U.S. patent application number 12/612405 was filed with the patent office on 2011-02-03 for methods to express recombinant proteins from lentiviral vectors.
Invention is credited to Jianmin Fang, Debbie Farson, Karin Jooss, Andrew Simmons.
Application Number | 20110028694 12/612405 |
Document ID | / |
Family ID | 37669529 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110028694 |
Kind Code |
A1 |
Fang; Jianmin ; et
al. |
February 3, 2011 |
METHODS TO EXPRESS RECOMBINANT PROTEINS FROM LENTIVIRAL VECTORS
Abstract
Lentivector constructs for expression of recombinant proteins,
polypeptides or fragments thereof and methods of making the same
are described. The lentivectors typically have a self-processing
cleavage sequence between a first and second protein or polypeptide
coding sequence allowing for expression of a functional protein or
polypeptide under operative control of a single promoter and may
further include an additional proteolytic cleavage sequence which
provides a means to remove the self-processing cleavage sequence
from the expressed protein or polypeptide. The vector constructs
find utility in methods relating to enhanced production of
biologically active proteins, such as immunoglobulins or fragments
thereof in vitro and in vivo.
Inventors: |
Fang; Jianmin; (Foster City,
CA) ; Jooss; Karin; (Bellevue, WA) ; Simmons;
Andrew; (San Mateo, CA) ; Farson; Debbie; (San
Francisco, CA) |
Correspondence
Address: |
ROPES & GRAY LLP
PATENT DOCKETING 39/361, 1211 AVENUE OF THE AMERICAS
NEW YORK
NY
10036-8704
US
|
Family ID: |
37669529 |
Appl. No.: |
12/612405 |
Filed: |
November 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11488568 |
Jul 18, 2006 |
7632509 |
|
|
12612405 |
|
|
|
|
60700298 |
Jul 19, 2005 |
|
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Current U.S.
Class: |
530/387.3 ;
435/320.1; 435/328 |
Current CPC
Class: |
C07K 2317/21 20130101;
C12N 2740/15043 20130101; C12N 15/86 20130101; C07K 16/2863
20130101; C12N 2510/02 20130101; C07K 2317/76 20130101 |
Class at
Publication: |
530/387.3 ;
435/320.1; 435/328 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10 |
Claims
1. A lentivector for expression of a recombinant immunoglobulin,
comprising: in the 5' to 3' direction, a promoter operably linked
to the coding sequence for a first chain of an immunoglobulin
molecule or a fragment thereof, an additional proteolytic cleavage
site, a sequence encoding a self-processing cleavage site and the
coding sequence for a second chain of an immunoglobulin molecule or
fragment thereof, wherein the sequence encoding the self-processing
cleavage site is inserted between the coding sequence for the first
chain and the coding sequence for the second chain of said
immunoglobulin molecule.
2. The lentivector according to claim 1, wherein the sequence
encoding the self-processing cleavage site comprises a 2A
sequence.
3. The lentivector according to claim 2, wherein the 2A sequence is
a Foot and Mouth Disease Virus (FMDV) sequence.
4. The lentivector according to claim 3, wherein the 2A sequence
encodes an oligopeptide comprising amino acid residues
LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1) or TLNFDLLKLAGDVESNPGP (SEQ ID
NO:2) or EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:9).
5. The lentivector according to claim 3, wherein the coding
sequence for the first chain of said immunoglobulin molecule or a
fragment thereof encodes an immunoglobulin heavy chain.
6. The lentivector according to claim 3, wherein the coding
sequence for the first chain of said immunoglobulin molecule or a
fragment thereof encodes an immunoglobulin light chain.
7. The lentivector according to claim 5, wherein the coding
sequence is the full length coding sequence of an immunoglobulin
heavy chain.
8. The lentivector according to claim 6, wherein the coding
sequence is the full length coding sequence of an immunoglobulin
light chain.
9. The lentivector according to claim 1, wherein said additional
proteolytic cleavage site is a furin cleavage site with the
consensus sequence RKRR (SEQ ID NO: 15).
10. The lentivector according to claim 3, wherein the promoter is
selected from the group consisting of an elongation factor 1-alpha
promoter (EF1 a) promoter, a phosphoglycerate kinase-1 promoter
(PGK) promoter, a cytomegalovirus immediate early gene promoter
(CMV), a chimeric liver-specific promoter (LSP) a cytomegalovirus
enhancer/chicken beta-actin promoter (CAG), a tetracycline
responsive promoter (TRE), a transthyretin promoter (TTR), a MND
promoter, a simian virus 40 promoter (SV40) and a CK6 promoter.
11. The lentivector according to claim 10, wherein said promoter is
a CAG hybrid promoter/enhancer.
12. The lentivector according to claim 10, wherein said promoter is
a CMV promoter/enhancer.
13. The lentivector according to claim 3, further comprising a
signal sequence.
14. The lentivector according to claim 3, wherein said heavy and
light chain immunoglobulin coding sequences are expressed in a
substantially equimolar ratio.
15. The lentivector according to claim 11, wherein said lentivector
comprises a CAG promoter operably linked to the coding sequence for
a first chain of an immunoglobulin molecule, a sequence encoding a
self-processing cleavage site and the coding sequence for a second
chain of an immunoglobulin molecule, wherein the sequence encoding
said self-processing cleavage site is inserted between the coding
sequence for the first chain and the coding sequence for the second
chain of said immunoglobulin molecule.
16. A producer cell transduced with the vector of claim 11.
17. A producer cell transduced with the vector of claim 12.
18. A producer cell transduced with the vector of claim 14.
19. A recombinant immunoglobulin molecule produced by a producer
cell infected with the lentivector of claim 11.
20. A recombinant immunoglobulin molecule produced by a producer
cell infected with the lentivector of claim 12.
21. A recombinant immunoglobulin molecule produced by a producer
cell infected with the lentivector of claim 14.
22-39. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of co-pending
U.S. application Ser. No. 11/488,568, filed Jul. 18, 2006, which
claims the priority benefit of U.S. Provisional Patent Application
No. 60/700,298, filed Jul. 19, 2005. The priority application is
expressly incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to novel lenti vector constructs
designed to express recombinant full-length proteins or fragments
thereof. The lenti constructs may be used for ex vivo or in vivo
expression of a heterologous protein coding sequence by a cell or
organ, or in vitro for the production of recombinant proteins by
lenti vector-transduced cells.
[0004] 2. Background of the Technology
[0005] Recombinant proteins as therapeutic modalities have found
increasing use in recent years. Numerous recombinant protein-based
therapies are in various stages of clinical development. One
limitation to widespread clinical application of recombinant
protein technology is the difficulty in production of proteins that
include two or more coding sequences or domains such that the
domains are expressed in the proper ratio with appropriate
post-translational processing resulting in production of a
functional heterodimeric molecule. A further limitation is the high
cost associated with the ability to produced adequate levels of
protein for clinical applications.
[0006] Monoclonal antibodies have been proven as effective
therapeutics for cancer and other diseases. Current antibody
therapy often involves repeat administration and long term
treatment regimens, which are associated with a number of
disadvantages, such as inconsistent serum levels, limited duration
of efficacy per administration such that frequent readministration
is required and high cost. The use of antibodies as diagnostic
tools and therapeutic modalities has also found increasing use in
recent years. One limitation to the widespread clinical application
of antibody technology is that typically large amounts of antibody
are required for therapeutic efficacy and the costs associated with
production are significant. Chinese Hamster Ovarian (CHO) cells,
SP20 and NSO2 myeloma cells are the most commonly used mammalian
cell lines for commercial scale production of glycosylated human
proteins such as antibodies. The yields obtained from mammalian
cell line production typically range from 50-250 mg/L for 5-7 day
culture in a batch fermentor or 300-1000 mg/L for 7-12 day cultures
in fed batch fermentors. High-level production often relies upon
gene amplification and selection of best performing clones that is
time consuming and further increases the cost of development and
production. In addition, stability issues with respect to
antibody-producing cell lines are often evident following multiple
passages.
[0007] Previous attempts to express full length recombinant
proteins with two or more domains or chains (and thus two or more
coding sequences or open reading frames (ORFs)) via recombinant DNA
technology have met with limited success, typically resulting in
unequal levels of expression of the two or more domains or chains
of the protein or polypeptide and more importantly, a lower level
of expression for the second coding sequence. In order to obtain
optimal expression of a fully functional and biologically active
protein or polypeptide that has two or more domains, substantially
equimolar expression of the two or more domains is required.
Conventional vectors that rely on dual promoter regulation of gene
expression are invariably affected by promoter interaction (i.e.,
promoter interference) that may compromise equimolar or
substantially equimolar expression of the genes.
[0008] Lentiviral vectors are a type of retroviral vector that can
infect both dividing and non-dividing cells. They can be used to
express protein from non-dividing or terminally differentiated
cells such as neurons, macrophages, hematopoietic stem cells,
retinal photoreceptors, muscle and liver cells, cell types for
which other vector systems cannot be used effectively.
[0009] There remains a need for improved gene expression systems
for production of recombinant proteins and polypeptides, in
particular proteins and polypeptides that have two or more domains
or chains, such that sufficient expression of a biologically active
recombinant protein or polypeptide is achieved at commercially
reasonable cost.
[0010] The present invention addresses this need by demonstrating
the feasibility and use of lentivector constructs for the
expression of functional recombinant proteins and polypeptides
which have two or more domains.
SUMMARY OF THE INVENTION
[0011] The present invention provides lentivector constructs for
expression of protein or polypeptide open reading frames from a
single cell and methods of using the same.
[0012] In one preferred approach, the vectors have a
self-processing cleavage sequence between the protein or
polypeptide coding sequences allowing for expression of more than
one functional protein or polypeptide using a single promoter. The
invention finds utility in production of two or more proteins or
polypeptides or a protein or polypeptide having two or more domains
(or chains) using a lentiviral vector where sustained expression
occurs in a single cell. Exemplary lentivector constructs comprise
a self-processing cleavage sequence and may further comprise an
additional proteolytic cleavage site for removal of the
self-processing cleavage sequence from the expressed protein or
polypeptide. The vector constructs find utility in methods relating
to enhanced production of biologically active proteins,
polypeptides or fragments thereof, in vitro and in vivo.
[0013] The invention relates to engineered lentiviral vectors that
encode two or more domains or chains of a multimeric protein. In
one aspect the multimeric protein is an immunoglobulin (i.e., an
antibody) and full-length antibody heavy and light chain coding
sequences are expressed using a lentivector comprising a single
open reading frame driven by a single promoter wherein the vector
comprises a self processing cleavage site or sequence between the
heavy and light chain coding sequences. In another aspect the
protein is a multimeric protein and the full-length coding
sequences are expressed using a lentivector comprising a single
open reading frame driven by a single promoter wherein the vector
comprises a self-processing cleavage site or sequence.
[0014] In yet another aspect, the invention relates to a method for
high level expression of recombinant protein using more than one
engineered lentiviral vector wherein each lentivector encodes a
single open reading frame of a multimeric protein driven by a
single promoter. For example, for expression of a full-length
antibody, individual lentivectors that encode the full-length
antibody heavy and light chain, respectively, are used to infect
the same cell such that high level expression of a biologically
active antibody results.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a schematic depiction of the process for
expression of a full-length immunoglobulin (antibody) using
constructs that include a self-processing cleavage site, such as
2A, and a furin cleavage site.
[0016] FIG. 2 is a schematic depiction of plasmid, lentivirus and
AAV expression cassettes comprising a self-processing cleavage site
(2A) for expression of immunoglobulin heavy (H) and light (L)
chains operatively linked to a CAG promoter (CAG), wherein the
vector may further include an additional proteolytic cleavage site
(Furin; "F").
[0017] FIG. 3 is a schematic depiction of the time line associated
for development of antibody-expressing clones with an illustration
of approximate clone development timelines, indicating the
advantage of lentivectors over the use of plasmids (transfection
and selection) in terms of time and antibody expression level.
[0018] FIG. 4 illustrates the expression of a full length rat
anti-VEGFR2 monoclonal antibody (CAG-DC101 IgG1) in different cell
lines (CHOD-, HuH7 and PerC6) following transduction with a
lentivector, wherein 2A refers to expression of DC101 via a single
vector including a self processing sequence.
[0019] FIG. 5 shows the results of a Southern blot analysis to
determine the number of integrated genomic copies of the lentiviral
vector for two 5.times. transfected clones expressing approximately
20-40 pg/cell/day of DC101 antibody. Samples containing known
genomic quantities of the lentiviral vector were used as a standard
for determining the number of integrated genomic copies.
[0020] FIG. 6 illustrates the antibody expression levels for 60
individual clones isolated from cells that had been transfected
4.times., 7.times. or 9.times. with the lentiviral 2A
DC101-encoding vector. The amount of antibody produced (in
pg/cell/day) is plotted against the number of clones that express
that amount of recombinant DC101 antibody from each population.
[0021] FIG. 7 illustrates the expression of a full length human CMV
anti-KDR IgG1 monoclonal antibody in different cell lines (CHOD-,
HuH7 and PerC6) following transduction with a lentivector, wherein
2A refers to expression of the heavy and light chain of KDR via a
single vector including a self processing sequence.
[0022] FIG. 8 illustrates the results of mass spectral analysis of
purifies IgG protein produced following transfection of an HF2AL
plasmid encoding into CHO cells, followed by furin cleavage and
treatment with carboxypeptidase.
[0023] FIG. 9 demonstrates the results of a stability study of
thirteen pancreatic clones transduced with a lentiviral vector
encoding human GM-CSF. The GM-CSF expression level of clones
expressing 500-2500 ng/10.sup.6 cells/24 hr GM-CSF was maintained
in continuous culture for 12 weeks with GM-CSF expression levels
tested at 3-week intervals.
[0024] FIG. 10 demonstrates the results of a stability study of
CT26 clones transduced with a lentiviral vector encoding murine
GM-CSF, where the GM-CSF expression level of the CT26 rodent cell
lines was shown to be stable for at least 9 weeks of continuous
culture.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The various compositions and methods of the invention are
described below. Although particular compositions and methods are
exemplified herein, it is understood that any of a number of
alternative compositions and methods are applicable and suitable
for use in practicing the invention. It will also be understood
that an evaluation of the protein or polypeptide expression
constructs (vectors) and methods of the invention may be carried
out using procedures standard in the art.
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology,
molecular biology (including recombinant techniques), microbiology,
biochemistry and immunology, which are known to those of skill in
the art. Such techniques are explained fully in the literature,
such as, Molecular Cloning: A Laboratory Manual, second edition
(Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait,
ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987);
Methods in Enzymology (Academic Press, Inc.); Handbook of
Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.);
Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P.
Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.
Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction,
(Mullis et al., eds., 1994); and Current Protocols in Immunology
(J. E. Coligan et al., eds., 1991).
DEFINITIONS
[0027] Unless otherwise indicated, all terms used herein have the
same meaning as they would to one skilled in the art and the
practice of the present invention will employ, conventional
techniques of microbiology and recombinant DNA technology, which
are within the knowledge of those of skill of the art.
[0028] The term "vector", as used herein, refers to a DNA or RNA
molecule such as a plasmid, virus or other vehicle, which contains
one or more heterologous or recombinant DNA sequences and is
designed for transfer between different host cells. The terms
"expression vector" and "gene therapy vector" refer to any vector
that is effective to incorporate and express heterologous DNA
fragments in a cell. A cloning or expression vector may comprise
additional elements, for example, the expression vector may have
two replication systems, thus allowing it to be maintained in two
organisms, for example in human cells for expression and in a
prokaryotic host for cloning and amplification. Any suitable vector
can be employed that is effective for introduction of nucleic acids
into cells such that protein or polypeptide expression results,
e.g. a viral vector or non-viral plasmid vector. Any cells
effective for expression, e.g., insect cells and eukaryotic cells
such as yeast or mammalian cells are useful in practicing the
invention.
[0029] The terms "heterologous DNA" and "heterologous RNA" refer to
nucleotides that are not endogenous (native) to the cell or part of
the genome in which they are present. Generally heterologous DNA or
RNA is added to a cell by transduction, infection, transfection,
transformation or the like, as further described below. Such
nucleotides generally include at least one coding sequence, but the
coding sequence need not be expressed. The term "heterologous DNA"
may refer to a "heterologous coding sequence" or a "transgene".
