggtacta acaaaaacag cacctccacc
120aaaaaaaccc ccttaaagag cggggcctca tccatcatcg acgcgggcgc
ttgcagtttc 180ctcttcttcg ccaataccct tatttgcctc ttctacctca
gctaactgag taa 233
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