Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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STREPTAVIDIN EXPRESSED GENE FUSIONS AND
METHODS OF USE THEREOF
TECHNICAL FIELD
The present invention relates generally to streptavidin expressed gene
fusion constructs, and more particularly, to genomic streptavidin expressed
gene
fusions and methods of using these constructs in diagnostic and therapeutic
applications.
BACKGROUND OF THE INVENTION
Streptavidin ("SA") is a 159 amino acid protein produced by
Streptomyces avidinii, and which specifically binds water-soluble biotin
(Chaiet et al.,
Arch. Biochem. Biophys. 106:1-5, 1964). Streptavidin is a nearly neutral
64,000 dalton
tetrameric protein. Accordingly, it consists of four identical subunits each
having an
approximate molecular mass of 16,000 daltons (Sano and Cantor, Proc. Natl.
Acad. Sci.
USA 87:142-146, 1990). Streptavidin shares some common characteristics with
avidin,
such as molecular weight, subunit composition, and capacity to bind biotin
with high
affinity (KD ~10-'S) (Green, Adv. Prot. Chem. 29:85-133, 1975). Further, while
streptavidin and avidin differ in their amino acid compositions, both have an
unusually
high content of threonine and tryptophan. In addition, streptavidin differs
from avidin
in that it is much more specific for biotin at physiological pH, likely due to
the absence
of carbohydrates on streptavidin. Various comparative properties and isolation
of
avidin and streptavidin are described by Green et al., Methods in Enzymology
184:51-
67, 1990 and Bayer et al., Methods in Enzymology 184:80-89, 1990.
The streptavidin gene has been cloned and expressed in E. coli (Sano
and Cantor, Proc. Natl. Acad. Sci. USA 87(1):142-146, 1990; Agarana, et al.,
Nucleic
Acids Res. 14(4):1871-1882, 1986). Fusion constructs of streptavidin, and
truncated
forms thereof, with various proteins, including single-chain antibodies, have
also been
expressed in E. coli (Sano and Cantor, Biotechnology (NY) 9(12):1378-1381,
1991;
Sano and Cantor, Biochem. Biophys. Res. Commun. 176(2):571-577, 1991; Sano, et
al.,
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Proc. Natl. Acad. Sci. USA 89(5):1534-1538, 1992; Walsh and Swaisgood,
Biotech.
Bioeng. 44:1348-1354, 1994; Le, et al., Enzyme Microb. Technol. 16(6):496-500,
1994;
Dubel, et al., J. Immunol. Methods 178(2):201-209, 1995; Kipriyanov, et al.,
Hum.
Antibodies Hybridomas 6(3):93-101, 1995; Kipriyanov, et al., Protein Eng.
9(2):203-
211, 1996; Ohno, et al., Biochem. Mol. Med. 58(2):227-233, 1996; Ohno and
Meruelo,
DNA Cell Biol. 15(5):401-406, 1996; Pearce, et al. Biochem. Mol. Biol. Int.
42(6):1179-
1188, 1997; Koo, et al., Applied Environ. Microbiol. 64(7):2497-2502, 1998)
and in
other organisms (Karp, et al., Biotechniques 20(3):452-459, 1996). Sano and
Cantor
(PNAS, supra) found that expression of full-length forms of streptavidin was
lethal to
E. coli host cells and, when capable of being expressed in truncated forms
(e.g., under a
T7 promoter system), only poor and varied expression was observed and the
protein
remained in inclusion bodies. However, there are also published reports of the
expression of soluble streptavidin in E. coli (Gallizia et al., Protein Expr.
Purif.
14(2):192-196, 1998; Veiko et al., Bioorg. Khim. 25(3):184-188, 1999). Those
of skill
in the art have frequently used "core streptavidin" (residues 14-136), or
similar
truncated forms, in the preparation of fusion constructs. The basis of the use
of core
residues 14-136 has been the observation that streptavidin preparations
purified from
the culture medium of S. avidinii have usually undergone proteolysis at both
the N- and
C-termini to produce this core structure, or functional forms thereof
(Argarana et al.,
supra).
Presently, preparations of streptavidin expressed gene fusions are
usually made by expressing a core streptavidin-containing construct in
bacteria,
wherein inclusion bodies are formed. Such production has several
disadvantages,
including the rigor and expense of purifying from inclusion bodies, the
necessity of
using harsh denaturing agents such as guanidine hydrochloride, and the
difficulty in
scaling up in an economical fashion. To a lesser extent, there has also been
reported
periplasmic expression of core streptavidin-containing constructs in soluble
form
(Dubel, et al., supra).
Therefore, there exists a need in the art for easy, cost effective, and
scaleable methods for the production of streptavidin fusion proteins.
Accordingly, the
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present invention provides several key advantages. For example, in one
embodiment, a
genomic streptavidin expressed gene fusion is expressed as a soluble protein
into the
periplasmic space of bacteria and undergoes spontaneous folding. Accordingly,
such
expression offers the advantage that the periplasm is a low biotin environment
and one
need not purify and refold the protein under harsh denaturing conditions that
may prove
fatal to the polypeptide encoded by a heterologous nucleic acid molecule fused
to the
genomic streptavidin nucleic acid molecule. The present invention fulfills
this need,
while further providing other related advantages.
SUMMARY OF THE INVENTION
The present invention generally provides expression cassettes and fusion
constructs encoded thereby comprising genomic streptavidin. In one aspect the
present
invention provides a vector construct for the expression of streptavidin
fusion proteins,
comprising a first nucleic acid sequence encoding at least 129 amino acids of
streptavidin (Figure 4), or a functional variant thereof, a promoter
operatively linked to
the first nucleic acid sequence, and a cloning site for, or with, insertion of
a second
nucleic acid sequence encoding a polypeptide to be fused with streptavidin,
interposed
between the promoter and the first nucleic acid sequence. Alternatively, the
second
nucleic acid may encode the streptavidin portion of the construct and the
first nucleic
acid encodes a polypeptide to be fused with streptavidin.
In certain embodiments, the promoter is inducible or constitutive. In
other embodiments, the first nucleic acid sequence encodes at least amino
acids 14 to
150, 14 to 151, 14 to 152, 14 to 153, 14 to 154, 14 to 155, 14 to 156, 14 to
157, or 14 to
158 of streptavidin, Figure 4. In yet other embodiments, the first nucleic
acid sequence
encodes at least amino acids 5 to 150-158 of Figure 4 or 1 to 150-158 of
Figure 4.
Host cells containing genomic streptavidin expression cassettes are also
provided as are fusion proteins expressed by the same. In certain embodiments
fusion
proteins comprising single chain antibodies are provided. In yet other
embodiments the
single chain antibodies are directed to a cell surface antigen. In yet other
embodiments
the single chain antibodies are directed to cell surface antigens, or cell-
associated
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stromal or matrix antigens, including, but not limited to, CD20, CD22, CD45,
CD52,
CD56, CD57, EGP40 (or EPCAM or KSA), NCAM, CEA, TAG-72, mucins (MUC-1
through MUC-7), (3-HCG, EGF receptor, IL-2 receptor, her2/neu, Lewis Y, GD2,
GM2,
tenascin, sialylated tenascin, somatostatin, activated tumor stromal antigen,
or
neoangiogenic antigens.
In other aspects of the present invention, methods for targeting a tumor
cell are provided, comprising the administration of a fusion protein, said
fusion protein
comprising at least a first and a second polypeptide joined end to end,
wherein said first
polypeptide comprises at least 129 amino acids of streptavidin (Figure 4), or
conservatively substituted variants thereof, wherein said second polypeptide
is a
polypeptide which binds a cell surface protein on a tumor cell, wherein the
fusion
protein binds the cell surface protein on a tumor cell and wherein the
streptavidin
portion of the fusion protein is capable of binding biotin. In certain
embodiments, the
second polypeptide is an antibody or antigen-binding fragment thereof.
In other aspects of the present invention, pharmaceutical compositions,
comprising genomic streptavidin fusion constructs are provided.
These and other aspects of the present invention will become evident
upon reference to the following detailed description and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of a heterologous protein-genomic
streptavidin expressed gene construct.
Figure 2 is a schematic representation of a single chain antibody-
genomic streptavidin fusion construct.
Figure 3 is a schematic representation of the pEX94B expression vector
containing a single chain antibody(huNR-LU-10)-genomic streptavidin fusion
construct.
Figure 4 is the sequence of genomic streptavidin (SEQ ID NO: 1)
including the signal sequence and predicted amino acid sequence (SEQ ID NO:
2).
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Figure 5 is a schematic representation of the construction of the
pKKlac/pelB vector.
Figure 6 is a schematic representation of the construction of the pEX-1
vector.
5 Figure 7 is a schematic representation of the construction of the pEX-
SA318 and pEX-scFv3.2.1 vectors.
Figure 8 is a schematic representation of the construction of the pEX94B
vector.
Figure 9 is a schematic representation of the construction of the pEX94B
neo vector.
Figure 10 represents the determined nucleic acid sequence (SEQ ID NO:
3) and predicted amino acid sequence (SEQ ID NO: 4) for the huNR-LU-10 single
chain antibody-genomic streptavidin fusion. The streptavidin regulatory
region, signal
sequence, and coding sequence are noted as are the various linkers and light
and heavy
chains of the single chain antibody.
Figures 11A and 11B are the determined nucleic acid (SEQ ID NO: 5)
and predicted amino acid sequences (SEQ ID NO: 6) of a B9E9 scFvSA fusion
construct, with the pKOD linker between VL and VH. Linkers are boxed and the
orientation is VL linker-VH-linker-Streptavidin.
Figure 11 C is an expression cassette comprising the nucleic acid
sequences (SEQ ID NO: 7) and predicted amino acid sequences (SEQ ID NO: 8) of
a
B9E9 scFvSA fusion construct encoding VH linker-VL linker-Streptavidin.
Figure 12 is a scanned image representing SDS-PAGE analysis of
huNR-LU-10 scFvSA.
Figure 13 is graphic representation of size exclusion HPLC analysis of
huNR-LU-10 scFvSA.
Figure 14 is a plot illustrating a competitive immunoreactivity assay of
huNR-LU-10 scFvSA (97-20.0 and 98-01.0) as compared to huNR-LU-10 mAb.
Figure 15 is a plot illustrating the rate of dissociation of DOTA-biotin
from huNR-LU-10 scFvSA (97-13.0) as compared to recombinant streptavidin (r-
SA).
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Figure 16 is a graph illustrating biodistribution of pretargeted huNR-LU-
scFvSA.
Figure 17 is a graph depicting blood clearance and tumor uptake of
huNR-LU-10 scFvSA versus a chemically conjugated form (mAb/SA).
5 Figure 18 is a bar graph illustrating biodistribution of pretargeted B9E9
scFvSA.
Figure 19 is a scanned image of SDS-PAGE analysis of scFvSA fusion
protein expression in the presence and absence of FkpA.
10 DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention, it may be helpful to an understanding
thereof to set forth definitions of certain terms that will be used
hereinafter.
"Core streptavidin," as used herein, refers to a streptavidin molecule
consisting of the central amino acid residues 14-136 of streptavidin of Figure
4 and also
of Figure 3 of U.S. Patent No. 4,839,293 and deposited at ATCC number X03591.
"Genomic streptavidin," as used herein, refers to a sequence comprising
at least 129 residues of the sequence set forth in Figure 4. Accordingly,
genomic
streptavidin refers to streptavidin molecules that have N-terminal, C-
terminal, or both
N- and C-terminal extensions of core streptavidin. The N- and C-terminal
extensions
may comprise any number of amino acids selected from 1 to 13, 137 to 159, and
in
some cases -1 to -24 of Figure 4.
The genomic streptavidin molecules of the subject invention also include
variants (including alleles) of the native protein sequence. Briefly, such
variants may
result from natural polymorphisms or may be synthesized by recombinant DNA
methodology, and differ from wild-type protein by one or more amino acid
substitutions, insertions, deletions, or the like. Variants generally have at
least 75%
nucleotide identity to native sequence, preferably at least 80%-85%, and most
preferably at least 90% nucleotide identity. Typically, when engineered, amino
acid
substitutions will be conservative, i.e., substitution of amino acids within
groups of
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polar, non-polar, aromatic, charged, etc. amino acids. With respect to
homology to the
native sequence, variants should preferably have at least 90% amino acid
sequence
identity, and within certain embodiments, greater than 92%, 95%, or 97%
identity.
Such amino acid sequence identity may be determined by standard methodologies,
including use of the National Center for Biotechnology Information BLAST
search
methodology available at www.ncbi.nlm.nih.gov. using default parameters. The
identity methodologies most preferred are those described in U.S. Patent
5,691,179 and
Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.
As will be appreciated by those skilled in the art, a nucleotide sequence
and the encoded genomic streptavidin or variant thereof may differ from known
native
sequence, due to codon degeneracies, nucleotide polymorphisms, or amino acid
differences. In certain embodiments, variants will preferably hybridize to the
native
nucleotide sequence at conditions of normal stringency, which is approximately
25-
30°C below Tm of the native duplex (e.g., SX SSPE, 0.5% SDS, SX
Denhardt's
solution, SO% formamide, at 42°C or equivalent conditions; see
generally, Sambrook et
al., Molecular Cloning.' A Laboratory Manual, 2nd ed., Cold Spring Harbor
Press,
1989; Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing,
1995). By way of comparison, low stringency hybridizations utilize conditions
approximately 40°C below Tm, and high stringency hybridizations utilize
conditions
approximately 10°C below Tm.
A "polypeptide," as used herein, refers to a series of amino acids of five
or more.
An "isolated nucleic acid molecule" refers to a polynucleotide molecule
in the form of a separate fragment or as a component of a larger nucleic acid
construct,
that has been separated from its source cell (including the chromosome it
normally
resides in) at least once, and preferably in a substantially pure form.
Nucleic acid
molecules may be comprised of a wide variety of nucleotides, including DNA,
RNA,
nucleotide analogues, or combination thereof.
The term "heterologous nucleic acid sequence", as used herein, refers to
at least one structural gene operably associated with a regulatory sequence
such as a
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promoter. The nucleic acid sequence originates in a foreign species, or, in
the same
species if substantially modified from its original form. For example, the
term
"heterologous nucleic acid sequence" includes a nucleic acid originating in
the same
species, where such sequence is operably associated with a promoter that
differs from
the natural or wild-type promoter.
An "antibody," as used herein, includes both polyclonal and monoclonal
antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate;
and
chimeric; and may be an intact molecule, a fragment thereof (such as scFv, Fv,
Fd, Fab,
Fab' and F(ab)'2 fragments), or multimers or aggregates of intact molecules
and/or
fragments; and may occur in nature or be produced, e.g., by immunization,
synthesis or
genetic engineering; an "antibody fragment," as used herein, refers to
fragments,
derived from or related to an antibody, which bind antigen and which ' in some
embodiments may be derivatized to exhibit structural features that facilitate
clearance
and uptake, e.g., by the incorporation of galactose residues. This includes,
e.g., F(ab),
F(ab)'2, scFv, light chain variable region (VL), heavy chain variable region
(VH), and
combinations thereof.
The term "protein," as used herein, includes proteins, polypeptides and
peptides; and may be an intact molecule, a fragment thereof, or multimers or
aggregates
of intact molecules and/or fragments; and may occur in nature or be produced,
e.g., by
synthesis (including chemical and/or enzymatic) or genetic engineering.