[0030] As used herein, the terms "protein" and "polypeptide" may be
used interchangeably and typically refer to "proteins" and
"polypeptides" of interest that are expresses using the self
processing cleavage site-containing vectors of the present
invention. Such "proteins" and "polypeptides" may be any protein or
polypeptide useful for research, diagnostic or therapeutic
purposes, as further described below.
[0031] The term "replication defective" as used herein relative to
a viral gene therapy vector of the invention means the viral vector
cannot independently further replicate and package its genome. For
example, when a cell of a subject is infected with rAAV virions,
the heterologous gene is expressed in the infected cells, however,
due to the fact that the infected cells lack AAV rep and cap genes
and accessory function genes, the rAAV is not able to
replicate.
[0032] As used herein, a "retroviral transfer vector" refers to an
expression vector that comprises a nucleotide sequence that encodes
a transgene and further comprises nucleotide sequences necessary
for packaging of the vector. Preferably, the retroviral transfer
vector also comprises the necessary sequences for expressing the
transgene in cells.
[0033] As used herein, "packaging system" refers to a set of viral
constructs comprising genes that encode viral proteins involved in
packaging a recombinant virus. Typically, the constructs of the
packaging system will ultimately be incorporated into a packaging
cell.
[0034] As used herein, a "second generation" lentiviral vector
system refers to a lentiviral packaging system that lacks
functional accessory genes, such as one from which the accessory
genes, vif, vpr, vpu and nef, have been deleted or inactivated.
See, e.g., Zufferey et al., 1997, Nat. Biotechnol. 15:871-875.
[0035] As used herein, a "third generation" lentiviral vector
system refers to a lentiviral packaging system that has the
characteristics of a second generation vector system, and further
lacks a functional tat gene, such as one from which the tat gene
has been deleted or inactivated. Typically, the gene encoding rev
is provided on a separate expression construct. See, e.g., Dull et
al., 1998, J. Virol. 72(11):8463-8471.
[0036] As used herein, "pseudotyped" refers to the replacement of a
native envelope protein with a heterologous or functionally
modified envelope protein.
[0037] The term "operably linked" as used herein relative to a
recombinant DNA construct or vector means nucleotide components of
the recombinant DNA construct or vector are functionally related to
one another for operative control of a selected coding sequence.
Generally, "operably linked" DNA sequences are contiguous, and, in
the case of a secretory leader, contiguous and in reading frame.
However, enhancers do not have to be contiguous.
[0038] As used herein, the term "gene" or "coding sequence" means
the nucleic acid sequence which is transcribed (DNA) and translated
(mRNA) into a polypeptide in vitro or in vivo when operably linked
to appropriate regulatory sequences. The gene may or may not
include regions preceding and following the coding region, e.g. 5'
untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer"
sequences, as well as intervening sequences (introns) between
individual coding segments (exons).
[0039] A "promoter" is a DNA sequence that directs the binding of
RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal
sequence sufficient to direct transcription. Promoters and
corresponding protein or polypeptide expression may be cell-type
specific, tissue-specific, or species specific. Also included in
the nucleic acid constructs or vectors of the invention are
enhancer sequences that may or may not be contiguous with the
promoter sequence. Enhancer sequences influence promoter-dependent
gene expression and may be located in the 5' or 3' regions of the
native gene.
[0040] "Enhancers" are cis-acting elements that stimulate or
inhibit transcription of adjacent genes. An enhancer that inhibits
transcription also is termed a "silencer". Enhancers can function
(i.e., can be associated with a coding sequence) in either
orientation, over distances of up to several kilobase pairs (kb)
from the coding sequence and from a position downstream of a
transcribed region.
[0041] A "regulatable promoter" is any promoter whose activity is
affected by a cis or trans acting factor (e.g., an inducible
promoter, such as an external signal or agent).
[0042] A "constitutive promoter" is any promoter that directs RNA
production in many or all tissue/cell types at most times, e.g.,
the human CMV immediate early enhancer/promoter region which
promotes constitutive expression of cloned DNA inserts in mammalian
cells.
[0043] The terms "transcriptional regulatory protein",
"transcriptional regulatory factor" and "transcription factor" are
used interchangeably herein, and refer to a nuclear protein that
binds a DNA response element and thereby transcriptionally
regulates the expression of an associated gene or genes.
Transcriptional regulatory proteins generally bind directly to a
DNA response element, however in some cases binding to DNA may be
indirect by way of binding to another protein that in turn binds
to, or is bound to a DNA response element.
[0044] As used herein, the term "sequence identity" means nucleic
acid or amino acid sequence identity between two or more aligned
sequences, when aligned using a sequence alignment program. The
terms "% homology" and "% identity" are used interchangeably herein
and refer to the level of nucleic acid or amino acid sequence
identity between two or more aligned sequences, when aligned using
a sequence alignment program. For example, 80% homology means the
same thing as 80% sequence identity determined by a defined
algorithm under defined conditions.
[0045] The terms "identical" or percent "identity" in the context
of two or more nucleic acid or protein sequences, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms described
herein, e.g. the Smith-Waterman algorithm, or by visual
inspection.
[0046] As used herein, an "internal ribosome entry site" or "IRES"
refers to an element that promotes direct internal ribosome entry
to the initiation codon, such as ATG, of a cistron (a protein
encoding region), thereby leading to the cap-independent
translation of the gene. See, e.g., Jackson R J, Howell M T,
Kaminski A (1990) Trends Biochem Sci 15(12):477-83) and Jackson R J
and Kaminski, A. (1995) RNA 1(10):985-1000. The examples described
herein are relevant to the use of any IRES element, which is able
to promote direct internal ribosome entry to the initiation codon
of a cistron. "Under translational control of an IRES" as used
herein means that translation is associated with the IRES and
proceeds in a cap-independent manner.
[0047] A "self-processing cleavage site" or "self-processing
cleavage sequence" is defined herein as a post-translational or
co-translational processing cleavage site or sequence. Such a
"self-processing cleavage" site or sequence refers to a DNA or
amino acid sequence, exemplified herein by a 2A site, sequence or
domain or a 2A-like site, sequence or domain. As used herein, a
"self-processing peptide" is defined herein as the peptide
expression product of the DNA sequence that encodes a
self-processing cleavage site, sequence or domain, which upon
translation mediates rapid intramolecular (cis) cleavage of a
protein or polypeptide comprising the self-processing cleavage site
to yield discrete mature protein or polypeptide products.
[0048] As used herein, the term "additional proteolytic cleavage
site", refers to a sequence which is incorporated into an
expression construct of the invention adjacent a self-processing
cleavage site, such as a 2A or 2A like sequence, and provides a
means to remove additional amino acids that remain following
cleavage by the self processing cleavage sequence. Exemplary
"additional proteolytic cleavage sites" are described herein and
include, but are not limited to, furin cleavage sites with the
consensus sequence RXK(R)R (SEQ ID NO: 10). Such furin cleavage
sites can be cleaved by endogenous subtilisin-like proteases, such
as furin and other serine proteases within the protein secretion
pathway.
[0049] As used herein, the terms "immunoglobulin" and "antibody"
may be used interchangeably and refer to intact immunoglobulin or
antibody molecules as well as fragments thereof, such as Fa, F
(ab')2, and Fv, which are capable of binding an antigenic
determinant. Such an "immunoglobulin" and "antibody" is composed of
two identical light polypeptide chains of molecular weight
approximately 23,000 daltons, and two identical heavy chains of
molecular weight 53,000-70,000. The four chains are joined by
disulfide bonds in a "Y" configuration. Heavy chains are classified
as gamma (IgG), mu (IgM), alpha (IgA), delta (IgD) or epsilon (IgE)
and are the basis for the class designations of immunoglobulins,
which determines the effector function of a given antibody. Light
chains are classified as either kappa or lambda. When reference is
made herein to an "immunoglobulin or fragment thereof", it will be
understood that such a "fragment thereof" is an immunologically
functional immunoglobulin fragment.
[0050] The term "humanized antibody" refers to an antibody molecule
in which one or more amino acids of the antigen binding regions of
a non-human antibody have been replaced in order to more closely
resemble a human antibody, while retaining the binding activity of
the original non-human antibody. See, e.g., U.S. Pat. No.
6,602,503.
[0051] The term "antigenic determinant", as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. Numerous regions of a protein or
fragment of a protein may induce the production of antibodies that
binds specifically to a given region of the three-dimensional
structure of the protein. These regions or structures are referred
to as antigenic determinants. An antigenic determinant may compete
with the intact antigen (i.e., the immunogen used to elicit the
immune response) for binding to an antibody.
[0052] The term "fragment", when referring to a recombinant protein
or polypeptide of the invention means a polypeptide which has an
amino acid sequence which is the same as part of but not all of the
amino acid sequence of the corresponding full length protein or
polypeptide, and which retains at least one of the functions or
activities of the corresponding full length protein or polypeptide.
The fragment preferably includes at least 20-100 contiguous amino
acid residues of the full-length protein or polypeptide.
[0053] The terms "administering" or "introducing", as used herein
refer to delivery of a vector for recombinant protein expression to
a cell or to cells and/or organs of a subject. Such administering
or introducing may take place in vivo, in vitro or ex vivo. A
vector for recombinant protein or polypeptide expression may be
introduced into a cell by transfection, which typically means
insertion of heterologous DNA into a cell by physical means (e.g.,
calcium phosphate transfection, electroporation, microinjection or
lipofection); infection, which typically refers to introduction by
way of an infectious agent, i.e. a virus; or transduction, which
typically means stable infection of a cell with a virus or the
transfer of genetic material from one microorganism to another by
way of a viral agent (e.g., a bacteriophage).
[0054] "Transformation" is typically used to refer to bacteria
comprising heterologous DNA or cells that express an oncogene and
have therefore been converted into a continuous growth mode such as
tumor cells. A vector used to "transform" a cell may be a plasmid,
virus or other vehicle.
[0055] Typically, a cell is referred to as "transduced",
"infected", "transfected" or "transformed" dependent on the means
used for administration, introduction or insertion of heterologous
DNA (i.e., the vector) into the cell. The terms "transduced",
"transfected" and "transformed" may be used interchangeably herein
regardless of the method of introduction of heterologous DNA. A
cell may be "transduced" by infection with a viral vector.
[0056] As used herein, the terms "stably transformed", "stably
transfected" and "transgenic" refer to cells that have a non-native
(heterologous) nucleic acid sequence integrated into the genome.
Stable transfection is demonstrated by the establishment of cell
lines or clones comprised of a population of daughter cells
containing the transfected DNA stably integrated into their
genomes. In some cases, "transfection" is not stable, i.e., it is
transient. In the case of transient transfection, the exogenous or
heterologous DNA is expressed, however, the introduced sequence is
not integrated into the genome and is considered to be
episomal.
[0057] As used herein, "ex vivo administration" refers to a process
where primary cells are taken from a subject, a vector is
administered to the cells to produce transduced, infected or
transfected recombinant cells and the recombinant cells are
readministered to the same or a different subject.
[0058] A "multicistronic transcript" refers to an mRNA molecule
that contains more than one protein coding region, or cistron. An
mRNA comprising two coding regions is denoted a "bicistronic
transcript". The "5'-proximal" coding region or cistron is the
coding region whose translation initiation codon (usually AUG) is
closest to the 5'-end of a multicistronic mRNA molecule. A
"5'-distal" coding region or cistron is one whose translation
initiation codon (usually AUG) is not the closest initiation codon
to the 5' end of the mRNA. The terms "5'-distal" and "downstream"
are used synonymously to refer to coding regions that are not
adjacent to the 5' end of an mRNA molecule.
[0059] As used herein, "co-transcribed" means that two (or more)
coding regions or polynucleotides are under transcriptional control
of a single transcriptional control or regulatory element.
[0060] The term "host cell", as used herein refers to a cell that
has been transduced, infected, transfected or transformed with a
vector. The vector may be a plasmid, a viral particle, a phage,
etc. The culture conditions, such as temperature, pH and the like,
are those previously used with the host cell selected for
expression, and will be apparent to those skilled in the art. It
will be appreciated that the term "host cell" refers to the
original transduced, infected, transfected or transformed cell and
progeny thereof.
[0061] As used herein, the terms "biological activity" and
"biologically active", refer to the activity attributed to a
particular protein in a cell line in culture or in a cell-free
system, such as a ligand-receptor assay in ELISA plates. The
"biological activity" of an "immunoglobulin", "antibody" or
fragment thereof refers to the ability to bind an antigenic
determinant and thereby facilitate immunological function.
[0062] As used herein, the terms "tumor" and "cancer" refer to a
cell that exhibits a loss of growth control and forms unusually
large clones of cells. Tumor or cancer cells generally have lost
contact inhibition and may be invasive and/or have the ability to
metastasize.
Internal Ribosome Entry Site (IRES)
[0063] IRES elements were first discovered in picornavirus mRNAs
(Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci
15(12):477-83) and Jackson R J and Kaminski, A. (1995) RNA
1(10):985-1000). Examples of IRES generally employed by those of
skill in the art include those described in U.S. Pat. No.
6,692,736. Examples of "IRES" known in the art include, but are not
limited to IRES obtainable from picornavirus (Jackson et al., 1990)
and IRES obtainable from viral or cellular mRNA sources, such as
for example, immunoglobulin heavy-chain binding protein (BiP), the
vascular endothelial growth factor (VEGF) (Huez et al. (1998) Mol.
Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2
(FGF-2), and insulin-like growth factor (IGFII), the translational
initiation factor eIF4G and yeast transcription factors TFIID and
HAP4, the encephelomycarditis virus (EMCV) which is commercially
available from Novagen (Duke et al. (1992) J. Virol 66(3):1602-9)
and the VEGF IRES (Huez et al. (1998) Mol Cell Biol
18(11):6178-90). IRES have also been reported in different viruses
such as cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine
leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
As used herein, "IRES" encompasses functional variations of IRES
sequences as long as the variation is able to promote direct
internal ribosome entry to the initiation codon of a cistron. An
IRES may be mammalian, viral or protozoan.
[0064] The IRES promotes direct internal ribosome entry to the
initiation codon of a downstream cistron, leading to
cap-independent translation. Thus, the product of a downstream
cistron can be expressed from a bicistronic (or multicistronic)
mRNA, without requiring either cleavage of a polyprotein or
generation of a monocistronic mRNA. Internal ribosome entry sites
are approximately 450 nucleotides in length and are characterized
by moderate conservation of primary sequence and strong
conservation of secondary structure. The most significant primary
sequence feature of the IRES is a pyrimidine-rich site whose start
is located approximately 25 nucleotides upstream of the 3' end of
the IRES. See Jackson et al.(1990).
[0065] In eukaryotic cells, translation is normally initiated by
the ribosome scanning from the capped mRNA 5' end, under the
control of initiation factors. However, several cellular mRNAs have
been found to have IRES structure to mediate the cap-independent
translation (van der Velde, et al. (1999) Int J Biochem Cell Biol.
31:87-106. An IRES sequence may be tested and compared to a 2A
sequence as shown in Example 1. In one exemplary protocol a test
vector or plasmid is generated with one transgene, such as PF-4 or
VEGF-TRAP, placed under translational control of an IRES, 2A or
2A-like sequence to be tested. A cell is transfected with the
vector or plasmid containing the IRES- or 2A-reporter gene
sequences and an assay is performed to detect the presence of the
transgene. In one illustrative example, the test plasmid comprises
co-transcribed PF-4 and VEGF-TRAP coding sequences
transcriptionally driven by a CMV promoter wherein the PF-4 or
VEGF-TRAP coding sequence is translationally driven by the IRES, 2A
or 2A-like sequence to be tested. Host cells are transiently
transfected with the test vector or plasmid by means known to those
of skill in the art and assayed for the expression of the
transgene.
[0066] For some time, in order to express two or more proteins from
a single viral or non-viral vector, an internal ribosome entry site
(IRES) sequence has been commonly used to drive expression of the
second, third, fourth gene, etc. Although the use of IRES is
considered to be the state of the art by many, when two genes are
linked via IRES, the expression level of the second gene is often
significantly reduced (Furler et al., Gene Therapy 8:864-873
(2001)). In fact, the use of an IRES to control transcription of
two or more genes operably linked to the same promoter can result
in lower level expression of the second, third, etc. gene relative
to the gene adjacent the promoter. In addition, an IRES sequence
may be sufficiently long to present issues with the packaging limit
of the vector, e.g., the eCMV IRES has a length of 507 base
pairs.
[0067] The present invention provides advantages over the use of an
IRES in that a vector for recombinant protein or polypeptide
expression comprising a self-processing peptide (exemplified herein
by 2A peptides) facilitates expression of two or more protein or
polypeptide coding sequences using a single promoter, wherein the
two or more proteins or polypeptides are expressed in a
substantially equimolar ratio.
[0068] Self-Processing Cleavage Sites or Sequences
[0069] The linking of proteins in the form of polyproteins in a
single open reading frame is a strategy adopted in the replication
of many viruses including picornaviridae. Upon translation,
virus-encoded proteinases mediate rapid intramolecular (cis)
cleavage of a polyprotein to yield discrete mature protein
products. Foot and Mouth Disease viruses (FMDV) are a group within
the picomaviridae that express a single, long open reading frame
encoding a polyprotein of approximately 225 kD. The full length
translation product undergoes rapid intramolecular (cis) cleavage
at the C-terminus of a self-processing cleavage site, for example,
a 2A site or region, located between the capsid protein precursor
(P1-2A) and replicative domains of the polyprotein 2BC and P3, with
the cleavage mediated by proteinase-like activity of the 2A region
itself (Ryan et al., J. Gen. Virol. 72:2727-2732, 1991); Vakharia
et al., J. Virol. 61:3199-3207, 1987). Similar domains have also
been characterized from aphthoviridea and cardioviridae of the
picornavirus family (Donnelly et al., J. Gen. Virol. 78:13-21,
1997).
[0070] A "self-processing cleavage site" or "self-processing
cleavage sequence" as defined above refers to a DNA or amino acid
sequence, wherein upon translation, rapid intramolecular (cis)
cleavage of a polypeptide comprising the self-processing cleavage
site occurs to result in expression of discrete mature protein or
polypeptide products. Such a "self-processing cleavage site", may
also be referred to as a post-translational or co-translational
processing cleavage site, exemplified herein by a 2A site, sequence
or domain. It has been reported that a 2A site, sequence or domain
demonstrates a translational effect by modifying the activity of
the ribosome to promote hydrolysis of an ester linkage, thereby
releasing the polypeptide from the translational complex in a
manner that allows the synthesis of a discrete downstream
translation product to proceed (Donnelly, 2001). Alternatively, a
2A site, sequence or domain demonstrates "auto-proteolysis" or
"cleavage" by cleaving its own C-terminus in cis to produce primary
cleavage products (Furler; Palmenberg, Ann Rev. Microbiol.
44:603-623 (1990)).
[0071] Although the mechanism is not part of the invention, the
activity of a 2A-like sequence may involve ribosomal skipping
between codons which prevents formation of peptide bonds (de Felipe
et al., Human Gene Therapy 11:1921-1931 (2000); Donnelly et al., J.
Gen. Virol. 82:1013-1025 (2001)), although it has been considered
that the domain acts more like an autolytic enzyme (Ryan et al.,
Virol. 173:35-45 (1989). Studies in which the Foot and Mouth
Disease Virus (FMDV) 2A coding region was cloned into expression
vectors and transfected into target cells showed FMDV 2A cleavage
of artificial reporter polyproteins in wheat-germ lysate and
transgenic tobacco plants (Halpin et al., U.S. Ser. No. 5,846,767;
1998 and Halpin et al., The Plant Journal 17:453-459, 1999); Hs 683
human glioma cell line (de Felipe et al., Gene Therapy 6:198-208,
1999); hereinafter referred to as "de Felipe II"); rabbit
reticulocyte lysate and human HTK-143 cells (Ryan et al., EMBO J.
13:928-933 (1994)); and insect cells (Roosien et al., J. Gen.
Virol. 71:1703-1711, 1990). The FMDV 2A-mediated cleavage of a
heterologous polyprotein has been shown for IL-12 (p40/p35
heterodimer; Chaplin et al., J. Interferon Cytokine Res.
19:235-241, 1999). The reference demonstrates that in transfected
COS-7 cells, FMDV 2A mediated the cleavage of a p40-2A-p35
polyprotein into biologically functional subunits p40 and p35
having activities associated with IL-12.
[0072] The FMDV 2A sequence has been incorporated into retroviral
vectors, alone or combined with different IRES sequences to
construct bicistronic, tricistronic and tetracistronic vectors. The
efficiency of 2A-mediated gene expression in animals was
demonstrated by Furler (2001) using recombinant adeno-associated
viral (AAV) vectors encoding .alpha.-synuclein and EGFP or Cu/Zn
superoxide dismutase (SOD-1) and EGFP linked via the FMDV 2A
sequence. EGFP and .alpha.-synuclein were expressed at
substantially higher levels from vectors which included a 2A
sequence relative to corresponding IRES-based vectors, while SOD-1
was expressed at comparable or slightly higher levels. Furler also
demonstrated that the 2A sequence results in bicistronic gene
expression in vivo after injection of 2A-containing AAV vectors
into rat substantia nigra.
[0073] For the present invention, the DNA sequence encoding a
self-processing cleavage site is exemplified by viral sequences
derived from a picornavirus, including but not limited to an
entero-, rhino-, cardio-, aphtho- or Foot-and-Mouth Disease Virus
(FMDV). In one preferred embodiment, the self-processing cleavage
site coding sequence is derived from a FMDV. Self-processing
cleavage sites include but are not limited to 2A and 2A-like sites,
sequences or domains (Donnelly et al., J. Gen. Virol. 82:1027-1041
(2001). Positional subcloning of a 2A sequence between two or more
heterologous DNA sequences in a vector construct allows the
delivery and expression of two or more open reading frames by
operable linkage to a single promoter. Preferably, self-processing
cleavage sites such as FMDV 2A sequences provide a unique means to
express and deliver from a single viral vector, two or more
proteins, polypeptides or peptides which can be individual parts of
for example, an immunoglobulin Factor VIII, a cytokine, or another
heterodimeric protein, an antibody, or a heterodimeric
receptor.
[0074] FMDV 2A is a polyprotein region that functions in the FMDV
genome to direct a single cleavage at its own C-terminus, thus
functioning in cis. The FMDV 2A domain is typically reported to be
about nineteen amino acids in length ((LLNFDLLKLAGDVESNPGP (SEQ ID
NO: 1); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 2); Ryan et al., J. Gen.
Virol. 72:2727-2732 (1991)), however oligopeptides of as few as
fourteen amino acid residues ((LLKLAGDVESNPGP (SEQ ID NO: 3)) have
also been shown to mediate cleavage at the 2A C-terminus in a
fashion similar to its role in the native FMDV polyprotein
processing.
[0075] Variations of the 2A sequence have been studied for their
ability to mediate efficient processing of polyproteins (Donnelly M
L L et al. 2001). Homologues and variant 2A sequences are included
within the scope of the invention and include but are not limited
to the sequences presented in Table 1, below:
TABLE-US-00001 TABLE 1 Table of Exemplary 2A Sequences (SEQ ID)
SEQUENCE NO: 1 LLNFDLLKLAGDVESNPGP NO: 2 TLNFDLLKLAGDVESNPGP NO: 3
LLKLAGDVESNPGP NO: 4 NFDLLKLAGDVESNPGP NO: 5 QLLNFDLLKLAGDVESNPGP
NO: 6 APVKQTLNFDLLKLAGDVESNPGP NO: 7
VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT LNFDLLKLAGDVESNPGP NO: 8
LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 9
EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP
[0076] Distinct advantages of self-processing cleavage sequences,
such as a 2A sequence or a variant thereof are their use in
generating vectors expressing self-processing polyproteins. This
invention includes lentivectors which comprise the coding sequence
for two or more proteins or polypeptides linked via self-processing
cleavage sites such that the individual proteins or polypeptides
are expressed in equimolar or close to equimolar amounts following
the cleavage of the polyprotein due to the presence of the
self-processing cleavage site. These proteins may be heterologous
to the vector itself, to each other or to the self-processing
cleavage site, e.g., FMDV. Thus the self-processing cleavage sites
for use in practicing the invention do not discriminate between
heterologous proteins or polypeptides and coding sequences derived
from the same source as the self-processing cleavage site, in the
ability to function or mediate cleavage.
[0077] The expression levels of individual proteins, polypeptides
or peptides from a promoter driving a single open reading frame
comprising more than two coding sequences are closer to equimolar
as compared to expression levels achievable using IRES sequences or
dual promoters. Elimination of dual promoters reduces promoter
interference that may result in reduced and/or impaired levels of
expression for each coding sequence.
[0078] In one preferred embodiment, the FMDV 2A sequence included
in a lentivector according to the invention encodes amino acid
residues comprising LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1).
Alternatively, a lentivector according to the invention may encode
amino acid residues for other 2A-like regions as discussed in
Donnelly et al., J. Gen. Virol. 82:1027-1041 (2001) and including
but not limited to a 2A-like domain from picornavirus, insect
virus, Type C rotavirus, trypanosome repeated sequences or the
bacterium, Thermatoga maritima.
[0079] The invention contemplates the use of nucleic acid sequence
variants that encode a self-processing cleavage site, such as a 2A
or 2A-like polypeptide, and nucleic acid coding sequences that have
a different codon for one or more of the amino acids relative to
that of the parent (native) nucleotide. Such variants are
specifically contemplated and encompassed by the present invention.
Sequence variants of self-processing cleavage peptides and
polypeptides are included within the scope of the invention as
well.
[0080] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), by the BLAST
algorithm, Altschul et al., J Mol. Biol. 215: 403-410 (1990), with
software that is publicly available through the National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov/), or by visual
inspection (see generally, Ausubel et al., infra) For purposes of
the present invention, optimal alignment of sequences for
comparison is most preferably conducted by the local homology
algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981).
See, also, Altschul, S. F. et al., 1990 and Altschul, S. F. et al.,
1997.
[0081] In accordance with the present invention, also encompassed
are sequence variants which encode self-processing cleavage
polypeptides and polypeptides themselves that have 80, 85, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity
to the native sequence.
[0082] A nucleic acid sequence is considered to be "selectively
hybridizable" to a reference nucleic acid sequence if the two
sequences specifically hybridize to one another under moderate to
high stringency hybridization and wash conditions. Hybridization
conditions are based on the melting temperature (Tm) of the nucleic
acid binding complex or probe. For example, "maximum stringency"
typically occurs at about Tm-5.degree. C. (5.degree. below the Tm
of the probe); "high stringency" at about 5-10.degree. below the
Tm; "intermediate stringency" at about 10-20.degree. below the Tm
of the probe; and "low stringency" at about 20-25.degree. below the
Tm. Functionally, maximum stringency conditions may be used to
identify sequences having strict identity or near-strict identity
with the hybridization probe; while high stringency conditions are
used to identify sequences having about 80% or more sequence
identity with the probe.
[0083] Moderate and high stringency hybridization conditions are
well known in the art (see, Sambrook, et al., 1989, Chapters 9 and
11, and in Ausubel, F. M., et al., 1993. An example of high
stringency conditions includes hybridization at about 42.degree. C.
in 50% formamide, 5.times.SSC, 5.times. Denhardt's solution, 0.5%
SDS and 100 .mu.g/ml denatured carrier DNA followed by washing
2.times. in 2.times.SSC and 0.5% SDS at room temperature and two
additional times in 0.1.times.SSC and 0.5% SDS at 42.degree. C. 2A
sequence variants that encode a polypeptide with the same
biological activity as the 2A polypeptides described herein and
hybridize under moderate to high stringency hybridization
conditions are considered to be within the scope of the present
invention.
[0084] As a result of the degeneracy of the genetic code, a number
of coding sequences can be provided which encode the same protein,
polypeptide or peptide, such as 2A or a 2A-like peptide. For
example, the triplet CGT encodes the amino acid arginine Arginine
is alternatively encoded by CGA, CGC, CGG, AGA, and AGG. Therefore
it is appreciated that such substitutions in the coding region fall
within the sequence variants that are covered by the present
invention.
[0085] Removal of Self-Processing Peptide Sequences
[0086] One concern associated with the use of self-processing
peptides, such as a 2A or 2A-like sequence is that the C terminus
of the expressed polypeptide contains amino acids derived from the
self-processing peptide, i.e. 2A-derived amino acid residues. These
amino acid residues are "foreign" to the host and may elicit an
immune response when the recombinant protein is expressed in vivo
(i.e., expressed from a viral or non-viral vector in the context of
gene therapy or administered as an in vitro-produced recombinant
protein or polypeptide) or delivered in vivo following in vitro or
ex vivo expression. In addition, if not removed, self-processing
peptide-derived amino acid residues may interfere with protein
secretion in producer cells and/or alter protein conformation,
resulting in a less than optimal expression level and/or reduced
biological activity of the recombinant protein.
[0087] The invention includes lenti-based expression constructs,
engineered such that an additional proteolytic cleavage site is
provided between a first protein or polypeptide coding sequence
(the first or 5' ORF) and the self processing cleavage site as a
means for removal of self processing cleavage site derived amino
acid residues that are present in the expressed protein
product.
[0088] Examples of additional proteolytic cleavage sites are furin
cleavage sites with the consensus sequence RXK(R)R (SEQ ID NO: 10),
which can be cleaved by endogenous subtilisin-like proteases, such
as furin and other serine proteases. The inventors have
demonstrated that self-processing 2A amino acid residues at the C
terminus of a first expressed protein can be efficiently removed by
introducing a furin cleavage site RAKR (SEQ ID NO: 18) between the
first polypeptide and a self-processing 2A sequence. In addition,
use of a plasmid containing a 2A sequence and a furin cleavage site
adjacent to the 2A sequence was shown to result in a higher level
of protein expression than a plasmid containing the 2A sequence
alone. This improvement provides a further advantage in that when
2A amino acid residues are removed from the C-terminus of the
protein, longer 2A- or 2A like sequences or other self-processing
sequences can be used. See, e.g., U.S. Patent Publication Nos.
20040265955 and 20050003482, expressly incorporated by reference
herein.
[0089] It is often advantageous to produce therapeutic proteins,
polypeptides, fragments or analogues thereof with fully human
characteristics. These reagents avoid the undesired immune
responses induced by proteins, polypeptides, fragments or analogues
thereof originating from different species. To address possible
host immune responses to amino acid residues derived from
self-processing peptides, the coding sequence for a proteolytic
cleavage site may be inserted (using standard methodology known in
the art) between the coding sequence for a first protein and the
coding sequence for a self-processing peptide so as to remove the
self-processing peptide sequence from the expressed protein or
polypeptide. This finds particular utility in therapeutic and
diagnostic proteins and polypeptides for use in vivo.
[0090] Any additional proteolytic cleavage site known in the art
that can be expressed using recombinant DNA technology may be
employed in practicing the invention. Exemplary additional
proteolytic cleavage sites which can be inserted between a
polypeptide or protein coding sequence and a self processing
cleavage sequence include, but are not limited to:
[0091] a). Furin consensus sequence or site: RXK(R)R (SEQ ID.
NO:10);
[0092] b). Factor Xa cleavage sequence or site: IE(D)GR (SEQ ID.
NO:11);
[0093] c). Signal peptidase I cleavage sequence or site: e.g.,
LAGFATVAQA (SEQ ID. NO: 12); and
[0094] d). Thrombin cleavage sequence or site: LVPRGS (SEQ ID. NO:
13).
[0095] As detailed herein, the 2A peptide sequence provides a
"cleavage" site that facilitates the generation of both chains of
an immunoglobulin or other protein during the translation process.