A. Streptavidin Genes and Gene Products
1. Streptavidin Nucleic Acid Molecules and Variants Thereof
The present invention provides streptavidin fusion constructs that
include streptavidin nucleic acid molecules of various lengths, which, in
certain
embodiments, are constructed from full-length genomic streptavidin nucleic
acid
molecules available in the art and specifically described in U.S. Patent Nos.
4,839,293;
5,272,254, and ATCC Accession number X03591.
Variants of streptavidin nucleic acid molecules, provided herein, may be
engineered from natural variants (e.g., polymorphisms, splice variants,
mutants),
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synthesized or constructed. Many methods have been developed for generating
mutants
(see, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press, 1989, and Ausubel, et al. Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley-Interscience, New York, 1995).
Briefly, preferred methods for generating nucleotide substitutions utilize an
oligonucleotide that spans the base or bases to be mutated and contains the
mutated
base or bases. The oligonucleotide is hybridized to complementary single
stranded
nucleic acid and second strand synthesis is primed from the oligonucleotide.
The
double-stranded nucleic acid is prepared for transformation into host cells,
typically E.
coli, but alternatively, other prokaryotes, yeast or other eukaryotes.
Standard methods
of screening and isolation and sequencing of DNA were used to identify mutant
sequences.
Similarly, deletions andlor insertions of the streptavidin nucleic acid
molecule may be constructed by any of a variety of known methods as discussed,
supra. For example, the nucleic acid molecule can be digested with restriction
enzymes
and religated, thereby deleting or religating a sequence with additional
sequences (e.g.,
linkers), such that an insertion or large substitution is made. Other means of
generating
variant sequences may be employed using methods known in the art, for example
those
described in Sambrook et al., supra; Ausubel et al., supra. Verification of
variant
sequences is typically accomplished by restriction enzyme mapping, sequence
analysis,
or probe hybridization. In certain aspects, variants of streptavidin nucleic
acid
molecules whose encoded product is capable of binding biotin, are useful in
the context
of the subject invention. In other aspects, the ability of the variant
streptavidin to bind
biotin may be increased, decreased or substantially similar to that of native
streptavidin.
In yet other embodiments, the ability to bind biotin is not required, provided
that the
variant form retains the ability to self assemble into a typical tetrameric
structure
similar to that of native streptavidin. Such tetrameric structures have a
variety of uses
such as the formation of tetravalent antibodies when fused to sequences
encoding an
antibody or fragment thereof.
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2. Genomic Streptavidin and Expression Cassettes Containing the Same
A genomic streptavidin fusion construct expression cassette of the
present invention may be generated by utilizing the full gene sequence of the
streptavidin gene, or variant thereof. In certain embodiments, the expression
cassette
5 contains a nucleic acid sequence encoding at least 129 contiguous amino
acids of
Figure 4 or functional variants thereof. In various other embodiments, the
nucleic acid
sequence encodes at least amino acid residues 14 to 140 of Figure 4. In a
further
embodiment, the nucleic acid sequence encodes at least amino acids 14 to 150,
14 to
151, 14 to 152, 14 to 153, 14 to 154, 14 to 155, 14 to 156, 14 to 157, 14 to
158, or 14 to
10 159 of streptavidin, Figure 4. In yet other embodiments, the nucleic acid
sequence
encodes at least amino acids 10 to 150-158 of Figure 4, or 5 to 150-158 of
Figure 4 or 1
to 150-158 of Figure 4. In yet other embodiments, the nucleic acid sequence
encodes at
least amino acid residues 1 to 159 of Figure 4. In still yet other
embodiments, the
expression cassette comprises a nucleic acid sequence that encodes at least 10
amino
1 S acids of residues -1 to -24 of Figure 4.
As noted above, the genomic streptavidin encoding nucleic acid
molecules of the subject invention may be constructed from available
streptavidin
sequences by a variety of methods known in the art. A preferred method is
amplification (e.g., polymerase chain reaction (PCR)) to selectively amplify
the
individual regions and place these in cloning vectors such as pCR2.1
(Invitrogen).
Moreover, such PCR reactions can be performed in a variety of ways such that
the
primers used for amplification contain specific restriction endonuclease sites
to
facilitate insertion into a vector.
Further, a variety of other methodologies besides PCR may be used to
attain the desired construct. For example, one skilled in the art may employ
isothermal
methods to amplify the nucleotide sequence of interest, using existing
restriction
endonuclease sites present in the nucleotide sequence to excise and insert
sequences, or
by the introduction of distinct restriction endonuclease sites by site-
directed
mutagenesis followed by excision and insertion. These and other methods are
described in Sambrook et al., supra; Ausubel, et al., supra. Briefly, one
methodology
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is to generate single-stranded streptavidin encoding DNA, followed by
annealing a
primer, which is complementary except for the desired alteration (e.g., a
small
insertion, deletion, or mutation such that a unique restriction site is
created between the
domains). Bacterial cells are transformed and screened for those cells which
contain
the desired construct. This construct is then digested to liberate the desired
sequences,
which can then be purified and relegated into the appropriate orientation.
One of skill in the art would recognize that the absolute length of the
genomic streptavidin is only a secondary consideration when designing an
expression
cassette, as compared to utilizing a form which is capable of binding biotin
and capable
of expressing into the periplasmic space of a bacterial host. Such constructs
can be
readily tested for their ability to bind biotin and maintain solubility in the
periplasmic
space by assays known in the art and those described herein. Accordingly,
experiments
such as, measuring biotin binding capacity and biotin dissociation rate are
well known
in the art and applicable in this regard. Briefly, such constructs can be
tested for their
ability to bind biotin by a variety of means, including labeling the fusion
protein with a
subsaturating level of radiolabeled biotin, then adding a 100-fold saturating
level of
biocytin to initiate dissociation. The free radiolabeled biotin is measured at
timed
intervals.
B. Vectors, Host Cells and Methods of Expressing and Producing Protein
The expression cassette of the present invention need not necessarily
contain a promoter, but upon insertion into a vector system the sequence
contained
within the cassette must be capable of being expressed once associated with a
promoter
or other regulatory sequences. In one embodiment, the expression cassette
itself
comprises a promoter. Further, the cassette preferably contains a cloning site
for the
insertion of a heterologous nucleic acid sequence to be fused/linked to the
genomic
streptavidin encoding sequence. One exemplary cassette is set forth in Figure
1.
However, it should be noted that the cloning site need not be 5' of the
genomic
streptavidin sequence, but could be placed 3' of the streptavidin sequence.
Thus, an
encoded fusion protein could contain the genomic streptavidin polypeptide
either N- or
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C- terminal to the encoded polypeptide fused thereto. Further, while it should
be noted
that a variety of other nucleic acid sequences can be linked to the genomic
streptavidin
encoding sequence, in one embodiment the sequence encodes an antibody
fragment,
and in certain embodiments a single chain antibody (scFv).
In addition to a cloning site, the cassette may include a linker molecule.
Linker molecules are typically utilized within the context of fusion proteins
and are
well known in the art. As exemplified in Figure 2, linkers are typically
utilized to
separate the genomic streptavidin sequence from the other sequences linked
thereto and
to separate the VH and the VL of the scFv. The linking sequence can encode a
short
peptide or can encode a longer polypeptide. Preferable linker sequences encode
at least
two amino acids, but may encode as many amino acids as desired as long as
functional
activity is retained. In the various embodiments, the linker sequence encodes
5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 35
amino acids. In
certain embodiments an encoded linker may be a standard linker such as
(Gly4Ser)x
where x may be any integer, but is preferably 1 to 10. The length and
composition can
be empirically determined to give the optimum expression and biochemical
characteristics. For example, the composition of the linker can be changed to
raise or
lower the isoelectric point of the molecule. Additionally, one of ordinary
skill in the art
will appreciate that the length of linker between variable light and heavy
chains need be
at least long enough to facilitate association between the two domains, while
the linker
between streptavidin and the antibody fragment may vary from zero amino acids
to 100
or more as long as functionality is maintained. Accordingly, the linker
between the
light and heavy chain is typically greater than five amino acids, and
preferably greater
than ten, and more preferably greater than fifteen amino acids in length.
The expression cassette may be used in a vector to direct expression in a
variety of host organisms. In certain embodiments, the genomic streptavidin
expressed
gene fusion is produced in bacteria, such as E. coli, or mammalian cells
(e.g., CHO and
COS-7), for which many expression vectors have been developed and are
available.
Other suitable host organisms include other bacterial species, and eukaryotes,
such as
yeast (e.g., Saccharomyces cerevisiae), plants, and insect cells (e.g., Sf~).
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In one embodiment, a DNA sequence encoding a genomic streptavidin
fusion protein is introduced into an expression vector appropriate for the
host cell. As
discussed above, the sequence may contain alternative codons for each amino
acid with
multiple codons. The alternative codons can be chosen as "optimal" for the
host
species. Restriction sites are typically incorporated into the primer
sequences and are
chosen with regard to the cloning site of the vector. If necessary,
translational initiation
and termination codons can be engineered into the primer sequences.
At a minimum, the vector will contain a promoter sequence. As used
herein, a "promoter" refers to a nucleotide sequence that contains elements
that direct
the transcription of a linked gene. At a minimum, a promoter contains an RNA
polymerase binding site. More typically, in eukaryotes, promoter sequences
contain
binding sites for other transcriptional factors that control the rate and
timing of gene
expression. Such sites include TATA box, CART box, POU box, AP 1 binding site,
and the like. Promoter regions may also contain enhancer elements. When a
promoter
is linked to a gene so as to enable transcription of the gene, it is
"operatively linked."
The expression vectors used herein include a promoter designed for
expression of the proteins in a host cell (e.g., bacterial). Suitable
promoters are widely
available and are well known in the art. Inducible or constitutive promoters
are
preferred. Such promoters for expression in bacteria include promoters from
the T7
phage and other phages, such as T3, T5, and SP6, and the trp, lpp, and lac
operons.
Hybrid promoters (see, U.S. Patent No. 4,551,433), such as tac and trc, may
also be
used. Promoters for expression in eukaryotic cells include the P10 or
polyhedron gene
promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Patent
Nos.
5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE
promoter, RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Patent
No. 4,870,009), ecdysone response element system, tetracycline-reversible
silencing
system (tet-on, tet-off), and the like.
The promoter controlling transcription of the genomic streptavidin
fusion construct may itself be controlled by a repressor. In some systems, the
promoter
can be derepressed by altering the physiological conditions of the cell, for
example, by
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the addition of a molecule that competitively binds the repressor, or by
altering the
temperature of the growth media. Preferred repressor proteins include, the E.
coli IacI
repressor responsive to IPTG induction, the temperature sensitive ~,cI857
repressor, and
the like.
Other regulatory sequences may be included. Such sequences include a
transcription termination sequence, secretion signal sequence (e.g.,
nucleotides 480-551
of Figure 2B of U.S. Patent No. 5,272,254), ribosome binding sites, origin of
replication, selectable marker, and the like. The regulatory sequences are
operationally
associated with one another to allow transcription, translation, or to
facilitate secretion.
The regulatory sequences of the present invention also include the upstream
region of
the streptavidin gene as described in U.S. Patent No. 5,272,254 (e.g., nucleic
acid
residues 174-551 depicted in Figures 2A-2B of U.S. Pat. No. 5,272,254).
Accordingly,
an upstream sequence of 100 to 300 base pairs may be utilized in expression
constructs
to facilitate secretion and/or expression. Such an upstream untranslated
region is
depicted in U.S. Patent No. 5,272,254 Figures 2A and 2B as nucleotides 174-
479. In
preferred embodiments nucleic acid residues 408-479 of those described above
are
utilized in the expression construct.
In other optional embodiments, the vector also includes a transcription
termination sequence. A "transcription terminator region" has either a
sequence that
provides a signal that terminates transcription by the polymerase that
recognizes the
selected promoter and/or a signal sequence for polyadenylation.
In one aspect, the vector is capable of replication in the host cells. Thus,
when the host cell is a bacterium, the vector preferably contains a bacterial
origin of
replication. Bacterial origins of replication include the fl-on and col El
origins of
replication, especially the on derived from pUC plasmids. In yeast, ARS or CEN
sequences can be used to assure replication. A well-used system in mammalian
cells is
SV40 ori.
The plasmids also preferably include at least one selectable marker that
is functional in the host. A selectable marker gene includes any gene that
confers a
phenotype on the host that allows transformed cells to be identified and
selectively
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grown. Suitable selectable marker genes for bacterial hosts include the
ampicillin
resistance gene (Amps, tetracycline resistance gene (Tc~ and the kanamycin
resistance
gene (Kan~. The ampicillin resistance and kanamycin resistance genes are
presently
preferred. Suitable markers for eukaryotes usually require a complementary
deficiency
5 in the host (e.g., thymidine kinase (tk) in tk- hosts). However, drug
markers are also
available (e.g., G418 resistance and hygromycin resistance).
The nucleotide sequence encoding the genomic streptavidin fusion
protein may also include a secretion signal (e.g., a portion of the leader
sequence, the
leader sequence being the upstream region of a gene including a portion of a
secretion
10 signal), whereby the resulting peptide is a precursor protein processed and
secreted.
The resulting processed protein may be recovered from the periplasmic space or
the
fermentation medium. Secretion signals suitable for use are widely available
and are
well known in the art (von Heijne, J. Mol. Biol. 184:99-105, 1985; von Heijne,
Eur. J.
Biochem. 133:17-21, 1983). Prokaryotic and eukaryotic secretion signals that
are
15 functional in E. coli (or other host) may be employed. The presently
preferred secretion
signals include, but are not limited to, those encoded by the following
bacterial genes:
streptavidin, pelB (Lei et al., J. Bacteriol. 169:4379, 1987), phoA, ompA,
ompT,
ompF, ompC, beta-lactamase, and alkaline phosphatase.
Other components which increase expression may also be included
either within the vector directing expression of the streptavidin fusion or on
a separate
vector. Such components include, for example, bacterial chaperone proteins
such as
SicA, GroEL, GroE, DnaK, CesT, SecB, FkpA, SkpA, etc.
One skilled in the art will appreciate that there are a wide variety of
vectors which are suitable for expression in bacterial cells and which are
readily
obtainable. Vectors such as the pET series (Novagen, Madison, Wisconsin), the
tac and
trc series (Pharmacia, Uppsala, Sweden), pTTQl8 (Amersham International plc,
England), pACYC 177, pGEX series, and the like are suitable for expression of
a
genomic streptavidin fusion protein. The choice of a host for the expression
of a
genomic streptavidin fusion protein is dictated in part by the vector.
Commercially
available vectors are paired with suitable hosts.
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16
A wide variety of suitable vectors for expression in eukaryotic cells are
also available. Such vectors include pCMVLacI, pXTl (Stratagene Cloning
Systems,
La Jolla, California); pCDNA series, pREP series, pEBVHis, pDisplay
(Invitrogen,
Carlsbad, California). In certain embodiments, the genomic streptavidin fusion
protein
encoding nucleic acid molecule is cloned into a gene targeting vector, such as
pMClneo, a pOG series vector (Stratagene Cloning Systems).
As noted above, preferred host cells include, by way of example,
bacteria such as Escherichia coli; mammalian cells such as Chinese Hamster
Ovary
(CHO) cells, COS cells, myeloma cells; yeast cells such as Saccharomyces
cerevisiae;
insect cells such as Spodoptera frugiperda; plant cells such as maize, among
other host
cells.
Insect cells are capable of high expression of recombinant proteins. In
this regard, baculovirus vectors, such as pBlueBac (see, e.g., U.S. Patent
Nos.
5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051 and
5,169,784;
available from Invitrogen, San Diego, California) may be used for expression
in insect
cells, such as Spodoptera frugiperda Sf~ cells (see, U.S. Patent
No.4,745,051).