In one exemplary embodiment, the C-terminus of the first protein,
for example the immunoglobulin heavy chain, contains approximately
13 amino acid residues that are derived from the 2A sequence
itself. The number of residual amino acids is dependent upon the 2A
sequence used. As set forth above, when a furin cleavage site
sequence, e.g., RAKR (SEQ ID NO: 18), is inserted between the first
protein and the 2A sequence, the 2A residues are removed from the
C-terminus of the first protein. However, mass spectrum data
indicates that the C-terminus of the first protein expressed from
the RAKR-2A construct contains two additional amino acid residues,
RA, derived from the furin cleavage site RAKR (SEQ ID NO: 18).
[0096] In one embodiment, the invention provides a method for
removal of these residual amino acids and a composition for
expression of the same. A number of novel constructs have been
designed that provide for removal of these additional amino acids
from the C-terminus of the protein. Furin cleavage occurs at the
C-terminus of the cleavage site, which has the consensus sequence
RXR(K)R (SEQ ID NO: 19), where X is any amino acid. In one aspect,
the invention provides a means for removal of the newly exposed
basic amino acid residues R or K from the C-terminus of the protein
by use of an enzyme selected from a group of enzymes called
carboxypeptidases (CPs), which include, but not limited to,
carboxypeptidase D, E and H (CPD, CPE, CPH). Since CPs are able to
remove basic amino acid residues at the C-terminus of a protein,
all amino acid resides derived from a furin cleavage site which
contain exclusively basic amino acids R or K, such as RKKR (SEQ ID
NO: 14), RKRR (SEQ ID NO: 15), RRRR (SEQ ID NO: 17), etc., can be
removed by a CP. A series of immunoglobulin expression constructs
that contain a 2A sequence and a furin cleavage site and which have
basic amino acid residues at the C terminus have been constructed
to evaluate efficiency of cleavage and residue removal. An
exemplary construct design is the following: H chain--furin (e.g,
RKKR (SEQ ID NO: 14), RKRR (SEQ ID NO: 15), RRKR (SEQ ID NO: 16) or
RRRR (SEQ ID NO: 17))-2A-L chain or L chain--furin (e.g, RKKR (SEQ
ID NO: 14), RKRR (SEQ ID NO: 15), RRKR (SEQ ID NO: 16) or RRRR (SEQ
ID NO: 17))-2A-H chain A schematic depiction of exemplary
constructs is provided in FIGS. 14 and 15, respectively of U.S.
Ser. No. 60/659,871, expressly incorporated by reference
herein.
[0097] As will be apparent to those of skill in the art, there is a
basic amino acid residue (K) at the C terminus of the
immunoglobulin heavy (H) chain (rendering it subject to cleavage
with carboxypeptidase), while the immunoglobulin light (L) chain,
terminates with a non-basic amino acid C. In one preferred
embodiment of the invention, an antibody expression construct
comprising a furin site and a 2A sequence is provided wherein the
immunoglobulin L chain is 5' to the immunoglobulin H chain such
that following translation, the additional furin amino acid
residues are cleaved with carboxypeptidase.
[0098] Immunoglobulins and Fragments Thereof
[0099] Antibodies are immunoglobulin proteins that are heterodimers
of a heavy and light chain and have proven difficult to express in
a full-length form from a single vector in mammalian culture
expression systems. Three methods are currently used for production
of vertebrate antibodies, in vivo immunization of animals to
produce "polyclonal" antibodies, in vitro cell culture of B-cell
hybridomas to produce monoclonal antibodies (Kohler, et al., Eur.
J. Immunol., 6: 511, 1976; Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988) and recombinant DNA technology
(described for example in Cabilly et al., U.S. Pat. No.
6,331,415).
[0100] The basic molecular structure of immunoglobulin polypeptides
is known to include two identical light chains with a molecular
weight of approximately 23,000 daltons, and two identical heavy
chains with a molecular weight 53,000-70,000, where the four chains
are joined by disulfide bonds in a "Y" configuration. The amino
acid sequence runs from the N-terminal end at the top of the Y to
the C-terminal end at the bottom of each chain. At the N-terminal
end is a variable region (of approximately 100 amino acids in
length) that provides for the specificity of antigen binding.
[0101] The present invention provides improved methods for
production of immunoglobulins of all types, including, but not
limited to full length antibodies and antibody fragments having a
native sequence (i.e. that sequence produced in response to
stimulation by an antigen), single chain antibodies which combine
the antigen binding variable region of both the heavy and light
chains in a single stably-folded polypeptide chain; univalent
antibodies (which comprise a heavy chain/light chain dimer bound to
the Fc region of a second heavy chain); "Fab fragments" which
include the full "Y" region of the immunoglobulin molecule, i.e.,
the branches of the "Y", either the light chain or heavy chain
alone, or portions, thereof (i.e., aggregates of one heavy and one
light chain, commonly known as Fab'); "hybrid immunoglobulins"
which have specificity for two or more different antigens (e.g.,
quadromas or bispecific antibodies as described for example in U.S.
Pat. No. 6,623,940); "composite immunoglobulins" wherein the heavy
and light chains mimic those from different species or
specificities; and "chimeric antibodies" wherein portions of each
of the amino acid sequences of the heavy and light chain are
derived from more than one species (i.e., the variable region is
derived from one source such as a murine antibody, while the
constant region is derived from another, such as a human
antibody).
[0102] The compositions and methods of the invention find utility
in production of immunoglobulins or fragments thereof wherein the
heavy or light chain is "mammalian", "chimeric" or modified in a
manner to enhance its efficacy. Modified antibodies include both
amino acid and nucleic acid sequence variants which retain the same
biological activity of the unmodified form and those which are
modified such that the activity is altered, i.e., changes in the
constant region that improve complement fixation, interaction with
membranes, and other effector functions, or changes in the variable
region that improve antigen binding characteristics. The
compositions and methods of the invention further include catalytic
immunoglobulins or fragments thereof.
[0103] A "variant" immunoglobulin-encoding polynucleotide sequence
may encode a "variant" immunoglobulin amino acid sequence that is
altered by one or more amino acids from the reference polypeptide
sequence. The variant polynucleotide sequence may encode a variant
amino acid sequence that contains "conservative" substitutions,
wherein the substituted amino acid has structural or chemical
properties similar to the amino acid which it replaces. In
addition, or alternatively, the variant polynucleotide sequence may
encode a variant amino acid sequence that contains
"non-conservative" substitutions, wherein the substituted amino
acid has dissimilar structural or chemical properties to the amino
acid that it replaces. Variant immunoglobulin-encoding
polynucleotides may also encode variant amino acid sequences that
contain amino acid insertions or deletions, or both.
[0104] The present invention contemplates immunoglobulin sequence
variants which encode biologically active immunoglobulins or
fragments thereof, wherein the immunoglobulin polypeptide sequence
or the nucleotide sequence encoding it has 80, 85, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the
native sequence.
[0105] Furthermore, a variant "immunoglobulin-encoding
polynucleotide" may encode the same polypeptide as the reference
polynucleotide sequence but, due to the degeneracy of the genetic
code, has a polynucleotide sequence altered by one or more bases
from the reference polynucleotide sequence. Immunoglobulin sequence
variants that encode a polypeptide with the same biological
activity as the immunoglobulin polypeptides described herein and
hybridize under moderate to high stringency hybridization
conditions are considered to be within the scope of the present
invention.
[0106] The term "fragment," when referring to a recombinant
immunoglobulin of the invention means a polypeptide which has an
amino acid sequence which is the same as part of but not all of the
amino acid sequence of the corresponding full length immunoglobulin
protein, which either retains essentially the same biological
function or activity as the corresponding full length protein, or
retains at least one of the functions or activities of the
corresponding full length protein. The fragment preferably includes
at least 20-100, 20-150 or 20-200 contiguous amino acid residues of
the full-length immunoglobulin.
[0107] The potential of antibodies as therapeutic modalities is
currently limited by long time frame needed to select clones that
produce commercially practical levels of immunoglobulin, the
production capacity and excessive cost of the current technology.
An improved v expression system for immunoblobulin production would
permit the expression and delivery of two or more coding sequences,
i.e., immunoglobulins with bi- or multiple-specificities from a
single vector. The present invention addresses these limitations
and is applicable to any immunoglobulin (i.e. an antibody) or
fragment thereof as further detailed herein, including engineered
antibodies such as single chain antibodies, full-length antibodies
or antibody fragments.
[0108] Antibody Production
[0109] In one example of the present invention, the coding sequence
for a first or second chain of a protein or polypeptide is the
coding sequence for the heavy chain or a fragment thereof for any
immunoglobulin, e.g., IgG, IgM, IgD, IgE or IgA. Alternatively, the
coding sequence for a first or second chain of a protein or
polypeptide is the coding sequence for the light chain or a
fragment thereof for an IgG, IgM, IgD, IgE or IgA. Genes for whole
antibody molecules as well as modified or derived forms thereof,
such as fragments, e.g., Fab, single chain Fv(scFv) and
F(ab').sub.2 are included within the scope of the invention. The
antibodies and fragments can be animal-derived, human-mouse
chimeric, humanized, Delmmunized.TM. or fully human. The antibodies
can be bispecific and include but are not limited to diabodies,
quadroma, mini-antibodies, ScBs antibodies and knobs-into-holes
antibodies.
[0110] In practicing the invention, the production of an antibody,
or variant (analogue) or fragment thereof using recombinant DNA
technology can be achieved by culturing a modified recombinant host
cell under culture conditions appropriate for the growth of that
host cell resulting in expression of the coding sequences. In order
to monitor the success of expression, antibody levels with respect
to the antigen may be monitored using standard techniques such as
ELISA, RIA, Western blot and the like. The antibodies are recovered
from the culture supernatant using standard techniques known in the
art. Purified forms of these antibodies can, of course, be readily
prepared by standard purification techniques, e.g., affinity
chromatography via protein A, protein G or protein L columns, or
based on binding to the particular antigen, or the particular
epitope of the antigen for which specificity is desired. Antibodies
can also be purified with conventional chromatography, such as an
ion exchange or size exclusion column, in conjunction with other
technologies, such as ammonia sulfate precipitation and
size-limited membrane filtration. Preferred expression systems are
designed to include signal peptides so that the resulting
antibodies are secreted into the culture medium or supernatant,
allowing for ease of purification, however, intracellular
production is also possible.
[0111] The production and selection of antigen-specific fully human
monoclonal antibodies from mice engineered with human Ig loci, has
previously been described (Jakobovits A. et al., Advanced Drug
Delivery Reviews Vol. 31, pp: 33-42 (1998); Mendez M, et al.,
Nature Genetics Vol. 15, pp: 146-156 (1997); Jakobovits A. et al.,
Current Opinion in Biotechnology Vol. 6, No. 5, pp: 561-566 (1995);
Green L, et al., Nature Genetics Vol. 7, No. 1, pp:
13-21(1994).
[0112] The production and recovery of the antibodies themselves can
be achieved in various ways known in the art (Harlow et al.,
Antibodies, A Laboratory Manual, Cold Spring Harbor Lab, 1988).
[0113] Protein Coding Sequences
[0114] As used herein, a "first protein coding sequence" refers to
a heterologous nucleic acid sequence encoding a polypeptide or
protein molecule or domain or chain thereof including, but not
limited to a chain of an antibody or immunoglobulin molecule or
fragment thereof, a cytokine or fragment thereof, a growth factor
or fragment thereof, a chain of a Factor VIII molecule, a soluble
or membrane-associated receptor or fragment thereof, a viral
protein or fragment thereof, an immunogenic protein or fragment
thereof, a transcriptional regulator or fragment thereof, a
proapoptotic molecule or fragment thereof, a tumor suppressor or
fragment thereof, an angiogenesis inhibitor or fragment thereof,
etc.
[0115] As used herein, a "second protein coding sequence" refers to
a heterologous nucleic acid sequence encoding: a polypeptide or
protein molecule or domain or chain thereof including, but not
limited to a chain of an antibody or immunoglobulin or fragment
thereof, a cytokine or fragment thereof, a growth factor or
fragment thereof, a chain of a Factor VIII molecule, a soluble or
membrane-associated receptor or fragment thereof, a viral protein
or fragment thereof, an immunogenic protein or fragment thereof, a
transcriptional regulator or fragment thereof, a proapoptotic
molecule or fragment thereof, a tumor suppressor or fragment
thereof, an angiogenesis inhibitor or fragment thereof, etc.
[0116] The lentivector constructs of the invention may comprise two
or more transgenes or heterologous coding sequences, e.g., a first
protein coding sequence, a second protein coding sequence, a third
protein coding sequence, etc. The two or more transgenes may be
delivered to a cell using one or more lentivectors. When a single
lentivector is employed the two or more transgenes are co-expressed
by operative linkage to a single promoter and a self processing
cleavage sequence such as 2A. Numerous transgenes may be employed
in the practice of the present invention and include, but are not
limited to, nucleotide sequences encoding one or more of the
proteins indicated below or a fragment thereof;
[0117] 1. A sequence encoding HIF-1.alpha. and HIF.beta. (HIF), p35
and p40 (IL-12), chain A and chain B of insulin, integrins such as,
but not limited to alpha V beta 3 or alpha V beta 5, antibody heavy
and light chains and the heavy and light chain of Factor VIII.
[0118] 2. A sequence encoding a soluble receptor, include but are
not limited to, the TNF p55 and p75 receptor, the IL-2 receptor,
the FGF receptors, the VEGF receptors, TIE2, the IL-6 receptor and
the IL-1 receptor;
[0119] 3. A sequence encoding a cytokine including, but not limited
to, any known or later discovered cytokine, for example, IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12,
IL-13, IL-18, IL-24, INF-.alpha., INF-.beta., INF-.gamma., GM-CSF,
G-CSF and erythropoietin.
[0120] 4. A sequence encoding a growth factor including, but not
limited to, VEGF, FGF, Angiopoietin-1 and 2, PDGF, EGF, IGF, NGF,
IDF, HGF, TGF-.alpha., TGF-beta.
[0121] 5. A sequence encoding a pro-apoptotic factor including, but
not limited to, Bad, Bak, Bax, Bcl2, Bcl-Xs, Bik, Caspases, FasL,
and TRAIL.
[0122] 6. A sequence encoding a tumor suppressor protein or cell
cycle regulator including, but not limited to, p53, p16, p19,-21,
p27, PTEN, RB1.
[0123] 7. A sequence encoding an angiogenesis regulator including,
but not limited to, angiostatin, endostatin, TIMPs, antithrombin,
platelet factor 4 (PF4), soluble forms of VEGFR1 (domains 1-7) and
VEGFR2 (domains 1-7) fused to an Fc segment of IgG1, VEGF-TRAP,
PEDF, PEX, troponin I, thrombospondin, tumstatin, 16 Kd
Prolactin.
[0124] Cloned sequences and full-length nucleotides encoding any of
the above-referenced biologically active molecules may be obtained
by well-known methods in the art (Sambrook et al., 1989). In
general, the nucleic acid coding sequences are known and may be
obtained from public databases and/or scientific publications.
[0125] Homologues and variants of heterologous protein and
polypeptide coding sequences are included within the scope of the
invention based on "sequence identity" or "% homology" to known
nucleic acid sequences which are available in public databases
and/or selective hybridization under stringent conditions to such
known nucleic acid sequences (as described above for self
processing cleavage sequences). Homologues and variants of
heterologous protein and polypeptide amino acid sequences and
nucleic acid sequences that encode them are further included within
the scope of the invention. Such sequences may be identified based
on "sequence identity" to known sequences using publicly available
databases and sequence alignment programs, as described above for
self-processing cleavage sequences.
[0126] The present invention contemplates heterologous protein and
polypeptide variants which encode biologically active proteins,
polypeptides or fragments thereof, wherein the protein or
polypeptide sequence or the nucleotide sequence encoding it has 80,
85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more
sequence identity to the native sequence.
[0127] Furthermore, a variant "heterologous protein or
polypeptide-encoding polynucleotide" may encode the same
polypeptide as the reference polynucleotide sequence but, due to
the degeneracy of the genetic code, has a polynucleotide sequence
altered by one or more bases from the reference polynucleotide
sequence. Heterologous protein and polypeptide sequence variants
that encode a polypeptide with the same biological activity as
heterologous protein or polypeptide described herein and hybridize
under moderate to high stringency hybridization conditions are
considered to be within the scope of the present invention.
[0128] Furthermore, a variant "immunoglobulin-encoding
polynucleotide" may encode the same polypeptide as the reference
polynucleotide sequence but, due to the degeneracy of the genetic
code, has a polynucleotide sequence altered by one or more bases
from the reference polynucleotide sequence. Immunoglobulin sequence
variants that encode a polypeptide with the same biological
activity as the immunoglobulin polypeptides described herein and
hybridize under moderate to high stringency hybridization
conditions are considered to be within the scope of the present
invention.
[0129] Protein Expression
[0130] It will be understood that the lentivectors of the invention
find utility in the expression of recombinant proteins and
polypeptides in any lentiviral-based protein expression system, a
number of which are known in the art and examples of which are
described herein.
[0131] Following expression, recombinant proteins are recovered
from the culture using standard techniques known in the art. The
production and recovery of recombinant proteins themselves can be
achieved in various ways numerous examples of which are known in
the art. For example, the production of a recombinant protein,
polypeptide, an analogue or fragment thereof, can be undertaken by
culturing the modified recombinant host cells under culture
conditions appropriate that host cell resulting in expression of
the coding sequence(s). In order to monitor the success of
expression, recombinant protein or polypeptide levels are monitored
using standard techniques such as ELISA, RIA, Western blot and the
like.
[0132] Purified forms of the recombinant proteins can, of course,
be readily prepared by standard purification techniques known in
the art, e.g., affinity chromatography. Recombinant proteins can
also be purified using conventional chromatography, such as an ion
exchange or size exclusion column, in conjunction with other
technologies, such as size-limited membrane filtration. The
expression systems are preferably designed to include signal
peptides so that the resulting recombinant proteins are secreted
into the medium, however, intracellular production is also
possible.
[0133] The operability of the present invention has been
demonstrated by expression of immunoglobulin heavy and light chains
using the self-processing cleavage sequence-containing lentivectors
of the present invention (See, e.g., Example 2). The advantages
associated with use of self-processing cleavage sequences are
enhanced by inclusion of an additional proteolytic cleavage site
between the coding sequence for a first protein or polypeptide and
the self-processing cleavage sequence in the vectors of the
invention, resulting in removal of amino acid residues associated
with the self-processing cleavage sequence. Efficient removal of 2A
residues by incorporation of a furin cleavage site in the vectors
of the invention is demonstrated U.S. Patent Publication Nos.
20040265955 and 20050003482.
[0134] Vectors for use in Practicing the Invention
[0135] Retroviral vectors are also a common tool for gene delivery
(Miller, Nature 357: 455-460, 1992). Retroviral vectors and more
particularly lentiviral vectors may be used in practicing the
present invention. Accordingly, the term "retrovirus" or
"retroviral vector", as used herein is meant to include
"lentivirus" and "lentiviral vectors" respectively. Retroviral
vectors have been tested and found to be suitable delivery vehicles
for the stable introduction of genes of interest into the genome of
a broad range of target cells. The ability of retroviral vectors to
deliver unrearranged, single copy transgenes into cells makes
retroviral vectors well suited for transferring genes into cells.
Further, retroviruses enter host cells by the binding of retroviral
envelope glycoproteins to specific cell surface receptors on the
host cells. Consequently, pseudotyped retroviral vectors in which
the encoded native envelope protein is replaced by a heterologous
envelope protein that has a different cellular specificity than the
native envelope protein (e.g., binds to a different cell-surface
receptor as compared to the native envelope protein) may also find
utility in practicing the present invention. The ability to direct
the delivery of retroviral vectors encoding one or more target
protein coding sequences to specific target cells is desirable in
practice of the present invention.
[0136] The invention relates to retroviral vectors, producer cells,
and producer cell lines. In particular, the invention relates to a
novel approach for the expression of multimeric, heterologous
coding sequences in a single mammalian cell using one or more
self-inactivating ("SIN") retroviral vectors that encode a
heterologous sequence. More particularly, the retroviral vectors
are SIN lentiviral vectors. The invention further relates to
methods of using SIN lentiviral vectors for making multimeric
recombinant proteins.
[0137] The present invention provides retroviral vectors that
include e.g., retroviral transfer vectors comprising one or more
transgene sequences and retroviral packaging vectors comprising one
or more packaging elements. In particular, the present invention
provides pseudotyped retroviral vectors encoding a heterologous or
functionally modified envelope protein for producing pseudotyped
retrovirus. Preferably, the heterologous env gene comprises a VSV-G
or baculoviral gp64 env gene, although those skilled in the art
will appreciate that other env genes may be employed.
[0138] One preferred method of vector production is transient
transfection of plasmids containing the viral packaging genes and
the transgene into a cell line. Alternatively, the vectors are
produced via transfection, transduction or infection into a
packaging cell line to make producer cells. Methods for
transfection, transduction or infection are well known by those of
skill in the art. Both transiently transfected and producer cells
are effective to generate viral particles that contain the
transgene. For either the stable or transient production method the
recombinant virus is recovered from the culture media, concentrated
and/or purified, and titrated by standard methods used by those of
skill in the art.
[0139] The core sequence of the retroviral vectors of the present
invention may be readily derived from a wide variety of
retroviruses, including for example, B, C, and D type retroviruses
as well as spumaviruses and lentiviruses (RNA Tumor Viruses, Second
Edition, Cold Spring Harbor Laboratory, 1985). An example of a
retrovirus suitable for use in the compositions and methods of the
present invention includes, but is not limited to, a lentivirus.
Other retroviruses suitable for use in the compositions and methods
of the present invention include, but are not limited to, Avian
Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus,
Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,
Reticuloendotheliosis virus and Rous Sarcoma Virus. Preferred
Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe,
J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC
No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey
Sarcoma Virus and Rauscher (ATCC No. VR-998), and Moloney Murine
Leukemia Virus (ATCC No. VR-190). Such retroviruses may be readily
obtained from depositories or collections such as the American Type
Culture Collection ("ATCC"; Rockville, Md.), or isolated from known
sources using commonly available techniques.
[0140] Preferably, a retroviral vector sequence of the present
invention is derived from a lentivirus. A preferred lentivirus is a
human immunodeficiency virus, e.g., type 1 or 2 (i.e., HIV-1 or
HIV-2, wherein HIV-1 was formerly called lymphadenopathy associated
virus 3 (HTLV-III) and acquired immune deficiency syndrome
(AIDS)-related virus (ARV)), or another virus related to HIV-1 or
HIV-2 that has been identified and associated with AIDS or
AIDS-like disease. Other lentiviruses include a sheep Visna/maedi
virus, a feline immunodeficiency virus (FIV), a bovine lentivirus,
simian immunodeficiency virus (SIV), an equine infectious anemia
virus (EIAV), and a caprine arthritis-encephalitis virus
(CAEV).
[0141] The various genera and strains of retroviruses suitable for
use in the compositions and methods are well known in the art (see,
e.g., Fields Virology, Third Edition, edited by B. N. Fields et
al., Lippincott-Raven Publishers (1996), see e.g., Chapter 58,
"Retroviridae: The Viruses and Their Replication, Classification",
pages 1768-1771, including Table 1.
[0142] The packaging system used to generate retroviral vectors is
composed of at least two packaging vectors, a first packaging
vector which comprises a first nucleotide sequence comprising a
gag, a pol, or gag and pol genes and a second packaging vector
which comprises a second nucleotide sequence comprising a
heterologous or functionally modified envelope gene. In a preferred
embodiment, the retroviral elements are derived from a lentivirus,
such as HIV. Preferably, the vectors lack a functional tat gene
and/or functional accessory genes (vif, vpr, vpu, vpx, nef). In a
further preferred embodiment, the system further comprises a third
packaging vector that comprises a nucleotide sequence comprising a
rev gene. The packaging system can be provided in the form of a
packaging cell that contains the first, second, and, optionally,
third nucleotide sequences.
[0143] The invention is applicable to a variety of systems, and
those skilled in the art will appreciate the common elements shared
across differing groups of retroviruses. The description herein
uses lentiviral systems as a representative example. However, all
retroviruses share the features of enveloped virions with surface
projections and containing one molecule of linear, positive-sense
single stranded RNA, a genome consisting of a dimer, and the common
proteins gag, pol and env.
[0144] Lentiviruses share several structural virion proteins in
common, including the envelope glycoproteins SU (gp120) and TM
(gp4l), which are encoded by the env gene; CA (p24), MA (p17) and
NC (p7-11), which are encoded by the gag gene; and RT, PR and IN
encoded by the pol gene. HIV-1 and HIV-2 contain accessory and
other proteins involved in regulation of synthesis and processing
virus RNA and other replicative functions. The accessory proteins,
encoded by the vif, vpr, vpu, vpx, and nef genes, can be omitted
(or inactivated) from the recombinant system. In addition, tat and
rev can be omitted or inactivated, e.g., by mutation or
deletion.
[0145] First generation lentiviral vector packaging systems provide
separate packaging constructs for gag/pol and env, and typically
employ a heterologous or functionally modified envelope protein for
safety reasons. See e.g., Miller and Buttimore, Molec. Cell. Biol.
6(8): 2895-2902 (1986). These modifications minimize the homology
between the packaging genome and the viral vector so that the
ability of the vector to form recombinants is reduced (see e.g.,
Miller and Rosman, BioTechniques 7(9):980-990 (1989)).
[0146] In second generation lentiviral vector systems, the
accessory genes, vif, vpr, vpu and nef, are deleted or inactivated
and the packaging functions are divided into two genomes: one
genome expresses the gag and pol gene products, and the other
genome expresses the env gene product (see e.g., Bosselman et al.,
Molec. Cell. Biol. 7(5):1797-1806 (1987); Markowitz et al., J.
Virol. 62(4):1120-1124 (1988); Danos and Mulligan, Proc. Nat'l.
Acad. Sci. (USA) 85:6460-6464 (1988)). This approach eliminates the
ability for co-packaging and subsequent transfer of the psi-genome
(containing the viral packaging element psi), as well as
significantly decreases the frequency of recombination due to the
presence of three retroviral genomes in the packaging cell that
must undergo recombination to produce RCR. In the event
recombinants arise, mutations or deletions within the undesired
gene products render recombinants non-functional (see e.g., Danos
and Mulligan, supra Danos and Mulligan, supra; Boselman et al.,
supra; and Markowitz et al., supra). In addition, the deletion of
the 3' LTR on both packaging function constructs further reduces
the ability to form functional recombinants.
[0147] Third generation lentiviral vector systems are preferred for
use in practicing the present invention and include those from
which the tat gene has been deleted or otherwise inactivated (e.g.,
via mutation). Compensation for the regulation of transcription
normally provided by tat can be provided by the use of a strong
constitutive promoter, such as the human cytomegalovirus immediate
early (HCMV-IE) enhancer/promoter. Other promoters/enhancers can be
selected based on strength of constitutive promoter activity,
specificity for target tissue (e.g., a liver-specific promoter), or
other factors relating to desired control over expression, as is
understood in the art. For example, in some embodiments, it is
desirable to employ an inducible promoter such as tet to achieve
controlled expression. The gene encoding rev is preferably provided
on a separate expression construct, such that a typical third
generation lentiviral vector system will involve four plasmids: one
each for gagpol, rev, envelope and the transfer vector. Regardless
of the generation of packaging system employed, gag and pol can be
provided on a single construct or on separate constructs.
[0148] Typically, the packaging vectors are included in a packaging
cell, and are introduced into the cell via transfection,
transduction or infection. Methods for transfection, transduction
or infection are well known by those of skill in the art. A
retroviral/lentiviral transfer vector of the present invention can
be introduced into a packaging cell line, via transfection,
transduction or infection, to generate a producer cell or cell
line. The packaging vectors of the present invention can be
introduced into human cells or cell lines by standard methods
including, e.g., calcium phosphate transfection, lipofection or
electroporation. In some embodiments, the packaging vectors are
introduced into the cells together with a dominant selectable
marker, such as neo, DHFR, Gln synthetase or ADA, followed by
selection in the presence of the appropriate drug and isolation of
clones. A selectable marker gene can be linked physically to genes
encoding by the packaging vector.
[0149] Stable cell lines, wherein the packaging functions are
configured to be expressed by a suitable packaging cell, are known.
For example, see U.S. Pat. No. 5,686,279; and Ory et al., Proc.
Natl. Acad. Sci. (1996) 93:11400-11406, which describe packaging
cells. Further description of stable cell line production can be
found in Dull et al., 1998, J. Virology 72(11):8463-8471; and in
Zufferey et al., 1998, J. Virology 72(12):9873-9880.
[0150] Zufferey et al., 1997, Nature Biotechnology 15:871-875,
teach a lentiviral packaging plasmid wherein sequences 3' of pol
including the HIV-1 envelope gene are deleted. The construct
contains tat and rev sequences and the 3' LTR is replaced with poly
A sequences. The 5' LTR and psi sequences are replaced by another
promoter, such as one that is inducible. For example, a CMV
promoter or derivative thereof can be used.
[0151] Preferred packaging vectors may contain additional changes
to the packaging functions to enhance lentiviral protein expression
and to enhance safety. For example, all of the HIV sequences
upstream of gag can be removed. Also, sequences downstream of the
envelope can be removed. Moreover, steps can be taken to modify the
vector to enhance the splicing and translation of the RNA.
[0152] Optionally, a conditional packaging system is used, such as
that described by Dull et al., J. Virology 72(11):8463-8471, 1998.
Also preferred is the use of a self-inactivating vector (SIN),
which improves the biosafety of the vector by deletion of the HIV-1
long terminal repeat (LTR) as described, for example, by Zufferey
et al., 1998, J. Virology 72(12):9873-9880. Inducible vectors can
also be used, such as through a tet-inducible LTR.
[0153] Any vector for use in practicing the invention will include
heterologous control sequences, such as a constitutive promoter,
e.g., the cytomegalovirus (CMV) immediate early promoter, the RSV
LTR, the MoMLV LTR, and the PGK promoter; tissue or cell type
specific promoters including mTTR, TK, HBV, hAAT, regulatable or
inducible promoters, enhancers, etc. Preferred promoters include
the LSP promoter (Ill et al., Blood Coagul. Fibrinolysis 852:23-30,
1997), the EF1-alpha promoter (Kim et al., Gene 91(2):217-23, 1990)
and Guo et al., Gene Ther. 3(9):802-10, 1996). Most preferred
promoters include the elongation factor 1-alpha (EF1a) promoter, a
phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus
immediate early gene (CMV) promoter, chimeric liver-specific
promoters (LSPs), a cytomegalovirus enhancer/chicken beta-actin
(CAG) promoter, a tetracycline responsive promoter (TRE), a
transthyretin promoter (TTR), a MND promoter, a simian virus 40
(SV40) promoter and a CK6 promoter. Other promoters/enhancers can
be selected based on strength of constitutive promoter activity,
specificity for target tissue or other factors relating to desired
control over expression, as is understood in the art. The sequences
of these and numerous additional promoters are known in the art.
The relevant sequences may be readily obtained from public
databases and incorporated into vectors for use in practicing the
present invention.
[0154] The invention uses lentiviral vectors, particles, packaging
systems and producer cells capable of producing a high titer
recombinant lentivirus capable of selectively infecting human and
other mammalian cells. In one embodiment, the recombinant
lentivirus of the invention has a titer of greater than
5.times.10.sup.5 infectious units/ml. Preferably, the recombinant
retrovirus has a titer of greater than 1.times.10.sup.6 infectious
units/ml. Typically, titer is determined by conventional
infectivity assay on 293T, HeLa or HUH7 hepatoma cells.
[0155] The present invention also contemplates the inclusion of a
gene regulation system for the controlled expression of the coding
sequence for two or more polypeptides or proteins of interest. Gene
regulation systems are useful in the modulated expression of a
particular gene or genes. In one exemplary approach, a gene
regulation system or switch includes a chimeric transcription
factor that has a ligand binding domain, a transcriptional
activation domain and a DNA binding domain. The domains may be
obtained from virtually any source and may be combined in any of a
number of ways to obtain a novel protein. A regulatable gene system
also includes a DNA response element that interacts with the
chimeric transcription factor. This element is located adjacent to
the gene to be regulated.