Expression in insect cells or insects is preferably effected using a
recombinant
baculovirus vector capable of expressing heterologous proteins under the
transcriptional
control of a baculovirus polyhedrin promoter. (e.g., U.S. Patent No. 4,745,051
relating
to baculovirus/insect cell expression system). Polyhedrin is a highly
expressed protein,
therefore its promoter provides for efficient heterologous protein production.
The
preferred baculovirus is Autographa californica (ACMNPV). Suitable baculovirus
vectors are commercially available from Invitrogen.
Also, the fusion construct of the present invention may be expressed in
transgenic animals. For example, the genomic streptavidin containing
expression
cassette may be operatively linked to a promoter that is specifically
activated in
mammary tissue such as a milk-specific promoter. Such methods are described in
U.S.
Patent No. 4,873,316 and U.S. Patent No. 5,304,498.
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17
The genomic streptavidin gene fusion may also be expressed in plants,
e.g., transgenic plants, plant tissues, plant seeds and plant cells. Such
methods are
described, e.g., in U.S. Patent No. 5,202,422.
Regardless of the particular system chosen, the design of systems
suitable for expression of recombinant proteins is well known and within the
purview
of one of ordinary skill in the art, as evidenced by the above-identified
references
relating to expression of recombinant fusion proteins.
Accordingly, as is evidenced by the text and examples herein, expression
of fusion proteins within the context of a genomic streptavidin expressed gene
fusion
construct provides several key advantages. For example, in one embodiment, the
genomic streptavidin fusion protein is expressed as soluble protein into the
periplasmic
space of bacteria and undergoes spontaneous folding. Accordingly, such
expression
offers the advantage that the periplasm is a low biotin, oxidizing environment
and
produces a soluble, functional molecule. This avoids having to purify and
refold the
protein under harsh denaturing conditions, which may prove fatal to the
polypeptide
encoded by the heterologous nucleic acid molecule.
The genomic streptavidin expressed gene fusion may be isolated by a
variety of methods known to those skilled in the art. However, preferably the
purification method takes advantage of the presence of a functional
streptavidin
molecule, by utilizing its high affinity binding to aid in purification.
Accordingly,
preferred purification methods are by the use of iminobiotin immobilized on a
solid
surface.
C. ANTIBODIES AS FUSION COMPONENTS
While a broad variety of genomic streptavidin expressed gene fusion
molecules may be designed by the methods described herein, a particularly
useful
fusion protein is that of an antibody and genomic streptavidin, in particular
an
antibody-genomic streptavidin expressed gene fusion (Ab-SA). In preferred
embodiments the expression construct encodes an Fv or scFv portion of an
antibody. In
a further preferred embodiment the construct encodes a Fab fragment or
functional
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18
derivative thereof, to which streptavidin may be linked via a terminus of
either the
heavy chain portion or light chain portion of the molecule. Accordingly, DNA
encoding the Fv regions of interest may be prepared by any suitable method,
including,
for example, amplification techniques such as polymerise chain reaction from
cDNA of
a hybridoma, using degenerate oligonucleotides, ligase chain reaction (LCR)
(see Wu
and Wallace, Genomics, 4:560, 1989, Landegren et al., Science, 241:1077, 1988
and
Barnnger et al., Gene, 89:117, 1990), transcription-based amplification (see
Kwoh et
al., Proc. Natl. Acid. Sci. USA, 86:1173, 1989), and self sustained sequence
replication
(see Guatelli et al., Proc. Natl. Acid. Sci. USA, 87:1874, 1990), cloning and
restriction
of appropriate sequences or direct chemical synthesis by methods such as the
phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the
phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862,
1981;
and the solid support method of U.S. Patent No. 4,458,066, as well as U.S.
Patent Nos.
5,608,039 and 5,840,300, as well as PCT Application No. WO 98/41641. DNA
encoding regions of interest, for example, Fab or scFv, may also be isolated
from phage
display libraries.
One of ordinary skill in the art would readily recognize that given the
disclosure provided herein, any number of binding pair members may be utilized
and
thus would not be limited to streptavidin/biotin binding. In this regard,
antibody/epitope pairs or any ligand/anti-ligand pair may be utilized. One of
ordinary
skill in the art would also appreciate that the present disclosure provides a
general
method for the preparation of tetravalent antibodies. Since the avidity of an
antibody
for its cognate antigen is generally a function of its valency, there are many
applications
in which a tetravalent antibody would be preferable to a divalent antibody.
Such
applications include, but are not limited to, immunoassays, immunotherapy,
immunoaffinity chromatography, etc.
Chemical synthesis may also be utilized to produce a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization
with a complementary sequence, or by polymerization with a DNA polymerise
using
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19
the single strand as a template. While it is possible to chemically synthesize
an entire
single chain Fv region, it is preferable to synthesize a number of shorter
sequences
(about 100 to 150 bases) that are later ligated together.
Alternatively, subsequences may be cloned and the appropriate
subsequences cleaved using appropriate restriction enzymes. The fragments may
then
be ligated to produce the desired DNA sequence.
Once the variable light (VL) and heavy chain (VH) DNA is obtained, the
sequences may be ligated together, either directly or through a DNA sequence
encoding
a peptide linker, using techniques well known to those of skill in the art. In
a preferred
embodiment, heavy and light chain regions are connected by a flexible
polypeptide
linker (e.g., (Gly4Ser)X, or the pKOD sequence, or others, provided, infra)
which starts
at the carboxyl end of the light chain Fv domain and ends at the amino
terminus of the
heavy chain Fv domain, or vice versa, as the order of the Fv domains can be
either
light-heavy or heavy-light. The entire sequence encodes the Fv domain in the
form of a
single-chain antigen binding protein.
A variety of methods exist for the recombinant expression of
immunoglobulins, the following references are representative of methods and
host
systems suitable for expression of recombinant immunoglobulins and fusion
proteins in
general: Weidle et al., Gene 51:21-29, 1987; Dorai et al., J. Immunol.
13(12):4232-
4241, 1987; De Waele et al., Eur. J. Biochem. 176:287-295, 1988; Colcher et
al.,
Cancer Res. 49:1738-1745, 1989; Wood et al., J. Immunol. 145(a):3011-3016,
1990;
Bulens et al., Eur. J. Biochem. 195:235-242 1991; Beggington et al., Biol.
Technology
10:169, 1992; King et al., Biochem. J. 281:317-323, 1992; Page et al., Biol.
Technology
9:64, 1991; King et al., Biochem. J. 290:723-729, 1993; Chaudary et al.,
Nature
339:394-397, 1989; Jones et al., Nature 321:522-525, 1986; Morrison and Oi,
Adv.
Immunol. 44:65-92, 1988; Benhar et al., Proc. Natl. Acad. Sci. USA 91:12051-
12055,
1994; Singer et al., J. Immunol. 150:2844-2857, 1993; Cooto et al., Hybridoma
13(3):215-219, 1994; Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033,
1989;
Caron et al., Cancer Res. 32:6761-6767, 1992; Dubel et al., J. Immunol.
Methods
178:201-209, 1995; Batra et al., J. Biol. Chem. 265:15198-15202, 1990; Batra
et al.,
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Proc. Natl. Acad. Sci. USA, 86:8545-8549, 1989; Chaudhary et al., Proc. Natl.
Acad.
Sci. USA, 87:1066-1070, 1990, several of which describe the preparation of
various
single chain antibody expressed gene fusions.
Accordingly, once a DNA sequence has been identified that encodes an
S Fv region which when expressed shows specific binding activity, fusion
proteins
comprising that Fv region may be prepared by methods known to one of skill in
the art.
The Fv region may be fused to genomic streptavidin directly in the expression
cassette
of the present invention or, alternatively, may be joined directly to genomic
streptavidin
through a peptide or polypeptide linker, thereby forming a linked product. The
linker
10 may be present simply to provide space between the Fv and the fused genomic
streptavidin or to facilitate mobility between these regions to enable them to
each attain
their optimum conformation. The genomic streptavidin-antibody expression
cassette,
typically, comprises a single vector which provides for the expression of both
heavy
and light variable sequences fused by an appropriate linker as well as a
linker fusing the
15 light and heavy chains with genomic streptavidin, thereby encoding a single
chain
antibody:genomic streptavidin (scFvSA) conjugate. In one embodiment the linker
connecting the variable light and heavy chains is of sufficient length or side
group
selection to allow for flexibility. In one embodiment the linker is a standard
linker such
as (Gly4Ser)x, described supra, while in another embodiment the linker is the
pKOD
20 linker (GlyLeuGluGlySerProGluAlaGlyLeuSerProAspAlaGlySerGlySer) (SEQ ID NO:
9). It should be understood that a variety of linkers may be used, but in some
embodiments it may be preferred that the linker separating the light and heavy
antibody
chains should allow flexibility and the linker attaching the scFv to the
genomic
streptavidin sequence can be fairly rigid or fairly flexible. Further, in
addition to
linkers, additional amino acids may be encoded by the addition of restriction
sites to
facilitate linker insertion and related recombinant DNA manipulation, as such
these
amino acids while not necessarily intended to be linkers may or may not be
included
within the constructs described herein, depending on the construction method
utilized.
Exemplary linkers are known by those of skill in the art. For example,
Fv portions of the heavy and light chain of antibodies held together by a
polypeptide
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21
linker can have the same binding properties as their full length two chain
counterparts
(Bird et al., Science, 242:423-26, 1988 and Huston et al., Proc. Natl. Acad.
Sci. USA,
85:5879-83, 1988). It has also been shown that, in some cases, fusion proteins
composed of single chain antibodies linked to toxins may retain the binding
capacity of
the single chain antibody as well as the activity of the toxin (Chaudary et
al., Nature,
339: 394-97, 1989; Batra et al., J. Biol. Chem., 265: 15198-15202, 1990; Batra
et al.,
Proc. Natl. Acad. Sci. USA 86: 8545-8549, 1989; Chaudary et al., Proc. Natl.
Acad.
Sci. USA 87:1066-1070, 1990). Exemplary fusion constructs containing
streptavidin
are described by Sheldon et al., Appl. Radiat. Isot. 43(11):1399-1402, 1992;
Sano and
Cantor, BiolTechnology 9:1378-1381, 1991; Spooner et al., Human Pathology
25(6):606-614, 1994; Dubel et al., J. Immun. Methods 178:201-209, 1995;
Kipriyanov et al., Protein Engineering 9(2):203-21 l, 1996. The DNA sequence
comprising the linker may also provide sequences, such as primer sites or
restriction
sites, to facilitate cloning or may preserve the reading frame between the
sequence
encoding the scFv and the sequence encoding genomic streptavidin. The design
of such
linkers is well known to those of skill in the art.
Further, one skilled in the art would find it routine to test the ability of
genomic streptavidin-antibody expressed gene fusions to bind the appropriate
ligand.
In contemplated embodiments, this ligand antigen may be a cell surface
antigen, cell-
associated stromal or matrix antigen, or cell-secreted antigens, including,
but not
limited to, CD19, CD20, CD22, CD25, CD33, CD45, CD52, CD56, CD57, EGP40 (or
EPCAM or KSA), NCAM, CEA, TAG-72, a mucin (MUC-1 through MUC-7), (3-HCG,
EGF receptor and variants thereof, IL-2 receptor, her2/neu, Lewis y, GD2, GM2,
Lewis
x, folate receptor, fibroblast activation protein, tenascin, sialylated
tenascin,
somatostatin, activated tumor stromal antigen, or a neoangiogenic antigen.
Moreover,
methods for evaluating the ability of antibodies to bind to epitopes of such
antigens are
known.
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22
D. APPLICABLE USES OF GENOMIC STREPTAVIDIN EXPRESSED
FUSION CONSTRUCTS
While any heterologous nucleic acid sequence can be joined to that
encoding genomic streptavidin and expressed, as described herein, particularly
useful
expressed fusion constructs are those comprising scFv linked to genomic
streptavidin,
referred to previously as scFvSA. Accordingly, in one aspect of the invention,
scFv
fragments of antibodies are useful as tools in methods for medical diagnostic
and
therapeutic purposes. A diagnostic or therapeutic method, is described for
detecting the
presence or absence of, or treating, a target site within a mammalian host.
When
determining the criteria for employing antibodies or antibody conjugates for
in vivo
administration for therapeutic purposes, it is desirable that the generally
attainable
targeting ratio is high and that the absolute dose of therapeutic agent
delivered to the
tumor is sufficient to elicit a significant tumor response. Methods for
utilizing such
antibodies described in the present invention can be found, for example, in
U.S. Patent
Nos. 4,877,868, 5,175,343, 5,213,787, 5,120,526, and 5,200,169. Upon in vivo
administration for therapeutic or diagnostic purposes, it is also desirable to
limit the
exposure of non-target tissues to the therapeutic agent.
One method for reducing non-target tissue exposure to a diagnostic or
therapeutic agent involves "pretargeting" the targeting moiety (e.g., scFvSA)
to a target
site, and then subsequently administering a rapidly clearing diagnostic or
therapeutic
agent conjugate that is capable of binding to the "pretargeted" targeting
moiety at the
target site. In this method, as generally described, an optional intermediate
step may
involve administration of a clearing agent to aid in the efficient removal of
the unbound
targeting moiety conjugate from the circulation prior to administration of the
active
agent conjugate. A description of embodiments of the pretargeting technique,
including
the descriptions of various clearing agents and chelates, such as DOTA, may be
found
in U.S. Patent Nos. 4,863,713, 5,578,287, 5,608,060, 5,616,690, 5,630,996,
5,624,896;
and PCT publication Nos. WO 93/25240, WO 95/15978, WO 97/46098, WO 97/46099,
which are incorporated herein in their entirety.
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23
In the pretargeting approach the pharmacokinetics of the active agent is
decoupled from that of the targeting moiety. The targeting moiety, conjugated
to a
member of a ligand/anti-ligand pair, is permitted to accrete to target sites.
Accordingly,
in one embodiment of the present invention, scFvSA is a conjugate (fusion) of
the
targeting moiety (scFv) and ligand (streptavidin). After accretion occurs and
a
substantial fraction of the non-target associated conjugate is cleared from
the recipient's
circulation, either by intrinsic clearance or via administration of a ligand
or anti-ligand
containing clearing agent, the active agent is administered as a conjugate to
the member
of the ligand/anti-ligand pair that is complementary to that of the targeting
conjugate
(e.g., biotin would be the complementary member in the exemplified
embodiment).
Preferably, the agent-ligand or agent-anti-ligand has a short serum half life
and is
excreted via the renal pathway. In this manner, the therapeutic agent either
accretes to
the target site where exertion of its therapeutic or diagnostic capability is
desired, or it
is rapidly removed from the recipient. This distribution of active agent
facilitates the
protection of normal tissues of the recipient from undesired toxicity. To
enhance renal
excretion, conjugation to a renal excretion-promoting biodistribution-
directing
molecule, may be employed. Essentially, such pretargeting methods are
characterized
by an improved targeting ratio or increased absolute dose to the target cell
sites in
comparison to conventional cancer diagnosis or therapy.