[0156] Exemplary gene regulation systems that may be employed in
practicing the present invention include, the Drosophila ecdysone
system (Yao et al., Proc. Nat. Acad. Sci., 93:3346 (1996)), the
Bombyx ecdysone system (Suhr et al., Proc. Nat. Acad. Sci., 95:7999
(1998)), the Valentis GeneSwitch.RTM. synthetic progesterone
receptor system which employs RU-486 as the inducer (Osterwalder et
al., Proc Natl Acad Sci 98(22):12596-601 (2001)); the Tet.TM. &
RevTet.TM. Systems (BD Biosciences Clontech), which employs small
molecules, such as tetracycline (Tc) or analogues, e.g.
doxycycline, to regulate (turn on or off) transcription of the
target (Knott et al., Biotechniques 32(4):796, 798, 800 (2002));
ARIAD Regulation Technology which is based on the use of a small
molecule to bring together two intracellular molecules, each of
which is linked to either a transcriptional activator or a DNA
binding protein. When these components come together, transcription
of the gene of interest is activated. Ariad has two major systems:
a system based on homodimerization and a system based on
heterodimerization (Rivera et al., Nature Med, 2(9):1028-1032
(1996); Ye et al., Science 283: 88-91 (2000)), either of which may
be incorporated into the vectors of the present invention.
[0157] Preferred gene regulation systems for use in practicing the
present invention are the ARIAD Regulation Technology and the
Tet.TM. & RevTet.TM. Systems.
[0158] Delivery Of Nucleic Acid Constructs Including Protein Or
Polypeptide Coding Sequences To Cells
[0159] The vector constructs of the invention comprising nucleic
acid sequences encoding heterologous proteins or polypeptides, and
a self-processing cleavage site alone or in combination with a
sequence encoding an additional proteolytic cleavage site may be
introduced into cells in vitro, ex vivo or in vivo for expression
of heterologous coding sequences by cells, e.g., somatic cells in
vivo, or for the production of recombinant polypeptides by
vector-transduced cells, in vitro or in vivo.
[0160] The vector constructs of the invention may be introduced
into cells in vitro or ex vivo using standard methodology known in
the art. Such techniques include transfection using calcium
phosphate, microinjection into cultured cells (Capecchi, Cell
22:479-488 (1980)), electroporation (Shigekawa et al., BioTechn.,
6:742-751 (1988)), liposome-mediated gene transfer (Mannino et al.,
BioTechn., 6:682-690 (1988)), lipid-mediated transduction (Felgner
et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), and
nucleic acid delivery using high-velocity microprojectiles (Klein
et al., Nature 327:70-73 (1987)).
[0161] For in vitro or ex vivo expression, any cell capable of
expressing a functional protein may be employed. Numerous examples
of cells and cell lines used for protein expression are known in
the art. For example, prokaryotic cells and insect cells may be
used for expression. In addition, eukaryotic microorganisms, such
as yeast may be used. The expression of recombinant proteins in
prokaryotic, insect and yeast systems are generally known in the
art and may be adapted for protein or polypeptide expression using
the compositions and methods of the present invention.
[0162] Exemplary host cells useful for expression further include
mammalian cells, such as fibroblast cells, cells from non-human
mammals such as ovine, porcine, murine and bovine cells, insect
cells and the like. Specific examples of mammalian cells include
COS cells, VERO cells, HeLa cells, Chinese hamster ovary (CHO)
cells, 293 cell, NSO cells, 3T3 fibroblast cells, W138 cells, BHK
cells, HEPG2 cells, DUX cells and MDCK cells.
[0163] Host cells are cultured in conventional nutrient media,
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences. Mammalian host cells may be cultured in a variety of
media. Commercially available media such as Ham's F10 (Sigma),
Minimal Essential Medium (MEM), Sigma), RPMI 1640 (Sigma), and
Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are typically
suitable for culturing host cells. A given medium is generally
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferring, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), antibiotics, trace elements, and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The appropriate culture conditions for a
particular cell line, such as temperature, pH and the like, are
generally known in the art, with suggested culture conditions for
culture of numerous cell lines for example in the ATCC Catalogue
available on line at
"www.atcc.org/SearchCatalogs/AllCollections.cfm"
[0164] The lentivectors of the invention may also be administered
in vivo via various routes (e.g., intradermally, intravenously,
intratumorally, into the brain, intraportally, intraperitoneally,
intramuscularly, into the bladder, etc.), to deliver multiple genes
to express two or more proteins or polypeptides in animal models or
human subjects. Dependent upon the route of administration, the
therapeutic proteins elicit their effect locally (e.g., in brain or
bladder) or systemically (other routes of administration). The use
of tissue specific promoters 5' to the open reading frame(s) for a
protein or polypeptide in the vectors of the invention may be used
to effect tissue specific expression of the two or more proteins or
polypeptides encoded by the vector.
[0165] Various methods that introduce recombinant lentivectors into
target cells in vitro, ex vivo or in vivo have been previously
described and are well known in the art. For example, in vivo
delivery of the recombinant vectors of the invention may be
targeted to a wide variety of organ types including, but not
limited to brain, liver, blood vessels, muscle, heart, lung and
skin. In the case of ex vivo gene transfer, the target cells are
removed from the host and genetically modified in the laboratory
using recombinant vectors of the present invention and methods well
known in the art.
[0166] The recombinant vectors of the invention can be administered
using conventional modes of administration including but not
limited to the modes described above. The recombinant vectors of
the invention may be provided in any of a variety of formulations
such as liquid solutions and suspensions, microvesicles, liposomes
and injectable or infusible solutions. The preferred form depends
upon the mode of administration and the therapeutic application. A
from appropriate to the route of delivery may be readily determined
using knowledge generally available to those of skill in the
relevant art.
[0167] Current methods of recombinant immunoglobulin production
rely on use of: CHO cells, derivatives thereof; NSO cells; PerC.6
cells; and HEK cells. Most currently used methods for recombinant
immunoglobulin production are based on use of vector constructs
which include an amplifiable gene such as dihydrofolate reductase
(DHFR). As a result the process of recombinant protein production
requires the steps of: (1) transfection; (2) selection by culture
in medium containing a drug such as Neomycin; (3) amplification of
the immunoglobulin coding sequence based on the presence of DHFR in
the construct and culture in medium containing a near lethal
concentration of methotrexate; (4) screening for viable cells; and
(5) further step-wise amplification in culture medium containing
increasing concentrations of methotrexate; and (6) further
screening at each amplification step to identify viable cells.
[0168] Using the DHFR system, the genomic copy number of the
nucleic acid encoding the immunoglobulin is increased by the
selective pressure of exposing cells to methotrexate, a drug that
blocks the activity of DHFR, resulting in higher levels of antibody
expression. After 2 to 3 weeks of exposure to methotrexate at a
near lethal concentration, the majority of cells die, but cells
that overproduce DHFR will survive. Multiple rounds of step-wise
amplification in methotrexate-containing medium are typically
required, which typically takes from 6 to 12 months, to select a
clone that produces at least 20 pg/c/day (pg per cell per day).
Frequently as many as 2000-4000 initial clones are screened in
order to select a clone that produces at least 20 pg/c/day.
[0169] The protein expression levels of different cell clones
obtained from step-wise methotrexate amplification can vary widely.
As a consequence, the identification of high-producer cell lines is
a tedious and labor-intensive process. Several methods for the
isolation of clones exist, the most popular being limiting dilution
cloning.
[0170] In contrast, the methods of the current invention do not
require amplification methotrexate-containing medium and hence
multiple rounds of successive culturing and screening are avoided.
The efficiency of infection and ability of the lentiviral vector to
re-infect cells multiple times allow for the rapid generation of
cell lines containing numerous genomic copies of the nucleic acid
encoding the antibody 2A fusion protein. As a result, 10-fold to
50-fold fewer clones are needed for screening to select a clone
that produces at least 20 pg/c/day, shortening the clonal selection
process by as much as about 10 months.
[0171] For products, like monoclonal antibodies, cell lines must
produce at least 20 pg/cell/day to be suitable candidates for
commercial production. The combination of fast growth and high
productivity makes a cell line a candidate for commercial
production. A further important consideration is the stability of
the cell line over extended periods of time and upon scale up.
[0172] Some clonal cell lines, such as GS-NS0 have been found to be
unstable after long term culture. In addition, the presence of
methotrexate in long term culture has been shown to result in
undesirable genetic heterogeneity in the cells.
[0173] The present invention provides advantages in that: 1) the
process does not rely on inclusion of an amplifiable gene such as
dihydrofolate reductase (DHFR) in the expression construct or use
of an agent such as methotrexate for amplification; 2) the process
is less expensive because of the fact that the resulting cell line
need not be methotrexate resistant thereby eliminating the need to
add exogenous methotrexate to the culture medium and allows for
additional cell lines capable of large scale culture to be employed
in the methods of the invention; 3) the screening time is
significantly reduced because the process requires less steps than
current commercial processes. The process only requires
transfection and selection in a medium containing a drug such as
neomycin. No amplification or subsequent rounds of screening are
required; 4) the cell line is preferably transfected or infected
multiple times over a short period of time to rapidly increase the
genomic copy number of the vector. The number of transfections or
infections can vary depending, in part, on the coding sequence of
the antibody to be expressed, strength of the selected promoter and
parent cell line used for expression. In certain embodiments, at
least 3 rounds of transfection or infection ("pings") are employed
to get optimal expression of a heterologous coding sequence by way
of a lentivirus vector; 5) the time for selection of high producer
clones is dramatically reduced from the typical 6-12 months to 1-2
months; and 6) the instability of the producer cell line over
extended periods of time and upon scale up is less likely to be a
problem due to the absence of methotrexate in the culture
medium.
[0174] In one preferred embodiment, for immunoglobulin production,
clonal cell lines of the invention produce at least 20 pg/cell/day,
preferably at least 25, 30, 35, 40, 45 or 50, 60, 70, 80, 90, 100,
125, 150 or 200 pg/cell/day. In another preferred embodiment, the
timing for selection of clones that produce at least 20 pg/cell/day
is less than 4 months, preferably less than 3 months and more
preferably less than 2 months. In yet another preferred embodiment
temperature for culture is at least 31.degree..
[0175] In one preferred embodiment, for immunoglobulin production,
clonal cell lines of the invention comprise at least 20, preferably
at least 25, 30, 35, 40, 45 or 50, 60, 70, 80, 90, 100 genomic
copies of the lentiviral vector comprising the nucleic acid
encoding the immunoglobulin 2A construct.
[0176] In yet another preferred embodiment, clonal cell lines of
the invention produce at least 20 pg/cell/day, preferably at least
25, 30, 35, 40, 45 or 50, 60, 70, 80, 90, 100, 125, 150 or 200
pg/cell/day and comprise at least 20 genomic copies of the
lentiviral vector comprising the nucleic acid encoding the
immunoglobulin 2A construct.
[0177] The many advantages of the invention to be realized in
recombinant protein and polypeptide production in vivo include
administration of a single vector for long-term and sustained
expression of two or more recombinant protein or polypeptide ORFs
in patients; in vivo expression of two or more recombinant protein
or polypeptide ORFs having biological activity; and the natural
posttranslational modifications of the recombinant protein or
polypeptide generated in human cells.
[0178] One preferred aspect is use of the recombinant vector
constructs of the present invention for the in vitro production of
recombinant proteins and polypeptides. Methods for recombinant
protein production are well known in the art and self processing
cleavage site-containing vector constructs of the present invention
may be utilized for expression of recombinant proteins and
polypeptides using such standard methodology.
[0179] In one exemplary aspect of the invention, lentivector
introduction or administration to a cell is carried out by:
[0180] (1) introduction or administration of the lentivector to a
cell by more than one round of transfection/transduction or
infection;
[0181] (2) culturing the infected cell under conditions that select
for a cell expressing the recombinant protein or polypeptide e.g.,
in medium containing a selection agent such as Neomycin;
[0182] (3) evaluating expression of the recombinant protein or
polypeptide; and
[0183] (4) collecting the recombinant protein or polypeptide.
[0184] In a preferred embodiment the cells are transfected or
infected at least 3 times, more preferably at least 4 or 5
times.
[0185] Methods and Compositions of the Invention
[0186] The invention relates to engineered lentiviral vectors for
expression of two or more domains or chains of a multimeric
protein. In one aspect the multimeric protein is an immunoglobulin
and full-length antibody heavy and light chain coding sequences are
expressed using a lentivector comprising a single open reading
frame driven by a single promoter wherein the vector comprises a
self-processing cleavage site or sequence between the heavy and
light chain coding sequences. In another aspect the protein is a
multimeric heterologous protein and the full-length coding
sequences are expressed using a lentivector comprising a single
open reading frame driven by a single promoter wherein the vector
comprises one or more self-processing cleavage sites or
sequences.
[0187] In yet another aspect, the invention provides a method for
high level expression of recombinant protein using more than one
engineered lentivector, wherein each lentivector encodes a single
open reading frame of a multimeric protein driven by a single
promoter. For example, for expression of a full-length antibody,
individual lentivectors that encode the full-length antibody heavy
and light chain, respectively, are used to infect the same cell
such that high-level expression of a biologically active antibody
results.
[0188] In one preferred embodiment, individual populations of host
cells are transduced with lentiviral transfer vectors wherein the
heterologous protein coding sequence encoded by the vectors is not
the same and wherein each lentivector comprises the coding sequence
for a single domain or chain of a heterologous protein operably
linked to an expression control sequence. For example, a population
of cells is transformed with a transfer vector comprising a
heterologous protein coding sequence, such as an immunoglobulin
light chain coding sequence operably linked to an expression
control sequence, and this population or clones derived from it are
transduced with a second transfer vector comprising a second
heterologous protein coding sequence, such as an immunoglobulin
heavy chain coding sequence operably linked to an expression
control sequence. The resulting population is cultured under
conditions suitable for production of the multimeric protein.
[0189] In another preferred embodiment, the transfer vectors
further comprise first a strong promoter (e.g. CMV, SV40, MND, or
CAG), followed by the antibody heavy chain sequence (H), a furin
cleavage site (F), a 2A self-processing sequence derived from the
Foot-and-Mouth Disease virus (2A), and an antibody light chain
sequence (L). The resulting construct is designated H-F-2A-L. The
2A peptide sequence provides a "cleavage" site that facilitates the
generation of two polypeptide chains of the antibody molecule
during the translation process as shown in FIG. 1. The furin
cleavage site provides a secondary cleavage during antibody
secretion pathway to remove 2A residues that are attached to the C
terminus of the first gene (i.e. heavy chain in this construct). In
yet another preferred embodiment, the transfer vectors further
comprise first a strong promoter, followed by the antibody light
chain sequence (L), a furin cleavage site (F), a 2A self-processing
sequence derived from the Foot-and-Mouth Disease virus (2A), and an
antibody heavy chain sequence (H). The resulting construct is
designated L-F-2A-H.
[0190] Recombinant lentiviral particles were generated and used to
transduce human or hamster cells in vitro. ELISA assays for
antibody expression from these supernatants revealed that the
antibody was produced at high levels in both 293 and CHO cells
transduced with CAG H-F-2A-L lentiviral particles. The ratio of the
heavy and light chains expressed from the H-F-2A-L construct was
approximately 1:1, and the final antibody retained full biological
activity based on antibody binding and neutralizing assays.
[0191] The present invention finds utility in expression of a
full-length monoclonal antibody (IgG, IgM, IgD, IgE, IgA) or
antibody fragments from mammalian cells transduced with lentiviral
vectors as well as expression of any heterodimeric protein. Given
the high transduction efficacy and gene expression level,
lentiviral vectors are able to rapidly generate stable cells lines
that express high levels of recombinant proteins such as
antibodies. In one preferred embodiment, the lentiviral vector
comprises a self-processing site or sequence.
[0192] The objects of the invention have been achieved by a series
of experiments, some of which are described by way of the following
non-limiting examples.
Examples
Example 1
Construction and Production of 2A Antibody Expression Constructs
Transfection Plasmids
[0193] In order to generate lentivector constructs encoding a rat
anti-mouse VEGFR2 and human anti-KDR antibody, DNA fragments that
encode the antibody heavy chain, furin cleavage site, 2A sequence,
and antibody light chain were linked together by PCR extension. A
DNA fragment including a furin cleavage site RAKR (SEQ ID NO: 18),
and the FMDV 2A sequence APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6),
were amplified from a cloned plasmid by PCR. The heavy and light
chain fragments were amplified from the cloned plasmids that encode
the full-length antibody heavy or light chains respectively. During
PCR, an EcoR I restriction endonucleotidase site was added to the
5' prime end of the heavy chain and the 3' prime end of the light
chain. The fused heavy chain-furin cleavage site-2A-light chain DNA
fragment was digested with EcoR I and purified via agarose gel. The
purified DNA fragment was inserted into the pDHFR plasmid at the
EcoR I site using T4 DNA ligase. The pDHFR contains a CAG promoter
operatively liked to the antibody coding sequences and an SV 40
promoter operatively liked to the DHFR gene. A native signal
peptide (leader) was included in the heavy or light chain,
respectively, to facilitate secretion of the polypeptides upon
synthesis. In addition, the construct also contains a polyA
sequence to ensure high-level gene expression (FIG. 2). A pDHFR
dual CAG Ab expression plasmid with a CAG promoter that drives the
antibody light chain and a second CAG promoter that drives the
antibody heavy chain was also constructed (FIG. 2). This plasmid
contains the same plasmid backbone as pDHFR but encodes two CAG
promoters. A multiple cloning site and a polyA signal sequence
follows each CAG promoter sequence. To generate the dual promoter
antibody plasmid, an antibody heavy chain or light chain coding
sequence was amplified from a cloned plasmid that contains the
antibody heavy or light chain sequence, respectively. The antibody
light chain sequence was inserted after the first CAG promoter, and
the heavy chain after the second CAG promoter using the multiple
cloning sites.
[0194] Lentiviral Plasmids: The nucleotide coding sequences
encoding the KDR and DC101 HF2AL antibodies were cloned into
3.sup.rd generation lentiviral transfer vectors using standard
molecular biology techniques routinely employed by those of skill
in the art. The 3.sup.rd generation lentiviral vector system has
previously been described (Dull et al., J. Virol. 72:8463-8471,
1998). Briefly, the transfer vector contains a 5' chimeric RSV/LTR
promoter, cPPT (Zennou et al., Cell 101:173-185, 2000), CAG
promoter (Miyazaki et al., Gene 79:269-277, 1989), and SIN LTR
(Zufferey et al., J. Virol. 72:9873-9880, 1998). For these studies,
the promoter driving the expression of the antibodies is comprised
of a CMV enhancer, the chicken beta-actin promoter and splice
donor, and the rabbit beta-globin splice acceptor (CAG). A
schematic of the lentiviral transfer vector is diagrammed in FIG.
2.
[0195] Lentivirus production: Vector production, concentration, p24
analysis, and titer assays were performed as previously described
(Dull et al., J. Virol. 72:8463-8471, 1998). Briefly, vectors were
prepared by transient transfection in a 10 cm dish with 6.5 ug of
pMDLg/pRRE, 2.5 ug of pRSV-Rev, 3.5 ug of pMD2.VSVG-Env, and 10 ug
of transfer vector. Vector particles were harvested after 24 hrs,
pooled, passed through a 0.2 um cellulose acetate filter, and
concentrated by ultracentrifugation for 2 hrs 20 min at 19,500 rpm
(50,000g) in a SW28 swinging bucket rotor. Pellets were resuspended
in PBS containing 40 mg/ml lactose and stored in aliquots at
-80.degree. C. Detection of the gag p24 protein was evaluated using
an Alliance HIV-1 p24 ELISA kit (Perkin Elmer).
[0196] AAV production: Recombinant AAV virus was prepared according
to standard procedures described in Snyder et al., 1996, In:
Current Protocols in Human Genetics, Seidman J S, (editor). John
Wiley & Sons: New York; 1-24. Briefly, sub-confluent 293 cells
were co-transfected with the vector construct pAAV-CAG-KDR
(2.13)-HF2AL, AAV helper plasmid pUC-ACG and Adeno helper plasmid
pXX6 using the calcium phosphate method. Eight hours after
transfection, media was replaced by fresh culture media and cells
were incubated for 72 hr, at which point cells were harvested and
lysed by three freeze/thaw cycles. Lysates were treated with
Benzonase (EM Industries, Hawthorne, N.Y.) for 15 min at 37.degree.
C. to digest nucleic acids, and centrifuged to remove the cellular
debris. The cleared cell lysate was fractionated by ammonium
sulfate precipitation and the rAAV virions were isolated on two
sequential CsCl gradients. The gradient fractions containing rAAV
were dialyzed against sterile PBS containing CaCl.sub.2 and
MgCl.sub.2, and stored at -80.degree. C. AAV titers were calculated
as genomic equivalents, following DNase I and proteinase K
treatment, by dot blot and by quantitative PCR as described in
Harding et al., 2004 Gene Ther (11): 204-213.
[0197] Transfection, selection and cloning: CHOD-cells were seeded
at 3.times.10.sup.6 in 10 cm plates 24 hr prior to transfection.
The transfection of 12 ug of DHFR-containing plasmid per plate was
performed with Fugene 6 reagent (Roche Molecular Biochemical)
according to the manufacturer's protocol in serum free OPTI-MEM I
medium (Invitrogen). 5-6 hours post transfection the medium was
replaced with regular growth medium (50:50 F12/DMEM medium
supplemented with 2 mM L-glutamine, 10 ug /ml glycine, 15 ug/ml
hypoxanthine, 5 ug/ml thymidine and 10% FBS) and incubated at
37.degree. C., 5% CO.sub.2. DHFR selection was carried out 48-72 hr
post transfection in IMDM medium (JRH) with 2 mM L-glutamine and
10% dialyzed FBS. 10 days post selection clones were picked into
96-well plates, and duplicate plates were made 2-3 days later.
24-hour supernatant was collected from one of the duplicate plates
and subjected to ELISA for antibody production and viable cell
numbers in each well were determined by CCK-8 proliferation assay
(Dojindo). The data from ELISA and CCK-8 assay was used to
determine the pg/cell/day antibody production level of each clone.
Clones>1 pg/cell/day were expanded from the second plate for
further characterization. Clones or populations that were selected
for Methotrexate (MTX) amplification were started at
5.times.10.sup.5 cells per 10 cm plate in MTX containing medium.
MTX concentration was increased from 25 nM to 50 nM, 100 nM, etc.
Cells in each selection were passaged 2-3 times before moving into
a higher concentration of selective medium and a population of
cells was banked (frozen) after each selection.
[0198] Lentivirus infection and cloning: CHOD-cells were seeded at
1.times.10.sup.5 cells per well in 6-well plates with 2 ml culture
medium containing appropriate amount of lentivector and polybrene
at 8 ug/ml. Medium was changed 24 hr post infection. Once the cells
were confluent they were expanded to a 10 cm plate. Successive
rounds of infections were performed at 2-7 day intervals.
Populations were subcloned by limiting dilution. Clones were picked
and screened as described in the transfection method.
[0199] Methods for making clones: In order to determine the
relative effectiveness of different vector systems, a comparison of
various methods of making cell lines that express a rat anti-mouse
VEGFR2 antibody (DC 101) and a human IgG1 anti-KDR antibody (2.13)
were evaluated including:
[0200] (1) transfection using a plasmid which includes a 2A
sequence wherein heavy and light chain are expressed under control
of a single promoter, followed by amplification with
methotrexate;
[0201] (2) transfection using a plasmid which includes dual
promoters to express antibody heavy and light chains, followed by
amplification with methotrexate;
[0202] (3) infection with an AAV construct which includes a 2A
sequence wherein heavy and light chains are expressed under control
of a single promoter; and
[0203] (4) infection with a lentivirus construct which includes a
2A sequence wherein heavy and light chains are expressed under
control of a single promoter.
Example 2
Comparing Different Methods for Making Stable Antibody-Producing
Cell Lines
[0204] Stable antibody expressing cell lines were made in
CHOD-cells by Fugene 6 transfection with a dual promoter anti-KDR
plasmid or a 2A-anti-KDR plasmid as described above. Alternatively,
CHOD-cells were infected with either an AAV-2A-anti-KDR vector or a
Lenti-2A-anti-KDR vector. In this example CHOD-cells were infected
5 times (5.times.) at one-week intervals with lenti-2A-KDR vector
supernatants containing 500 ng p24. AAV-2A-KDR infections were
performed 3 times at an MOI of 10.sup.5 particles/cell. The
transfected populations, lenti 4.times. and 5.times. infected
populations, and AAV transduced populations were all subcloned.
Clones were picked, screened and expanded as described above. Table
2 compares these four methods.
TABLE-US-00002 TABLE 2 Evaluation of anti-KDR antibody expressing
clones produced by different methods. Total clones >1 pg/cell/
>10 pg/cell/ >10 pg/cell/ Method examined day 96-well day
6-well day 10-cm Transfection 373 2 0 0 2A-KDR Transfection 373 11
0 0 dual-KDR AAV-2A-KDR 800 2 0 0 Lenti-2A- KDR 260 260 >41 20
4X pop Lenti-2A-KDR 180 180 >14 6 5X pop
[0205] The results in Table 3 indicate that transfection of
CHOD-cells with a plasmid which comprises an antibody heavy and
light chain coding sequence operatively linked to a single promoter
and further including a self processing 2A sequence did not show an
advantage over dual promoter controlled expression of antibody
heavy and light chain coding sequences following transfection and
screening. Alternatively, four or five rounds of
transfection/transduction of CHOD-cells with lentivector constructs
which comprise an antibody heavy and light chain coding sequence
operatively linked to a single promoter and further including a
self processing 2A sequence resulted in transduction of 100% of
clones, yielding higher numbers of clones with significantly
greater levels of antibody production compared to the other
methods. This approach also reduced the number of clones necessary
to screen and significantly reduced the time necessary for
isolation of high expressing clones (FIG. 3). Multiple rounds of
transduction with a lentivector also increased the copy number in a
producer cell resulting in high level protein expression. The
twelve lenti clones with the highest expression levels were plated
at 1.times.10.sup.7 cells/10 cm plate and evaluated for antibody
expression at 31.degree. C. (Table 3). These data show that all of
the individual clones evaluated express high levels of
antibody.
TABLE-US-00003 TABLE 3 Lenti-2A-KDR CHOD- expressing clones. # of
clone # infections pg/cell/day 22 4X 18.2 30 4X 26.2 61 4X 18.6 62
4X 1 5.0 70 4X 18.2 87 4X 26.7 15 5X 20.1 65 5X 21.9 66 5X 16.0 80
5X 10.7 88 5X 10.9 89 5X 9.9
[0206] A similar experiment comparing methods of producing clones
was performed using the rat anti mouse DC101 antibody.
CHOD-transfections were performed as described above to compare
protein expression following transfection with the 2A DC101 plasmid
relative to the dual promoter DC101 plasmid. Alternatively, five
serial infections of CHOD-cells were performed at 1-week intervals
with the lenti-2A-DC101 vector (200 ng p24/infection). In all three
cases the clones were isolated and analyzed for antibody
expression. Table 4 compares the expression levels of the 2A-DC101
plasmids with the dual promoter (H+L) plasmids. There was no
apparent advantage to the 2A construct in transfection experiments.
In general, expression levels of the anti-DC101 antibody are higher
than the anti-KDR antibody. The antibody expression in the
lenti-2A-DC101 population was much higher than the transfected
populations. Again, fewer clones had to be screened due to higher
antibody expression levels, and the greater frequency of positive
clones as compared to the other systems tested. For example, when
expanded to 6 well plates all of the lentiviral clones produced
greater than 1 p/c/d of antibody. Ten of the highest producing
lenti-2A clones from 6-well plates were expanded to 10-cm plates
for further analysis (Table 6). While only 8/10 clones exceeded 10
p/c/d, two of the clones, #45 and 51, were exceptionally high
producers, again demonstrating that serial rounds of transduction
with lenti 2A vectors results in the need to perform screening of
fewer clones in order to obtain high expressing clones. This method
also eliminates the need for an amplification step, e.g., with
DHFR.
TABLE-US-00004 TABLE 4 Comparison of antibody expression levels in
CHOD- clones transfected with 2A-DC101 or dual promoter DC101
plasmids. # clones # clones Population >1 p/c/d >1 p/c/d
Plasmid pg/cell/day Total clones 96-well 6-well H + L DC101 1.7 400
137 120/137 2A-DC101 0.55 400 84 75/84 Lenti 5X 8.17 130 130
60/60
TABLE-US-00005 TABLE 5 Antibody expression in CHOD- clones infected
5X with Lenti-2A-DC101. Clone # p/c/d 8 13.69 22 13.77 31 15.78 38
8.49 39 7.57 45 45 47 9.96 51 40 55 14.09 59 11.48
[0207] The number of integrated genomic copies in cells transfected
with the lentiviral vector comprising the immunoglobulin 2A
construct encoding the DC101 antibody was also examined by Southern
Blot analysis (FIG. 5). Genomic DNA was isolated from naive CHO
cells and from two 5.times. transduced clones that express
approximately 20-40 pg/cell/day (Clones 45 and 51). The genomic DNA
was digested using the restriction enzyme EcoRI using standard
conditions and resolved by electrophoresis on a 1% agarose gel in
the presence of known, increasing genomic amounts of control DNA to
estimate copy number. The resolved DNA fragments were transferred
to nylon filter for further analysis. The filter hybridized to a
2.2 Kb radiolabeled DNA fragment comprising the full-length
nucleotide sequence encoding the DC101 antibody. The position of
the bound probe was visualized using autoradiography and
quantitated using a phosphorimager (Molecurar Dynamics, Sunnyvale,
Calif.).
[0208] As shown in FIG. 5, the radiolabeled probe hybridizes to a
single 2.2 Kb fragment containing the nucleotide sequence encoding
the DC101 antibody. Clones 45 and 51 also exhibit only a single 2.2
Kb band and comprise approximately 33 genomic copies of the
lentiviral vector construct. This number is consistent with the
Taq-man results presented above. Thus, within a 5-week period, a
high producer cell line comprising 33 copies of the antibody coding
sequences may be prepared without drug-based amplification.
[0209] CHOD-cells were transfected with 200 ng of p24 of the
lentiviral vector encoding DC101 4.times., 7.times. or 9.times. at
3 day intervals. Populations from the transfected cells were
subcloned and approximately 100-160 clones were expanded into 96
well plates and the supernatants screened by ELISA to determine
DC101 expression levels. Cell number was determined using a CCK8
assay. Approximately 50 high expressing clones were chosen,
expanded in 6-well plates and the amount of antibody produced
(pg/cell/day) was determined for each clone (FIG. 6). As the number
of transfections was increased, there was a concomitant increase in
the number of cells that express increasing levels of antibody,
e.g., compare pg/cell/day of antibody production for 4.times. and
9.times. clones (FIG. 6). Furthermore, approximately 50% of the
cells transfected in the 9.times. population expressed greater that
30 pg/cell/day thereby greatly reducing the time and number of
clones required to be screened to identify cell lines capable of
expressing commercially-relevant amounts of recombinant
antibody.
Example 3
Demonstration of Antibody Expression Levels from Different Cell
Types Following Transduction with Lentiviral Constructs
[0210] Antibody expression levels were evaluated for lenti human
anti-KDR and lenti rat anti-VEGFR2 (DC101) while looking at a
number of variables: different cell types, different promoters, and
different lenti constructs. The different cell types examined were
CHOD-cells, PerC6 cells and HuH7 cells. The different promoters
were CAG, CMV and MND.
[0211] For the assays CHOD-cells, PerC6 cells, or HuH7 cells were
seeded at 1.times.10.sup.5 cells per well in 6-well plates
containing 2 ml of culture medium including an appropriate amount
of lentivector and polybrene at 8 ug/ml. Infection was allowed to
proceed as described above. The medium was changed 24 hr post
infection. Cells were maintained in 6-well plates until they
reached 80% confluence. The medium was then refreshed and at 24 hr
and supernatant collected from each well. The cell numbers in each
well were determined by hemocytometer. The antibody expression
level was determined by ELISA.