In one embodiment of the pretarget methodology, the targeting moiety
will comprise an antibody fusion of the present invention specific for a
particular
antigen associated with the target cells of interest. In related embodiments
the antigen
marker may be associated with a cancer, including, but not limited to, the
following:
lymphoma (e.g., CD20); leukemia (e.g., CD45); prostate (e.g., TAG-72); ovarian
(e.g.,
TAG-72); breast (e.g., MUC-1); colon (e.g., CEA); and pancreatic (e.g., TAG-
72). For
example, the CD20 antigen may be targeted for the treatment of lymphoma
wherein the
ligand/anti-ligand binding pair may be biotin/avidin (e.g., streptavidin-gene
fusion
(scFvSA)), and the active agent will be a radionuclide in pretargeting
methods. Further,
a variety of antigens may be targeted, such as CD45 antigen targeting for
pretargeted
radioimmunotherapy (PRIT) to treat patients having any one of a broad range of
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24
hematologic malignancies by employing antibody-mediated targeting to the CD45
antigen. CD45 is the most broadly expressed of the known hematopoietic
antigens,
found on essentially all white blood cells and their precursors, including
neutrophils,
monocytes and macrophages, all lymphocytes, myeloid and lymphoid precursors,
and
about 90% of acute myelogenous leukemia (AML) cells. Accordingly, as the
antigens
available for targeting for diagnostic or therapeutic purposes are numerous,
the present
invention may be used to facilitate targeting to any of these antigens.
An optional step in pretarget methods, including those identified above,
comprises the initial administration of a non-conjugated targeting moiety (i.
e., not
conjugated to a ligand or anti-ligand) or, alternatively, administering this
non-
conjugated targeting moiety concurrently with the conjugated form in the first
step, thus
blocking those targets contacted initially. Such blocking may be especially
useful, for
example, in the treatment of non-Hodgkin's lymphoma, where the first set of
targeted
tissues may be the spleen, while most tumors are found in the deep lymph
nodes. Such
1 S pre-blocking allows for substantial protection of the spleen cells from
later treatment
with the active agent. While the non-conjugated targeting agent need not
necessarily
bind the same epitope, to be effective it should preclude binding by the
targeting moiety
conjugate.
One skilled in the art could use multiple targeting moiety conjugates
comprising different antibodies that also bind to the same cell type to
enhance the
therapeutic effect or diagnostic utility. U.S. Patent No. 4,867,962 issued to
Abrams
describes such an improved method for delivering active agent to target sites,
which
method employs active agent-targeting moiety conjugates. Briefly, the Abrams
method
contemplates administration to a recipient two or more active agent-targeting
moiety
conjugates, wherein each conjugate includes a different antibody species as
the
targeting moiety. Each of the utilized antibody species is reactive with a
different target
site epitope (associated with the same, or a different, target site antigen),
while the
patterns of cross-reactivity of the antibody species with non-target tissues
are non-
overlapping. In this manner the different antibodies accumulate additively at
the desired
target site, while fewer than the total species accumulate at any type of non-
target
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
tissue. A higher percentage of the administered agent, therefore, becomes
localized in
vivo at target sites than at non-target tissues. The present invention
encompasses
approaches similar to this, as well as in pretargeting formats. In one
embodiment, for
example, two or more species of targeting conjugates (fusion) with antibodies
directed
5 to different epitopes and having non-overlapping cross-reactivity, each
prepared
according to the present invention, are administered according to the
pretarget method
so as to improve the diagnostic or therapeutic utility. A further embodiment
utilizes the
property that streptavidin monomers naturally associate to form tetramers.
Thus, two or
more antibodies each conjugated (fusion) to the monomeric form of
streptavidin, are
10 selectively combined and, upon formation of tetrameric streptavidin, yield
single
species with specificity for multiple epitopes at the target site.
It should be understood that the methods described may be modified and
still achieve the desired effect. For example, two antibodies specific for the
same
antigen or cell type, regardless of their respective cross-reactivity, may be
used. All that
15 is necessary for these methods is that the targeting moiety-ligand/anti-
ligand conjugate
preferentially binds the target cells and that the active agent-conjugate
substantially
localizes to the pretargeted cells and is otherwise substantially cleared from
circulation.
Alternatively, antibody-based or non-antibody-based targeting moieties
may be employed to deliver a ligand/anti-ligand to a target site bearing an
unregulated
20 antigen. Preferably, a natural binding agent for such an unregulated
antigen is used for
this purpose.
Pretarget methods as described herein optionally include the
administration of a clearing agent. The dosage of the clearing agent is an
amount which
is sufficient to substantially clear the previously administered targeting
moiety-
25 ligand/anti-ligand conjugate from the circulation. Generally, the
determination of when
to administer the clearing agent depends on the target uptake and endogenous
clearance
of the targeting moiety conjugate. Particularly preferred clearing agents are
those which
provide for Ashwell receptor-mediated clearance, such as galactosylated
proteins, e.g.,
galactosylated biotinylated human serum albumin, and small molecule clearing
agents
containing N-acetylgalactosamine and biotin.
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26
Types of active agents (diagnostic or therapeutic) useful herein include
radionuclides, toxins, anti-tumor agents, drugs, genes, and cytokines. For
example, as
described above, conjugates of such agents to biotin may be useful in the
pretargeting
approach. In this regard, a therapeutic antibody (e.g., an antibody that
induces
apoptosis or inhibits angiogenesis) may be used in a therapeutic modality such
as
pretargeting. With regard to diagnostic agent fusions, in contrast to
therapeutic agent
fusions, enhanced target cell internalization is disadvantageous if one
administers
diagnostic agent-targeting moiety conjugates. Internalization of diagnostic
conjugates
results in cellular catabolism and degradation of the conjugate. Upon
degradation,
small adducts of the diagnostic agent or the diagnostic agent per se may be
released
from the cell, thus eliminating the ability to detect the conjugate in a
target-specific
manner.
Diagnostic or therapeutic agents useful herein include radionuclides,
drugs, anti-tumor agents, toxins, genes, and cytokines. Radionuclides useful
within the
present invention include gamma-emitters, positron-emitters, Auger electron-
emitters,
X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters
preferred for
therapeutic use. Radionuclides are well-known in the art and include '23I,
'ZSI, l3oh 131h
133I' l3sl' 47SC' 72AS' 72Sre' 90Y' 88Y' 97Ru' 100Pd' lolm~' 119sb' 128Ba'
197Hg' 211At' 212Bi,
153Sm' 169Eu' 212Pb' 109Pd' 111In' 67Ga' 68Ga' 64Cu' 67Cu' 75Br' 76Br' 77Br'
99mTC' 11C' 13N' 15p
'66Ho and'sF. Preferred therapeutic radionuclides include'ssRe, 's6Re,
Z°3Pb, z'zBi, Z'3Bi,
lo9Pd~ 64Cu' 67Cu' 90Y' 125I' 131I' 77Br' 211At' 97Ru' IOSn7 ' l9sAu and'99Ag,
166H0 or'77Lu.
As one of ordinary skill in the art can readily appreciate the above
streptavidin gene fusions may be utilized in combination therapies, such as
when
"pretargeting" is combined with the use of radiation-sensitizing agents. Such
radiation
sensitizing agents include, but are not limited to, gemcitabine, 5-
fluorouracil, paclitaxel,
and the like.
Several of the potent toxins useful within the present invention consist
of an A and a B chain. The A chain is the cytotoxic portion and the B chain is
the
receptor-binding portion of the intact toxin molecule (holotoxin). Because
toxin B
chain may mediate non-target cell binding, it is often advantageous to
conjugate only
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the toxin A chain to a targeting moiety (e.g., molecule). However, while
elimination of
the toxin B chain decreases non-specific cytotoxicity, it also generally leads
to
decreased potency of the conjugated toxin A chain, as compared to the
conjugate of the
corresponding holotoxin.
Preferred toxins in this regard include holotoxins, such as abrin, ricin,
modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussis toxin, Shiga
toxin, and
bryototoxin; and A chain or "A chain-like" molecules, such as ricin A chain,
abrin A
chain, modeccin A chain, the enzymatic portion of Pseudomonas exotoxin A,
Diphtheria toxin A chain, the enzymatic portion of pertussis toxin, the
enzymatic
portion of Shiga toxin, gelonin, pokeweed antiviral protein, saporin, tritin,
barley toxin
and snake venom peptides. Ribosomal inactivating proteins (RIPS), naturally
occurring
protein synthesis inhibitors that lack translocating and cell-binding ability,
are also
suitable for use herein. Highly toxic toxins, such as palytoxin and the like,
are also
contemplated for use in the practice of the present invention. However,
therapeutic
drugs may themselves facilitate internalization of the complex.
Therapeutic drugs, administered as targeted conjugates, are also
encompassed herein. Again, the goal is administration of the highest possible
concentration of drug (to maximize exposure of target tissue), while remaining
below
the threshold of unacceptable normal organ toxicity (due to non-target tissue
exposure).
Unlike radioisotopes, however, therapeutic drugs need to be taken into a
target cell to
exert a cytotoxic effect. In the case of targeting moiety-therapeutic drug
conjugates, it
would be advantageous to combine the relative target specificity of a
targeting moiety
with a means for enhanced target cell internalization of the targeting moiety-
drug
conj ugate.
Therapeutic drugs suitable for use herein include conventional chemo-
therapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-
fluorouracil,
6-thioguanine, cytarabine, cyclophosphamide and cis-platinum, as well as other
conventional chemotherapeutics including those described in Cancer: Principles
and
Practice of Oncology, 2d ed., V.T. DeVita, Jr., S. Hellman, S.A. Rosenberg,
J.B.
Lippincott Co., Philadelphia, Pennsylvania, 1985, Chapter 14, and analogues of
such
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drugs where the analogue has greater potency that the parent molecule. Another
drug
within the present invention is a trichothecene. Other preferred drugs
suitable for use
herein as a diagnostic or therapeutic active agent in the practice of the
present invention
include experimental drugs including those as described in NCIInvestigational
Drugs,
Pharmaceutical Data 1987, NIH Publication No. 88-2141, Revised November 1987.
Other anti-tumor agents, e.g., agents active against proliferating cells,
are administerable in accordance with the present invention. Exemplary anti-
tumor
agents include pro-apoptotic antibodies, anti-angiogenic antibodies,
cytokines, such as
IL-2, tumor necrosis factor or the like, lectin inflammatory response
promoters
(selectins), such as L-selectin, E-selectin, P-selectin or the like, and
similar molecules.
One skilled in the art, based on the teachings in this application and the
applications referenced herein, can readily determine an effective diagnostic
or
therapeutic dosage and treatment protocol. This will depend upon factors such
as the
particular selected therapeutic or diagnostic agent, the route of delivery,
the type of
target site(s), affinity of the targeting moiety for the target site of
interest, any cross-
reactivity of the targeting moiety with normal tissue, condition of the
patient, whether
the treatment is effected alone or in combination with other treatments, among
other
factors. A therapeutic effective dosage is one that treats a patient by
extending the
survival time of the patient. Preferably, the therapy further treats the
patient by arresting
the tumor growth and, most preferably, the therapy further eradicates the
tumor.
All the references, including patents and patent applications, discussed
throughout, are hereby incorporated by reference in their entirety.
The present invention is further described through presentation of the
following examples. These examples are offered by way of illustration and not
by way
of limitation.
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EXAMPLES
EXAMPLE I
CONSTRUCTION OF HUNR-LU-lO SINGLE CHAIN ANTIBODY-GENOMIC STREPTAVIDIN
S FUSION
Generically, a single chain Fv/streptavidin (scFvSA) fusion protein is
expressed from the genetic fusion of the single chain antibody of the variable
regions
(scFv) to the genomic streptavidin of Streptomyces avidinii. The scFv gene
consists of
the variable regions of the light (VL) and heavy (VH) chains separated by a
DNA linker
sequence (e.g., Figure 2). The streptavidin coding sequence is joined to the
3' terminus
of the scFv gene, and the two genes are separated in-frame by a second DNA
linker
sequence. The signal sequence from the streptavidin gene is fused at the 5'
terminus of
the scFvSA gene to direct expression to the E. coli periplasmic space. The
scFvSA
gene is under control of the lac promoter, and the expressed fusion protein is
extracted
and purified from E. coli and forms a soluble tetramer of about 172,000
molecular
weight.
Plasmid pKK233-2 (Amersham Pharmacia Biotech, Piscataway, NJ)
was digested with BamHI and Ncol to remove the trc promoter. The lac promoter
was
amplified from pBR322 by polymerise chain reaction (PCR) and cloned into the
BamHllNcol site of pKK233-2. In the process an EcoRI site was introduced
immediately 5' to the Ncol site. The plasmid was digested with Ncol and Pstl
and
ligated with oligonucleotides encoding the pelB leader sequence. The accepting
Ncol
site on the plasmid was not regenerated and a new Ncol site was introduced in
the 3'
area of the pelB encoding sequence. The resulting plasmid was referred to as
pKK-
lac/pelB (Figure 5). pKK-lac/pelB and pUCl8 were digested with Pvul and PvuII.
The
2.9 kb fragment of pKK-lac/pelB containing the lac promoter and mufti-cloning
site
was ligated to the 1.4 kb fragment of pUCl8 containing the origin of
replication to
form plasmid pEX-1 (Figure 6).
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The streptavidin and huNR-LU-10 scFv genes (a monoclonal antibody
that binds the antigen EGP40 or EPCAM, epithelial glycoprotein, 40 kD) were
cloned
onto separate plasmids prior to construction of the huNR-LU-10 scFvSA gene.
The
streptavidin gene, signal sequence and approximately 300 by of upstream
sequence
5 were PCR-amplified from Streptomyces avidinii (ATCC 27419) genomic DNA and
cloned into pEX-1 as an EcoRllHindIII fragment to form pEX318 (Figure 7). The
huNR-LU-10 scFv was derived from the humanized antibody plasmid pNRX451
(Graves et al., Clin. Cancer Res., 5:899-908, 1999). The heavy and light chain
variable
regions were PCR-amplified separately from pNRX451 and then combined in a
10 subsequent PCR. Oligonucleotides used in this process were designed to
introduce a
(Gly4Ser)3 linker between the leading VL and the trailing VH. The resulting
PCR
product was cloned into pEX-1 as a NcollHindIll fragment forming the plasmid
pEX-
scFv3.2.1 (Figure 7). The scFv and streptavidin genes were PCR-amplified from
pEX-
scFv3.2.1 and pEX318, respectively, and combined into a fusion, as illustrated
in
15 Figure 8. The oligonucleotides used in these reactions created an overlap
between the
3' end of the leading scFv and the S' end of the trailing streptavidin, which
encoded a
five amino acid linker (GSGSA). The fragments were joined by PCR using the
outside
primers. The resulting 1.25 kb fragment was cloned into the Ndel and BamHI
sites of
vector pET3a (Novagen), generating pET3a-41B. This plasmid was digested with
Xhol
20 and Hindlll, and the 1.3 kb fragment containing the VH-SA coding region and
transcription terminator was ligated to a 4.6 kb XhollHindlll fragment of pEX
scFv3.2.1 containing the VL coding region, lac promoter, and ampicillin
resistance gene
(pYL256). The streptavidin regulatory region and signal sequence were PCR-
amplified
from pEX318 and cloned into the EcoRIlNcoI sites of pYL256 to form pEX94B
(Figure
25 8).
The Tn5 kanamycin resistance gene (neo) was inserted into the huNR-
LU-10 scFvSA expression plasmid pEX94B as follows (Figure 9): plasmid pNEO
(Amersham Pharmacia) was digested with BamHI, blunt-ended with nucleotides
using
Pfu polymerase (Stratagene, La Jolla, CA), then further digested with HindIII.
The
30 1494 by fragment containing the kanamycin resistance gene was ligated to
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HindIIIlScal digested pEX94B plasmid, generating plasmid pEX94Bneo. The DNA
sequence of the 1.6 kb EcoRI to BamHl fragment of plasmids pEX94B and
pEX94Bneo
is shown in Figure 10.