[0212] FIG. 4 compares the antibody production from three cell
lines (CHOD-, PerC6, HuH7) infected with lenti-2A-anti-DC101. FIG.
7 demonstrates that antibody is expressed in the same cell lines
following infection with lenti-2A-anti-KDR using a CMV
promoter.
Example 4
Alteration in the Amount of Lentivector and Changes in the
Infection Intervals can Increase Antibody Expression Levels and
Decrease Time for Identifying Clones
[0213] CHOD-cells were serially infected with lenti-2A-DC101
vector. Transductions done at one week intervals were compared with
transduction is done at 2-3 day intervals. Infections were
performed as described above. After each infection, the populations
were saved, expanded, and assayed for antibody expression as
described. Additionally, each cell population was analyzed for the
number of lenti copies/cell by TaqMan PCR (Table 7).
TABLE-US-00006 TABLE 6 Comparison of antibody expression and
lentivector copy number in CHOD- populations infected with
Lenti-2A-DC101 at different time intervals. Experiment #1
Experiment #2 Day of Lenti Population Day of Lenti Population
infection copies/cell pg/cell/day infection copies/cell pg/cell/day
P2ng/ml 50 1 7 1.60 * * * 100 1 37 2.26 * * * 200 (1X) 1 20 3.62 1
20 4.58 200 (2X) 8 40 7.07 3 41 7.62 200 (3X) 15 36 7.50 5 58 8.05
P4ng/ml 200 (4X) 22 43 8.64 8 97 9.46 200 (5X) 29 77 8.17 11 94
11.30 200 (6X) * * * 14 75 12.54 200 (7X) * * * 17 145 14.49 200
(8X) * * * 20 135 13.98 200 (9X) * * * 23 125 15.09
[0214] Taken together, the results suggest that successive
transductions with lentivectors increases the number of antibody
producer clones that generate high levels of antibodies. Integrated
lentivector copies increase with each transduction and correlates
with increasing antibody expression in the populations. It is
interesting to note that decreasing the time between infections had
a positive effect on antibody expression levels. Additionally, by
decreasing the interval between infections from 1 week to 2-3 days,
antibody expression was increased approximately 2-fold and the
overall infection process was decreased by a week.
Example 8
Removal of Extra Amino Acids Derived from Furin Cleavage Site at
C-Terminus by Carboxypeptidases
[0215] It has been shown that after furin cleavage newly exposed
basic amino acids at the C-terminus of proteins can be removed by
carboxypeptidases, we hypothesized that all additional amino acid
derived from the furin cleavage site at the C-terminus of antibody
heavy chain can be removed by using a furin cleavage site that
consists of exclusively basic amino acids, i.e. R or K. In testing
a number of furin cleavage sites with all basic amino acids, one
construct was developed wherein the last amino acid K at the
C-terminus of antibody heavy chain was deleted and the antibody
heavy and light chains were linked with a furin cleavage site RKRR
(SEQ ID NO: 15) and a 2A sequence. The plasmid DNA was transfected
into CHO cells in 10 cm tissue culture dish and the antibody
protein was purified from the supernatant by protein A affinity
chromatography. The purified IgG protein was separated in SDS-PAGE
and the heavy chain band was sliced out from the gel. The heavy
chain protein was then cleaved by Cyanogen Bromide (CNBr) and the
resulting peptides were analyzed by mass spectrometer. Mass
spectral analysis showed a strong peak that corresponds to the
C-terminal fragment of the antibody heavy chain (FIG. 8). No peaks
were observed in the spectrum that would represent the C-terminal
fragment plus any amino acid(s) derived from the furin cleavage
site. This data strongly suggested that the RKRR (SEQ ID NO: 15)
site facilitates efficient removal of extra amino acids at the
C-terminus of the antibody heavy chain in HF2AL constructs,
resulting in expression of the antibody heavy chain without any
extra amino acid residues.
Example 9
Stable Long-Term Expression of Lentivectors in Human Pancreatic and
Mouse CT26 Cells
[0216] Lentiviral vectors expressing human GMCSF from a CAG
promoter were prepared by transient transfection, as described.
Pancreatic cells were cultured and 3.times.10.sup.4 cells were
spinoculated with 1 ml of lentivirus and 8 ug/ml polybrene for 4
hrs at 3400 rpm, then plated in a 6 well plate with fresh medium. A
total of three infections were performed at 2 week intervals. The
populations from each infection were labeled "1.times., 2.times.,
and 3.times.." The 3.times. population was dilution cloned, and
1280 clones were picked and transferred to 96 well plates.
Supernatants from these clones were screened for GM-CSF expression
by ELISA. Nearly all clones were positive for human GM-CSF
expression, and 88 clones were expanded to 6-well plates and
re-assayed. Thirteen clones expressing 500-2500 ng/10.sup.6
cells/24 hr GM-CSF were expanded to 10 cm plates, reassayed again,
and put into a stability study. For the stability study the cells
were maintained in continuous culture for 12 weeks with GM-CSF
expression levels checked at 3 week intervals. FIG. 9 demonstrates
that in 13 clones lentivector GM-CSF expression was stable for 12
weeks of continuous culture in medium that did not contain any
selection.
[0217] In a second lentivector expression stability study,
long-term expression of mouse GM-CSF-producing clones was evaluated
using CT26 cells (mouse adenocarcinoma cells) transduced with
lentiviral vector expressing mouse GM-CSF. The CT26 cells were
spinoculated and cloned as described. Five clones expressing GM-CSF
were selected for a stability study. At weeks 1, 3, 5, 7 and 9, the
cells were assayed for GM-CSF expression by ELISA. The results
shown in FIG. 10 show that 5 clones exhibited stable GM-CSF
expression over the course of the study.
[0218] Both the pancreatic and CT26 GM-CSF expression experiments
demonstrate that lentivector generate clones are stable in
long-term culture.
Example 10
Stable Long-Term Expression Of Lentivectors In CHO-S Cells
[0219] Lentiviral vectors expressing human DC101-IgG1 or KDR-IgG4
from a CAG promoter were prepared. The lentivector construct
contained a 33 amino acid sequence from Foot and Mouth Disease
Virus (FMDV) designated as 2A14 (SEQ ID NO: 9) and an optimized
furin sequence RKRR (SEQ ID NO: 15), referred to herein as the
"Lenti-DC101 vector". Suspension adapted, serum-free CHO-S cells or
suspension adapted NSO cells were transfected with the lentivector
construct. The DC101 antibody coding sequence was modified so that
the C terminal contains a methionine that can be cleaved for Mass
Spec analysis.
[0220] CHO-S cells were serially infected with 200 ng P24 of the
Lenti-DC101 vector at 2 to 3 day intervals. The populations were
assayed for DC101 expression by ELISA (Table 1), and the data
demonstrate that a steady increase in antibody production resulted
following each infection, with no impact on the doubling times.
TABLE-US-00007 TABLE 7 DC101 Expression from CHO-S Cells Infected
with Lenti-DC101-furin/2A14 by ELISA. # of infections Doubling time
(hrs) pg/cell/day 1x 18 7 2x 18 9 3x 19 19 4x 19 18 5x 17 16 6x 18
22 7x 19 24
[0221] Populations of cell that were infected 4.times. and
7.times., respectively, were subcloned and analyzed for DC101
production. When compared, the clones derived from the population
infected 7.times. generally had higher levels of DC101 expression
than the clones from the population infected 4.times.. (Table
2).
TABLE-US-00008 TABLE 8 DC101 expression by CHO-S cells infected 4x
or 7x with Lenti-DC101-furin/2A14. Range of pg/cell/day 4X
population 7X population 1 1 0 10 18 4 20 19 13 30 5 12 40 3 7 50 0
3 60 0 3 70 0 3 80 0 1 90 0 0 100 0 0 110 0 0 120 0 1 Total Clones
46 47
[0222] A panel of clones isolated from the 2', 3.times. and
4.times. infected populations were subjected to continuous culture
in 10 ml shaker flasks for 12 weeks and evaluated at one week
intervals for stability of DC101 production. The clones were found
to be stable (Table 3).
TABLE-US-00009 TABLE 9 Long Term DC101 expression by CHO-S cells
infected with Lenti-DC101-furin/2A14. Clones: pg/cell/day weeks
4x-4 4x-3 4x-2 4x-1 3x-3 2x-8 2x-5 1 61.8 71.7 45.6 34.9 22.4 28.7
23.3 2 76.2 59.3 51.0 34.8 24.1 25.6 23.2 3 89.3 79.0 101.2 66.5
41.9 37.8 45.4 4 67.2 73.6 58.0 35.9 43.7 65.4 62.0 5 61.8 75.7
67.9 21.1 23.8 24.8 16.2 6 99.5 62.6 49.8 47.4 30.4 30.8 23.3 7
58.3 57.4 56.9 45.5 24.4 24.4 23.5 8 63.4 44.2 54.7 23.9 35.3 21.2
22.2 9 62.0 36.9 57.4 23.2 20.7 27.6 22.7 10 64.2 42.7 57.3 30.0
20.8 18.6 23.5 11 54.6 48.1 38.3 24.6 22.9 22.9 28.0 12 55.7 52.4
31.4 26.2 24.5 27.8 33.9
[0223] the doubling times stabilized at less than 20 hours (Table
4).
TABLE-US-00010 TABLE 10 Doubling Time of DC101 expressing CHO-S
cells infected with Lenti-DC101-furin/2A14. Clones: Doubling time
(hrs) weeks 4x-4 4x-3 4x-2 4x-1 3x-3 2x-8 2x-5 1 17.1 25.5 16.2
15.5 16.6 16.2 14.0 2 21.2 30.1 17.7 16.7 17.1 16.7 16.2 3 21.2
35.7 21.2 21.2 18.1 18.1 18.7 4 21.2 29.1 21.2 16.2 22.4 29.4 22.9
5 17.1 19.4 16.2 15.5 16.4 15.9 15.2 6 18.1 19.4 15.8 16.2 15.9
15.9 14.9 7 20.2 19.4 16.6 18.1 16.2 15.5 14.9 8 17.2 18.1 16.6
15.9 17.8 15.2 15.9 9 18.1 17.2 17.1 16.2 16.4 15.2 15.5 10 16.6
17.1 17.2 14.9 16.0 15.1 15.3 11 19.7 16.6 16.2 15.7 15.6 15.5 15.9
12 19.1 18.1 15.9 16.2 16.2 15.9 14.7
[0224] Clones 4.times.-3 and 4.times.-4 were analyzed for genetic
stability by Southern blot. DNA was extracted from both clones at
weeks 1 and 7, digested with restriction enzymes EcoR I or Nsi I
overnight, run on a 1% agarose/TBE gel and probed with a 592 by
fragment from the HC region. The standard is a serial dilution of
EcoR I-digested DC101 plasmid in a background of 10 mg of genomic
CHO-S DNA. The EcoR I releases a 2.2 kb fragment in both clones,
and Clone 4.times.-4 appears to have several partial integrants.
With both restriction enzymes the two clones have stable
integration patterns from 1 to 7 weeks, demonstrating genetic
stability.
[0225] Binding and neutralization assays were done to establish
that antibodies from these clones were biologically equivalent to
antibody produced from a hybridoma. In the binding assays 96-well
plates were coated with recombinant Flk-1, incubated with serial
dilutions of supernatants from clone 7.times.-24, the DC101
hybridoma, or a control, and quantified with anti-rat IgG1 HRP. The
results show that the antibodies bind equivalently (Table 5).
TABLE-US-00011 TABLE 11 Binding of DC101 expressed from CHO-S cells
infected with Lenti- DC101-furin/2A14 relative to DC101 Produced by
a hybridoma. ng/ml Mock CHO-S Hybridoma 1000 0.152 1.144 1.121 333
0.149 0.993 0.894 111 0.152 0.616 0.724 37 0.121 0.486 0.585 12
0.120 0.412 0.403 4 0.104 0.274 0.312 0 0.118 0.129 0.117
[0226] A neutralization assay was carried out using supernatants
from Clone 7.times.-24, a DC101 hybridona and a negative control.
Each supernatant was serially diluted and mixed with Flk-1, then
incubated on 96-well plate pre-coated with human VEGF and read with
anti-Flk-1-HRP. The antibodies generated from the 7.times.-24 clone
neutralized the Flk-1 binding at a level equivalent to the
hybridoma-produced DC 101 antibody (Table 6).
TABLE-US-00012 TABLE 12 Neutralization Assay of DC101 antibody
produced by clone 7X-24 versus hybridoma-produced DC101. ng/ml Mock
CHO-S Hybridoma 1000 1.278 0.184 0.193 333 1.28 0.237 0.357 111
1.203 0.43 0.635 37 1.195 0.826 0.834 12 1.134 0.945 0.931 4 1.152
1.043 0.992 0 1.089 1.069 1.105
[0227] The clones were tested for replication competent lentivirus
by a TaqMan assay for VSV.G sequences. To establish the sensitivity
of the assay, a cell line known to contain 1 copy of VSV-G envelope
was spiked into a background of CHO cells ranging from 10-10,000
cells. DNA samples were prepared and assayed for the presence of
VSV.G sequences. The sensitivity of this assay allows for detection
of one VSV.G sequence in a background of 10,000 cells. The three
clones and the CHO-S control cells were all negative for
replication-competent lentiviral sequences.
TABLE-US-00013 TABLE 13 Sequences for Cell 164.1. (SEQ ID) SEQUENCE
NO: 1 LLNFDLLKLAGDVESNPGPSEQUENCE NO: 2 TLNFDLLKLAGDVESNPGP NO: 3
LLKLAGDVESNPGP NO: 4 NFDLLKLAGDVESNPGP NO: 5 QLLNFDLLKLAGDVESNPGP
NO: 6 APVKQTLNFDLLKLAGDVESNPGP NO: 7
VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT LNFDLLKLAGDVESNPGPSEQUENCE
NO: 8 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 9
EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 10 Furin consensus sequence
or site RXK(R)R NO: 11 Factor Xa cleavage sequence or site: IE(D)GR
NO: 12 Signal peptidase I cleavage sequence or site: e.g.,
LAGFATVAQA NO: 13 Thrombin cleavage sequence or site: LVPRGS NO: 14
Furin site-RKKR NO: 15 Furin site-RKRR NO: 16 Furin site-RRKR NO:
17 Furin site-RRRR
Sequence CWU 1
1
19119PRTFoot-and-mouth disease virus 1Leu Leu Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15Pro Gly
Pro219PRTFoot-and-mouth disease virus 2Thr Leu Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15Pro Gly
Pro314PRTFoot-and-mouth disease virus 3Leu Leu Lys Leu Ala Gly Asp
Val Glu Ser Asn Pro Gly Pro 1 5 10417PRTFoot-and-mouth disease
virus 4Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro
Gly 1 5 10 15Pro520PRTFoot-and-mouth disease virus 5Gln Leu Leu Asn
Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15Asn Pro
Gly Pro 20624PRTFoot-and-mouth disease virus 6Ala Pro Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 1 5 10 15Asp Val Glu
Ser Asn Pro Gly Pro 20758PRTFoot-and-mouth disease virus 7Val Thr
Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro 1 5 10
15Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys
20 25 30Ile Val Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys
Leu 35 40 45Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 50
55840PRTFoot-and-mouth disease virus 8Leu Leu Ala Ile His Pro Thr
Glu Ala Arg His Lys Gln Lys Ile Val 1 5 10 15Ala Pro Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 20 25 30Asp Val Glu Ser
Asn Pro Gly Pro 35 40933PRTFoot-and-mouth disease virus 9Glu Ala
Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu 1 5 10
15Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly
20 25 30Pro105PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Arg Xaa Lys Arg Arg 1
5115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Ile Glu Asp Gly Arg 1 51210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Leu
Ala Gly Phe Ala Thr Val Ala Gln Ala 1 5 10136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Leu
Val Pro Arg Gly Ser 1 5144PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Arg Lys Lys Arg
1154PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Arg Lys Arg Arg 1164PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Arg
Arg Lys Arg 1174PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Arg Arg Arg Arg 1184PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Arg
Ala Lys Arg 1195PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 19Arg Xaa Arg Lys Arg 1 5
* * * * *