EXAMPLE II
CONSTRUCTION OF B9E9 SCFVSA FUSIONS
Additional single chain antibodies containing genomic streptavidin were
constructed in a similar manner as noted above. A scFvSA version of the anti-
CD20
mAb, B9E9, was constructed in the VLVH orientation with either a (Gly4Ser)3
(SEQ ID
NO: 10)linker or a linker termed pKOD (amino acids GLEGSPEAGLSPDAGSGS)
(SEQ ID NO: 9). Briefly, B9E9-1D3 hybridoma cells (1 X 10')(from Bioprobe BV,
Amstelveen, The Netherlands) were harvested, and total RNA was prepared. The
cDNAs for kappa chain and heavy chain of B9E9 were obtained by a reverse
transcriptase reaction using primers RX207 and RX215, respectively. PCR
fragments
of variable regions of kappa chain and heavy chain were obtained using above
cDNAs
and pairs of oligos (RX207 and NX54 for kappa chain; RX215 and NX50 for heavy
chain). The PCR fragments were digested with EcoRl and Notl and subsequently
cloned into a pPICaA vector (Invitrogen, Sorrento Valley, CA), previously
restricted
with EcoRl and Notl. The resultant plasmids C58-1 and C58-16 carried B9E9
kappa
chain and heavy chain, respectively. The two chains were further cloned out
from C58-
1 and C58-16 by PCR using pairs of oligos (RX468 and RX469 for kappa chain;
RX470 and RX471 for heavy chain). The kappa chain fragment was digested with
Ncol and Bglll and the heavy chain was digested with Xhol Sacl, respectively.
The
kappa chain was cloned into pEX94B (Ncol Bglll) as vector and heavy chain was
cloned at Xhol Sacl sites in pEX94B. The resultant plasmids (C74-2 for kappa
chain
and C76-10 for heavy chain) were digested with Xhol and HindIII. The small
fragment
from C76-10 was ligated into C74-2 vector restricted with the same enzymes. A
resultant plasmid (C87-14) carried B9E9 scFvSA fusion protein with a (GQS)3
(SEQ ID
NO: 10) linker between kappa chain and heavy chain. The C87-14 was further
digested
with BgIII and Xhol and ligated with a pKOD linker prepared with two oligos
(pInewS'
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and pInew3') to generate C136-1. Figures 11A and 11B illustrate the determined
nucleic acid sequence and predicted amino acid sequence of B9E9pKOD scFvSA.
Another version of B9E9 scFvSA was constructed in the VHVL orientation with
an extended 25mer (Gly4Ser)5 (SEQ ID NO: 11) linker. The Ncol Sacl fragment of
C87-14 containing scFv was further subcloned by PCR using a pair of primers
(RX633
and RX471) to add a serine residue in the VL region. The PCR fragment was
digested
with Ncol and Sacl and cloned into the pEX94B vector restricted with Ncol and
Sacl.
The resultant plasmid D59-3 was subject to subcloning to generate the VH or VL
fragments by PCR using RX781 and RX782 or RX729 and RX780, respectively. The
VH PCR fragment was digested with Ncol and Bglll and cloned into the pEX94B
vector
at the same sites to form D142-6. The VL PCR fragment was digested with Xhol
and
Sacl and cloned into the pEX94B vector at the same sites to form D142-1. A
Xhol
Hindlll fragment from D142-1 was isolated and replaced a Xhol HindIII fragment
of
D142-6 to generate D148-1 (VH-VL scFvSA). A HindIIl BamHl fragment, (blunted
at
BamHl side) containing a neo gene as described previously, was used to replace
a
HindIIl Scal fragment of D148-1 to form D164-13. The D148-1 was also digested
with
BgIII and Xhol to remove the linker fragment and ligated with a 25mer linker
(annealed
with RX838 and RX839) to form ES-2-6. A EcoRI Hindlll fragment of ES-2-6
containing VH-VL scFvSA was excised and ligated with the D164-13 vector
previously
restricted with EcoRI and HindIII to form E31-2-20. Both plasmids ES-2-6
(carbenicillin-resistant) and E31-2-20 (kanamycin-resistant) express the B9E9
scFvSA
fusion protein. Figure 11 C illustrates the nucleic acid sequence and
predicted amino
acid sequence of B9E9 scFvSA (VH VL 25-mer).
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All oligonucleotide primers, as listed below, were synthesized by Operon
'Technologies,
Inc. (Alameda, CA).
NX50 (SEQ ID NO: 12)
S TGCCGTGAATTCGTSMARCTGCAGSARTCWGG
NX54 (SEQ ID NO: 13)
TGCCGTGAATTCCATTSWGCTGACCARTCTC
RX207 (SEQ ID NO: 14)
TAGCTGGCGGCCGCCCTGTTGAAGCTCTTGACAAT
RX215 (SEQ ID NO: 15)
TAGCTGGCGGCCGCTTTCTTGTCCACCTTGGTGC
RX468 (SEQ ID NO: 16)
TTACGGCCATGGCTGACATCGTGCTGCAGTCTCCAGCAATCCTGTCT
RX469 (SEQ ID NO: 17)
CACCAGAGATCTTCAGCTCCAGCTTGGTCCCA
RX470 (SEQ ID NO: 18)
CGGAGGCTCGAGCCAGGTTCAGCTGGTCCAGTCAGGGGCTGAGCTGGTGAA
G
RX471 (SEQ ID NO: 19)
GAGCCAGAGCTCACGGTGACCGTGGTCCCTGCGCCCCA
pInewS' (SEQ ID NO: 20)
GATCTCTGGTCTGGAAGGCAGCCCGGAAGCAGGTCTGTCTCCGGACGCAGG
TTCCGGC
pInew3' (SEQ ID NO: 21 )
TCGAGCCGGAACCTGCGTCCGGAGACAGACCTGCTTCCGGGCTGCCTTCCA
GACCAGA
RX633 (SEQ ID N0:22)
TTACGGCCATGGCTGACATCGTGCTGTCGCAGTCTCCAGCAATCCTGTCT
RX779 (SEQ ID NO: 23)
TTCCGGCTCGAGCGACATCGTGCTGTCGCAGTCTCCA
RX780 (SEQ ID NO: 24)
GAGCCAGAGCTCTTCAGCTCCAGCTTGGTCCC
RX781 (SEQ ID NO: 25)
TTACGGCCATGGCTCAGGTTCAGCTGGTCCAGTCA
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RX782 (SEQ ID NO: 26)
AGACCAGAGATCTTGCTCACGGTGACCGTGGTCCC
RX838 (SEQ ID NO: 27)
GATCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGCTC
GGGTGGTGGTGGGTCGGGCGGCGGCGGC
RX839 (SEQ ID NO: 28)
TCGAGCCGCCGCCGCCCGACCCACCACCACCCGAGCCGCCGCCACCCGACC
CACCACCGCCCGAGCCACCGCCACCAGA
EXAMPLE III
1 S EXPRESSION OF HUNR-LU-1 O scFvSA arrD B9E9 scFvSA PROTEINS
Transformants of E. coli strain XL1-Blue (Stratagene, La Jolla, CA)
containing plasmids pEX94B (huNR-LU-10 scFvSA) or ES-2-6 (B9E9 scFvSA) were
grown overnight at 30°C in Terrific broth (20 ml; Sigma) containing
carbenicillin (50
pg/ml). The culture was diluted 100-fold into fresh medium and grown in a
shaking
incubator at 30°C. When the culture attained an A6oo of 0.3-0.5, IPTG
(Amersham
Pharmacia Biotech, Piscataway, NJ) was added to a final concentration of 0.2
mM, and
incubation was continued overnight. Periplasmic extracts were prepared for
qualitative
analysis of the scFvSA expression level. Cells were resuspended in an ice-cold
solution of 20% sucrose, 2 mM EDTA, 30 mM Tris, (pH 8.0), and lysozyme (2.9
mg/ml) and were incubated on ice for 30 min. Supernatants were analyzed on 4-
20%
Tris-glycine SDS-PAGE gels (Novex) under non-reducing, non-boiled conditions,
and
gels were stained with Coomassie Blue. Expression in shake flasks was
optimized by
testing different environmental parameters, such as IPTG concentration and
timing,
temperature, media, or carbon source, or testing genetic factors, such as
different
promoters or signal sequences.
Clones were further grown in an 8L fennentor and analyzed for
expression level. The primary inoculum (50 ml) was grown overnight at
30°C in shake
flasks containing Ternfic broth plus SO pg/ml kanamycin (plasmids pEX94Bneo or
E31-2-20) or carbenicillin (plasmids pEX94B or ES-2-6), depending on the
selectable
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marker of the plasmid. The culture was then diluted 100-fold into the same
medium
and grown at 30°C for an additional 4-5 h. This secondary inoculum (0.5
liter) was
transferred to a 14 liter BioFlo 3000 fermentor (New Brunswick Scientific)
containing
8 liters of complete E. coli medium [per liter: 6 g NaZHP04, 3 g KHZP04, 0.5 g
NaCI, 3
5 g (NH4)ZS04, 48 g yeast extract (Difco), 0.25 ml Mazu DF204 antifoam (PPG
Industries
Inc., Pittsburgh, PA), 0.79 g MgS04 -7Hz0, 0.044 g CaCl2-2Hz0, and 3 ml of
trace
elements (per liter: 0.23 g CoCl2, 0.57 g H3B03, 0.2 g CuCl2-2Hz0, 3.5 g FeCl3-
6HZ0,
4.0 g MnClz-4H20, 0.5 g ZnCI, 1.35 g thiamine, and 0.5 g NazMo04-2H20)]. The
medium contained an initial 5 g/liter galactose as carbon source plus 50 pg/ml
of
10 kanamycin or carbenicillin for plasmid retention. The culture was grown at
30°C and
induced with IPTG (0.2 mM) at 6 h post-inoculation. The pH was maintained at
7.0 by
the automatic addition of either phosphoric acid or NaOH. Dissolved oxygen
concentration was maintained at or above 30% throughout the run using
agitation
speeds of 400-800 rpm and oxygen supplementation as necessary. A galactose
solution
15 (50%) was fed over a 9 h period after exhaustion of the initial galactose
present in the
medium to a total of 20-25 g per liter. Cells were harvested at 24-26 h post-
inoculation
(for B9E9 scFvSA) or 48-56 h post-inoculation (for huNR-LU-10 scFvSA) in a
continuous flow centrifuge (Pilot Powerfuge, Carr Separations, Franklin, MA),
washed
with PBS (10 mM sodium phosphate, 150 mM NaCI, pH 7.2), and pelleted by
20 centrifugation. A typical fermentation produced 80-90 g of cells (wet wt)
per liter
culture medium.
For determining expression levels, cells were washed twice in PBS,
resuspended to the original volume, and disrupted either by sonication on ice
(Branson
Ultrasonics, Danbury, CT) or through two cycles of microfluidization
(Microfluidics
25 International, Newton, MA). Two assays were used for quantitating fusion
protein in
the supernatent of a centrifuged sample of crude lysate. Initially, an ELISA
assay was
used in which biotinylated albumin (100 ng per well in PBS) was coated
overnight in
96-well plates at 4°C and incubated with serial two-fold dilutions of
either HPLC-
purified fusion protein (200 ng/ml) or test samples. Detection was using
peroxidase-
30 labeled goat anti-streptavidin polyclonal antibody (Zymed, So. San
Francisco, CA) and
ABTS (Sigma) substrate buffer. Plates were read at 415/490 nm with a dual
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wavelength automated plate reader. A first order, log x/log y regression
analysis was
performed for quantitation of the fusion protein.
Alternatively, a rhodamine-biotin HPLC assay was devised that
provided faster results. The fusion protein in centrifuged lysates was
complexed with
excess rhodamine-derivatized biotin, which was prepared as follows: 5-(and-6-)
carboxytetramethylrhodamine, succinimidyl ester (Molecular Probes, Eugene OR)
was
coupled to biocytin (Pierce, Rockford IL) through the formation of a stable
amide bond.
The reaction mixture was purified by HPLC using a Dynamax semi-preparative C-
18
column (Rainin Instrument Co., Woburn, MA). The effluent was monitored at 547
nm
and peak fractions collected and analyzed by mass spectrometry. Fractions
corresponding in molecular weight to biocytin-rhodamine conjugate were pooled
and
concentrated by roto-evaporation (Buchii, Switzerland). An excess of purified
biocytin-rhodamine conjugate was added to the clarified crude lysate and
analyzed by
size exclusion chromatography using a Zorbax GF-250 column (MAC-MOD, Chadds
Ford PA) equilibrated in 20 mM sodium phosphate containing 15% DMSO at 1.0
ml/min flow rate. The effluent was monitored at 547 nm using a Varian Dynamax
PDA-2 detector, and the peak area corresponding to fusion protein elution was
determined using a Varian Dynamax HPLC Data System (Walnut Creek, CA). The
concentration of fusion protein in the crude lysate was calculated by
comparison to a
standard analyzed under the same conditions. The molar extinction coefficient
for the
fusion protein standard was calculated using a previously described method
summing
the relative contributions of amino acids absorbing at 280 nm (Gill and von
Hippel,
Analyt. Chem. 182:319-326, 1989).
Expression levels in fermentor-grown cells were 100-130 mg/liter for huNR
LU-10 scFvSA, 40 mg/liter for B9E9pKOD scFvSA, and 270-300 mg/liter for B9E9
scFvSA (VH-VL 25-mer).
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EXAMPLE IV
EXPRESSION OF B9E9 SCFVSA USING VARIOUS LINKERS AND SIGNAL SEQUENCES
A number of genetic variants were constructed that contained linkers of
different lengths and composition and the variable regions in different order
(Table 1).
These constructs were initially grown and induced in shake flask cultures and
qualitatively assessed for expression by visualizing periplasmic proteins on
Coomassie-
stained, non-reducing, SDS gels. High-expressing constructs were further
tested in an
8L fermentor using a galactose fed-batch protocol, and their expression levels
were
quantitatively determined by size exclusion HPLC using rhodamine-derivatized
biotin.
The construct that best fulfilled these criteria contained a 25-mer Gly4Ser
linker with
the scFv in the VHVL orientation.
Table 1. Summary of expression levels of B9E9 scFvSA genetic variants.
° Linker sequences (L1):
15 mer G4S: GGGGSGGGGSGGGGS (SEQ ID NO: 10)
18 mer G4S: GGGGSGGGGSGGGGSGGS (SEQ ID NO: 29)
mer G4S: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ m NO: 11)
mer G4S: 7 copies of GGGGS (SEQ ID NO: 30)
18 mer pKOD: GLEGSPEAGLSPDAGSGS (SEQ ID NO: 9)
25 18 mer pKOD2: GLEGSPEAGLSPDAGSDS (SEQ ID NO: 31)
b The expression levels of the fusion proteins were based on 8L fermentor runs
except for those
qualitative data, designated as plus symbols, based on SDS-PAGE analysis of
shake-flask cultures.
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EXAMPLE V
INCREASED EXPRESSION OF SCFV SA FUSION PROTEINS IN PERIPLASM OF E. COLI
The E. coli fkpA gene is a member of the family of FK506-binding
S proteins (FKBPs) and is one of the periplasmic components involved in
protein folding.
It is expressed in the E. coli periplasm and has peptidyl-prolyl isomerase
(PPIase)
activity. The PPIase-independent chaperone activity of the FkpA gene product
has also
been demonstrated both in vivo and in vitro. The FkpA chaperone protein is
involved
in a protein-folding process by stabilizing the folding intermediates in the
periplasm. It
was tested whether co-expression of the single chaperone gene (fkpA) was able
to
stimulate the expression of scFvSA fusion proteins, especially among those
that had not
previously expressed well in E. coli.
In order to clone the DNA fragment of the fkpA gene, chromosomal
DNA was extracted from E. coli XL1-Blue cells (Stratagene) and digested with
XhoI.
Thirty-five cycles of PCR were performed using a pair of oligonucleotides
(RX1229:
ACGACGGTTGCTGCGGCGGTC (SEQ ID NO: 32); RX1231: AGGCTCATTAAT
GATGCGGGT (SEQ ID NO: 33); both obtained from Operon Technologies, Inc.) and
300 ng of the digested genomic DNA as a template. The PCR mixture was subject
to a
second round of PCR (30 cycles) using a pair of nested oligonucleotides
(RX1230:
GGATCCAAGCTTACGATCACGGTCATGAACACG (SEQ ID NO: 34); RX1232:
CTCGAGAAGCTTTAACTAAATTAATACAGCGGA) (SEQ ID NO: 35). The PCR
fragments were resolved on a 1 % agarose gel, and the 1.6-kb fragment was
isolated.
The extracted DNA was cloned into the TA vector (Invitrogen), and the sequence
was
confirmed by DNA sequencing. The clone was digested with HindIII, using a site
that
was incorporated into oligonucleotides RX1230 and RX 1232 and was ligated with
HindIII-digested vector E84-2-8 (NeoRx Corp.), harboring the anti-CEA T84.66
scFvSA fusion gene (T84.66 cDNA from City of Hope, Duarte, CA). The resultant
plasmid (Fl 15-1-1) was used to transform XL1-Blue E. coli for shake-flask
expression.
The periplasmic components were extracted and analyzed on 4-20% SDS-PAGE. For
electrophoretic analysis, 20 ~1 of the solution of scFvSA periplasmic fusion
proteins
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were loaded in each lane of the gel. Following electrophoresis, the gel was
stained with
Coomassie Blue 8250. The FkpA protein, with a molecular weight of about
30,000,
was prominently present in all samples carrying the fkpA gene (+), while
absent in those
lacking the gene (-), as shown, for example, in Figure 19. The molecular
weights of the
seven components in the SeeBlue molecular standard marker (M), obtained from
Novex, listed in order of increasing size, from the bottom of the gel, are
16,000;
30,000; 36,000; 50,000; 64,000; 98,000; and 250,000. As seen in Figure 19,
expression
of the T84.66 scFvSA fusion protein increased dramatically when co-expressed
with
the FkpA chaperone protein, in comparison to the parent construct (E84-2-8)
lacking
the fkpA gene. Additional scFvSA fusions were constructed by moving NcoI-SacI
fragments to the F115-1-1 vector, which had previously been restricted with
NcoI and
SacI. The resultant plasmids were tested in E. coli XL1-Blue shake flask
cultures.
Upon electrophoretic analysis, several showed increased fusion protein
expression, as
demonstrated in Figure 19 and Table 2. The results summarized here involve
only the
Vh V,-SA fusion configuration incorporating the (Gly4Ser)5 (SEQ ID NO:
11)linker. As
summarized in Table 2, the expression levels of fusion proteins in the shake
flask
experiments were estimated qualitatively, with the highest level assigned a
level of
+++++.
Table 2. Qualitative expression of scFvSA fusion proteins in E. coli.
Antigen scFvSA SEQ ID Ex ression
level
NO. FkpA FkpA+
CEA T84.66 36 - ++
Col-1 37 - +
PRlA3 38 - -
MFE-23 39 ++++ ++
Nrco-2 40 +++ +
Ta -72 CC49 41 ++ ++++
MUC 1 BrE-3 42 - -+
c-erbB2 ICR12 43 - -
CD20 B9E9 44 +++ +++++
C2B8 45 - -
CD45 BC8 46 + +++
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WO 00/75333 PCT/US00/15595
EXAMPLE VI
PURIFICATION OF HUNR-LU-1 O SCFVSA AND B9E9 SCFVSA PROTEINS
The iminobiotin affinity matrix was prepared by reacting epoxide-
activated Macro-prep matrix (BioRad, Hercules CA) with 112 ~m N-(3-amino-
propyl)-
1,3 propane diamine (Sigma) per g of matrix in 0.2 M carbonate buffer. The
reaction
10 was stopped after 8 h by filtering the slurry through a scintered glass
funnel and rinsing
the matrix with distilled water. Residual epoxides were inactivated by
reacting the
matrix with 0.1 M sulfuric acid for 4 h at 80°C, and the matrix was
again rinsed. The
amine-derivitized matrix was suspended in PBS, and the pH increased to 8.5 by
the
addition of 10% volume of 0.5 M sodium borate, pH 8.5. NHS-iminobiotin
(Pierce)
15 was dissolved in DMSO and added to the suspended matrix at a ratio of 2.6
mg/g of
matrix. Following a 4 h reaction, the matrix was rinsed with distilled water
followed by
several alternating washes with pH 11 sodium carbonate buffer and pH 4 sodium
acetate buffer and a final rinse with distilled water. The matrix was stored
as a slurry in
20% ethanol.
20 Cells (650-750 g, wet wt) were washed twice in PBS, resuspended to 10-
20% weight per volume with ice-cold 30 mM Tris, 1 mM EDTA, pH 8, and disrupted
through two cycles of microfluidization. The lysate was adjusted to 50 mM
glycine,
450 mM NaCI, pH 9.6, with a conductivity range of 46-48 mSe per cm, and then
centrifuged at 12,000 rpm for 90 min. The supernatant was filtered (0.2 p.m),
then
25 affinity purified over immobilized iminobiotin. The iminobiotin matrix was
packed in
a column and equilibrated in 50 mM glycine, 500 mM NaCI, pH 9.6 with a
conductivity of 46-48 mSe per cm. Capacity using recombinant streptavidin
(Roche
Biochemical, Indianapolis, IN) was 2 mg per ml of bed volume under a flow of 2
ml/cm2/min. The 0.2 p,m filtered cell homogenized supernatant was pumped at
room
30 temperature at 2 ml/cm' per min using 80 ml of bed volume per 100 g of
cells. After
washing with 20 bed volumes of column equilibrating buffer, the scFvSA fusion
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41
protein was eluted with 0.2 M sodium acetate, 0.1 M NaCI, pH 4.0, neutralized
with
Tris buffer, and then exhaustively dialyzed in refrigerated PBS.
To reduce protein aggregation, purified scFvSA was treated with 10%
DMSO for 5-7 h at room temperature and dialyzed in PBS. The purified protein
was
concentrated using an Amicon YM30 membrane apparatus and filter-sterilized for
aseptic storage at 4°C. At concentrations of 2-3 mg/ml, purified
preparations typically
contained ca. 5-8% aggregate.
Typical recoveries from iminobiotin chromatography were 50-60% with
less than 5% appearing in the flow-through and wash. The residual remained as
aggregate/entrapped material on the column. Addition of DMSO to the eluting
buffer
yielded <5% additional purified protein. Use of a variety of ionic and
nonionic
detergents did not improve recoveries. HPLC size exclusion analysis of the
eluted
fusion protein showed that up to 40% of the protein was in an aggregated form.
Light
scattering HPLC indicated aggregate sizes between 400,000 and 4 million.
Treatment
with 10% DMSO for several hours resulted in the slow de-aggregation of the
fusion
protein, yielding >92% tetrameric species that remained so when stored
refrigerated in
PBS at a concentration of <3 mg/mL.
EXAMPLE VII
BIOCHEMICAL CHARACTERIZATION OF HUNR-LU-lO SCFVSA AND B9E9 SCFVSA
PROTEINS
SDS-PAGE Analysis. Purified fusion proteins were analyzed on 4-20%
Tris-glycine SDS-PAGE gels (Novex, San Diego, CA) under nonreducing
conditions.
Before electrophoresis, samples were mixed with SDS-loading buffer and
incubated at
either room temperature or 95°C for 5 min. Gels were stained with
Coomassie blue.
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SDS-PAGE demonstrated that the fusion proteins were purified to >
95% homogeneity after iminobiotin chromatography (Figure 12, lanes 2 & 3; huNR-
LU-10 data only). The major band migrated at the expected molecular weight of
173
kDa with minor isoforms evident. These isofonns were also detected with
polyclonal
anti-streptavidin antibody on Western gel analysis (data not shown). However,
all
bands resolved into a single species of ~ 43 kDa when the protein was boiled
prior to
electrophoresis, consistent with a single protein entity dissociable into its
homogeneous
subunit (Figure 12, lanes 4 & 5). The molecular weights of the seven
components in
the SeeBlue molecular standard marker (Figure 12, lane 1), available from
Novex, are
described in Example V.
Size exclusion HPLC and Laser Light Scattering Analysis. Purified
protein preparations were analyzed by size exclusion HPLC performed on a
Zorbax
GF-250 column with a 20 mM sodium phosphate/0.5 M NaCI mobile phase. The
molecular weight of the fusion construct was measured using this Zorbax system
connected in series with a Varian Star 9040 refractive index detector and a
MiniDawn
light scattering instrument (Wyatt Technologies, Santa Barbara, CA). A dn/dc
value of
0.185 for a protein in an aqueous buffer solution was used in the molecular
weight
calculations.
HPLC size exclusion chromatography exhibited a major peak with a
retention time appropriate for the huNR-LU-10 tetramer with a minor (<8%)
aggregate
peak (Figure 13). B9E9 scFvSA showed a very similar profile (graph not shown).
These analyses demonstrated that all of the purified protein was tetrameric or
an
aggregate thereof. Light scattering analysis of huNR-LU-10 scFvSA indicated a
molecular weight of 172,600, as predicted for the tetrameric protein.
Amino-terminal sequencing. Automated amino acid sequencing was
performed using a Procise 494 sequenator (Applied Biosystems, Inc., Foster
City, CA).
This revealed that the leader sequences of both huNR-LU-10 scFvSA and B9E9
scFvSA were cleaved at the expected signal peptidase site adjacent to the
first amino
acid of the variable region.
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Molecular weight determination of B9E9 scFvSA. Liquid
chromatographic separation was conducted with an Hewlett Packard series 1100
system, fitted with a Jupiter C18 column (300 0, 3.2 x 50 mm, 5 ~) and C18
"SafeGuard" column (Phenomenex, Torrance, CA) at a flow rate of 500 ~1/min.
The
mobile phase was composed of water/1% formic acid (buffer A) and
acetonitrile/1%
formic acid (buffer B). The gradient applied was 2% B for 3 min rising to 99%
B
within 7 min. B9E9 scFvSA (10 ~1) was eluted at a retention time of 8.7 min.
The
analytical column was interfaced with a Thermoquest/Finnigan ESI LCQ ion trap
mass
spectrometer (San Jose, CA). The instrument was calibrated with myoglobin and
operated in the positive ion mode with the heated capillary set to
200°C and 5.1 kV
applied to the electrospray needle. The data were acquired in a full scan MS
mode (m/z
[500-2000 Da/z]) using automated gain control with 3 microscans and a maximum
ion
time of 500 ms.
The mass spectrum of the B9E9 monomer showed a deconvoluted
molecular weight of 43,401, which is in agreement with the calculated most
abundant
mass of 43,400.
HuNR LU 10 Competitive Immunoreactivity ELISA. Serial dilutions
of the humanized NR-LU-10 whole antibody or the huNR-LU-10 fusion protein were
allowed to compete with peroxidase-labeled murine NR-LU-10 whole antibody for
binding to an 0.1 % NP40 membrane extract from the human carcinoma cell line,
LS-
174 (ATCC #CL188). Following a log-logit transformation of the data in which
curves
were fit to the same slope, the concentration of competitor antibody that gave
50%
inhibition (k) was calculated. Percent immunoreactivity was determined
according the
formula: k (fusion protein standard)/k (whole antibody standard) x 100. The
huNR-
LU-10 fusion protein was found to possess immunoreactivity superior 0225%) to
the
intact divalent humanized antibody (Figure 14).
B9E9 Competitive Immunoreactivity FA CS Assay. Immunoreactivity
was assessed in a competitive binding assay using flow cytometry that measured
the
binding of fluorescein-labeled B9E9 to the CD20-positive Ramos cell line
(Burkitt's
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lymphoma; ATCC CRL-1596) in the presence of various concentrations of
unlabeled
antibody. B9E9 mAb was labeled using fluorescein N-hydroxysuccinimidate, and
an
optimized amount of this conjugate was mixed with serial dilutions (3-200
ng/ml) of
B9E9 mAb standard or molar equivalents of B9E9 scFvSA and incubated with 1 x
106
cells at 4°C for 30 minutes. Samples were washed and then analyzed on a
single laser
FACSCalibur (Becton Dickinson). After gating on single cells, the geometric
mean
fluorescence intensity was determined from a histogram plot of fluorescence.
The
concentration of competitor antibody required for 50% inhibition (ICS°)
of fluorescein
B9E9 binding was calculated using nonlinear regression analysis for one-site
binding.
Percent immunoreactivity = [ICS° scFvSA/ ICS° mAb] x 100.
The scFvSA was about twice as immunoreactive 0185%) as the
divalent B9E9 antibody on a molar basis, and nearly equivalent (~93%) to B9E9
mAb
when adjusted for tetravalency (graph not shown).
B9E9 scFvSA Avidity. Avidity was determined using saturation binding
experiments that measure specific binding of radiolabeled mAb or fusion
protein
(0.025-50 ng/ml) at equilibrium in the presence of excess antigen (10' cells).
Nonspecific binding was determined in the presence of excess cold mAb or
fusion
protein (50 ~g/ml). Mixtures were incubated and centrifuged as described
above. The
equilibrium dissociation constant (Kd) was calculated from nonlinear
regression
analysis of nM bound vs. nM radioligand using immunoreactivity-adjusted
antibody
concentrations. The B9E9 fusion protein retained the same relative nanomolar
avidity
as the B9E9 mAb, as determined by radiolabeled binding to Ramos cells (Table
3).
Table 3. Avidity of B9E9 mAb and scFvSA fusion protein.
Antibody Kd (nM)a Ka (x 1 O8 M-' )
B9E9 mAb 9.75 1.02
B9E9 scFvSA (25-mer) 12.44 0.80
aAntibody concentrations were adjusted for immunoreactivity (67% and 79% for
mAb
and scFvSA, respectively). Kd and Ka were calculated using nonlinear
regression
analysis of nM bound vs. nM radioligand.
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Biotin Binding and Dissociation. Biotin binding capacity was
determined by incubation of a known quantity of fusion protein with a 9-fold
molar
excess of [3H]biotin (NEN Research Products, Boston, MA). After removal of
uncomplexed biotin using streptavidin-immobilized beads (Pierce Chemical;
Rockford,
S IL), the amount of [3H]biotin associated with the fusion protein was
determined.
HuNR-LU-10 scFvSA and B9E9 scFvSA were capable of binding an
average of 3.0 and 3.6 biotins, respectively, as compared to 4 biotin binding
sites for
recombinant streptavidin.
For huNR-LU-10 scFvSA, the rate of DOTA-biotin dissociation was
10 assessed at 37°C in 0.25 M phosphate, 0.15 M sodium chloride, pH 7.0
containing
either 10 pM fusion protein or recombinant streptavidin and a subsaturating
level of
[9°Y]DOTA-biotin. A 100-fold saturating level of biocytin (Sigma) was
added to
initiate the dissociation measurement. At timed intervals, aliquots of
incubate were
diluted in PBS containing 0.5% bovine serum albumin. In order to precipitate
the
15 protein, zinc sulfate was added to each diluted aliquot, followed by sodium
hydroxide,
each to yield a final concentration of 0.06 M. Following microcentrifugation,
free
[9oY]DOTA-biotin in the supernatant was assessed using a Hewlett Packard beta
counter. The DOTA-biotin dissociation rate of huNR-LU-10 scFvSA was comparable
to that of recombinant streptavidin (t"z of 58 min for huNR-LU-10 scFvSA vs.
47 min
20 for recombinant streptavidin; Figure 15).
For B9E9 scFvSA, biotin dissociation was measured as described above,
except [3H]biotin was used instead of [9°Y]DOTA-biotin. The calculated
t"2 for biotin
dissociation was 379 min for B9E9 scFvSA vs. 364 min for recombinant
streptavidin
(graph not shown).
EXAMPLE VIII
ANALYSIS OF BIODISTRIBUTION OF ~~IIN-DOTA-BIOTIN AFTER PRETARGETING WITH
HuNR-LU-10 scFvSA
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The expressed huNR-LU-10 scFvSA gene fusion was tested in a full
pretarget protocol in female nude mice bearing SW-1222 human colon cancer
xenografts (100-200 mg), subcutaneously implanted on the right flank. In these
experiments, 575 ~g of 'ZSI-labeled fusion protein was injected intravenous
(iv) and
allowed to circulate for 18 hours prior to iv injection of 100 ~g of synthetic
clearing
agent (sCA) (See e.g., PCT Publication Nos. WO 97/46098 and WO 95/15978).
Three
hours after the sCA injection, there was an injection of 1.0 ~g of "'In-DOTA-
biotin,
essentially a chelating agent containing a radionuclide, conjugated to biotin
(see U.S.
Patent Nos. 5,578,287 and 5,608,060). Mice were sampled for blood, then
sacrificed
and dissected at 2, 24, 48, and 120 hours after "'In-DOTA-biotin injection.
The concentration of 'ZSI-huNR-LU-10 scFvSA radioactivity in blood
and most well-perfused soft tissues was very low, due to the low blood pool
concentration induced by the sCA complexation and subsequent hepatic
clearance. The
exceptions were liver and tumor. Liver uptake and retention of fusion protein
was due
to the mechanism of clearing agent action, and the somewhat retarded
degradation of
the streptavidin-containing fusion protein, which was consistent with similar
results
observed in studies of both streptavidin and the chemical conjugate of huNR-LU-
10
and streptavidin (huNR-LU-10/SA) (data not shown). The 'ZSI-huNR-LU-10 scFvSA
exhibited evidence of in vivo immunoreactivity by the retention of relatively
high
radiolabel concentration at the tumor (both stoichiometrically and relative to
blood pool
concentration) at all time points. The ratio of tumor concentration to blood
concentration continuously increased from 23 to 143 hours. The lower blood
pool
values induced by clearing agent have led to a dramatic increase in the ratio,
achieving
average values over twice those observed in the absence of clearing agent
(data not
shown).
The pretargeted "'In-DOTA-biotin biodistribution is shown in Figure
16. Consistent with pretargeting results employing the chemical conjugate
huNR-LU-10/SA, the concentration of "'In-DOTA-biotin radioactivity in blood
and all
non-xenograft soft tissues was very low. Despite the high concentrations of
fusion
protein in the liver noted above, "'In-DOTA-biotin uptake and retention in
this organ
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was not evident, indicating that the fusion protein had been efficiently
internalized and
was unavailable to bind the subsequently administered radiobiotin. The highest
concentration of "'In-DOTA-biotin was at the tumor at all time points. (The
tissues in
order in Figure 16 are blood, tail, lung, liver, spleen, stomach, kidney,
intestine, and
tumor.) The rapid uptake, achieving peak concentrations at the earliest time
point
sampled, is a hallmark of pretargeting. Efficient, consistent delivery and
retention of
"'In-DOTA-biotin at the tumor was also observed. Peak concentrations of "'In-
DOTA-biotin at the tumor were within the range consistently achieved by use of
the
chemical NR-LU-10/SA conjugate (20-25 % injected dose/g) (data not shown).
EXAMPLE IX
ANALYSIS OF BLOOD CLEARANCE AND TUMOR UPTAKE OF HUNK-LU-1 O SCFVSA
VERSUS HUNK-LU-lO/STREPTAVIDIN CHEMICAL CONJUGATE
1S
Tumor to blood ratios of huNR-LU-10 scFvSA increased from nearly
100, two hours after DOTA-biotin injection, to several thousand by 24 hours.
Comparative results for the huNR-LU-10/SA chemical conjugate and fusion
protein,
showing the efficiency of radiobiotin delivery to tumor and corresponding area-
under-
the-curve (AUC) values for blood, and tumor are shown in Figure 17.
The overall tumor AUC using the fusion protein was somewhat less than
that of the chemical conjugate (1726, for the time interval between 0-120
hours, versus
2047 for a typical chemical conjugate experiment). However, there was a
dramatic
difference in the concentration of "'In-DOTA-biotin in the blood pool, with
the
concentration in the fusion protein group consistently lower at all time
points. The
greatest ramification of this decreased retention of radioactivity in the
blood is that
animals treated with the fusion protein experience a higher therapeutic index
(tumor/blood) than those treated with the chemical conjugate.
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EXAMPLE X
PRETARGETED BIODISTRIBUTION OF B9E9 sCFVSA
Pretargeted radioimmunotherapy studies were conducted in female nude
mice bearing well-established Ramos human cancer xenografts (100-400 mg).
Tumored BkI:BALB/c/nu/nu nude mice were obtained by implanting 5-25 x 106
cultured cells subcutaneously in the side midline 10-25 days prior to study
initiation.
Mice received intravenous injections of the'25I-labeled B9E9 scFvSA (600 ~.g),
and 20
hours later were injected intravenously with 100 ~g of synthetic clearing
agent. "'In-
labeled DOTA-biotin (1.0 p.g) was injected intravenously into each mouse 4
hours after
clearing agent. Groups of three mice per time point were bled and sacrificed
at 2, 24,
and 48 hours after injection of "'In-DOTA-biotin. Whole organs and tissue were
isolated, weighed, and counted for radioactivity using a gamma counter.
As shown in Figure 18, the "'In-DOTA-biotin radioactivity in blood and
all non-xenograft soft tissues was below 2% of the injected dose/g. Further,
"'In-
DOTA-biotin uptake and retention in liver is not seen, indicating that the
fusion protein
has been efficiently internalized by the liver, via the added clearing agent,
and is
unavailable to bind the subsequently administered radiobiotin. Stable delivery
and
retention of "'In-DOTA-biotin at the tumor were observed. The highest
concentration
of radiobiotin at all time points was at the tumor (both stoichiometrically
and relative to
blood pool concentration). Peak concentrations of "'In-DOTA-biotin at the
tumor were
17-24 % of injected dose/g (mean 21.66, s.d. 3.17). Tumor to blood ratios
increased
from about 90, 2 hours after DOTA-biotin injection, to greater than 700 by 24
hours. In
these experiments no effort was made to optimize the dose of the fusion
protein,
clearing agent, or DOTA-biotin, nor was any effort made to optimize the
schedule of
administration of these components. (In Figure 18, the tissues in order are
blood, tail,
lung, liver, spleen, stomach, kidney, intestine, and tumor.)
CA 02376192 2001-12-04
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1
SEQUENCE LISTING
<110> Goshorn, Stephen C.
Graves, Scott Stoll
Schultz, Joanne Elaine
Lin, Yakankg
Sanderson, James A.
Reno, Jonh M.
<120> STREPTAVIDIN EXPRESSED GENE FUSIONS AND
METHODS OF USE THEREOF
<130> 690022.547PC
<140> PCT
<141> 2000-06-05
<160> 46
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CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
2
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Met Arg Lys Ile Val Val Ala Ala Ile Ala Val Ser Leu Thr Thr Val
1 5 10 15
Ser Ile Thr Ala Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
3
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Gly Ile Arg Gly Asn Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Gly Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Leu Asn Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Tyr Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
100 105 110
Arg Asn Ala Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
115 120 125
Lys Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
145 150 155 160
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe,Asn Ile
165 170 175
Lys Asp Thr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
180 185 190
Gln Trp Met Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Ser Asp
195 200 205
Leu Ser Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn
210 215 220
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val
225 230 235 240
Tyr Tyr Cys Ser Arg Glu Val Leu Thr Gly Thr Trp Ser Leu Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Gly Ser Ala
260 265 270
Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly
275 280 285
Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr
290 295 300
Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly
305 310 315 320
Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro
325 330 335
Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys
340 345 350
Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr
355 360 365
Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser
370 375 380
Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp
385 390 395 400
Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys
405 410 415
Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln
420 425 430
<210> 5
<211> 1239
<212> DNA
<213> Streptomyces avidinii
<400> 5
gacatcgtgc tgtcgcagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
4
atgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccagga120
tcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgc180
ttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaa240
gatgctgccacttattactgccagcagtggattagtaacccacccacgttcggtgctggg300
accaagctggagctgaagatctctggtctggaaggcagcccggaagcaggtctgtctccg360
gacgcaggttccggctcgagccaggttcagctggtccagtcaggggctgagctggtgaag420
cctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaat480
atgcactgggtaaagcagacacctggacagggcctggaatggattggagctatttatcca540
ggaaatggtgatacttcctacaatcagaagttcaaaggcaaggccacattgactgcagac600
aaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtc660
tattactgtgcaagagcgcaattacgacctaactactggtacttcgatgtctggggcgca720
gggaccacggtcaccgtgagctctggctctggttcggcagacccctccaaggactcgaag780
gcccaggtctcggccgccgaggccggcatcaccggcacctggtacaaccagctcggctcg840
accttcatcgtgaccgcgggcgccgacggcgccctgaccggaacctacgagtcggccgtc900
ggcaacgccgagagccgctacgtcctgaccggtcgttacgacagcgccccggccaccgac960
ggcagcggcaccgccctcggttggacggtggcctggaagaataactaccgcaacgcccac1020
tccgcgaccacgtggagcggccagtacgtcggcggcgccgaggcgaggatcaacacccag1080
tggctgctgacctccggcaccaccgaggccaacgcctggaagtccacgctggtcggccac1140
gacaccttcaccaaggtgaagccgtccgccgcctccatcgacgcggcgaagaaggccggc1200
gtcaacaacggcaacccgctcgacgccgttcagcagtaa 1239
<210> 6
<211> 412
<212> PRT
<213> Streptomyces avidinii
<400> 6
Asp Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ile Ser Asn Pro Pro Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ile Ser Gly Leu Glu Gly
100 105 110
Ser Pro Glu Ala Gly Leu Ser Pro Asp Ala Gly Ser Gly Ser Ser Gln
115 120 125
Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser
130 135 140
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn
145 150 155 160
Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly
165 170 175
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys
180 185 190
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
195 200 205
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe Asp Val Trp Gly Ala
225 230 235 240
Gly Thr Thr Val Thr Val Ser Ser Gly Ser Gly Ser Ala Asp Pro Ser
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
245 250 255
Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly
260 265 270
Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala
275 280 285
Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu
290 295 300
Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp
305 310 315 320
Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr
325 330 335
Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly
340 345 350
Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr
355 360 365
Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr
370 375 380
Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly
385 390 395 400
Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln
405 410
<210>
7
<211>
1280
<212>
DNA
<213> tomyces
Strep avidinii
<400>
7
ccatggctcaggttcagctggtccagtcaggggctgagctggtgaagcctggggcctcag60
tgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaa120
agcagacacctggacagggcctggaatggattggagctatttatccaggaaatggtgata180
cttcctacaatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagca240
cagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaa300
gagcgcaattacgacctaactactggtacttcgatgtctggggcgcagggaccacggtca360
ccgtgagcaagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggct420
cgggtggtggtgggtcgggcggcggcggctcgagcgacatcgtgctgtcgcagtctccag480
caatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtg540
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatg600
ccacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacct660
cttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagc720
agtggattagtaacccacccacgttcggtgctgggaccaagctggagctgaagagctctg780
gctctggttcggcagacccctccaaggactcgaaggcccaggtctcggccgccgaggccg840
gcatcaccggcacctggtacaaccagctcggctcgaccttcatcgtgaccgcgggcgccg900
acggcgccctgaccggaacctacgagtcggccgtcggcaacgccgagagccgctacgtcc960
tgaccggtcgttacgacagcgccccggccaccgacggcagcggcaccgccctcggttgga1020
cggtggcctggaagaataactaccgcaacgcccactccgcgaccacgtggagcggccagt1080
acgtcggcggcgccgaggcgaggatcaacacccagtggctgctgacctccggcaccaccg1140
aggccaacgcctggaagtccacgctggtcggccacgacaccttcaccaaggtgaagccgt1200
ccgccgcctccatcgacgcggcgaagaaggccggcgtcaacaacggcaacccgctcgacg1260
ccgttcagcagtaaggatcc 1280
<210>
8
<211>
423
<212>
PRT
<213> tomyces
Strep avidinii
<400>
8
Met Ala Val Gln Ser Gly Glu Leu Lys Pro
Gln Leu Val Ala Val
Gln
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
6
1 5 10 15
Gly Ala Ser Va1 Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu
35 40 45
Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln
50 55 60
Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
65 70 75 80
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Lys Ile Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu Ser Gln Ser Pro Ala
145 150 155 160
Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala
165 170 175
Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser
180 185 190
Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val
195 200 205
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
210 215 220
Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
225 230 235 240
Trp Ile Ser Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
245 250 255
Lys Ser Ser Gly Ser Gly Ser Ala Asp Pro Ser Lys Asp Ser Lys Ala
260 265 270
Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln
275 280 285
Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr
290 295 300
Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu
305 310 315 320
Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
325 330 335
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser
340 345 350
Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile
355 360 365
Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp
370 375 380
Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser
385 390 395 400
Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn Gly Asn
405 410 415
Pro Leu Asp Ala Val Gln Gln
420
<210> 9
<211> 18
<212> PRT
<213> Artificial Sequence
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
7
<220>
<223> pKOD linker
<400> 9
Gly Leu Glu Gly Ser Pro Glu Ala Gly Leu Ser Pro Asp Ala Gly Ser
1 5 10 15
Gly Ser
<210> 10
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Linker used to create a scFvSA version of
anti-CD20mAb, B9E9 in the VLVH orientation
<400> 10
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 11
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Linker used to create a version of B9E9 scFvSA in
the VHVL orientation
<400> 11
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 12
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 12
tgccgtgaat tcgtsmarct gcagsartcw gg 32
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 13
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
8
tgccgtgaat tccattswgc tgaccartct c 31
<210> 14
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 14
tagctggcgg ccgccctgtt gaagctcttgacaat 35
<210> 15
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 15
tagctggcgg ccgctttctt gtccaccttggtgc 34
<210> 16
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 16
ttacggccat ggctgacatc gtgctgcagtctccagcaat cctgtct 47
<210> 17
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 17
caccagagat cttcagctcc agcttggtccca 32
<210> 18
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 18
cggaggctcg agccaggttc agctggtccagtcaggggct gagctggtga ag 52
<210> 19
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
9
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 19
gagccagagc tcacggtgac cgtggtccctgcgcccca 38
<210> 20
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 20
gatctctggt ctggaaggca gcccggaagcaggtctgtct ccggacgcag gttccggc58
<210> 21
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 21
tcgagccgga acctgcgtcc ggagacagacctgcttccgg gctgccttcc agaccaga58
<210> 22
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 22
ttacggccat ggctgacatc gtgctgtcgcagtctccagc aatcctgtct 50
<210> 23
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 23
ttccggctcg agcgacatcg tgctgtcgcagtctcca 37
<210> 24
<211> 32
<212> DNA
<213> Artificial Sequence
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
<220>
<223> Oligonucleotide primer
<400> 24
gagccagagc tcttcagctc cagcttggtccc 32
<210> 25
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 25
ttacggccat ggctcaggtt cagctggtccagtca 35
<210> 26
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 26
agaccagaga tcttgctcac ggtgaccgtggtccc 35
<210> 27
<211> 79
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 27
gatctctggt ggcggtggct cgggcggtggtgggtcgggt ggcggcggct cgggtggtgg60
tgggtcgggc ggcggcggc 79
<210> 28
<211> 79
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 28
tcgagccgcc gccgcccgac ccaccaccacccgagccgcc gccacccgac ccaccaccgc60
ccgagccacc gccaccaga 79
<210> 29
<211> 18
<212> PRT
<213> Artificial Sequence
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
11
<220>
<223> Linker sequence
<400> 29
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Ser
<210> 30
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Linker sequence
<400> 30
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser
<210> 31
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Linker sequence pKOD2
<400> 31
Gly Leu Glu Gly Ser Pro Glu Ala Gly Leu Ser Pro Asp Ala Gly Ser
1 5 10 15
Asp Ser
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 32
acgacggttg ctgcggcggt c 21
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
12
<400>
33
aggctcattaatgatgcgggt 21
<210>
34
<211>
33
<212>
DNA
<213>
Artificial
Sequence
<220>
<223> primer
Oligonucleotide
<400>
34
ggatccaagcttacgatcacggtcatgaacacg 33
<210>
35
<211>
33
<212>
DNA
<213>
Artificial
Sequence
<220>
<223> primer
Oligonucleotide
<400>
35
ctcgagaagctttaactaaattaatacagcgga 33
<210>
36
<211>
783
<212>
DNA
<213>
Streptomyces
avidinii
<400>
36
gaggttcagctgcagcagtccggggcagagcttgtggagccaggggcctc agtcaagttg60
tcctgcacagcttctggcttcaacattaaagacacctatatgcactgggt gaagcagagg120
cctgaacagggcctggaatggattggaaggattgatcctgcgaatggtaa tagtaaatat180
gtcccgaagttccagggcaaggccactataacagcagacacatcctccaa cacagcctac240
ctgcagctcaccagcctgacatctgaggacactgccgtctattattgtgc tccgtttggt300
tactacgtgtctgactatgctatggcctactggggtcaaggaacctcagt caccgtctcc360
tcaaagatctctggtggcggtggctcgggcggtggtgggtcgggtggcgg cggctcgggt420
ggtggtgggtcgggcggcggcggctcgagcgacattgtgctgacccaatc tccagcttct480
ttggctgtgtctcttgggcagagggccactatgtcctgcagagccggtga aagtgttgat540
atttttggcgttgggtttttgcactggtaccagcagaaaccaggacagcc acccaaactc600
ctcatctatcgtgcatccaacctagaatctgggatccctgtcaggttcag tggcactggg660
tctaggacagacttcaccctcatcattgatcctgtggaggctgatgatgt tgccacctat720
tactgtcagcaaactaatgaggatccgtacacgttcggaggggggaccaa gctggaaata780
aag 783
<210>
37
<211>
786
<212>
DNA
<213>
Streptomyces
avidinii
<400>
37
gaagttcagctgcagcagtctggggcagaacttgtgcgttcaggggcctcagtcaaaatg 60
tcctgcaccgcttctggcttcaacattaaagattactatatgcattgggtgaaacagcgt 120
ccggaacagggcctggaatggattggttggattgatccggaaaatggtgataccgaatat 180
gccccgaaattccagggcaaagccacgatgaccaccgatacctcctccaacaccgcctac 240
ctgcagctcagcagcctgacctctgaagataccgccgtctattactgtaatacccgtggt 300
ctatctaccatgattacgacgcgttggttcttcgatgtctggggcgcagggaccacggtc 360
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
13
accgtctccaagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggc 420
tcgggtggtggtgggtcgggcggcggcggctcgagcgatattgtgctgacccagtctccg 480
gcttccttaaccgtatctctgggtctgcgtgccaccatctcatgccgtgccagcaaaagt 540
gtcagtgcatctggctatagttatatgcattggtaccaacagcgtccgggtcagccgccg 600
aaactcctcatctatcttgcatccaacctacaatctggtgtcccggcccgtttcagtggc 660
agtgggtctgggaccgatttcaccctcaacatccatccggtggaagaagaagatgctgca 720
acctattactgtcagcatagtcgtgaacttccgacgttcggtggtggcaccaaactggaa 780
atcaag 786
<210>
38
<211>
771
<212>
DNA
<213>
Streptomyces
avidinii
<400>
38
caggtgaagctgcagcagtcaggtccggagttgaagaagccgggtgagaccgtcaagatc 60
agctgcaaggcttctggttataccttcaccgtgtttggtatgaactgggtgaagcaggct 120
ccgggcaagggtttaaagtggatgggctggattaacaccaaaactggtgaagcaacctat 180
gttgaagagtttaagggtcgctttgccttctctttggagacctctgccaccactgcctat 240
ttgcagatcaacaacctcaaaaatgaggacacggctaaatatttctgtgcacgttgggac 300
ttctatgattacgtggaagctatggattactggggccaagggaccacggtcaccgtctcc 360
aagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggt 420
ggtgggtcgggcggcggcggctcgagcgatattgtgatgacccagtctcaacgtttcatg 480
tccacttcagtaggtgatcgtgtcagcgtcacctgcaaagccagtcagaatgtgggtacg 540
aatgttgcctggtatcaacagaaaccgggtcaatccccgaaagcactgatttactcggca 600
tcctaccgttacagtggtgtcccggatcgcttcaccggcagtggttctgggaccgatttc 660
acgctcaccatcagcaatgtacagtctgaagacttggcggagtatttctgtcatcaatat 720
tacacctatccgttattcacgttcggctcggggaccaagttggaaatgaag 771'
<210>
39
<211>
762
<212>
DNA
<213>
Streptomyces
avidinii
<400>
39
caggtgaaactgcagcagtctggtgcagaacttgtgcgttcagggacctcagtcaaattg 60
tcctgcaccgcttctggcttcaacattaaagattcctatatgcattggttgcgtcagggt 120
ccggaacagggcctggaatggattggttggattgatccggagaatggtgatactgaatat 180
gcaccgaagttccagggcaaagccacctttactaccgatacctcctccaacaccgcctac 240
ctgcagctcagcagcctgacctctgaagatactgccgtctattattgtaatgaagggact 300
ccgactggtccgtactactttgattactggggtcaagggaccacggtcaccgtctccaag 360
atctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggtggt 420
gggtcgggcggcggcggctcgagcgaaaatgtgctcacccagtctccggcaatcatgtct 480
gcatctccgggtgagaaagtcaccattacctgcagtgccagctcaagtgtaagttacatg 540
cattggttccagcagaaaccgggtacttctccgaaactctggatttatagcacctccaac 600
ctggcttctggtgttccggctcgcttcagtggcagtggttctgggacctcttactctctc 660
accatcagccgtatggaagctgaagatgctgccacttattactgccagcaacgtagtagt 720
tatccgctcacgttcggtgctggcaccaaactggaactgaag 762
<210>
40
<211>
765
<212>
DNA
<213>
Streptomyces
avidinii
<400>
40
caggtccaactacagcagtcagggggagacttagtgaagcctggagggtccctaaaattc 60
tcctgtgcagcctctggattccctttcaatcgctatgccatgtcttgggttcgccagact 120
ccagagaagaggctggagtgggtcgcattcattagtagtgatggtatcgcctactatgca 180
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
14
gacagtgtgaagggccgattcaccatctccagagataatgccaggaacatcctgtaccta 240
caaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagagtttattac 300
tacggtagtagttactttgactactggggccaagggaccacggtcaccgtgagcaagatc 360
tctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggtggtggg 420
tcgggcggcggcggctcgagcgacatccagatgactcagtctccaaaattcatgcccaca 480
tcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgcgggtactaatgta 540
gcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcatcgtct 600
cggaacagtggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctc 660
accatcagcaatgtgcagtctgaagacttggcagagtatttctgtcagcaatataacagc 720
tatcctctggtcacgttcggtgctgggaccaagctggaaataaag 765
<210>
41
<211>
768
<212>
DNA
<213>
Streptomyces
avidinii
<400>
41
caggttcagttgcagcagtctgatgctgaattggtgaaaccgggtgcttcagtgaaaatt 60
tcctgcaaagcttctggctacaccttcaccgatcatgcaattcattgggtgaaacagaac 120
ccggaacagggcctggaatggattggttatttctctccgggtaatgatgatttcaaatac 180
aatgaacgtttcaaaggcaaagccacgctgaccgcagataaatcctccagcaccgcctac 240
gtgcagctcaacagcctgacgtctgaagattctgcagtgtatttctgtacgcgttccctg 300
aatatggcctactggggtcaaggtacctcagtcaccgtctccaagatctctggtggcggt 360
ggctcgggcggtggtgggtcgggtggcggcggctcgggtggtggtgggtcgggcggcggc 420
ggctcgagcgatattgtgatgtcacagtctccgtcctccctaccggtgtcagttggcgaa 480
aaagttaccttgagctgcaaatccagtcagagccttttatatagtggtaatcagaaaaac 540
tacttggcctggtaccagcagaaaccgggtcagtctccgaaactgctgatttactgggca 600
tccgctcgtgaatctggggtcccggatcgcttcaccggcagtggttctgggaccgatttc 660
accctctccatcagcagtgtgaaaaccgaagacctggcagtttattactgtcagcagtat 720
tatagctatccgctcacgttcggtgctgggaccaaactggtgctgaag 768
<210>
42
<211>
765
<212>
DNA
<213>
Streptomyces
avidinii
<400>
42
gaagtgaaacttgaagagtctggtggtggcttggtgcaaccgggtggctccatgaaactc 60
tcttgtgctgcttctggcttcacctttagtgatgcctggatggattgggtccgccagtct 120
ccggagaaagggcttgaatgggttgctgaaattcgtaacaaagccaataatcatggtacc 180
tattatgatgagtctgtgaaagggcgcttcaccatctcacgtgatgattccaaaagtcgt 240
gtgtacctgcaaatgattagcttacgtgctgaagataccgggctttattactgtaccggg 300
gaatttgctaactggggccaggggacgctggtcaccgtctctaagatctctggtggcggt 360
ggctcgggcggtggtgggtcgggtggcggcggctcgggtggtggtgggtcgggcggcggc 420
ggctcgagcgatgttgtgatgacccaaactccgctctccctgccggtcactcttggtgat 480
caagcttccatctcttgccgttctagtcagaaccttgtacataacaatggtaacacctat 540
ttatattggttcctgcagaaatcaggccagtctccgaaactgctgatttatcgcgcatcc 600
atccgcttttctggtgtcccggatcgcttcagtggcagtggttcagaaaccgatttcacg 660
ctcaagatcagccgtgtggaagctgaagacctgggtgtttatttctgctttcaaggtacg 720
catgttccgtggacgttcggtggtggcaccaaactggaaatcaag 765
<210>
43
<211>
741
<212>
DNA
<213>
Streptomyces
avidinii
<400>
43
caggtgcagcttcaggagtcaggacctggccttgtgaaaccctcacagtcactctccctc 60
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
acctgttccgtcactggttactccatcactactgattactggggctggatccggaagttc 120
ccaggaaataaaatggagtggatgggatacataagctacagtggtagcactggctacaac 180
ccatctctcaaaagtcgaatctccattactagagacacatcgaagagtcagttcttcctg 240
cagttgaactctgtaactactgaggacacagccacatattactgtgcaagatacagtagc 300
cttgattactggggccgaggagtcatggtcgcagtctccaagatctctggtggcggtggc 360
tcgggcggtggtgggtcgggtggcggcggctcgggtggtggtgggtcgggcggcggcggc 420
tcgagcgatgttgtgatgacccagacaccaccgtctttgtcggttgccattggacaatca 480
gtctccatctcttgcaagtcaagtcagagcctcgtatatagtgatggaaagacatatttg 540
cattggttattacagagtcctggcaggtctccgaagcgcctaatctatcaggtgtctaat 600
ctgggctctggagtccctgacaggttcagtggcactggatcacagaaagattttacactt 660
aaaatcagcagagtggaggctgaggatttgggagtttactactgcgcgcaaactacacat 720
tttcctctcacgttcggttcg 741
<210>
44
<211>
765
<212>
DNA
<213>
Streptomyces
avidinii
<400>
44
caggttcagctggtccagtcaggggctgagctggtgaagcctggggcctcagtgaagatg 60
tcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagaca 120
cctggacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctac 180
aatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctac 240
atgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagagcgcaa 300
ttacgacctaactactggtacttcgatgtctggggcgcagggaccacggtcaccgtgagc 360
aagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggt 420
ggtgggtcgggcggcggcggctcgagcgacatcgtgctgtcgcagtctccagcaatcctg 480
tctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttac 540
atgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatcc 600
aacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactct 660
ctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggatt 720
agtaacccacccacgttcggtgctgggaccaagctggagctgaag 765
<210>
45
<211>
765
<212>
DNA
<213>
Streptomyces
avidinii
<400>
45
caggttcagctgcaacagccaggggctgagctggtgaagcctggggcctcagtgaagatg 60
tcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagaca 120
cctggacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctac 180
aatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctac 240
atgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagaagcacc 300
tattacggcggtgattggtacttcaacgtctggggcgcagggaccacggtcaccgtgagc 360
aagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggt 420
ggtgggtcgggcggcggcggctcgagccagatcgtgctgtcgcagtctccagcaatcctg 480
tctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttac 540
attcactggtttcagcagaagccaggatcctcccccaaaccctggatttatgccacatcc 600
aacctggcttctggagtccctgtgcgcttcagtggcagtgggtctgggacctcttactct 660
ctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggacc 720
agtaacccacccacgttcggtggcgggaccaagctggagatcaag 765
<210>
46
<211>
780
<212>
DNA
<213>
Streptomyces
avidinii
CA 02376192 2001-12-04
WO 00/75333 PCT/US00/15595
16
<400>
46
caggttcagctggtggaatcaggaggtggcctggtgcagcctggaggatccctgaaactc 60
tcctgtgcagcctcaggattcgatttcagtagatactggatgagttgggtccggcaggct 120
ccagggaaagggctagaatggattggagagattaatccaactagcagtacgataaacttt 180
acgccatctctaaaggataaagtcttcatctccagagacaacgccaaaaatacgctgtac 240
ctgcaaatgagcaaagtgagatccgaggacacagccctttattactgtgcaagagggaac 300
tactataggtacggagatgctatggactactggggtcaaggaacctcagtcaccgtgagc 360
aagatctctggtggcggtggctcgggcggtggtgggtcgggtggcggcggctcgggtggt 420
ggtgggtcgggcggcggcggctcgagcgacatcgtgctgacccagtctcctgcttcctta 480
gctgtatctctgggacagagggccaccatctcatgcagggccagcaaaagtgtcagtaca 540
tctggctatagttatctgcactggtaccaacagaaaccaggacagccacccaaactcctc 600
atctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtct 660
gggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattac 720
tgtcagcacagtagggagcttccattcacgttcggctcggggacaaagttggaaataaag 780
